Deciphering D4Z4 CpG methylation gradients in fascioscapulohumeral muscular dystrophy using nanopore sequencing

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 due to 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.

Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing

“…Thus, the separately computed methylation levels show greater precision than the overall methylation level computed for the two 4qA alleles. Nanopore CRISPR/Cas9-targeted resequencing has been proposed for diagnosing FSHD [20,21]. Targeted sequencing of chromosome 4q/10q regions with high sequencing depths is a cost-effective method.…”

“…The third-generation single-molecule sequencing technology developed by Oxford Nanopore Technologies (ONT) is promising for diagnosing FSHD because of its long sequencing length and ability to simultaneously detect methylation [17][18][19]. Nanopore CRISPR/Cas9-targeted resequencing has also been applied to accurately measure the number of D4Z4 RUs and associated methylation status in patients with FSHD [20,21]. However, for the FSHD2 subtype, DNA bisulfite sequencing (BSS) or next-generation sequencing is still needed for diagnosis.…”

Exchange of subtelomeric regions between chromosomes 4q and 10q reverts the FSHD genotype and phenotype

“…As DUX4 expression is known to be affected by the chromatin state of the D4Z4 locus (28), we have also checked the methylation status of the D4Z4 units in the control, FSHD, and t(4;10) myoblasts. Consistent with previous reports (28)(29)(30), the D4Z4 array of the control AB1190 myoblasts was more heavily methylated than that of the FSHD AB1080 myoblasts. Nevertheless, we did not observe any significant changes in D4Z4 methylation in the maternal versus translocation-bearing clones (fig.…”

Post-transcriptional RNA stabilization of telomere-proximal RNAs FRG2, DBET, D4Z4 at human 4q35 in response to genotoxic stress and D4Z4 macrosatellite repeat length

“…Studies investigating D4Z4 chromatin have detected multiple repressive modifications, including DNA methylation [29][30][31][32][33][34][35], di/trimethylation of histone H3 at lysine 9 (H3K9me2/3) [36][37][38], trimethylation of histone H3 at lysine 27 (H3K27me3) [39], and association of the related chromatin proteins CBX3/HP1γ and EZH2 [39,40]. Moreover, D4Z4 recruits a multi-protein repressor complex, the D4Z4 Recognition Complex (DRC), whose removal is associated with increased expression of 4q35 genes [41].…”