Abstract
Fascioscapulohumeral muscular dystrophy (FSHD) is caused by a unique genetic mechanism that relies on contraction and hypomethylation of the D4Z4 macrosatellite array on the Chromosome 4q telomere allowing ectopic expression of the DUX4 gene in skeletal muscle. Genetic analysis is difficult because of the large size and repetitive nature of the array, a nearly identical array on the 10q telomere, and the presence of divergent D4Z4 arrays scattered throughout the genome. Here, we combine nanopore long-read sequencing with Cas9-targeted enrichment of 4q and 10q D4Z4 arrays for comprehensive genetic analysis including determination of the length of the 4q and 10q D4Z4 arrays with base-pair resolution. In the same assay, we differentiate 4q from 10q telomeric sequences, determine A/B haplotype, identify paralogous D4Z4 sequences elsewhere in the genome, and estimate methylation for all CpGs in the array. Asymmetric, length-dependent methylation gradients were observed in the 4q and 10q D4Z4 arrays that reach a hypermethylation point at approximately 10 D4Z4 repeat units, consistent with the known threshold of pathogenic D4Z4 contractions. High resolution analysis of individual D4Z4 repeat methylation revealed areas of low methylation near the CTCF/insulator region and areas of high methylation immediately preceding the DUX4 transcriptional start site. Within the DUX4 exons, we observed a waxing/waning methylation pattern with a 180-nucleotide periodicity, consistent with phased nucleosomes. Targeted nanopore sequencing complements recently developed molecular combing and optical mapping approaches to genetic analysis for FSHD by adding precision of the length measurement, base-pair resolution sequencing, and quantitative methylation analysis.