Population Haplotypes of Exon ORF15 of the Retinitis Pigmentosa GTPase Regulator Gene in Germany
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Population Haplotypes of Exon ORF15 of the Retinitis Pigmentosa GTPase Regulator Gene in Germany Implications for Screening for Inherited Retinal Disorders Daniela Karra,1 Felix K. Jacobi,2 Martina Broghammer,1 Nikolaus Blin1 and Carsten M. Pusch1 ¨ Division of Molecular Genetics, Institute of Anthropology and Human Genetics, University of Tubingen, ¨ Tubingen, Germany 2 Department of Ophthalmology, University of Giessen, Giessen, Germany
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Abstract
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.
The retinitis pigmentosa GTPase regulator gene (RPGR) has been demonstrated to be involved in 60–90% of cases of X-linked retinitis pigmentosa (XLRP);[1-3] however, only 20–25% of affected patients originally showed mutations in this gene.[4-7] This discrepancy suggested that other mutations might be located in yet unknown exons.[3] Expression studies in mice, dogs, and humans indicated the presence of alternative splice variants,[3,8-10] some of them expressed in a tissue-specific manner. In addition to the original 19 exons, five new exons (15 b1, 15 b2, 15 a, ORF14, and ORF15) have been determined.[3,9] The exon ORF15 is phyloge-
netically highly conserved and ubiquitously expressed with significant expression levels in the retina.[3,10,11] Exon ORF15 encodes 567 amino acid residues containing a repetitive domain rich in glycine and glutamic acid residues.[3] The distinctly high mutation rate indicates a mutational hot spot that is likely to b
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