FSHD Syndrome: DNA Repeats, Methylation, & Chromatin

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DESCRIPTION (provided by applicant): Fascioscapulohumeral muscular dystrophy (FSHD) is an unusual autosomal dominant syndrome caused by the loss of some copies of a complex repeat (D4Z4) in a subtelomeric region (4q35) of one chromosome 4 homologue. The number of copies of this 3.3-kb repeat at 4q35 is polymorphic. Unaffected individuals have 11 to about 95 copies on each allelic 4q35. In contrast, more than 90% of FSHD patients have less than 10 copies at one of these allelic 4q subtelomeric regions. It has been proposed by many investigators that normally this region is heterochromatic but that when the number of tandem copies of D4Z4 is less than 10, the region loses its condensed chromatin structure, This loss of heterochroma-tinization, in turn, is hypothesized to induce inappropriate gene expression in the affected muscle cells. However, there have been no reports about studies of the chromatin structure in this region for normal or FSHD cells. In the planned research, immunochemical, cytochemical, and immunocytochemical methods will be used to examine whether this region is indeed heterochromatic and whether it loses the heterochromatic structure when it contains the FSHD deletion. Myoblast cultures and lymphoblastoid cell lines from normal individuals and FSHD patients will be studied. These experiments will include analysis of histone acetylation and binding of heterochromatin 1 beta protein to the D4Z4 chromatin region. Also, we will determine whether this region is late-replicating in normal cells, as is the case for heterochromatin. Consistent with the proposed heterochromatic nature of this region, it has recently been shown that this repeat is highly methylated. The preliminary study of methylation of the D4Z4 repeat will be expanded to examine whether this repeat is no longer hypermethylated in the deletion-containing chromosome 4 in FSHD cells. It has recently been shown that cells from another genetic syndrome, ICF (a DNA methyltransferase-deficiency and chromosome instability syndrome), are undermethylated in this repeat. Because abnormal hypomethylation can favor chromosome rearrangements, ICF and normal cell lines will be compared for the frequency of rearrangements in this region. The proposed research should help elucidate the molecular etiology of the enigmatic FSHD syndrome.