Spectrum of Mutations of Familial Mediterranean Fever Gene in Iranian Population

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1 Research Article Spectrum of Mutations of Familial Mediterranean Fever Gene in Iranian Population Tayebeh Sabokbar 1,2, Ali Malayeri 2, Cyrus Azimi 2, Seyyed Reza Raeeskarami 3,4, Vahid Ziaee 3,4, Yahya Aghighi 3,4, Abbas Shakoori 2,5 Abstract Objective: Familial Mediterranean fever is a genetic disease that is caused by the MEFV gene. Our study was based on clinical manifestations of recurrent attacks of fever and inflammation and distribution of MEFV mutations in an Iranian patient using a comprehensive mutation detection method. Methods: Eighty-five clinically diagnosed FMF patients from university hospitals who were referred to the Department of Genetics for molecular diagnosis of FMF. Results: Mutations in one allele and in both alleles were identified in 33 (39%) and 34 (40%) patients respectively and no mutation could be detected in 18 (21%). The most common mutation was M694V (21%). Other mutations accounted for a further 38% of the alleles: E148Q (11%), M694I (11%), M680I (6%), V726A (4%), R761H (5%). In this study, two rare mutations were found in two patients from Zanjan province in exon 10 in codons: A744S and K695R with the frequency of 1.1% each. Conclusions: The most frequent mutations were detected in three codons: M694V and M694I in exon 10 with 27.3% and 16.6% respectively and codon E148Q in exon 2 with 15.4%. Our data suggests that E148Q has a more dominant pattern of inheritance with reduced penetrance and M680I, M694V and M694I mutations have a more recessive pattern of inheritance. Key words: FMF, MEFV Gene, Iranian Ethnicity 1 Neurology and Neuroscience Research Center, Qom University of Medical Science, Qom, Iran 2 Department of Medical Genetics, Cancer Institute, Tehran University of Medical Sciences Tehran, Iran, (TUMS), 3 Department of Pediatrics, TUMS, 4 Pediatric Rheumatology Research Group, Rheumatology Research Center, TUMS, 5 Occupational Sleep Research Center, TUMS. Corresponding Author: Abbas Shakoori, Medical Genetic Department, Faculty of Medicine, Cancer Institute Hospital, Keshavarz Blv, Tehran, Iran shakooria@sina.tums.ac.ir Received: Oct 30, 2013 Accepted: Nov 18, 2013 Ann Paediatr Rheum 2014;3:11-17 Introduction Familial Mediterranean fever (FMF) is a hereditary inflammatory disorder caused by a mutation in MEFV, a gene which encodes a 781-amino acid protein called pyrin. The classic clinical picture consists of recurrent febrile episodes that are usually of an acute onset, variable frequency, sometimes without a recognized triggering factor, but often occurring with menstruation, emotional stress or strenuous physical activity. FMF is usually an autosomal recessive disorder. The disease affects populations of Mediterranean origin: Ashkenazi Jews, North African Jews, Armenians, Arabs, Italians, and Turks. The gene causing FMF was cloned in The gene responsible for FMF (MEFV) is located on the short

2 12 Sabokbar T et al arm of chromosome 16 (16p13) and was independently identified by two positional cloning consortia and four missense mutations in exon 10, namely M694V, V726A, M694I and M680I. These four mutations and E148Q in exon 2 are the most common MEFV mutations among the 59 putative mutations identified to date and are believed to be associated with the disease. Additional less common mutations have been found subsequently in exons 3, 5, 9 and 10, bringing the current total of known mutations to 23. Exon 10 remains the major site of mutations, with a smaller cluster in exon 2 ( There is also an autosomal dominant form of FMF (134610), which is caused by a heterozygous mutation in the MEFV gene. The FMF carrier rate can be as high as one in three in the commonly affected ethnic groups, raising the possibility of selective heterozygote advantage. Homozygous or compound heterozygous mutations in the MEFV gene result in classic familial Mediterranean fever (FMF; ), which shows autosomal recessive inheritance. The MEFV transcript is expressed in granulocytes that play an essential role in the inflammatory response. Pyrin, the protein encoded by the MEFV gene, has already been analyzed by crystallography [Fig. 1]. Because the disease has a wide spectrum of signs and symptoms, molecular techniques are helpful tools for verification of the diagnosis. Molecular analysis of MEFV is useful in clinical practice, mainly in an atypical form of the diseases. Colchicine is the preferred drug of choice. There is a report that mutational hotspots in codons 680 and 694 are associated with severity of disease, hospital admission, early onset, high frequency of attacks, the necessity of high dose of colchicine to control attacks, and frequent occurrence of amyloidosis in untreated patients. This study is to determine the mutation frequency in the clinically diagnosed FMF patients of an Iranian population. We report here on the distribution of MEFV mutations in FMF patients among an Iranian subethnic group. Methods Study design and patients Routine molecular diagnosis of FMF in our laboratory began in Currently there are 120 patients in the registry, 85 of whom were included in this study. The diagnosis of FMF in these patients was made according to established clinical criteria by pediatricians. Eighty-five unrelated subjects (31 males and 54 females) clinically diagnosed with FMF were recruited from university hospitals and private pediatric clinics after referral by a physician to the Department of Genetics for confirmation of diagnosis of FMF. The study was designed to determine the spectrum of mutations in the Mediterranean fever gene in an Iranian population with FMF and to consider their clinical symptoms. Mutation Analyses A 3ml blood sample was collected from all subjects, and our method was based on the FMF strip assay (ViennaLab Diagnostics GmbH) for detecting mutations. Genomic DNA was extracted from 200µL peripheral blood samples obtained from the patients by using the Gene extract extraction method, and was stored at 20 C until use. Then, patients were studied using a reverse hybridization test strip-based assay Figure 1. Crystallography of the protein encoded by the MEFV gene. Annals of Paediatric Rheumatology Year 2014 Volume:3 Issue:

3 FMF in Iranain population 13 (FMF Strip Assay) that allows detection of the 12 most frequent MEFV mutant alleles located in exons 2, 3, 5, and exon 10. The procedure includes three steps: DNA isolation, PCR amplification and hybridization. Results Mutation analysis showed that identified missense mutations accounted for all of the 170 independent FMF alleles. The mutation detection protocol revealed in patients eight different previously reported mutations located in exon 10 (M680I, M694I, M694V, K695R, V726A, A744S, R761H) and exon 2 (E148Q). There aren t any mutations in exon 3 (P369S), exon 5 (F479L) and exon 10 (I692del) in our detection. The most common mutation was M694V (21%). Other mutations accounted for a further 38% of the alleles: M694I (11%), M680I (6%), E148Q (11%), V726A (4%), and R761H (5%) [Table 1]. Mutations in both alleles were identified in 33 of the 85 patients (39%): seven (9%) were homozygous for the same mutation, and 26 (30%) were compound heterozygous for different combinations of the mutations. Thirty-four (40%) were found to be heterozygous for one mutation. In 18 patients (21%), no mutation could be detected. Out of the 85 patients, 67 (79%) carried at least one mutation [Table 2]. The numbers of each allele in patients in whom at least one mutation was detected are shown in two groups: heterozygote and homozygote or compound heterozygote [Fig. 2]. Two rare mutations (A744S and K695R) were present at Figure 2. Number of each allele in patients that at least one mutation was detected is shown in two groups: heterozygote and homozygote or compound heterozygote (total alleles: 170). Figure 3. Number of alleles for each ethnicity among Iranian population of patients with FMF. an allele frequency <1% in Zanjan, one of Azari provinces, and three mutations in exon 3 (369S), exon 5 (F479L) and exon 10 (I692del) were not detected in any of our patients [Table 1]. All of the cases in this study were from different parts of Iran, mostly from Azari provinces (Turk ethnicity) (28%), north provinces (Alborz area) (25%), Tehran province (20%), and 27% of patients were from the rest of Iran. The most frequent mutations among all patients as well as the patients from Azari provinces (Turk ethnicity), north provinces (Alborz area) and Tehran provinces were M694V, while the most frequent mutations among Kurdish were M694I and among central provinces were E148Q [Figure 3]. The clinical characteristics of the patients were as follows: joint problems were observed in 17% and abdominal pain in 52%. Other clinical data for chest pain, peritonitis, vasculitic rash, and amyloidosis were not available in our lab registry at this time. Age of disease onset was 27% of the patients under one year old and was 35% and 38% for patients under and above 6 years old, respectively. The average age of onset among all patients was 8 years old. Almost all patients had fever and two patients had a history of amyloidosis. Thirtythree (39%) patients had a history of at least one admission to hospital. The percentages of the frequency of each mutation were almost the same among admitted versus non-admitted patients, except for E148Q mutation, which was 24% among admitted patients and 15% among non-admitted patients, and R761H mutation, which was 4.5% among admitted patients

4 14 Sabokbar T et al Table 1. Frequency of the MEFV gene mutations in 85 Iranian FMF patients. Exon Nucleotide Mutation Protein Mutation Number of alleles Frequency (%) Exon 2 C<c.442G E148Q 19 11% Exon 3 T<c.1105C P369S 0 0 Exon 5 G,c.2040G F479L 0 0 Exon 10 C<c.2040G M680 I 10 6% Exon 10 Del2078<c.2076 I692del 0 0 Exon 10 A<c.2082G M694I 19 11% Exon 10 G<c.2080A M694V 36 21% Exon 10 c.2084a>g K695R % Exon 10 C<c.2177T V726 A % Exon 10 c.2230g>t A744S % Exon 10 A<c.2282G R761H 8 5% Unknown 70 41% Total % Table 2. Ratio of heterozygote vs. homozygote patients. Heterozygote 34 40% 40% Compound Heterozygote % Homozygote 7 9% 39% Unknown 18 21% Total % and 11% among non-admitted patients. Among the two cases with amyloidosis, both of them were heterozygous for E148Q mutation. Thirty-nine percent of patients received drugs for treatment and the patient without detectable mutations also received treatment. We identified three adults, one with R761H homozygote mutation and two with compound heterozygote mutations of R761H/ V726A and V726A/M694V, who had the age onset of over 35 years old. The 67 patients who carry at least one mutation have classical clinical symptoms, with fever lasting from a few hours to 1 week, which was not different from the 18 patients in whom no mutations were identified. The clinical picture in all of the patients is summarized. A comparison between the phenotypic features of FMF patients and the genotypes of all patients is shown in [Table 3]. Discussion To our knowledge, this is the first comprehensive study of FMF in the Iranian population that examines the spectrum and distribution of MEFV mutations among affected individuals. In this study, we have determined the mutation frequencies of 12 common mutations in the MEFV gene in 85 Iranian FMF patients. The M694V mutation, which is found in 21% of populations, and another seven mutations were found in about 38% of Iranian FMF alleles [Table 1]. Mutations in both alleles were identified in 33 of the 85 patients (39%): seven (9%) were homozygous for the same mutation, and 26 (30%) were compound heterozygous for different combinations of the mutations [Table 2]. In Turkish people the mutations in both alleles were identified in 263 of the 450 patients (58.44%): 179 (39.77%) were homozygous for the same mutation, and 84 (18.66%) were compound heterozygous for different combinations of the mutations. Eighty-three (18.44%) were found to be heterozygous for one mutation and no mutation could be detected in 104 patients (23.11%). Out of the 85 patients, 67 (79%) carried at least one mutation (43 males and 24 females). While all patients carried at least one mutation, they are not expected to carry mutant chromosomes [Table 1, Table 2]. The most common mutation was M694V (21%). Annals of Paediatric Rheumatology Year 2014 Volume:3 Issue:

5 FMF in Iranain population 15 Table 3. Clinical data. Num. Percent Age < 10 years old 44 52% 10 to % 20 to % 30 years old < 5 6% Disease Onset < 1 year old 17 20% 2 to % 10 to % 20 years old < 6 3.5% Unknown 22 26% Ethnicity Kurdish 7 8% South 8 9.5% Central 8 9.5% Tehran 17 20% North 21 25% Turkish 24 28% Hospital Admission Admitted 33 39% Not admitted % Unknown % Num. Percent Sex Female 31 36% Male 54 64% Genotype Homozygote 7 9% Compound Heterozygote 26 30% Heterozygote 34 40% Unknown 18 21% Fever Fever 79 93% Unknown 6 7% Pain Abdominal pain 35 41% Joints pain 7 8% Abd. & Joints pain 7 8% Amiloidosis & pain 2 3% Unknown 34 40% Drugs Corticosteroids 1 1% NSAIDs & AB 34 40% Colchicines 33 39% Unknown 17 20% Other mutations accounted for a further 38% of the alleles: M694I (11%), M680I (6%), E148Q (11%), V726A (4%), and R761H (5%). Three mutations in exon 3 (P369S), exon 5 (F479L) and exon 10 (I692del) were not detected in any of our patients [Table 1]. The third, fourth, fifth and sixth commonest mutations in our study were E148Q (11%), M680I (6%), V726A (4%) and R761H (5%), respectively. These results were near to the Algerian study in which the third, fourth and fifth commonest mutations were E148Q (12%), A744S (10%) and M680I (8%), respectively. But A744S was present at an allele frequency <1% in Zanjan, one of the Azari provinces. Here, mutation analysis showed that six identified missense mutations accounted for 58% of the 180 independent FMF alleles [Table 1]. This study confirms that M694V is the most common MEFV mutation in the Iranian population, but shows a new spectrum and distribution that are different from what have previously been reported. In our data, E148Q mutations show 30% under one year of disease onset, but show 14% above one year of disease onset, suggesting that E148Q mutations act as an early onset disease factor and show their deleterious effect on MEFV gene function. The E148Q mutation is a dominant inheritance (AD) pattern with reduced penetrance most of the time. In future functional studies, we should provide answers to the questions: What is the biologic basis of the heterozygote adwww.aprjournal.org

6 16 Sabokbar T et al vantage?, What are the modifier genes? and How does amyloidosis work? Also, allele frequency of each geographic area in Iran needs to perform more analyses in our future plan. The proportion of unknown alleles (41%) in our study [Table 1], which are carrying rare mutations, needs to be analyzed for unscreened exons. Conclusion In conclusion, our study describes the MEFV mutational spectrum and distribution in the Iranian population by studying FMF patients and controls and employing a comprehensive mutation detection strategy. Our future strategy for mutation analysis is to perform detection analysis for all unknown as well as heterozygous patients by sequencing the promoter region (~500 bp upstream of the ATG translation), the entire coding sequence, and all exon intron boundaries (50 bp of the exon flanking intronic sequences) in forward and reverse directions after PCR amplification. Acknowledgment: This research has been made possible with the financial assistance of the Department of Medical Genetics, Cancer Institute (TUMS). Competing interests: The authors declared no competing interest. Funding: None. Provenance and peer review: Not commissioned; externally peer reviewed. References 1. Siegal S. Benign paroxysmal peritonitis. Gastroenterology ;12: Samuels J, Aksentijevich I, Torosyan Y, Centola M, Deng Z, Sood R, et al. Familial Mediterranean fever at the millennium. Clinical spectrum, ancient mutations, and a survey of 100 A merican referrals to the National Institutes of Health. Medicine (Baltimore) ;77: Aksentijevich I, Centola, M., Deng, Z. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. The International FMF Consortium. Cell ;90: Alain Bernot CC, Corinne Dasilva. A candidate gene for familial Mediterranean fever. Nat Genet ;17: Booth DR, Gillmore JD, Booth SE, Pepys MB, Hawkins PN. Pyrin/marenostrin mutations in familial Mediterranean fever. QJM ;91: Bernot A, da Silva C, Petit JL, Cruaud C, Caloustian C, Castet V, et al. Non-founder mutations in the MEFV gene establish this gene as the cause of familial Mediterranean fever (FMF). Hum Mol Genet ;7: Touitou I. The spectrum of Familial Mediterranean Fever (FMF) mutations. Eur J Hum Genet ;9: Gershoni-Baruch R, Shinawi M, Leah K, Badarnah K, Brik R. Familial Mediterranean fever: prevalence, penetrance and genetic drift. Eur J Hum Genet ;9: Kogan A, Shinar Y, Lidar M, Revivo A, Langevitz P, Padeh S, et al. Common MEFV mutations among Jewish ethnic groups in Israel: high frequency of carrier and phenotype III states and absence of a perceptible biological advantage for the carrier state. Am J Med Genet ;102: Stoffman N, Magal N, Shohat T, Lotan R, Koman S, Oron A, et al. Higher than expected carrier rates for familial Mediterranean fever in various Jewish ethnic groups. Eur J Hum Genet ;8: Yilmaz E, Ozen S, Balci B, Duzova A, Topaloglu R, Besbas N, et al. Mutation frequency of Familial Mediterranean Fever and evidence for a high carrier rate in the Turkish population. Eur J Hum Genet ;9: Al-Alami JR, Tayeh MK, Najib DA, Abu-Rubaiha ZA, Majeed HA, Al-Khateeb MS, et al. Familial Mediterranean fever mutation frequencies and carrier rates among a mixed Arabic population. Saudi Med J ;24: Hiller S, Kohl A, Fiorito F, Herrmann T, Wider G, Tschopp J, et al. NMR structure of the apoptosis- and inflammation-related NALP1 pyrin domain. Structure ;11: Sohar E, Gafni J, Pras M, Heller H. Familial Mediterranean fever. A survey of 470 cases and review of the literature. Am J Med ;43: Langevitz P, Livneh A, Neumann L, Buskila D, Shemer J, Amolsky D, et al. Prevalence of ischemic heart disease in patients with familial Mediterranean fever. Isr Med Assoc J ;3: Miller SA, Dykes DD, Polesky HF. A simple salting out Annals of Paediatric Rheumatology Year 2014 Volume:3 Issue:

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