DGGE and SSCPE screening of the gene for the gamma subunit of cone transducin in patients with cone disease

Purpose: We have been screening the gene for the gamma subunit of cone transducin (GNAG8) in patients with autosomal recessive and simplex "cone disease" (cone dystrophy, cone-rod degeneration, Bardet Biedl syndrome or macular degeneration) for point mutations that may be related to their disease. Methods: Exon 1 (containing the putative promoter) and the coding exons 2 and 3 from the DNAs of more than 100 unrelated patients were amplified by PCR with primers that include 18-24 bases of intronic flanking sequence along with each exon. The amplicons were screened for sequence variants by DGGE and SSCPE, and any that were found were sequenced directly. DNAs from more than 50 controls were also screened for any sequence variants found in patients. Results: To date, three different sequence variants have been found in exon 2 and one in exon 3. One of the exon 2 variants has been found in 2 recessive patients, 5 simplex patients and 2 controls; the other exon 2 variants have each been found in one control and one simplex patient. The exon 3 variant has been found only in one recessive patient so far. However, in no patient were two variants found (i.e. one variant in each allele). Sequencing of these variants is underway. Conclusions: None of the heterozygous polymorphisms alone can explain disease in the autosomal recessive patients. However, there is a possibility that heterozygous single sequence variants could be responsible for disease in simplex patients if the sequence variant predicted a significant change in the protein and was absent from controls. Further screening and sequencing is underway to complete the analysis of the GNAG8 exons in these patients with simplex or recessive cone disease.