Familial Mediterranean fever with a single MEFV mutation: where is the second hit?

Abstract

Objective: Familial Mediterranean fever (FMF) has traditionally been considered an autosomal-recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only 1 demonstrable MEFV mutation. The purpose of this study was to perform an extensive search for a second MEFV mutation in 46 patients diagnosed clinically as having FMF and carrying only 1 high-penetrance FMF mutation.

Methods: MEFV and other candidate genes were sequenced by standard capillary electrophoresis. In 10 patients, the entire 15-kb MEFV genomic region was resequenced using hybridization-based chip technology. MEFV gene expression levels were determined by quantitative reverse transcription-polymerase chain reaction. Pyrin protein levels were examined by Western blotting.

Results: A second MEFV mutation was not identified in any of the patients who were screened. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between patients with a single mutation and those with a double mutation; however, FMF patients of both types showed higher protein expression as compared with controls and with non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare mutations in a small number of patients, suggesting the possibility of digenic inheritance.

Conclusion: Our data underscore the existence of a significant subset of FMF patients who are carriers of only 1 MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of the most common mutations and detection of a single mutation appears to be sufficient in the presence of clinical symptoms for the diagnosis of FMF and the initiation of a trial of colchicine.

Figure 1

Mean relative expression levels of MEFV (exons 9–10) comparing FMF patients with 1 MEFV mutation, patients with 2 mutations, and healthy controls. All results are relative to the mean of the healthy control group. No significant difference in expression level was observed between any of the groups. Values represent fold change and were generated using β2 microglobulin (B2M) as the endogenous control. Error bars indicate standard error of the mean.

Figure 2

A. Western blots of granulocyte lysates from FMF patients with one or two MEFV mutations and healthy controls probed with Abs to pyrin or GAPDH. B. Densitometry analysis of pyrin bands. Values represent pyrin expression relative to GAPDH in each individual and are shown as pyrin’s percentage of GAPDH (pyrin/GAPDH×100). Values are normalized to the mean pyrin expression in the healthy control group within each individual blot. Patient groups exhibited significantly higher levels of pyrin as determined by a Mann-Whitney U test (P = 0.007). There was no significant difference in pyrin levels between patients with one or two MEFV mutations. Error bars indicate standard error of the mean.

Figure 3

A. Western blots of granulocyte lysates from non-FMF patients with active inflammation (ID), FMF patients, and healthy controls probed with Abs to pyrin or GAPDH. B. Data analysis was done as described in Figure 2. FMF patients exhibited significantly higher levels of pyrin as determined by a Mann-Whitney U test (FMF vs. patients with active inflammation P=0.0286, FMF patients vs. controls P=0.0095). There was no significant difference in pyrin levels between patients with active inflammation and controls. Error bars indicate standard error of the mean.

Figure 4

A modeled structure for the CARD domain of ASC (residues 109–195). This homology model, based on the CARD domain structures of RAIDD and Apaf-1, describes the ASC CARD domain as a 6-helix structure adopting a Greek key fold. The mutation W171X would truncate the final two alpha-helices from the C-terminus, and these are highlighted in blue.