Analysis of mefv variations, expression and pyrin levels in familial mediterranean fever disease

Familial Mediterranean Fever (FMF) is an autosomal recessively inherited autoinflammatory disease which is characterized mainly by periodic attacks of recurrent fever, serotisis, joint, chest, and abdominal pain. The vast majority of FMF patients belong toTurkish, Armenian, Arab, or North African Jew. Mediterranean fever gene (MEFV) is located on the short arm of chromosome 16 at position 13.3 and consists of ten exons. More than 300 sequence variations have been reported in the gene and majority of exonic variations are associated with the disease. The most common variations that appear to be present in more than 70% of patients are located in exon 2 (E148Q, R202Q) and in exon 10 (M680I, M694V, M694I, and V726A) of the MEFV gene. However, screening of MEFV pathological variations in patients to improve diagnosis is not enough to definitely define FMF in mentioned populations with high prevalence, due to high carrier rate of the variations. Moreover, there are FMF patients showing typical clinical manifestation who do not have any identified MEFV pathological variation. This finding is in contradiction with a pattern that is observed in a typical autosomal recessive Mendelian inherited disease. To this end we have been studying the transcriptional and related epigenetic regulation of the MEFV gene. Our initial findings suggested that exon 2 methylation of the MEFV gene produces an alternatively spliced variant, and in cell culture models and in patients leucocytes which further translates into protein isoform in the cytoplasm. Full-length MEFV encodes Pyrin/Marenostrin protein formed by 781 amino acids (a.a.). MEFV-d2 (MEFV Δ2) is one of the isoform of MEFV gene encoding 570 a.a. protein in length and it is formed by removal of exon 2. There are also other identified pyrin isoforms. In this thesis, we wanted to study and correlate MEFV gene variations with its transcript and protein levels in FMF patients and controls. Previous studies have shown that MEFV gene is expressed in dendritic cells, synovial fibroblasts, neutrophils, eosinophils and cytokine activated monocytes. Recently, it has been shown that MEFV gene expression level is associated with type of the MEFV mutations. It has been shown that FMF patients have lower mRNA level than healthy controls and in healthy carriers level of the MEFV transcripts were intermediate which indicates association of number of mutations with abundance of mRNA. Furthermore, type of the mutations was shown to alter level of MEFV transcript. It was found that M694V mutation, which leads to severe disease, has lowest mRNA expression when compared with other mutations and number of M694V mutations affects the mRNA level in the manner of decreasing. It was also shown that in comparison with other mutations, mRNA expression level is highest in the patients carrying E148Q mutation giving rise to milder disease. Our previous published studies have also shown that MEFV transcript levels overall decreases in FMF patients, however exon 2 alternatively spliced transcript increases compared to controls. Nonetheless, the role of pyrin in the disease and inflammation has not been clarified yet. In this context, the aim of the study is to analyze MEFV gene expression and pyrin levels in FMF patients having different genotypes using case-control methodology. To this end, MEFV variation analysis was conducted in 69 FMF patients and 28 healthy controls in order to determine genotype of the study group. 8/69 (11%) of the FMF patients have no pathogenic variations; whereas 30 (43%) patients have variations within exon 2 region of the gene, and 31 (44%) patients have pathogenic variations within exon 10 region of the gene, which are specified as pathogenic. Expression of alternatively spliced MEFV transcripts were also quantified in leukocytes of all samples using SYBR-Green based quantitative Real-Time PCR method with 3 sets of primers targeting the junction of exon 1-3, exon 2-3, and exon 4-5. In order to establish differences of pyrin levels between the patients having different genotypes and healthy individuals, western blot analysis was also conducted with anti-pyrin antibodies using proteins isolated from leukocytes. Semi quantitative analyses of the gels were performed with volume densitometry using Image Lab Software. In contrast to previous studies, no differences in the level of MEFV gene expression have been found between patients and healthy controls. Variations in the study design and amplified regions of the gene imply that differential expression of splice variants in different studies may lead to variable results about expression pattern of the gene. However, it is also important to note that sample size of the present study is larger than the previous studies. In order to study the role of MEFV gene at the level of pyrin expression, pyrin levels of 48 FMF patients, mostly with pathological variations, were also compared with 24 healthy individuals and significantly higher pyrin levels were found in FMF patients (p=0,0092) indicating high level of pyrin protein might lead to disease phenotype in FMF patients having recurrent inflammatory attacks. The findings in this thesis support the hypothesis for the function of pyrin as an activator of inflammation. In the present study, no correlation between MEFV transcript level and pyrin level was found. This finding was thought that post-transcriptional modifications might have a role in FMF. All together, the present study suggests that MEFV gene might contributes FMF phenotype at protein level. As a future direction, cell type specific analysis should be applied at both transcript and protein levels; additionally, transcript and protein isoforms should be studied separately. Furthermore, including patients in the study during in attack periods and investigation of other genetic and epigenetic factors will lead us better understanding about pathogenesis of FMF.