EXCLUSION OF EPIDERMAL GROWTH-FACTOR AND HIGH-RESOLUTION PHYSICAL MAPPING ACROSS THE RIEGER SYNDROME LOCUS

We have evaluated the 4q25-4q26 region where the autosomal dominant di sorder Rieger syndrome has been previously mapped by linkage. We first excluded epidermal growth factor as a candidate gene by carrying out SSCP analysis of each of its 24 exons using a panel of seven unrelated individuals with Rieger syndrome, No evidence for etiologic mutations was detected in these individuals, although four polymorphic variants were identified, including three that resulted in amino acid changes, We next made use of two apparently balanced translocations, one famil ial and one sporadic, to identify a narrow physical localization likel y to contain the gene or to be involved in regulation of gene function . Somatic cell hybrids were established from individuals with these ba lanced translocations, and these hybrids were used as a physical mappi ng resource for, first, preliminary mapping of the translocation break points using known sequence tagged sites from chromosome 4 and then, a fter creating YAC and cosmids contigs encompassing the region, for fin e mapping of those breakpoints. A cosmid contig spanning these breakpo ints was identified and localized the gene to within similar to 150 kb of D4S193 on chromosome 4. The internal between the two independent t ranslocations is similar to 50 kb in length and provides a powerful re source for gene identification.