EMQN best practice guidelines for genetic testing in dystrophinopathies

Abstract

Dystrophinopathies are X-linked diseases, including Duchenne muscular dystrophy and Becker muscular dystrophy, due to DMD gene variants. In recent years, the application of new genetic technologies and the availability of new personalised drugs have influenced diagnostic genetic testing for dystrophinopathies. Therefore, these European best practice guidelines for genetic testing in dystrophinopathies have been produced to update previous guidelines published in 2010. These guidelines summarise current recommended technologies and methodologies for analysis of the DMD gene, including testing for deletions and duplications of one or more exons, small variant detection and RNA analysis. Genetic testing strategies for diagnosis, carrier testing and prenatal diagnosis (including non-invasive prenatal diagnosis) are then outlined. Guidelines for sequence variant annotation and interpretation are provided, followed by recommendations for reporting results of all categories of testing. Finally, atypical findings (such as non-contiguous deletions and dual DMD variants), implications for personalised medicine and clinical trials and incidental findings (identification of DMD gene variants in patients where a clinical diagnosis of dystrophinopathy has not been considered or suspected) are discussed.

Ethnicity-related DMD Genotype Landscapes in European and Non-European Countries

“…When the MLPA result suggested a single exon deletion or duplication, the result was always confirmed by an alternative molecular technique, which was PCR and sequencing for single-deleted exons (including the 5′ and 3′ adjacent exons) and exon-specific real-time PCR for single exon duplications, according to current DMD diagnosis guidelines. 8 , 9 …”

“…When the MLPA result suggested a single exon deletion or duplication, the result was always confirmed by an alternative molecular technique, which was PCR and sequencing for singledeleted exons (including the 59 and 39 adjacent exons) and exonspecific real-time PCR for single exon duplications, according to current DMD diagnosis guidelines. 8,9 Glossary ACMG = American College of Medical Genetics and Genomics; BMD = Becker muscular dystrophy; CK = creatine kinase; DMD = Duchenne muscular dystrophy; IGV = Integrative Genomics Viewer; LGMD = limb-girdle muscular dystrophy; NGS = next-generation sequencing; OMIM = Online Mendelian Inheritance in Man; VUS = variant of uncertain/unknown significance; WES = whole-exome sequencing; WGS = whole-genome sequencing.…”

249th ENMC International Workshop: The role of brain dystrophin in muscular dystrophy: Implications for clinical care and translational research, Hoofddorp, The Netherlands, November 29th–December 1st 2019

“…Pathogenic mutations in the DMD gene, mainly deletions (65%), duplications (10%) and small mutations (25%), cause DMD and BMD. Very accurate mutation detection is currently available via MLPA (multiplex ligation-dependent probe amplification) or full sequencing, mainly using next generation sequencing methods [11] . DMD encodes the dystrophin protein, a sub-sarcolemma protein involved in sarcolemma integrity, force transduction, muscle signalling and regeneration.…”

Identification and Characterization of Splicing Defects by Single-Molecule Real-Time Sequencing Technology (PacBio)

“…Alternatively, the sole transcript of interest can be analyzed when its coding gene is strongly associated with the observed phenotype and DNA analysis (usually focused on the coding region) has not identified any causative variant. For example, in patients with a clinical suspicion of Duchenne muscular dystrophy, when dystrophin expression is abnormal/absent and no mutation in the DMD gene is found with MLPA or DNA sequencing, the transcript needs to be analyzed (12,13). Multiple RT-PCRs, followed by a direct Sanger sequencing on purified RT-PCR products, are performed to identify possible splicing defects (14).…”