Familial Mediterranean Fever (FMF) is a hereditary autoinflammatory disorder characterized by episodes of inflammation in the absence of high-titer autoantibodies or antigen-specific T cells. The MEFV (Mediterranean Fever) gene located on chromosome 16p13.3, which encodes the 781–amino-acid protein pyrin, is the causative gene for this monogenic Mendelian disease. This study presents the molecular analysis of an MEFV gene mutation screen of 5,518 Turkish individuals with clinical diagnoses of FMF. Patients were genetically diagnosed using the FMF StripAssay and DNA sequencing analysis. Contrary to the results achieved by the FMF StripAssay, DNA sequencing analysis identified large-scale coding and noncoding novel sequence variants, together with a significant group (76%) of individuals who were receiving colchicine and had a single heterozygous mutation, despite the recessive inheritance of FMF. In conclusion, sequence analysis, unlike other routine laboratory techniques, may enable screening for a broad range of nucleotide variations and may prevent less common, population-restricted, novel sequence variants from being overlooked. Familial Mediterranean Fever (FMF, MIM249100) is the prototype of a group of disorders termed ‘‘systemic autoinflammatory diseases’’ and is characterized by unprovoked episodes of inflammation in the absence of high-titer autoantibodies or antigen-specific T cells (Sohar et al., 1967). Familial Mediterranean Fever is common among Mediterranean populations; however, previous genetic diagnostic reports have confirmed its presence worldwide. It has been described in Mediterranean populations, including Italian, Spanish (Domingo et al., 2000), Portuguese, French, and Greek, as well as in patients from Northern Europe and Japan. Nevertheless, only rare occurrences have been reported throughout the general population because of the low frequency of the causative allele (Kastner et al., 2005; Booty et al., 2009; Sugiura et al., 2008; Touitou, 2001).
In 1997, the MEFV (Mediterranean Fever) gene, located on chromosome 16p13.3, was found to be the causative gene for this monogenic Mendelian disease. This critical gene contains 10 exons and encodes the 781-amino-acid protein pyrin (The French FMF Consortium, 1997; The International FMF Consortium, 1997). Pyrin is a component of the inflammasome complex, whose exact function in the inflammatory response is still unknown. Depending on the experimental conditions, both up- and down-regulatory effects on neutrophils were reported for pyrin in the regulation of IL-1β and NF-kB activation (Chae et al., 2003; Yu et al., 2006). To date, mutations have been identified in exons 1, 2, 3, 5, 9 and 10 of the MEFV gene, and exons 2 and 10 constitute the so-called mutational `hot spots' (Bernot et al., 1998; Touitou, 2001). According to INFEVERS (fmf.igh.cnrs.fr/infevers), the database of hereditary autoinflammatory disorder mutations, out of 195 registered FMF sequence variants, 81 have been clinically associated with the typical FMF phenotype. The remarkably wide clinical variability of the disease, as indicated by previous reports, has been linked to the MEFV allelic heterogeneity that underlies genotypic and phenotypic heterogeneity (Shohat et al., 1999; Cazeneuve et al., 2000; Gershoni-Baruch et al., 2003), and this has made detailed mutation screening critically important. This study enrolled 5,518 individuals from Western, Eastern, Northern, and Southern Turkey (age range 2 months to 67 years; 2,909 females and 2,609 males), including first- and second-degree relatives of individuals with FMF clinical diagnoses (including suspicious, possible, and definitive cases discovered by family screening) who were referred to the Molecular Medicine Laboratory for genetic diagnosis between 2002 and 2009. In this work, using either FMF StripAssay or DNA Sequencing analysis, we aim to characterize the allelic heterogeneity of 5518 Turkish pateints with a first stage clinical diagnosis of FMF. Further, the ability of FMF StripAssay and DNA Sequencing methods to detect false negatives was compared.
Materials and Methods
Subjects
The Tel-Hashomer and Livneh criteria were used for the clinical diagnosis of FMF based on the model of major, minor, and supportive criteria, which stipulates the presence of either 1 major or 2 minor criteria or 1 minor and 5 supportive criteria for a diagnosis. A simple set of criteria for the diagnosis of FMF required 1 or more major and/or 2 or more minor criteria (Livneh et al., 1997). Clinical features included recurrent, acute, self-limited episodes of fever and peritonitis, as well as attacks of pleuritis, arthritis, and an erysipelas-like skin disease. None of the patients with FMF had an immunological disorder or another rheumatic disease. Active clinical presentations (fever, abdominal pain, arthritis, and myalgia) and laboratory parameters (high levels of serum amyloid A [SAA], C-Reactive Protein [CRP], fibrinogen, white blood cell [WBC] counts and erythrocyte sedimentation rates [ESR]) were determined for each patient. For the healthy control group, 250 individuals who did not have any symptoms or positive familial history of FMF were enrolled in the study.
Experimental design
Mutation analysis of the MEFV gene was performed in two distinct patient groups by two separate methods. Following genomic DNA preparation and quantitation, molecular screening of 3,378 individuals (1,795 females and 1,583 males) was performed using the FMF reverse hybridization StripAssay, while the remaining 2,140 patients (1,115 females and 1,025 males) were genotyped using DNA sequencing analysis.
Genomic DNA preparation and quantitation
DNA from 2 ml of peripheral blood that had been collected into ethylenediaminetetraacetic acid (EDTA)-anticoagulated tubes by the standard venipuncture method was extracted using the QIAamp DNA Blood Isolation kit (Qiagen GmbH, Hilden, Germany) following the manufacturer’s instructions. The DNA concentration was determined using a Thermo Scientific NanoDrop spectrophotometer (Wilmington, USA).
MEFV Gene Molecular Analysis
FMF StripAssay: Reverse Hybridization Multiplex PCR (Polymerase Chain Reaction)
The FMF StripAssay reverse hybridization assay was performed according to the manufacturer’s instructions (FMF StripAssay, Viennalab Labordiagnostika GmbH). Briefly, in the first step, multiplex PCR was performed using biotinylated primers for exons 2, 3, 5, and 10. PCR products were selectively hybridized to a test strip presenting a parallel array of allele-specific oligonucleotide probes that included 12 common MEFV mutations (E148Q, P369S, F479L, M680I [G/C], M680I [G/A], I692del, M694V, M694I, K695R, V726A, A744S, and R761H).
PCR and DNA Sequencing Analysis
The 4 regions (exons 10, 5, 3 and 2) examined for MEFV mutations were analyzed by PCR amplification followed by automated DNA sequence analysis. One microliter (100 ng) of genomic DNA was added to amplification buffer containing 20 mM Tris (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 0.2 mM each of dATP, 2’-deoxycytidine 5’-triphosphate, dGTP, and 2’-deoxythymidine 5’-triphosphate; 10 pmol each of reverse and forward primers provided by Invitrogen; and 1.0 U of PlatiniumTaq DNA Polymerase (Invitrogen, Carlsbad, CA) in a total volume of 25 µl. The cycling conditions included a hot-start denaturation step at 95°C for 10 min, followed by 35 amplification cycles of denaturation at 95°C for 30 s, annealing at 61°C for exon 10, 58°C for exons 2 and 3, or 57°C for exon 5 for 40 s, and elongation at 72°C for 45 s; a final extension was performed at 72°C for 7 min (the oligonucleotide sequences are available upon request). Prior to sequencing, PCR products were purified using an ExoSAP-IT PCR Product Clean-Up kit. BigDye Terminator chemistry (Applied Biosystems) was used in cycle sequencing reactions. Cycle sequencing of PCR products followed purification with the BigDye Terminator v3.1 Cycle Sequencing Kit (ABI PRISM, San Diego, California, USA), and the data were analyzed using an ABI 3130xl Genetic Analyzer. DNA sequencing was performed in both directions, initiated from the forward and reverse primers that were used in the initial PCR reaction. SeqScape 2.0 sequence analysis software (ABI PRISM, San Diego, California, USA) was employed for sequence evaluation. Samples that were determined to be mutation-negative by the FMF StripAssay were further analyzed by DNA sequencing. Additionally, DNA sequencing was performed for exons 1, 7, 8 and 9 of the MEFV gene for patients who lacked mutations in exons 2, 3, 5, and 10.
Results
Two separate techniques for the detection of mutations in the MEFV gene were used in two distinct patient groups. We analyzed 12 point mutations by the FMF StripAssay and entire regions by DNA sequencing. The frequencies of the 12 common mutations were similar between the two assays, with minor differences being due to sample size and the techniques used.
FMF StripAssay Analysis Results
Of the 3,378 patients enrolled in the StripAssay group, only 1,674 (49.5%) were found to carry at least one of the 12 detectable mutations, whereas the remaining 1,704 (50.5%) individuals did not carry any mutation (Table 1). Among the group of carriers of MEFV mutations, for a single mutation, 1,091 were heterozygous, 205 were homozygous, and 372 were compound heterozygotes. Five different complex MEFV genotypes were identified in 6 patients. Carriers of a single mutation in the homozygous or heterozygous state among the patient group were found with remarkable frequency (1,296 patients). In this group, the most common genotype was M694V/Wt (43%), followed by E148Q/Wt (19%), V726A/Wt (11%), and M680I(G/C)/Wt (11%). Among the analyzed mutations, the most common was the P369S mutation (4.6%), followed by R761S (2.7%), K695R (2.3%), M680I(G/A) (2.2%), both F479L and M694I (0.7%), A744S (0.2%), and I692del (0.04%) (Table 2). Remarkably, rare homozygous F479L mutations were identified in 3 patients. Individuals who were determined to be mutation-negative by the FMF StripAssay were further examined by DNA sequencing.
DNA Sequencing Analysis Results
DNA sequencing analysis revealed novel coding and noncoding sequence variants in exons 2, 3, 5, 9, and 10 of MEFV that were not found in any of the 500 chromosomes analyzed in the healthy controls. Novel sequence variants found among the 2,140 FMF patients were as follows: p.R151S (c.453G>C, exon 2); p.G340R (c.1018G>C, exon 3); p.P350R (c.1049C>G, exon 3); p.E456D (c.1368A>C, exon 5); p.Y471X (c.1413C>A, exon 5); p.S503C (c.1508C>G, exon 5); p.I506V (c.1516A>G, exon 5); p.P588P (c.1764G/A, exon 9); p.K695N (c.2085G>C, exon 10); and p.L709R (c.2126T>G, exon 10).
The distribution of MEFV genotypes was stratified according to the existence of common and rare mutations, as depicted in Table 3. Missense disease-causing mutations and synonymous polymorphisms accounted for 37% (802/2,140) and 55% (1,174/2,140) of MEFV chromosomes, respectively. Mutations in both alleles were identified in 267 of the 802 (33%) individuals with missense mutations, of whom 82 were homozygous for the same mutation, while 185 were compound and complex heterozygous for various combinations of mutations. Additionally, 535 patients were found to be heterozygous for a single mutation. DNA sequence analysis revealed no disease-causing mutations or synonymous/non-synonymous polymorphisms in 164 patients (7%). Table 4 depicts the distribution of 1,065 mutant alleles among 802 patients. M694V accounted for the majority of FMF chromosomes (38%), followed by E148Q (21%), V726A (11%), M680I (10%), P369S (4%), R408Q (3%), K695R (2%), M694I and R761H (1.6%), A744S (1.4%), and F479L (0.09%). Among the mutation-positive patients, the total homozygosity was 10%, and the total heterozygosity was 66%. The total compound and complex heterozygosity was 23%. Among the homozygous group, the homozygosity of M694V was 68%, followed by M680I at 13%, E148Q at 8.5%, and V726A at 2.4%. The most frequent compound heterozygous mutation was M680I/M694V (3.8%). The homozygosity of M694V (58 patients) was not more frequent than the conjunction of this mutant allele with other alleles on the other chromosome (67 patients). Sequence analysis also revealed that 55% of the patients (1,174/2,140) shared single nucleotide polymorphisms (SNPs) that were not known to cause disease, either alone or combined with other SNPs.
In addition, 612 patients from the 802 members of the mutation-positive group (76%) were found to have a single mutation (only one mutated site in MEFV), and while 76 of them (12%) were unique, 536 of the 612 (87%) shared 16 common SNPs, all of which occur in the coding region and include the following: exon 2- D102D, D103D, P124P, G138G, A165A, P180P, and R202Q; exon 3- R314R; exon 5- E474E, Q476Q, R501R, I506I, and D510D; exon 9- P588P; and exon 10- P706P. Nevertheless, 458 of the 535 individuals with single heterozygous mutations, 143 of the 185 with compound-complex heterozygous mutations and 78 of the 82 with homozygous mutations were found to present with accompanying SNPs near mutations. With 679 patients with accompanying SNPs and 1,174 patients with solo SNPs, a total of 1,853 concomitant SNP alleles was found in 88% of the study group.
Among the Turkish general population, the most frequent healthy heterozygous carrier mutation was E148Q (6.9%), and the carrier rate was 16%, with a mutation frequency of 8% (Table 5). FMF carrier frequencies were remarkably high in all populations examined. The frequencies of the four most common MEFV mutations in different ethnic groups within the general population are shown in Table 6. The frequencies of E148Q, V726A, M694V, M680I, M694I, and A744S in the Turkish general population were 0.038 (19/500) (95% CI=0.02-0.06), 0.012 (95% CI=0.0-0.02), 0.01 (95% CI=0.0-0.01), 0.008 (95% CI=0.0-0.02), 0.006 (95% CI=0.0-0.01), and 0.006 (95% CI=0.0-0.01), respectively. E148Q was the most common mutation in healthy carriers in this study, consistent with the results of studies performed in Jewish and Arab populations (p<0.0001).
A comparison of the distribution of MEFV mutations between FMF patients and healthy carriers is depicted in Table 7. E148Q was the most common mutation identified among healthy carriers and the second most common among FMF patients (p<0.0001). With regard to founder mutation distributions in different ethnic groups (Table 8), the most frequent mutation was M694V in all studied groups. E148Q was the second most common mutation identified in the present study, in the Turkish FMF study group and Armenians (p<0.0001). Additionally, nearly 80% of the individuals found to be mutation-negative by the FMF StripAssay were discovered to have sequence variants (disease- causing mutation/synonymous/non-synonymous polymorphism) that were not detected by the StripAssay (data not shown).
Discussion
In this study, to increase the chances of detecting uncommon and novel mutations, we enlarged the patient group to include atypical and probable FMF cases involving relatives of patients screened according to published criteria (Livneh et al., 2007). Because all possible mutations, including novel and rare variants, are detected by DNA sequencing, which is more comprehensive than the 12 primary mutations indexed by the StripAssay, all comparisons were made using the DNA sequencing results. In this assay, the most frequent mutation was M694V/M694V (37%), but the detection frequency for this mutation was different from those of the StripAssay (43.9%) used in this study and from other recent studies (51.4% in Turkish FMF Study Group data, 2005; 57% by Touitou et al., 2001; 52.2% by Ozturk et al., 2008; 36.1% by Solak et al., 2008; 20.7% by Sahin et al., 2008; 51.6% by Yılmaz et al., 2001). The apparent differences in the distribution of the same mutation in Turkish patients are thought to be the result of different sample sizes and techniques and are possibly due to the increasing rate of non-consanguineous marriages in Turkey. The second most common mutation was E148Q, which is consistent with the results for Armenians (Sarkisian et al., 2005) but contrary to the findings in the Turkish FMF Study Group data (2005). R761H and M694I were the most frequent rare mutations. M694I was rare in Turkish patients, consistent with data from Jews and Armenians but inconsistent with data from Arabs. The restriction of the R761H mutation to Turkish FMF patients was previously reported (Dode et al., 2000; Medlej-Hashim et al., 2005; Sarkisian et al., 2005; Stoffman et al., 2000; Sabbagh et al., 2008; Demirkaya et al., 2008). Among the rare mutations detected by DNA sequencing analysis, L110P, E230K, and R241K were homozygous in three patients.
Additionally, sequence analysis revealed that there was a single FMF-associated mutation in the MEFV coding region of 76% of the Turkish individuals studied, and 80% of these individuals initiated colchicine treatment following molecular diagnosis. The prevalence of a single mutation in patients experiencing a pathogenic effect in Turkey (76%) is contrary to the expected pattern of autosomal recessive inheritance and does not support the ‘’heterozygous advantage’’ selection theory. However, the expression of the FMF phenotype may be influenced by other candidate modifier gene loci, autoinflammatory pathway genes or FMF-like diseases (Booty et al., 2009; Ozen S., 2009). For this reason, genome-wide association studies involving more patients should be performed and the data included in future investigations covering critical coding and noncoding gene SNPs for Turkish FMF patients.
The majority of FMF patients in classically affected populations are screened by routine methods that are limited to the detection of common mutations. These tests primarily target the most prevalent MEFV mutations to rule out asymptomatic cases in at-risk populations. Therefore, while searching for the common mutations that underlie typical FMF symptoms, we should primarily consider the entire coding sequence of the MEFV gene before analyzing other recurrent fever genes. In conclusion, by using sequencing analysis, we can prevent less common, population-restricted, novel sequence variants from being overlooked. This has implications for the characterization of typical and atypical FMF; screening for the most common mutations by routine methods is sufficient for the initial laboratory diagnosis of FMF in Turkish patients; however, the results should be confirmed by specific DNA sequencing of all coding exons and exon-intron flanking regions.