AMP 2025: Oxford Nanopore-based structural and epigenetic profiling of D4Z4 arrays in FSHD

In this Corporate Workshop Day presentation from AMP 2025, Scott Hickey (Oxford Nanopore Technologies) demonstrates the performance and utility of any read length nanopore sequencing in identifying challenging medically relevant genes including D4Z4/DUX4 in facioscapulohumeral muscular dystrophy (FSHD) samples.

Abstract: Facioscapulohumeral muscular dystrophy (FSHD) is caused by contractions in the D4Z4 repeat array on chromosome 4 and epigenetic dysregulation of the DUX4 gene. Current diagnostic methods rely on a combination of optical genome mapping, methylation-sensitive Southern blotting, and NGS. These approaches can be labor-intensive and have limited resolution. Here, we present an Oxford Nanopore sequencing-based approach that accurately resolves both short pathogenic and long wild-type D4Z4 arrays, using 30 kb reads to generate haplotype-resolved local assemblies. Ultra-long (100 kb+) reads that span the entirety of some arrays were used to confirm the results, up to 58 repeat units. In addition, methylation analysis revealed hypomethylation in short affected alleles, and hypermethylation in unaffected copies, revealing additional mechanistic confirmation in FSHD1. Finally, FSHD2-like samples with DNMT3B mutations showed global reduction in D4Z4 methylation, demonstrating pathogenic variant detection, D4Z4 measurement, and methylation readout from a single assay. This workflow offers a scalable, high-resolution alternative to traditional methods, with potential for future streamlined clinical FSHD analysis.

This talk was originally presented at AMP 2025, Boston.