Meeting Reports 2005

FMF and Beyond
The Fourth International Congress on Systemic Autoinflammatory Diseases
(FMF 2005 - Abstracts)

November 6-10, 2005

Complete List of Abstracts Submitted

  Generation of Mice with TRAPS-associated Mutations

Hirsh  D. Komarow1, Jae J. Chae1, Nina Raben2, Wood Geryl1, and Kastner L. Daniel1           

1Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 2IRP Connective Tissue, NIAMS, NIH, Bethesda, MD

Heterozygous mutations in the extracellular domain of the 55-kDa tumor necrosis factor (TNF) receptor (TNFRSF1A), a key regulator of inflammation, define a periodic fever syndrome, TRAPS (TNF receptor-associated-periodic syndrome) that is characterized by attacks of fever, sterile peritonitis, arthralgia, myalgia, skin rash and/or conjunctivitis; some patients also develop amyloidosis. The autoinflammatory phenotype has been associated with impaired downregulation of membrane TNFRSF1A and diminished shedding of potentially antagonistic soluble receptor. To facilitate additional study of the effects of these mutations on TNFRSF1A, we generated a mouse model for TRAPS.
Two (of the 33 reported) mutations generated by site-directed mutagenesis were utilized for the generation of knock-in mice: Cys33Tyr (C33Y) and Thr50Met (T50M). Heterozygous and homozygous animals for T50M and C33Y have been generated. Current investigations defining their phenotype include observation, mouse core body temperature studies, gene expression studies, Tnfr-1 receptor shedding assays, inflammatory cytokine profiles and histologic studies.
Mice homozygous for T50M and C33Y demonstrate p55 gene and protein expression. Surprisingly though, they appear to be phenotypically similar to tnfsfr1a knockout mice showing resistance to lethal doses of lipopolysaccharide, decreased cell surface receptor and inflammatory cytokines expression. Endotoxin induced hypothermia, a characteristic inflammatory response however, was more pronounced in T50M heterozygous mice. Although mice with TRAPS-associated mutations do not appear to be a model of the human disease, they may reveal important insights into Tnf receptor biology and it’s role in health and disease.

  Not a Usual Sunday

Donald P. Goldsmith               

Drexel University College of Medicine-Section of Rheumatology, St. Christopher's Hospital for Children, Philadelphia, PA

"We always knew that he wasn't going to make it." This sounded oddly almost as a single voice, even rehearsed, although I knew that it couldn't possibly be so. I hadn't seen them for several years. There is no easy way to cope with the loss of a child yet with strong personal family and social support it had seemed then, outwardly at least, that they had moved through the bereavement process somewhat seamlessly. They came now to my strangely quiet office on this Sunday morning because I had thought, and they had agreed, that they might like to know more about all the recent NOMID/CINCA scientific and therapeutic break throughs. We had once been so joined together, they as young parents of an always sick child, and I as a young pediatrician just starting a subspecialty career in rheumatology. What was this hideous disease that was slowly but inexorably stealing their child's ability to walk, hear, and see? The relentless rash and fever had finally been controlled at least partially with corticosteroids but at such a high biologic cost. The three of us always had been awed by his remarkably even temperment as if he deemed us all never to be gloomy, and for me not to seem so helpless in the face of his decline. "His temperment right up the very end was something truly special," we all agreed. Those words abruptly revived memories of the last few days and hours of his life. A case of chicken pox which had suddenly recast itself into hyperpyrexia and unconsciousness. He died at the age of nine, in 1983, in the transport helicopter racing to our emergency department.

I inquired about his older sister who been inordinately traumatized, being the index family case of chicken pox. As his hearing had failed, she, along with her parents, learned to "sign", forever solidifying her attachment to him. She now lived in Colorado, living out a childhood dream in a small mountain community, continuing to be a great joy to her parents, and also soon to be married.

Years ago his parents knew that there was a scattering of families with more than one affected person with NOMID/CINCA. Two more younger children had been perfectly okay as were many of their nieces and nephews. But would their grandchildren-to-be have NOMID/CINCA?
When a child dies it's not something that parents get over, they just adjust somehow. We talked about missense mutations, the promise of anti-cytokine therapy, the new categories of disease, and how their strong marriage and support systems benefited them over the years. They seemed astonished that there now was a parent-based NOMID/CINCA chat room. They weren't yet ready for that. A few days later we spoke again on the telephone. They were grateful. I knew there would always be something more to talk about, and that was comforting.
  A functional defect of TNFRSF1A signaling in R92Q leukocytes suggest that this low penetrance variant is not a neutral polymorphism

Belinda Nedjai1, Mark D. Turner1, Michael McDermott2, and Graham A. Hitman1            

1Departement of diabetes and metabolic medicine, Institute of cell and molecular sciences. 4 newark street. Whitechapel. E1 2AT London; 2Clinical Science BuildingSt. James's University Hospital. University of Leeds. Beckett Street Leeds LS9 7TF, UK

Tumour necrosis factor-alpha (TNF-alpha) is a key molecule in inflammatory disease, mediating its actions through two receptors, TNF receptor 1 and 2 (TNFR1 and TNFR2, also known as TNFRSF1A and TNFRSF1B). We have previously described 25 TNFRSF1A mutations which lead to the pro-inflammatory disorder TNFR-associated periodic fever syndrome (TRAPS) including R92Q variant. R92Q was found in two unrelated patients, including one with a family history of recurrent inflammatory disease. The presence of this variant in the normal control population has been reported in ~1% control chromosomes. This finding raises the question as to whether it is a true disease associated mutation. However, the increased frequency of R92Q among patients with TRAPS compared to controls supports the notion that R92Q is a low penetrance mutation rather than a benign polymorphism. Familial segregation of R92Q with disease has also been described, together with the development of amyloidosis, in a Dutch family

The purpose of the current study was to investigate whether the R92Q variant of TNFRSF1A causes defective TNFRSF1A signaling and NF-kappaB activation, which might in turn lead to the pathophysiology of TRAPS and other inflammatory diseases.

Nuclear and cytosolic fractions were generated from peripheral blood leukocytes (PBLs) from 2 TRAPS patients (TNFRSF1A R92Q, mutation) and 7 healthy controls. Western blot and ELISA analyses were performed using antibodies against NF-kappaB subunits, and electromotility shift assay (EMSA) analysis was performed using a 32P-labelled NF-kappaB probe. Statistical significance was determined using T-test.

The pattern of subcellular RelA (p65) NF-kappaB subunit localisation from control cells and both R92Q variants was primarily cytosolic, as expected in normal cells. Western blotting, using an antibody against IκB-alpha revealed a similar localisation of this protein to the cytosolic fraction of R92Q cells and controls. Surprisingly, when we perform an EMSA using a 32P-labelled NF-kappaB probe, we found elevated levels of NF-kappaB in the nucleus of R92Q leukocytes. Therefore we perform an Elisa and confirm the results we obtained with EMSA.

These findings suggest that the R92Q TNFRSF1A variant leads to a defect in NF-kappaB signaling. We hypothesise that altered NF-kappaB subcellular localization may explain some of inflammatory processes which occur in TRAPS and could therefore open new routes for therapeutic disease intervention.

  Investigation of TNF Receptor Signalling in Peripheral Blood Mononuclear Cells in TNF-Receptor 1 Associated Periodic Syndrome (TRAPS)

Elizabeth Drewe1, Mark B. Frank2, Igor Dozmorov2, Michael Centola2, Richard J. Powell1, and Patrick J. Tighe1          

1Clinical and Molecular Immunology, University Hospital Nottingham, UK; 2Oklahoma Medical Research Foundation, Microarray Research Facility, Oklahoma City, USA

BACKGROUND: TRAPS is an autosomal dominant autoinflammatory disease characterised by recurrent episodes of fever with localised sites of inflammation. TRAPS is genetically defined by mutations in the extracellular domain of TNFR1. Skin and muscle biopsies in TRAPS demonstrate lymphocytic and monocytic infiltrates. Defects in TNFR1 shedding are implicated in the pathogenesis of TRAPS but this is dependant on both mutation and cell type studied, suggesting other mechanisms may be involved. We sought to investigate whether TNFR signalling is attenuated in TRAPS by studying gene expression by gene arrays and NF-kB protein levels by TransFactor assays.
METHOD: Gene arrays: RNA was prepared from peripheral blood mononuclear cells of 30 samples from 8 subjects with C33Y TRAPS and 11 healthy individuals. Cy-3-dUTP labelled cDNA was produced and hybridized to 21,329 gene human oligonucleotide microarrays. Cy3 intensity was assessed using ImaGene 5 software and results imported in to PathwayAssist superimposing data on pathways examining TNFR1 signalling. TransFactor assays: Nuclear extracts were prepared from 11 subjects (38 samples) with C33Y TRAPS and 8 healthy controls. ELISA was performed on plates coated with DNA consensus binding sequence for NF-kB p65.
RESULTS: Gene arrays: Differences in gene expression between TRAPS subjects and controls were subtle. FADD expression was reduced 1.8 fold in TRAPS compared to controls but caspase 8 levels were unchanged. NF-kB1 and Jun mRNA were reduced 1.5 fold and 1.3 fold respectively in TRAPS. SODD expression was increased 1.2 fold in TRAPS. TransFactor assays: NF-kB p65 protein levels were significantly reduced in TRAPS compared to controls, Mann Whitney test p=<0.0001.
CONCLUSION: TRAPS peripheral blood mononuclear cells exhibit evidence of reduced TNFR1 signalling compared to controls. It is unclear whether this represents an inhibitory effect of TNFR1 mutation on cell signalling or downregulation of signalling pathways secondary to chronic stimulation.

  Resistance of SAA 1.5 to Degradation by MMP-1 as an Explanation for Protection Against Amyloidosis

Jeroen C. van der Hilst1, Toshiyuki Yamada2, Evelien J. Bodar1, Joost P. Drenth3, Jos W. van der Meer1, and Anna Simon1          

1Department of General Internal medicine, Univerity Medical Center Nijmegen st Radboud, Nijmegen, the Netherlands; 2Clinical Laboratory Medicine, Jichi Medical School, Minamikawachi, Tochigi, Japan; 3Department of gastroenterology and hepatology, Univerity Medical Center Nijmegen st Radboud, Nijmegen, the Netherlands

Introduction
Reactive amyloidosis is a serious complication of the hereditary periodic fever syndromes. Amyloid fibrils are derived from cleaved serum amyloid A (SAA) proteins. Polymorphisms in the SAA1 gene lead to three isoforms: SAA 1.1, SAA 1.3, and SAA 1.5, which are associated with different relative risks for developing amyloidosis. Matrix metalloproteinases (MMP) have been implicated in the pathogenesis of amyloidosis.

Aim of the study:
To investigate the degradation of the different SAA isoforms by MMPs.

Material and Methods:
Recombinant SAA1.1, 1.3, -and 1.5 were produced in an E.coli expression system and exposed to MMP-1, -2, and –3. After various time intervals, the reaction was stopped and samples were subjected to Tris-Tricine SDS polyacrylamide gel electrophoresis and western-blotting. Fragments of degradation were analysed with MALDI-TOF mass spectrometry.

Results:
MMP-1 was the most potent in degrading SAA (MMP-1 37% degradation, MMP-2 4%, MMP-3 25% ). SAA1.5 was relatively resistant to degradation by MMP-1 compared to SAA 1.1 and -1.3.(SAA 1.1 48%, SAA .1.3 54%, SAA 1.5 8% p=0.01) . Western blot analysis revealed two bands at 6 and 6.5 kd in the SAA 1.1 and SAA 1.3 samples not found in SAA 1.5. Mass spectrometry showed that these bands corresponded to fragments 1-57 and 57-104.

Conclusion:
Isoform SAA 1.5, which is associated with the lowest risk for amyloidosis, is resistant to degradation by MMP-1. The 1-57 fragment that is formed from SAA 1.1 and 1.3 might enhance amyloidosis directly by incorporation into fibrils or may act as Amyloid Enhancing Factor.

  Mechanism of colchicine anti inflammatory effect in FMF and other rheumatic diseases: A possible new outlook through microarray analysis

Eldad Ben-Chetrit1, Sven  Bergmann2, and Ruchira  Sood1             

11Department of Biochemistry, School of Medicine, Stanford University, California, USA,; 21Department of Biochemistry, School of Medicine, Stanford University, California, USA,; 3Current address: Hadassah University Hospital, Jerusalem, Israel 12000

Background: Colchicine is an alkaloid that is used to alleviate acute gout and to prevent acute attacks of Familial Mediterranean Fever (FMF). However, it is not beneficial when given during the occurrence of an acute FMF episode. It is believed that colchicine exerts its anti inflammatory effect through direct interaction with microtubules.
Objective: To study the molecular basis of colchicine action by analyzing the effect of this drug on global gene expression of HUVEC cell line.
Methods: HUVEC cells were exposed to various concentrations of colchicine and were harvested at different time points. RNA was extracted, amplified, reverse transcribed and hybridized to cDNA microarrrays containing more than 40,000 probes to human ESTs. This approach enabled us to have a global look on the transcriptional response induced by colchicine treatment.
Results: Colchicine changed the expression of many genes in HUVEC cells following exposure to 100 ng/ml or higher concentration. Following short exposure (30 or 120 minutes) colchicine affected genes known to be involved in the cell-cycle and its regulation. However, change in expression of genes involved in neutrophil migration or other inflammatory processes were observed mainly after 12 to 24 hours.
Conclusions: The anti-inflammatory effect of colchicine may be mediated not only through direct interaction with microtubules, but also through changes at the transcriptional level. This latter effect apparently requires a higher concentration and a longer time to occur. This can explain the observation that colchicine does not have an immediate effect when given during an acute FMF attack.


  Ex Vivo Treatment with IL-1ra Inhibits TNF-alpha and IL-6 Production by PBMCs from HIDS Patients.

L.M. Kuijk1, J. Frenkel1, S.H.L. Mandey2, H.R. Waterham2, and G.T. Rijkers1           

1Paediatric Immunology, University Medical Centre Utrecht, Utrecht, The Netherlands; 2Laboratory Genetic Metabolic diseases, Academic Medical Centre, Amsterdam, The Netherlands

Background: Hyper IgD and periodic fever syndrome (HIDS) is an auto-inflammatory syndrome characterised by recurrent episodes of fever and inflammation. The enzyme affected is mevalonate kinase, which plays a role in cholesterol and non-sterol isoprenoid biosynthesis. Somehow, this defiency leads to fever and inflammation. We have previously shown that IL-1beta plays an important role. However, several other proinflammatory cytokines (gamma-IFN, TNF-alpha and IL-6) may be involved. We investigated whether activation of these cytokines is a consequence of IL-1beta action or an independent process.
Methods: LPS-stimulated peripheral blood mononuclear cells (PBMCs) from HIDS patients (n=3) were cultured in presence or absence of recombinant human interleukin-1 receptor antagonist (IL-1ra) and the production of cytokines was measured using multiplex bead analysis.
Results: LPS greatly induced production of IL-1beta, TNF-alpha and IL-6, but not of gamma-IFN. Presence of IL-1ra slightly reduced IL-1beta levels and decreased levels of both TNF-alpha and IL-6 significantly. Levels of IL-1alpha, IL-8 and IL-10 also increased after LPS stimulation, but were not reduced in the presence of IL-1ra.
Conclusion: These data suggest that in HIDS most, but not all proinflammatory cytokine activation is IL-1beta dependent. Treatment of patients with anakinra could therefore be expected, not only to reduce the effect of IL-1 alpha and beta, but also of TNF-alpha and IL-6, making it an attractive candidate drug.

  Inhibition of Amyloidosis by Lovastatin

Jeroen C. van der Hilst1, Barbara Kluve-Beckerman2, Joost P. Drenth3, Evelien J. Bodar1, Jos W. van der Meer1, and Anna Simon1          

1Department of general internal medicine, Univerity Medical Center Nijmegen st Radboud, Nijmegen, the Netherlands; 2Amyloid Research Group, Indiana University School of Medicine, Indianapolis, IN; 3Department of gastroenterology and hepatology, Univerity Medical Center Nijmegen st Radboud, Nijmegen, the Netherlands

Introduction
Amyloidosis is a severe complication of the hereditary periodic fever syndromes. However, in Hyper Ig-D and periodic fever syndrome (HIDS), amyloidosis is rare despite a vigorous acute-phase response. HIDS is caused by a mutation in the gene coding for mevalonate kinase, an enzyme of the isoprenoid pathway.

Aim of the study
To investigate if inhibition of the isoprenoid pathway influences amyloidogenesis in a cell culture system

Material and methods
Human monocytes were isolated from healthy volunteers and grown in 96-well plate. Cells were exposed to recombinant serum amyloid A (SAA) and amyloid enhancing factor for 10 days. Lovastatin, a HMG-CoA reductase inhibitor, or vehicle were added to the cultures in increasing concentrations. After 10 days cells were collected, fixated on a cover slide and stained with Congo red. Images of samples were scanned and the area covered with Congo Red was calculated by computer.

Results
The amount of amyloid formed in the cell culture system was dependent on the concentration of SAA in the cell culture medium and duration of incubation. Addition of lovastatin to the medium resulted in a dose dependent reduction of amyloid formed (33% reduction at 1 mM (p<0.05), 41% at 5mm (p<0.05), 54% at 10 mM (p=0.01)).

Conclusion
Inhibition of isoprenoid metabolism by lovastatin decreases the formation of amyloid depositions.

  Gene Expression Profiling in Pyrin-Null Mice

Silvia Stojanov1, Hong-Wei Sun2, James Balow1, Ivona Aksentijevich1, Daniel L. Kastner1, and Jae Jin Chae1          

1Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 2Biodata Mining and Discovery Section, Office of Science and Technology, NIAMS, NIH, Bethesda, MD

The familial Mediterranean fever (FMF) protein, pyrin/marenostrin, is encoded by the MEFV gene and involved in the regulation of inflammation and apoptosis. Human pyrin consists of an N-terminal pyrin domain (PYD), followed by a B box, coiled-coil and a C-terminal B30.2 domain, which is largely missing in mice. In order to study the physiologic role of pyrin in vivo, we initially developed a mouse model expressing a hypomorphic truncated form of pyrin. These pyrin-truncated mice exhibit heightened sensitivity to endotoxin and a defect of macrophage apoptosis. However, the function of pyrin cannot be fully addressed in this system due to the retention of a functional PYD that can interact with the apoptosis related speck-like protein with CARD (ASC). Thus we produced pyrin-null mice (pyrin -/-) by removing exon 1 and exon 2 of MEFV. Similar to pyrin-truncation mice, pyrin -/- animals were produced at the expected Mendelian frequency, developed normally, and reproduced as well as wild-type (WT) littermates. We studied the gene expression profile in resident peritoneal macrophages of 7.5 to 9 week- and 13 to 16 week- old male C57BL/6 pyrin -/- and WT mice. Labeled cRNA prepared from total RNA of peritoneal macrophages was hybridized to the MOE430A 2.0 GeneChip (Affymetrix), containing 22690 transcripts. For statistical analysis we generated four-step filtering criteria. In both age groups of the pyrin -/- mice, 85 genes were found to be differentially expressed. The largest functional group of mRNAs that showed altered expression comprised genes involved in inflammation and the immune response. Consistent with our previous results for pyrin truncation mice, pyrin -/- mice showed an up-regulation of several members of the interleukin-1 (IL-1) family, which was confirmed by Western blot for IL-1 beta. In addition, a number of other genes were upregulated, including cryopyrin, chemokines, Fc receptors, and genes involved in the stress response, further suggesting that reduction in pyrin activity leads to autoinflammation. Nevertheless, underlining the complexity of the regulatory network, genes involved in host defense to pathogens were downregulated relative to WT mice. Studies are currently in progress to further validate our findings by alternative methods, such as quantitative real-time RT-PCR, Western blot, and ELISA.
  Pyrin, product of the MEFV locus, interacts with Siva

Banu Peynircioglu12, Ann Staubach2, Neil Richards2, and Deborah Gumucio2            

1Department of Medical Biology, Faculty of Medicine, Hacettepe University, Ankara, Turkey.; 2Department of Cell and Developmental Biology, 5704 Medical Science II, University of Michigan, Ann Arbor, MI 48109-0616.

Familial Mediterranean Fever (FMF) is a recessive autoinflammatory disorder, which commonly affects families of Mediterranean decent. As the most common hereditary periodic fever sydrome, symptoms of FMF occur as brief episodes of fever, severe localized inflammation and occasionally, amyloidosis. The cause of this disease was recently attributed to mutations in MEFV (MEditerranean FeVer), the gene encoding the protein product pyrin. In order to elucidate the role of pyrin in inflammation, we are studying the molecular interactions mediated by the various domains of pyrin. We carried out a yeast two-hybrid screen of a cDNA library generated from neutrophils aspirated from a knee joint of a patient with gout. Both ASC and PSTPIP1 were isolated from this library. A third protein, Siva, was also isolated and confirmed by immunoprecipitation to be a pyrin-interacting protein. Siva (named for the Hindu God of Death) exists as two different splice variants: Siva-1 is the full length protein containing four exons while Siva-2 lacks exon 2, the central region of the protein that displays weak homology to a death domain. Previous work demonstrated that the N and C terminal regions of Siva bind the cytoplasmic portion of CD27, a tumor necrosis factor (TNF) family member, and trigger an apoptotic signaling cascade in T lymphocytes. The N-terminus of Siva contains a 20-amino acid amphipathic helical region (SAH), which localizes both Siva variants to the cell nucleus while the C-terminus contains zinc fingers. To learn more about Siva and its interaction with pyrin, we conducted colocalization and immunoprecipitation studies. We find that both Siva-1 and Siva-2 bind pyrin, but the interaction may be more robust with Siva-2. Preliminary data suggest that exon 10 of pyrin is important in the binding of Siva. This is particularly interesting since the majority of mutations in pyrin occur within exon 10. Thus, the three pyrin-interacting proteins (ASC, PSTPIP1 and Siva) interact with three different regions of the pyrin molecule (exon 1, containing the pyrin domain; exons 3-5, containing the B-box and a portion of the coiled coil region; and exon 10, encoding the rfp or B30.2 domain).
  The Structure of the SPRY Domain – Implications for the Pyrin Protein and Familial Mediterranean Fever (FMF)

Seth L. Masters, Shenggen Yao, Tracy A. Willson, Jian-Guo Zhang, Kirsten R. Palmer, Brian J. Smith , Jeffrey J. Babon, Nicos A. Nicola, Raymond S. Norton, and Sandra E. Nicholson     

1The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Australia

The SPRY domain is a prevalent protein-protein interaction motif. With more than 300 proteins containing this domain now known, and 76 alone coded for by the human genome, it is surprising that a structure for the SPRY domain has yet to be determined. We have solved the solution structure of the SPRY domain-containing SOCS box protein-2 (SSB-2) by NMR. The structure of the SPRY domain was observed to adopt a novel fold, and this provides for a classification of the SPRY and related B30.2 domains, which are not currently well defined. Due to the conserved nature of secondary structure elements among SPRY domain proteins, we argue that this structure will also apply to other SPRY domain proteins, such as in pyrin, where mutations causing Familial Mediterranean Fever (FMF) cluster within this domain.

We have also performed structure/function analysis of the SSB-2 SPRY domain, as the structure was suggestive of a rigid central core, with flexible loop regions dictating binding specificity. This documented conserved binding residues required to bind the receptor for hepatocyte growth factor (HGF), c-Met, and a novel interaction partner, Prostate Apoptosis Response protein-4 (Par-4). A structure for the SPRY domain of pyrin was modelled on that from SSB-2, and the point mutations causing FMF were analysed. Indeed almost all of these affected the flexible loop regions associated with protein binding. Most interestingly, this also provides some evidence towards a structural explanation for the putative genotype/phenotype correlation presenting due to FMF. This would implicate M694V/I and M680I as the most severe and penetrant mutations while V726A is associated with a less penetrant disease with a better prognosis. In the modelled structure of pyrin, the SPRY domain residues M694 and M680 both fall on loop regions that when mutated in SSB-2 lead to an abrogation of binding both Par-4 and c-Met. In contrast, when the residue analogous to pyrin V726 was mutated, SSB-2 was still able to bind both Par-4 and c-Met. Therefore if FMF is due to the inability of pyrin to bind a target protein via its SPRY domain, V726 may not be as critical for the interaction, or be required for a different interaction than M964 and M680.

In conclusion we have documented the first structure of a SPRY domain as a part of the protein SSB-2, and found that it adopts a novel fold with conserved residues required to bind Par-4 and c-Met. A structure for the SPRY domain of pyrin was modelled on this, and disease causing mutations were found to map on loop regions associated with protein binding that perhaps provide an explanation for the genotype/phenotype correlation observed in FMF.

  The Effect of NC-503 (Fibrillex; 1,3-propanedisulfonate; 1,3-PDS) in the Treatment of Amyloid A (AA) Amyloidosis in Patients with FMF: a Sub-Group Analysis of an International, Randomized, Placebo-Controlled Study

Avi Livneh1, Ahmet Gul2, Huri Ozdogan2, Haner Direskeneli3, Philip N. Hawkins4, Helen J. Lachmann4 , Laura M. Dember5, Martha Skinner5, Eric Hachulla6, Giampaolo Merlini7, Laura Obici7, Merrill D. Benson8, Peter D. Gorevic9, Wendy Hauck10, and Denis Garceau10

1Heller Institute of Medical Research, Sheba Medical Center, Tel Hashomer, Israel 52621; 2University of Istanbul, Istanbul, Turkey; 3Marmara University, Istanbul, Turkey; 4Royal Free and University College Medical School, London, United Kingdom; 5Boston University, Boston, MA; 6Hopital Claude Huriez, Lille, France; 7University Hospital San Matteo, Pavia, Italy; 8Indiana University, Indianapolis, IN; 9 Mount Sinai Medical Center, New York, NY; 10 Neurochem Inc., Laval, Canada

Background: The outcomes in patients with familial Mediterranean fever (FMF) complicated by AA amyloidosis are disappointing. Despite colchicine treatment, >50% of FMF-amyloidosis patients with nephrotic syndrome, and all those with a serum creatinine (SCr) >1.5mg% will progress to chronic renal failure/dialysis. NC-503 inhibits interactions between amyloid and glycosaminoglycans, thus interfering with amyloidogenesis.
Aim: To determine the efficacy and safety of NC-503 in the treatment of AA amyloidosis in the FMF patient sub-group.
Methods: Adults with a biopsy confirmed diagnosis of AA amyloidosis and having renal involvement were randomized 1:1, receiving either placebo or NC-503 for 2 years. The primary composite endpoint classified disease as ‘worsened’ (using a stringent definition of deterioration: either doubling of SCr, ≥50% reduction in creatinine clearance [CrCl], progression to dialysis/ESRD, or death), ‘improved’ (≥50% increase in CrCl and none of the parameters of worsened disease), or ‘stable’, relative to baseline. A separate statistical analysis in the FMF patient sub-group is presented here.
Results: One hundred eighty three patients with AA amyloidosis, due to various underlying inflammatory conditions, were recruited. After 2 years, a total of 24 of the 89 patients in the NC-503 group (27%) fulfilled the primary endpoint criteria for deterioration, as compared with 38 of the 94 patients in the placebo group (40%). Cox proportional hazards regression analysis showed that NC-503 was associated with a 42% reduction in risk of renal deterioration or death (95%C.I. 37-93%, p=0.025). The 2-year median change in the glomerular filtration rate (GFR, as measured by 24 hour urine collection), NC-503 vs placebo, was -11.7 vs -17.2 (p=0.025). The FMF sub-group, which included a total of 36 recruited FMF patients (15 on NC-503 vs 21 on placebo), showed a reduction in deterioration with NC-503: 2 vs 6 patients (risk reduction of 55%, p=0.32). After 2 years, the median change in GFR (mL/min/1.73 m2) was –7.2 with NC-503, as compared with –15.8 with placebo (p=0.63). For FMF patients with nephrotic syndrome at baseline (25%), the median treatment difference in GFR was –19.2 mL/min/1.73 m2 (p=0.093; –2.0 with NC-503 vs –21.2 with placebo).
Conclusions: NC-503 demonstrated clinically meaningful beneficial effects on renal disease progression in AA amyloidosis patients in this first, large, therapeutic trial of an anti-amyloid compound. In the FMF sub-group, results also suggest a benefit of NC-503 over placebo, but the trend failed to reach significance, probably due to the small size of the FMF patient group.
  A role for isoprenoid biosynthesis in IL-1beta secretion

Saskia H. Mandey1*, Loes M. Kuijk2*, Joost Frenkel2, and Hans R. Waterham1            

1Laboratory Genetic Metabolic Diseases, Departments of Clinical Chemistry and Pediatrics, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 2Departments of General Pediatrics and Pediatric Immunology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands ; *These authors contributed equally to this work.

Mevalonate kinase deficiency (MKD) is an autosomal recessive auto-inflammatory disorder characterized by recurring episodes of inflammation. Mevalonate kinase catalyzes an early step in isoprenoid biosynthesis. Although the precise mechanism by which defective activity of MK may lead to inflammation is still unknown, it is believed that it is due to a temporary shortage of prenylated proteins.
Ex vivo, LPS-stimulated peripheral blood mononuclear cells (PBMCs) from MKD patients, secrete elevated levels of interleukin (IL)-1beta. We studied whether this high IL-1beta secretion could be explained by a reduced isoprenoid biosynthesis.
Treatment of PBMCs from controls with an HMG-CoA reductase inhibitor, a farnesyl pyrophosphate synthase inhibitor or a geranylgeranyltransferase inhibitor led to an increase in IL-1beta secretion. In contrast a farnesyltransferase inhibitor did not affect IL-1beta secretion indicating that the increase in IL-1beta secretion in impaired isoprenoid biosynthesis in control PMBCs is due to a lack of geranylgeranylated proteins. This was supported by experiments in PBMCs of patients in which the IL-1beta secretion was reduced in the presence of geranylgeranylpyrophosphate (GGPP). When we incubated PBMCs of MKD patients and controls with a squalene synthase inhibitor the (simvastatin and MKD-induced) increase in IL-1beta secretion was lowered suggesting that a shortage of non-sterol products leads to elevated IL-1beta secretion.
To evaluate the possible role of elevated mevalonate levels in the inflammation associated with MK deficiency disorders, we investigated the inflammatory response of peripheral blood mononuclear cells (PBMCs) to exposure with a concentration range of mevalonate in the presence or absence of lipopolysaccharides (LPS). An increase in IL-1beta secretion was only observed after incubations of PBMCs of controls with excessive, non-physiological amounts of mevalonate. Mevalonate reduced the elevated IL-1beta secretion in PBMCs of MKD patients.
From these observations we conclude that mevalonate is unlikely to cause inflammation in MK deficiency and that it is a shortage of geranylgeranylated proteins that contributes to increased IL-1beta secretion by PBMCs of MKD patients.

  Human Monocytes Have Complete Capacity to Initiate Pathogenesis of Reactive Amyloidosis

Nadine Magy1, Barbara Kluve-Beckerman1*, and Merrill D. Benson1             

1Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN USA 46202; 2Department of Veterans Affairs Richard L. Roudebush VA Medical Center, Indianapolis, IN USA 46202 ; *These authors contributed equally to this work.

Reactive amyloidosis is a systemic protein deposition disease that develops in association with chronic inflammatory conditions including FMF, TRAPS, and Muckle-Wells syndrome. The deposits are composed of extracellular, fibrillar masses of amyloid A (AA) protein, which is an N-terminal fragment of the acute phase serum protein serum amyloid A (SAA). The pathogenic conversion of SAA into amyloid has been studied in two human cell culture models, peritoneal cells and peripheral blood monocytes. Human monocyte cultures proved more robust than either mouse or human peritoneal cells at initiating amyloid formation in the absence of a preformed nidus such as amyloid-enhancing factor and particularly well-suited for examination of individual cells engaged in nascent amyloid formation. Monocyte cultures engaged in amyloid formation were examined by Congo red (amyloid), Alcian blue (glycosaminoglycans), and immunocytochemical (SAA/AA, CD68, CD14, Lamp-1, EEA1) staining and by direct fluorescence confocal microscopy (SAA). These studies showed that AA amyloid fibrils are generated intracellularly, exocytosis of fibrils takes place via lysosome-derived vesicles, and amyloid formation triggers dramatic production of glycosaminoglycans. Cytotoxic effects were not observed at any stage of amyloid formation. Amyloid was composed of C-terminally processed fragments of SAA, with the smallest one being approximately 9 kD. In summary, human monocytes demonstrate totipotency for carrying out the initial phase of amyloidogenesis, i.e., intracellular SAA accumulation, nucleation, fibril initiation, re-location of nascent amyloid to extracellular sites, incorporation of glycosaminoglycans into amyloid , and C-terminal cleavage of SAA to AA.
  Beneficial Response to Interleukin-1-receptor Antagonist (IL-1ra) in Patients with the Hyper IgD and Periodic Fever Syndrome (HIDS).

Evelien J. Bodar1, Anna Simon1, Jeroen C. van der Hilst1, Joost P. Drenth2, and Jos  W. van der Meer1           

1Department of General Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands; 2Department of Gastroenterology, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands

Interleukin-1 (IL-1) is one of the central pro-inflammatory cytokines, and it has been implicated as an important pathogenic factor in several autoinflammatory syndromes, including the Hyper-IgD and periodic fever syndrome (HIDS). Good results with interleukin-1 receptor antagonist (IL-1ra) treatment have been reported in patients with Muckle Wells syndrome, TRAPS, CINCA, Schnitzler’s syndrome and other autoinflammatory syndromes. This prompted us to try anakinra in HIDS patients.
Three HIDS patients, aged between 39 and 42 years, with confirmed mevalonate kinase gene mutations, and with an inflammatory episode frequency of more than once a month were treated with 100 mg subcutaneous anakinra for four days at the time of an inflammatory episode. Within two days after starting anakinra treatment we saw a rapid decline in CRP levels, subsiding of fever and relief of symptoms reported by the patients. Thus anakinra lead to a decrease in number of symptomatic days when compared with untreated attacks. The optimal treatment duration and effects on the asymptomatic period in between two attacks are still unclear and warrant further observation.

  Cytokine Regulation and Post-translational Processing of Pyrin in Human Synovial Fibroblasts.

Arturo Diaz1, Chunbo Hu2, Daniel L. Kastner3, and Deborah L. Gumucio2            

1Department of Internal Medicine/Rheumatology, University of Michigan Health System, Ann Arbor, MI, USA; 2Department of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA; 3Genetics and Genomics Branch, NIAMS-NIH, Bethesda, MA, USA

OBJECTIVE. In our quest to understand the role of pyrin in the regulation of the inflammatory process in the synovial environment, we have investigated its expression in human synovial fibroblasts (SF) derived from osteoarthritis (OA) and rheumatoid arthritis (RA) joints under the stimuli of pro- and anti-inflammatory cytokines. Pyrin expression was analyzed by real-time PCR and Western blot. SF were incubated with pro-inflammatory (IL-1b, TNFa, LPS, IFNg, IL-6, IL-8 IL-17, IL-18), anti-inflammatory (IL-4, IL-10, IL-13) or immunoregulatory (IL-12, IL-15) cytokines either alone or in combination.

RESULTS. Four OA and five RA SF primary cultures were analyzed. Reall-time PCR analysis showed that pyrin mRNA was consistently upregulated by IL-1b, TNFa, LPS and IFNg. Anti-inflammatory cytokines did not upregulate pyrin mRNA, nor did they blunt the LPS effect. Rheumatoid SF expressed lower levels of pyrin mRNA under all conditions. Western blot analysis revealed that IFNg induced a robust accumulation of a 55 kDa species of the pyrin protein. LPS induced a lower accumulation of the 55 kDa protein, and this induction was abrogated by dexamethasone. Induction by IL-1b and TNFa was negligible. The IFNg effect was seen at a concentration as low as 1 unit/ml (50 fg/ml), was detected at 8 hours and lasted for at least 96 hours. Analysis of separate cytosolic and nuclear extracts revealed that the accumulation of the 55 kDa pyrin protein was restricted to the cytosol; the level of full-length pyrin was unchanged by treatments and restricted to the nuclei.

CONCLUSIONS. These data indicate that in human SF pyrin is upregulated by bacterial products (LPS) and by pro-inflammatory cytokines in the context of a TH-1 response. In addition, pyrin is subject to post-translational processing as well as subcellular compartmentalization, suggesting that pyrin may serve multiple context-dependent roles in the inflammatory network.

  Clinical improvement with infliximab in a child with amyloidosis secondary to familial Mediterranean fever

Selcuk Yuksel1, Fatoş Yalçınkaya1, Z.Birsin Özçakar1, Banu Acar1, and Mesiha Ekim1           

1Pediatric Nephrology, Ankara University School of Medicine, Ankara, Turkey 06100

Systemic AA amyloidosis is the most serious complication of familial Mediterranean fever (FMF) leading to end-stage renal disease (ESRD). Amyloidosis can also affect the gastrointestinal tract (resulting malabsorbtion), liver, spleen and the other organ systems. Particularly after the onset of nephrotic syndrome due to renal amyloidosis related with FMF, majority of patients developed ESRD insipite of colchicine usage. A new promising therapeutic alternative is represented by anti-tumor necrosis factor-α drugs particularly in patients with amyloidosis secondary to chronic inflammatory disease such as rheumatoid arthritis, ankylosing spondylitis, Chron’s disease. In this report we present a girl with AA amyloidosis secondary to FMF who was treated with infliximab.
A 12-year-old girl was admitted to the hospital with nephrotic syndrome in June 2002. Her past history revealed recurrent attacks of fever and abdominal pain since 1994. The pain and swelling of right ankle added to these complaints in 1998. She had received combination therapy with steroid, sulphasalazine and naproxen with presumed JRA for 3 years. In 2001, genetic analysis for MEFV gene was performed and showed compound heterozygous mutations, M680I /M694V. Colchicine was started as an additional therapy.When she was referred to our clinic in June 2002, she had got serious clinical findings of renal involvement gastrointestinal involvement and protracted arthritis. Laboratory results revealed serum urea 33mg/dl, creatinine 0.6mg/dl, albumin 1.1g/dl and 24 hour urine protein 6g/day. Renal, antral and duodenal biopsy revealed AA amyloidosis. Although she received colchicine and naproxen, protracted arthritis, recurrent vomiting, diarrhea, recurrent abdominal attacks and heavy proteinuria persisted. The requirement of hospitalization and albumin infusion therapy gradually increased to two times monthly in the last three months during the period between 2002 and 2003. In November 2003 laboratory examinations revealed urea 44mg/dl, creatinine 0.7mg/dl albumin 1g/dl and heavy proteinuria of 10g/day. A trial of infliximab 3mg/kg was started and applied at weeks 0, 2, 6 and then every 8 weeks. After the third dose of infliximab, her gastrointestinal complaints disappeared. During the ensuing 3 months her condition got better with decreased proteinuria (3g/day). After 22 months of therapy with infliximab, gastrointestinal symptoms and protracted arthritis completely improved and proteinuria stabilized. Moreover, her serum albumin and total protein levels were increased (2.9g/dl and 5.4g/dl, respectively) and she did not receive any albumin infusion after the onset of infliximab therapy.
In conlusion, we suggested that infliximab could be beneficial in the treatment of amyloidosis secondary to FMF.


  Intravenous Interleukin-1-receptor Antagonist in a Patient with Severe TNF-receptor Associated Periodic Syndrome (TRAPS)

Evelien J. Bodar1, Anna Simon1, Theo J. Fiselier2, Jeroen C. van der Hilst1, Joost P. Drenth3, and Jos W. van der Meer1          

1Department of General Internal Medicine, Radboud University Medical Centre, Nijmegen, the Netherlands; 2Department of Pediatrics, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands; 3Department of Gastroenterology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands

Introduction: TRAPS is an auto-inflammatory disorder characterized by recurrent incapacitating episodes of fever and a generalized inflammatory response. The responsible genetic defect is located in the TNF receptor type-1 gene (TNFRSF1A). We report a new therapeutic option as observed in a case of severe TRAPS.
Case: A 20 year old female patient who experienced fever attacks since childhood was diagnosed with TRAPS, confirmed by a C43Y mutation in the TNFRSF1A gene. She was almost continuously severely incapacitated by very painful inflammatory episodes. Numerous therapies were tried with disappointing results, including non-steroidal anti-inflammatory drugs (NSAIDs), methotrexate, cyclosporin, sirolimus and etanercept. Prednisone, at least 30 mg daily, was needed to relieve symptoms. Anakinra, given subcutaneously at a dose of 100 mg daily did result in a complete remission of symptoms and acute phase response as reported previously, and prednisone could be withdrawn entirely. Unfortunately after six months symptoms relapsed. The effect of subcutaneous anakinra was markedly diminished. Trials of intermittent treatment with anakinra and even combined with etanercept did not improve her condition and high dose prednisone had to be reintroduced. Administration of the intravenous TNF-receptor antagonist infliximab resulted in additional exacerbation of symptoms. We then decided on the use of intravenous anakinra 300mg a day for two days. Within a few hours the patient’s clinical condition improved. Complete remission was reached within two days and lasted for one month.
Conclusion: in this severe case of TRAPS, effectiveness of subcutaneous use of Anakinra diminished after continuous use for six months. Subsequent intravenous administration of Anakinra again lead to complete remission. The cause of this discrepancy is still unclear.

  Phenotype and response to treatment of 22 patients with autoinflammatory disease associated with mutations in TNFRSF1A

Mark Offer1, Helen J. Lachmann1, Alison Bybee1, Dorota M. Rowczenio1, Hugh J. Goodman1, and Philip N. Hawkins1          

1National Amyloidosis Centre, Royal Free and University College Medical School, London, UK

We have characterized the phenotype and response to treatment of 21 patients with mutations in TNFRSF1A who had clinical features consistent with autoinflammatory disease. The patients collectively had 8 different variants, of which R92Q was commonest accounting for 9 (43%) cases; the others were F60L in a British father and daughter, and in a Turkish father and son, T50M in 2 unrelated British patients, T37I in a British/Polish father and daughter, and one case each of Del42, C29F, R92P and a splice site mutation all in British Caucasians. The sex distribution was 1:1.3 male: female, with onset of symptoms at a median of 8 years (0.5-55 years). The phenotype was variable; all but 3 patients described episodic attacks (median 3 attacks/year) mostly lasting 1-3 weeks. All patients were febrile during attacks, 66% had severe myalgia and/or arthralgia, 61% reported peritonitic abdominal pain, 57% maculopapular rash, 24% pleurisy and 19% developed cervical lymphadenopathy. Despite apparently discrete attacks clinically, only 4 patients regularly had a completely normal plasma SAA concentration when they were asymptomatic. Patients with R92Q generally had the mildest disease, with median age at onset of 16 years and pre-treatment median SAA 4mg/L between attacks and 89mg/L during attacks, compared with 36mg/L and 491mg/L respectively in patients with other mutations. 6 (29%) of patients had AA amyloidosis, of whom two have received renal transplants and one remains on dialysis. No patient with R92Q had amyloidosis. Prior to receiving cytokine blocking therapies, a third of patients had been managed with analgesia alone, and another third had been treated with corticosteroids; 5 had received colchicine, with apparent clinical benefit in 3 cases. Five patients with R92Q and 2 others continue to have sufficiently mild or infrequent attacks to be managed just symptomatically. Eight patients have received cytokine blocking agents; two patients with R92Q received infliximab which in one case aborted attacks but was followed by a major inflammatory flare, and in the other was associated with substantial and prolonged exacerbation of his TRAPS symptoms. The latter patient has subsequently had an excellent and sustained response to etanercept. Four other patients have received etanercept with good effect, although the response has declined or been lost completely in two cases after 2-3 years. Two patients have been treated with anakinra, one of whom remains in complete remission on just 33mg/day, the other requiring 100mg/day to suppress his symptoms and acute phase response.
  Efficacy of Methylprednisolon Infusion in the Treatment of Acute FMF Attacks

Eren Erken1, Huseyin T. Ozer1, Berna Bozkurt2, Ramazan Gunesacar1, and Ertugrul G. Erken2           

1Department of Rheumatology-Immunology, Cukurova University Faculty of Medicine, Balcali, Adana, 01330, Turkey; 2Department of Internal Medicine, Cukurova University Faculty of Medicine, Balcali, Adana, 01330, Turkey

Treatment of acute FMF attacks still remains to be a major problem in the management of the disease. Efficacy of colchicine therapy has not been proven in acute attacks; and limited effect was observed by interferon-alpha treatment. Corticosteroid effect in attack period has not been evaluated extensively. In this particular study efficacy of methylprednisolon infusion in FMF attacks was investigated.

Twenty-two FMF patients (13 female, 9 male) in first 24 hrs of attacks were included into the study. Forty mg methylprednisolon infusion in 250 ml saline in two hours was performed to 17 patients; and saline was administered as placebo to 5 patients. Clinical evaluation was done before infusion (0) and 6, 24, 48 and 72 hrs after commencement of infusion. Serial serum samples were obtained for the measurements of interleukin (IL)-6, C-reactive protein (CRP), serum amyloid A (SAA) and tumor necrosis factor-alpha (TNF-alpha) along with each clinical evaluation.

Decreases in abdominal pain in 0-6, 0-24, 0-48 and 0-72 hr intervals in corticosteroid (CS) group were significantly more pronounced than those of the placebo group (p=0.019, p=0.006, p=0.011, p=0.024, respectively). Abdominal tenderness was also significantly decreased in 0-6, 0-24 and 0-48 hr intervals in CS group than those of the placebo controls (p=0.013, p=0.003, p=0.005). Rapid decline of IL-6 levels in the CS group was observed at the 6th hr of the infusion (mean:144.15±202.61 pg/ml to 28.43±84.95 pg/ml, p=0.001) and reached to lower levels at 24 hr (17.95±22.25 pg/ml, p=0.034, for 0-24 hr). When the groups are compared, changes in IL-6 levels at 0-6 and 6-24 hr intervals were significantly more pronounced in CS group than those of the placebo group ( p=0.001, p=0.045). Decline in CRP levels in 0-48, 6-48 and 24-48 hr intervals in the CS group was significantly more pronounced than that of the placebo group (p=0.043, p=0.028, p= 0.043). Decrease in CRP levels from 24 to 48 hr (mean:95.86±74.19 mg/L to 21.11±24.77 mg/L, p=0.043) was significant in CS group. Changes in the serial serum SAA and TNF-alpha measurements showed no significant difference between the two group. Mean TNF-alpha levels in CS group significantly declined at the 6th hr (34.77±11.37 to 27.68±3.14 pg/ml, p= 0.09), however a significant increase was observed at 24 hr (41.85±39.93 pg/ml, p=0.019, 6-24hr). No adverse event was observed during the study.

Our result indicate that patients may benefit from methyprednisolon infusion in early phases of acute FMF attacks.

  Somatic mosaicism of CIAS1 in a CINCA syndrome patient

Megumu Saito1*, Akihiro Fujisawa2*, Ryuta Nishikomori1, Naotomo Kambe2, Mami Nakata-Hizume1, Momoko Yoshimoto1 , Katsuyuki Ohmori3, Ikuo Okafuji1, Takakazu Yoshioka1, Takashi Kusunoki1, Yoshiki Miyachi2, Toshio Heike1, and Tatsutoshi Nakahata1  

1Department of Pediatrics, Kyoto University graduate school of medicine, Kyoto, Japan; 2Department of Dermatology, Kyoto University graduate school of medicine, Kyoto, Japan; 3Department of Laboratory Medicine, Kyoto University graduate school of medicine, Kyoto, Japan ; *These authors contributed equally to this work.

Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome is a severe inflammatory disease that was recently found to be associated with mutations in CIAS1. However, CIAS1 mutations have only been detected in half of the CINCA syndrome patients and it remains unclear which genes are responsible for CINCA syndrome in the remaining patients. We describe a CINCA syndrome patient who exhibits CIAS1 somatic mosaicism.
We analyzed the CIAS1 gene in various blood cells and the buccal mucosa and found that our patient has one heterologous single nucleotide polymorphism 587G>A (S196N) and one heterologous mutation 1709A>G (Y570C) in exon 3 of CIAS1. The latter mutation was found to occur as somatic mosaicism. Next we measured IL-1b production by peripheral blood mononuclear cells (PBMCs) by ELISA and found that PBMCs from the patient produced a large amount of IL-1b in the absence of stimulation, unlike those from normal controls or from his mother, who also bears the S196N polymorphism. Finally we assessed the ability of his mutant CIAS1 gene to enhance apoptosis-associated speck-like protein containing CARD (ASC)-dependent NF-kB activation to confirm that the mutations of CIAS1 found in this patient are responsible for his clinical manifestations of the CINCA syndrome. The Y570C mutation (with or without the S196N polymorphism) elevated the ability of CIAS1 to induce ASC-dependent NF-kB activation, unlike the wild type gene or the gene bearing the S196N polymorphism alone.
From this CINCA syndrome patient with somatic mosaicism of CIAS1 gene, we have learned that somatic mosaicism of CIAS1 gene is enough to cause CINCA syndrome although the clinical phenotype is less severe and that somatic mosaicism is one reason why CIAS1 mutations have not been detected in some CINCA syndrome cases.

  The influence of polymorphisms in MDR1 on colchicine unresponsiveness in Familial Mediterranean Fever

Ruth  Gershoni-Baruch1, Yael Peretz1, Lidar  Merav2, Efrat Dagan1, Jean-Michel Scherrmann3, and Avi Livneh2          

11 Institute of Human Genetics, Rambam Medical Center, Haifa, Israel; 22Heller Institute of Medical Research, Sheba Medical Center, Tel-Hashomer, Israel; 33INSERM U26, Hopital Fernand Widal, Paris, France

Background & Aims: The human multidrug resistance 1 (MDR1) gene product P-glycoprotein (PGP) is a transmembrane efflux transporter that contributes to the elimination of therapeutic agents and xenobiotics. Two single-nucleotide polymorphisms (SNP) in the human MDR1gene (G2677T/A in exon 21 and C3435T in exon 26) reportedly predict expression and function of PGP in human enterocytes and lymphocytes. The aim of the study as to investigate the association of these two major MDR1 SNPs with unresponsiveness to colchicine in patients with familial Mediterranean fever.
Methods: Allele frequencies and genotype distributions of the C3435T and the G2677T/A (Ala893Ser/Thr) SNPs of MDR1 were investigated in 105 FMF patients sub-grouped on account of their being responders (n = 47) or non-responders (n = 58) to a daily dose of 2 mg colchicine and comparable with respect to gender, age, duration and onset of disease, various demographic parameters and MEFV status.
Results: The distributions of the C/C, T/T, and C/T genotypes of the C3435T polymorphism were 26,7%, 20%, and 53.3%, respectively. Significantly increased frequency of the 3435TT genotype was observed in non-responders compared to non-responders (3435T: P = 0.049; odds ratio, 1.4; 95% confidence interval, 1.02–1.94; 3435TT: P = 0.045; odds ratio, 2.03; 95% confidence interval, 1.04–3.95). Colchicine concentration in the lymphocytes, significantly higher among responders compared to non-responders, was positively associated with the 3435C allele and the 3435CC genotype. PMN’s colchicine concentrations, although not found to be significantly higher among responders compared to non-responders, was equally positively associated with the 3435C allele and the 3435CC genotype. The distributions of the GG, GT, TT and AT genotypes of the G2677T/A polymorphism were 27.8%, 55.7%, 14.4% and 2.1%, respectively and did not differ in responders compared to non-responders.
Conclusion – Colchicine treatment failure associated with inadequate, lymphocyte & PMN’s colchicines, concentration, is related to the 3435T allele in the MDR1 gene. The results are controversial and in discordance with those reported in the literature.


  Pyrin, product of the MEFV locus, interacts with elements of the cytoskeletal machinery

Andrea L. Waite1, Philip Schaner2, Neil Richards1, Banu Peynircioglu1, and Deborah Gumucio1           

1Department of Cell & Developmental Biology, University of Michigan, Ann Arbor, MI 48109; 2Program in Cell & Molecular Biology, University of Michigan, Ann Arbor, MI, 48109

Familial Mediterranean fever (FMF) is characterized by attacks of generalized fever and pain highly localized to the joints, abdomen, chest, or the skin. FMF is caused by mutations in the MEFV locus, which encodes pyrin, a protein expressed in myeloid cells and several fibroblastic cell types. Exon 1 of pyrin encodes a death domain-related structure, now known as a pyrin domain (PyD). At least 35 different PyD containing proteins have been identified, most of which seem to be involved in inflammation and/or apoptosis pathways. To learn more about the role of pyrin in native neutrophils and monocytes, we carried out immunolocalization studies. Although epitope-tagged versions of pyrin had been shown to localize exclusively to the cytosol in transfected cells, we found that pyrin in neutrophils is nuclear. In monocytes, both nuclear and cytoplasmic pyrin was seen; cytosolic pyrin appeared to be localized in membrane ruffles and in leading edge lamellipodia. Since these regions are sites of active actin polymerization, we tested whether pyrin is drawn specifically to foci of actin polymerization using the Listeria rocket assay. Listeria monocytogenes recruits host machinery to polymerize an actin-based “rocket” behind itself, providing the bacterium with the mobility to invade neighboring cells. We found that, in transfected cells, pyrin is localized to Listeria rockets. We mapped the specific domain responsible for this interaction to the B box and coiled coil region of pyrin. Using a yeast two-hybrid screen of a library prepared from neutrophils, we identified a pyrin-binding protein, PSTPIP1, a known actin-associated protein. Previous studies in the Kastner lab also isolated PSTPIP1 from monocytes as a pyrin-interacting protein. Mutations in PSTPIP1 are responsible for PAPA syndrome, characterized by sterile pauciarticular arthritis, pyoderma granulosum and severe cystic acne. Though these data tempted the speculation that PSTPIP1 could be responsible for the recruitment of pyrin to Listeria rockets, we found that PSTPIP1 does not associate with these actin structures in transfected cells. Thus, another factor must be responsible for the recruitment of pyrin to sites of actin polymerization. We are currently testing by immunoprecipitation whether pyrin binds directly to proteins known to be involved in actin polymerization, including: actin, Arp3, VASP, alpha-actinin, profilin, cofilin, gelsolin, and capping protein.
  Personal Statement of Ryuta Nishikomori, MD, PhD

Ryuta  Nishikomori*               

Kyoto University Hospital ; *These authors contributed equally to this work.

I am working at one of few clinical facilities doing research on periodic fever syndromes (PFS) in Japan and performing genetic analysis of them. We have published the paper regarding the common etiology of early onset sarcoidosis shared with Blau syndrome, which is caused by
NOD2 mutations (Kanazawa et al, Blood, 105, 1195). Now I am interested in the pathophysiologic mechanism how constitutive active NOD2 can induce such granuloma lesions and working on mouse models of Blau syndrome.
We are also publishing a CINCA syndrome case with somatic mosaicism of
CIAS1 in Arthritis and Rheumatism (in press). I am presenting data on CINCA syndrome case with CIAS1 mosaicism. I believe the somatic mosaicism is one of possible mechanism why some CINCA syndrome patients did not show any CIAS1 mutations. We are now working on how we can identify such CIAS1 mosaicism cases.
In Japan, Anakinra is not sold commercially, so we are in a big trouble to treat CINCA syndrome patient with it. I would like to explore the possible way of getting anakinra for Japanese CINCA syndrome patients (at least 4) at the meeting.
In summary, I would like to attend the meeting to communicate with the world experts on PFS and learn from them as a clinician, and also deepen my understanding of the disease mechanism of PFS from the scientific point of view.

I hope this would help me registered for FMF and beyond.

  A Mutation in pstpip2 Causes an Autoinflammatory Phenotype in the cmo Mouse.

Polly  J. Ferguson1, Hatem  I. El-Shanti1, Xinyu Bing1, Mohammed A. Vasef2, Luis A. Ochoa1, Amar Mahgoub1 , Tom J. Waldschmidt2, Lorraine  T. Tygrett2, Michael Carlson2, and Annette  J. Schlueter2     

1Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA; 2Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA


Chronic recurrent multifocal osteomyelitis (CRMO) is a chronic inflammatory disease that primarily affects children. It presents with multiple inflammatory bone lesions with or without fever. Affected patients often have an associated non-osseous chronic inflammatory condition such as psoriasis, palmar-plantar pustulosis or inflammatory bowel disease. Multiple lines of evidence suggest that CRMO has a genetic component to its etiology including: 1) There are reports of affected siblings in the literature, 2) There is a published report of a susceptibility locus for CRMO on human chromosome 18q, 3) Majeed syndrome, an autosomal recessive syndrome in which CRMO is associated with a congenital dyserythropoietic anemia, is caused by homozygous mutations in LPIN2 and 4) There is a similar disorder in the mouse (cmo, chronic multifocal osteomyelitis) resulting from a spontaneous mutation that is inherited as an autosomal recessive trait. The murine locus is syntenic to the human CRMO susceptibility locus on human 18q.

Utilizing a backcross breeding strategy, we refined the cmo locus to a 1.3Mb region on chromosome 18. Direct sequencing of the genes in the refined interval revealed a single base pair change c.293T>C in the pstpip2 gene resulting in a highly conserved leucine at amino acid 98 being replaced by a proline (L98P). No mutations were found in the remaining 8 genes. In addition to the inflammatory bone lesions that had previously been described in the cmo mice, our laboratory has identified abnormalities of the lymph node, spleen, nails, auricular cartilage and dermal tissues of the ear. The T and B cell compartments in cmo mice are grossly normal as assessed by flow cytometry. Reciprocal bone marrow transfer experiments demonstrate that hematopoietically derived cells transfer the inflammatory phenotype. Rag1 deficient cmo mice develop tail kinks and foot deformities, demonstrating that the phenotype does not require the adaptive immune system. We conclude that homozygous mutations in pstpip2 cause the autoinflammatory phenotype seen in cmo mice and that the inflammatory phenotype is mediated by cells of the innate immune system.



  Manipulation of Isoprenoid Biosynthesis as Potential Therapeutic Intervention in Mevalonate Kinase Deficiency

Marit S. Schneiders, Linda Henneman, Sander M. Houten, Ronald J. Wanders, and Hans R. Waterham           

1Laboratory Genetic Metabolic Diseases, Department of Pediatrics/ Emma Chlidren's Hospital and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

Mevalonate kinase deficiency (MKD) is an autosomal recessive auto-inflammatory disorder characterized by recurring episodes of high fever associated with headache, arthritis, nausea, abdominal pain, diarrhea, and skin rash. Originally, two distinct syndromes had been defined, classic mevalonic aciduria (MA) and hyper-IgD and periodic fever syndrome (HIDS), which, after the discovery of the underlying biochemical and molecular cause, are now recognized as the severe and mild ends of MKD. MKD is caused by mutations in the MVK gene, resulting in depressed activity of the encoded enzyme MK. As a consequence, MK has become the rate-limiting enzyme in the isoprenoid biosynthesis pathway in MK-deficient patients, determining the flux through the pathway. As this implies that upregulation of the residual MK activity in these patients would be a therapeutic option, we studied in vitro the effect of two inhibitors of specific isoprenoid biosynthetic enzymes on the residual MK activity. Treatment of cultured fibroblasts of MK-deficient patients with either simvastatin, an inhibitor of HMG-CoA reductase, or Zaragozic Acid A, an inhibitor of squalene synthase, led to a marked increase in residual MK enzyme activity due to an increased MVK gene transcription. This effect was enhanced when the cells were cultured in lipoprotein-depleted medium. However, when the flux towards non-sterol isoprenoid end product synthesis was studied in MK-deficient cells, we found that simvastatin reduced the amount of membrane-bound RhoA and Rac1, indicating a decreased pathway flux. This negative effect on the pathway flux could be counterbalanced by co-incubation with Zaragozic Acid A. Our results indicate that manipulation of isoprenoid biosynthesis may be an interesting therapeutic option to treat MK deficiency.
  "Early Innate Inflammatory Changes in Familial Mediterranean Fever Patients Undergoing Structured Treatment Interruption of Colchicine"

Nona T. Colburn1, Douglas Mogul1, Jae-Jin Chae1, Katharina Richard1, James  Balow, Jr1, Beverly  Barham2 , Grace Park2, Geryl Wood1, Marta Haverkamp3, Hong Wei Sun4, Ivona Aksentijevich1, and Daniel Kastner1   

1Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 2Office of the Clinical Director, NIAMS, NIH, Bethesda, MD; 3NIAID, NIH, Bethesda, MD; 4Biomining Data, NIAMS, NIH, Bethesda, MD

Purpose: Familial Mediterranean fever (FMF) is a recessively inherited periodic fever syndrome characterized by recurrent episodes of fever, rash, serositis and arthralgia. FMF is unique in its therapeutic response to the microtubule inhibitor colchicine. Pyrin, the protein product of the FMF gene, colocalizes with microtubules and may modulate leukocyte effector functions. Moreover, mutations in the C-terminal B30.2 domain may lead to increased cytokine production and NF-kappaB activation. Our purpose was to detect the earliest molecular and cellular changes that can occur in FMF without the influence of colchicine.

Methods: Seven patients who met clinical criteria for FMF were recruited by telephone. Patient demographics and acute phase reactants including erythrocyte sedimentation rate, C-reactive protein (CRP), haptoglobin, fibrinogen, leukocyte count, and serum amyloid A (SAA) were measured. Five healthy volunteers with no family history of FMF comprised the control group. All assays were performed on peripheral venous blood samples obtained at baseline and 24, 48, and 72 hours off colchicine. Gene expression profiles were studied with Affymetrix microarrays. For cleavage analysis of endogenous pyrin, Western blots were probed with anti-pyrin and anti-I kappaB alpha antibodies and analyzed by densitometry. Proinflammatory cytokines from cultured monocytes were serially measured using the Cytometric Bead Array. Activation and adhesion markers were measured on monocytes, neutrophils, and lymphocyte subpopulations by flow cytometry.

Results: Colchicine levels at 72 hours were less than 0.05 ng/ml. Five of seven patients had no clinical symptoms or change in acute phase reactants. Two patients reported an attack at 48 hours, with a significant elevation in at least one acute phase reactant. Candidate genes identified by microarray were found to participate in apoptotic, growth factor, and NF-kappaB-mediated pathways. Consistent with previous data from our laboratory, patients typically demonstrated a higher ratio of cleaved pyrin to full-length pyrin as compared to controls. Furthermore, the proportion of cleaved pyrin was reciprocally related to levels of I kappaB alpha an inhibitor of NF-kappaB, further supporting the hypothesis that pyrin might regulate leukocyte activation through the NF-kappaB pathway. A stepwise increase in neutrophil activation was seen during the first 48 hours, with no change in lymphocyte subpopulations. As compared to baseline, stimulated monocytes at 72 hours produced more TNF and significantly greater levels of IL-12p70.
Conclusion: These studies suggest that early cellular and molecular manifestations of FMF off colchicine include increased pyrin cleavage, upregulation of proinflammatory cytokines, including Il-12p70 and TNF, as well as neutrophil activation.

  Gene Expression Studies in FMF Patients

James E. Balow, Jr.1, Ivona Aksentijevich1, Hong-wei Sun2, Chris Cheadle3, Nona Colburn4, Beverly Barham5 , and Daniel Kastner1        

1Genomics Section/Microarray Unit, Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 2Biodata Mining and Discovery, Office of Science and Technology, NIAMS, NIH, Bethesda, MD; 3Cellular Biochemistry Section, NCI, NIH, Bethesda, MD; 4Inflammatory Biology Section, Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 5Office of the Clinical Director, NIAMS, NIH, Bethesda, MD

Familial Mediterranean fever (FMF) is characterized by recurrent bouts of fever and inflammation usually lasting 1-3 days. It is caused by mutations in MEFV, which encodes a protein denoted pyrin, that is involved in the regulation of inflammation and apoptosis. In order to further elucidate the cellular and molecular consequences of FMF-associated pyrin mutations, we have utilized DNA microarrays to compare global gene expression patterns between FMF patients and healthy controls.

Towards this aim, we extracted RNA from PBMCs that were harvested from blood samples obtained from 26 FMF mutation positive patients on colchicine therapy and 37 healthy controls. The purified RNA was reverse transcribed and amplified using an in vitro transcription reaction to generate target cRNA that was subsequently hybridized to Affymetrix U133A DNA microarrays. To examine the genes that are differentially expressed between our patient and control cohorts we filtered the results of statistical analysis (T-test assuming equal variance) performed on data that was processed by two independent signal quantitation algorithms, namely RMA and MAS5.0.

Preliminary data analysis has identified several classes of genes that show decreased levels of expression in FMF patients relevant to controls. These classes include genes involved in the regulation of transcription (NR4A2, EGR1, C21orf7, EGR3), apoptosis (BCL-2), the inflammatory response (CXCL5, CD69, CD160) and the structural development of muscle cells (MYOM2, CALD1). Since all the FMF patient samples were obtained during treatment with colchicine and since colchicine is known both to block FMF-mediated inflammation and to block microtubule polymerization by binding to tubulin it is plausible that colchicine may preferentially downregulate the expression of these structural genes. By focusing on those genes that are downregulated in FMF patients relevant to controls, we may gain some insight into the direct mechanism of colchicine action. However, additional experiments are needed to determine whether these genes are downregulated as a result of colchicine treatment or as a result of their disease state. Finally, despite the fact that there were very few differentially upregulated genes in FMF patients relative to controls at our chosen fold-change threshold, we did find some interesting upregulated genes involved in the defense response (MPO, DEFA4, DEFA1) and the inflammatory response (TNFA1P6). Experiments are currently in progress to validate these target genes using independent technologies.

  Genetic Susceptibility to Neonatal-Onset Multisystem Inflammatory Disease (NOMID/CINCA): Novel CIAS1 Mutations and Genomic Strategies to Resolve Locus Heterogeneity

Ivona Aksentijevich1, James Balow Jr.1, Janet Jones2, Natalie Dailey2, Scott Canna2, Elaine F. Remmers1 , Raphaela Goldbach-Mansky2, and Daniel L. Kastner1       

1Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 2Office of Clinical Director, NIAMS, NIH, Bethesda, MD

Neonatal-onset multisystem inflammatory disease (NOMID/CINCA) is a rare autoinflammatory disease characterized by fever, chronic meningitis, uveitis, sensorineural hearing loss, urticarial rash, and a characteristic deforming arthropathy. NOMID is caused by missense mutations in CIAS1 (also known as NALP3, PYPAF1) that encodes cryopyrin. NOMID is allelic with Muckle-Wells syndrome (MWS) and familial cold autoinflammatory syndrome (FCAS), two dominantly inherited disorders with some similarities to NOMID. Currently, over 35 disease-associated mutations have been found and all are clustered in the NACHT domain of the protein. We previously reported 6 mutations in 13 NOMID cases. Subsequently, we analyzed 23 new patients and collected PBMC from 14 patients for gene expression studies. In 6 patients we identified new missense changes: V262A, L264F, V351L, G326E, F443L, and F523C, while 8 patients carried previously described mutations. Thus, including our previous report we evaluated 36 NOMID patients and we found mutations in 20/36 (55%). There was no clinical difference between mutation positive and negative patients and they both responded dramatically to anakinra treatment. We used Affymetrix U133A 2.0 microarrays to identify disease-specific gene expression profiles in PBMC of 12 NOMID patients at baseline and during their treatment with the IL-1RN (anakinra). Among the genes that are at least 1.5 fold down-regulated in patients after the treatment at a significance level of p=< 0.01 are several cytokine receptors (IL-1RN, INFGR1, INFGR2, IL6R, IL8R, IL10RB, IL17R, TNFRSF1A), genes that are involved in the immune response and regulation of NF-kapaB pathway (CD59, IFITM2, DAF, NCF4, TLR1, TLR2, TLR5, TLR8, CR1, IKBKG, NKIRAS2), cell adhesion and chemotaxis (ICAMs, CCR1, CCR2, C3AR1, C5R1), regulation of apoptosis (CFLAR, DAP, BAX inhibitor, MCL1, BAG1), and leukotriene biosynthesis (ALOX5, ALOX5AP, ALOX12). Given these genes are down-regulated in patients following treatment with anakinra and the fact that these NOMID patients respond very well to anakinra, these genes are likely to be involved in the pathogenesis of disease. Validation studies are in progress. Thus, the preliminary analysis of the gene expression profile in NOMID patients substantiates in-vitro data suggesting a role of the CIAS1 gene in the regulation of IL-1 and NF-kapaB pathways, and apoptosis. In addition, through these studies we hope to uncover novel candidate genes for the screening of CIAS1 mutation negative patients.



  Modelling of TNFRSF1A-TRAPS related mutants exhibit conformational changes consistent with their functional abnormalities

Susie Rebelo1, Rasoul  Kashipaz1, Paul Radford1, Sue Bainbridge1, Richard  Powell1, Ian  Todd1 , and Paddy Tighe1        

1Inflammation and Clinical Immunology Research Group, School of Molecular Medical Sciences, University of Nottingham, Nottingham, UK, NG7 2UH

Mutations in the TNFRSF1A gene cause the autosomal dominant, auto-inflammatory tumour necrosis factor receptor associated periodic syndrome (TRAPS). We have established an in vitro system with two transfected human cell lines (SK-HEP-1 & HEK-293) in which the cellular consequences of the mutations can be defined in ways that may be relevant to the pathophysiology of TRAPS. TRAPS-associated mutants behave differently to wild-type (WT) TNFRSF1A and have different localisation properties within the cell as a direct result of the mutations in the ectodomains of TNFRSF1A. From a structural point of view, mutants with a predicted conformational stability similar to that of the WT protein (e.g. R92Q) behave similarly to WT in cell surface expression and TNF binding, whereas forms of TNFRSF1A with mutations predicted to destabilise the protein structure more drastically (e.g. cysteine mutations), show defects in cell surface expression and TNF binding. We hypothesised that the TNFRSF1A TRAPS mutants result in conformationally defective TNFRSF1A that may play a role in the pathogenesis of TRAPS. This prompted modelling of the 3-D structure of TRAPS-associated mutants of TNFRSF1A using the crystal structure of TNFRSF1A as a template. Model structures of the different mutant TNFRSF1A proteins show that even a single amino acid substitution at a residue not involved in receptor-ligand binding can have an overall impact on protein folding. In addition the overall structural organisation of the protein is strained and as a result of the single amino acid mutations in the ectodomains of TNFRSF1A. The results obtained from the in vitro experiments in combination with the modelled structures suggest that the phenotype and clinical differences between different TNFRSF1A TRAPS mutants may result from different conformations of the TNFRSF1A mutant protein.
  MEFV Gene Mutation Distribution in Ege Region of Turkey One Single Center Expierency

Afig Berdeli, Sevgi Mir, Elif Ozalkaya, Fatma  Mutlubas, Yilmaz Tabel, Ebru Yilmaz , and Alphan Cura        

1Ege University Medical Faculty Department of Pediatrics,Molecular Medicine Laboratory,Izmir/Turkey,35100; 2Ege University Medical Faculty Department of Pediatrics,Pediatric Nephrology,Izmir/Turkey,35100; 3Ege University Medical Faculty Department of Pediatrics,Izmir/Turkey,35100; 4Ege University Medical Faculty Department of Pediatrics,Pediatric Nephrology,Izmir/Turkey,35100; 5Ege University Medical Faculty Department of Pediatrics,Pediatric Nephrology,Izmir/Turkey,35100; 6Ege University Medical Faculty Department of Pediatrics,Pediatric Nephrology,Izmir/Turkey,35100; 7Ege University Medical Faculty Department of Pediatrics,Pediatric Nephrology,Izmir/Turkey,35100

The MEFV gene related to Familial Mediterranean Fever (FMF) was discovered in 1997 by two independent consortiums . Diagnostic criteria for FMF haven’t been changed even after molecular diagnosis has been introduced and molecular analysis was not included in the diagnostic criteria. It is not clear which of the MEFV mutations, currently numbers of which reached to about 98, could be linked with phenotype of FMF and amyloidosis. To reveal phenotype-genotype relationship in EGE region west of Turkey by establishing the distribution of MEFV mutations among FMF patients, their healthy relatives and control groups and to re-establish the indications of the treatment in the light of genotype.
MEFV mutations were analyzed in 1653 blood samples taken from the patients with presumed diagnosis of FMF and their relatives. These patients were classified according to Tel- Hashomer criteria; patients having to 2 major or 1 major and 2 minor criteria were accepted as definite FMF and 1 major and 1 minor criteria were accepted as probable FMF. Patient having 1 major or 1 minor criteria were accepted suspected FMF. The patients which included in our study are different from Turkish FMF Study Group’s patients. 165 healthy individuals without symptoms were also included in the study as a control group. Mutation analyses for MEFV gene in the 2nd, 3rd, 5th and 10th exons were studied by PCR-RFLP, dot-blot hybridization and direct DNA sequencing methods.
MEFV gene mutation was found in 933 ( 56.5%) of 1653 FMF subjects. And 720 (43.5%) subjects were negative for MEFV gene mutation. Mutation distrubition in exons were following; in 725 subjects (77.8%) mutation in exon 10; 204 subjects in exon 2; in 64 subjects exon 3 and in 24 subjects exon 5. In control group 37 subjects (22%) were found positive for MEFV mutation whom were all heterozygotes and the frequency of M694V mutation was 1.2%. Biopsy proven amyloidosis were diagnosed in 26 patients,19( 73%) of them with M694V mutation.
As a conclusion, MEFV mutation distribution in this study was found similar to Turkish FMF Study Group’s data which had published previously. High frequency of detecting mutations arrived from inclusion of the results of mutation analyses of exon 3 and exon 5 in the present study. Additionally, the fact that the number of the patients with amyloidosis decreased dramatically might be attributed to early diagnosis of FMF with molecular analyses and succesfull application of colcihine- therapy.

  Association of MCP1 Gene-2518G/A Polymorphisms with FMF Independent of MEFV Gene Mutation.

Afig Berdeli*, Gulgun  Asar*, and Sevgi  Mir             

1Ege University Medical Faculty Department of Pediatrics,Molecular Medicine Laboratory,Izmir/Turkey,35100; 2Ege University Medical Faculty Department of Pediatrics,Izmir/Turkey,35100; 3Ege University Medical Faculty Department of Pediatrics,Pediatric Nephrology,Izmir/Turkey,35100 ; *These authors contributed equally to this work.

The chemokine MCP-1 is thought to be important for the recruitment of mononuclear cells and the maintenance of inflammation in different tissue. Recently, single nucleotide polymorphisms (SNPs) in the MCP-1 regulatory region have been identified and in vitro study demonstrated that the SNP at position -2518 of the MCP-1 gene affected transcription of the gene. In the 5' flanking region of the MCP-1 gene, a guanine (G)/adenine (A) transition identified at position -2518 upstream from the transcription site was reported to be associated with circulating levels. We investigated whether the G/A polymorphism was associated with FMF and the biological significance of this polymorphism.
Mutation analyses for MEFV gene in the 2nd, 3rd, 5th and 10th exons were studied by PCR-RFLP, dot-blot hybridization methods. MCP-1 gene -2518G/A polymorphism was determined by the PCR-RFLP method. In this case-control study, we present our analysis of 667 FMF patients (318/349 M/F). FMF mutations were found in one or more alleles in 62% of patients who were examined for MEFV gene analysis. No mutation was detected in 38% of patients. Genotype distribution and allele frequencies of MCP1 gene polymorphism were shown in the table.

Genotypes Alleles

Groups GG-n (%) AG- n (%) AA-n (%) G-n(%) A-n(%)

Clinically diagnosed
FMF- 667 316 (47.4) 295 (44.2) 56 (8.4) 927(69.5) 407(30.5)
Control -
151 90 (59.6) 54 (35.8) 7 (4.6) 214 (77.5) 88 (22.5)
FMFmut(+)
415 206 (49.6) 181 (43.6) 28 (6.7) 593 (71.4) 237 (28.6)
FMFMut(-)
252 110 (43.7) 114 (45.2) 28 (11.1) 334 (66.3) 170 (33.7)
As shown in the table ; -2518A allele frequency of MCP1 gene is increased in FMF patients group compared to healty controls. -2518A allele frequency in patients who displayed without mutation in MEFV gene doesn’t show difference with patients who have MEFV gene mutation.
These results suggest that a genetic polymorphism in the 5' flanking region of the MCP-1 gene would be associated with FMF through modulating MCP-1 expression that independent from MEFV gene mutations.


  G241R Polymorphisms in Exon 4 of ICAM1 Gene Associated with FMF.

Sevgi Mir*, Gulgun  Asar*, Alphan  Cura*, and Afig Berdeli*            

1Ege University Medical Faculty Department of Pediatrics,Pediatric Nephrology,Izmir/Turkey,35100; 2Ege University Medical Faculty Department of Pediatrics,Pediatric Nephrology,Izmir/Turkey,35100; 3Ege University Medical Faculty Department of Pediatrics,Izmir/Turkey,35100; 4Ege UniVersity Medical Faculty Molecular Medicine Laboratory, Izmir/Turkey,35100 ; *These authors contributed equally to this work.

Familial Mediterranean fever is an autosomal recessive disease characterized by recurrent attacks of fever and serositis. The disease is caused by mutations in the MEFV gene, presumed to act as a down-regulator of inflammation within the polymorphonuclear cells.
The interaction between cells is mediated by different families of adhesion molecules. ICAM-1 participates in trafficking of inflammatory cells, in cell-cell interactions during antigen presentation and in signal transduction through outside-in signaling events.
The aim of this study was to examine the association of G241R polymorphism of ICAM-1 gene with FMF and its severity.
In this case-control study, we examined 872 patients affected by FMF. Mutation analyses for MEFV gene in the 2nd, 3rd, 5th and 10th exons were studied by PCR-RFLP, dot-blot hybridization and direct DNA sequencing methods. ICAM-1 gene polymorphism was determined by the PCR-RFLP based method.
FMF mutations were found in one or more alleles in 60% of patients were examined for MEFV gene analysis. Genotype distribution and allele frequencies of ICAM1 gene G241R polymorphism were shown in the table.

Genotypes Alleles
Groups RR-n ( %) GR- n ( %) GG -n ( %) R -n( %) G -n( %)
FMF-(872) 14 (1.7) 461 (52.8) 397 (45.5) 489 (28) 1255 (72)
Control-(108) 00 25 (22) 83 (78) 25 (12) 191 (88)
Mut(+)-520 10 (1.9) 296 (56.9) 214 (41.2) 316 (30.4) 724 (69.6)
Mut(-) 352 4 (1.1) 165 (46.9) 183 (52.0) 173 (24.6) 531 (75.4)

As shown in the table ; 241R allele frequency of ICAM1 gene in clinically diagnosed FMF was significantly different from the healty control group(p=0.001). Besides this, the patients who were carrying a mutation in the Mediterranean fever gene had a higher frequency of 241Arg allele from the patients who had not been determined with mutation in MEFV gene (p=0.008). In this cohort of patients , there are associations between carrying R allele at 241 position of ICAM1 gene with clinically diagnosed FMF, particullary carrying MEFV mutations.
  The prevalence of MEFV gene mutations in patients with rheumatic heart disease

Afig Berdeli*, Gulgun  Asar*, Sevgi  Mir*, Ruhi Ozyurek*, and Alphan Cura*           

1Ege University Medical Faculty Department of Pediatrics,Molecular Medicine Laboratory,Izmir/Turkey,35100; 2Ege University Medical Faculty Department of Pediatrics,Izmir/Turkey,35100; 3Ege University Medical Faculty Department of Pediatrics,IPediatric Nephrology,Izmir/Turkey,35100; 4Ege University Medical Faculty Department of Pediatric Cardiology, Izmir/Turkey,35100; 5Ege University Medical Faculty Department of Pediatrics, Pediatric Nephrology, Izmir/Turkey,35100 ; *These authors contributed equally to this work.

Rheumatic fever is an inflammatory disease and follows 0.3% of cases of group A beta-hemolytic streptococcal pharyngitis in children. It can affect many of the body's connective tissues — especially those of the heart, joints, brain or skin. Rheumatic fever is thought to result from an autoimmune response, but the exact pathogenesis remains unclear. The role of genetic factors in the pathogenesis of RF is not clearly known. In our previous publications we found significantly association of RF and TLR2 and FCGR2A gene polymorphism. Individuals having MEFV mutations might be more prone to the late complications of streptococcal infections such as RF.
In order to test this hypothesis, we examined mutation analysis for MEFV gene, which was carried out in group of patients with RHD without clinical signs of FMF.
The study group included sixty six Turkish children (33 female, 33male) with RHD . Patients' ages ranged between 4–17 years (mean 10.9 years). The modified Jones criteria provide guidelines for the diagnosis of rheumatic heart disease. None of the patients had the diagnosis of FMF and family history for FMF. 132 chromosomes from patients were analysed.
The 4 hot spots exons (exons 10, 5, 3 and 2) for MEFV mutations were investigated by sequence analysis were carried out using the ABI PRISM dye terminator cycle sequencing kit, and the products were analyzed on an ABI 310 DNA sequencer. DNA sequencing was performed in both directions, initiated from the forward and the reverse primers used in the initial PCR described by Bernot et al.
We found approximately seven times greater MEFV mutation prevalence in patients with RHD than in the normal population in Turkey. FMF mutations were found in both alleles in 6 patients and in a single allele in 17 patients. No mutation was detected in forty three patients. E148Q was the most common mutation, followed by M694V and V726A. The mutation distribution was different from that in patients with FMF in various studies from Turkey.
The presence of MEFV mutation appears to be the risk factor for ARF development upon streptococcal pharyngitis.

  Reduced Level of MEFV mRNA in Peripheral Blood Leukocytes is Associated with Acute Inflammation

Fatih Selcukbiricik1, Duran Ustek2, Cumhur G. Ekmekci2, Burcak Vural2, Hakan Yanar3, Korhan Taviloglu3 , Ugur Ozbek2, and Ahmet Gul1       

1Division of Rheumatology, Department of Internel Medicine, Istanbul Faculty of Medicine, Istanbul 34390, Turkey; 2Department of Genetics, Institute for Experimental Medical Research, Istanbul University, Istanbul 34390, Turkey; 3Department of Surgery, Istanbul Faculty of Medicine, Istanbul 34390, Turkey

PURPOSE: The pathogenic mechanisms of the disease-related mutations in the MEFV gene has not yet been identified in familial Mediterranean fever (FMF). Recently, reduced expression of MEFV mRNA in peripheral blood leukocytes (PBL) of FMF patients during remission compared with healthy individuals was reported. This study aimed to investigate the mRNA expression of MEFV gene in PBL in patients with FMF during an abdominal attack to explore expression dynamics.
METHODS: We studied 19 patients with FMF during an attack of acute peritonitis. The study group also consisted of 20 otherwise healthy individuals, who were operated with a diagnosis of acute appendicitis, as non-FMF controls. Blood samples were collected both during the acute inflammation and following the resolution of all inflammatory findings. mRNA levels in PBL were detected with real-time RT-PCR method using LightCycler. We used 2 sets of primers for the MEFV gene (exons 7-10 and exons 2-3). We also analyzed the expression of CIAS1 and PSTPIP1 genes, and all expression levels were compared with an internal control of β2-microglobulin.
RESULTS: MEFV expression was found to be reduced in FMF patients during remission compared with non-FMF controls (MEFV 7-10 1.38 ± 1.11 vs. 2.78 ± 1.72, P=0.003; MEFV 2-3 3.24 ± 2.32 vs. 6.84 ± 3.44, P<0.001). We observed a further decrease of MEFV 2-3 expression in FMF patients during the abdominal attack (1.90 ± 0.77 vs. 3.24 ± 2.32, P=0.02). However, a reduced MEFV expression was also noted in the preoperative period of individuals with acute appendicitis compared with their non-inflammatory periods (MEFV 2-3 4.05 ± 3.46 vs. 6.84 ± 3.44, P=0.02). The MEFV expression pattern of non-FMF controls who were identified as heterozygous carriers of MEFV mutations was found to be similar to that of FMF patients. CIAS1 expression in PBL of patients with FMF was also found to be lower than that of healthy controls (1.02 ± 0.83 vs. 2.08 ± 1.01, P=0.001). However, CIAS1 expression did not change with acute inflammation in both groups. PSTPIP1 expression was similar in both FMF patients and healthy controls. We observed a lower expression in FMF patients during acute attack despite a non-significant increase in patients with acute appendicitis.
CONCLUSIONS: These observations suggest that reduced MEFV expression in PBL is associated with acute inflammation. Lower expression of MEFV in FMF patients even in the remission period supports a continuous subclinical inflammatory activity in those patients. Reduced expression of CIAS1 in PBL of patients with FMF may indicate a new homeostatic mechanism to compensate the lower MEFV expression.
  A Survey of Co-morbidity and Socioeconomical Status in Patients with Familial Mediterranean Fever (FMF)

Filiz Akcay1, Emire Seyahi2, Selda  Celik2, and Huri Ozdogan2            

1University of Istanbul, Cerrahpasa Medical Faculty, Department of Internal Medicine, Division of Rheumatology; 2University of Istanbul, Cerrahpasa Medical Faculty, Department of Internal Medicine, Division of Rheumatology

Objective: To describe the frequency of co-morbidities in addition to socioeconomical and educational status in patients with FMF comparing with diseased controls and controls from the community.
Patients and Methods: Consecutive 80 patients with FMF (40 M/ 40 F; mean age: 33 ± 9 years), 35 with systemic lupus erythematosus (SLE), (1 M/34 F; mean age 37 ± 13), 50 male patients with Behçet’s syndrome (BS), (mean age: 34 ± 9) and 47 healthy controls (30 M/ 17 F; mean age: 38 ± 12) selected among hospital workers, all aged 18 years and over, participated in the study. A formal questionnaire was prepared to assess the co-morbid conditions, socioeconomical and educational status of the study groups. In addition, history of these co-morbid conditions and deaths among first-degree relatives were also questioned.
Results: FMF patients were relatively younger compared to SLE patients and controls (p= 0.06). Marital and educational status was similar among study groups. The rate of employment, which was assessed only among males, was also similar among patients with FMF, BS and controls (P = 0.2). The monthly income among patients with FMF (1247 ± 689 USD) was higher than that of the patients with BS (725 ± 288 USD), (P <0.001), and similar to that of the patients with SLE (1227 ± 793) and controls (1149 ± 655 USD).
The rate of diabetes mellitus, chronic pulmonary disease (CPD), recurrent upper respiratory tract infections, ischemic or valvular heart disease, stroke, hyperlipidemia, peptic/duodenal ulcer (PDU), psoriasis, allergic skin disorders, deafness, hepatitis, tuberculosis and cancer (CA) was not different among the study groups. The rate of hypertension in FMF (11%) and SLE (20 %) were comparable (p =0.2), whereas higher when compared to BS (2%) and controls (2 %) (P = 0.008). The rate of urinary tract infections was higher among FMF and SLE patients however diarrhea (>3/year) was prevalent only in FMF patients (25 %). Family history of FMF was positive in 30 of the 80 FMF patients (35 %) and negative in the rest of the study population. There were 2 family members each, in FMF and SLE groups, with BS but only one in the BS group. Besides the family history of CPD and PDU, which were more frequent in the FMF group, no difference with regard to above listed co-morbidities were noted among the family members of the study groups.
Conclusions
According to the preliminary results of this ongoing study, we may say that there is no increase in the frequency of various co-morbidities including IHD, DM, CA and infections in patients with FMF and their first degree relatives compared to that of SLE, BS and healthy controls.

  The Frequency of Entesopathy Is Not Increased in FMF

Gulen Hatemi1, Koray  Tascilar1, Huri Ozdogan1, and Izzet Fresko1            

1Department of Rheumatology, Cerrahpasa Medical School, Istanbul University, Turkey

Objective: An association between Familial Mediterranean Fever (FMF) and spondylitis has been reported, but the prevalence of enthesopathies is not known. Ultrasonography (USG) is a reliable method for detecting enthesitis. This study aims to determine the frequency of entheseal abnormalities among patients with FMF using B mode and Power Doppler USG.

Methods: Thirty-five FMF patients, 19 AS patients and 20 healthy controls underwent USG. Probes with frequencies of 8.5-10 MHz, 11.5-13.5 MHz and 12.5-15 MHz were used. Power Doppler was also performed and a positivity was defined as an image obtained at 750 MHz and a gain of 24 dB. Five entheseal sites (superior and inferior poles of patella, tibial tuberosity, superior and inferior poles of calcaneus) on both lower limbs were examined. Tendon thicknesses and the presence of bursitis, bony erosions, entesophytes and positive Power Doppler signals were assessed and a composite score was calculated using an enthesitis scoring system, giving a total of 36 points. This score was compared by ANOVA in FMF patients and healthy and diseased controls. A second examiner performed USG on the same day to 22 of these patients. Interobserver reliability was assessed by intraclass correlation coefficient.

Results: Total enthesitis score over a maximum possible total of 36 was 9.2 ± 4.2 among AS patients, 4.2 ± 2.6 among FMF patients and 4.2 ± 1.9 among healthy controls. This score was significantly higher among AS patients when compared to both FMF patients (p=0.001) and healthy controls (p<0.000). There was no dıfference between FMF patients and healthy controls.
Among the 22 patients who were evaluated by 2 examiners, findings were compatible for 824/1012 (83%) entheseal abnormality parameters examined. Intraclass correlation coefficient was significant (r = 0.538, p<0.0001).

Conclusion: Frequency of entheseal abnormalities is not increased in FMF patients when compared to healthy controls.


  Alternative Treatments in Colchicine-Resistant Patients with Familial Mediterranean Fever (FMF)

Emire Seyahi1, Hasan Yazici1, and Huri Ozdogan1             

1University of Istanbul, Cerrahpasa Medical Faculty, Department of Internal Medicine, Division of Rheumatology

Background:
About 5-10 % of patients with FMF may experience attacks under regular colchicine (COL) treatment (1). We had previously reported the beneficial effect of thalidomide (TD) in controlling attacks refractory to COL, in a patient with FMF (2). Here we present 6 colchicine-resistant cases who received TD and / or etanercept (ETC) subsequently in addition to COL treatment.
Patients
Six (5 M/ 1 F) FMF patients with a median age of 36 years (range: 27-49) experiencing 1-4 attacks per month (median 2), while taking COL 2 mg/d regularly were offered a trial of thalidomide 100 mg/d. Informed consent was taken and possible side effects were explained in detail. COL 2 mg/day was continued. Three of these patients either due to ineffectiveness or side effects later were switched to receive ETC sc injections (25 mg, twice weekly). Patients were followed prospectively by monthly physical examinations and laboratory tests.
Results:
The duration of TD treatment ranged from 2 months to 2 years. Treatment was stopped in one patient after 20 days because of subjective complaints like shortness of breath and fear of death. One other patient discontinued treatment in the second month due to inefficacy and lethargy. The next patient who was reported previously in detail (2) had used TD at a dose of 200 mg/day for 1.5 years with a successful outcome. He continued a further 6 months with a reduced dose of 100 mg/d and had to stop TD because of the intense pain and numbness of the legs and an increase in the frequency of the attacks. The remaining 3 patients are still on TD. First patient is free of attacks for 5 months, next one had no abdominal attacks but 2 short episodes of joint attacks within 2 months, and the third one had 2 abdominal attacks compared to an expected 8 during the 2 months on TD treatment.
Three patients (2 M/ 1F) who stopped TD were put on ETC. One developed an allergic reaction during the 3rd injection and the treatment had to be discontinued. The other 2 are on ETC 25 mg twice weekly for 3 months. No significant decrease in the frequency, duration and the severity of the attacks has been observed up to now.
Conclusions:
If tolerated, TD seems to work better than etanercept in controlling the colchicine-resistant attacks of FMF. Close monitoring is required for thalidomide side effects.
References:
1) Lancet 1998; 351: 659-64.
2) Clin Exp Rheumatol 2002; 20 (4 Suppl 26):S43-4.

  Subclinical Atherosclerosis in Familial Mediterranean Fever (FMF)

Emire Seyahi1, Serdal Ugurlu2, Rana Cumali3, Huriye Balci4, Hasan Yazici1, and Huri Ozdogan1          

1University of Istanbul, Cerrahpasa Medical Faculty, Department of Internal Medicine, Division of Rheumatology; 2University of Istanbul, Cerrahpasa Medical Faculty, Department of Internal Medicine; 3University of Istanbul, Cerrahpasa Medical Faculty, Department of Internal Medicine, Radiology Laboratory; 4University of Istanbul, Cerrahpasa Medical Faculty, Central laboratory

Background: Chronic inflammation may play an important role in the initiation and progression of atherosclerosis and ischemic heart disease. FMF is an auto-inflammatory disease with evidence of subclinical inflammation during attack-free periods and increased acute phase response in first-degree relatives. However the frequency of ischemic heart disease (IHD) was found to be compatible with normal population (1). Atherosclerosis has not been formally assessed in FMF patients. In this study, we investigated surrogate markers of atherosclerosis by B-mode ultrasonography in FMF patients and age and sex matched healthy controls.
Patients and Methods: Fifty-five patients with FMF, (30 M/ 25 F; mean age 39 ± 7 yr), and 55 healthy controls (HC) (30 M/ 25 F; mean age 39 ± 6 yr) were studied. Subclinical atherosclerosis was assessed by investigating atherosclerotic plaques and measuring intima media thickness (IMT) from common, internal and external carotid and common femoral arteries using B-mode USG. Traditional atherosclerotic risk factors were evaluated. Patients and controls with a history of amyloidosis, chronic renal failure, myocardial infarction and ischemic stroke were not included into the study.
Results: The mean disease duration of FMF patients was 20 ± 10 years. The mean age and atherosclerotic risk factors were similar among all patients and HC, except HDL levels, which were significantly lower among FMF patients, both male and female, compared to that of HC (p < 0.001). The overall prevalence of atherosclerotic plaques in carotid and / or femoral artery was similar among patients with FMF (n=9; 16 %) and HC (n=8; 15 %) (P = 0.3). Also the mean IMT of carotid and femoral arteries was not different among patients with FMF (0.68 ± 0.07 mm) and HC (0.67 ± 0.09) (P = 0.7). This was also true when males and females were evaluated separately. Multiple logistic regression analyses showed that the age [for 1 year increase: OR= 1.2; 95 % CI (1.0-1.4); p= 0.023] and family history of premature IHD [OR= 26; 95 % CI (3.1-225.1); p= 0.003] were associated with plaque presence in FMF patients, whereas increased IMT remained as the only predictive factor for atherosclerotic plaque in HC.
Conclusion: These findings suggest that despite the evidence of recurrent inflammation, colchicine-treated FMF patients are not more predisposed to IHD than the HC. Low levels of HDL among FMF patients deserve further evaluation.
Reference:
1) Langevitz P et al. Prevalence of ischemic heart disease in patients with familial Mediterranean fever. Isr Med Assoc J 2001; 3: 9-12.

  Transaminase Elevations in FMF Patients During Colchicine Treatment May Be Caused by Muscle Toxicity

Koray  Tascilar1, Gulen Hatemi1, and Huri Ozdogan1             

1Department of Rheumatology, Cerrahpasa Medical School, Istanbul University, Turkey

Objective: Elevation of transaminase levels is frequently encountered in FMF patients during colchicine treatment. It is not clear whether these elevations are caused by liver or muscle toxicity. The aim of this study is to determine the presence and scope of muscle enzyme elevation in patients with FMF on colchicine treatment.

Method: Patient charts of consecutive FMF patients followed in a tertiary rheumatology outpatient clinic and a rheumatologist’s private practice were retrospectively reviewed with regards to elevated creatine kinase (CK) levels, alanine and aspartate transaminase levels, colchicine dose and duration of use. The action taken in case of elevated enzyme levels and the outcome were also recorded.

Results: Two-hundred-twenty-seven patient charts (91 charts from the outpatient clinic and 136 charts from the private practice) were reviewed. Nine of the 227 patients (4%; 5 men, 4 women, mean age 32 ± 8 years) had elevated CK levels during their follow up. Median duration of colchicine treatment was 2 years (range 1-12 years) in these patients. Patients were on 1 to 2 mg of colchicine per day at the time of enzyme elevations.
Six of these were mild elevations, below 2 times the upper limit of normal. Remaining 3 patients had CK levels within 3, 4 and 5 times the upper limit of normal. Six of the patients had concommitant mild to moderate unexplained transaminase level elevations which had lasted for months before CK was checked. Colchicine was discontinued in two of the patients and the dose was reduced in the other 7. High CK levels persisted for 1 to 13 months (median 4 months) and did not always respond promptly to dose reduction or discontinuation of the drug.
Elevated aminotransferase levels in the range of 2-5 times the upper limit of normal was observed in 22/227 (10%) patients. Increased CK levels accompanied the elevation in aminotransferase levels in 6/22 patients. These patients are among the 9 patients with elevated CK levels, described above.
None of the patients with CK elevation had weakness or muscle pain as a primary complaint. EMG and muscle biopsy was performed to one of the patients who had persistant CK elevation. EMG showed mild myopathy in the proximal limb muscles and muscle biopsy did not reveal any inflammatory or toxic changes.

Conclusion: Some of the transaminase elevations during colchicine treatment may be accompanied by an elevation in CK suggesting muscle toxicity. CK should be included in the list of routine investigations asked from patients on prophylactic colchicine treatment. Prospective data are needed to determine the real prevalance and clinical significance of muscle enzyme elevation in FMF patients receiving colchicine treatment.


  Lack of association of the CD14 C159T polymorphism with the development of secondary amyloidosis in Familial Mediterranean Fever

Ozlem  Keskin2, Engin Yilmaz3, Aysin Bakkaloglu1, Fatos  Yalcinkaya4, Mustafa Arici5, Rezan  Topaloglu1 , Nesrin  Besbas1, Birsin  Ozcakar4, and Seza Ozen1      

1Dept. of Pediatrics, Hacettepe University, Ankara, Turkey; 2Dept. of Allergy Laboratory, Hacettepe University, Ankara, Turkey; 3Molecular Biology, Hacettepe University, Ankara, Turkey; 4Dept. of Pediatrics, Ankara University, Ankara, Turkey; 5Dept. of Internal Medicine, Hacettepe University, Ankara, Turkey

The presence of environmental risk factors for the development of secondary amyloidosis in Familial Mediterranean Fever (FMF) has been an intriguing question. CD14 which is a pathogen-recognition receptor complex, is critical to develop responses to microbes. An increased expression of CD14 enhances the generation of T-cell responses. The aim of this study was to investigate whether the CD14 C-159T polymorphism, which is associated with higher CD14 expression resulting in more robust T-cell responses, was associated with the development of amyloidosis.
The study population consisted of four groups: The secondary amyloidosis group consisted of 34 children with FMF, all who had developed amyloidosis before their 16th birthday. There were two FMF patient groups, the first group being 24 adult patients in whom the diagnosis was missed during childhood despite symptoms, but had not developed amyloidosis and 99 children with FMF all who have started treatment at childhood. 92 healthy adults serving as controls, constituted the final group.
The distribution of each of the genetic variants met the conditions of the Hardy-Weinberg equilibrium. There were no significant differences in the genotype distributions between either of the groups.
This study attempted to investigate a possible role of environment on a genetic determinant, however, we have failed to associate this polymorphism with the development of amyloidosis. We suggest that the complex genetic interactions necessitate a broader study of the relevant pathways.

  Is there a Defect in the Neuroendocrine Immune System in Familial Mediterrandean Fever?

Rezan Topaloglu1, Yelda Bilginer1, Nazlı Kara2, Nesrin Besbas1, Seza Ozen1, and Aysin Bakkaloglu1          

1Pediatric Nephrology and Rheumatology, Department of Pediatrics, Hacettepe University Faculty of Medicine, Ankara Turkiye ; 2Pediatric Nephrology and Rheumatology, Department of Pediatrics, Social Security Hospital, Ankara Turkiye

The aim was to determine the interactions between neuroendocrine and immune system in patients with Familial Mediterranean Fever (FMF) and investigate the role of neuroendocrine system in the acute inflammation process. Demographic characteristics, disease activity, mutation analysis and duration of the disease were defined in 15 FMF patients (7 female,8 male; mean age ± SD 9.1 ± 4.2 years ). The diagnosis is based on Tel-Hashomer criteria. Ten healthy voluenteers and 21 active juvenile idiopathic arthritis (JIA) patients composed the control groups. Furthermore 10 of these 15 patients with FMF were also studied during the attack free period. Erythrocyte sedimentation rate, C-reactive protein (CRP), fibrinogen, ACTH, cortisol, prolactin, IGF-1, IGFBP3, free T3, free T4, TSH, urine cortisol levels, IL-1β, IL-6 and TNF-α were evaluated in FMF patients with attack and attack free period. The median levels of ACTH (12.7 pg/ml) and cortisol (12 ug/dl) at 08.00 a.m. were lower in FMF patients with attack than attack free period, this did not reach the statistical significance. On the other hand the median levels of ACTH and cortisol were significantly lower in attack than in healthy control group (p<0.05). The median level of IGF-1 (118.5 ng/ml) was significantly lower in FMF attack than attack free period (p<0.05). There was a negative correlation between IGF-1 and CRP (r=-0.69). The median level of IL-6 was 18.1 pg/dl in FMF attack and significantly higher than the attack free period and healthy control groups (p<0.05). There was a negative correlation between cortisol levels at 08.00 am and IL-6 (r = -0.55). When we compare JIA with FMF patients in attack inappropriately low secretion of adrenal cortisol and ACTH and impaired diurnal rythm of cortisol were more pronounced in JIA than FMF. Although it is more prominent in chronic inflammation neuroendocrine immune system is impaired in relation to acute inflammation in FMF.
  Quality of Life (QOL) of Patients with Familial Mediterranean Fever

Fatih Saygili1, Baris Akinci1, Serkan A. Yener1, Yildiz Akvardar2, Yucel Demiral3, Servet Akar4 , and Mehmet Tunca1        

1Dokuz Eylul University School of Medicine Department of Internal Medicine, Izmir Turkey; 2Dokuz Eylul University School of Medicine Department of Psychiatry, Izmir Turkey; 3Dokuz Eylul University School of Medicine Department of Public Health, Izmir Turkey; 4Dokuz Eylul University School of Medicine Department of Rheumatology, Izmir Turkey

Introduction. The aim of this study is to assess the impairment of QOL of patients with FMF and the features that cause this impairment by using Short Form-36 (SF-36) scale.

Methods. One hundred and fifty three patients were compared to 150 healthy controls by using SF-36 scale. SF-36 includes 8 main items and two general items which are calculated by using the eight major items. This scale was chosen because it was previously employed in several rheumatological disorders with satisfactory results.

Results. The mean physical and mental scores of the patients were significantly lower than the control group: physical mean score: 47,36±8,5 vs 52,6±8,8 (p<0,001), mental mean score: 43,67±9,92 vs 51,7±5,6 (p<0,001). Also there was significant impairment of scores through all eight items in the study group. Many factors that show the severity of disease activity were related with both mental and physical mean scores. Multiple regression analysis showed that the number of attacks in the last 6 month was associated with the impairment of physical health and the number of abdominal pain in the last 6 month was found to be associated with impairment of mental health.

Conclusions. The QOL of FMF patients was found to be impaired compared to healthy controls. This impairment may be parallel to the disease severity.





  Serum amyloid A level as a tool in the diagnosis and treatment of familial Mediterranean fever.

Yackov  Berkun1, Shai  Padeh1, Brian Reichman1, Nurit  Zaks2, Einat  Rabinovitz2, Anat  Doron2 , and Avi Livneh2        

1Department of Pediatrics, Safra Childrens Hospital, Sheba Medical Center, Tel-Hashomer, Israel 52621; 2Heller Institute for Medical Research, Sheba Medical Center, Tel-Hashomer, Israel 52621

Background: Familial Mediterranean fever (FMF) is a hereditary disease characterized by recurrent episodes of inflammation and manifested by fever, peritonitis, pleuritis, arthritis and high serum amyloid A (SAA), a sensitive acute phase reactant and a major component of amyloid deposits.
Objectives: To evaluate the role of SAA levels in the diagnosis and management of FMF.
Patients and Methods: Medical files of 204 patients from our FMF center, in whom SAA measurements were taken, were retrospectively reviewed. SAA levels, main clinical features, treatment and diagnosis decisions were analyzed.
Results: SAA level was determined for diagnosis in 28.9% of the patients. In the remaining patients, the SAA measurement was used for adjusting colchicine treatment, which was changed in 29.9% of the patients. Treatment was initiated in 9.8% of all studied patients, and the dose of colchicine was increased in 13.2% of the patients. The use of SAA level was most effective in colchicine treated patients with proteinuria or with atypical attacks, leading to an increase in dosage in 50% and 43% of the patients in these subgroups, respectively. The use of SAA level was least effective in asymptomatic patients treated with low dosage and in patients who remained symptomatic despite maximal colchicine dosage. Increased SAA levels during attack-free periods were detected in 23% of all patients. The highest rate was in patients with proteinuria and in non-compliant patients (60% and 40%, respectively), while the lowest rate was in patients treated by low dosage colchicine who remained asymptomatic (10.4%).
Conclusions: SAA measurement plays an important role in FMF for the adjustment of colchicine dose, determination of therapeutic response and diagnosis.

  Peritoneal adhesions and intestinal obstructions in patients with familial Mediterranean fever – are they more frequent?

Yackov  Berkun2, Eli  Ben-Chetrit3, Aharon Klar4, and Eldad  Ben-Chetrit1            

1FMF Clinic, Department of Medicine, Hadassah University Hospital, Jerusalem, Israel; 2Department of Pediatrics, FMF Clinic, Safra Children's Hospital, Sheba Medical Center, Tel-Hashomer, Israel 52621; 3Department of Medicine, Shaare Zedek Medical Center, Jerusalem, Israel; 4Department of Pediatrics, Bikur Cholim Hospital, Jerusalem, Israel

Background: Familial Mediterranean fever FMF) is characterized by recurrent episodes of peritonitis. A controversy exists whether intestinal obstruction is common in FMF patients due to peritoneal adhesions induced by these attacks.
Objective: To estimate the rate of spontaneous or post surgical small bowel obstruction (SBO) in FMF patients.
Patients and Methods: We searched the English medical literature for reports on SBO - due to peritoneal adhesions - in FMF patients. We reviewed the charts of 560 FMF patients followed in our clinic for spontaneous SBO. We also looked for post appendectomy intestinal obstruction among 88 FMF patients compared with 208 individuals without FMF who had appendectomy in the same Medical Center, during the same period.
Results: Eight out of 560 FMF patients (1.4%) developed spontaneous SBO requiring laparotomy and adhesiolysis. Three out of 88 (3.4%) FMF patients who underwent appendectomy developed SBO requiring surgery. None of the non-FMF patients developed SBO.
Conclusions: FMF patients are at higher risk than healthy individuals to develop small bowel obstruction either spontaneously or as a post surgical complication.


  Common FMF Alleles Increase the Risk for Developing Vascular-Behcet's Disease

Einat Rabinovich, Pnina Langevitz, Merav  Leiba, Yael Shinar, and Avi  Livneh           

1“Heller Institute for Medical Research, Sheba Medical Center, Tel-Hashomer, Israel 52621”

Background: Behcet's disease (BD) is an inflammatory disorder of unknown cause, associated with vasculitis. Arterial or venous thrombosis occurs in about 25 % of the patients. Familial Mediterranean fever (FMF) is another inflammatory disorder, which stems from mutations in the FMF gene (MEFV) and shares a number of features with BD.
Objective:MEFV analysis in patients with BD suggests that mutated MEFV may act as a susceptibility gene in BD. We studied the rate and the clinical correlates of MEFV mutations in Israeli BD patients.
Methods: Included were 54 BD patients, who satisfied the International Study Group criteria for BD. All BD patients were genotyped using polymerase chain reaction and restriction enzyme analysis for the 3 common MEFV mutations (M694V, V726A, E148Q). The association between BD manifestations and MEFV alleles was analyzed.
Results: Twenty one BD patients were found to carry a single MEFV mutation and an additional 3 were compound heterozygotes, a frequency significantly higher than that expected for ethnically matched healthy individuals. There were no statistically significant differences between carriers and non-carriers with respect to gender, frequency of HLA B5 antigen, cutaneus lesions, joint disease and severity score. However, carriers did suffer more often from thrombosis (54 %vs 17%, p<0.005) and less often from uveitis (20% vs 40%, p<0.05).
Conclusions: MEFV appears to be a susceptibility and modifier gene in BD.
  MEFV mutation analysis in patients with IgA nephropathy and other types of glomerulonephritis

Lesya  Koukoui1, Juri  Kopolovic2, Alex  Blau1, Aharon  Ben-David1, Dani  Lotan3, Shaked  Meital4 , Yael  Shinar5, Pnina Langevitz5, and Avi  Livneh5      

1Department of Nephrology and Hypertension, Sheba Medical Center, Tel-Hashomer, 52621,Israel; 2Department of Pathology, Sheba Medical Center, Tel-Hashomer, 52621,Israel; 3Pediatric Nephrology unit, Safra Children Hospital, Tel-Hashomer, 52621,Israel; 4Department of Nephrology, Sourasky Medical Center,Tel Aviv, Israel; 5Heller Institute of Medical Research, Sheba Medical Center, Tel-Hashomer, 52621,Israel

Background and aim - IgA nephropathy (IgAN) is the most common cause of glomerulonephritis. This disease may be expressed with a wide spectrum of manifestations changing between asymptomatic microhematuria to an end-stage kidney disease. The etiology of the disease is unclear and involves a variety of environmental and genetic factors. The disease seems to be common in familial Mediterranean fever (FMF). Pyrin, the gene of FMF, was found to predispose and affect several inflammatory diseases. The aim of the present study was to discover if MEFV gene mutations constitute a risk factor for IgAN and if they modify the course of this disease.
Methods – The study group consisted of 40 patients with biopsy proven IgAN. The control group consisted of 22 patients with other types of glomerulonephritides. Patients of the two groups were interviewed, examined and donated blood samples for MEFV mutation analysis. The severity of IgAN was classified as mild, moderate, or severe based on the findings in the urine and the serum creatinine level. MEFV mutation analysis was carried out using PCR amplification and restriction enzyme digestion.
Results - The rate of the MEFV mutations in the study and control groups was comparable (12:40 vs. 5:22). However, the frequency of MEFV mutations in IgAN was three times higher than expected in the ethnically matched general population (p<0.05). The severity of IgAN, however, was much lower in carriers of mutations (p<0.05), even though kidney pathology and urine analysis were comparable.
Conclusion – carrying a MEFV mutation predisposes the development of IgAN, but with a mild phenotype.


  Liver Involvement Associated With Familial Mediterranean Fever(FMF)

Véronique Hentgen1*, Muriel Girard2*, Monique  Fabre3, Philippe  Reinert4, and Olivier Bernard2           

1Departement of pediatrics, hôpital Mignot, Le Chesnay, France 78150; 2hépatologie pédiatrique, hôpital Bicêtre, Le Kremlin-Bicêtre , France 94270; 3Anatomie pathologique, hôpital Bicêtre, Le Kremlin-Bicêtre , France 94270; 4Departement of pediatrics, CHIC, Créteil, France 94000 ; *These authors contributed equally to this work.

Liver involvement has been very rarely reported in patients with FMF and the relationship between both conditions is still a matter of debate. We report a child in whom the diagnosis of FMF was made on the occasion of several episodes of hepatitis with liver failure. This girl is the first child of unrelated parents of Sephardic origin. FMF is present in 3 relatives on the mother’s side. From age 12 months she presented every 2-3 weeks with 48 h bouts of fever, malaise and pain. During one such episode at age 19 m serum transaminases ALT were found to be 10xN with gammaGT 1.5xN, prothrombin time (PT) 50% and normal bilirubin; CRP was 174mg/l, platelets 510.000/cumm, IgG 9.6 g/l and fibrinogen 4.4 g/l. PT normalised in 3 days and ALT decreased to 2.5xN. 2 similar episodes occurred over the following month with PT 59 and 49 %, factor VII 31 and 21 % and ALT 8xN and 7xN respectively. No signs of a known viral, autoimmune, biliary, metabolic or toxic cause of liver disease could be found. Liver histology showed portal fibrosis and polymorph inflammation with slight piecemeal necrosis. Testing of the MEFV gene displayed a homozygous M694V mutation. Colchicin therapy was started and ALT returned to normal. She presented over the next 6 months with only 2 episodes of FMF with ALT 3.5xN and 20xN respectively but normal PT. Over the following two years there have been 2-3 yearly episodes of FMF and ALT remained normal. Although the fortuitous association of a serologically negative autoimmune hepatitis cannot be excluded, the coincidence of the signs of liver involvement and of the clinical episodes of FMF and the improvement of both condition under colchicin therapy raises the possibility of a link between FMF and liver disease in this child.
  Searching for Genes of Periodic Fevers of Unknown Origin

Francesca Ancarani1, Micaela La Regina1, Marialuisa Diaco1, Monica Vastola1, Elena Campobasso1, Sara Dalvai1 , Giovanni Neri2, Maria Grazia Pomponi2, Raffaele Manna1, and Giovanni Gasbarrini1     

1Centre of Periodic Fevers, Policlinic A. Gemelli, Catholic University of Sacred Heart, Rome, Italy; 2Institute of Human Genetics, Catholic University of Sacred Heart, Rome, Italy

The marenostrin/pyrin-encoding gene (MEFV), mapped to the short arm of chromosome 16, has been proposed as a candidate gene for Familial Mediterranean Fever (FMF) on the basis of the identification of mutations clustered in few exons as 10 and 2. Over 50 MEFV mutations have been identified so far in FMF database of INFEVERS website. In addition to disease-causing mutations, several polymorphisms have been detected. The G>A substitution at codon 605 (R202Q) probably represents a common polymorphism of MEFV, since it has been found frequently in association with M694V mutations in non-carrier chromosomes as well as in controls unrelated to FMF families. It has been already reported that R202Q is not associated with FMF and does not confer a significant risk for the disease.
Among patients affected by periodic fevers of unknown origin (P-FUO), collected in our centre since 1997, that underwent to the complete MEFV sequencing, 146 had a certain diagnosis FMF according to Tel Hashomer criteria. R202Q substitution was present in 16% of patients included in this series, according to the literature. No correlation genotype-phenotype was found.
Among the remaining 83 patients with P-FUO, other than FMF, TRAPS and HIDS, 31 carried the R202Q substitution (37,3%), showing a percentage double than expected. We tried to evaluate if there was a correlation between R202Q and the level of MEFV transcript or if there was an interference with its biological activity. Expression levels of marenostrin/pyrin in 3 patients affected by P-FUO and in 3 matched controls were determined based on 2 independent sets of RT-PCR. In homozygous M694V we observe the lowest expression (0.53); similarly in the homozygous R202Q patient we obtained reduced MEFV mRNA levels (0.63), whereas in the heterozygous R202Q patient we found increased levels of mRNA (1.23), as we can observe in the heterozygous l’E148Q; we can hypothysise that a protein instability can trigger a feedback transcription. Therefore the role of R202Q in these patients deserves further investigations. It would be useful to extend the research by microarrays tools and/or determine expression levels of marenostrin/pyrin in a wider number of P-FUO patients in order to correlate MEFV mRNA levels to specific clinical features. R202Q prevalence in Italian healthy subject is currently under investigation too.

  Genomic Differential Expression of FMF Patients without MEFV Mutations by Microarray-Bioinformatics Analysis

Francesca Ancarani1, Claudia Cerquaglia1, Natalie Saulnier1, Micaela La Regina1, Marialuisa Diaco1, Antonio Gasbarrini1 , Raffaele Manna1, and Giovanni Gasbarrini1       

1Periodic Fevers Research Centre, Department of Internal Medicine, Catholic University of Sacred Heart, Rome, Italy

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of fever and serositis. Other manifestations include pleuritis, arthritis, and erysipelas-like skin disease. Mutations in the pyrin/marenostrin gene (MEFV) located in chromosome 16p13,3, have been identified in the majority of FMF patients; moreover about 30% of certain FMF patients does not have any known mutation, renforcing the idea that probably other important genes play a role. The wide clinical variability is partly attributed to MEFV allelic heterogeneity, and partly influenced by environmental and genetic factors, many of which remain unknown.
Recent advances in bioinformatics and high-throughput technologies such as microarray analysis are bringing about a revolution in our understanding of the molecular mechanisms underlying normal and dysfunctional biological processes. Gene expression profiling has enabled the measurement of thousands of genes in a single RNA sample.
Therefore we are trying to analyse the genomic differential expression among FMF patients without any known mutation using microarrays gene technology.
The study cohort, consisting of 15 patients with FMF according to Tel-Hashomer criteria, without known MEFV mutations, was divided into three groups, according to the presence of fever, serositis and articoular pain (n = 4)(group A), of fever alone (n = 5)(group B), or else of fever with arthritis (n = 6) (group C). The results were then compared with 8 controls.
RNA was isolated from the samples, using standard procedures, from peripheral leukocytes. Then it was retro-transcribed into cDNA, amplified and labeled during an in vitro transcription reaction. Human Focus arrays (counting about 8400 genes) were hybridized with about 15 ug of cRNA and read with a scanner. Data analysis was performed with GeneSpring software 7.2. Data were normalized with the Robust Multichip Analysis (GC-RMA). Finally, analysis of the multiple data was made with parametric tests (ANOVA Test).
Among the results, we focused our attention on the common genes between the groups: in particular, we identified statistically significant gene expression signatures that characterize group 1, 2 and 3. The FMF gene expression signature is enriched for four genes that are functionally associated with inflammation, induction of apoptosis, such as FADD, HEM 1, BCL 10 and ARFGEF1.
The differential examination between FMF patients and healthy control subjects shows that the former have upregulated some genes involved in the induction of apoptosis via death domain receptors and related to antimicrobial inflammation response. Differential expression analysis between groups requires further investigations.


  Looking for Colchicine Analogues: Basic Chemical Studies and Tests in vitro

Claudia Cerquaglia1*, Giuseppe La Regina3*, Marialuisa Diaco1, Micaela La Regina1, Sara Dalvai1, Valentina Curigliano1 , Pasquale De Sole2, Cristina Rossi2, Romano Silvestri3, and Raffaele Manna1     

1Periodic Fevers Research Centre, Department of Internal Medicine, Chatolic University of Sacred Heart, Rome, Italy; 2Institute of Clinical Biochemistry, Chatolic University of Sacred Heart, Rome, Italy; 3Institute of Pharmaceutical Chemistry, La Sapienza University, Rome, Italy ; *These authors contributed equally to this work.

INTRODUCTION: Colchicine, the unique available drug for Familial Mediterranean Fever (FMF), acts by preventing activation of neutrophils. It inhibits assembly of microtubules and mitotic spindle formation binding beta-tubulin and making beta-tubulin-colchicine complexes. Moreover its mode of action includes modulation of chemokines, prostanoids production, inhibition of neutrophils and endothelial cell adhesion molecules. Unfortunately, colchicine have low therapeutic index, easy toxicity and sometimes ineffectiveness (10-15% of FMF patients according to literature data).
We tried to identify colchicine analogues with better therapeutic effects and less adverse effects testing their efficacy and toxicity in vitro.
MATERIALS AND METHODS: Chemical procedures: we selected for biological tests some small molecules colchicine and other very-well known tubulin inhibitors related, previously synthesized in our laboratories. To investigate the possible binding mode of the most active compounds, we carried out docking studies in the colchicine binding site of tubulin, which was obtained from the recently reported 3D structure of tubulin cocrystallized with a colchicine analogue: N-deacetyl-N-(2-mercaptoacetyl) colchicine (DAMA-colchicine).
Tests in vitro: we show the results obtained with 34 new colchicine analogues tested out on the respiratory burst of neutrophils. The ability of the different analogues to inhibit the neutrophils respiratory burst was analysed by measuring their effects on luminol-dependent chemiluminescence of neutrophils stimulated by zymosan or phorbol ester (PMA).
Toxicity was investigated by exposure of colonic adenocarcinoma cell lines (WiDr) to the five colchicine analogues that seemed more active by chemiluminescence of neutrophils.
Subsequently, we had analysed inhibition of cell lines growth curves.
RESULTS: Any of the colchicine analogues we used, showed a strong inhibition by neutrophils chemiluminescence. Instead toxicity test showed that 2 of 5 colchicine analogues (RS 1228, RS 1363) caused a lower inhibition level of cell lines growth that colchicine.
CONCLUSIONS: Our study shows that the 34 colchicine analogues used would not be better than colchicine according to chemiluminescence test in vitro, whereas the analogues RS 1228 and RS 1363 show a profile of reduced toxicity on exposure of colonic adenocarcinoma cell lines. Further investigations are ongoing to confirm these results as well as to identify new analogues.

  Geno-phenotypical Correlations in Italian People Afflicted with Familial Mediterranean Fever (FMF)

Claudia Cerquaglia1, Micaela La Regina1, Marialuisa Diaco1, Ferruccio D'Onofrio1, Grazia Pomponi2, Giovanni Neri2 , Grazia Maria Marino2, Alessandra Terracciano2, Isabelle Touitou3, Giovanni Gasbarrini1, and Raffaele Manna1    

1Periodic Fevers Research Centre, Department of Internal Medicine, Catholic University of Sacred Heart, Rome, Italy; 2Institute of Human Genetic, Catholic University of Sacred Heart, Rome, Italy; 3Laboratory of Genetic, A. Villeneuve Hospital, Montpellier, France

In the registry of our Centre of Periodic Fevers 146 adult patients with FMF are recorded; according to Tel-Hashomer criteria, 122 patients had a diagnosis of certain FMF and 22 probable.
Complete MEFV sequencing was performed in 144 Italians with FMF diagnosis; 2 patients refused to perform genetic test.
From genetic test 54/144 (37,5%) patients had two MEFV mutation, 31/144 (21,5%) patients had least one MEFV mutation, 46/144 (32%) patients had not any known MEFV mutation. We found 10/144 (6,9%) patients with R202Q in heterozygosis and 6/144 (4,2%) patients with R202Q in associations with other MEFV mutations.
M694V, in heterozygosis or homozygosis, was the most frequent mutation (41%), followed by M680I (21%), M694I (20%), E148Q (19%), V726A (13%), R761H (7%) and M680I G>A (5%). The prevalence of M694I, a mutation frequently encountered in Maghrebins, and of the rare form of M680I (M680I G>A) was relatively high in Italians (20% and 5% respectively). Other rare exon 10 mutations were also found: A744S, S675N, C280A, E167D, P369S, R408Q, K695R, I369S, P369Q and a stop codon mutation Y688X. No complex allele was detected.
The results of geno-phenotypical correlations are: 1. homozygotes for M694V and M694I are associated with more severe phenotype (mean severity score 9 and 8,6 respectively) than heterozygotes at these codons (mean severity score 7.1 and 6,2 respectively); 2. homozygotes and heterozygotes at codons 680 are associated with a moderate phenotype (mean severity score 7.5); 3. homozygotes at codons 148 show a little more mild phenotype (mean severity score 5) than heterozygotes (mean severity score 6,9); 4. patients with only R202Q or with this polymorphism in association with in other mutations show moderate phenotype (mean severity score 6,4 and 7,6 respectively); 5. patients with other mutations in heterozygosis or homozygosis are not a phenotypically homogeneous group, with a severity score ranging from 2 to 13; 6. patients without any known mutation show moderate phenotype (mean severity score 7).
In conclusion, Italians with FMF show a moderate phenotype independently from the genotype, probably due to the modulator effect of other genes. Really, 55,5% of our patients have moderate phenotype, 28,8% mild and only 15,7% severe. We would point out 32% patients with severe phenotype have M694V and 32% (among them 3 cases of renal amyloidosis) does not have any known mutation, renforcing the idea that probably other genes play a role: investigations by microarrays are currently ongoing.


  Unresponsiveness to Colchicine: Why?

Valentina Curigliano1*, Elena Verrecchia1*, Claudia Cerquaglia1, Ferruccio D'Onofrio1, Francesca Ancarani1, Monica Vastola1 , Massimo Montalto1, Raffaele Manna1, and Giovanni Gasbarrini1      

1Periodic Fevers Research Centre, Department of Internal Medicine, Catholic University of Sacred Heart, Rome, Italy ; *These authors contributed equally to this work.

INTRODUCTION: Mainstay of FMF treatment is colchicine and its adverse effects are well known. Among gastrointestinal involvement, the mucosal injury can induce secondary lactose intolerance, with a consequent osmotic diarrhea. We suppose that the condition could lead to a modified colchicine absorption and incomplete effect. We report a case of a FMF patient, with colchicine unresponsiveness and lactose intolerance.
CASE REPORT: We describe the case of a young woman, 20 years, from south-Italy. Since she was 2 years old, she had fever, abdominal and thoracic pain, vomiting and constipation, lasting 24 hours, occurring monthly. During those manifestations, acute phase proteins increased, while were normal in the attack-free periods. The attacks were often exacerbated during menstruations. Genetic analysis of MEFV identified homozygosis for M680I mutation, that is FMF-related. The patient started colchicine therapy, 1 mg/d, with no effectiveness. Successively, we increased the dose to 2 mg/d, but no changes were observed in disease manifestations. She was investigated for occult inflammatory process, without any results. Since lactose intolerance realized, we recommended a diet without milk and dairy products. Successively, she had no more FMF attacks.
DISCUSSION: Colchicine could lead to a secondary lactase deficiency. It has been demonstrated that colchicine could affect the gastrointestinal mucosa by inhibition of Na+/K+ exchange pump regulating water and electrolytes transport or with a direct bowel mucosal damage. Since the condition of lactose intolerance causes osmotic diarrhea, we can suppose that the amount of absorbed drug is reduced. Consequently, the bioavaibility of the drug could be modified, leading to an incomplete colchicine effectiveness. According this observation, we can consider that colchicine unresponsiveness in FMF patients, in absence of any trigger factors, may due to conditions that interfere with drug-absorption, as lactose intolerance.

  Clinical Features of Familial Mediterranean Fever (FMF) in Italians: an Overview

Marialuisa Diaco1, Claudia Cerquaglia1, Elena Verrecchia1, Francesca Ancarani1, Micaela La Regina1, Valentina Curigliano1 , Alessandra Costa1, Giuliana De Socio1, Raffaele Manna1, and Giovanni Gasbarrini1     

1Periodic Fevers Research Centre, Department of Internal Medicine, Catholic University of Sacred Heart, Rome, Italy

In the current study we analyzed clinical data of 146 patients (pts) (81 males; 65 females), aged from 3 to 81, affected by FMF, attending the adult section of a Periodic Fevers Research Centre in the last 8 years. Clinical diagnosis was established by Tel-Hashomer criteria; genetic testing by complete MEFV sequencing in 144/146 pts was performed. All pts can contact us by mail or telephone and understand better the disease through a website (www.conosciamocimeglio.it). A database file of genetic and clinical recordings of pts is available in Excel, Word and papery format.
Most pts come from the south and the centre of Italy, probably because of the geographical position of Italy and the migratory changes of its population during last 20 centuries. Some pts come from near countries (Malta, Syria, Tunisia).
The age of onset is under 20 in 65% of the pts. The frequency of the attacks goes from less than 1 attack/month (23,3%) to more than 2 attack/month (26,7%). According to the severity score a mild disease was documented in 28,8%, a moderate disease in 55,5% and a severe one in 15,8% of pts.
The most typical attack presents fever and abdominal pain (80,8%) followed by fever and articular pain (65,1%) and by fever and thoracic pain (41,8%). Only 3 pts have abdominal and thoracic pain or abdominal and articular pain or abdominal, thoracic and articular pain without fever. Thoracic, pleural, pericardial or pleuro-pericardial pain affects 66 pts.
97 pts had joints involvement: acute arthritis in 23, arthralgias in 13 and chronic arthritis in 3 pts; 11 complained of myalgias.
Abdominal pain affects 121 pts with nausea (33,6%), vomiting (33,6%) and in few cases diarrhoea or constipation. 20 pts have a renal function compromised and the damage goes from microalbuminuria to renal failure; 2 pts carry a phenotype 2. Menses and physical/psychological stress triggered the attacks in 23 and 9 pts, respectively. 127 pts received colchicine treatment (86,6% good effectiveness; 9,4% mild effectiveness; 3,9% no response).
The collected data suggest that the characteristics of this Italian series are: a less severe disease, low prevalence of amyloidosis, higher incidence of late onset, high rate of colchicine responders.
Moreover, the foundation of a centre devoted to periodic autoinflammatory diseases allows frequent consultations and monitoring of the patients. Moreover investigations about new genes, early diagnosis, reduction of cost of diagnosis, disease complications and colchicine analogues are currently ongoing.

  9-Base Insertion in MEFV Gene in a Patient with Familial Mediterranean Fever

Micaela La Regina1, Marialuisa Diaco1, Francesca Ancarani1, Elena Verrecchia1, Elena Campobasso1, Gabriella Nucera1 , Grazia Pomponi2, Giovanni Neri2, Giovanni Gasbarrini1, and Raffaele Manna1     

1Periodic Fevers Research Centre, Department of Internal Medicine, Catholic University of Sacred Hearth, Rome, Italy; 2Institute of Human Genetic, Catholic University of Sacred Hearth, Rome, Italy

After gene cloning, over 50 mutations, including three 3-base deletions and one 1-base insertion, have been associated to clinical phenotypes of Familial Mediterranean fever (FMF; OMIM #249100).
We describe a patient with FMF phenotype and a 9-base insertion within exon 2 on one allele and a polymorphysm on the other one.
Male, 42 years old, coming from central Italy, reports a 25-year history of short attacks of fever and arthralgias, followed by headache and diarrhoea with increased acute phase reactants and leucocytosis. Clinically, after the exclusion of other causes of fever and arthralgias, the patient started colchicine (1,5 mg/d) successfully.
The complete sequencing of MEFV (Gene Bank Access number XM_007971) shows a frameshift on exon 2 in the proband and his mother and a G>A substitution at codon 605 in his father (R202Q). His parents are asymptomatic.
Gene cloning from aminoacidic position 92 to 270, by Topo-cloning kit, and the following sequencing, shows an insertion of 9 bases from codon 390 (GAGGGGAAC) on one allele either in the proband and his mother. The proband carries the same G>A substitution of his father (R202Q) on the other allele. The 9-base insertion causes the insertion of 3 amino-acids (glutamic acid, glycine and asparagine) respectively on position 131, 132 and 133 of the pyrin.
Our hypothesis is that the insertion of the 9 bases and, subsequently, of the 3 aminoacids can cause reduced level of transcript or interfere with its biological activity. Expression levels of pyrin were determined based on 2 independent sets of RT-PCR. Reference subjects (homozygous for M694V and healthy volunteer) were included in each PCR round.
We obtained the following MEFV: GAD ratios: 1 in healthy controls (wild-type genotype), 0.6 in homozygotes for M694V, 1.10 in the healthy carrier of the insertion, 1.7 in the proband (9-base insertion and R202Q). RT-PCR failed to demonstrate a reduced MEFV mRNA in the carriers of the 9-base insertion.
On the basis of these results, we can exclude that the 9-base insertion can affect MEFV mRNA stability or alter the tridimensional conformation of the DNA and the access of transcriptional factors as M694V is likely to do. It would be useful to determine whether the increased number of transcripts corresponds to an increased number of protein copies. However probably the 9-base insertion within exon 2 altering the function of pyrin could affect the clinical picture.

  Searching for Trigger Factors in Patient with Familial Mediterranean Fever: Role of Small Bowel Bacterial Overgrowth

Elena Verrecchia1, Claudia Cerquaglia1, Valentina Curigliano1, Alessandra Costa1, Giuliana de Socio1, Monica Vastola1 , Massimo Montalto1, Raffaele Manna1, and Giovanni Gasbarrini1      

1Periodic Fevers Research Centre, Department of Internal Medicine, Catholic University of Sacred Hearth, Rome, Italy

INTRODUCTION: Familial Mediterranean Fever (FMF) is an autosomal recessive disease due to a genetically determined unbalance of innate immune response. The age of onset of FMF can vary, independently from genotype for unknown reasons. Indeed genetic factors MEFV-independent have been identified only for some complications such susceptibility to renal amyloidosis. Therefore the periodic attacks could derive from an interplay of genetic and environmental factors: indeed it has been proposed that some triggers can stimulate FMF attacks. Recently it has been demonstrated that Helicobacter pylori infection in patients with FMF increases the frequency and severity of attacks and its eradication reduces significantly symptomatology. We have hypothesized that in some cases intestinal bacterial overgrowth could trigger FMF attacks.
CASE REPORT: We describe our experience with 3 patients, males of 17, 48 and 52 years, who came from south-Italy. They complained with fever and abdominal pain, begun respectively at 15, 18 and 47 years. The incidence of febrile attacks was respectively every 15 days, once a month and every two months. History, physical examination, laboratory data and genetic analysis were suggestive for FMF. All patients started colchicine treatment, at the dose of 2 mg/d. After 4 months of therapy, no changes were observed in disease manifestations. They were investigated for occult infections or inflammatory processes. Since lactulose H2breath test was positive in all patients, they were treated with intestinal antibiotics and probiotics. After bacterial overgrowth eradication, FMF-attacks did not recur.
DISCUSSION: The exact role of pyrin is not well-known, probably it acts as a suppressor of inflammation. It has been demonstrated that in pyrin-truncation mice there is a higher sensitivity to endotoxin. A transient bacteremia in patients with an abnormal pyrin, as in Helicobacter pylori infection, or in other similar conditions, might provoke exaggerated IL-1 production and thereby a systemic inflammatory response to events ordinarily innocuous. Since small bowel bacterial overgrowth has been associated with bacterial translocations, as in spontaneous peritonitis in hepatic cyrrhosis, we suppose that bacterial overgrowth could be considered as trigger of FMF attacks.
CONCLUSION: We suggest that the presence of trigger factors of different origin (intestinal, respiratory, dental or urinary) could be the reason for the different clinical manifestations, including differences of age onset. Furthermore patients with unresponsiveness to colchicine treatment should be investigated for small bowel bacterial overgrowth by a fair and simple test as lactulose H2breath.

  Familial Mediterranean Fever in the Syrian Population: Gene Mutation Frequencies, Carrier Rates and Phenotype-Genotype Correlation

Myrna Medlej-Hashim1, Hanadi  Mattit2, Muhidin  Joma 2, Salwa  Al-Cheikh 3, Mohammed  El-Khateeb 4, Nabiha  Salem1 , Valérie  Delague1, and André  Megarbané1       

1Medical Genetics Unit, Faculty of Medicine, Saint Joseph University, Beirut, Lebanon. ; 2Biochemistry department, Faculty of pharmacy, Damascus University, Damascus, Syria. ; 3Internal medicine department, Faculty of Medicine, Damascus University, Damascus, Syria. ; 4National Center for Diabetes, Endocrinology and Genetics, and Faculty of Medicine, Jordan University, Amman, Jordan.

Background: Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disease mainly affecting populations of the Mediterranean region, particularly Arabs, Non-Ashkenazi Jews, Armenians, and Turks. The gene responsible for FMF, MEFV, has been located on chromosome 16 short arm, cloned and over 50 mutations in the coding region have been identified. The prevalence of FMF mutations in certain Arab populations has already been reported [2,12,13], but as far as we know, not in the Syrian population.
Objective: To identify the distribution and the frequency of the MEFV gene mutations in Syrian FMF patients and population and perform a genotype/phenotype correlation in the patients’ cohort.
Patients and Methods: The study was carried out on 83 clinically diagnosed Syrian FMF patients and 242 healthy subjects. The tested individuals were screened for the most common 5 MEFV mutations (M694V, M694I, M680I, V726A and E148Q) by Restriction fragment length polymorphism (RFLP). Sequencing of exon 10 was performed only for the patients’ DNA where just one or no mutation was detected.
Results and discussion: Of the 83 patients studied, 74 (89%) were positive either for one, two or three mutations and 9 (11%) had no mutations detected. Of those positive for mutations, 25 were homozygous, 30 were compound heterozygotes, 3 had complex alleles, and 16 patients had only one mutation. The M694V, V726A, M694I, M680I and E148Q mutations accounted for 45.8%, 26%, 13.9 %, 4.8% and 6% of the alleles respectively. The carrier rate in the Syrian population for the tested mutations was 17.5% (one in 5.7), E148Q being the most common mutation, followed by V726A and M694V. M694I and M680I were undetected among the healthy adult cohort. The severity of the disease and development of amyloidosis seem to have an association with M694V, the most common mutation in Syrian FMF patients.

  Study of MEFV Mutations in the Iranian Population by Means of Reverse-Hybridization Teststrips

Christian Oberkanins1, Sayyed H. Amini2, Babak Moghimi3, Alireza Ghamari4, Navid Almadani2, Kimia Kahrizi4 , Gernot Kriegshaeuser1, and Hossein Najmabadi4       

1ViennaLab Labordiagnostika GmbH, Vienna, Austria; 2Kariminejad/Najmabadi Genetic and Pathology Center, Tehran, Iran; 3Medical School, Tehran University of Medical Sciences, Tehran, Iran; 4Genetics Research Center, Social Welfare and Rehabilitation Sciences University, Tehran, Iran

Familial Mediterranean Fever (FMF) is a hereditary inflammatory disorder caused by mutations in the MEFV gene. Carrier rates are known to be high among Turks, Armenians and Arab populations, whereas no data on the frequency and the spectrum of MEFV mutations were so far available from neighbouring Iran.
We have applied reverse-hybridisation teststrips (FMF StripAssay) to simultaneously analyse twelve common MEFV mutations in 208 asymptomatic Iranians from different regions and ethnic groups. The overall frequency of mutant alleles in our study population (15.6%) was moderate compared to Armenia, but exceeded the values known from Turkey and Iraq. The most common variant E148Q was identified in 9.6% of MEFV genes. Five other mutations (P369S, M694V, V726A, A744S, R761H) were observed with lower prevalence.
In addition, we studied the case of an 8 year old boy with short recurrent fever attacks and abdominal pain from a small village in the northwest of Iran. He turned out to be homozygous for MEFV mutation M694V. His parents were M694V/N and M694V/R761H. Several other members of this large family were found to be affected by typical symptoms of FMF and to carry MEFV mutations. Among 30 asymptomatic inhabitants of this village, who consented to participate in our study, we identified six different variants (E148Q, P369S, M680I(G/C), M694V, V726A, R761H) in a total of 13 mutant MEFV genes. Given the high frequency of MEFV mutations in Iran, the awareness for FMF and the availability of testing needs to increase significantly.
(oberkanins@viennalab.co.at)
  Hereditary Angioedema (HAE) and Familial Mediterranean Fever (FMF): Coexistence in a Turkish Child Leads to Early Manifestation of Edema and Increases the Attack Frequency

Kristina Huss1, Gundula Notheis1, Peter Lohse2, Monika M. Deml3, Silvia Stojanov4, Uwe  Wintergerst1 , Michaela Hamm1, Florian Hoffmann1, and Bernd H. Belohradsky1      

1Department of Infectious Diseases and Immunology, University Children's Hospital, Munich, Germany; 2Department of Clinical Chemistry - Grosshadern, University of Munich, Munich, Germany; 3Department of Pediatric Haemostaseology, University Children's Hospital, Munich, Germany; 4Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD

Hereditary angioedema (HAE) is a rare autosomal dominant disease. The mutated SERPING1 gene on chromosome 11q12 leads to a quantitative and/or qualitative deficiency of C1 esterase inhibitor (C1-INH), affecting the activation of complement, the coagulation system, and fibrinolysis. The disease is characterized by episodic edema of subcutaneous and mucosal tissue, often beginning at school-age and resulting from several known triggers.
Familial Mediterranean fever (FMF), on the other hand, is an autosomal recessive disorder due to mutations in the MEFV gene on chromosome 16p13. As a consequence of an inappropriate activation of inflammation, FMF manifests with recurrent attacks of fever, erythema, monoarthritis, and sterile peritonitis, starting at age under ten years.
We present the case of a five-year-old Turkish girl, who was diagnosed with asymptomatic HAE at the age of 20 months due to a positive family history. The patient’s C1-INH serum activity was reduced to 29%. Ten months later, she developed monoarthritis, recurrent episodes of high fever without pain, and recurrent attacks of limb and facial swelling without breathing problems. Molecular genetic analyses of the patient confirmed the diagnosis of FMF due to a homozygous Met694->Val substitution encoded by exon 10 of the MEFV gene and of HAE due to a heterozygous Gly162->Arg exchange encoded by exon 3 of the SERPING1 gene. The fever attacks seemed to trigger and intensify the symptoms of HAE. Therapy with colchicine was initiated.
To our knowledge, a patient simultaneously suffering from FMF and HAE has not been described so far. Based on the presented case, we hypothesize that hereditary autoinflammatory diseases such as FMF might influence the clinical manifestation of hereditary angioedema, leading to an early onset of episodic edema and increasing the frequency of attacks.

  Molecular Genetic Study of Familial Mediterranean Fever in the Hellenic population of Greece and Cyprus

Elena P. Rossou1, P Pratsidou-Gertsi2, Anastasia Kouvatsi1, Florentia Kanakoudi-Tsakalidou2, and Constantinos C. Deltas3           

1Department of Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki, Greece; 2A Department of Pediatrics, Aristotle University, Hippokration Hospital, Thessaloniki, Greece.; 3Department of Biological Sciences, University of Cyprus

Familial Mediterranean Fever (FMF) is a hereditary inflammatory disease with autosomal recessive transmission. Typically it presents as acute episodes of periodic fever accompanied by abdominal pain, chest pain, or joint pain. Appearance of renal amyloidosis indicates severe prognosis. About 40 mutations have been identified so far, some of them being very frequent. Molecular investigation of the Cypriot population reveals that about 1:8 is a carrier of one of four mutations, E148Q being the most frequent (1:12). Despite the high frequency of E148Q, only 9 of 91 patients carried it, supporting its mild nature. Among 186 Cypriot MEFV chromosomes analysed, the results are: V726A 28.5 %; M694V 22%; F479L 20.4 %; E148Q 5.4 %; M694I 2.2%; R761H 3.2%; M680I 1.6%; unknown 16.7%. Mutation F479L is rather rare in other populations. Preliminary evidence suggests that this frequent, in the Cypriot population, mutation is associated with later age of onset of symptoms, the most debilitating of which is strong and frequent abdominal pain, with or without fevers and arthralgias. After investigation it was found that the patients – carriers of the MEFV mutation F479L shared a common haplotype, the same that Armenian patients - carriers of this mutation share. This findings lead to the conclusion that the common haplotypes shared by Greek Cypriots who carry mutation F479L is the result of a founder effect, the founder perhaps being of Armenian origin, as in previous historical periods there were massive waves of Armenians finding refuge in Cyprus. In a number of Hellenic samples from Greece tested, the F479L mutation was not present, whereas in a cohort of patients with childhood onset of FMF, F479L was found in only one patient in heterozygosity. In this same cohort the severe mutations M694I, M694V and Μ680Ι were highly represented, in accordance with the childhood onset of disease. Among the Greek childhood patients M694V was the most frequent (32.1 %) followed by M680I (15.5 %) and V726A (7.1%). It is our experience as well as the experience of others, that in a relatively high percentage of FMF patients no mutations are found in the MEFV gene, while there is no evidence for the existence of a second MEFV gene. One explanation is that unknown mutations still exist in areas of the gene that are not routinely tested, or these are patients who suffer from a similar but different disease.
  Renal Manifestations Other than Amyloidosis in Familial Mediterranean Fever (FMF)

Marina Papazyan 1, Tamara Sarkisian2, Ara Babloyan 1, Ernst Leumann 3, Armen Sanamyan 4, Ariana Gaspert 5 , and Ashot Sarkissian 1        

1Nephrology department, Arabkir Joint Medical Centre, Yerevan, Armenia 375014; 2Center of Medical Genetics, National Academy of Sciences, Yerevan, Armenia 375010; 3University Children’s Hospital, Zurich , Switzerland 8032; 4Republican Centre of Pediatric Pathology, Yerevan, Armenia 375014; 5Institute of Clinical Pathology, University Hospital, Zurich, Switzerland 8091

Background: The classical complication of FMF is amyloid nephropathy. The diagnostic role of renal biopsy in FMF patients with renal involvement is therefore disputed.
Purpose: To evaluate all renal biopsies done in paediatric patients with FMF in Armenia, particularly those without amyloid deposits, and to study the clinical/histological correlation.
Patients and methods: From 2001 to 2004 biopsies of native kidneys were performed in 60 children (age < 18 years). Of these, 21 patients (age 6-18 years; 13 males) had FMF. Diagnosis of FMF was based on clinical history and genetic analysis in all of them. Amyloid deposits were determined by Congo red stain. Biopsies with other pathology were additionally examined by light and electron microscopy in Zurich.
Results: Twelve patients were nephrotic (renal failure in 1), 4 were nephritic (renal failure in 1) and 5 had proteinuria (isolated or with microscopic haematuria). Renal amyloidosis was found in 14 patients, but one third (7) had nephropathies other than amyloidosis: Minimal change (MC) nephrotic syndrome (2), FSGS (1), and acute post-infectious glomerulonephritis (APGN; 2); two had extrarenal vasculitis. One of the latter with a purpuric rash had a GN with few fibrocellular crescents and no immune complexes, consistent with ANCA-associated GN, and the other with protracted myalgia had crescentic membrano-proliferative GN type I. Altogether patients with amyloidosis had earlier onset of FMF and were mostly nephrotic. In contrast, patients without amyloidosis had more haematuria and higher serum creatinine. However, the clinical presentation in 3 patients – 2 with MC and 1 with FSGS – was not distinguishable from that seen in amyloid nephropathy.
Conclusions: 1) Renal diseases other than amyloidosis were more frequent than expected, 2) FMF is known to be weakly associated with various forms of GN, but in at least five (MC, FSGS, APGN) it was probably coincidental, 3) Renal biopsy is important in FMF, also in patients presenting with pure nephrotic syndrome.

  Opportunity of the prevention of amyloidosis in the asymptomatic and atypical variants of familial Mediterranean fever in Armenia

Alexander Ayvazyan               

Yerevan State Medical University; 2Medical Centre “Erebouni

BACKGROUND: The prevalence of familial Mediterranean fever (FMF) in Republic of Armenia (RA) makes about 1,6 %. The population of RA is about 3 million. FMF uncured with colchicine in conditions of RA reduces average duration of life to 31 years. The phenomenon of phenotype II of FMF is described for a long time. Phenotype II in FMF is the onset of amyloidosis before the onset of FMF with its typical attacks, or as an isolated finding in a member of an FMF family. According to authors phenotype II is met in 10-27 % of cases. In RA presence of phenotype II is denied and by physicians (Ajvazjan A.A., 1982), and by pediatricians (Astvatsatrjan V.A., Torosjan E.H., 1989).
METHODS: The genealogical research in the families of patients with renal amyloidosis without serositic attacks of FMF registered in nephrology departments of all clinics of Yerevan for 1977-2000 (171 cases) is carried out.
RESULTS: Among siblings of patients with renal amyloidosis without serositic attacks of FMF the incidence of typical cases of FMF is 21,2 % (vs 20,1 % among siblings of patients with typical clinic of FMF).
CONCLUSIONS: Thus, among all cases of amyloidosis in RA, the non serositic variants of FMF make hardly less than 16,4 % (171 cases among 1046 cases of amyloidosis). For revealing and preliminary preventive treatment with colchicine of an appreciable part of these patients in RA it is necessary to carry out screening of all inhabitants on presence of determined mutations of the MEFV gene (sensitivity of the PCR-method in RA is about 85 %). It will allow rescuing only at one generation of inhabitants of RA approximately 1264800 years of life.

  Dosage of colchicine in elderly patients with familial Mediterranean fever

Alexander Ayvazyan               

Yerevan State Medical University; 2Medical Centre “Erebouni

BACKGROUND: Familial Mediterranean fever (FMF) is wide-spread among Armenians. Amyloidosis is the basic danger menacing to life of these patients. In the literature there are no data on age dynamics of probability of amyloidosis, dynamics of frequency of attacks of FMF and efficacy of their suppression with colchicine that is very important at selection of a dosage of colchicine in elderly patients.
METHODS: Catamnestic observation of patients with FMF, suffering from classic forms of disease (4167 cases). Ninety four of them were born till 1920, 217 - during 1921-1930, 458 - 1931-1940, 470 - 1941-1950, 977 - 1951-1960, 988 - 1961-1970.
RESULTS: The frequency of manifestation of amyloidosis within separate decades of life of patients with FMF is the following: II - 6,67, III - 9,93, IV - 10,8, V - 13,4, VI - 14,1, VII - 16,1, VIII - 11,5 %, the percentage of patients with often attacks - accordingly 40,0, 50,7, 52,4, 48,2, 37,8 and 26,9 %. With the years beneficial effect of colchicine on course of attacks of FMF strengthens. For example, the percentage of FMF patients with often attacks on a background of regular therapy with colchicine in a dosage of 1,0 mg daily during the second decade of life has made 40,0 %, and during the sixth - 0 %.
CONCLUSIONS: As at "natural" course of FMF the probability of development of amyloidosis with the years does not drop, but frequency of attacks of FMF and "answer" of the last on therapy with colchicine decreases, with the years advisable not to reduce a dosage of colchicine. At the beginning of therapy with colchicine in elderly patients, and also at presence of assemble of risk factors of amyloidosis and (or) preservation of the increased parameters of inflammation, it is expedient to apply a dosage of colchicine, one step above what helps clinically.

  Influence of different diseases or syndromes on probability of development of amyloidosis in patients with familial Mediterranean fever. Report I. The influence of separate general syndromes and pathologies of muscles and joints, respiratory and cardiovascular systems

Alexander Ayvazyan2, Artem Ayvazyan1, Sergey Ayvazyan1, Margarita Abrahamyan1, and Aida Zakaryan1           

1Yerevan State Medical University; 2Medical Centre “Erebouni

BACKGROUND: Familial Mediterranean fever (FMF) is met in 1-2 % of the population of Yerevan and tends to growth. The basic complication extremely lowering the life expectancy of FMF patients is amyloidosis, meets with various frequencies in representatives of different ethnic groups. We have studied the influence of various diseases or syndromes on probability of development of amyloidosis in patients with FMF.
METHODS: Catamnestic observation of patients with FMF, suffering from classic forms of illness (4167 cases).
RESULTS: In patients who alongside with the basic serositic attacks had also periodic feverish attacks, rather low probability of development of amyloidosis was observed only during the forth decade of life (2,9 %). Significant differences with the general contingent of FMF patients were not revealed in patients with low body mass (on decades of life, beginning from the second: 11,1%; 5,9%; 7,1%), with adiposity (9,1%; 0%), constantly high ESR (7,7%; 0%; 9,1%), destructive arthritis (13,1%, 10,3%; 19,5%; 33,3%), ancylosing spondyllitis (3,6%; 12,0%) and in patients with polymyositis (7,4%; 18,2%). In patients with expressed myalgy of crural muscles only within the second decade of life development of amyloidosis was observed more often, than in general contingent of FMF patients (13,3%; 8,2%). At the mentioned diseases of the muscles and jonts taken altogether, in FMF patients the higher probability of amyloidosis was observed significantly within the second, the fourth and the fifth decades of life (11,6%; 10,8%; 23,6%; 28,6%; 30,4%). Within the fourth decade of life in patients with allergic predisposition (3,0%; 6,3%; 3,6%; 11,8%), and within the third decade in patients with SLE-like syndrome (5,7%; 2,2%; 13,6%) FMF was complicated with amyloidosis less often, that was not observed within other decades of life and also in FMF patients with polyarteritis nodosa (4,4%) or lymphadenopathy (4,2%). In FMF patients with chronic bronchitis during the second (1,9%; 4,6%; 11,8%; 17,4%; 11,1%), and in obstructive variant of the last - within the third decade of life (4,6%; 0%; 6,7%) comparatively low probability of amyloidosis was observed.

  Influence of different diseases or syndromes on probability of development of amyloidosis in patients with familial Mediterranean fever. Report II. The influence of different gastroenterological, endocrinological, gynecological, infectious and surgical diseases and/or syndromes

Alexander Ayvazyan2, Artem Ayvazyan1, Sergey Ayvazyan1, Margarita Abrahamyan1, and Aida Zakaryan1           

1Yerevan State Medical University; 2Medical Centre “Erebouni”

BACKGROUND & METHODS: See first report.
RESULTS: In patients suffered from familial Mediterranean fever (FMF) with previous history of the expressed gastritis within the II, III and IV decades of life lower probability of amyloidosis is observed (on decades of life, beginning from the second: 2,9%; 4,84%; 5,0%; 17,3%; 19,2%), than in the general contingent of patients with FMF. The same situation is in case of duodenal ulcer (1,2%; 2,7%; 12,7%; 15,4%; 25,0%), chronic expressed colitis (1,6%; 3,4%; 7,9%; 9,3%; 9,8%). In case of cholelithiasis (0%; 0%; 5,9%; 23,1%; 21,1%) and chronic cholecystitis without calculus of gallbladder (1,9%; 3,1%; 8,9%; 16,6%; 9,9%) amyloidosis is manifested less frequent during the II and III decades, viral hepatitis - during the II (2,9%; 12,8%; 28,6%), and in case of pancreatitis – during the III decades of life (2,9%; 0%; 5,9%). In patients with chronic hepatitis (3,9%; 6,0%; 14,5%; 22,0%; 25,0%), and also in patients with cirrhosis of liver (7,7%) significant differences are not revealed. Hepatosplenomegaly sharply raises probability of amyloidosis during all decades of life, especially in the II and the IV ones (14,8%; 18,3%; 45,3%; 46,7%). Diabetes mellitus results in lower probability of amyloidosis during the IV decade of life (3,5%; 3,7%; 0%; 16,7%). Sterility (5,0%; 11,7%; 20,0%; 15,6%; 15,8%), dysmenorrhea (3,9%; 0%), and uterine myoma (0%; 5,6%) do not significantly influence on the investigated parameter. The expressively low frequency of acute respiratory viral diseases is associated with lower frequency of amyloidosis during the III and the IV decades of life of patients with FMF (4,2%; 3,3%; 4,3%; 10,0%). The children's infections result in a greater frequency of amyloidosis during the II, and lower - during the III decade of life (14,8%; 1,9%; 9,4%). The operative interventions (5,4%, 8,3%, 11,3%, 16,7%, 11,1%) and chronic tonsillitis (7,6%; 7,6%; 9,4%; 12,5%) do not influence on the development of amyloidosis in patients with FMF.

  The death rate from neoplastic diseases in patients suffering from familial Mediterranean fever in Armenia

Alexander Ayvazyan               

Yerevan State Medical University; 2Medical Centre “Erebouni”

BACKGROUND: Familial Mediterranean fever (FMF) is met in 1-2 % of the population of Yerevan and tends to growth. The basic complication extremely lowering the life expectancy of FMF patients is amyloidosis, meeting with various frequencies at representatives of various ethnic groups. The myocardial infarction is met in FMF patients also more often, than in the general population. Studying of occurrence of neoplastic diseases (ND) at a contingent of patients with Shepard Jewish origin has shown, that at the regularly accepting colchicine FMF patients, ND met with the same frequency, as in the general population. There was a question, and whether it is consequence of the different directed influences of illness and colchicine, or neither it, nor another one influence on the frequency of ND.
METHODS: We investigated the frequency of ND at FMF patients and at their siblings.
RESULTS: It was shown that compared groups of patients within any decade of life do not significantly differ concerning frequency of death from ND (on decades of life: IV - 0,462 vs 0,512 %; V - 1,203 vs 2,149 %; VI - 1,841 vs 4,027 %; VII - 8,00 vs 6,94 %).
CONCLUSION: Thus, FMF itself does not influence on the risk of ND, hence and therapy with colchicine at FMF cannot raise the risk of ND.

  Incidence of amyloidosis during life of patients with familial Mediterranean fever in Armenia. Report I. Common data

Alexander Ayvazyan               

Yerevan State Medical University; 2Medical Centre “Erebouni”

BACKGROUND: Familial Mediterranean fever (FMF) is wide-spread among Armenians. Amyloidosis is the basic danger menacing to life of these patients. In the literature there are no data on age dynamics of probability of amyloidosis during decades of life.
METHODS: Catamnestic observation of patients with FMF, suffering with classical forms of illness (4167 cases). Ninety four of them were born till 1920, 217 - during 1921-1930, 458 - 1931-1940, 470 - 1941-1950, 977 - 1951-1960, 988 - 1961-1970.
RESULTS: On our data, frequency of manifestation of amyloidosis during the decades of life of patients suffering from attacks of familial Mediterranean fever (FMF) in Armenia is the following: II - 6,67; III - 9,93; IV - 10,8; V - 13,4; VI - 14,1; VII - 16,1; VIII - 11,5 %. In elderly persons the probability of manifestation of amyloidosis with the years does not decrease. At young age the course of FMF is complicated with amyloidosis more often by the abdominal, than by the mixed forms of FMF (on decades of life: II - 8,35 vs 6,41 %; III - 9,99 vs 10,8 %; IV - 12,4 vs 10,7 %; V - 13,8 vs 13,5 %; VI - 13,9 vs 14,9 %), and these two forms are greatly more hazardous than the thoracic one (1,65; 2,67; 2,06; 9,09; 8,33 %). However, in elderly patients this difference is not such big. Average age of demonstration of amyloidosis has made 29,7 years, and in patients with mixed form of FMF - 29,7, abdominal - 29,5, and thoracic one - 33,7 years. We quite often observe amyloidosis in patients with many years arrest of serositic attacks (34 cases). In males the frequency of manifestation of amyloidosis is higher within almost all decades of life both in patients with mixed (II: 7 vs 4; III: 10 vs 7; IV: 12 vs 7; V: 16 vs 10 %), and abdominal forms of FMF (II: 8 vs 7; III: 12 vs 6; IV: 13 vs 8; V: 19 vs 7 %).

  Incidence of amyloidosis during life of patients with familial Mediterranean fever in Armenia. Report II. Influence of different factors

Alexander Ayvazyan               

Yerevan State Medical University; 2Medical Centre “Erebouni”

BACKGROUND & METHODS: See first report.
RESULTS: The age of manifestation of attacks significantly influences on the frequency of manifestation of amyloidosis during the proximate years after manifestation of serositic attacks of familial Mediterranean fever (FMF). In case of the manifestation of serositic attacks in 1-5 years old age, the frequency of manifestation of amyloidosis during the II, III, IV and V decades of life is 17, 21, 18, and 21 %, in case of the manifestation years 6-10 – 11, 18, 11, and 17 %, 11-20 – 3, 9, 13, 16 %, respectively. Only during the II decade of life of patients with mixed form of FMF the initial localization of attacks (abdominal or thoracic) influences on the frequency of manifestation of amyloidosis (6 vs 1 %). Patients who have often attacks of FMF in the age of till 10 years, have also higher probability of the amyloidosis manifestation at least up to 40-years age (during II, III, IV and V decades of life 17, 24, 17, and 22 % vs 4, 8, 11, and 14 %). Expressed joint syndrome in the age of till 10 years has been more expressed and has long prognostic value (12, 19, 18, and 22 % vs 4, 7, 9, and 12 %). The east or west parentage of ancestors of patients does not influence on the probability of incidence of amyloidosis (6, 10, 9, and 11 % vs 6, 10, 8, and 14 %).
-years age (during II, III, IV and V decades of life 17, 24, 17, and 22 % vs 4, 8, 11, and 14 %). Expressed joint syndrome in the age of till 10 years has been more expressed and has long prognostic value (12, 19, 18, and 22 % vs 4, 7, 9, and 12 %). The east or west parentage of ancestors of patients does not influence on the probability of incidence of amyloidosis (6, 10, 9, and 11 % vs 6, 10, 8, and 14 %).
  A case of asymptomatic carrier of two mutations of MEFV gene one of which was absent in both parents

Artem Ayvazyan               

Yerevan State Medical University

Presence of two mutations of MEFV gene in asymptomatic carriers is not a very rare phenomenon (Cazeneuve C. et al., 1999, Ayvazyan A., 2001, Ajrapetyan H. et al., 2001). However in the literature is not described the similar phenomenon that one of these mutations would be absent in parents. We observed case of familial Mediterranean fever (FMF) with the similar phenomenon in family. Our proband, woman born in 1995, suffered from adnominal attacks of FMF with arthropathy. Parents of proband deny the cases of FMF among relatives. The mutation analysis which has been carried out in Center of Medical Genetics of Academy of Sciences of Republic of Armenia (chief – professor Sarkisian T.), has shown at her the mutation genotype M680I/R761H. Father of proband is practically able-bodied with the mutation genotype R761H/0. The grandmother on a paternal line also is practically able-bodied with the mutation genotype 0/0. The grandfather on a paternal line has already died on the causes which were not linked with FMF. Mother of proband is practically able-bodied, but appeared asymptomatic carrier of two mutations of MEFV gene (E148Q/M680I). The grandfather on a maternal line is practically able-bodied with the mutation genotype M680I/0. The grandmother on a maternal line also is practically able-bodied with the mutation genotype 0/0. It is obtained, that mother of ours proband did not inherit the mutation E148Q neither from the father, nor from mother! If to leave in the side the extremely improbable explanations about possible substitution of the child in the maternity home, or about possible methodical error in genetical laboratory there is only one scientific explanation of the given phenomenon - virus transfer of the mutated MEFV gene. We explain the given paradox by means of a hypothesis of the viral transfer of mutated MEFV gene in one of embryonal cells, with the further formation of an organism with genetic defect only in a part of mesotheliocytes (Ayvazyan A., 2001). That creates an opportunity of the presence of mutation of MEFV gene in gametes, but not in the genomes of leukocytes, in DNA of which the search of mutations of MEFV gene is carried out.
  Efficiency of Hypothalamic Proline-rich Polypeptide (PRP) in the Phosphoinositide Cycle Regulation at Famillial Mediterranean Fever

Petros Ghazaryan1*, and Nune  Mkrtchyan2*              

1Center of Haematology, Armenia, Yerevan; 2Yerevan Statte Medical University ; *These authors contributed equally to this work.

There was studied the intensity of (U14C)-glucose incorporation (in vitro) into the structure of phosphatidylinosites, phosphatic acids, phosphatidilcholines (PC), lysophosphatidilcholines (LPC) and other fractions of phospholipids, monoacyl-, diacylglycerides and glycerolphosphate, the changes in the activity of phospholipase A2 in the lymphocytes at Familial Mediterranean Fever (FMF). In experiments it is used the blood of 45 children (in the age of 2-14) with various types of FMF at fits, at interictal periods, on the background of colchicine treatment, and after combined use of colchicine with a new hypothalamic cytokine PRP (proline-rich polypeptride). It is established that at FMF fit there is observed a noticeable decrease of 14C-glucose incorporation rate in the contents of PC, and increase of that in LPC. Simultaneously, it is observed a sharp increase of phospholipase A2 activity. At moderate and medium levels of the disease the incorporation of the radioactive label to the contents of PC decreases for 60% and 85%, respectively, with simultaneous increase of LPC radioactivity and phospholipase A2 activity. At moderate and medium levels of the disease the indicated indices tend to norm during interictal period and on the background of regular colchicines treatment. At high level of the FMF there is observed a significant decrease of 14C-glucose incorporation intensity in the PC contents, and a sharp increase of it in LPC. Hence, phospholipase A2 activity increases over 3 fold. The above-mentioned indices are not leveled both at interictal period and on the background of colchicine treatment. When colchicines is used in combination with PRP, there is observed almost full normalization of the level for the mentioned indices. It indicates the membrane stabilizing properties of that treatment.
  Effects of Colchicine Therapy on Immunological Parameters in Familial Mediterranean Fever

Gohar Mkrtchyan1, Anna Boyajyan1, Alexandr Ayvazyan2, and Eduard Nazaretyan2            

1"Laboratory of Macromolecular Complexes, Institute of Molecular Biology, Armenian National Academy of Sciences, Yerevan, Armenia"; 2"The First Department of Internal Medicine of the Yerevan State Medical University, Yerevan, Armenia"

The present study evaluates the role of the inflammatory immune reactions in the pathogenesis of Familial Mediterranean fever (FMF) and the influence of colchicine therapy on these reactions. As the indicators of the inflammatory response, the levels of circulating immune complexes (CIC), the total hemolytic activity of the complement, and the activities of the individual complement components, C1, C2, C3 and C4, were determined in the blood serum of 54 patients with familial Mediterranean fever and 28 healthy subjects.
Based upon the regularity of colchicine therapy, the patients were divided into 3 groups following groups: patients receiving regular colchicine treatment (n=19); patients receiving irregular colchicine treatment (n=17); patients not receiving colchicine treatment for at least 1 year prior to hospitalization (n=18).
In comparison to the healthy subjects, a significant increase of the total hemolytic activity of the complement and the hemolytic activities of the C1, C2, C3 and C4 complement components was detected in the serum of colchicine-free patients (n=10). In the serum of those patients, who were receiving irregular colchicine treatment (n=12), significant changes were detected only in case of the total hemolytic activity of the complement and the hemolytic activity of the C2 complement component: both parameters were higher than that of the healthy subjects. No significant changes in the hemolytic activities of the C1, C2, C3 and C4 complement components were found between the healthy subjects and those FMF patients, who were receiving regular colchicine treatment (n=10). Regarding CIC data, the results obtained indicate an increase of this parameter in colchicines-free patients and those who were receiving colchicine irregularly. However, no significant difference was obtained when comparing the patients receiving regular colchicine treatment and the healthy subjects.
The results obtained, confirming the efficiency of colchicine treatment and underline the possible anti-inflammatory effect of colchicine therapy. It is proposed that colchicine might be effectively used as potential immunomodulating agent. However, to draw final conclusion further investigations are required.

  Assesment of the Course of Familial Mediterranean Fever in Children by the State of Peripheral Blood Lyphocyte Membranes

Nune  Mkrtchyan1*, Petros Ghazaryan2*, and Vilen Astvatsatryan3*             

1National Pediatric Center for FMF of Arabkir Joint Medical Center – Institute of Child and Adolescent Health of Armenia, ; 2Center of Haematology, Yerevan, Armenia; 3National Pediatric Center for FMF of Arabkir Joint Medical Center – Institute of Child and Adolescent Health of Armenia, ; *These authors contributed equally to this work.

The phospholipase A2 activity and lipid peroxidation (LP) intensity have been studied in peripheric blood lymphocytes of 66 (27 girls, 39 boys) patients aged from 2 to 18 years suffering from various clinical forms of Familial Mediterranean Fever (FMF).
It was established that in patients with mild and moderate gravity degrees of FMF during the attack a 2 fold increase of phospholipase A2 activity and a 1,5 fold increase of LP intensity are observed. The abovementioned indices tend to normal both in interictal period as well as on the background of regular colchicine therapy.
In case of a severe disease an acute 4 fold increase of phospholipase A2 activity and over 2,5 fold of LP intensity is observed. In all patients of the investigated group there have been observed frequent attacks, in 90% the onset of the disease was stated at the age under 5 years, in 46,3 - under 1 year, erysipelas-like erythema has been observed in 55,4%, acute arthritis in 87%, in 35,4 % of them it has been of a protracted form. The biochemical indices in this group of patients have normalized neither during the interictal period nor on the background of colchicines treatment. The combined treatment including colchicine and a new hypothalamic prolin rich polipeptide (PRP) in vitro has almost completely normalized the levels of the indicated indices, which testifies to the membrane stabilizing effect of PRP and its possible effectiveness at FMF.

  Pathogenicity and Clinical Symptoms of Heterozygote Individuals in Familial Mediterranean Fever (FMF) in Armenian Population

Mike  M. Moradian 2 3, Tamara Sarkisian1, Hasmik Ajrapetyan1, Gohar Shahsuvaryan1 , Erik AvanissAghajani2, and Ara Hartoonian2       

Center of Medical Genetics, NAS of Armenia, 5/1 Zakiyan Street, 375010, Yerevan, Armenia; 2Primex Clinical Laboratory Inc. 16742 Stagg st. Van Nuys, CA ; 3University of California Los Angeles, Los Angeles, CA

Symptoms and pathogenicity in patients with Familial Mediterranean Fever (FMF) varies significantly among heterozygote individuals of Armenian descent from various regions in Republic of Armenia and Karabakh. We have determined the symptoms associated with 9 different mutations in MEFV gene in heterozygote patients, believed to be suffering from FMF, and compared them with affected homozygotes and compound heterozygotes. We have analyzed 414 homozygote recessives, 980 compound heterozygotes, and 317 heterozygotes. Our data indicate that the only homozygote recessive cases are from M694V, V726A, M680I, and R761H mutations. When clinical symptoms of these mutations from heterozygote patients are compared to homozygotes a strong correlation, over 90%, is observed. These correlations are of great importance since they take into account each clinically significant symptom and different combination of them, which cover almost all of the reported cases. In heterozygote patients the most prevalent and severe cases are caused by the presence of a single M694V mutation, which is associated with fever, abdominal, thoracic, and joint pain, skin eruptions, myalgia (protracted fibril), and amyloidosis. However, less prevalent mutations such as V726A, M680I, R761H, and E148Q still cause clinically significant symptoms in heterozygote individuals and are treated by Colchicin. We also found that mutations such as M694I and R42W are only present in compound heterozygote patients and nowhere else. Therefore heterozygote individuals for these mutations indicate no significant clinical symptoms and they seem to be healthy. Hence we suggest that in Armenian population heterozygote individuals for M694V mutation suffer from a milder version of the FMF. The same suggestion is, to a lesser extent, valid for V726A, M680I, R761H and E148Q heterozygotes.
We also investigated the effects of Colchicin on homozygote, compound heterozygote, and symptomatic heterozygote patients. We found that on average, in almost 90% of the cases, Colchicin is effective to keep the inflammation under control. Resistance to Colchicine may be caused by individual’s response or endurance to the drug, non-efficient scheme of treatment, quality of the medication, and social behaviors such as smoking and alcohol consumption.


  A novel and de novo CARD15/NOD2 gene missense mutation in a Spanish patient with Early-Onset Sarcoidosis.

Juan I. Arostegui1, Rosa Merino2, Julia Garcia-Consuegra2, Josefa Rius1, Susana Plaza1, Jordi Vives1 , and Jordi Yague1        

1Immunology Department. Hospital Clínic. Barcelona.; 2Pediatric Rheumatology Department. Hospital La Paz. Madrid.

INTRODUCTION
Blau syndrome (BS) and Early-Onset Sarcoidosis (EOS) are two different names usually employed for the CARD15/NOD2 gene-associated systemic granulomatosis, being used the former when there is familial history of disease and the later when there is not familial history. In both cases, age at disease onset is under 4 years of age, and skin, joint and eyes are the most severely affected organs. Other different clinical manifestations, such as recurrent fever, cranial neuropathies or hypertension have been previously reported. All patients showed multiple non-caseating granulomata in the organs affected, and they usually showed a successful response to steroid. From 2001 it has been established a relationship between mutations on CARD15/NOD2 gene and BS/EOS, enabling a more accurate assessment of patients with a clinical suspicion of BS/EOS and patients with atypical forms of systemic-onset JIA.
CASE REPORT
Herein we report the case of a 20 year-old man with a first clinical diagnosis of sarcoidosis, without familial history of disease. His disease onset was at 4th month of life, with recurrent fever, hepatosplenomegaly, generalized maculo-papular skin rash, headache and a symmetrical polyarthritis. His joint disease became a chronic arthropathy, with synovial thickening and joint deformities. At 4 years of age ocular involvement was detected as bilateral synecchiae. Altogether, these clinical symptoms caused him a slowness in his normal growth and school attendance. Biopsies of skin, synovial and hepatic tissues showed the presence of multiple non-caseating granulomata. With regard to treatments administered, only partial successful response were obtained with NSAID, steroid and methotrexate.
GENETIC ANALYSES
Since a differential diagnosis between sarcoidosis and EOS should be made in this case, a genetic analysis of CARD15/NOD2 gene was performed. It was detected in exon 4 the G-to-A transition at 1484 position, resulting in the previously unreported missense variant C495Y in heterozygous state. In order to elucidate if this variant was a disease-associated mutation or a polymorphism, two complementary approaches were performed. First, the CARD15/NOD2 gene analysis in his parents showed its absence. Paternity was confirmed by means of HLA haplotypes segregation. These data agreed with the absence of familial history of disease, and allowed us to state that it was a de novo genetic variant. Secondly, we analyzed it among 115 healthy unrelated individuals, and we did not found it in none, suggesting that the C495Y variant was a new true disease-associated mutation.

  Pediatric Granulomatous Arthritis and CARD 15 mutations: A status report on the International Registry.

Tammy  M. Martin1, Carine  H. Wouters3, Silvia   . Meiorin4, and Carlos  D. Rose2            

1Casey Eye Institute, Oregon Health System, Portland Oregon, USA; 2Tomas Jeffferson University, Philadelphia, Pennsylvania, USA; 3Gasthuisberg University Hospital, Leuven, Belgium; 4Hospital de Ninos JM Gutierrez, Buenos Aires, Argentina

Blau syndrome (BS), the familial form of childhood granulomatous arthritis is associated with substitutions in the NACHT domain of CARD 15 in 50-100% of the cases. The sporadic form of the disease, known as Early Onset Sarcoid arthritis (EOS) was shown by us and others to be associated with the same mutation. This finding was expected since both diseases share similar phenotype. Isolated reports suggest that the spectrum of the disease is wider than the triad: arthritis-dermatitis-uveitis to involve systemic granulomatous inflammation and large vessel vasculitis. Other mutations or polymorphisms are expected and the identity of the disseminated forms is still undefined. An International Registry was established in the spring of 2005 with the goal of improving clinical definition and expanding understanding of genetic variants. Exon 4 of CARD 15 is being investigated in probands and relatives. De-identified clinical information is collected. RESULTS: 10 familes from Europe, 7 from NorthAmerica and 2 from LatinAmerica have been collected. Mutation work has been completed in 11 families. Except for one all probands have histopatologic documentation of non-caseating granulomas. Arthritis was observed in 16, uveitis in 12, dermatitis in 13 index cases. 8 were familial and the rest sporadic. 2 presented disseminated disease, 2 had liver granuloma and 2 erythema nodosum. Average age of onset 2 years (1 mos-4 years). Substitutions at 334 were found in 7/11completed. Polymorphisms P268S and R459R were found in a family with disseminated disease.
  Mutation Detection in PAPA syndrome

Carol A. Wise1, Saralove S. Swaney1, Marilynn Punaro2, Virginia Pascual2, and Dongping Zhang1           

1Seay Center for Musculoskeletal Research, Texas Scottish Rite Hospital for Children, Dallas, TX; 2Arthritis Service, Texas Scottish Rite Hospital for Children, Dallas, TX

Pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome (OMIM #604416) is a dominantly inherited autoinflammatory disease characterized by influx of neutrophils into affected joints. Patients may develop cystic lesions of the skin that can progress to frank pyoderma gangrenosum (PG). Severe adolescent cystic acne that persists into adulthood is also seen in some patients. We previously identified the gene responsible for PAPA syndrome, PSTPIP1 (also known as CD2BP1). Two mutations were identified in two families, 904G-A predicting an A230T substitution, and 964G-C predicting an E250Q substitution. Since our initial report we have performed mutational analysis of PSTPIP1 on a research basis by direct DNA sequencing. A total of eight cases were referred to us with a tentative diagnosis of PAPA syndrome. These cases originated in Spain, Italy, USA, New Zealand, Canada, and France. Ethnicities were undetermined. In the first five independent cases we detected the 904G-A mutation twice (samples from Spain, Canada), no mutation in screens of the complete PSTPIP1 coding sequence in two cases, and no mutation in screens of exons 10 and 11 encompassing nucleotides 904 – 964 in the fifth case. One of the three negative cases reported only severe PG without associated early onset arthritis or strong family history; a second negative case reported only clinical features “consistent with PAPA syndrome”. We subsequently modified acceptance criteria for PSTPIP1 screening to include only cases with three or more of the following: inflammatory arthritis; skin findings including pyoderma gangrenosum, cystic lesions, or severe acne; strong family history of inflammatory arthritis, and early disease onset. Exons 10 and 11 only were screened in the next three consecutive cases, and one mutation (964G-C) was detected in a case referred from New Zealand. These data are too limited to draw firm conclusions, and our PCR-based methods would not have detected microdeletions or insertions. Aside from this, it is clear that 904G-A and 964G-C mutations explain several apparently independent cases of PAPA syndrome, and it is reasonable to conduct initial screens for these changes. Negative cases with multiple findings consistent with PAPA syndrome may be worthy of further PSTPIP1 screening. Our clinical group has had good success with the IL1Ra agent Anakinra in controlling arthritis associated with PAPA syndrome. This information is offered to physicians referring cases with confirmed PSTPIP1 mutations.
  Schnitzler Syndrome: an Acquired Autoinflammatory Disorder

Heleen D. de Koning1, Anna Simon1, Evelien  J. Bodar1, and Jos W M. van der Meer1            

1Dept. of General Internal Medicine, Radboud University Medical Centre Nijmegen, the Netherlands, 6500 HB

Schnitzler syndrome is a rare disorder characterised by urticarial rash, periodic fever, arthralgia or arthritis, and bone pain, accompanied by a monoclonal gammopathy. We reviewed 80 cases published in literature and seen in our own clinic and retrieved follow-up data from 40 of them.
The mean age of onset is 52 years. All patients suffer from episodes of seemingly unprovoked urticarial rash, in more than 80% of cases accompanied by intermittent fever and in more than 60% by arthralgia. The frequency of attacks ranges from daily to twice a year. Bone pain is reported in half of the cases. In all cases a monoclonal gammopathy is found, mostly of IgM type, sometimes IgG.
The pathophysiology is still unknown; autoantibodies or antigen-specific T-cells are lacking. No mutation in the cryopyrin gene exon 3 was found in two of our patients.
Schnitzler syndrome follows a chronic, relapsing course, and no spontaneous complete remissions have been reported. Ten out of the 80 cases have developed Waldenström’s macroglobulinemia.
Many different treatments, including antihistamines and NSAIDs, have proved ineffective. Serious side effects limited the use of high-dose corticoids and thalidomide. Recently, however, we have reported rapid remission with the use of interleukin-1 receptor antagonist (anakinra) in three patients (Ann Rheum Dis online first 11 August 2005 doi:10.1136/ard.2005.045245).
Conclusion: Schnitzler syndrome is an acquired disorder with clear resemblance to cryopyrin-associated periodic syndromes, including a phenotype of urticaria, periodic fever and arthralgia, and a dramatic response to IL-1ra treatment. In view of this phenotype and the lack of high-titer autoantibodies or antigen-specific T-cells, we propose that Schnitzler syndrome should be incorporated in the group of systemic autoinflammatory syndromes.

  Clinical Response and Outcome Criteria in Adult-Onset Still’s Disease

John J. Cush1, Andres Quiceno1, and Richard  Stern1             

1Presbyterian Hospital of Dallas, Dallas, TX 75235; 2St. Paul University Medical Center, Dallas, TX 75235

Introduction. Still’s disease is a systemic inflammatory disorder that preferentially affects children and and young adults with quotidian fevers, evanescent rashes and arthritis. While the diagnosis has become less problematic, there is a lack of validated outcome and response measures. At issue is how to longitudinally assess systemic disease activity. This report will propose the utility of the Still’s Disease Inflammatory Index (SDII) in the outcomes of adult-onset Still’s disease (AOSD) patients
Methods. Based on literature review and empiric experience we have proposed the Stills Disease Inflammatory Index (SDII) based on 10 sensitive measures of inflammatory activity. Components of the Adult Still’s Disease Inflammatory Index (SDII)+ : 1) Fever > 102oF; 2) Sore Throat (reported within 2 weeks of other SD symptom or findings); 3) Still’s Rash# (erythematous, evanescent, nonfacial); 4) Weight loss# (>10% from basal weight); 5) Serositis (pleuritis or pericarditis or peritonitis); 6) Reticuloendothelial System involvement (hepatomegaly or splenomegaly or generalized lymphadenopathy); 7) Inflammatory synovitis of 2 or more joints; 8) Elevated WBC > 12.5 x 103 cells/mm3; 9) Acute phase reactant (ESR > 50 mm/hr, CRP > 2.0 mg/dl); or 10) Elevated Hepatic Enzymes (AST or ALT > 1.5xULN). This index is appropriate for the serial assessment of adult or juvenile Still’s disease patients with active systemic inflammatory activity (new onset, early, chronic or intermittent disease). +Scoring: The presence of each feature is one point, with a maximum (Total) ASDII= 10 points. Remission: SDII score = 0. Major improvement (for patients with score > 2): SDII drop by >50% (for patients with initial SDII of 1 or 2, only a SDII score of 0 qualifies as major improvement). Flare: Increase in SDII by > 2 points. Major Flare: increased SDII > 5 points.
Results. We will describe the outcomes of 4 AOSD patients according to SDII criteria. All patients had >4 points at entry and all showed significant improvement with therapy (steroids, anankinra). All patients demonstrated major improvement or remission.
Conclusion. SDII proved to be a useful and dynamic tool in patients showing dramatic responses to prednisone or anakinra treatment. We believe that such an instrument will foster drug development, and establish the efficacy, utility and safety of existing regimens against potentially novel therapies. Although these criteria may also prove useful in patients with chronic inflammatory arthritis, chronic synovitis may best be assessed using either the ACR20 or DAS28 response criteria.

  Nod1-Dependent Control of Tumor Growth

Jean da Silva Correia1, Yvonne Miranda1, Nikki Austin-Brown1, Jenny Hsu1, John Mathison1, Rong Xiang1 , Huamin Zhou2, Qinxi Li2, Jihuai Han1, and Richard Ulevitch1     

1The Scripps Research Institute, Department of Immunology, La Jolla, CA, 92037, USA; 2Xiamen University, School of Life Sciences, Xiamen 361005, Fujian, China

Nod1 is a cytosolic protein that senses components derived from bacterial peptidoglycans and therefore it has been linked to innate immune responses to bacteria. Recently, complex insertion/deletion polymorphisms in Nod1 have been linked to susceptibility to inflammatory bowel disease as well as asthma and elevated IgE levels. Here we describe a new function for Nod1 whereby it controls tumor formation. The human breast cancer epithelial cell line, MCF-7, was used in a SCID mouse xenograft model to characterize a novel pathway linking Nod1 to the growth of estrogen-sensitive tumors. In MCF-7 cells the absence of Nod1 or blockade of its downstream signaling correlates with tumor growth, an increased sensitivity to estrogen-induced cell proliferation and a failure to undergo Nod1-dependent apoptosis. These data provide support for our contention that there is a link between Nod1 and the control of tumor cell growth.
  Favorable Response to Anakinra in Two Patients with Refractory Adult-Onset Still’s Disease

Tom Pettersson1, Merja Aarnio2, Tapani  Helve2, Riitta Luosujärvi2, Dan  Nordström1, and Marjatta Leirisalo-Repo2          

1Division of Internal Medicine, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland; 2Division of Rheumatology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland

Proinflammatory cytokines including TNF-alpha, IL-1, IL-6 and IL-18 are thought to play a major role in inducing the multiplicity of clinical manifestations in adult-onset Still’s disease (AOSD). In AOSD refractory to conventional therapy, blockade of cytokines or cytokine receptors has been proposed. We report positive responses to the IL-1 receptor antagonist anakinra in two patients with refractory AOSD.

Patient 1. A 20-year-old man developed spiking fever, sore throat, rash, arthralgias and myalgias. Investigations revealed neutrophilia, a serum CRP level of 200 mg/l, elevated liver enzyme levels, a pleural effusion and a moderate enlargement of the spleen. Antimicrobial serology was negative and no autoantibodies were detected. A diagnosis of AOSD was made and prednisone 80 mg/day was started. He stayed in remission with prednisone and cyclosporine but 2 years later there was a relapse with a serum ferritin level of 3,206 mcg/l. Despite addition of methotrexate and podophyllotoxin the disease remained active. Infliximab was initiated and was continued for 3 years and 8 months but there were only short and partial responses. Etanercept and, three months later, adalimumab were tried but the inflammation persisted. At this stage, subcutaneous injections of anakinra 100 mg/day were started and there was a good initial response and a complete response within 3 weeks. Nine months later he stays in remission with anakinra and prednisone 10 mg/day.

Patient 2. A 56-year-old woman presented with spiking fever, sore throat, rash, arthralgias and myalgias. Investigations revealed neutrophila, thrombocytosis, a serum CRP level of 347 mg/l and elevated liver enzyme levels. The diagnostic work-up showed no signs of infectious or autoimmune disease. The serum ferritin level was 13,400 mcg/l. AOSD was diagnosed and prednisone 80 mg/day was started. Five months later, because of recurring and persistent symptoms, methotrexate was added to the drug regimen. There was an initial response but six months later her symptoms recurred and they persisted despite a weekly methotrexate dose of 17.5 mg and high-dose prednisone. After addition of anakinra 100 mg/day she became free from fever within a day and rapidly recovered. Nine months later she remains in remission

Anakinra seems to induce a favorable response at least in some patients with AOSD refractory to conventional therapy and to TNF-alpha blocking agents. The results indicate that the effects of IL-1 and the family of IL-1 receptors are important in the development of AOSD.

  Macrophage migration inhibitory factor in hereditary periodic fevers

Donato Rigante1, Andrea Flex2, Gilda Federico1, Marcello Candelli3, Roberto Pola2, Raffaele Manna3 , Anna Lisa Pugliese1, Claudia Cerquaglia3, Alessandra Costa3, and Achille Stabile1     

1Dept. of Pediatric Sciences, Università Cattolica Sacro Cuore, Rome, Italy; 2Lab. of Vascular Biology and Genetics, Università Cattolica Sacro Cuore, Rome, Italy; 3Dept. of Internal Medicine, Università Cattolica Sacro Cuore, Rome, Italy

Background: Macrophage migration inhibitory factor (MIF) plays a critical role in the regulation of many broad-spectrum immunologic responses. A single-nucleotide G-to-C polymorphism at position -173 in the MIF gene (MIF) has been associated with increased serum MIF levels and poorer therapeutic response in systemic onset-juvenile idiopathic arthritis. The potential role of MIF in hereditary periodic fevers (HPF) such as Familial Mediterranean fever (FMF) and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS) has not yet been investigated.
Objective: To examine MIF-173 polymorphism and serum MIF concentrations in a group of Italian patients with HPF during a free-symptom phase of their disease in comparison with healthy controls of identical ethnic origin.
Methods: Genomic DNA for MIF and serum MIF concentrations were evaluated in 22 patients with HPF recruited in the Centre of Periodic Fevers of our University (12 males, 10 females, 17 with FMF, 5 with HIDS, mean age: 28.8±26.4 years, mean time since diagnosis: 24.1±24.3 years). No patient had current evidence of infection or was treated with steroids; patients with FMF were treated with colchicine. The control group consisted of 169 healthy Italian individuals with no history of chronic or infectious disease; in 41 of them serum MIF levels were also dosed. Genotype and allele frequencies were analyzed using the X2 test or Fisher’s exact test; serum MIF concentrations were studied with Students’ T test for unpaired data. Statistical significance was set at p<0.05.
Results: Frequency of MIF-173 CG genotype has resulted higher in HPF patients than in controls (36.4% versus 12.4%, p<0.005): the C allele was more frequent in the HPF group (p<0.05). The CG genotype and C allele frequencies were higher in patients with FMF than in controls; no correlation was found both for genotype and allele frequencies in HIDS patients. Serum MIF concentrations have resulted higher in HPF patients than in controls with a remarkable statistical significance (79.81±29.02 ng/ml versus 6.032±13.29 ng/ml, p<1•10-11). There was no significant difference in serum MIF concentrations between patients with FMF and HIDS. Serum MIF concentrations appeared unrelated to specific MIF genotypes both for HPF patients and specifically in those with FMF.
Conclusions: MIF 173-*C allele is more represented and likewise MIF serum concentrations appear to be remarkably elevated in patients with HPF. These preliminary data support the hypothesis that MIF might be involved in the pathophysiology of autoinflammatory syndromes and justify further research into the definition of the exact role of MIF in HPF.

  Behcet's disease in adults Druze of north Israel :the influence of ethnic origin on disease expression and severity.

Michael Rozenbaum1*, Nina Boulman1, Gleb  Slobodin1, Itzhak  Rosner1, Davy Zisman2, Reuven  Mader3 , Ana Yankevitch4, and Avraham Weinberger4       

1Department of rheumatology, Bnai Zion Medical Center,Rappaport Faculty of Medicine, technion,Haifa, Israel ; 2Internal Medicine A, Carmel Medical Center, Haifa, Israel. ; 3Rheumatic Disease Unit, Haemek Medical Center, Afula, Israel.; 4Department of Medicine B, Rabin Medical Center, Petach Tikva, Israel ; *These authors contributed equally to this work.


OBJECTIVE: to evaluate the type and frequency of clinical features of Behcet's disease(BD) in a population of adults Druze patients in north Israel.

METHODS: we retrospectively studied 23 Druze patients with BD (15 males and 8 females, mean age:39,3 years).Disease expression was compared between druzes and 30 Arabs with BD ( 20 males and 10 females, mean age:39,8 years).All patients fulfilled the classification criteria developed by the international study group for BD.

RESULTS: The most frequent manifestation was recurrent oral aphtae ( 95,6 %) and genital aphtae ( 60,9 %) versus ( 100 %) and ( 53,3 %) in Arabs patients ,followed by inflammatory ocular involvement ( 65,2 %) versus in Arabs BD (53,3 %).Anterior uveitis occurs in nine patients ( 47,8 %) and panuveitis in 4 patients and no case of blindness in comparison to Arabs BD (30 %) with anterior uveitis , panuveitis in 4 patients and 4 cases of blindness ( p:0,040).One Druze BD patient had deep vein thrombosis versus 8 Arabs patients ( p:0,017).No pulmonary embolism ,aneurysm ,valvular involment were report in Druze BD versus one case of every one cardiovascular complication in Arab BD.Arthritis was seen in ( 39,1% ) Druze patients and (26,6 % in Arabs.).No Neuro Behcet's were reported in Druze,there were 6 cases of Neuro Behcet in Arabs BD (p: 0,023).The prevalence of HLA B 51 in Druze,there was not significant difference in both groups.One druze BD was associated with moderate familial Mediterranean fever disease(FMF) .


CONCLUSION: Druze BD patients in Israel have a relatively mild course of the disease,like in FMF .Our results suggested an ethnic influence on expression of BD ,not related to HLA B 51.Environmental factors may be responsible for the low severity in Druze BD patients.





  The Use of Anti-TNF Therapy in Systemic-Onset Juvenile Arthritis with a Systemic Course: an Observational, Open-label Study

Ricardo A. Russo1, and María  M. Katsicas1              

1Servicio de Inmunología, Hospital de Pediatría "Prof. Dr. Juan P. Garrahan", Buenos Aires, Argentina 1245

Background: Use of anti-TNF drugs has become the mainstay of therapy in patients with refractory juvenile arthritis. However, efficacy in patients with systemic-onset juvenile arthritis (SOJA) appears to be not as satisfactory as in other forms of juvenile arthritis. Children with SOJA with a systemic course have been consistently excluded from most clinical trials and information about the utility of anti-TNF therapy in such patients is lacking.
Objective: To assess the long-term effectiveness and safety of anti-TNF treatment in patients with systemic-course SOJA.
Methods: Thirteen patients with refractory systemic-course SOJA who showed active polyarthritis were included. Median age at disease onset was 4.0 years; median disease duration at entry was 3.0 years. All patients had received methotrexate at ≥ 20 mg/m2/week, had a negative tuberculin test, and were followed regularly. Patients initially received etanercept 0.4 mg/kg/dose twice weekly or infliximab 5 mg/kg/dose, at weeks 0,2,6, and every 4 weeks thereafter, combined with methotrexate. Dosages were increased up to 1 mg/kg and 10 mg/kg respectively if improvement was not achieved. Each anti-TNF agent was switched to the other one or to adalimumab if inefficacy or loss of efficacy was observed, or if toxicity precluded its use. The primary efficacy outcome was the ACR pediatric 30 criteria for improvement. Achievement of ACR 50, ACR 70; remission; disappearance of fever and rash; decrease of steroid dosage ≥ 50 %; improvement and treatment duration; and occurrence of adverse events were recorded. Twelve children with polyarticular-course SOJA who were similarly treated served as a control group.
Results: Ten patients (77 %) achieved ACR 30 criteria, 8 ACR 50, and 5 (10 controls, p< 0.02) ACR 70; only 4 patients (9 controls, p< 0.03) sustained improvement criteria for ≥ 6 months. After 1 year, 4 patients (0 controls) had discontinued therapy due to inefficacy or toxicity. After 2 years, 2 patients remained in the study, 1 (6 controls, p< 0.02) achieved complete remission. Fever and rash subsided in 2 patients and steroid dosage was tapered in 5 children. Toxicity leading to withdrawal occurred in 6 patients (0 controls, p< 0.009), treated with infliximab (peri-infusional events).
Conclusions: in this study, anti-TNF therapy was efficacious in 77 % of patients with systemic-course SOJA in the first year of treatment, but improvement was short-lived or toxicity limited its continuous use in most cases. Patients with a systemic course show a poorer response than children with a polyarticular course.

  Incidence of amyloidosis during life of patients with familial Mediterranean fever in Armenia. Report III. Influence of genealogical and genetic factors

Alexander Ayvazyan               

Yerevan State Medical University; 2Medical Centre “Erebouni”

BACKGROUND & METHODS: See first report.
RESULTS: The presence of familial Mediterranean fever (FMF) among the first and the second-degree relatives has "negative" influence on the frequency of manifestation of amyloidosis, i.e. amyloidosis in such patients manifests less often (during the II, III, IV and V decades of life: 6, 9, 10, 11 % vs 7, 12, 13, 18 % concerning the presence/absence of FMF in the first-degree, and 6, 9, 7, 7 % vs 7, 11, 13, 16 % - in the second-degree relatives). At presence of FMF among the third-degree relatives this law is persisted only during the fourth decade of life (5, 14, 4, 11 % vs 7, 10, 12, 15 %). The presence of amyloidosis among siblings of patients with FMF makes more probable the manifestation of amyloidosis only during the second decade of life of proband, and during other decades the percentage of manifestation of amyloidosis does not significantly depend on the presence of amyloidosis among siblings of patients with FMF (9, 10, 7, 7 % vs 4, 6, 5, 9 %). On our data, sharp differences in frequency of demonstration of amyloidosis between different mutant genotypes are found only during the second decade of life (M694V/M694V - 29 %; M694V/M680I - 7 %; M694V/V726A - 0 %; M694V/U - 0 %). The same parameters for the third decade have made 8; 8; 0 and 0 %, and for the fourth - 0; 12; 7 and 25 % accordingly.

  The differences of course of familial Mediterranean fever in different regions of Yerevan. Report I. The distribution of clinical forms of disease and average age of manifestation of attacks.

Alexander Ayvazyan2, Amalya Voscanyan3, Eduard Nazaretian1, and Knarik Ginosyan1            

1Yerevan State Medical University; 2Medical Centre “Erebouni”; 3Medical Centre Nr 2 of Yerevan

BACKGROUND: Investigations of the differences of course of familial Mediterranean fever in different regions of Yerevan with the same Armenian population residing with identical climatic conditions, but with different geographical origin of their ancestors.
METHODS: The catamnestic information on 1894 patients is obtained, among whom 1050 suffered from the mixed (55,4 %), 694 – from the abdominal (36,6 %) and 150 – from the thoracic (7,9 %) forms of FMF. Women were 741 (39,1 %).
RESULTS: The big differences of course of familial Mediterranean fever (FMF) in representatives of different ethnic groups are known. But even in the same Armenian population, at the same place of residing with identical climatic conditions (city Yerevan), the course of familial Mediterranean fever in different regions of the city Yerevan which are formed during the various periods of development of city, and are populated with refugees from various parts of historical Armenia is different. First it concerns to distribution of clinical forms of FMF. For example, in region Arabkir patients with mixed form of FMF make 51,1, and in region Zeytoun-Kanaker - 74,6 % of patients' contingent. The average age of manifestation of attacks of FMF also depends on region of a residence of patients in Yerevan (for example, in Center - 14,1 vs 9,8 years old in Achapnyak). At various clinical forms of FMF these laws also are different (for example, in the case of the above-stated regions by the abdominal form - 17,0 vs 10,1, and by the mixed one - 11,4 vs 9,1 years old).

  The differences of course of familial Mediterranean fever in different regions of Yerevan. Report II. The frequency of typical attacks of disease, the expressiveness of an articular syndrome, and the probability of development of amyloidosis.

Alexander Ayvazyan2, Amalya Voscanyan3, Eduard Nazaretian1, and Knarik Ginosyan1            

1Yerevan State Medical University; 2Medical Centre “Erebouni”; 3Medical Centre Nr 2 of Yerevan

BACKGROUND & METHODS: See first report.
RESULTS: The frequency of typical attacks of FMF also depends on region of a residence of patients in Yerevan (for example, the percentage of patients with often attacks of FMF in the case of the abdominal form has made during I-IV decades of life 30,3; 48,3; 37,1 and 40,0 % in Arabkir, and 34,3; 25,0; 66,6 and 33,3 % - in Erebouni). The expressiveness of an articular syndrome at FMF is different also in dependence on a region of residence of patients in Yerevan (for example, in the case of the abdominal form in Norki Zangvatsner within the first and the four decades of life of patients with the expressed articular syndrome have made 22,8; 31,5; 29,2 and 26,0 %, and in the Harav-Arevmtyan Zangvats - 55,5; 44,4; 22,2 and 20,0 % accordingly). The probability of development of amyloidosis at FMF also depends on region of a residence of patients in Yerevan (for example, during the II-IV decades of life the probability of development of amyloidosis has made by the mixed form 10,0 %; 2,0 % and 3,6 % in Norki Zangvatsner, and 6,9 %; 10,3 %; 3,9 % - in Center). These researches show, that genetic factors play very important role in aspect both clinical course of FMF, and probabilities of development of amyloidosis.

  SLE-like syndrome in patients with familial Mediterranean fever in Armenia

Alexander Ayvazyan, Laura Hovsepyan, Aida Zavgorodniaia, and Knarik Ginosyan            

1Yerevan State Medical University; 2Medical Centre “Erebouni”

BACKGROUND: According to investigations of Israel colleagues it is observed the low incidence and the mild course of systemic lupus erythematosus (SLE) in familial Mediterranean fever (FMF) patients (Langevitz P. et al., 2002). According to investigations of Seza Ozen and Aysin Bakkaloglu in Turkey none of over 1000 patients in their pediatric registry had SLE. Conversely, none of their patients with SLE had association with FMF (Ozen S., Bakkaloglu A., 2005). The same findings are reported also in more large investigation of about 3000 Turkish patients with FMF (Tunca M. et al., 2005). AA amyloidosis in SLE is also an unusual complication (Queffeulou G. et al., 1998; Ridley M.G. et al., 1984).
METHODS: Catamnestic observation of patients with FMF, suffering from classic forms of illness (4167 cases). The diagnosis of SLE was determined according to criteria of ARA.
RESULTS: Contrary to data of our above mentioned colleagues we observed 14 patients with the concomitant diagnosis of SLE among our 4167 FMF patients (0,36 +/- 0,090%), that is the increased frequency of SLE in our population. Among these patients women were 11 (78,5 %). The average age at the diagnosis of SLE was 23,6 +/- 2,25 years. The mean follow-up was 12 +/- 2,11 years. During the follow-up we observed only three lethal cases, and all 3 due to chronic renal failure. The postmortem examination show renal amyloidosis in all 3 cases. The rest of patients did not continue steroidal therapy more than one year, and only two patients received daily colchicine prophylaxis during about 5 years, moreover, one of these patients had begun colchicine treatment since 13 years after SLE diagnosis.
CONCLUSIONS: Thus, SLE in Armenian FMF patients has been found at increased frequency, but the course of SLE in FMF patients in Armenia is in majority of cases very mild, and does not corresponds to classic progressive course of SLE. We suggest that cases of FMF fulfilled the criteria of SLE are not the classic cases of SLE, and we have to call these cases as SLE-like syndrome in FMF, and we have also follow-up these patients, but have not begun to treat them with prednisolone immediately.

  Familial Mediterranean fever in inhabitants of Karabakh. Report I. Prevalence, peculiarities of inheritance and clinical forms of disease

Alexander Ayvazyan2, Karine Atayan3, and Eduard Nazaretian1             

1Yerevan State Medical University; 2Medical Centre “Erebouni”; 3Medical Centre of Stepanakert

BACKGROUND: All scientific works devoted to a problem of familial Mediterranean fever (FMF) were carried out basically without taking into account regional peculiarities of illness. The data on Karabakh in this respect are especially important, because the climatic conditions and ethno-geographical origin of the population of Karabakh differ from ones at Republic of Armenia (RA). The population of Karabakh is presented mainly as Armenians of east origin.
METHODS: The catamnestic information on 244 patients is obtained. Two hundred nineteen patients of them suffered from classic (serositic) forms of FMF among whom 83 suffered from the mixed (37,9 %), 119 –the abdominal (54,3 %) and 17 – the thoracic (7,8 %) forms of FMF. Women were 117 (52,2 %, in RA - 40,3 %).
RESULTS: Prevalence of FMF in Karabakh estimates in range 1,0 - 1,4 %, that significantly does not differ from prevalence of FMF in Yerevan (1,1 %). Frequency of FMF among siblings in Karabakh appeared 6,9 % (in RA - 8,9 %), that is significantly lower than in siblings of probands of west Armenian origin (11,0 %, p <0.01), and does not differ from similar percentage in probands of east Armenian origin (7,4 %, NS). In Karabakh a sharp difference in percentage of FMF among siblings in case of the various clinical forms of FMF in proband (mixed - 6,8 %, abdominal - 8,1 %), that is observed in RA (10.6 % and 6.1 % respectively) isn’t observed. Probability of a birth of children with FMF from parents with FMF in Karabakh is very low at the thoracic form in parent (0 %, in RA - 6,0 %), but is higher in general contingent (13,6 vs 6,8 %), and this law basically is observed in case of the parents sick with mixed form of FMF (18,5 vs 7,2 %). Abdominal form of FMF is significantly met more often among patients of Karabakh in comparison with patients of RA with west Armenian (39,8 vs 26,7 %), but not with east Armenian origin (40,3 %).

  Familial Mediterranean fever in inhabitants of Karabakh. Report II. Peculiarities of clinical course of disease

Alexander Ayvazyan2, Karine Atayan3, and Eduard Nazaretian1             

1Yerevan State Medical University; 2Medical Centre “Erebouni”; 3Medical Centre of Stepanakert

BACKGROUND & METHODS: See I report.
RESULTS: Middle age of the manifestation of attacks of familial Mediterranean fever (FMF) in Karabakh makes 11,1 years (in Republic of Armenia [RA] - 14,3); in case of the mixed form - 9,3 (in RA- 12,1), abdominal - 12,1 (in RA - 16,9), thoracic - 14,8 years (in RA - 18,1). In Karabakh only in the first decade of life at mixed form of FMF patients with often attacks are met greatly more often (51,4 %), than at other forms of FMF, and in the same decade of life patients with often attacks among patients with abdominal form of FMF are met a bit more often (33,0 %), than among patients with the thoracic form (12,5 %). The peak of frequency of attacks of FMF in inhabitants of Karabakh, as well as in inhabitants of RA, is in the age of 20-40 years. On the contrary to RA in the age of 41-50 years among inhabitants of Karabakh there were less patients with often attacks of FMF (18,8 vs 53,4 %). The expressiveness of an articular syndrome in patients with FMF in Karabakh, as against frequency of the basic attacks of illness, significantly does not vary in the various decades of life (36-43 %) and significantly does not differ at various clinical forms of FMF. And in Karabakh and in RA the course of articular syndrome of FMF significantly does not differ. In Karabakh the sharp peak of a case rate of amyloidosis in the age of 21-30 (19,6 %) is observed which is not observed in inhabitants of RA, and such peak of a case rate is observed only at mixed form of FMF. In Karabakh the average life expectation at FMF without colchicine therapy has made 39,9 years, as in RA. The difference between sexes concerning the rate of frequent serositic attacks of FMF during separate decades of life of patients is more expressed in Karabakh than in Republic of Armenia (RA), and is significant only in the second and the third decades of life, and it is observed only at the mixed form of FMF, and frequency of attacks was higher at women of the specified age groups (61,5 vs 30,0 % and 88,9 vs 33,3 %). The difference between sexes from the point of view of an expressiveness of articular syndrome and probability of amyloidosis in Karabakh is not significant.

  Familial Mediterranean fever in inhabitants of Karabakh. Report III. Peculiarities of interference of some factors on clinical course of disease

Alexander Ayvazyan2, Karine Atayan3, and Eduard Nazaretian1             

1Yerevan State Medical University; 2Medical Centre “Erebouni”; 3Medical Centre of Stepanakert

In Karabakh the frequency of manifestation of amyloidosis within separate decades of life of patients suffering from familial Mediterranean fever (FMF) significantly does not differ at various age of the manifestation of attacks. Often attacks of FMF during the first decade of life "positively" influence on frequency of manifestation of amyloidosis only during the second and the third decades of life (71,7 vs 21,6%, and 72,7 vs 37,4%). The course of articular syndrome in the first decade of life has also the prognostic value on the course of serositic attacks during the decades of life of patients with FMF (II: 71,7 vs 18,3%; III: 85,7 vs 25,7%; IV: 84,0 vs 27,3%; V: 63,6 vs 8,3). The prognostic value of the expressed articular syndrome on the course of the same syndrome during the next decades is also very big (II: 95,0 vs 6,73; III: 92,9 vs 6,76%; IV: 96,0 vs 13,6%; V: 90,9 vs 12,5). However prognostic value of an expressiveness of the articular syndrome is not so expressed, and is not significant from the point of view of amyloidosis. The presence of the first-degree relatives who had FMF «positively» influences on the course of serositic attacks of FMF, i.e. patients with frequent attacks of FMF in the certain decades of life (II: 45,3 vs 34,2%; III: 61,5 vs 40,3%; IV: 66,7 vs 35,7%; V: 40,0 vs 15,0%) are more often met, however the difference in Karabakh is significant only in the third and the fourth decades of life. The same factor influences also on the course of articular syndrome, however, the difference is significant only in the second decade of life (52,8 vs 32,4%) and does not influence on the probability of manifestation of amyloidosis. Studying of the influence of the presence of FMF among the second-degree relatives on the course of serositic attacks has shown, that in Karabakh the presence of FMF among the second-degree relatives considerably rises the probability of presence of often attacks of FMF at least during the second and the third decades of life of patients (66,7 vs 34,9%; 75,0 vs 44,2%), even despite of considerably smaller number of observations in comparison with cases with the presence of FMF among first-degree relatives. In Karabakh it is not revealed a significant difference of the course of articular syndrome and the frequency of manifestation of amyloidosis at presence or absence of FMF among the second-degree relatives.
  Influence of change of course of attacks of familial Mediterranean fever and an articular syndrome on probability of amyloidosis

Alexander Ayvazyan2, Amalya Voscanyan3, Eduard Nazaretian1, and Tamara Bayramyan1            

1Yerevan State Medical University; 2Medical Centre “Erebouni”; 3Medical Centre Nr 2 of Yerevan

AIM: Influence of the change of course of attacks of familial Mediterranean fever (FMF) and an articular syndrome on probability of amyloidosis in patients living in same clinical conditions (city Yerevan).
METHODS: The catamnestic information on 1894 patients is obtained, among whom 1050 suffered from the mixed (55,4 %), 694 – the abdominal (36,6 %) and 150 – the thoracic (7,9 %) forms of FMF. Women were 741 (39,1 %).
RESULTS: Studying of probability of living without amyloidosis up to 20-years age in dependence on course of attacks during the first and the second decades of life has shown, that in inhabitants of Yerevan on the probability of amyloidosis influences basically the course of attacks of FMF during the first decade of life even if after often attacks in the first decade of life there comes the term of infrequent attacks during the second decade. Moreover, improvement of course of attacks of FMF even reduces chances of surviving without amyloidosis (67,7 vs 85,3%). Course of articular syndrome during the first decade of life has important role on the probability of amyloidosis and in case of persistence of expressive articular syndrome during the second decade of life (89,1%) and in case of absence of those in the second decade (86,1% vs 96,7% in case of absence of articular syndrome during the first and the second decades of life). Even presence of the expressed articular syndrome from the second till the fourth decades of life in case of that absence in the first decade of life, is more favorable course of FMF. The probability of living without amyloidosis up to 40-years age in those cases are higher in comparison with cases with expressed articular syndrome from the first decade of life (100 vs 79,6 %). Moreover, improvement of articular symptomatology after 2 or 3 decades of the expressed articular syndrome reduces chances of surviving without amyloidosis up to 40-years boundary (54,6 and 52,94 vs 79,6%).

  Nitric Oxide, IL 6 and IL 10 Level in Blood Plasma of Children with Familial Mediterranean Fever as Markers of Treatment Efficacy of Some Adaptogenes.

Gayane G. Amaryan1, Marine K. Ambardzumjan2, and Vilen A. Astvatsatryan1             

1 Chair of Pediatrics,Yerevan State Medical University,Yerevan,Armenia, 375025; 2Expert Analytical Laboratory, Branch of Drug and Technological Research Centre, Yerevan, Armenia,375025

Considering anti-inflammatory efficacy of andrographolide we studied the effect of new herbal drug - adaptogen ImmunoGuard on Nitric Oxide, IL-6 and IL-10 level in blood plasma of children with FMF.
The results of this study of FMF patients showed that the basal level of NO in blood of these patients during attack-free periods was not higher than that found in blood of healthy subjects. This finding is quite unusual in chronic inflammatory disorders which are typically characterized by elevated plasma NO levels. Even more surprising is the our
finding that the level of NO in blood of FMF patients significantly decreased in inflammatory attacks. As an explanation of our observations with FMF patients, we suggest that the decrease in NO level during attacks is a defensive compensatory response intended to decrease the inflammation. However, this decrease in NO also leads to an increase in the production of pro-inflammatory cytokines such as IL-6 and neutrophil accumulation in serous tissues, thus triggering fever in FMF patients. It has to be also suggested that a stimulus decreasing the activity of iNO-synthase in FMF patients plays a crucial role in initiation of fever. In our study we also found that ImmunoGuard increases NO in blood of FMF patients in attack free periods. It has normalizing effect on NO, IL-6 and IL-10 blood levels in FMF patients during attacks, which become less severe and shorter than before the treatment or in control group of placebo treated patients.That clearly indicates on the relationship between beneficial effect of ImmunoGuard, reducing severity of inflammatory attacks in FMF patients and their NO blood level. It can be speculated that decrease of the blood NO level can provoke an inflammatory attack in FMF patients.
Taking into consideration all above mentioned, we suppose that the main curative effect of ImmunoGuard relates to his an active principle, i.e. andrographolide, presents in extracts of A.paniculata. which has a long-standing reputation in Scandinavia for prevention and treatment of the common cold as an anti-inflammatory agent, particularly, in adaptogen KanJamg.. Apart from an anti-inflammatory component, an immunostimulatory action has also been attributed to andrographolide.
Consequently our preliminary study encourages further investigation of mechanisms of action of andrographolide of some adaptogenes and its effect on FMF patients.

  Tendency to Increasing the Frequency of Early Manifestation of Familial Mediterranean Fever in Children in Armenia

Gayane G. Amaryan1, Sahakanoush S. Aroustamyan1, Nune G. Mkrtchyan1, Vilen A. Astvatsatryan1, Tamara F. Sarkissian2, and Hasmik Hayrapetyan2          

1Republican Pediatric Centre of FMF, "Arabkir" Medical Centre, Chair of Pediatrics, Yerevan State Medical University, Yerevan, Armenia, 375025; 2Centre of Medical Genetics, National Academy of Sciences,Yerevan, Armenia, 375025

Familial Mediterranean Fever (FMF) being an endemic disease for the Armenian population remains a crucial medical-social problem. Since 2003 a program on early diagnosis and treatment of FMF in children in Armenia has been initiated by the Republican Pediatric Center of FMF, which aims to improve the management of children with FMF.
Resuming two-year activity of the program, it should be noted the substantially increasing of the number of newly diagnosed FMF cases. Also total number of patients increased from 510 to 960. That testifies as about the wide spreading of the disease in children in Armenia, as well as about the improvement of medical care at FMF. There is a significant increase in the number of children with early manifestation of FMF up to 5 years from 3, 2% to 10, 9%, especially before age of 1 year. Among 103 patients of FMF at ages of 5 years, 56,3% of them revealed the first symptoms up to 1year.The positive familial history was observed in 44,8% of these cases. The clinical peculiarities of FMF in children before age of 1 year usually include atypical fits expressed by repeated episodes of anxiety, colics and/or fever. Genetic analysis of MEFV gene mutations has revealed the predominance of homozygous type of inheritance by mutation M694V (39, 6%) and compound-heterozygotes by mutations M694V/ V726A(27.9%). Treatment with colchicine was less efficient.

  A Variety of Corticosteroids Suppresses the Second Phase of Amyloid Formation in Mice

Shmuel Shtrasburg1, Mordechai  Pras1, Clara  Pariente2, Rivka Gal3, and Avi  Livneh1           

1Heller Institute for Medical Research, Sheba Medical Center, Tel-Hashomer 52621, Israel; 2Department of Obstetrics and Gynecology, Sheba Medical Center, Tel-Hashomer 52621, Israel; 3Department of Pathology, Rabin Medical Center, Golda Campus, Hasharon Hospital, Petach-Tikva, Israel.

Background: Steroid treatment of amyloidosis was previously studied in human and murine models of reactive amyloidosis, but with limited success and with conflicting results.
Aim: To determine whether steroids may indeed inhibit amyloidogenesis, and to study the mechanism of their effect.
Methods: Induction of amyloidosis was performed in Swiss male mice, using our enhanced model of a single i.v. AEF injection and three successive daily s.c. AgNO3 injections, in 3 to 6 day long experiments. This procedure shortens amyloidogenesis by skipping the first stage of the amyloidogenic process. Suppression of amyloid formation was studied using various commonly used steroid preparations. The amount of splenic amyloid deposition was determined using the crush and smear technique, with a 5 grade scale.
Results: All steroids examined were found to suppress amyloidogenesis, but revealed differences in the degree and the duration of inhibition. The most potent was dexamethazone, particularly in the 4 day regimen, where methyl-prednisolone was found to have the lowest activity. Hydrocortisone was found to be the most potent in the 3 day experiments. Its effect continued for 3 additional days after it was discontinued.
Conclusions: Corticosteroids were found to inhibit the second phase of amyloidogenesis. This finding encourages steroid use to supplement different anti-amyloidosis treatment protocols.

  Appendectomy in Familial Mediterranean Fever Patients

Merav  Lidar1, Pnina Langevitz1, Nurit  Zaks1, Anat Doron1, Einat  Rabinovich1, and Avi  Livneh1          

1Heller Institute for Medical Research, Sheba Medical Center, Tel-Hashomer 52621, Israel

Aim: Acute appendicitis is the most common reason for emergency abdominal surgery, and must be distinguished from other causes of abdominal pain. Severe abdominal pain is the most common attack manifestation in familial Mediterranean fever (FMF), often leading to an unnecessary appendectomy. The frequency, characteristics and results of appendectomy in FMF have been hitherto scarcely studied, and therefore constitute the aim of the present study.
Methods: We surveyed the abdominal attack manifestations of 182 consecutive FMF patients visiting the outpatient clinic for routine follow-up. The prevalence of appendectomies was quantified and their outcome assessed.
Results: Of the 182 FMF patients included in the study, 104 (57.1%) were females, and 71 (39%) had undergone an appendectomy (2-fold higher than the reported 12-25% frequency in the general population). Only 13 (18.3%) of the histologic specimens showed evidence of acute inflammation in the appendix (significantly less than the reported 85% in the general population). Periappendicitis was evident in 6 cases (8.4%), while no evidence of inflammation was noted in 29 (40.8%) of the cases. Pathological data was not available for 19 (26%) of the patients, and in 4 (5.6%), the appendectomy was elective. There was no gender or age difference between operated and non-operated patients. The age of onset and diagnosis were comparable in both groups. Moreover, MEFV mutations were similarly distributed among the groups. Most (60%) of the operated patients suffered from diffuse, (not localized to the right lower quadrant) abdominal pain during the attack that led to the appendectomy. There was a similar proportion of white appendectomies performed in peripheral vs. tertiary centers (41% in both).
Conclusion: These findings suggest that surgeons’ capability to cope with acute abdomen of FMF and avoid unnecessary operation is grossly limited.

  The Prodrome as a Prominent yet Overlooked, Pre-Attack Manifestation of Familial Mediterranean Fever

Merav Lidar1, Marina Yaqubov1*, Nurit  Zaks1, Shomron Ben-Horin1, Pnina  Langevitz1, and Avi Livneh1          

1Heller Institute for Medical Research, Sheba Medical Center, Tel-Hashomer 52621, Israel ; *These authors contributed equally to this work.

Objective – To identify and characterize pre-attack symptoms (prodrome) in familial Mediterranean fever (FMF) patients.
Methods – 48 FMF patients whose attacks are preceded by a prodromal period comprised the study population. Clinical, demographic and genetic characteristics of the study group were compared to those of a control group of 48 FMF patients whose attacks begin without a premonitory phase. Patients of both groups were recruited consecutively, during their routine follow-up visit to the FMF clinic.
Results – A prodrome was found to be a common manifestation of FMF, experienced by about 50% of patients. Overall, demographic, clinical and genetic parameters were comparable between the study and control groups. In patients experiencing a prodrome, it recurs in most attacks, lasts around a mean of 20 hours and manifests with either a mildly unpleasant sensation in the site of the forthcoming spell (discomfort prodrome), or with a spectrum of various physical, emotional and neuropsychological complaints (variant prodrome). The two types of prodromata were frequently accompanied by a myriad of constitutional symptoms.
Conclusions – A prodromal period heralding attacks is a newly defined and a reliable FMF manifestation that reproducibly predicts attacks and may be advantageously used.

  Colchicine Therapy and the Cognitive Status of Elderly Patients with Familial Mediterranean Fever

Arthur  Leibovitz1, Merav Lidar2, Yehuda  Baumoehl1, Avi  Livneh 2, and Rafael  Segal1           

1Shmuel-Harofeh Geriatric Medical Center, Beer-Yaakov, Israel; 2The Heller Institute of Medical Research, Sheba Medical Center, Tel-Hashomer 52621, Israel.

Background - For more than 30 years, colchicine has been the mainstay of treatment for familial Mediterranean fever (FMF). Studies in animal models have shown ill effects from colchicine to the central nervous system, including cognitive functions. Thus, concern arises as to a possible detrimental effect on cognitive function of FMF patients, due to long-term use of colchicine. In this study we evaluated the cognitive status of elderly FMF patients who have been receiving continuous colchicine treatment for a long time.
Methods - The study group consisted of 55 FMF patients, aged 74±5 years, who are being followed in our clinic and who have been on colchicine treatment for 25±9 years. Cognitive function was evaluated using the mini-mental state examination (MMSE). Patient's scores were compared with the accepted population based norms, and with the scores of another 56 subjects, matched for age and education, who formed the control group.
Results - Compared to population-based norms, adjusted by age and education, the study group had significantly higher mean MMSE scores (27.2±2.2 vs. 25.5±2.4, p<0.001). The mean control group’s score was also somewhat higher than expected, but not significantly so. Moreover, all colchicine treated FMF patients were scored way above the normal lower limit (also called the cut-off point) of an age and education corrected sample of the general population, with a mean MMSE score exceeding the “normal” cut-off scores by 5±1.6 points (p<0.001).
Conclusions - Our results do not support the view that prolonged colchicine treatment may be associated with cognitive impairment. On the contrary, it is possible that long-term colchicine treatment may protect FMF patients from cognitive decline.


  Successful Treatment of Protracted Arthritis of FMF with INFLIXIMAB

Riva  Brik1, and Ruth Gershoni- Baruch2              

1Department of Pediatrics and Pediatric Rheumatology service,Meyer Children's Hospital of Haifa, Israel,31096; 2Institute of Human Genetics, Rambam Medical Center, Haifa ,Israel, 31096

The most common musculoskeletal manifestation of FMF consists of acute episodes of monoarthritis which, in most cases, leave no permanent sequelae. In about 5% of the patients, however, protracted arthritis develops, mostly in the hips or knees. We report a girl with FMF and protracted knee arthritis who responded very favorably to Infliximab therapy.
A 13-year-old girl suffered from FMF since the age of 4, featuring attacks of fever and abdominal pain. Acute phase reactants in her serum were raised and genetic studies revealed homozygosity for the V726A/E148Q complex in the FMF gene. The patient received colchicine and responded with substantial diminution in the frequency and severity of the attacks. About 2 years ago, while on colchicine treatment she began to suffer from acute episodes of arthritis of both knees and one elbow, which only partially responded to various NSAID’s. Later on, the right knee gradually became chronically inflamed and was constantly swollen, painful and limited in motion. Treatment with repeated intra-articular corticosteroids, oral prednisone, Salazopyrine and MTX, failed to induce a satisfactory response. On January 2004, the girl underwent arthroscopy of the knee, which revealed synovial hypertrophy with intact ligaments. Partial synovectomy was performed but several weeks later the synovitis recurred. In view of to the lack of efficacy of conventional treatment, Infliximab was given intravenously and was followed by a prompt resolution of the arthritis. She received a total of 3 doses of Infliximab and was able to stop her corticosteroids and NSAID treatment. However, arthritis recurred 4 months after last dose of Infliximab. She is now successfully treated with weekly MTX and Infliximab as needed.
We described a patient with clinically and genetically confirmed FMF, who developed protracted arthritis of her knee. Although the coexistence of juvenile idiopathic arthritis (JIA) cannot be excluded, the course of the disease is not very characteristic for pauciarticular JIA. Our patient’s first arthritic episode occurred when she was 11-yeas old and she did not have uveitis or a positive ANA.
Colchicine is the main therapy in FMF, however, colchicine is not always effective in preventing the musculoskeletal manifestations of FMF, namely the exertional leg pains, protracted febrile myalgia syndrome, or the chronic protracted arthritis that might occur in about 5% of patients . Our patient received colchicine and other treatment modalities for chronic arthritis, including synovectomy without availe.
Our report calls for the consideration of anti-TNF therapy of FMF manifestations that are unresponsive to colchicine.







  The Chinese Shar-Pei Dog: a Canine Model of Human Autoinflammatory Disease

Francesca  Puppo1, Linda Tintle2, Austin Wong1, Ivona Aksentijevich1, Zac Moak1, Elaine Ostrander3 , Anne Avery4, Elaine Remmers1, and Daniel Kastner1      

1Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 2Wurtsboro Veterinary Clinic, Wurtsboro, NY; 3Comparative Genetics Section, NHGRI, NIH, Bethesda, MD; 4College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO

Chinese Shar-Pei dogs suffer from a disorder characterized by recurrent episodes of fever, arthritis, and rash that may begin as early as two months of age. Later, some affected animals develop systemic AA amyloidosis eventually leading to renal failure and death. Pedigree analyses are most consistent with a single-gene autosomal recessive mode of inheritance, and together with clinical signs suggest that “Shar-Pei fever" most closely resembles human familial Mediterranean fever (FMF), although a mutational screen in the dog ortholog by another laboratory was reported negative.
In order to characterize this naturally occurring canine model of human autoinflammatory disease, we are cloning and screening the canine orthologs of the human genes mutated in FMF (MEFV), TRAPS (TNFRSF1A), HIDS (MVK), NOMID (CIAS1), and PAPA (PSTPIP1) for disease-associated mutations.
Preliminary data suggest a strong conservation in exon size, and in nucleotidic and aminoacidic sequences for most of them.
In the event mutational studies are negative, we will perform a linkage analysis in Shar-Pei pedigrees using a genome-wide set of canine microsatellite markers. Recent results from a pilot study in a limited number of pure-bred and mixed breed unrelated Shar-Pei dogs indicate the polymorphism rate is high enough to collect pedigrees and perform linkage analysis.
Finally, for the selection of positional candidate genes, we will compare gene expression profiles of Shar-Pei and normal animals using a canine microarray.
In this way we hope to establish the Shar-Pei as a model of one of the known human autoinflammatory diseases, or to identify a novel genetic basis for Shar-Pei fever that may shed light on the molecular basis of heretofore mutation-negative fever syndromes in man.
  RNA interference: A potential for the study of pyrin

Geryl M. Wood1, Nitza G. Shoham2, James Balow, Jr3, and Daniel L. Kastner4            

1Inflammatory Biology Section, Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 2Allergy Immunology Laboratory, Meir Hospital, Kfar-Saba, Israel; 3Microarray Section, Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 4Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by episodes of fever and intense inflammation. The gene causing FMF, MEFV, encodes a protein, pyrin, consisting of 781 amino acids that is expressed at high level in granulocytes and monocytes and in some myeloid leukemia cell lines, THP.1 and U-937. Although pyrin appears to play a role in the regulation of IL-1 Beta and NFkappaB activation, its function has not been completely defined. One of the potential roadblocks for elucidating this function is the cell type in which pyrin is endogenously expressed. Studying mononuclear cells in the past has been challenging due to low transfection efficiency and the difficulty in obtaining stably transfected clones. With the establishment of RNAi as a powerful tool for identifying gene function and the relative small size of the duplex RNA, it has been shown that RNAi is a functional pathway in human myeloid leukemia cell lines. We investigate the potential usefulness of this method in understanding the function of pyrin. Initial data suggest that MEFV siRNA can be efficiently transfected into THP.1 (>75%). The high percentage of transfection leads to a significant decrease in the endogenous levels of pyrin as compared to a scramble control siRNA (SC). To provide molecular insight into pyrin function, we compared expression profiles between SC and siMEFV using microarray. We identified a total of 344 genes with 1.5 fold difference and p < 0.05 of which 252 genes had lower expression (down regulated) and 92 genes had higher expression (up regulated) relative to control. A gene list of 15 down regulated and 3 up regulated genes involved in inflammation, immune response and signal transduction were identified. qRT-PCR was performed to validate the microarray data. Thus far we have validated two genes down regulated by MEFV siRNA, S100A8 and CD36. Taken together, our findings indicate that siRNA technique can be useful in the study of pyrin function in native cells. Further research is necessary to validate remaining gene list and do some functional studies of the genes identified.
  Colchicine use in children and adolescents with Familial Mediterranean fever: A proposal for evidence based treatment guidelines
A Turkish - German consensus


Tillmann Kallinich1, Dieter Haffner2, Tim Niehues3, Kristina Huss4, Elke Lainka5, Ulrich Neudorf5 , Christof Schaefer6, Silvia Stojanov4, Christian Timmann7, Rolf  Keitzer1, Huri Ozdogan8, and Seza  Ozen9   

1Department of Pediatric Pulmonology and Immunology, Charité Campus Virchow-Klinikum, Universitaetsmedizin Berlin, Germany ; 2Department of Pediatrics, University Hospital Rostock, Germany; 3Department of Pediatric Oncology, Hematology and Immunology Heinrich Heine University Düsseldorf, Germany; 4Department of Infectious Diseases and Immunology, Children’s University Hospital Munich, Germany; 5Department of Pediatrics, University Duisburg-Essen, Germany; 6Pharmakovigilanz- und Beratungszentrum für Embryonaltoxikologie, Berliner Betrieb für Zentrale Gesundheitliche Aufgaben, Berlin, Germany; 7Department of Molecular Medicine, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; 8Cerrahpasa Faculty of Medicine, University of Istanbul, Cerrahpasa, Turkey; 9 Department of Pediatric Nephrology and Rheumatology, Hacettepe University Faculty of Medicine, Turkey

Background: Familial Mediterranean fever (FMF) is an autosomal recessive inherited disorder characterized by short recurrent febrile attacks associated with serositis. Although, daily application of colchicine is the standard therapy for the prophylaxis of attacks and the development of amyloid depositions, no evidence- based guidelines with respect to colchicine treatment have been established.
Objectives: This work analyses the grade of evidence for the use of colchicine in children and adolescents with FMF in order to develop evidence-based guidelines. These are intended to improve the quality of colchicine therapy and the outcome of these patients.
Methods: Data were obtained from an extensive literature search using PubMed MEDLINE and the keyword “Familial Mediterranean fever” in the age group 0 – 18 years, studies performed in adults, databases and text books. Three consensus meetings were held. Evidence and recommendation levels were graded according to Feldman [1].
Results: The continuous use of colchicine for prophylaxis of attacks and amyloidosis is recommended [IIA]. It should be introduced as soon as the diagnosis has been established and continued life-long [IIIA]. Colchicine is also recommended for the treatment of already established amyloidosis [IIA]. For monitoring of disease activity and side effects regulary clinical check-ups including laboratory tests should be performed on a regular bases [IIIA]. At present, no alternative to colchicine can be recommended [IIIC]. Colchicine intoxication requires hospitalisation and application of activated charcoal [IIIC].
Conclusions: Although several questions remain open, evidence-based recommendations can be given with respect to indication and application of colchicine in children and adolescents with Familial Mediterranean fever.

  Are Carriers for MEFV Mutations “Healthy”?

Mukaddes Kalyoncu1, Banu Celiker Acar2, Nilgun Cakar3, Aysin Bakkaloglu1, Nesrin Besbas1, Rezan Topaloglu1 , Selda Ozturk3, Mesiha Ekim2, Emel Dereli2, Mehmet Tunca4, Ozgur Kasapcopur5, Fatos Yalcinkaya2, and Seza Ozen1  

1Hacettepe University Faculty of Medicine, Department of Pediatrics, Pediatric Nephrology and Rheumatology Unit, Ankara, TURKEY; 2University Faculty of Medicine, Department of Pediatrics, Pediatric Nephrology and Rheumatology Unit, Ankara, TURKEY; 3Ankara Diskapi Children’s Hospital, Department of Pediatric Nephrology, Ankara, TURKEY; 4Dokuz Eylül University Faculty of Medicine, Department of Internal Medicine, İzmir, TURKEY ; 5İstanbul University, Cerrahpasa Faculty of Medicine, Department of Pediatrics, Pediatric Nephrology and Rheumatology Unit, Ankara, TURKEY

Abstract
Objective: We aimed to compare whether carriers for the MEFV mutations display an increase or decrease in certain features. We compared the frequency of a number of inflammatory symptoms and diseases in carriers and a control population.
Methods: A questionnaire was designed to be applied to parents of children with FMF and a control group of parents. Clinical features and some diseases including the frequency of febrile episodes, abdominal pain, arthralgia, prophylaxis with penicilline, acute rheumatic fever, rheumatoid arthritis, vasculitis, spondyloarthropathy, urinary tract infection, asthma, allergy, irritable bowel disease, appendectomy and tonsillectomy were inquired. 676 parents of 336 children with FMF were surveyed in this study. Controls (n: 774) were selected as parents of healthy children.
Results: The presence of febrile episodes more than four per year, arthralgia, past diagnosis for acute rheumatic fever, rheumatoid arthritis and prophylaxis of penicillin, acute rheumatic fever, and rheumatoid arthritis were significantly higher in obligate carriers for the MEFV mutations compared to controls. The frequency of allergy was found to be significantly lower in the obligate carriers as compared to controls. There was no significant difference at the frequency of urinary tract infection and tonsillectomy between the parents of the patents and controls.
Conclusions: We suggest that one MEFV mutation may indeed be conferring a heightened inflammation as suggested by the increased frequency in inflammatory symptoms. The carrier status for MEFV mutations seem to be unique, in that they cause an alteration in the state of “health”.

  Potential Interactions Between Neutrophil Apoptosis and Oxidative Damage in Familial Mediterranean Fever (FMF)

F.Guldal Kirkali1, Miral Dizdaroglu2, Halil Resmi3, Hayri Ozsan4, Pınar Tuncel1, Halil Ates4 , Sermin  Genc3, Tuncay Kume5, Ismail Sarı6, Zahide Cavdar3, Omer Binicier7, Ozgur Kadicesme1, Ozcan M.Ali4, Servet Akar6, and Mehmet Tunca7

1Dokuz Eylul University School of Medicine Department of Biochemistry, Inciraltı -35340 İzmir, Turkey ; 2National Institut of Standards and Technology, DNA Technologies Group, Gaithersburg, MD 20899-8311, USA; 3Dokuz Eylul University School of Medicine ARLAB Division, İnciraltı-35340 İzmir, Turkey ; 4Dokuz Eylul University School of Medicine Department of Hematology, İnciraltı-35340 İzmir, Turkey ; 5Dokuz Eylul University Central Laboratory, İnciraltı-35340 İzmir, Turkey ; 6Dokuz Eylul University School of Medicine , Department of Rheumatology, İnciraltı-35340 İzmir, Turkey ; 7Dokuz Eylul University School of Medicine Department of Internal Medicine, İnciraltı-35340 İzmir, Turkey

There is a massive influx of neutrophils into the serosal membranes during the acute phase of FMF. It is known that “oxidative burst” caused by persistently activated neutrophils may cause oxidative damage in cell macromolecules.
The first aim of this study is to investigate the level of oxidative stress and antioxidant capacity in FMF, which are thought to be important in the inflammatory pathway and to determine the effect of oxidative stress on neutrophil apoptosis. These parameters will also be analysed according to the genotypes of the patients.
The study group is composed of 60 adult patients (30 homozygotes- 30 heterozygotes), and 30 healthy controls. In these cases, neutrophil activation (myeloperoxidase, complement 5a); DNA base damage (8- hydroxy-deoxyguanin); lipid peroxidation (malondialdehyde, 8-isoprostane, hydroperoxides) and protein damage (total tiol groups, nitrotyrosine) will be investigated. The levels of antioxidant enzymes (superoxide dismutase, glutathione peroxidase) and antioxidant compounds (reduced and oxidized glutathione, vitamin E) will also be determined. Annexin-V, caspase-1 and caspase-8 activity measurements will be performed in order to assess the effect of oxidative damage on neutrophil apoptosis.
This study was activated on September 1, 2005 and we expect to retrieve most of the results by the end of October 2005 and the final deadline is January 2006.
The results of this study will enable us to understand if the cause of cellular damage in FMF is due to oxidative stress. We think that this information may explain the differences between the genotypes and the phenotypic expressions observed in clinical practice.

This study was supported by a grant (No. 104S336) from The Scientific and Technological Research Council of Turkey

  Comparison of Phenotypes of Wild-type and other Genotypes of the FMF Patients

Ismail  Sari1, Guldal  Kirkali2, and Mehmet Tunca3             

1Dokuz Eylul University School of Medicine Department of Rheumatology, Izmir Turkey; 2Dokuz Eylul University School of Medicine Department of Biochemistry, Izmir Turkey; 3Dokuz Eylul University School of Medicine Department of Internal Medicine, Izmir Turkey

OBJECTIVE
Despite intensive sequencing, a substantial number of FMF patients display none of the known mutations on the MEFV gene. The aim of this study was to investigate if there was any phenotypic difference between the wild-type and other genotypes.

PATIENTS AND METHODS
We studied the 212 patients who were previously genotyped. Exon 10 was fully sequenced in all cases and the mutation encoding pyrin E148Q was sought by gel electrophoresis of the 5 prime exon 2 amplicon after MvaI digestion. Following data were collected and analyzed: sex, age, disease duration, delay of diagnosis, genotype status, number of attacks per year, presence of fever, abdominal pain, pleuritic pain, arthritis, erysipelas-like erythema (ELE) and inflammatory back pain (IBP).

RESULTS
Among the 212 patients who were genotyped none of the searched mutations were detectable in 23 (10.85%) of them. There was no significant difference between patients with various mutations and wild genotype regarding the above-mentioned demographical and clinical parameters. We then compared this group with M694V homozygotes (n: 34) who are universally accepted as a “severe” phenotype, and M680I homozygous (n: 12) patients whose phenotypic expression is more controversial. Homozygous M694V cases had significantly more pronounced pleuritic pain (78.8 vs 52.2%), arthritis (82.4 vs 26.1%) and ELE (57.6 vs 9.5%) than wild-type FMF patients, while the latter displayed a phenotypic expression in parallel with the M680I homozygotes.

CONCLUSION
Wild type phenotype is essentially similar to patients in general. Since 10-15 percent of FMF patients fit in this category, genotyping for diagnostic purposes will only complicate the clinical decision making process.

  Comparison of Phenotypes and Genotypes of FMF Patients from Eastern Anatolia and Balkan Countries

Ismail Sari1, Guldal Kirkali2, and Mehmet Tunca3             

1Dokuz Eylul University School of Medicine Department of Rheumatology, Izmir Turkey; 2Dokuz Eylul University School of Medicine Department of Biochemistry, Izmir Turkey; 3Dokuz Eylul University School of Medicine Department of Internal Medicine, Izmir Turkey

OBJECTIVE
The prevalence of FMF is relatively lower in western regions of Turkey than Eastern Anatolia. Likewise, the disease is quite uncommon in Greece and other Balkan countries. It is also presumed that patients from Eastern Anatolia have more severe phenotypic expression. If this presumption is relevant, underdetection of milder cases would contribute to observed unequal distribution of FMF. We compared the clinical characteristics of our patients according to their ancestral origins.
PATIENTS AND METHOD
Among the 453 adult FMF patients registered in the computer files of our institute there were 113 cases whose ancestors were either strictly from Balkan countries and coastal regions of Aegean Sea (n: 27) or Eastern Anatolia (n: 86). Patients with mixed ancestors were not included. The following data were analyzed: geographical origin of the parents, sex, age, disease duration, delay of diagnosis, genotype status, number of attacks per year, presence of fever, abdominal pain, pleuritic pain, arthralgias, arthritis, myalgias, erysipelas-like erythema (ELE) and inflammatory back pain (IBP).
RESULTS
Comparisons of the clinical and demographical features of these groups did not reveal significant difference in any of the analyzed parameters. Further analysis for severity scoring was considered irrelevant. Genotype analyses were available in 63 patients. Four founder mutations (M694V, E148Q, V726A, M680I) were accounted for 88.9% of all mutations in these patients and the remaining 11.1% were wild type. Numbers of mutations were as follows: 46 for M694V, 9 for E148Q, 21 for V726A, 19 for M680I and 17 for wild type. The mutation frequencies were comparable between the groups, including the E148Q which is presumed to be more prevalent among the westerners.
CONCLUSION
The comparison of the Turkish FMF patients from Balkan countries and eastern Anatolia revealed no statistically significant difference regarding to their clinical and demographical features and genotypes. Probably phenotypic presentation is independent of ancestral origins of the patients.

  Serum Amyloid A Levels in the Patients with Familial Mediterranean Fever (Do they really predict the development of amyloidosis?)

Fatos Yalcinkaya1*, Nilgun Cakar2*, Banu Acar1, Haluk Guriz1, Ercan Tutar1, Selda Ozturk2 , Aydan Kansu1, Erdal Ince1, Atilla Elhan1, Derya Aysev1, Mesiha Ekim1, Semra Atalay1, Nurten Girgin1, and Ulker Dogru1 

1Ankara University School of Medicine; 2Ankara Diskapi Children's Hospital ; *These authors contributed equally to this work.

In order to determine the role of levels of acute phase proteins (APPs) for the development of amyloidosis in FMF patients, the levels of serum amyloid A (SAA), C reactive protein (CRP), fibrinogen and erythrocyte sedimentation rate (ESR) were measured in paired sera of 36 FMF patients during and in between acute attacks, 39 of their healthy parents (obligate heterozgotes), and 15 patients with FMF associated amyloidosis. To compare the levels of APPs, 39 patients with chronic infections or inflammatory diseases who may develop secondary amyloidosis, 20 patients with acute infections who are known to have elevated APR but will never develop amyloidosis and 19 healthy controls were included. The median levels of all APPs are increased in the patients with FMF during attacks and a significant decrease was observed after the attack was over. The level of SAA was above reference range in all FMF patients during the attack free period and the level of at least one other APP was also above normal in 64% of the patients. Both CRP and SAA levels were found to be higher in obligate heterozygotes compared to controls. The levels of SAA in patients with FMF during the attack-free period, obligate heterozygotes and patients with FMF-amyloidosis were found to be similar. The levels in each group were found to be higher than SAA levels found in healthy controls yet lower than the levels measured in the patients with acute infections and patients with chronic inflammation or chronic infections. In conclusion, our results show that SAA level reflects subclinical inflammation with high sensitivity but its value for the prediction of amyloid formation process seems to be low.


  The Possible Effect of Subclicical Inflammation on Daily Life in Familial Mediterranean Fever

Birsin Ozcakar , Fatos Yalcinkaya, Selcuk Yuksel, Banu Acar, Derya Gokmen, and Mesiha Ekim          

1Ankara University School of Medicine, Ankara TURKEY

Familial Mediterranean fever (FMF) is an autosomal recessive disease, characterized by recurrent, self limited attacks of fever with serositis involving the peritoneum, pleura and joints. Acute phase reactants (APR) are generally elevated during the attacks of FMF and return to normal with clinical remission. Recently it was shown that in some patients APR could remain high during the intervals between the attacks. This has led to the suggestion that subclinical inflammation continue during the attack-free periods but the influence of subclinical inflammation on daily life has not been investigated previously. This study was performed to investigate the attack free complaints of patients with familial Mediterranean fever (FMF), the impact of colchicine on these symptoms and on subclinical inflammation.
A questionnaire that includes information about the disease course and symptoms during the attack free period was applied to the parents of 50 FMF patients. For the evaluation of attack free period four items were asked about daily activities of children -weakness, lack of appetite, sleep problems and decreased activity. The respondants have rated the items and the total score was taken as the sum of all the specific items. The laboratory values were noted from the patients’ files. During the attack free period patients with mild disease had higher total scores, higher weakness and decreased activity scores than patients with moderate disease. When we compared the daily activity scores before and after colchicine therapy, statistically significant increase was observed in the total scores and in all of the specific item scores. Also significant decrease was seen in the ESR and white blood cell counts, a significant increase was seen in the hemoglobin levels during attack free period after colchicines usage. So regression of inflammation together with the improvement in daily activities was observed.
Familial Mediterranean fever patients seem to have complaints during the attack free period that may be related with subclinical inflammation. Moreover, colchicine besides preventing the FMF attacks and the dangerous complication of amyloidosis, also seem to hinder the symptoms of attack free period in children with FMF.

  Prevalence of MEFV Mutations in Children with PAN not-Associated with FMF

Fatoş Yalçınkaya1, Z.Birsin Özçakar1, Ayşenur Öztürk2, Özgür Kasapçopur3, Nejat Akar2, Engin Yılmaz4 , Ayşin Bakkaloğlu5, Nil Arısoy3, Seza Özen5, and Mesiha Ekim1     

1Pediatric Nephrology, Ankara University School of Medicine, Ankara, Turkey 06100; 2Pediatric Genetics, Ankara University School of Medicine, Ankara, Turkey 06100; 3Pediatric Nephrology and Rheumatology, İstanbul University Cerrahpaşa Medical School, İstanbul, Turkey; 4Pediatric Genetics, Hacettepe University Medical School, Ankara, Turkey; 5Pediatric Nephrology and Rheumatology, Hacettepe University Medical School, Ankara, Turkey

Objectives: Polyarteritis nodosa (PAN) is one of the most important but rare vasculitis of childhood. The pathogenesis is unknown and the role of both genetic and environmental factors are suggested. Based on the fact that increased frequency of PAN in the patients with FMF we assessed the prevalence and significance of MEFV mutations in children with PAN who do not have FMF.
Study Design: A questionnaire was used for the present survey. The setting was 3 referral centers from Turkey. Eleven boys and 10 girls who were diagnosed as PAN were interviewed and asked to denote blood. The diagnosis of PAN was established according to the Chapel Hill consensus criteria for microscopic polyarteritis or classic PAN. Mutations (M694V, M680I, V726A, E148Q K695R) in the MEFV gene were studied.
Results: The age at diagnosis of PAN ranged from 2.5 to 16 years. All patients had constitutional symptoms, elevated acute phase reactants, and myalgia at the time PAN was diagnosed. The diagnosis of PAN was confirmed by angiography in 5 patients and biopsy in 13 patients. Ten MEFV mutations were identified in 42 chromosomes. Gene frequency was 1/ 4.2 and that was approximately 7.5 times higher than the estimated gene frequency of 1/32.7 in Turkey. Six out of 21 patients were found heterozygous for one of the sequenced MEFV mutations; M694V in two patients, K695R, V726A, M680I, E148Q each in one patient. Two patients had homozygous M694V mutation.
Conclusion: The presence of MEFV mutations might play a role in the development of childhood PAN by causing an augmented inflammatory response and asymptomatic FMF cases who bear two MEFV mutations might be present among the children with PAN.


  Interferon Alpha (IFN) for the treatment of FMF attacks

Nurit Tweezer-Zaks1, Einal Rabinovitz2, and Avi Livneh3             

1Heller Institute for Medical Research, Sheba Medical Center, Tel-Hashomer, Israel 52621

Introduction and Aim
While colchicine is considered the gold standard therapy for FMF, some 20% of all patients only partially respond to this treatment and continue to suffer from frequent and severe FMF attacks, leading to suffering, disability and absence from work or school. Currently there is no effective medication for suppressing these attacks. Studies on the use of IFN α in acute FMF attacks demonstrated conflicting data with regards to the efficacy of this medication. The aim of this study was to determine the effectiveness of IFN α in colchicine resistant FMF patients.
Materials and Methods
We conducted a prospective, open label, self controlled, observational study on the effect of IFN α. Three MIU injections were given early in FMF attacks in a cohort of 10 colchicine non responsive FMF patients. The patients were asked to write reports on IFN treated, or IFN – untreated attacks, describing the severity (estimated by visual analogue scale) and the length of the attacks. These data were used to evaluate the efficacy of the drug.
Results
Most patients had severe, multi-focal disease, with frequent attacks despite treatment with 2-3mg of colchicine daily. Most individuals were homozygous to the M694V mutation of MEFV. A total of 58 IFN α injections were given. While 83% of patients reported an over 20% improvement in pain severity, only 17% enjoyed a dramatic, higher than 70% improvement of pain. The time to attack resolution with IFN α injection showed a greater than 70% improvement in more than 70% of the attacks. Side effects from the injection were common, usually tolerable and included malaise, fever, headaches, nausea, myalgias, chills, nervousness, apathy and dizziness.
Conclusions
IFN α is a moderately effective, affordable (with regard to side effects) treatment for use in severely affected FMF patients who are non responsive to colchicine.

  Cryptogenic cirrhosis in a cohort of patients with familial Mediterranean fever

Nurit Tweezer-Zaks , Anat Doron-Libner, Shomron  Ben-Horin, Perez Weiss, and Avi Livneh           

1Heller Institute of Medical Research and Department of Gastroenterology, Sheba Medical Center, Tel Hashomer 52621 Israel

Background and aim
Even though the association between Familial Mediterranean fever (FMF) and chronic liver disorders is not well characterized, hepatomegaly, splenomegaly and transient elevations of liver enzymes are often encountered in FMF patients. The aim of this study is to describe FMF patients with cryptogenic cirrhosis and discuss the possible relationship between the two entities.
Methods
Using a hospital computerized search we crossed between the terms FMF and terms related to liver abnormalities (elevated liver enzymes, hepatomegaly, splenomegaly, ascites, hepatitis, cirrhosis, hepatic failure and gastrointestinal bleeding). The clinical, histological and laboratory parameters of the patients identified were retrieved from the hospital's out and in patient charts and from the primary care physician. The diagnosis of cirrhosis was defined by typical histology or clinical, imaging and laboratory findings, strongly suggestive of cirrhosis. The diagnosis of cryptogenic cirrhosis was determined by excluding other etiologies for the disease. Patients with known systemic AA amyloidosis or positive Congo red staining of liver biopsies were excluded.
Results
In our registry of 6000 patients at the time of the study, 84 FMF patients with findings suggestive of liver disease were found. Of these, 9 patients had cryptogenic cirrhosis. The remaining had liver disease due to other etiologies (69 patients), or lack of solid evidence for chronic liver disease. A rate of 9/6000 (0.15%) is 10-20 fold greater than expected in the Israeli population, even in the most stringent calculations. Most patients with cryptogenic cirrhosis had moderate to severe FMF, associated with attacks in 2-4 sites. The MEFV genotype was M694V/M694V in 5/7 patients. Colchicine dose was less than 1.5mg per day in 7/9 patients. The mean duration of FMF at the diagnosis of liver disease was 23 years. The mean duration of liver disease at the study was 6 years. At that time, all but one patient had upper GI bleeding and the Child-Pugh score was suggestive of a mild disease.
Conclusions
Cryptogenic cirrhosis is relatively common in FMF. Whether this finding is due to chronic inflammation or treatment with colchicine remains to be determined.

  Erysipelas like Erythema as the first presentation of Familial Mediterranean Fever.

Anat Libner-Doron, Nurit Tweezer-Zaks, Aviv Barzilai, Avi Livneh, and Pnina Langevitz           

1Heller Institute for Medical Research and Dermatology , Sheba Medical Center, Tel-Hashomer, Israel 52621

Introduction
Erysipelas like erythema (ELE) is an erysipelas resembling rash. Lesions are red, warm, tender, raised and well demarcated, pathognomonic for FMF, and reported in 2%-20% of patients. This rash is predominantly distributed over the lower extremities, often accompanied by fever, and resolves within 24-72 hours. Histological examination reveals edema of the superficial dermis and sparse perivascular infiltrate composed of few neutrophils, lymphocytes and nuclear dust. The aim of the study was to further characterize ELE in FMF, in a group of patients with ELE as the first presenting sign of FMF.
Methods
Disease characteristics of patients with ELE as the first presenting sign (study group) were compared to two control groups: patients with FMF and ELE not at first presentation (control group I), and FMF patients without ELE in their disease course (control group II).
Results
ELE as the first presentation of FMF is rare and was detected in 0.13% of our patients. The study group was characterized by delayed diagnosis of FMF as compared to the control group I (p= 0.001), higher frequency of the M694V mutation, and increased disease severity greater than control group II (p=0.008). a wide range of age and heterogeneous ethnic origin distribution were noted in the study group.
Conclusion
ELE as the first manifestation of FMF is rare, and is associated with delayed diagnosis and a more severe course.


  A Novel, Iranian Jewish Mutation in the B30.2 Domain of the FMF Gene: Clinical and Conformational Significance.

Yael Shinar1, Sophia Menasherow1, Irena Kuchuk1, Mirit Kolet1, Pnina Langevitz1, Elon Pras2 , and Avi Livneh1        

1Heller Institute for Medical Research, Sheba Medical Center, Tel-Hashomer, Israel 52621; 2Institute of Human Genetics, Sheba Medical Center, Tel-Hashomer, Israel 52621

Background: Unlike Iraqi Jews, FMF in Iranian Jews is rare (IJ, 1:5000), and its clinical expression and underlying MEFV mutations have not been studied.
Aim: To screen the B30.2 domain of MEFV for rare or new IJ mutations, to evaluate their clinical significance and to predict their effect on the secondary structure of the B30.2 domain of pyrin.
Methods: FMF patients with two (N=10) or one PJ parent (N=10) were interviewed and examined for their manifestations. Exon 10 of MEFV encoding for the B30.2 domain of pyrin was sequenced in 6 patients. The entire coding region was sequenced in three patients. A new mutation was identified and screened for by restriction site analysis in the remaining patients and in 52 unaffected IJ and 44 non-Jewish Baha'i Iranians. The secondary structure of B30.2 domain variants was predicted using the Structure Prediction Meta Server (Bioinfo.PL).
Results: One novel mutation, G632S, was found in five IJ male patients from three unrelated families. Two were compound heterozygous (G632S/V726A and G632S/E148Q) and 3 heterozygous. Compared to the remaining PJ FMF patients (N=15) the manifestations of these patients were atypical in that only one patient suffered from peritonitis and two had only febrile episodes. The disease severity ranked mild. The G632S mutation was found only once (in a female) in 104 alleles of unaffected IJ and was not found in 88 alleles of non-Jewish Baha'i Iranians.
The predicted secondary structure of wild type B30.2 was modified by the G632S mutation (using SAM-T02-DSSP, performing with over 80% accuracy) as by the M694V and V726A mutations.
Conclusion: clinical, genetic and structural considerations suggest that the G632S mutation is a true, rare PJ mutation causing mild, male biased FMF.

  Idiopathic Recurrent Acute Pericarditis: Lack of Familial Mediterranean Fever Mutations

Yael Shinar1, Antonio Brucato2, Lilach Ashtamker1, Livia  Robbiolo2, LiGiovanni  Brambilla2, and Avi Livneh1          

1Heller Institute for Medical Research, Sheba Medical Center, Tel-Hashomer, Israel 52621; 2Department of Medicine, Niguarda Hospital Milano, Italy

Background: Idiopathic recurrent acute pericarditis (IRAP) is thought to be an autoimmune disease. However, based on the good response of IRAP to colchicine and several reports of recurrent pericarditis in patients with familial Mediterranean fever (FMF) it has been suggested that FMF may be the underlying disease in at least some patients with IRAP. In Italy, FMF is not a rare disease and it was found to associate with a substantial number of known mutations in the Mediterranean fever gene.
Aim: To screen Italian patients suffering from IRAP for mutations in the Mediterranean fever gene, MEFV.
Methods: Stringent criteria were used for the diagnosis of idiopathic recurrent pericarditis, All 23 patients included in this study were Italians not from the Armenian, Arab, Jewish or Turkish ancestry. Their DNA samples were PCR amplified using specific primers for exon 10 of the FMF gene and the amplified products were sequenced. In addition, the E148Q mutation of exon 2 was checked by restriction site analysis.
Results: Mutations linked to FMF were absent in exon 10 of all our 23 Italian IRAP patients, and there were no novel mutations. The E148Q mutation was not present either.
Conclusions: This study suggests that MEFV mutations are not a likely cause of IRAP in a substantial group of patients and that other, autoimmune based pathogenic mechanisms should be considered.

  Genetic Investigations of FMF in Armenia

Tamara F. Sarkisian*, Hasmik S. Ajrapetyan*, and Gohar R. Shahsuvaryan*             

1Center of Medical genetics of NAS of Armenia, Yerevan, Armenia, 375010 ; *These authors contributed equally to this work.

The activities of Center for Medical Genetics of NAS of Armenia are focused on genetic and clinical investigations with the main goal of determining the frequency and structure of the hereditary pathology in Armenian population.

Familial Mediterranean Fever (FMF, MIM 249100), or Periodic Disease, is a condition prevalent in populations with Mediterranean decent. FMF is a recessively inherited and ethnically restricted condition which has been notoriously difficult to diagnose until 1997 when the mutations in the MEFV gene were determined, and they turned out to be tremendous help for diagnosis of complicated cases. The prevalence of FMF in Armenians and some other Mediterranean populations is as high as 1 in 5 individuals. Genetic testing is helpful for carrier screening and pregnancy planning since certain mutations have been shown to have significant correlation with amyloidosis, the worst possible manifestation of MEFV mutations. We have demonstrated the correlation between spectrum of MEFV mutations and clinical severity of the disease, including development of renal amyloidosis. The screening of 12 mutations in MEFV gene in more than 5000 patients with a clinical diagnosis of FMF (according to established criteria) indicated that almost 10% of patients have no mutated alleles. We didn’t detect MEFV mutations in 444 individuals. According to the Tel Hashomer criteria, among these 98 cases characterized as “probable FMF” with recurrent febrile attacks, These results revealed that there may exist FMF-like syndromes without MEFV mutations. We were also able to demonstrate the pedigree analysis of more than 1250 family cases and suggest that parent-to-offspring inheritance of mutations was autosomal recessive in 91.5% and pseudo-dominant in 8.49%.

Colchicine largely prevents the development of renal amyloidosis in FMF; however in a few cases the effect of colchicine remains controversial. The homozygous genotype M694V/M694V (exon 10) is associated with a severe phenotype of FMF. We demonstrated that genotype may assist predicting the response to colchicines treatment given to children with renal amyloidosis. FMF patients homozygous for M694V mutation not only present a more severe phenotype but also show a limited response to colchicine at the nephrotic stage of renal amyloidosis. In contrast, FMF patients with other genotypes still have a good chance to enter remission of the nephrotic syndrome and to maintain renal function.

In conclusion, our investigation on more than 5000 individuals since 1997, confirms the informativity of screening the MEFV mutations for prevention, treatment and prognosis of the development of FMF complications.

  Approach to Genetic Analysis in Diagnosis of Hereditary Autoinflammatory Syndromes

Anna Simon1, Jos W. van der Meer1, Richard Vesely2, Urban Myrdal3, Kayoko Yoshimura4, Pieter Duys5 , and Joost P. Drenth6        

1Department of General Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands; 2Department of Pediatrics, Faculty Hospital, Kosice, Slovakia; 3Pediatric and Adolescent Clinic, Central Hospital Vastmanland, Vasteras, Sweden; 4Department of Pediatrics, Tosa Municipal Hospital, Tosa City, Japan; 5Department of Pediatrics, Gemini Hospital, Den Helder, the Netherlands; 6Department of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands

Hereditary autoinflammatory syndromes are characterized by recurrent episodes of fever and inflammation. Several subtypes have been described, caused by mutations in at least 4 different genes. Apart from a common phenotype of lifelong recurrent inflammatory attacks, all of them have distinct features and specific therapeutic options, which emphasizes the need for a specific diagnosis in each case.
Objectives: to examine whether genetic screening would allow classification of previously unclassified patients, and whether individual patients suffering from an autoinflammatory syndrome carry additional mutations in one of the other autoinflammatory genes.
Methods: We included 60 patients with an unclassified autoinflammatory syndrome, 87 patients diagnosed with either hyper-IgD syndrome (HIDS), familial Mediterranean fever (FMF) or TNF-receptor associated periodic syndrome (TRAPS), and 50 healthy controls. DNA samples were screened for the most prevalent mutations in the MEFV, TNFRSF1A, MVK and CIAS1 genes. Results: This yields only one possible diagnosis of FMF in the 60 previously unclassified patients. Two low-penetrance mutations were found in equal numbers in the groups of patients and controls.
Conclusions: screening of highly prevalent mutations in known genes involved in these disorders does not yield additional relevant information. Differential diagnosis of hereditary autoinflammatory syndromes can be made by thorough clinical examination followed by targeted genetic analysis of one or two most likely syndromes. High prevalence, low penetrant mutations from autoinflammatory genes do not occur more frequently in hereditary autoinflammatory patients compared to the general population.
  Immunoglobulin D and A1 O-glycosylation in HIDS – a Pilot Study

Jacob F. de Wolff1, Alice C. Smith2, Anna  Simon3, Jos W. van der Meer3, and Jonathan Barratt2           

1Department of Medicine, Chase Farm Hospital, Enfield, Middx, United Kingdom ; 2John Walls Renal Unit, Leicester General Hospital, Leicester, United Kingdom; 3Department of General Internal Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands

A high concentration of immunoglobulin D (IgD) is a characteristic feature of the hyper-IgD and periodic fever syndrome (HIDS). It is often accompanied by a raised concentration of IgA, mainly IgA1. The cause of this high IgD level is still unclear. IgD and IgA1 are the only immunoglobulin subclasses characterised by a proline-rich hinge region and O-glycosylation of serine and threonine residues in this region. Two disease entities, IgA nephropathy and possibly Henoch-Schönlein Purpura, have been shown to be associated with increased levels of IgA1 caused by a deficiency of O-sialylation of IgA1.
Aim: to investigate whether a reduction of IgD O-glycosylation could explain its high serum concentration in HIDS patients by studying the O-glycosylation status of IgD and IgA1.
Methods: in a pilot study, serum samples of 5 HIDS patients with known mevalonate kinase mutations were compared with control samples. Serum IgA1 and IgD levels were measured by ELISA, and sera were diluted as needed. O-glycosylation status was studied by lectin binding assays using Vicia villosa (VV), Helix aspersa (HA) and peanut agglutinin (PNA).
Results and discussion: Compared to IgA1, native IgD has much higher PNA binding, but virtually no VV or HA binding; all binding is increased by desialylation. This may indicate that IgD is more heavily galactosylated than IgA1. As expected, the HIDS samples all had very high serum IgD levels compared to controls, and modestly raised serum IgA1. The five HIDS samples tested all fall within the control range for binding of all three lectins to native and sialyl IgA1 and IgD. However, mean binding of PNA to native IgA1 and IgD tends to be higher in HIDS, although this small pilot study is not conclusive (0.418 A492 PNA/A492 anti-IgD vs. 0.29, p=0.057 and 0.136 A492 PNA/A492 anti-IgA1 vs. 0.104, p=0.051). This might indicate low sialylation, either as a consequence or a primary cause of the high concentrations. More samples will be measured to further investigate this.
  Characterisation of the Genes Associated with Hereditary Periodic Fevers Syndromes in Patients with AA Amyloidosis of Undetermined Aetiology

Helen J. Lachmann1, Alison Bybee1, Dorota M. Rowczenio1, Hugh J. Goodman1, and Philip N. Hawkins1           

1National Amyloidosis Centre, Royal Free and University College Medical School, London, UK

AA amyloidosis is a frequent and serious complication of inherited periodic fever syndromes (IPFS). Among a series of 370 consecutive patients referred to our centre with AA amyloid, 33 (9%) had a recognised IPFS, apparently over-representing this rare population as compared with the 64% of cases who had inflammatory arthritis underlying their amyloidosis. The nature of the chronic inflammatory disorder was not evident at diagnosis of AA amyloidosis in a further 22 patients, in whom we speculatively performed limited screening of the four recognised IPFS genes MEFV, TNFRSF1A, NALP3, and MVK.

In total, 9 patients (41%) were identified to have mutations in their IPFS genes. Two men were MEFV compound heterozygotes, one who presented at age 70 years with F479L/V726A associated with apparent phenotype II FMF; the other had M694V/E148Q and failed to respond to colchicine, and a Sephardi woman was heterozygous for M694V and also did not respond to colchicine. Two men had NALP3 variants, T350M and A439V, and mild symptoms compatible with this in retrospect; a further patient had the A349V variant plus the known polymorphism Q703K, and the latter was also detected as a solitary finding in another asymptomatic patient. Two young men were MVK compound heterozygotes in whom the significance of symptoms compatible with HIDS had not been recognised. A British woman had the TNFRSF1A variant R92P; another woman had the TRAPS polymorphism P46L plus MEFV E148Q, and one of the 2 HIDS patients also carried TNFRSF1A R92Q.

Apart from the 2 patients with HIDS, in whom this rare disorder simply has not been considered, clinical features in the other 7 cases were either very mild and non-specific, and/or atypical for the respective IPFS. Four patients died before the possibility of an IPFS had been established, 2 patients with MEFV variants did not respond to colchicine, and very interestingly the woman with R92P responded completely to IL-1 blockade.

Mutations in the genes responsible for IPFS were common in this series of patients with AA amyloidosis of undetermined aetiology, although their significance remains unclear in many cases. These findings suggest that the phenotypic spectrum of these particular IPFSs may be wider than is presently recognised, and/or that these allelic variants may be modifiers of other inflammatory disorders.

  An Orally-Active ICE/Caspase-1 Inhibitor, VX-765, Reduces Inflammatory Biomarkers and Symptoms in Patients with Muckle-Wells Syndrome

Helen J. Lachmann1, Philip N. Hawkins1, Ramon Mohanlal2, Molly  Opferman 2, Cheryl  Eaton2, and John  C. Randle2          

1National Amyloidosis Centre, Royal Free and University College Medical School, London, UK ; 2Vertex Pharmaceuticals, Inc., Cambridge MA, USA

Interleukin-1beta (IL-1beta) is a key inflammatory mediator in Muckle-Wells Syndrome (MWS), as highlighted by the clinical response to treatment with IL-1 receptor antagonist (IL-1Ra) and IL-1trap. IL-1beta activation requires cleavage by the IL converting enzyme (ICE, caspase-1) and CIAS1/NALP3/PYPAF1, the mutated gene associated with MWS, encodes an accessory protein, cryopyrin, involved in the regulation of ICE. Therefore, inhibition of ICE may be a promising therapeutic approach in MWS. VX-765 is an orally active ICE inhibitor in clinical development as an anti-inflammatory agent. We conducted an open-label study of VX-765 in patients with MWS to obtain a preliminary assessment of the safety and tolerability, pharmacodynamics and clinical response.
Six MWS patients (3M/3F) were enrolled in the study. The subjects were all naïve to anti-IL-1 therapies. VX-765 (900 mg) was administered orally three (4 subjects) or four (2 subjects) times daily for 14 days. Assessments included safety and tolerability, and serum inflammatory markers (IL-18, IL-6, SAA, CRP). The subjects reported symptoms (fever/chills, rash, joint/muscle pain, eye discomfort, fatigue) and the investigator noted disease signs (rash, joint swelling/tenderness, conjunctivitis) on a 5-point severity scale.
VX-765 was well tolerated with only mild-moderate adverse events reported by 3/6 subjects. For each subject, VX-765 was confirmed to inhibit the release of IL-1beta from whole blood stimulated with LPS, but possibly less potently than in healthy volunteers (HV) or FCAS patients (studied separately). Baseline serum IL-18 was only modestly elevated relative to healthy volunteers and was reduced by >50% by VX-765 treatment. IL-6 levels were also generally elevated and were reduced by approximately 40% during VX-765 treatment. SAA and CRP levels were elevated in all subjects and decreased, on average, by approximately 70% and 60%, respectively. Four subjects reported moderate-severe baseline symptoms and a 1.5-2.5 point reduction in symptom scores, while two subjects had minimal-mild baseline symptoms and reported more modest improvements. Improvement was greatest for fever/chills and joint/muscle pain. The Physician’s Assessment was also improved during VX-765 treatment. All assessments rapidly returned to pre-treatment levels when VX-765 was discontinued.
We conclude that VX-765 reduced serum inflammatory markers and appeared to improve signs and symptoms in patients with MWS. Nevertheless, the anti-inflammatory effects of VX-765 were incomplete. It is unclear whether this reflects a limitation of the ICE/caspase-1 inhibitory mechanisms of action in MWS patients, or if more complete disease control might be achieved with an optimized VX-765 dosing regimen.

  Genetic Screening of Patients Presenting with Clinical Features Suggesting the Possibility of Muckle-Wells (MWS) or CINCA/NOMID

Alison  Bybee1, Ebun Aganna2, Helen J. Lachmann1, Belinda  Nedjai2, Michael F. McDermott3, and Philip N. Hawkins1          

1National Amyloidosis Centre, Royal Free and University College Medical School, London, UK; 2Barts and The London, Queen Mary's School of Medicine and Dentistry, University of London, UK; 3Academic Unit of Musculoskeletal Disease, St. James's University Hospital, University of Leeds, UK

Patients with a variety of clinical features raising the possibility of either Muckle-Wells (MWS) or CINCA/NOMID syndromes were screened for NALP3/CIAS1 variants. Genomic DNA from 102 unrelated patients referred for genetic testing was screened for mutations in exon 3 of NALP3 by PCR and automated DNA sequencing. At least one other hereditary periodic fever syndrome (HPFS) gene (MEFV, TNFRSF1A and/or MVK) was also screened in 78% of cases by the same methods.

Heterozygous missense substitutions in NALP3 were found in 10 of the 102 patients (10%). Three novel substitution mutations were identified: g.944C>T (p.P315L), g.1051G>A (p.V351M), and g.1709A>T (p.Y572F). Another 7 patients (from 6 families) had the previously reported variants V198M, D303N, T436I, A439V, T350M (2 siblings) and G569R. All 3 patients with novel mutations were referred as possible cases of CINCA, as was the patient with D303N. The G569R variant was associated with an overlapping CINCA/MWS phenotype associated with progressive CNS involvement; the remaining variants were associated with a phenotype consistent with MWS, including urticarial rash in all but one, and deafness was present in 50%, but without overt CNS involvement or skeletal abnormalities. The patient with V198M had severe knee arthropathy due to epiphyseal overgrowth, in addition to substantial acute phase activity but no other recognised clinical features of MWS or CINCA; interestingly, this patient also carried the low-penetrance MEFV variant E148Q. A total of 13 patients without mutations in NALP3 were found to have allelic variants, often of questionable significance, in MEFV, TNFRSF1A or MVK. However, the majority (~80%) of patients referred for HPFS testing, including several referred specifically for possible CINCA or MWS, had no amino acid variation in any of the candidate genes tested, supporting phenotypic and/or genotypic heterogeneity.

  AA Amyloidosis in Two Patients with Previously Undiagnosed HIDS

Helen  J. Lachmann1, Hugh  J. Goodman1, Alison  Bybee1, Dorota  M. Rowczenio1, and Philip  N. Hawkins1           

1National Amyloidosis Centre, Royal Free and University College Medical School, London, UK

In contrast to certain other periodic fever syndromes, the risk of AA amyloidosis seems to be extremely low in patients with the hyperimmunoglobulin D with periodic fever syndrome (HIDS), and only one case has been reported. We describe here two further patients with HIDS complicated by AA amyloidosis.

The first patient is a British male who presented aged 19 years with end stage renal failure, and whose kidney biopsy demonstrated AA amyloidosis. A history was obtained of periodic fevers with an onset in his first year of life and which occurred every 6-8 weeks lasting for 7-14 days. Fevers were accompanied by cervical and axillary lymphadenopathy, nausea, vomiting and diarrhoea, apthous ulcers, anorexia and transient arthralgia. Immunisations precipitated florid attacks. His serum IgD concentration was elevated at 337mg/L, and he had an older brother with similar symptoms but so far without amyloidosis. DNA sequencing demonstrated that both siblings were MVK compound heterozygotes V377I/I268T. Sequencing of MEFV and TNFRSF1A revealed no mutations. His autoinflammatory disease remained very active whilst he was on dialysis (median SAA 37mg/L), and did not respond convincingly to treatment with anakinra. He received a live related renal transplant in August 2005 and his early post-operative course has been without complication.

The second patient is a German man who presented at the age of 22 with nephrotic syndrome due to AA amyloidosis. His renal function declined and he commenced dialysis within 3 years. He gave a history of periodic febrile attacks from infancy, occurring every 6-8 weeks which were more marked in winter. Accompanying symptoms characteristically included headache, cervical and inguinal lymphadenopathy, diarrhoea and vomiting. He was found to have IgG2/4 subclass deficiencies at the age of 9 years and his attacks had been thought to represent recurrent infections, but he had received intravenous immunoglobulin supplementation without benefit. His serum IgD concentration was elevated at 104mg/L. DNA sequencing demonstrated not only MVK V337I/L234P but also the TNFRSF1A variant R92Q. He improved clinically with etancercept therapy, following which his median SAA concentration has been 8mg/L. A live related renal transplant is planned.

The median duration of inflammatory disease preceding the development of AA amyloidosis generally is 17 years, possibly contributing the low incidence of amyloidosis in HIDS which typically ameliorates after adolescence. AA amyloidosis nevertheless evidently can occur, and the course of our patients’ disease following renal transplantation remains to be determined.

  Clinical Significance of Pyrin I591T

Dorota M. Rowczenio1, Helen J. Lachmann1, Alison Bybee1, Hugh J. Goodman1, Frank Bridoux2, and Philip N. Hawkins          

1National Amyloidosis Centre, Royal Free and University College Medical School, London, UK; 2Department of Nephrology, University Hospital, Poitiers, France

The role of pyrin I591T in the pathogenesis of FMF and/or atypical inflammatory disease is unclear. It has been reported as an apparent low penetrance FMF-causing variant in a Spanish family, and we identified it as the solitary genetic finding in a French woman with a late onset atypical chronic febrile disorder characterized by severe anaemia and attacks of headache and drenching night sweats lasting for 24 hours, recurring regularly every four days, but which remitted completely following introduction of colchicine.

We report here the features of four further patients who had the MEFV exon 9 mutation encoding pyrin I591T. In two cases, one of whom was French and one of mixed Turkish-Cypriot/British ancestry, I591T was the only mutation identified following analysis of exons 1, 2, 3, 5, 9 and 10 of MEFV. The two further patients, of Maltese/Irish and Sephardic origin respectively, were both compound heterozygotes for I591T/M694V. Both of the compound heterozygotes and the Turkish-Cypriot/British simple I591T heterozygote had classical FMF including a complete response to prophylactic colchicine therapy.

The clinical features of the remaining French heterozygote were very unusual, in that she present in her fifth decade with renal AA amyloidosis on the background of a longstanding but very non-specific chronic inflammatory disorder. Comprehensive sequencing of MEFV, NALP3, TNFRSF1A and MVK failed to identify any other mutations and she has responded to colchicine.

These findings suggest that pyrin I591T may contribute to the pathogenesis of classical FMF, and either cause or contribute to serious atypical inflammatory processes in some individuals.

  Defective Apoptosis of Peripheral Blood Lymphocytes in Hyper-IgD and Periodic Fever Syndrome

Evelien J. Bodar1, Jeroen C. van der Hilst1, Waander van Heerde2, Jos W. van der Meer1, Joost P. Drenth3, and Anna Simon1          

1Department of General Internal Medicine, Division of Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands; 2Department of Haematology, Division of Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands; 3Department of Gastroenterology, Division of Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands

Introduction: Hereditary periodic fever syndromes are characterized by incapacitating attacks of fever and generalized inflammation. Three of these syndromes are the hyper-IgD syndrome (HIDS), caused by mutations in the mevalonate kinase gene, TNF-Receptor Associated Periodic Syndrome (TRAPS), caused by mutations in the type 1 TNF receptor gene and Familial Mediterranean Fever (FMF), caused by mutations in the MEFV gene encoding pyrine. Their pathogenesis remains unclear.
Aim of the study: To investigate apoptosis in periodic fever patients as a possible pathogenic factor.
Methods: We measured anisomycin-induced apoptosis with annexin V flowcytometry in peripheral blood lymphocytes from symptom-free patients with HIDS (n=7), TRAPS (n=7) and FMF (n=2).
Results and conclusion: HIDS lymphocytes showed a decreased percentage of apoptosis during remission compared to controls (12 vs 50%, p=0,0023), whereas no difference was observed in TRAPS or FMF lymphocytes. This decreased apoptosis of lymphocytes may be a central pathogenic mechanism in HIDS since dysfunction of one of the inhibitory mechanisms to curtail the immunological response to trivial insults could lead to the excessive generalized inflammation seen during a HIDS attack.

  phenotype/genotype correlation in 30 MAPS patients

severine guillaume1, valentine brousse1, laurence cuisset2, marie-odile rolland3, daniel rabier4, pierre quartier1 , and anne-marie prieur1        

1Pediatric rheumatology unit, Necker Hospital, Paris, France; 2Genetic department, Cochin Hospital, Paris, France; 3Biochemistry unit, Debrousse Hospital, Lyon, France; 4Biochemistry unit, Necker Hospital, Paris, France

Introduction
Mevalonate kinase Associated Periodic Syndromes (MAPS) have mainly been described in french and dutch populations. We here report on the french cohort of 30 MAPS patients in order to study phenotype/genotype correlation.
Results
There were 8 cases of mevalonate aciduria (MA) and 22 cases of hyper-IgD syndrome (HIDS) with male/female ratios being 5/3 and 8/14, respectively. Among MA patients, two kindreds (4 patients) originated from north african consanguineous parents. In MA, disease onset was earlier, syndromic crisis shorter and recurrences less frequent. There were 3 deaths in MA patients (pts) and 1 death in HIDS pts, the last proven to be unrelated to MAPS. Dysmorphic and neurological features, as well as anemia and failure to thrive were only reported on MA pts. Diarrhea was twice more frequent in MA than in HIDS pts, may be partly explaining growth defects. Other clinical features were equally distributed in MA and HIDS. Urinary mevalonate ranged 189 to 6000 and 3-60 umol/mmol creatinine in MA and HIDS, respectively. Mean mevalonate kinase activity was 0,3% in MA and 5,3% in HIDS. Homozygous DNA mutations were found in 5/8 MA pts but never in HIDS pts. The V377I mutation was found in 94% of HIDS pts who were always compound heterozygotes.
Conclusion
Phenotype of MA pts is very severe, correlating with high urinary excretion of mevalonate and dramatic decrease in mevalonate kinase activity, compared to HIDS pts. Genetics allows to find a high rate of homozygocy in MA pts, while double heterozygocy is constant in HIDS pts, mostly involving the mild V377I mutation. Thus phenotype/genotype correlation is strong in MAPS.
  In Vivo Effect of Anakinra Treatment in Two HIDS Patients.

L.M. Kuijk1, J. Frenkel1, A.M. van Furth2, E. Bodar3, M. Cuppen4, and W. Kuis1          

1University Medical Centre Utrecht, Utrecht, The Netherlands; 2Free University Medical Centre, Amsterdam, The Netherlands; 3University Medical Centre St. Radboud, Nijmegen, The Netherlands; 4Slingeland Hospital, Zutphen, The Netherlands

Purpose: To report the effect of anakinra (interleukin-1 receptor antagonist) in severe inflammatory disease due to mevalonate kinase deficiency.
Patients and methods: Two patients with intractable therapy resistant disease received anakinra 1-2 mg/kg/day subcutaneously. Disease activity was recorded on a standardized form.
Results: Patient 1, a fourteen year old boy, had been affected since the age of 6 months. Every 2-3 weeks, he had 3-6 days of fever, with rash, lymphadenopathy, headache, abdominal pain, vomiting, and diarrhoea, followed by another 4-8 days of monoarthritis. At the age of 12 years, anakinra, 1 mg/kg/day subcutaneously was started. He entered complete remission, until, after 7 months, he wished to discontinue anakinra. Two weeks later, he developed a full blown attack and a second followed in less than 2 weeks.
Patient 2, a 23 month old girl, had been ill since birth with 3-7 days of fever every 2-4 weeks, accompanied by chills, irritability, vomiting, abdominal discomfort, diarrhoea, rash, stomatitis, hepatosplenomegaly and lymphadenopathy. From the age of 10 months, a painful non-erosive polyarthritis and anaemia of chronic illness persisted between attacks. Her disease affected both growth and development. At the age of 23 months, anakinra, 1 mg/kg/day was started. Within 24 hours she became afebrile, but after 9 weeks intermittent inflammation resumed. Yet, the patient was generally much better than before treatment, with no active arthritis and steadily improving joint function. She displayed catch-up development of both gross motor function and speech. Her hemoglobin rose from 6.5 mg/dl to 10.2 mg/dl. Raising the dose of anakinra to 2 mg/kg led to a further subjective improvement, but inflammatory episodes continued to recur.
Conclusion: Anakinra is not uniformly effective in mevalonate kinase deficiency. It is, however, a promising treatment option for otherwise therapy resistant patients.

  Proteolytic Activation and Export of Interleukin-1beta and -18 Rise in Impaired Isoprenoid Biosynthesis.

L.M. Kuijk1*, S.H.L. Mandey2*, J. Frenkel1, H.R. Waterham2, and G.T. Rijkers1           

1Paediatric Immunology, University Medical Centre Utrecht, Utrecht, The Netherlands; 2Laboratory Genetic Metabolic diseases, Academic Medical Centre, Amsterdam, The Netherlands ; *These authors contributed equally to this work.

Objective: To study the mechanism of increased interleukin-1beta (IL-1beta) secretion in impaired isoprenoid biosynthesis.
Methods: We studied the effect of impaired isoprenoid biosynthesis on transcription, translation and secretion of (pro-)caspase-1 and (pro-)IL-1beta. This was done in LPS-stimulated THP-1 cells. Simvastatin was used to artificially impair isoprenoid synthesis. The isoprenyl intermediates farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) were used to restore the synthesis downstream from mevalonate kinase. We quantified caspase-1 and (pro-)IL-1beta mRNA and protein in cell extracts and culture supernatants.
Results: LPS-stimulated THP-1 cells secrete 25-fold higher amounts of mature IL-1beta (mIL-1beta) and 6-fold higher amounts of IL-18 in the presence of simvastatin. This effect was reversed by restoring downstream isoprenoid biosynthesis with GGPP or FPP. No changes were observed in caspase-1 mRNA levels. Although simvastatin treatment did induce an increase in pro-IL-1beta mRNA and the corresponding pro-IL-1beta protein, this effect was not reversed by the isoprenylpyrophosphates. Isoprenylpyrophosphates prevent maturation and export of IL-1beta, as reflected by an intracellular accumulation of pro-IL-1beta.
Conclusion: Isoprenylpyrophosphates reduce the amount of mIL-1beta secreted by LPS-stimulated THP-1 cells that are treated with simvastatin. This reduction is due to a decrease in both maturation and secretion of IL-1beta, not to a decrease in transcription or translation of either caspase-1 or IL-1beta. Maturation and secretion of mIL-1beta are linked processes, as pro-IL-1beta, not mIL-1beta, accumulates intracellularly in the presence of GGPP or FPP.

  Clinical features suggesting CINCA and FCU in a Swedish family

Stefan Berg, Anders Fasth, Monika Leissner, Christina Åhs, Alison Bybee1, and Philip N. Hawkins1          

1National Amyloidosis Centre, Department of Medicine, Royal Free and University College Medical School, London, NW3 2PF

We describe a 3 generation Swedish family, with a wide spectrum of features consistent with cryopyrin associated periodic syndrome (CAPS). The family includes a girl born in 2004, with clinical features of chronic, infantile, neurological, cutaneous and articular syndrome (CINCA). Her mother, 2 aunts and grandmother have clinical features consistent with familial cold urticaria (FCU). The affected child was noted to have symptoms at 6 months of age, comprising fever, a non-pruritic urticarial rash, chronic meningitis and an inflammatory arthropathy affecting the knees, elbows ankles, wrists and fingers. The knees and hands are severely affected. There has subsequently been slight growth retardation, and possibly impaired neurological development. Interestingly, she has improved with anti-TNF treatment (etanercept 5 mg/week), though treatment with anakinra remains under consideration. The mother developed symptoms at the age of 12 years, comprising a non-pruritic urticarial rash, arthralgia, myalgia and fatigue that follows cold exposure. The episodes each last for about 12 hours, and the attack frequency is variable, usually with just a few attacks per year. The other affected family members have symptoms that are similar to those of the mother. Mutational analysis of the NALP3/CIAS1 gene is underway, but results so far have been negative. This family is of interest in that the phenotypes of affected individuals appear to span the full spectrum of CAPS disease, and yet no mutation has yet been identified. Although a substantial proportion of children diagnosed to have CINCA do not have identifiable mutations, the reported literature is far less clear on the existence and nature of NALP3/CIAS1 mutation-negative FCU.
  Abnormal Neutrophil Chemotaxis in a patient with NOMID/MWS

Mary A. Lokuta1, Kate M. Cooper2, Ivona Aksentijevich3, Daniel L. Kastner3, and Anna Huttenlocher4           

1Department of Pediatrics, University of Wisconsin, Madison, WI; 2Cellular and Molecular Biology, University of Wisconsin, Madison, WI; 3Genetics and Genomics Branch, NIAMS, NIH, Bethesda, MD; 4Departments of Pharmacology and Pediatrics, University of Wisconsin, Madison, WI

NOMID/CINCA is an autoinflammatory disease characterized by urticarial rash, arthropathy, and central nervous system inflammation. We describe a 13-year-old girl with overlapping symptoms of both NOMID and Muckle-Wells syndrome (MWS) who has a mutation in cryopyrin (NALP3), a protein expressed in neutrophils. Neutrophil defects in chemotactic migration were found to a variety of chemoattractants including interleukin-8 (IL-8), f-Met-Leu-Phe (fMLP), C5a, and leukotriene B4. Her neutrophils exhibited elevated basal levels of ERK MAPK activation and a diminished induction upon stimulation with IL-8, fMLP, and C5a. This study is the first to demonstrate defects in neutrophil chemotaxis and MAPK signaling in neutrophils from a NOMID/MWS patient with a cryopyrin mutation.
  Phenotype and response to treatment of 23 patients with recurrent fever associated with mutations in CIAS1/NALP3

Kieron S. Leslie1, Helen J. Lachmann2, Ilze Bruning3, John A. McGrath3, Philip N. Hawkins2, Philip F. Roberts4 , and Clive E. Grattan5        

1Department of Dermatology, University of California, San Francisco, USA; 2Royal Free and University College Medical School, Royal Free Hospital, London,UK; 3Genetic Skin Disease Group, St John’s Institute of Dermatology, GKT, London, UK; 4Department of Pathology, Norfolk & Norwich University Hospital, Norwich, UK.; 5Department of Dermatology, Norfolk & Norwich University Hospital, Norwich, UK.

We characterized the phenotype of 23 patients from 14 unrelated families who had inflammatory-type symptoms in association with mutations in exon 3 of CIAS1/NALP3. The most common variants were T348M in seven individuals from five families; R260W in nine individuals from four families; V200M in four individuals from two families; A439V in two individuals from two families, and one individual had G571R. Twenty patients reported daily febrile symptoms with a circadian rhythm, in most cases worse during the evening; in four of them, symptoms were precipitated by exposure to cold. Eighteen of these patients had been symptomatic from birth, and two other patients, both with R260W, began to have symptoms during adolescence. Twenty-two patients had urticarial type skin rashes, and although 19 patients reported arthralgia only one had a deforming arthropathy; finger clubbing was present in four cases. Significant sensorineural deafness was present in 13 patients, affecting patients with all variants except A439V. Nine patients had headaches consistent with elevated intracranial pressure, seven of whom had chronic papilloedema. Six patients, five of whom had R260W, are known to be sub-fertile. Lab markers of inflammation were measured serially in 19 patients, and were substantially elevated in each case (median serum amyloid A protein (SAA) and C-reactive protein (CRP) 141 and 38mg/L respectively). Six patients had AA amyloidosis resulting in ESRF in three men, one of whom had a renal transplant. Fifteen patients have received anakinra therapy, all of whom achieved complete remissions (median SAA 2.8 mg/L, CRP 1mg/L).
In this series, there was marked variation in the severity of symptoms including patients with the same NALP3 variant, and substantial overlap of clinical features that have been considered characteristic in FCU, MWS and CINCA/NOMID. Notably, rash was completely absent in one patient and very minimal in others. Rapid and complete response to anakinra in all treated patients suggests that this criterion may contribute to the clinical diagnosis of variant NALP3 related inflammatory disease.

  "Initial Characterization of Transgenic Mutant hCIAS1 Mice"

James L. Mueller 2 3, Brian L. Niehaus1, David R. Greaves4, Ella Kothari2 , Felix Karp2, Nissi M. Varki2, and Hal M. Hoffman1 2 3    

Department of Pediatrics, University of California at San Diego, La Jolla, CA, USA, 92093; 2Department of Medicine, University of California at San Diego, La Jolla, CA, USA, 92093; 3Ludwig Institute of Cancer Research, University of California at San Diego, La Jolla, CA, USA, 92093; 4Sir William Dunn School of Pathology, University of Oxford, Oxford, UK, OX1 2JD

Mutations in human CIAS1 are responsible for a continuum of autoinflammatory disorders including familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal onset multisystem inflammatory disease. CIAS1 codes for the protein cryopyrin, which has been implicated in the regulation of inflammatory cytokines such as interleukin-1. There is in vitro evidence suggesting that mutations in CIAS1 lead to a gain of function for cryopyrin resulting in increased interleukin-1, but this has not been studied in vitro.
Transgenic mice were generated using wild type and L353P mutant hCIAS1 cDNA in a CD68 promoter vector. Expression levels in peripheral blood and tissues were measured by qPCR. Hematology indices were obtained and serum cytokine levels were measured by ELISA. Tissues were fixed with formalin and stained for histologic analysis.
The transgenic mutant mouse line with the highest expression of hCIAS1 by qPCR developed a persistent erythematous rash within the first month of life whereas mutant lines with lower expression and wild type hCIAS1 transgenic lines demonstrated no obvious phenotype. All of the transgenic mice were viable and had apparent normal reproductive function. Significant hCIAS1 RNA levels were detected in bone marrow and spleen, which is consistent with CD68 specific myeloid expression, but was also significantly elevated in the joints of mutant animals. A higher percentage of neutrophils were observed on peripheral blood smears in mutant mice, but the absolute neutrophil counts were not significantly different between mutant and wild type mice. Many, but not all of the mutant mice also had elevated baseline serum IL-1 levels, whereas IL-1 was not detected in serum from wild type transgenic or low expressing transgenic mutant mice. Perivascular neutrophil infiltration was observed in lung tissue and granuloma formation was observed in liver tissue from mutant mice, but not wild type mice.
Initial phenotype analysis suggests that these transgenic mutant mice may serve as a useful model of CIAS1 mediated disease observed in patients. Further studies with these mice and other mouse models will allow for better elucidation of in vitro function of mutant and wild type cryopyrin.

  Muckle-Wells Syndrome Associated with a Novel CIAS1 Complex Allele in an Italian Family

Laura Obici1, Cesare Danesino2, Antonio Lazzaro3, Carla Olivieri2, Sabrina Marciano1, Simona Donadei1 , and Giampaolo  Merlini1        

1Biotechnology Research Laboratories, IRCCS Policlinico San Matteo, Pavia, Italy; 2Medical Genetics, IRCCS Policlinico San Matteo and University of Pavia, Pavia, Italy; 3Medical Oncology and Haematology, Ospedale Saliceto, Piacenza, Italy

Mutations of CIAS1 are associated with the occurrence of a group of autosomal dominant autoinflammatory syndromes including FCAS, MWS and CINCA. These disorders are characterized by recurrent inflammatory attacks starting at birth or in early childhood and represent a spectrum of signs and symptoms of increasing severity, with CINCA being the more severe disorder. More 40 CIAS1 variants have been proved to be pathogenic and all but one are located within exon 3, predominantly in the sequence corresponding to the highly-conserved NACHT domain of cryopyrin. Although no clear-cut genotype-phenotype relationship could be established, several observations suggest that some variants, such as V198M and R260W, are predominantly associated with less severe conditions (either FCAS or MWS or intermediate phenotypes) while others have been found exclusively in CINCA patients.
We describe an Italian family with five members over four generations presenting with recurrent fever, arthralgia, urticaria, and moderate sensorineural deafness, not triggered by cold exposure. Clinical and laboratory evaluation disclosed persistent elevation of inflammatory parameters but no evidence of systemic AA amyloidosis. Automated sequencing of exon 3 of CIAS1 showed the presence of both the V198M and R260W variants in all affected family members studied. Both mutations were not found in 200 Italian control chromosomes.
To our knowledge, this is the first Italian family genetically diagnosed with MWS. In this kindred the disease segregates with a complex V198M-R260W CIAS1 allele, that has never been reported before. Unexpectedly, this double mutant of cryopyrin is not associated with a severe disease expression, but it otherwise presents with signs and symptoms of MWS, without the occurrence of AA amyloidosis. These two variants have been predicted to belong to the same functional domain of cryopyrin (Neven et al, Blood 2004; 103:2809). Our observation favours previous speculation that phenotypic expression of CAPS may be related to the cryopyrin domain affected by the mutation and to the resulting impairment of protein function and expression.

  Is the CIAS1 Q705K Mutation Pathogenic? Three Case Presentations

Philip J. Hashkes               

Dept. of Rheumatic Diseases, Cleveland Clinic Foundation, Cleveland, OH 44195

Background: More than 50 mutations of the CIAS1 gene have been described. The clinical significance for some, including the Q705K CAG/AAG glutamine to lysine substitution, is not clear, since it is present in 5-11% of the population. I describe 3 cases that suggest this mutation may be pathogenic in some patients.
Case reports: 1. A 3.5 year old Caucasian male of German ancestry who developed from the age of 1 year a rash and arthropathy, with erythematous nodules. He also has episodic fever with worsening of the rash and arthropathy with marked increases in inflammatory indices (normal between episodes). Episodes last 2-14 days. At age 15 months he developed spastic diplegia and ataxia after MMR vaccine that is gradually improving. Father has an undiagnosed arthropathy. ANA was positive; other autoantibodies were negative and an extensive work-up for other arthropathy causes and genetic conditions was negative. CIAS1 testing revealed Q705K and T221 (threonine to threonine) mutations. His arthropathy is well controlled with anakinra.
2. A 12 year-old Caucasian male of German-Italian ancestry who from age 9 years had recurrent episodes of fever, sore throat, mouth sores, abdominal pain, erythematous hive-like rash, arthralgia and myalgia with elevation of inflammatory indices during episodes. Episodes last 2-6 weeks. In an early episode he had knee swelling and was diagnosed with seronegative enthesopathy arthropathy syndrome and treated for 2 years with methotrexate. After 2 years of treatment he developed 3 episodes. Extensive work-up was negative except finding Q705K and A244G (alanine to alanine) mutations. Since his genetic diagnosis he has had no episodes and anakinra was not started.
3. A 17 year-old Caucasian female of German-Irish-Scottish ancestry developed at age 5 months a left parietal and thalamic stroke and has since had recurrent rash, canker sores and arm swelling. At 14 years she developed labyrinthitis, at 16 years she developed left retinal occlusion and at 17 years had a left TIA. ESR and CRP are persistently elevated and serum amyloid A was markedly elevated. Comprehensive inflammatory work-up was negative except finding Q705K, T221T and IVS3+59g/a-mutations.
Conclusion: The Q705K CIAS1 mutation may be pathogenic in some patients, especially if accompanied by additional heterozygote mutations. The clinical manifestations are not those typically described for cryopyrin syndromes but may include neurologic manifestations. It is possible that the second mutation (although not resulting in a substitution) may have influence on the pathogenicity of the Q705K mutation.

  Clinical and genetic characterization of CINCA/NOMID syndrome reveals a wide heterogeneity in the Italian Registry patients

Loredana Lepore1, Alessandra Pontillo2, Marco Gattorno3, Sergio Crovella2, Andrea D'Osualdo4, Laura Travan1 , Mariolina Alessio5, Achille Stabile6, Francesco Caroli4, Alberto Tommasini1, and Isabella Ceccherini4    

1Dipartimento di Pediatria, IRCCS Burlo Garofolo e Università di Trieste, Trieste, Italy; 2Dipartimento di Genetica, IRCCS Burlo Garofolo e Università di Trieste, Trieste, Italy; 3U.O.Pediatria II, Istituto G. Gaslini, Genova, Italy; 4Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genova, Italy.; 5Dipartimento di Pediatria, Ospedale Federico II, Napoli, Italy; 6Dipartimento di Scienze Pediatriche, Università Cattolica Sacro Cuore, Roma, Italy

Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome is a severe autoinflammatory disease associated with mutations of CIAS1, a gene which plays a pivotal role in the inflammasome complex assembly. Aim of the present study was to analyze the clinical and genetic characteristics of the patients focusing on the possible differences in the clinical presentation, disease course and response to anti-IL-1 treatment between CIAS1-mutated and non-mutated patients.
The whole coding region and intronic flanking sequences of the CIAS1/PYPAF1/NALP3 and ASC genes, as well as a portion of the CIAS1 promoter region (1000bp upstream 5’UTR) were directly sequenced with appropriate primers in 12 patients presenting clinical features consistent with CINCA/NOMID. Seven patients (58 %) carried CIAS1 missense mutation, localized within the nucleotide binding domain of cryopyrin. Three previously described mutations and four new heterozygous CIAS1 missense mutations were identified. On the other hand, we could not find any other mutation in the ASC gene or the CIAS1 promoter in the remaining five patients. In the present series, no substantial differences in the clinical picture were found between CIAS1 mutated and not-mutated patients. The only exception regards the higher incidence of relevant perinatal events in patients carrying CIAS1 mutations. In seven patients, 4 CIAS1-mutated and 3 not mutated, anti-IL1 therapy (Anakinra) treatment lead to a dramatic improvement of the disease. In both CIAS1 mutated and not-mutated patients fever and rash completely disappeared after few days from the beginning of the treatment and a general improvement of other disease-associated symptoms was observed.
Finally, comparison between severity of the most frequent clinical features, time of symptoms onset and also effect of the Anakinra treatment has not revealed any correlation with the possible presence of a CIAS1 mutation in the whole cohort of Italian CINCA/NOMID patients thus analyzed.
  Distribution of TNFRSF1A gene mutations among patients affected with autoinflammatory disorders correlates with different degrees of symptoms severity

Andrea D'Osualdo1, Francesca Ferlito2, Laura Obici3, Antonella Meini4, Francesco Zulian5, Allessandra Pontillo6 , Fabrizia Corona7, Roberto Barcellona8, Marco Di Duca1, Giuseppe Santamaria1, Francesco Traverso2, Paolo Picco2, Alberto Martini2, Marco Gattorno2, and Isabella Ceccherini1

1Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genova, Italy; 2U.O.Pediatria II, Istituto G. Gaslini, Genova, Italy; 3Laboratorio di Biotecnologie, IRCCS Policlinico S. Matteo, Pavia, Italy; 4Immunoreumatologia pediatrica, Università di Brescia, Brescia, Italy; 5Dipartimento di Pediatria, Università di Padova, Padova, Italy; 6Servizio di Genetica Medica, IRCCS Burlo Garofalo, Trieste, Italy; 7Clinica Pediatrica, Università di Milano, Milano, Italy; 8Clinica Pediatrica, Università di Palermo, Palermo, Italy

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory disease associated with mutations of the extracellular domain of p55 tumor necrosis factor receptor (TNFR), whose underlying pathogenetic mechanisms still need to be clarified. This study reports clinical and genetic observations on an italian series of 21 patients, selected among 265 affected with autoinflammatory syndrome for the presence of mutations of the TNFRSF1A gene. We have subdivided these 21 patients into two groups according to kind of mutation detected, occurrence also within the control population and association with specific disease sign.

A total of eight patients presented six missense substitutions, among which four affecting cysteine residues and the already known T50M substitution, one already reported splicing mutation and one novel in frame interstitial deletion. This latter mutation, affecting the most proximal portion of the extracellular region, might be involved in the receptor shedding, interfere with folding of the extracellular portion or affect other receptor functions. Missense substitutions of cysteine or threonine residues presented the more aggressive disease course. The associated disease phenotype is represented in these cases by a homogeneous combination of typical TRAPS symptoms, with only a few not significant exceptions.

By contrast, the remaining thirteen patients, representing more than 50% of those carrying a defect of the TNFRSF1A gene, showed the R92Q substitution, a missense mutation already reported in both patients affected with TRAPS and control individuals. In our case, among 530 affected chromosomes, we could detect an allele frequency of 2.45% which does not differ significantly from the allele frequency of 2.25% found in 400 normal chromosomes (P=0.84). It is worth noting that patients carrying the R92Q mutation displayed a very heterogeneous picture of autoinflammatory disease, quite different from the typical TRAPS phenotype, with a milder disease course in term of duration of flares, intensity of disease-associated symptoms and responsiveness to steroids treatment. According to such a view, for a number of “R92Q” patients, a PFAPA-like disorder can be envisaged.

In this light, and also based on functional evidence recently obtained in vivo from patients’cells (Gattorno et al., this conference), we are tempted to propose that the R92Q is not causing a TRAPS phenotype and that each of these patients is in fact suffering from a different still undefined clinical entity, whose molecular basis does not depend on the TNFRSF1A genetic defect but is due to mutation(s) in other still unidentified disease gene(s).
  Distribution of TNFRSF1A gene mutations among patients affected with autoinflammatory disorders correlates with different degrees of symptoms severity

Andrea D'Osualdo1, Francesca Ferlito2, Laura Obici3, Antonella Meini4, Francesco Zulian5, Alessandra Pontillo6 , Fabrizia Corona7, Roberto Barcellona8, Marco Di Duca1, Giuseppe Santamaria1, Francesco Traverso2, Paolo Picco2, Alberto Martini2, Marco Gattorno2, and Isabella Ceccherini1

1Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genova, Italy; 2U.O.Pediatria II, Istituto G. Gaslini, Genova, Italy; 3Laboratorio di Biotecnologie, IRCCS Policlinico S. Matteo, Pavia, Italy; 4Immunoreumatologia pediatrica, Università di Brescia, Brescia, Italy; 5Dipartimento di Pediatria, Università di Padova, Padova, Italy; 6Servizio di Genetica Medica, IRCCS Burlo Garofalo, Trieste, Italy; 7Clinica Pediatrica, Università di Milano, Milano, Italy; 8Clinica Pediatrica, Università di Palermo, Palermo, Italy

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory disease associated with mutations of the extracellular domain of p55 tumor necrosis factor receptor (TNFR), whose underlying pathogenetic mechanisms still need to be clarified. This study reports clinical and genetic observations on an italian series of 21 patients, selected among 265 affected with autoinflammatory syndrome for the presence of mutations of the TNFRSF1A gene. We have subdivided these 21 patients into two groups according to kind of mutation detected, occurrence also within the control population and association with specific disease sign.
A total of eight patients presented six missense substitutions, among which four affecting cysteine residues and the already known T50M substitution, one already reported splicing mutation and one novel in frame interstitial deletion. This latter mutation, affecting the most proximal portion of the extracellular region, might be involved in the receptor shedding, interfere with folding of the extracellular portion or affect other receptor functions. Missense substitutions of cysteine or threonine residues presented the more aggressive disease course. The associated disease phenotype is represented in these cases by a homogeneous combination of typical TRAPS symptoms, with only a few not significant exceptions.
By contrast, the remaining thirteen patients, representing more than 50% of those carrying a defect of the TNFRSF1A gene, showed the R92Q substitution, a missense mutation already reported in both patients affected with TRAPS and control individuals. In our case, among 530 affected chromosomes, we could detect an allele frequency of 2.45% which does not differ significantly from the allele frequency of 2.25% found in 400 normal chromosomes (P=0.84). It is worth noting that patients carrying the R92Q mutation displayed a very heterogeneous picture of autoinflammatory disease, quite different from the typical TRAPS phenotype, with a milder disease course in term of duration of flares, intensity of disease-associated symptoms and responsiveness to steroids treatment. According to such a view, for a number of “R92Q” patients, a PFAPA-like disorder can be envisaged.
In this light, and also based on functional evidence recently obtained in vivo from patients’cells (Gattorno et al., this conference), we are tempted to propose that the R92Q is not causing a TRAPS phenotype and that each of these patients is in fact suffering from a different still undefined clinical entity, whose molecular basis does not depend on the TNFRSF1A genetic defect but is due to mutation(s) in other still unidentified disease gene(s).
  Intra-Abdominal Abscess in a Patient with Tumor Necrosis Factor Receptor-Associated Periodic Syndrome

Susanna Stjernberg-Salmela1, Arto Kivisaari3, Pauli Puolakkainen4, Martti Färkkilä2, Esko Kemppainen4, Annamari Ranki1 , and Tom Pettersson2        

1Department of Dermatology, Helsinki University Central Hospital, Helsinki, FIN-00029 HUCH, Finland; 2Department of Medicine, Helsinki University Central Hospital, Helsinki, FIN-00029 HUCH, Finland; 3Department of Radiology, Helsinki University Central Hospital, Helsinki, FIN-00029 HUCH, Finland; 4Department of Surgery, Helsinki University Central Hospital, Helsinki, FIN-00029 HUCH, Finland

Tumor necrosis factor (TNF) receptor-associated periodic fever syndrome (TRAPS) is an autoinflammatory disorder characterized by periodically occurring attacks of fever, peritonitis, pleurisy, pericarditis, arthritis, myalgia and a migratory skin rash. Mutations in the gene coding for the TNF type I receptor (TNFRSF1A) often cause defective shedding of the receptor, resulting in an inappropriate and prolonged inflammation.
A 16-year old female patient with a F112I missense mutation in exon 4 of the TNFRSF1A gene, confirmed by mutational screening, was admitted to hospital because of fever and abdominal pain. The patient has suffered from febrile attacks involving abdominal pain since the age of 11. Typically, the attacks recur once or twice a year, last from one to three weeks, and the pain localizes to the right side of the lower abdomen. The abdominal pain has been relieved by NSAIDs and occasional courses of corticosteroids, at an initial dose of 30 mg prednisolone per day and tapering within four to six weeks by 5-10 mg a week. The same mutation was revealed in the patient’s symptomatic mother and maternal grandfather, as well as in two symptom free half-siblings of the patient’s mother. The patient’s medical history and the initial investigations led to interpret the symptoms as manifestations of another TRAPS attack. As CRP-levels rose to 385 mg/l and the abdominal pain intensified, an enteric bacterial infection was considered probable, and intravenous cefuroxime and metronidazole-treatment was commenced. Initially, CRP-levels decreased, only to rise again, and treatment with corticosteroids was initiated, as an autoinflammatory reaction associated with TRAPS could not be ruled out. The abdominal pain and nausea worsened, and elevated urinary levels of amylase of up to 8580 U/l (normal 60-2000 U/l) were measured. A computer tomography (CT) of the abdomen revealed no signs of perforation, and no abnormalities in the internal organs. Echocardiography of the heart showed no signs of valvular vegetations or pericarditis. The patient’s condition worsened, until a repeated computer tomography of her abdomen revealed an intra-abdominal abscess, which necessitated urgent surgical intervention.
It is possible that an initial attack of TRAPS may have caused intestinal paralysis and an increase in intra-intestinal pressure. A subsequent increase in epithelial cell permeability and bacterial extravasation, possibly accelerated by the action of pro-inflammatory cytokines, may thereby have resulted in the formation of an intra-abdominal abscess. This case stresses the importance of differential diagnostic vigilance when dealing with patients with rare genetic diseases
  A novel TNFRSF1A H105P mutation causing colchicine-sensitive TRAPS

Danielle S. Wendling1, Kristina Huss2, Florian Hoffmann2, Bernd Belohradsky2, and Peter Lohse3           

1Department of Pediatric Surgery, Children's Hospital, University of Munich, Germany; 2Department of Immunology and Infectious Disease, Children's Hospital, University of Munich, Germany; 3Department of Clinical Chemistry-Großhadern, University of Munich, Germany

Tumor necrosis factor receptor-associated syndrome (TRAPS) is an autosomal dominantly inherited disease characterized by periodic fever and abdominal pain. Arthralgia, myalgia, and erythema are also common. Conjunctivitis with or without periorbital edema may be present in addition.
Here, we report a German family with a novel TNFRSF1A mutation. A 7-year-old boy presented with abdominal pain and fever to the emergency room. The family history revealed that the boy’s father had also experienced recurring episodes of fever and pain since the age of 6 years. At the age of 33 years, he had been diagnosed with systemic amyloidosis that had made a kidney transplant necessary. Since the biopsies had shown type AA amyloid suggesting a chronic inflammation, the presumptive diagnosis had been familial Mediterranean fever, and the patient had therefore been treated with colchicine for the past 13 years. As a consequence, he did not have any more fever attacks except for one episode that had occurred when the treatment with colchicine had been discontinued once. However, no molecular genetical analyses had been conducted to confirm the tentative diagnosis.
Due to the boy’s family history, a DNA sequence analysis of all 10 exons and of the adjacent intronic sequences of the MEFV gene was performed, which did not reveal a mutation. To exclude a TRAPS, sequencing of exons 2-4 and 6 of the TNFRSF1A gene was carried out in addition. This led to the detection of a novel heterozygous A→C substitution (c.401A>C) in exon 4, resulting in a histidine (CAT)→proline (CCT) substitution at amino acid position 105 (H105P). The father was subsequently also analyzed, and he was found to carry the identical genetic defect. Analysis of both grandparents demonstrated that the mutation had occurred de novo in the paternal germline.
The finding that colchicine treatment effectively reduced disease activity in the father is unusual since colchicine is generally assumed to be ineffective in TRAPS patients.