Reverse hybridization vs. DNA sequencing in the molecular diagnosis of Familial Mediterranean fever

Familial Mediterranean fever (FMF) is an autosomal recessive inflammatory disorder predominantly affecting people living in or originating from areas around the Mediterranean Sea. It is caused by a number of mutations within the MEFV gene, which differently affect the severity of the disease phenotype. Because patients usually present with rather nonspecific clinical symptoms, MEFV genotyping can confirm and refine FMF diagnosis and improve treatment of affected individuals. We have performed a method comparison study on 100 Lebanese FMF patients to evaluate the potential of a rapid reverse-hybridization teststrip-based assay (FMF StripAssay) to serve as a first-line screening test for our population. When results obtained by reverse-hybridization and DNA sequencing of exons 2, 3, 5, and 10 were compared, the FMF StripAssay identified 144/149 mutations, and correctly typed all 12 different MEFV mutations covered. We conclude that reverse-hybridization provides a very rapid, accurate and easy-to-perform screening method, and, in combination with more comprehensive diagnostic methods, represents an efficient strategy for FMF genotyping.     

Prevalence and distribution of MEFV mutations among Arabs from the Maghreb patients suffering from familial Mediterranean fever

Familial Mediterranean fever (FMF) is an autosomal recessive inherited disease caused by mutations in MEFV. This disease is characterized by recurrent episodes of fever accompanied with topical signs of inflammation. Some patients can develop renal amyloidosis. We prospectively investigated MEFV mutations in a cohort of 209 unrelated Arab patients from Maghreb (85 Algerians, 87 Moroccans, and 37 Tunisians) with a clinical suspicion of FMF. FMF is the main cause of periodic fever syndrome in Maghreb. The most frequent MEFV mutations in this cohort were M694V and M694I. These mutations account for different proportions of the MEFV mutations in Algeria (5%, 80%), Morocco (49%, 37%), and Tunisia (50%, 25%) patients. M694I mutation is specific to the Arab population from Maghreb. Other rare mutations were observed: M680L, M680I, A744S, V726A, and E148Q. We estimated the frequency of MEFV mutation carriers among the Arab Maghrebian population at around 1%, which is significantly lower than in non-Ashkenazi Jews, Armenians or Turks.     

Mutational analysis of the PRYSPRY domain of pyrin and implications for familial mediterranean fever (FMF)

Familial Mediterranean fever (FMF) is an autosomal, recessively inherited disease, characterized by recurrent fever and serositis that affects mainly patients of the Mediterranean basin. The gene responsible for FMF, named MEFV, was cloned and several missense mutations were found to be responsible for the disease. Based on a recent molecular analysis of MEFV gene mutations in 43 patients from Crete aiming to correlate specific genotypes and clinical manifestations of FMF, we were prompted to construct a three-dimensional model (3-D model) of the PRYSPRY domain of pyrin. The majority of the known MEFV mutations located on this domain have been classified, according to disease severity, and localized on this 3-D model. The functional consequences of these mutations and their implications on disease severity are discussed. Moreover, we report a putative novel missense mutation, S702C, which we identified in exon 10 of the MEFV gene and localized on the constructed 3-D model.     

Two novel mutations R653H and E230K in the Mediterranean fever gene associated with disease

Familial Mediterranean fever (FMF) is an autosomal recessive disorder caused by mutations in the Mediterranean fever gene (MEFV). We describe two novel missense mutations in MEFV, R653H and E230K. Both were found in compound heterozygosity with the mutation M694V in single Turkish patients with clinical syndromes characteristic for FMF. DNA sequencing and PCR-RFLP typing of the families confirmed the mutations and verified recessive modes of inheritance.     

Mutation and haplotype studies of familial Mediterranean fever reveal new ancestral relationships and evidence for a high carrier frequency with reduced penetrance in the Ashkenazi Jewish population

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever with serositis or synovitis. The FMF gene (MEFV) was cloned recently, and four missense mutations were identified. Here we present data from non-Ashkenazi Jewish and Arab patients in whom we had not originally found mutations and from a new, more ethnically diverse panel. Among 90 symptomatic mutation-positive individuals, 11 mutations accounted for 79% of carrier chromosomes. Of the two mutations that are novel, one alters the same residue (680) as a previously known mutation, and the other (P369S) is located in exon 3. Consistent with another recent report, the E148Q mutation was observed in patients of several ethnicities and on multiple microsatellite haplotypes, but haplotype data indicate an ancestral relationships between non-Jewish Italian and Ashkenazi Jewish patients with FMF and other affected populations. Among approximately 200 anonymous Ashkenazi Jewish DNA samples, the MEFV carrier frequency was 21%, with E148Q the most common mutation. Several lines of evidence indicate reduced penetrance among Ashkenazi Jews, especially for E148Q, P369S, and K695R. Nevertheless, E148Q helps account for recessive inheritance in an Ashkenazi family previously reported as an unusual case of dominantly inherited FMF. The presence of three frequent MEFV mutations in multiple Mediterranean populations strongly suggests a heterozygote advantage in this geographic region.     

Comparison of amplification refractory mutation system and polymerase chain reaction-restriction fragment length polymorphism techniques used for the investigation of MEFV gene exon 10 point mutations in familial Mediterranean fever patients living in Cukurova region (Turkey)

Familial Mediterranean fever (FMF) is an autosomal recessive inherited disease characterized by recurrent fever, serositis and arthritis. The disease is highly prevalent in Mediterranean basin populations. Recently, the gene responsible for FMF (MEFV) was cloned and at least 40 MEFV gene mutations have been identified. The most frequently observed mutations in the MEFV gene are M694V, M694I, M680I, and V726A. These occur within exon 10 of the gene, and account for 85% of the known MEFV alleles. In this study, the reliability and economical aspects of amplification refractory mutation system (ARMS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques were compared for analyzing the frequencies of the major point mutations of 90 unrelated patients with FMF from the Cukurova region in Turkey. Both techniques yielded similar results: The ratio of independent alleles of 90 patients carrying one of the tested mutations was 81.1%; patients consisted of 12 different genotypes. In 64 of 90 patients (71.1%) mutations were observed in both alleles. Thirty-six patients (40%) were homozygous for the same mutation, 28 (31.1%) were heterozygous for different mutations. Eighteen patients (20%) were heterozygous for one allele with one of the four mutations but the other allele was unknown. In 8 patients (8.8%) no mutation could be detected. The most frequently observed mutation was M694V (51.66%), followed by M680I (17.22%), V726A (10.55%), and M694I (1.66%). In conclusion ARMS and PCR-RFLP techniques were equally reliable to detect the mutations in Turkish FMF patients. However, the ARMS technique was found to be more rapid and economical than the PCR-RFLP techniques.     

Screening of common and novel familial mediterranean fever mutations in south-east part of Turkey

Familial mediterranean fever (FMF) is an autosomal recessive autoinflammatory disorder (MIM# 249100), particularly common in populations of Mediterranean extraction. MEFV gene, responsible for FMF, encoding pyrin has recently been mapped to chromosome 16p13.3. In the present study, 3,341 unrelated patients with the suspicion of FMF in south-east part of Turkey between the years 2009 and 2013 were enrolled and genomic sequences of exon 2 and exon 10 of the MEFV gene were scanned for mutations by direct sequencing. We identified 43 different type of mutations and 9 of them were novel. DNA was amplified by PCR and subjected to direct sequencing for the detection of MEFV gene mutations. Among the 3,341 patients, 1,598 (47.8 %) were males and 1,743 (52.1 %) were females. The mutations were heterozygous in 806 (62.3 %), compound heterozygous in 188 (14.5 %), homozygous in 281 (21.8 %) and mutations had complex genotype in 17 (1.32 %) patients. No mutation was detected in 2,051 (61.4 %) patients. The most frequent mutations were M694V, E148Q, M680I(G/C) and V726A. We could not find any significant differences between the two common mutations according to the gender. Molecular diagnosis of MEFV is a useful tool in clinical practice, thus a future study relating to genotype/phenotype correlation of FMF in more and larger group in Turkish population involving the whole MEFV gene mutations is necessary.     

Frequency of alterations in the MEFV gene and clinical signs in familial Mediterranean fever in Central Anatolia, Turkey

Familial Mediterranean fever is a recessive autoinflammatory disease that is frequent in Armenians, Jews, Arabs, and Turks. The MEFV gene is responsible for this disease. We looked for MEFV gene variations (polymorphism and mutations) in a population that resides in Central Anatolia, Turkey. DNA was extracted from peripheral blood leukocytes of 802 familial Mediterranean fever patients. The DNA sequence data were examined for approximately 150 different mutations and polymorphisms, including single nucleotide polymorphisms in different exons of the MEFV gene. The male:female ratio of these patients was 1.44:1. Mutations were detected in 48.1% of the patients; 7.5% were homozygous, 11.1% were compound heterozygous and 31.5% had only one identifiable mutant allele. No mutations were detected in 51.9% of the patients. The main clinical characteristics of the patients were: abdominal pain in 20.6%, arthritis in 22.9% and amyloidosis in 4.6%. Sixty-six percent of patients had a family history of familial Mediterranean fever; 19.4% of the patients were found to have parental consanguinity. We conclude that the genetics of familial Mediterranean fever is more complex than has previously been reported; heterozygous patients presenting a severe phenotype should be further analyzed for less common secondary MEFV mutations, using gene sequencing.     

MEFV-Gene analysis in armenian patients with Familial Mediterranean fever: diagnostic value and unfavorable renal prognosis of the M694V homozygous genotype-genetic and therapeutic implications.

Familial Mediterranean fever (FMF) is a recessively inherited disorder that is common in patients of Armenian ancestry. To date, its diagnosis, which can be made only retrospectively, is one of exclusion, based entirely on nonspecific clinical signs that result from serosal inflammation and that may lead to unnecessary surgery. Renal amyloidosis, prevented by colchicine, is the most severe complication of FMF, a disorder associated with mutations in the MEFV gene. To evaluate the diagnostic and prognostic value of MEFV-gene analysis, we investigated 90 Armenian FMF patients from 77 unrelated families that were not selected through genetic-linkage analysis. Eight mutations, one of which (R408Q) is new, were found to account for 93% of the 163 independent FMF alleles, with both FMF alleles identified in 89% of the patients. In several instances, family studies provided molecular evidence for pseudodominant transmission and incomplete penetrance of the disease phenotype. The M694V homozygous genotype was found to be associated with a higher prevalence of renal amyloidosis and arthritis, compared with other genotypes (P=.0002 and P=.006, respectively). The demonstration of both the diagnostic and prognostic value of MEFV analysis and particular modes of inheritance should lead to new ways for management of FMF-including genetic counseling and therapeutic decisions in affected families.     

Mediterranean fever gene mutations: correlation with cytotoxic T‐lymphocyte‐associated antigen 4 gene polymorphism

Mutations in the Mediterranean fever (MEFV) gene lead to familial Mediterranean fever (FMF), a pro‐inflammatory state characterized by outbursts of inflammatory cytokines. The aims of this study were to identify the common mutations of MEFV gene in Egyptian patients with FMF, to study cytotoxic T lymphocyte associated antigen 4 (CTLA‐4) gene polymorphism and to evaluate correlations between CTLA4–1661 polymorphisms and MEFV mutations and clinical symptoms. Four hundred and twenty‐four patients with clinical pictures suspicious of FMF were enrolled in this study. Mutations in MEFV gene were confirmed by reversed hybridization. Patients with homozygous and compound heterozygous mutations and 120 healthy controls were investigated for polymorphism of ?1661 CTLA4 gene and the findings correlated with disease incidence and clinical symptoms of the disease. Ninety‐seven patients had single heterozygous mutations and 78 had compound heterozygous or homozygous MEFV gene mutations. M694I/V726A was the most common genotype (14.1%), followed by homozygous M694I. There was no statistically significant difference between patients and controls in incidence of ?1661 A/G single nucleotide polymorphism CTLA4 (P = 0.189), nor any significant correlation with any of the clinical symptoms of FMF and MEFV gene mutations.

     

Common Familial Mediterranean Fever gene mutations in a Turkish cohort

Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disorder with the responsible gene of MEFV which primarily affects Jewish, Armenian, Turkish and Arab populations. The FMF gene (MEFV) has recently been cloned to chromosome 16p, which encodes pyrin. In the present study, we enrolled 2,067 unrelated patients with the suspicion of FMF in Middle Anatolia between the years 2006–2009 and identified the 12 MEFV mutations. DNA was amplified by PCR and subjected to reverse hybridization for the detection of MEFV gene mutations. Among the 2,067 patients, 866 (41.9%) were males and 1,201 (58.1%) were females. The mutations were homozygous in 176 (16.85%) patients, compound heterozygous in 314 (30.1%) patients, heterozygous in 546 (52.25%) patients and the other forms of mutations were found in 8 patients (0.76%). No mutation was detected in 1,023 (49.5%) patients. The most frequent mutations were M694V, M680I (G/C), E148Q and V726A. We could not find any significant differences between the two common mutations according to the gender. The high incidence of MEFV gene mutations in the Turkish population indicated that newborn screening may be discussed in the future. Because of the ethnic origin of Anatolia, larger serial analyses are necessary to investigate the rate and coexistence of these mutations.     

Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5'-Cyclopurine-2'-deoxynucleosides and 8-hydroxy-2'-deoxyguanosine

Mutations in breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 predispose women to a high risk of these cancers. Here, we show that lymphoblasts of women with BRCA1 mutations who had been diagnosed with breast cancer are deficient in the repair of some products of oxidative DNA damage, namely, 8-hydroxy-2'-deoxyguanosine and 8,5'-cyclopurine-2'-deoxynucleosides. Cultured lymphoblasts from 10 individuals with BRCA1 mutations and those from 5 control individuals were exposed to 5 Gy of ionizing radiation to induce oxidative DNA damage and then allowed to repair this damage. DNA samples isolated from these cells were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure 8-hydroxy-2'-deoxyguanosine, (5'-S)-8,5'-cyclo-2'-deoxyadenosine, (5'-R)-8,5'-cyclo-2'-deoxyguanosine, and (5'-S)-8,5'-cyclo-2'-deoxyguanosine. After irradiation and a subsequent period of repair, no significant accumulation of these lesions was observed in the DNA from control cells. In contrast, cells with BRCA1 mutations accumulated statistically significant levels of these lesions in their DNA, providing evidence of a deficiency in DNA repair. In addition, a commonly used breast tumor cell line exhibited the same effect when compared to a relevant control cell line. The data suggest that BRCA1 plays a role in cellular repair of oxidatively induced DNA lesions. The failure of cells with BRCA1 mutations to repair 8,5'-cyclopurine-2'-deoxynucleosides indicates the involvement of BRCA1 in nucleotide-excision repair of oxidative DNA damage. This work suggest that accumulation of these lesions may lead to a high rate of mutations and to deleterious changes in gene expression, increasing breast cancer risk and contributing to breast carcinogenesis.     

Clinical and molecular analysis of common MEFV gene mutations in familial Mediterranean fever in Sivas population

Familial Mediterranean fever (FMF) is the most frequent hereditary inflammatory disease characterized by self-limited recurrent attacks of fever and serositis. The aim of the current study is to determine the frequency of the mutations in 365 suspected FMF patients and to reveal whether there is a correlation between genotype and phenotype of these patients. All patients were clinically examined according to Tell-Hashomer FMF criteria and were screened genetically in terms of common 12 Mediterranean fever gene (MEFV) mutations. Various point mutations were detected in 270 (74%) patients. The most frequent mutation was M694V (26.85% of the alleles) and was followed by E148Q (15.55%), M680I (G/C) (9.62%) and V726A (7.96%). Patients who bear M694V homozygous mutation had most severe disease phenotype and high risk for amyloidosis (P = 0.04). Our results indicate that Sivas population has a wide range of heterozygous mutated carriers of MEFV gene and there is a high frequency of E148Q allele when compared to the other Mediterranean groups.     

In vitro effect of gliclazide on DNA damage and repair in patients with type 2 diabetes mellitus (T2DM)

Type 2 diabetes mellitus is associated with elevated level of oxidative stress, which is one of the most important factors responsible for the development of chronic complications of this disease. Moreover, it was shown that diabetic patients had increased level of oxidative DNA damage and decreased effectiveness of DNA repair. These changes may be associated with increased risk of cancer in T2DM patients, since DNA damage and DNA repair play a pivotal role in malignant transformation. It was found that gliclazide, an oral hypoglycemic drug with antioxidant properties, diminished DNA damage induced by free radicals. Therefore, the aim of the present study was to evaluate the in vitro impact of gliclazide on: (i) endogenous basal and oxidative DNA damage, (ii) DNA damage induced by hydrogen peroxide and (iii) the efficacy of DNA repair of such damage. DNA damage and DNA repair in peripheral blood lymphocytes of 30 T2DM patients and 30 non-diabetic individuals were evaluated by alkaline single cell electrophoresis (comet) assay. The extent of oxidative DNA damage was assessed by DNA repair enzymes: endonuclease III and formamidopyrimidine-DNA glycosylase. The endogenous basal and oxidative DNA damages were higher in lymphocytes of T2DM patients compared to non-diabetic subjects and gliclazide decreased the level of such damage. The drug significantly decreased the level of DNA damage induced by hydrogen peroxide in both groups. Gliclazide increased the effectiveness of DNA repair in lymphocytes of T2DM patients (93.4% (with gliclazide) vs 79.9% (without gliclazide); P< or =0.001) and non-diabetic subjects (95.1% (with gliclazide) vs 90.5% (without gliclazide); P< or =0.001). These results suggest that gliclazide may protect against the oxidative stress-related chronic diabetes complications, including cancer, by decreasing the level of DNA damage induced by reactive oxygen species.     

Prevalence of known mutations in the MEFV gene in a population screening with high rate of carriers

The Familial Mediterranean Fever (FMF) shows an autosomal recessive pattern of inheritance and affects certain ethnic groups. Disease is caused by mutations in MEFV gene and more than 180 mutations have been defined in affected individuals. Current study aimed to determine the frequency-type of the mutations for MEFV gene in Sivas??middle Anatolian city. The cohort was composed of 3340 patients. MEFV gene mutations were studied by multiplex PCR based reverse hybridization stripAssay method. Patients?? clinical features were; family history: 68%, erysipelas-like erythema: 17.6%, fever: 89.9%, abdominal pain: 84.2%, peritonitis: 90.2%, arthritis: 33%, pleuritis: 14.2%, parental consanguinity: 21.2%. Current results revealed that M694V is the most frequent mutation (43.12%), followed by E148Q (20.18), M680I(G/C) (15.00%) and V726A (11.32%). The study population has a high rate of carriers and the E148Q mutation frequency was found to be highest when compared to the other regions of Turkey and other Mediterranean groups.     

Reprint of: gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.     

<Emphasis Type="Italic">MEFV</Emphasis> gene mutations spectrum among Lebanese patients referred for Familial Mediterranean Fever work-up: Experience of a major tertiary care center

Familial Mediterranean Fever (FMF) is an autosomal recessive inflammatory disorder predominantly affecting people living in or originating from areas around the Mediterranean Sea, mainly Jews, Armenians, Turks, and Arabs. It is characterized by recurrent attacks of inflammation of serosal membranes and fever resulting in acute abdominal, chest, or joint pain. Over 50 MEditerranean FeVer (MEFV) mutations and polymorphisms have been identified in FMF patients. The objective of this study was to analyze the distribution and frequencies of 12 MEFV mutations in 266 referred Lebanese patients using a reverse-hybridization assay. Of the 266 patients, 129 (48.5%) were positive for at least one mutation and 137 (51.5%) had no mutations detected. Of the 129 patients with mutations, 35 were homozygous, 41 were compound heterozygous and 53 were heterozygous. The five most common mutations M694V, E148Q, V726A, M694I and M680I (G/C) accounted for 26.1, 22.2, 21.3, 9.6 and 7.7%, respectively. The A744S, F479L, R761H and I692del were encountered in 2.9% of patients; P369S and M680I (G/A) were found in 1.2% of patients while K695R was absent. The spectrum of the MEFV mutations among our sampled Lebanese FMF patients shows the high heterogeneity at the allelic level when compared to Arab and non-Arab populations. The most important feature was the relatively high frequency of the E148Q in our study group that allows us to question it as a mutation rather than a polymorphism. Further studies should be conducted to evaluate the role of the E148Q allele.     

The role of the pro-apoptotic protein Siva in the pathogenesis of Familial Mediterranean fever: A structural and functional analysis

Familial Mediterranean fever (FMF) is an autosomal, recessive disease, attributed to mutations in MEFV gene encoding pyrin, which is characterized by recurrent, acute and self-limiting attacks of fever as well as an increased neutrophil and monocyte apoptosis. Most disease-associated mutations in MEFV gene reside on the C-terminal PRYSPRY (B30.2) domain of pyrin, an area found to interact with the pro-apoptotic protein Siva. Because apoptotic events may be contributing to endogenous inflammation we hypothesized that mutations in pyrin may affect Siva-mediated apoptosis. The confirmation of this hypothesis would be of a great biological significance since it would be demonstrated a connection between apoptosis and inflammation. We used homology modeling to construct a 3-D model of Siva protein and the constructed model of Siva defined structural elements with potential of binding other proteins to induce apoptosis. Given that Siva protein binds pyrin as shown by transfection and immunoprecipitation experiments, apoptosis was assessed by FACS and Western blotting. No differences in rates of apoptosis in myeloid cells (THP-1) upon transfection with either wt pyrin or mutant forms of pyrin were found. Patients with FMF did not display any mutations in the Siva-1 (full length) gene. Siva-1 was not linked to pyrin in the major predicted FMF gene network constructed using a literature-curated gene signature for FMF. These results suggest that Siva-mediated unprovoked apoptosis is not likely to be involved in the pathogenesis of FMF.     

Gene susceptibility to oxidative damage: from single nucleotide polymorphisms to function

Oxidative damage to DNA can cause mutations, and mutations can lead to cancer. DNA repair of oxidative damage should therefore play a pivotal role in defending humans against cancer. This is exemplified by the increased risk of colorectal cancer of patients with germ-line mutations of the oxidative damage DNA glycosylase MUTYH. In contrast to germ-line mutations in DNA repair genes, which cause a strong deficiency in DNA repair activity in all cell types, the role of single nucleotide polymorphisms (SNPs) in sporadic cancer is unclear also because deficiencies in DNA repair, if any, are expected to be much milder. Further slowing down progress are the paucity of accurate and reproducible functional assays and poor epidemiological design of many studies. This review will focus on the most common and widely studied SNPs of oxidative DNA damage repair proteins trying to bridge the information available on biochemical and structural features of the repair proteins with the functional effects of these variants and their potential impact on the pathogenesis of disease.