Sex reversal of the 46, XY type can have several causes. Besides mutations in the testis determining gene SRY, autosomal genes have also been implicated. One of the chromosomal regions involved in such disorders is located at the distal end of the short arm of chromosome 9 (9p). Patients with deletions of this region of chromosome 9 present with a number of congenital malformations, including various degrees of sex reversal ranging from ambiguous genitalia to complete sex reversal. The present proposal is to isolate one or more genes responsible for sex reversal from the distal 9p region by positional cloning. Previous work by the PI has identified a small deletion of chromosome 9p which will be combed for expressed sequence. These sequences will then be searched for mutations in patients with sex reversal but no visible abnormalities of chromosome 9. In the first aim, the PI proposes to extend her mapping resources within the 9p critical region that was previously delineated. Previously identified cosmid and YAC probes will be used to screen genomic libraries, initially focusing on cosmid and P1 page libraries and chromosome 9- specific cosmid libraries. If necessary YAC and BAC libraries will also be screened. The probes using interphase FISH followed by determination of overlapping regions using dot blot hybridization, vector/insert function sequencing and fingerprinting. In the second aim, candidate sex determining gene will be searched for within the contig corresponding to the critical region of 9p. Strategies proposed include screening for sequences homologous to two genes, implicated in other forms of sex reversal. SOX9 and SRY, using PCR amplification with HMG hox specific primers. In addition, cDNA libraries will be screened for clones in the region using pools of genomic sequences from the contig as probes. Elimination of false positives and redundant clones will be followed by sequencing and database searches. A third approach is to use magnetic bead capture of relevant cDNAs with multiple cycles of enrichment. In the third aim, a search for mutations in specific genes from the critical region will be conducted in 46, XY females with no apparent deletion of chromosome 9. Patients will be recruited through collaborative efforts and by advertisement in genetic journals. At present, 10 patients have been collected. A combination of approaches, including FISH and LOH analyses will be done to search for deletions. Microsatellite markers will be generated from the new cDNAs isolated to carry out the LOH study. Sequence analysis or SSCP assays on specific genes will be done to look for mutations in the cDNAs isolated in aim 2.
