Enhanced exon 2 skipping caused by c.910G>A variant and alternative splicing of MEFV genes in two independent cases of familial Mediterranean fever

Abstract

Most reported cases of familial Mediterranean fever (FMF) involve missense mutations of MEFV concentrated within exon 10. We experienced two independent pedigrees of a unique variant in the MEFV gene that might cause excessive exon 2 skipping due to enhanced alternative splicing. In this study, we tried to elucidate the molecular mechanism of the MEFV variant as a cause of the FMF phenotype. Peripheral blood was obtained from volunteers and two patients with homozygous c.910G>A variant of the MEFV gene. MEFV messenger RNA (mRNA) expression patterns in mononuclear cells and granulocytes were compared using forward and reverse primers from exons 1 and 3, respectively. Expression profiles of pyrin were examined by transfecting wild-type and variant MEFV genes into HEK293T cells. Expression of normal-sized mRNA was extremely reduced in these patients, whereas that of aberrant short mRNA, deleting exon 2 (Δex2), was significantly increased. Immunohistochemical and immunoblotting analyses revealed a truncated immunoreactive pyrin protein in cells transfected with Δex2 cDNA. The MEFV gene c.910G>A variant results in accelerated aberrant splicing with abnormal protein size, presumably leading to anomalous pyrin function. This is the first report to show that an MEFV variant other than missense mutation is responsible for the FMF phenotype.

Alternatively spliced MEFV transcript lacking exon 2 and its protein isoform pyrin-2d implies an epigenetic regulation of the gene in inflammatory cell culture models

“…These variations appeared to have a role in the pathogenesis of TRAPS disease ( Rittore et al , 2014 ). Furthermore, in another study, Tone et al (2011) suggested that c.910G > A variant (rs75977701) leads to the skipping of MEFV exon 2. This rare variant, which has a Minor Allele Frequency (MAF) of T = 0.0074/37 in the Thousands Genomes Project, was absent in our FMF cohort.…”

Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants

“…Conversely, defects affecting only a fraction of the transcripts produced and mostly evidenced on sequencing electrophoregrams are more difficult to interpret in terms of disease causality. This is even more problematic when a VUS seems to strengthen an alternative splicing pattern, as inappropriate levels of isoforms can be damaging to the cells [Brezin et al, 2011; Tone et al, 2012]. Quantitative evaluation of partial splicing defects therefore cannot be based on inspection of sequencing electrophoregrams.…”

Role of E148Q in familial Mediterranean fever with an exon 10 mutation in MEFV

“…The MEFV gene underwent direct sequencing, as described previously. 6 Serum concentrations of cytokines were determined using enzyme-linked immunosorbent assay kits: neopterin (IBL, Hamburg, Germany); IL-6; soluble tumor necrosis factor (TNF) receptor type 1 (sTNF-RI) and type 2 (sTNF-RII; R&D Systems, Minneapolis, MN, USA); and IL-18 (MBL Co., Ltd, Nagoya, Japan). 7 All samples were collected during the afebrile phase in patients with FMF and their family members.…”