Identification, genomic structure, and screening of the vacuolar proton-ATPase membrane sector-associated protein M8-9 gene within the COD1 critical region (Xp11.4)

Purpose: Our goal is to identify the gene responsible for X-linked cone-rod dystrophy (COD1) that has been localized to a limited region of Xp11.4. Methods: A complete physical contig of the COD1 region was partially sequen ced and subjected to BLAST searches to identify homologies with GenBank EST s. ESTs were analyzed for overlapping or related cDNA sequences and retinal expression by PCR screening of multiple human retina cDNA libraries. RACE was performed to complete the missing 5' end of the transcripts. Transcript s were compared with genomic sequences to specify intron-exon boundaries. G enomic DNAs from COD1-affected males from 3 families were screened for muta tions using direct PCR sequencing of the exons. Results: The vacuolar proton-ATPase membrane sector-associated protein M8-9 (APT6M8-9) gene was identified within our critical region. We confirmed it s retinal expression and its genomic location in our physical contig. Eight exons (with flanking intronic sequences) were characterized from partial c DNA sequence and genomic sequence data. An additional 5' end exon was ident ified using RACE. No mutations were found in the COD1-affected males. Conclusions: The combination of disease mapping and information from the Hu man Genome project has enabled us to identify candidate genes within the CO D1 region, including APT6M8-9 gene. We found no evidence that this gene is responsible for COD1 in our families, but it may be an important candidate for other diseases that have been mapped to this region of the X chromosome .