Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder
caused by complex genetic and epigenetic mechanisms. Previous studies showed that transcription de-
repression of double homeobox protein 4 (DUX4) due to epigenetic changes in the D4Z4 region causes
FSHD. The epigenetic changes are caused by either contraction of the D4Z4 array from 11-150 repeat
units in unaffected individuals to 1-10 repeat units in roughly 95% of patients (FSHD1) or mutations in
epigenetic regulators of the region (FSHD2). Current genetic testing for FSHD is labor intensive and
does not assess DNA methylation status, which has been suggested to be the primary determinant of
disease severity in FSHD1. In this application, we propose to use a novel approach, Nanopore long-
read sequencing, to determine the copy number and methylation level of D4Z4 region in a single test.
Our preliminary data showed that optical mapping can accurately provide sizing for D4Z4 repeat region.
In aim 1 of the project, we will first develop a Nanopore long-read sequencing assay to evaluate the
D4Z4 arrays on both chromosome 4 and chromosome 10 and built a data analysis workflow for the
data. In aim 2, we will use the approach to determine D4Z4 repeat size and DNA methylation status of
FSHD DNA samples and DNA samples from unaffected individuals collected from a recent study of
early onset FSHD; then determine the correlation between the methylation status and disease onset
and severities. Development of a single test that could assess both genetic and epigenetic causes of
FSHD will significantly transform the molecular diagnosis of FSHD as well as provide a roadmap for
researcher to investigate how genetic and epigenetic variations in subtelomeric region of 4q35 affect
FSHD onset and severity.