Key result
A novel multiplex fluorescent primer-extension method successfully detected 4 common dysferlin gene mutations in approximately 8 hours with complete concordance to direct sequencing.
A newly developed multiplex fluorescent primer extension method provides a rapid, 8-hour screening tool for the four most common dysferlin gene mutations in Japanese patients.
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May accelerate dysferlinopathy screening in Japanese patients; leaves open broader validation before clinical adoption.
OBJECTIVE: Mutations in the dysferlin gene cause limb-girdle muscular dystrophy (LGMD) 2B and Miyoshi myopathy (MM), which are collectively named dysferlinopathy. Dysferlinopathy is the most frequent type of LGMD in the Japanese population. Molecular genetic analysis is essential for the diagnosis of dysferlinopathy because of its variable immunohistochemical patterns of biopsied muscles, including patterns similar to normal controls. The analysis of the entire dysferlin gene however, is time-consuming and laborious; therefore a simple and rapid screening method to detect hot spot mutations in the dysferlin gene is essential for the diagnosis of dysferlinopathy. METHODS: We previously showed that 4 mutations, c.937+1G>A, c.1566C>G, c.2997G>T and c.3373delG account for 50% of all the mutations identified in Japanese dysferlinopathy patients. We performed a one-tube multiplex PCR, followed by extension of primers for each mutation with a fluorescence-labeled dideoxynucleotide to screen the 4 hot spot mutations. RESULTS: The multiplex primer-extension reaction was developed on samples of known mutations. The extension products were represented as 4 different peaks that corresponded to a mutated nucleotide on electropherogram. Using the developed screening method, we were able to detect mutations in these hot spots in 3 samples out of 8 clinically suspected LGMD2B/MM patients in only approximately 8 hours. These 3 cases were definitely diagnosed as LGMD2B/MM by exonic sequencing. CONCLUSION: We have developed a simple and rapid screening method which could facilitate the definitive diagnosis of dysferlinopathy, contributing to an understanding of the genotype-phenotype correlations for dysferlinopathy.
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Hayashi et al. (2010) studied Dysferlinopathy (LGMD2B/Miyoshi myopathy) (n=22). Fluorescent primer extension (multiplex PCR) vs. Direct exonic sequencing was evaluated on Detection of 4 hot spot mutations in the dysferlin gene. A novel multiplex fluorescent primer-extension method successfully detected 4 common dysferlin gene mutations in approximately 8 hours with complete concordance to direct sequencing.
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