Facioscapulohumeral muscular dystrophy (FSHD) is the second most common muscular dystrophy in adults, and it is associated with local D4Z4 chromatin relaxation, mostly via the contraction of the... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is the second most common muscular dystrophy in adults, and it is associated with local D4Z4 chromatin relaxation, mostly via the contraction of the D4Z4 macrosatellite repeat array on chromosome 4q35. In this study, we aimed to investigate the use of Optical Genome Mapping (OGM) as a diagnostic tool for testing FSHD cases from the UK and India and to compare OGM performance with that of traditional techniques such as linear gel (LGE) and Pulsed-field gel electrophoresis (PFGE) Southern blotting (SB). A total of 6 confirmed and 19 suspected FSHD samples were processed with LGE and PFGE, respectively. The same samples were run using a Saphyr Genome-Imaging Instrument (1-color), and the data were analysed using custom EnFocus FSHD analysis. OGM was able to confirm the diagnosis of FSHD1 in all FSHD1 cases positive for SB (n = 17), and D4Z4 sizing highly correlated with PFGE-SB (p < 0.001). OGM correctly identified cases with mosaicism for the repeat array contraction (n = 2) and with a duplication of the D4Z4 repeat array. OGM is a promising new technology able to unravel structural variants in the genome and seems to be a valid tool for diagnosing FSHD1. Show less
Lemmers, R.J.L.F.; Butterfield, R.; Vliet, P.J. van der; Bleecker, J.L. de; Pol, L. van der; Dunn, D.M.; ... ; Maarel, S.M. van der 2023
Facioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation... Show moreFacioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation facilitates inappropriate expression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded by a retrogene that is embedded within the distal region of the D4Z4 repeat array. In the European population, the D4Z4 repeat array is usually organized in a single array that ranges between 8 and 100 units. D4Z4 chromatin relaxation and DUX4 derepression in FSHD is most often caused by repeat array contraction to 1-10 units (FSHD1) or by a digenic mechanism requiring pathogenic variants in a D4Z4 chromatin repressor like SMCHD1, combined with a repeat array between 8 and 20 units (FSHD2).With a prevalence of 1.5% in the European population, in cis duplications of the D4Z4 repeat array, where two adjacent D4Z4 arrays are interrupted by a spacer sequence, are relatively common but their relationship to FSHD is not well understood. In cis duplication alleles were shown to be pathogenic in FSHD2 patients; however, there is inconsistent evidence for the necessity of an SMCHD1 mutation for disease development.To explore the pathogenic nature of these alleles we compared in cis duplication alleles in FSHD patients with or without pathogenic SMCHD1 variant. For both groups we showed duplication-allele-specific DUX4 expression. We studied these alleles in detail using pulsed-field gel electrophoresis-based Southern blotting and molecular combing, emphasizing the challenges in the characterization of these rearrangements. Nanopore sequencing was instrumental to study the composition and methylation of the duplicated D4Z4 repeat arrays and to identify the breakpoints and the spacer sequence between the arrays. By comparing the composition of the D4Z4 repeat array of in cis duplication alleles in both groups, we found that specific combinations of proximal and distal repeat array sizes determine their pathogenicity. Supported by our algorithm to predict pathogenicity, diagnostic laboratories should now be furnished to accurately interpret these in cis D4Z4 repeat array duplications, alleles that can easily be missed in routine settings.Facioscapulohumeral muscular dystrophy (FSHD) is caused by shortening of the D4Z4 repeat array. Occasionally duplications of this array are found with unknown pathogenicity. Lemmers et al. have developed a formula to predict pathogenicity of these duplications and have unravelled the mechanism by which they cause FSHD. Show less
Neuromuscular diseases (NMDs) affect similar to 15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However,... Show moreNeuromuscular diseases (NMDs) affect similar to 15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However, most affected families are in low-to-middle income countries (LMICs) with limited access to DNA-based diagnosis. Most (86%) published genetic data is derived from European ancestry. This marked genetic data inequality hampers understanding of genetic diversity and hinders accurate genetic diagnosis in all income settings. We developed a cloud-based transcontinental partnership to build diverse, deeply-phenotyped and genetically characterized cohorts to improve genetic architecture knowledge, and potentially advance diagnosis and clinical management.We connected 18 centres in Brazil, India, South Africa, Turkey, Zambia, Netherlands and the UK. We co-developed a cloud-based data solution and trained 17 international neurology fellows in clinical genomic data interpretation. Single gene and whole exome data were analysed via a bespoke bioinformatics pipeline and reviewed alongside clinical and phenotypic data in global webinars to inform genetic outcome decisions.We recruited 6001 participants in the first 43 months. Initial genetic analyses 'solved' or 'possibly solved' similar to 56% probands overall. In-depth genetic data review of the four commonest clinical categories (limb girdle muscular dystrophy, inherited peripheral neuropathies, congenital myopathy/muscular dystrophies and Duchenne/Becker muscular dystrophy) delivered a similar to 59% 'solved' and similar to 13% 'possibly solved' outcome. Almost 29% of disease causing variants were novel, increasing diverse pathogenic variant knowledge. Unsolved participants represent a new discovery cohort. The dataset provides a large resource from under-represented populations for genetic and translational research.In conclusion, we established a remote transcontinental partnership to assess genetic architecture of NMDs across diverse populations. It supported DNA-based diagnosis, potentially enabling genetic counselling, care pathways and eligibility for gene-specific trials. Similar virtual partnerships could be adopted by other areas of global genomic neurological practice to reduce genetic data inequality and benefit patients globally.Wilson et al. present the findings of an international partnership established to study genetic causes of neuromuscular diseases in under-represented diverse populations from 12 low-middle income sites. A genetic cause was identified in similar to 55% of cases and similar to 30% of variants were novel, improving understanding of neuromuscular disease genetics. Show less
