Efforts to determine the molecular basis of VCFS

Velo-cardio-facial syndrome (VCFS) is a congenital anomaly disorder associated with hemizygous 22q11 deletions. VCFS is characterised by craniofacial anomalies, heart defects and learning disabilities. It is likely that haploinsufficiency of a gene(s) in 22q11 is responsible for its aetiology. Many additional clinical findings occur in association with VCFS with varying levels of phenotypic expression. Haploinsufficiency or uncovering of a recessive mutation of other genes in 22qll, may contribute to the associated findings of the disorder. To define the region deleted in VCFS patients, we developed a genetic and physical map of the region. Fifteen consecutive genetic markers were used to genotype 151 VCFS patients and perform haplotype analysis on 105 of them to define the extent of deletion in each patient. Over 90% of the patients had a similar large 3 Mb deletion. Most of the remaining patients had a nested distal deletion breakpoint resulting in a 1.5 Mb region. A lew rare patients had unique deletions. Individual genes have been isolated from the 1.5 Mb region by the hybridization selection methodology as well as a variety of other approaches. Over 20 genes from this interval have been isolated to date, however their role in the aetiology of VCFS has not been established. To determine the molecular basis of VCFS, we are developing mouse models that contain hemizygous deletions of the orthologous region on mouse chromosome 16. Using the cre/loxP system, we have generated delelions in embryonic stem cells and obtained chimeric animals. We will complement the deletion by mating the mice that carry the deletion with BAG transgenic mice containing human genes that map to the 22ql1 region. A set of transgenic lines containing the human genes PNL'TL, GP1BB and TBX1, have been generated and analysis of the animals underway. Equally as important as identifying the VCFS gene(s). is to determine the molecular basis of the 22ql 1 deletion. The fact that most VCFS patients have a similar deletion of 3 Mb in size, suggests that sequences at the chromosome breakpoints confer susceptibility to rearrangement. To determine the molecular basis of the common deletion, it is necessary to clone and sequence the deletion breakpoint junction. To accomplish this goal, we cloned the interval con-taining the proximal and distal chromosome breakpoints. A 300 kb tandem duplication of sequences that surrounded the chromosome breakpoints was discovered. The duplication included the genes/pseudogenes for BCR-L, POM-121, GGT-rel. GGT1 and LSPL. Low copy repeat clusters have been implicated in mediating chromosome rearrangements for other disorders, supporting the hypothesis that low copy repeats mediate recombination events. To determine whether intraand inter-chromosome recombination leads to the common deletion, haplotype analysis was performed on VCFS patients as well as their unaffected parents and grandparents. Analysis of these patients as well as a patient with a duplication of the same 3Mb region that is deleted in VCFS patients revealed that both mechanisms lead to the deletion.