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.
Facioscapulohumeral muscular dystrophy (FSHD) is a disease with complex underlying genetic and epigenetic mechanisms. The proposed studies aim at developing a novel tool to determine D4Z4 repeat number and investigate DNA methylation status at the D4Z4 region, which can potentially be used for molecular diagnosis for FSHD. The tool will be used to evaluate a unique patient cohort with early onset FSHD.