Zuoming Deng

Ancient missense mutations in a new member of the RoRet gene family are likely to cause Familial Mediterranean Fever

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever and serosal inflammation. This report describes the cloning of the gene likely to cause FMF from a 115-kb candidate interval on chromosome 16p. Three different missense mutations were identified in affected individuals, but not in normals. Haplotype and mutational analyses disclosed ancestral relationships among carrier chromosomes in populations that have been separated for centuries. The novel gene encodes a 3.7-kb transcript that is almost exclusively expressed in granulocytes. The predicted protein, pyrin, is a member of a family of nuclear factors homologous to the Ro52 autoantigen. The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory responses.Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever and serosal inflammation. This report describes the cloning of the gene likely to cause FMF from a 115-kb candidate interval on chromosome 16p. Three different missense mutations were identified in affected individuals, but not in normals. Haplotype and mutational analyses disclosed ancestral relationships among carrier chromosomes in populations that have been separated for centuries. The novel gene encodes a 3.7-kb transcript that is almost exclusively expressed in granulocytes. The predicted protein, pyrin, is a member of a family of nuclear factors homologous to the Ro52 autoantigen. The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory responses.

FAMILIAL MEDITERRANEAN FEVER AT THE MILLENNIUM CLINICAL SPECTRUM, ANCIENT MUTATIONS, AND A SURVEY OF 100 AMERICAN REFERRALS TO THE NATIONAL INSTITUTES OF HEALTH

Regarded as the most common and best understood of the hereditary periodic fever syndromes, familial Mediterranean fever (FMF) is a recessively inherited disease of episodic fever with some combination of severe abdominal pain, pleurisy, arthritis, and a characteristic ankle rash. The flares typically last for up to 3 days at a time, and most patients are completely asymptomatic between attacks; if untreated with prophylactic colchicine, some patients later develop amyloidosis and renal failure. The recent cloning of the FMF gene on the short arm of chromosome 16p, and the subsequent finding that its tissue expression is limited to granulocytes, has helped to explain the dramatic accumulation of neutrophils at the symptomatic serosal sites; the wild-type gene likely acts as an upregulator of an anti-inflammatory molecule or as a downregulator of a pro-inflammatory molecule. For nearly half a century, FMF was thought to cluster primarily in non-Ashkenazi Jews, Arabs, Armenians, and Turks, although the screening of the 8 known mutations in an American cohort has identified substantial numbers of people from the Ashkenazi Jewish and Italian populations in the United States who also have this disease. Nevertheless, the symptoms often go unrecognized and patients remain undiagnosed for years, not receiving the highly efficacious colchicine therapy; their histories often include multiple laparotomies, laparoscopies, and psychiatric evaluations. The combinations of clinical manifestations among FMF patients are quite heterogeneous, but our American cohort did not establish any connections between individual mutations and specific clinical pictures--as is seen in other diseases like cystic fibrosis, in which distinct genotypes target certain organ systems. Specifically, the data from our American series are insufficient to evaluate the hypothesis that the M694V/M694V genotype confers a more severe phenotype, or increases the risk of amyloidosis; but both our data and the recent literature (160) indicate that amyloidosis can occur in FMF patients with only 1 copy, or no copies, of the M694V mutation. It appears that specific MEFV mutations are probably not the sole determinants of phenotype, and that unknown environmental factors or modifying genes act as accomplices in this disease. Although we hope the discovery of the FMF gene will allow the diagnosis of FMF to become genetically accurate, the reality is that both clinical and genetic tools must still be used together unless mutations are identified on both of a patients chromosomes. Physicians should be careful not to rule out the diagnosis in patients of high-risk ethnic backgrounds just because of atypical clinical features, as our data indicate that MEFV mutations are sometimes demonstrable in such patients. At the same time, physicians cannot yet rely solely on a genetic diagnosis because we have not yet identified a sufficient spectrum of mutations, and it is not currently feasible to examine every patients full DNA sequence for the entire gene; screening an ethnically consistent and clinically positive patient for the 8 known mutations frequently identifies a mutation on only 1 chromosome, and genetic analysis of other classic cases will often reveal none of the 8 mutations. Still, our data suggest that ethnic background is an important predictor of finding 1 of the presently known mutations, and the knowledge of ancestries atypical for FMF can suggest the diagnosis of other hereditary periodic fever syndromes. As the list of FMF-associated MEFV mutations is expanded, and/or new sequencing technologies permit more rapid screening, the value and interpretation of genetic testing for FMF will become more straightforward. Moreover, as the pathophysiology of this disorder becomes less of a hypothesis and more of an understood entity, it is likely that treatment options will broaden beyond the use of daily prophylactic colchicine. (ABSTRACT TRUNCATED)

Diagnosis of Familial Mediterranean Fever by a Molecular Genetics Method

Familial Mediterranean fever, or recurrent polyserositis, is a recessively inherited disorder that affects Sephardic Jews, Turks, Armenians, and Arabs. In these populations, the carrier frequency has been estimated to be as high as 1 in 5 persons [1, 2]. The disorder is characterized by recurrent episodes of unprovoked inflammation involving the joints; the pleural and peritoneal cavities; and, less frequently, the skin. Familial Mediterranean fever peritonitis, the most common manifestation of this disease, may resemble acute abdomen, leading to laparotomy and appendectomy that reveal only an inflamed peritoneum and a neutrophilic exudate. If a surgical procedure is avoided, the attack resolves spontaneously [3-7]. Patients are treated with colchicine, a neutrophil-suppressive agent that has been shown to decrease the frequency and severity of attacks when administered on a long-term basis [8, 9]. Some patients develop amyloidosis that can also be prevented by prophylactic colchicine administration [10]. The diagnosis of familial Mediterranean fever requires a high index of suspicion and is based on the clinical criteria of acute, reversible serosal attack and family history, when available. Until recently, the only specific laboratory test for this disease was the documentation of C5a-inhibitor deficiency in serosal or synovial fluid, a laborious assay that requires an invasive procedure [11-13]. The gene responsible for familial Mediterranean fever, designated MEFV, was recently cloned [14, 15]. Its protein product pyrin-marenostrin was found by computer alignment to be homologous with previously described nuclear factors that may play a role in the regulation of inflammatory processes. Several missense mutations were identified, accounting for a large percentage of patients with familial Mediterranean fever, but these mutations were absent in all normal persons studied. As a result, it is highly likely that mutations of MEFV are responsible for familial Mediterranean fever. We describe a rapid and accurate method for establishing the molecular diagnosis of familial Mediterranean fever, based on polymerase chain reaction (PCR) amplification of three common mutations-M680I, M694V, and V726A-in this newly cloned gene. Methods Patients and DNA Samples We obtained DNA samples from 107 persons. A total of 74 persons from 44 families gave specimens at the Sheba Medical Center in Tel Hashomer, Israel, as part of the project to identify the familial Mediterranean fever gene by positional cloning. Forty-three of these families were of non-Ashkenazi Jewish ancestry, and one family was Druze. Of the 74 family members, 19 had familial Mediterranean fever according to established clinical criteria [16], 48 were unaffected but were familial Mediterranean fever carriers according to haplotype analysis, and 7 were unaffected and were noncarriers according to haplotype analysis. Two persons (1 affected person and 1 asymptomatic carrier) from 1 Armenian-American family also gave blood as part of the same positional cloning project at the Cedars-Sinai Medical Center. Thirty-one additional persons gave or sent samples to the National Institutes of Health for genetic testing after the familial Mediterranean fever gene had been identified. Of these 31 persons, 27 had undiagnosed fever syndromes and 4 were unaffected family members. By DNA sequencing, 1 person was an M694V homozygote, 2 were M694V/M680I compound heterozygotes, 6 were symptomatic but had only one copy of the three mutations, and 4 were asymptomatic heterozygote carriers. The remaining 18 persons were negative for all three mutations by DNA sequencing. Informed consent of the participants in the study was obtained after approval by the human experimentation committee at each institution. Standard techniques were used to extract DNA from whole blood or from Epstein-Barr virus-transformed lymphocytes [17]. Mutation Detection by the Amplification Refractory Mutation System The amplification refractory mutation system (ARMS) assay comprises two complementary reactions, each conducted with the same substrate DNA. One reaction includes an ARMS primer specific for the normal DNA sequence and cannot amplify mutant DNA at a given locus. The second reaction includes a mutant-specific primer and cannot amplify normal DNA. The same common primer is used in both reactions [17, 18]. The lack of PCR products according to use of a specific mutation primer set in patients suspected of carrying the mutation for familial Mediterranean fever suggests that the patient in question is not carrying the mutation being probed. However, an appropriate internal PCR control should be run to show that the DNA is amplifiable. Therefore, the complementary reaction with the normal primer set serves as an internal control for PCR amplification and allows discrimination of homozygotes from heterozygotes. Mutations were assessed by amplifying the genomic DNA template with three sets of normal and mutant-specific ARMS primers designed to selectively amplify the normal or altered sequence of each of the three MEFV mutations. Each set of primers consisted of three oligonucleotides. For mutation M680I, the sequences were 5-TTAGACTTGGAAACAAGTGGGAGAGGCTGC-3 (common), 5-ATTATCACCACCCAGTAGCCATTCTCTGGCGACAGAGCG-3 (mutant), and 5-ATTATCACCACCCAGTAGCCATTCTCTGGCGACAGAGCC-3 (normal); for M694V, they were 5-TGACAGCTGTATCATTGTTCTGGGCTCTCCG-3 (common), 5-TCGGGGGAACGCTGGACGCCTGGTACTCATTTTCCTTCCC-3 (mutant), and 5-TCGGGGGAACGCTGGACGCCTGGTACTCATTTTCCTTCCT-3 (normal); and for V726A, they were 5-TGGAGGTTGGAGACAAGACAGCATGGATCC-3 (common), 5-TGGGATCTGGCTGTCACATTGTAAAAGGAGATGCTTCCTG-3 (mutant), and 5-TGGGATCTGGCTGTCACATTGTAAAAGGAGATGCTTCCTA-3 (normal). Each DNA sample was tested for the three mutations. The PCR amplification was performed in a final volume of 25 L containing 100 ng of purified genomic DNA, 0.04 U of Ampli Taq Gold (Perkin-Elmer, Branchburg, New Jersey) and its 1x PCR buffer (contains 15 mmol of MgCl2 per L), 0.2 mmol of deoxynucleoside 5-triphosphate mix per L (Gibco BRL, Gaithersburg, Maryland), and 1 pmol of each primer. Amplification conditions were kept the same for all of the ARMS tests, and the procedure was carried out as follows. The reaction was heated to 94C for 9 minutes for denaturation, followed by 35 cycles with denaturation at 94C for 10 seconds, annealing at 60C for 10 seconds, and extension at 72C for 30 seconds. Final extension was done for 10 minutes at 72C. The amplified products were separated by electrophoresis on a 2% agarose gel. Ethidium bromide staining of the agarose gel was used to detect the amplified fragments. Results Samples of DNA from persons affected by familial Mediterranean fever, their relatives, and normal controls were screened to determine mutation status. The results of a representative assay for each mutation are shown in Figure 1. Figure 1. Detection of three MEFV mutations by amplification refractory mutation system (ARMS) assay. Top. Middle. Bottom. A summary of the results obtained for the study group is shown in the (Table 1). For 82 samples that were studied, all three mutations were found in accordance with mutation status. None of these mutations was identified in the remaining 25 samples, as predicted by previous sequencing analysis. The most frequent mutation in our predominantly non-Ashkenazi Jewish panel was M694V. All persons with the M680I mutation were of Armenian ancestry, whereas the V726A mutation was found in Armenians, Ashkenazi Jews, and Iraqi Jews. No false-positive or false-negative results were obtained by using the three sets of primers for each sample, indicating a sensitivity and specificity of 100% for this assay. Table 1. Mutation Status Detected in the Study Group* Discussion We describe a simple, rapid, and highly reliable method for establishing the molecular diagnosis of familial Mediterranean fever, a disease primarily diagnosed on a clinical basis [3-7]. The results were validated by analyzing previously genotyped samples and proved to be totally accurate. For 107 samples that were independently genotyped by automatic sequencing as part of the project to identify the familial Mediterranean fever gene by positional cloning [14], no false-positive or false-negative results were obtained with the ARMS assay. The ARMS assay used for molecular diagnosis in our study allows accurate detection of haplotypes with mutations involving single-base changes or small deletions [18, 19]. We developed a single ARMS assay for detection of the three most common MEFV point mutations described during the cloning of the familial Mediterranean fever gene [14, 15]. In addition to the reliability of the ARMS assay, several practical considerations associated with this assay may be of interest. First, genomic DNA or, alternatively, crude cell lysate of leukocytes is used as a source of template DNA. Second, it is not necessary to prepare high-quality DNA suitable for restriction enzyme digestion. Finally, the use of radioactive materials is not required. Moreover, additional mutations could be studied by using the same method; the three disease-associated mutations discussed here do not account for all familial Mediterranean fever carrier chromosomes, and at least eight additional mutations are under study ([20]; Aksentijevich et al. In preparation). It seems that about 70% of carrier chromosomes from non-Ashkenazi Jews in Israel bear the M694V or V726A mutation. A population study that should clarify this issue is already under way. In a recent study of Turkish familial Mediterranean fever [21], the three mutations presented here accounted for 29 of 34 disease alleles. The occurrence of these mutations in the United States may be somewhat lower [20]. In persons with suggestive clinical and family history but no documented common mutation, an ARMS assay with additional sets of primers designed for other mutations or complete sequencing of the MEFV gene i

GG-10 Imagine SLE: international multi-site assessment of genetics and inflammation in early onset and familial systemic lupus erythematosus

Background Systemic Lupus Erythematosus (SLE) is a severe, multisystem autoimmune disease. Twin and sibling studies indicate a strong genetic contribution (44–69%) to SLE. Although numerous recent GWAS studies have identified gene variants, few have been linked to causal polymorphisms in SLE. It may be that few, rare variants could have large impact on SLE risk. Paediatric SLE patients have earlier onset of disease, suffer aggressive course of illness, and may have a stronger genetic risk than adults. Studying aggressive disease in paediatrics has led to myriad breakthroughs in disease pathogenesis, as demonstrated by familial hypercholesterolemia and atherosclerosis, and fever syndromes and autoinflammation. Whole exome sequencing (WES) is a powerful tool to identify rare coding variants for complex phenotypes such as that of SLE. We have established a multisite international paediatric SLE collaboration at four sites: USA, Canada, South Africa, and Mexico. We will use WES to investigate the genetic variants which may give insight into molecular pathways contributing to SLE. Materials and methods Paediatric SLE patients at sites in the USA, Canada, South Africa and Mexico will be consented. Whole exome capture/sequencing will be performed on patients with paediatric-onset SLE age ≤10 years and/or SLE with strong familial aggregation, defined as > one first degree relative or two second degree relatives with SLE. Patient and parent samples will be processed and analysed as trios. We will collect standard information on all cohorts, including demographic information, clinical history, family history, medications, exam findings, laboratory values, SLEDAI and SLICC-DI. Organ damage will be defined as end stage renal disease or SLICC-DI>0. Raw data will be processed by Whole Exome Sequencing using Illumina HiSeq2500. Bioinformatic analysis will be performed at NIH. We will develop an SLE specific bioinformatics pipeline to process data and analyse variants. Results will be filtered against known variants and parental samples. Results We currently have access to 50 pSLE patients in the US, 75 pSLE patients in SA, 200 pSLE patients in Mexico, and 500 pSLE patients in Canada from which to recruit patients. We anticipate analysis of 160 samples (20 patient/parent trios at NIH, 50 in Canada) to be complete at the time of presentation. We expect to recruit 30 SA trios, 135 Mexican trios, 40 US trios, and 200 Canadian trios during the total course of the study. Novel rare variants identified will be reviewed. Conclusions Novel rare variants identified will be reviewed.