The population of patients with conotruncal cardiac malformations at-risk for deletions of 22q11.21 to q11.23 is large. Therefore, a rapid DNA- based screening method to detect individuals at high risk for deletions will be required. Thus, we propose to develop and apply a multiplex PCR- based screening assay using highly polymorphic DNA markers. Individuals at risk for deletions will be further screened by fluorescence in situ hybridization (FISH) utilizing 22q11.2-specific cosmid clones which span and flank the cardiac critical region. Utilization of multi-colored fluors for simultaneous hybridization of multiple probes to interphase nuclei will help us to estimate the extent of deletions for selected individuals. We will test the hypothesis that differences in size, location, parent of origin, and etiology of the deletions within 22q11 account for the phenotypic variability observed between CCHD patients with deletions. Furthermore, we suggest that variability in the clinical manifestations between affected members of families may result from change in the size of an inherited deletion during meiosis. To test this hypothesis, we will correlate the size and location of the deletions with the clinical findings the genes discovered and their expression pattern. We will compare the size of deletions in multiple affected family members and test for imprinting effects by determining the parent of origin in patients with deletions. Finally, we propose that 22q11 may rearrange by centromeric exchange with other acrocentric chromosomes. We will examine the prevalence of such """"""""cryptic translocations"""""""" as a mechanism associated with generating 22q11.2 deletions. Finally, we propose that the conotruncal heart defects may be caused by abnormalities of a single gene in the cardiac critical region. To test this hypothesis, we will examine the evidence for the presence of a cardiac critical region in 22q11.21 to 22q11.23 by examining locus associations in CHD families or looking for mutations in specific """"""""candidate"""""""" genes in non-deleted patients.
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