Multiplex Molecular Diagnosis of MEFV Mutations in Patients with Familial Mediterranean Fever by LightCycler Real-Time PCR

Familial Mediterranean fever (FMF) is an autoinflammatory disease inherited as an autosomal recessive condition and characterized by recurrent episodes of fever, abdominal pain, pleuritis, and arthritis (1)(2). The most dangerous manifestation is renal amyloidosis and end-stage renal failure. Many researchers have associated the M694V mutation (3)(4)(5) as well as the α allele of serum amyloid A protein I with significantly higher risk for amyloidosis (6).FMF is very prevalent in non-Ashkenazi Jews, Armenians, Arabs, and Turks, all of whom have ancestors of Mediterranean origin. The carrier frequency in these populations can reach up to 1:5, rendering it the most frequent autosomal recessive condition. Currently, it is considered frequent in other Mediterranean countries, including Cyprus (7)(8)(9)(10)(11). The gene associated with FMF, MEFV , which encodes pyrin/marenostrin, a protein of 781 amino acids (12)(13), belongs to the pyrin family of genes that play a role in autoinflammatory diseases and inflammatory pathways (14)(15). More than 40 mutations have been identified, but 4–6 mutations usually account for a high percentage of MEFV chromosomes in different ethnic populations, emphasizing the importance of molecular epidemiologic studies for identifying the various molecular defects in the population of interest. The detection of known mutations in MEFV can be accomplished by various methods, including direct DNA sequencing. These methods are time-consuming and prone to carryover PCR contamination because of the repetitive work in a diagnostic setting. Using fluorescence resonance energy transfer (FRET) on a LightCycler® instrument (Roche), we developed a rapid method that detects point mutations and small deletions by use of fluorescent hybridization probes. Because of the close proximity of several mutations on exon 10, we designed a single pair of fluorescent probes with multiplex melting profiles that permits …