Project 2 will expand our knowledge of FSHD biomarkers to enable identification of FSHD disease genes and genetic and epigenetic mechanisms responsible for FSHD muscle pathology, and facilitate pre-clinical studies to develop FSHD therapeutics.
In Aim 1, biomarkers for FSHD will be identified and validated using whole transcriptome sequencing (RNA-seq) and qRT-PCR analyses of muscle and myogenic cells from affected and unaffected subjects in FSHD family cohorts and in nonmanifesting carriers. Our large collection of muscle and cells available in the Cell Core will enable statistically powered studies to identify quantitative differences in the expression of coding and non-coding RNAs and splicing variants and candidate modifiers of disease severity, also being investigated in Project 1 by exome sequencing. The expression and functions of candidate disease biomarkers and modifier genes and their encoded proteins will be investigated in FSHD cells (Aim 1) and humanized mouse xenograft models (Project 3), and by using morpholino antisense inhibition to evaluate their functions in FSHD pathophysiology (Aim 3).
Aim 2 will investigate epigenetic and regulatory mechanisms controlling the expression of DUX4-fl and non-coding RNAs transcribed from the D4Z4 FSHD disease locus. Investigations focus on nonmanifesting carriers and unaffected members of FSHD families to test the hypothesis that disruptions in the DNA methylation status of the D4Z4 locus are early modifiers of genetic and regulatory disruptions leading to FSHD disease.
Aim 3 is a collaboration with our industry partner, Genzyme, to develop antisense oligo morpholino technology for investigating the disease functions of DUX4-fl and other candidate FSHD disease genes identified in Aim 1. The initial objectives are to develop and validate DUX4-fl morpholinos for RNA-seq studies to identify downstream targets of DUX4-fl regulation in FSHD cells as candidate FSHD disease genes. Cell morpholino studies will enable use of morpholinos in pre-clinical drug development studies in humanized mouse xenograft models in Project 3 to investigate the roles of DUX4-fl and other candidate disease genes in FSHD muscle pathophysiology.
A complete molecular signature of FSHD disease provides biomarker tools for investigating and understanding the underlying genetic, epigenetic and pathophysiological mechanisms responsible for FSHD muscle disease, for development therapeutics in pre-clinical studies, and for monitoring efficacy of therapeutic agents in clinical trials
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