Molecular combing reveals complex 4q35 rearrangements in Facioscapulohumeral dystrophy

• Published in: Human mutation Vol. 38; no. 10; pp. 1432 - 1441
• Main Authors: Nguyen, Karine, Puppo, Francesca, Roche, Stéphane, Gaillard, Marie‐Cécile, Chaix, Charlène, Lagarde, Arnaud, Pierret, Marjorie, Vovan, Catherine, Olschwang, Sylviane, Salort‐Campana, Emmanuelle, Attarian, Shahram, Bartoli, Marc, Bernard, Rafaëlle, Magdinier, Frédérique, Levy, Nicolas
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.10.2017; Wiley
• Subjects: Alleles; Biochemistry, Molecular Biology; Bisulfite; Cellular Biology; Chromosomal Proteins, Non-Histone - genetics; Chromosome 4; Chromosome Aberrations; Chromosomes, Human, Pair 10 - genetics; Chromosomes, Human, Pair 4 - genetics; D4Z4; DNA methylation; DNA Methylation - genetics; DNA sequencing; Dystrophy; Female; FSHD; Genetic diversity; Genetic Predisposition to Disease; Genetic Variation; Genetics; Genomics; Haplotypes; Haplotypes - genetics; High-Throughput Nucleotide Sequencing; Human genetics; Human health and pathology; Humans; Life Sciences; Male; methylation; minigene complementation assay (pCAS); Molecular biology; Molecular Combing; Muscular Dystrophy, Facioscapulohumeral - diagnosis; Muscular Dystrophy, Facioscapulohumeral - genetics; Muscular Dystrophy, Facioscapulohumeral - physiopathology; Mutation; Mutation - genetics; Neurobiology; Neuromuscular diseases; Neurons and Cognition; Pathology, Molecular; SMCHD1; Sodium; Subcellular Processes; FSHD; minigene complementation assay (pCAS); SMCHD1; D4Z4; Molecular Combing; methylation; Epigenetics
• ISSN: 1059-7794; 1098-1004; 1098-1004
• DOI: 10.1002/humu.23304

Facioscapulohumeral dystrophy (FSHD), one of the most common hereditary neuromuscular disorders, is associated with a complex combination of genetic variations at the subtelomeric 4q35 locus. As molecular diagnosis relying on Southern blot (SB) might be challenging in some cases, molecular combing (MC) was recently developed as an additional technique for FSHD diagnosis and exploration of the genomic organization of the 4q35 and 10q26 regions. In complement to the usual SB, we applied MC in a large cohort of 586 individuals with clinical FSHD. In 332 subjects, the two 4q alleles were normal in size, allowing exclusion of FSHD1 while we confirmed FSHD1 in 230 patients. In 14 patients from 10 families, we identified a recurrent complex heterozygous rearrangement at 4q35 consisting of a duplication of the D4Z4 array and a 4qA haplotype, irresolvable by the SB technique. In five families, we further identified variations in the SMCHD1 gene. Impact of the different mutations was tested using a minigene assay and we analyzed DNA methylation after sodium bisulfite modification and NGS sequencing. We discuss the involvement of this rearrangement in FSHD since all mutations in SMCHD1 are not associated with D4Z4 hypomethylation and do not always segregate with the disease. By using molecular combing and a ‘morse’ bar code corresponding to the 4q35 subtelomeric locus we identified a novel and recurrent rearrangement consisting in the duplication of the D4Z4 array and flanking qA allele in individuals affected with Facio‐Scapulo Humeral Dystrophy. These results reveal the variability of molecular data among subjects and within families and highlight the complexity one might face in analyzing FSHD families for molecular diagnosis but also genetic counseling.