SEARCH FOR THE FSHD GENE USING CDNA SELECTION IN A REGION SPANNING 100-KB ON CHROMOSOME-4Q35

Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions o f 3.3-kb tandemly repeated units contained within a large polymorphic EcoRI fragment close to the telomere of chromosome 4q. Since the rearr angements were assumed to interfere with the structure or function of the putative FSHD gene, the gene search was focused on cosmids contain ing these repeat units and, in addition, cosmids spanning 75 kb of ups tream sequences. cDNA selection hybridization was applied to four over lapping cosmid clones, yielding a total of 150 putative cDNA clones. T hese clones showed a random distribution across the cosmid contig, exc ept for three regions which contained a much larger number of clones. Nine cDNA clones hybridized to a 2.2-kb EcoRI fragment, located 22 kb centromeric to the 3.3-kb repeated units. This 2.2-kb fragment showed evolutionary conservation, and analysis of the sequence by ''GRAIL'' p redicted the presence of several exons. Transcripts homologous to this fragment could be identified but none of them originated from the 4q3 5 locus. Strikingly, most clones revealed 4-10 homologous loci, and no single copy clones could be isolated. These findings are in line with earlier observations by fluorescent in situ hybridization (FISH) show ing hybridization of individual cosmid clones to multiple chromosomes. The presence of homologous regions on other chromosomes seriously com plicates the cloning of the FSHD gene. (C) 1995 John Wiley and Sons, I nc.