Abstract

IntroductionFacioscapulohumeral muscular dystrophy 1 (FSHD1) is a relatively common autosomal dominant adult muscular dystrophy with variable disease penetrance. The disease is caused by shortening of a D4Z4 repeat array located near the telomere of chromosome 4 at 4q35. This causes activation of a dormant gene DUX4, permitting aberrant DUX4 expression which is toxic to muscles. Molecular diagnosis of FSHD1 by Southern blot hybridization or FISH combing is difficult and time consuming, requiring specialist laboratories. As an alternative, we apply a novel approach for the diagnosis of FSHD1 utilizing single‐molecule optical mapping (SMOM).MethodsLong DNA molecules with BssS1 enzyme marking were subjected to SMOM on the Bionano Genomics platform to determine the number of D4Z4 repeats. Southern blot and molecular combing were used to confirm the FSHD1 haplotypes.ResultsIn a study of a five‐generation FSHD1 pedigree, SMOM correctly diagnosed the disease and normal haplotypes, identifying the founder 4qA disease allele as having 4 D4Z4 repeat units. Southern blot and molecular combing analysis confirmed the SMOM results for the 4qA disease and 4qB nondisease alleles.ConclusionBased on our findings, we propose that SMOM is a reliable and accurate technique suitable for the molecular diagnosis of FSHD1.

Highlights

  • Facioscapulohumeral muscular dystrophy 1 (FSHD1) is a relatively common autosomal dominant adult muscular dystrophy with variable disease penetrance

  • single‐molecule optical mapping (SMOM) is capable of analyzing DNA fragments from hundreds of kilobases to nearly a megabase in size, and here, we demonstrate successful application of our approach by analyzing clinical DNA samples from a well‐characterized multigeneration FSHD1 pedigree

  • A summary of the SMOM run statistics for the nine selected members of the FSHD1 pedigree that were analyzed in this study is summarized in Supporting Information Table S1

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Summary

| INTRODUCTION

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant adult muscular dystrophy, with a population incidence of ~1 in 20,000 (Scionti et al, 2012; Tawil, Maarel, Padberg, & Engelen, 2010). The 4qA/B haplotype of the last D4Z4 repeat is important for the development of FSHD since the telomeric flanking region of D4Z4 in the 4qA allele contains the 3′ UTR of DUX4 ( called the pLAM region) It is the presence of this polyadenylation signal that allows stable DUX4 expression and subsequent disease manifestation (Richards et al, 2012). Molecular analysis of FSHD1 using available methods is complicated by the length of the repeat structure (D4Z4), even in reduced array form in affected individuals, as well as the variable size of the subtelomeric region of chromosome 4 in different individuals. More recent approaches have used long range PCR or the more technical pulse field gel electrophoresis (PFGE) followed by Southern hybridization (Wijmenga et al, 1993) to identify array changes. SMOM is capable of analyzing DNA fragments from hundreds of kilobases to nearly a megabase in size, and here, we demonstrate successful application of our approach by analyzing clinical DNA samples from a well‐characterized multigeneration FSHD1 pedigree

| MATERIALS AND METHODS
| RESULTS
Findings
| DISCUSSION
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