Sankhavaram R. Patanjali

Molecular Definition of 22q11 Deletions in 151 Velo-Cardio-Facial Syndrome Patients

Velo-cardio-facial syndrome (VCFS) is a relatively common developmental disorder characterized by craniofacial anomalies and conotruncal heart defects. Many VCFS patients have hemizygous deletions for a part of 22q11, suggesting that haploinsufficiency in this region is responsible for its etiology. Because most cases of VCFS are sporadic, portions of 22q11 may be prone to rearrangement. To understand the molecular basis for chromosomal deletions, we defined the extent of the deletion, by genotyping 151 VCFS patients and performing haplotype analysis on 105, using 15 consecutive polymorphic markers in 22q11. We found that 83% had a deletion and >90% of these had a similar approximately 3 Mb deletion, suggesting that sequences flanking the common breakpoints are susceptible to rearrangement. We found no correlation between the presence or size of the deletion and the phenotype. To further define the chromosomal breakpoints among the VCFS patients, we developed somatic hybrid cell lines from a set of VCFS patients. An 11-kb resolution physical map of a 1,080-kb region that includes deletion breakpoints was constructed, incorporating genes and expressed sequence tags (ESTs) isolated by the hybridization selection method. The ordered markers were used to examine the two separated copies of chromosome 22 in the somatic hybrid cell lines. In some cases, we were able to map the chromosome breakpoints within a single cosmid. A 480-kb critical region for VCFS has been delineated, including the genes for GSCL, CTP, CLTD, HIRA, and TMVCF, as well as a number of novel ordered ESTs.

Locus Specific Identification of Transcribed Sequences Using Yacs and Whole Yeast Genomic DNA

A large fraction of mammalian genomes contain DNA which appears to be very poor in biologic information. Unless or until nucleic acid sequencing procedures emerge that are capable of a much higher throughput of good quality sequence at a lower cost than the present protocols, it appears attractive to approach characterization of these genomes by focussing attention on transcribed regions and immediately adjacent sequences. The advantages gained by such an approach are that nearly all the genes of man will be identified and probes for these genes made available long before the first human genome is sequenced. Our laboratory has been concentrating on hybridization selection approaches (1) to identify transcribed sequences in yeast artificial chromosomes (YACs) or other cloned genomic DNA, and on affinity selection procedures to identify cDNAs that embed particular motifs.