P.4.13 Central core disease (CCD): Improving the screening for mutations in RYR1 gene

Abstract

Central core disease (CCD) is a congenital myopathy, characterized by the presence of central core-like areas in muscle fibers. Patients with RYR-related CCD usually have mild or moderate axial and proximal weakness, hypotonia and motor developmental delay. CCD is associated with susceptibility to malignant hyperthermia (MH), and both conditions have been linked to mutations in human RYR1 gene, which encodes a calcium release channel known as ryanodine receptor (RyR1). RYR1 mutational spectrum linked with CCD includes more than 200 described mutations mostly heterozygous dominant missense mutations and minor deletions or duplications. Definite diagnosis of CCD is done by clinical history and evaluation, histopathological analyses of muscle biopsy and, recently, genetic testing of gene’s “hot spots”, because RYR1 is a large gene and direct sequencing of each exon is laborious. Here, we tested Multiplex Ligation-dependent Probe Amplification (MLPA) as an alternative and simple method for screening patients with histological diagnosis or family history of CCD. This technique uses high-performance PCR to quantify up to 45 transcripts in small human DNA samples. It is based on annealing of two hemi-probes into its complementary sequence and was designed to detect the 33 most common mutations in RYR1 gene. From 41 patients diagnosed with CCD in three different institutions, 24 (58,5%) had mutations found in direct sequencing and 11 (26,8%) in MLPA. Seventeen patients (41,5%) had consistent results: 6 (14,6%) had their mutation diagnosed with sequencing and MLPA and neither method was able to find mutations in 11 patients (26,8%). Mutations in exon 101 were the most prevalent in sequencing (11 patients) and MLPA (4 patients). MLPA had shown to be a relevant tool to help diagnosing CCD; moreover, it is especially useful in diagnosing family members of patients with known mutations detectable by MLPA. Financial support: FAPESP-CEPID, CNPQ-INCT, FINEP.