The phenotypic mapping of the cat-like cry observed in the cri-du-chat syndrome and molecular characterization of the proposed critical region

The cri-du-chat syndrome results in children born with deletions or unbalanced translocations involving the short arm of chromosome 5. The syndrome is defined at birth by microcephaly, low birth weight, hypotonia, a collection of distinct facial features, and an unusual high-pitched, cat-like cry. Children born with cri-du-chat syndrome exhibit severe mental and developmental delay. With an incidence of 1/50,000 live births, it is among the most common monosomies in humans. Preliminary phenotypic, cytogenetic, and molecular analyses of patients who are hemizygous for material of the short arm of chromosome 5 (5p; 5p-) have suggested the existence of a critical region for the severe delay and typical facies in 5p15.2, while the region involved in the cat-like cry appears to be distinct and maps more distally in 5p15.3. Patients must have deletions encompassing both regions to have the full syndrome. The diagnostic and prognostic implications of this data makes it important to further refine the locations of these critical regions through the analysis of additional patients with atypical phenotypes using fluorescence in situ hybridization and somatic cell hybrid techniques. Results of such additional patient analyses is described in this work, and does confirm the presence of a distinct critical region for the cat-like cry in 5p15.3, which is approximately 1 Mb in size. The construction of a physical map or the region was implemented through the identification of sequence-tagged sites (STSs) and short tandem repeats (STRs) which mapped in and around the cat-like cry critical region. The availability of these markers allowed for the subsequent identification and isolation of large-insert genomic clones from yeast artificial chromosome (YAC) and cosmid libraries which mapped to the region The identification of these clones resulted in the formation of a comprehensive physical map, in which the entire region was cloned into YACs and approximately 80-85% of the region is encompassed by cosmid contigs. Additional physical characterization of the region was performed by creating a rare-cutting restriction enzyme map, which indicated the region to be about 800 Kb in size and assisted in determining the sizes of the cosmid contigs and the gaps between them. Exon-trapping, a technique which identifies coding sequences based on the presence of flanking splice-acceptor and splice-donor sites, was performed on pools of highly overlapping cosmids. This experiment resulted in a high level of artifactual clones, but did however yield one promising clone, 1-C2, which is comprised of two small exons with open reading frames that were spliced together. The genomic DNA flanking these exons was cloned, sequenced, and analyzed by computer programs which identify coding sequences and characteristics often associated with such sequences. These analyses suggest the presence of additional exons in this genomic region of $\sim$3 Kb. In addition, the potential locations for CpG islands throughout the cat-like cry critical region were identified by mapping the sites of rare-cutting restriction enzymes which primarily cut at CpG islands. (Abstract shortened by UMI.