Population haplotypes of exon ORF15 of the retinitis pigmentosa GTPase regulator gene in Germany : implications for screening for inherited retinal disorders.

BACKGROUND: Mutations in exon ORF15 of the retinitis pigmentosa GTPase regulator gene (RPGR) within chromosomal region Xp21.1 are a significant cause of a number of retinal disorders. The high mutation rate is ascribed to the highly repetitive, purine-rich tracts within the exon ORF15 sequence. Importantly, all exon ORF15 mutations observed to date represent protein-truncating mutations (nonsense and frameshift mutations). Because of its repetitive motifs, mutation screening of the hot-spot region by direct DNA sequencing is a technically challenging task.
METHODS: We devised a screening strategy for exon ORF15 mutations that reserves DNA sequencing for precise sizing and base-order assessment of detected mutations. The screening strategy is based on a PCR/restriction fragment length polymorphism (RFLP) analysis of exon ORF15 and comparison with population-specific RFLP haplotypes. The latter were constructed from PCR/RFLP analysis of DNA samples from 100 healthy German male individuals. Mutational alterations of normal RFLP haplotype patterns were predicted.
RESULTS: Six distinct RFLP haplotypes (founder alleles H1-H6) were observed with frequencies ranging from 2% to 63%. All natural variations of exon ORF15 were in-frame alterations ranging in size between 3bp and 36bp. Prediction of mutation-specific RFLP patterns indicated a high detection rate of mutations.
CONCLUSION: A new strategy has been developed using routine protocols for mutation screening of difficult-to-sequence, highly repetitive exon ORF15 of the RPGR gene in a German population.