The gene for facioscapulohumeral muscular dystrophy (FSHD) will be cloned. cDNAs in the FSHD gene region will be identified by """"""""direct selection"""""""" using several YACs. The chromosomal localization of the cDNAs will be determined by hybridization to hybrid cell lines. If the cDNAs map to the 4q35-4qter region, they will be tested for evolutionaiy conservation by hybridization to Southern blots with DNA from a variety of different species. In addition, they will be hybridized to mRNA on Northern blots to determine the size and tissue-specific expression of candidate genes. cDNA libraries will be screened to isolate full length cDNAs and these cDNAs will be sequenced. The sequence will be analyzed for the presence of open reading frames and homologies to known genes. The DNA sequences from normal and affected individuals in the candidate gene region will be compared to identify differences using SSCP, DGOE and sequencing. Detection of sequence changes will implicate the candidate gene as being responsible for FSHD. When sequence differences are found in a gene, members of all of the FSHD families will be tested for changes in the same gene. In addition, the expression of the FSHD gene will be examined in affected muscle tissue by performing RT-PCR on RNA from biopsies from normal and affected muscle. A candidate with both mutations that are inherited with the disease and altered expression in mutant muscle will be considered proof that we have identified the FSHD gene. As mutant alleles are found in the families, PCR-based diagnostic tests will be developed for each allele. Polyclonal antibodies will be raised to the FSHD protein. Peptides or fusion proteins will be used as antigens to ensure success. The antibodies will be used to examine tissue and cell localization. When different therapies such as gene replacement are tested to reverse the effects of FSHD, the anti-FSHD antisera will help determine the effect of the treatment. Throughout the duration of the project, new disease families with unusual features will continue to be identified and collected. For example, families with sporadic cases of FSHD, early onset (juvenile), or families with affected members with a wide range of severity will be studied. These families may play a key role in identifying the location of the FSHD gene and/or in defining the functional portions of the FSHD protein.
