Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy in Patients Clinically Suspected of FSHD Using Optical Genome Mapping

BACKGROUND AND OBJECTIVES: Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common muscular dystrophy in the general population and is characterized by progressive and often asymmetric muscle weakness of the face, upper extremities, arms, lower leg, and hip girdle. In FSHD type 1, contraction of the number of D4Z4 repeats to 1–10 on the chromosome 4–permissive allele (4qA) results in abnormal epigenetic derepression of the DUX4 gene in skeletal muscle. In FSHD type 2, epigenetic derepression of the DUX4 gene on the permissive allele (4qA) with normal-sized D4Z4 repeats (mostly 8–20) is caused by heterozygous pathogenic variants in chromatin modifier genes such as SMCHD1, DNMT3B, or LRIF1. We present validation of the optical genome mapping (OGM) platform for accurate mapping of the D4Z4 repeat size, followed by diagnostic testing of 547 cases with a suspected clinical diagnosis of FSHD and next-generation sequencing (NGS) of the SMCHD1 gene to identify cases with FSHD2. METHODS: OGM with Bionano Genomics Saphyr and EnFocus FSHD analysis software was used to identify FSHD haplotypes and D4Z4 repeat number and compared with the gold standard of Southern blot–based diagnosis. A custom Agilent SureSelect enrichment kit was used to enrich SMCHD1, followed by NGS on an Illumina system with 100-bp paired-end reads. Copy number variants were assessed using NxClinical software. RESULTS: We performed OGM for the diagnosis of FSHD in 547 patients suspected of FSHD between December 2019 and December 2022, including 301 male (55%) and 246 female patients (45%). Overall, 308 of the referred patients were positive for D4Z4 contraction on a permissive haplotype, resulting in a diagnosis of FSHD1. A total of 252 of 547 patients were referred for concurrent testing for FSHD1 and FSHD2. This resulted in the identification of FSHD2 in 9/252 (3.6%) patients. In our FSHD2 cohort, the 4qA allele size ranged from 8 to 18 repeats. Among FSHD1-positive cases, 2 patients had biallelic contraction and 4 patients had homozygous contraction and showed early onset of clinical features. Nine of the 308 patients (3%) positive for 4qA contraction had mosaic 4q alleles with contraction on at least one 4qA allele. The overall diagnostic yield in our cohort was 58%. DISCUSSION: A combination of OGM to identify the FSHD haplotype and D4Z4 repeat number and NGS to identify sequence and copy number variants in the SMCHD1 gene is a practical and cost-effective option with increased precision for accurate diagnosis of FSHD types 1 and 2.