A novel approach for investigating the D4Z4 region in facioscapulohumeral muscular dystrophy

Translational Research
Virtual
Yi-Wen Chen, DVM, PhD, Children's National Hospital, George Washington University
Alexander Liu, BS, Children's National Research Institute

Facioscapulohumeral muscular dystrophy (FSHD) is caused by aberrant expression of double homeobox 4 (DUX4) due to epigenetic changes in the D4Z4 macrosatellite array at chromosome 4q35. The number of the D4Z4 repeat number and DNA methylation of the region are used for disease diagnosis. Currently no single assay examines both simultaneously. The goal of this study is to develop a cost-effective long-read sequencing-based assay that can be used to determine repeat copy number and DNA methylation of the D4Z4 region. In this study, a CRISPR/Cas9-based enrichment protocol in combination with the Nanopore long-read sequencing was used to specifically target the D4Z4 region. We targeted regions upstream and downstream of the D4Z4 array and successfully obtained complete D4Z4 arrays spanning from the p13e11 region to the pLAM region. We developed bioinformatics workflow to determine the array size, distinguish the chromosomal origin of the D4Z4 arrays, and the allele types (e.g. 4qA and 4qB). In addition, we were able to determine DNA methylation of the region and showed various methylation patterns among different arrays as well as among different repeats. The approach can be further developed into an assay for FSHD diagnosis.