Abstract
Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant myodystrophy. Approximately 95% of cases of FSHD are caused by partial deletion of the D4Z4 macrosatellite tandem repeats on chromosome 4q35. The existing FSHD1 diagnostic methods are laborious and not widely used. Here, we present a comprehensive analysis of the currently used diagnostic methods (Southern blotting and molecular combing) against a new qPCR-based approach for FSHD1 diagnosis. We observed 93% concordance between the results obtained by the new qPCR-based approach, reference Southern blotting and molecular combing methods. Applying the qPCR-based approach in the studied population, we observed a prevalence (64.9%) of the permissive alleles in the range of 3–6 D4Z4 units for a group of patients, while in a group of carriers, the permissive alleles were mostly (84.6%) present in the range of 6–9 D4Z4 units. No prevalence of disease penetrance depending on gender was observed. The results confirmed the earlier established inverse correlation between permissive allele size and disease severity, disease penetrance. The results suggest the applicability of the qPCR-based approach for FSHD1 diagnosis and its robustness in a basic molecular genetics laboratory. To our knowledge, this is the first study of FSHD1 permissive allele distribution in a Russian population.
Highlights
Facioscapulohumeral dystrophy (FSHD) is a progressive skeletal muscle dystrophy with a prevalence from 1:20,000 to 1:8500 in observed populations, making it the third most common muscle dystrophy after Duchenne muscular dystrophy and myotonic dystrophy.FSHD is uniquely linked with subtelomeric macrosatellite tandem D4Z4 repeats of chromosome 4q35
As FSHD1 accounts for more than 95% of cases, we focused on comprehensive analysis of methods for FSHD1 molecular diagnostics, such as Southern blotting and molecular combing [17,18,19] versus a newly developed qPCR-based method
In order to avoid hybridization during the steps used in the blotting and in the molecular combing, the following approach for chromosome 4 D4Z4 array length estimation was used: agarose-embedded DNA preceded treatment by EcoRI endonuclease; the obtained restriction fragments were separated by pulsed-field gel electrophoresis (PFGE) in a 0.8% agarose gel; the gel was photorepeats are highlighted. (C) Sequence of PCR products obtained by Rep1F + Re1R1seq primers pair and native gDNA
Summary
Facioscapulohumeral dystrophy (FSHD) is a progressive skeletal muscle dystrophy with a prevalence from 1:20,000 to 1:8500 in observed populations, making it the third most common muscle dystrophy after Duchenne muscular dystrophy and myotonic dystrophy. 4q35 D4Z4 array chromatin in cis with the 4qA haplotype and activation of the ectopic expression of DUX4 from the most distal D4Z4 repeat [16]. As FSHD1 accounts for more than 95% of cases, we focused on comprehensive analysis of methods for FSHD1 molecular diagnostics, such as Southern blotting and molecular combing [17,18,19] versus a newly developed qPCR-based method. The qPCR-based FSHD1 diagnostic test allows avoiding the laborious step of hybridization used in Southern blotting and molecular combing, making FSHD1 diagnostic available for a broad spectrum of diagnostic laboratories. To our knowledge, this is the first genotype study representing permissive allele distribution of FSHD1 patients and their unaffected relatives from Russia
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