A Streamlined Protocol for Molecular Testing of the DMD Gene within a Diagnostic Laboratory: A Combination of Array Comparative Genomic Hybridization and Bidirectional Sequence Analysis

Purpose. The aim of this study was to develop a streamlined mutation screening protocol for the DMD gene in order to confirm a clinical diagnosis of Duchenne or Becker muscular dystrophy in affected males and to clarify the carrier status of female family members. Methods. Sequence analysis and array comparative genomic hybridization (aCGH) were used to identify mutations in the dystrophin DMD gene. We analysed genomic DNA from six individuals with a range of previously characterised mutations and from eight individuals who had not previously undergone any form of molecular analysis. Results. We successfully identified the known mutations in all six patients. A molecular diagnosis was also made in three of the four patients with a clinical diagnosis who had not undergone prior genetic screening, and testing for familial mutations was successfully completed for the remaining four patients. Conclusion. The mutation screening protocol described here meets best practice guidelines for molecular testing of the DMD gene in a diagnostic laboratory. The aCGH method is a superior alternative to more conventional assays such as multiplex ligation-dependent probe amplification (MLPA). The combination of aCGH and sequence analysis will detect mutations in 98% of patients with the Duchenne or Becker muscular dystrophy. 1. Introduction The dystrophinopathies are a group of muscle disorders that are caused by mutations in the DMD gene [1]. The DMD gene encodes dystrophin, a glycoprotein that is present principally in muscle cells and forms part of the complex linking the cytoskeleton with the extracellular matrix [2]. Mutations that lead to a complete lack of dystrophin expression tend to cause the more severe Duchenne muscular dystrophy (DMD) phenotype, whereas mutations that lead to an abnormal quality or quantity of dystrophin result in the Becker muscular dystrophy (BMD) [3]. In addition, DMD-related X-linked dilated cardiomyopathy (DCM) occurs as a result of mutations that lead to a lack of functional dystrophin in cardiac muscles due to altered tissue-specific transcription or alternative splicing [4]. More than 5,000 mutations have been identified in individuals with DMD or BMD [5, 6]. These pathogenic mutations are highly variable and run the full gamut from deletion of the entire gene, to deletion or duplication of one or more exons, to small deletions or insertions, and to single-base pair changes. Deletions and duplications account for 60–70% and 5–10%, respectively, of all cases [3]. Sequence variants (point mutations and small indels) are responsible for