Positional candidate cloning of genes causing mouse mutations homologous to human birth defects

The positional candidate approach to identifying the genetic basis of an inherited disorder utilizes chromosomal mapping to examine the candidacy of genes implicated by virtue of congruent function. Numerous mouse developmental mutations exist, are models for human birth defects, and have been precisely mapped on mouse chromosomes. We have undertaken an effort to define genes responsible for human birth defects using a positional candidate approach in the mouse. Embryospecific cDNA clones were selected from a 10.5 day mouse embryo library, on the basis of absent hybridization with adult mouse liver cDNA, and sequenced. Candidacy for developmental mutations was examined by chromosomal assignment of 17 novel cDNAs. Clones were mapped using segregation analysis of unique RFLPs on Southern blots derived from a panel of interspecific backcross mice. These novel cDNA clones appeared to be clustered within genomic regions rich in mouse developmental mutations; 15 of 17 clones mapped adjacent to spontaneous mouse mutants. Candidacy for genetically coincident mutations was further evaluated by whole mount in situ hybridization of cDNA-derived riboprobes to midgestation mouse embryos. Two clones demonstrated highly restricted expression patterns which closely corresponded to the anatomical distribution of defects described in genetically coincident mutants. Clone 33 was assigned to chromosome 14, adjacent to wabbler-lethal (wl), and was expressed in spinal and cranial ganglia, and in the sensory horn of the spinal cord. wl mice exhibit tremor and abnormal gait, and show axonal degeneration and myelin dissolution in vestibulocerebellar and spinocerebellar tracts, basal ganglia, spinal cord and cerebellum. Expression of clone 75, which mapped adjacent to loop-tail (Lp) on distal chromosome 1, was restricted to the limb and tail buds. Lp is associated with abnormalities of the axial skeleton, cerebral ventricles and neural tube. The positional candidate approach appears to be an effective method for identification of genes with potential regulatory functions in mammalian embryogenesis, and may greatly assist our understanding of the molecular mechanisms underlying human birth defects.