Genetic Linkage Localization And Mutation Analysis Of Chinese X-linked Retinitis Pigmentosa Pedigrees

BackgroundRetinitis pigmentosa (RP) is among the most common causes of inherited blindness, which affects about 1 per 3500 people worldwide. Most patients with RP typically experience night blindness, a gradual loss of peripheral visual field and eventual loss of central vision due to degeneration of rod photoreceptor cells followed by cone cells. RP is heterogeneous, and can be inherited in various forms including adRP (autosomal dominant RP), arRP (autosomal recessive RP), XLRP (X-linked RP) and mitochondria-linked RP. Of these, XLRP, which accounts for 6 to 20% of all RP cases, is the most severe form with early age of onset and rapid progression. Molecular genetic study of this disease can elucidate its molecular defects, which would be of great value for the application of molecular diagnosis and efficient therapy to this disorder. So far, six RP loci have been identified in human X chromosome, including RP2, RP3, RP6, RP23, RP24 and the newly reported RP34. According to the linkage analysis, RP3 is considered to account for 70-90% of XLRP while RP2 account for 10-20%. These two major loci have already been cloned.RP3 gene, also called Retinitis Pigmentosa GTPase Regulator (RPGR), has different transcripts derived from alternative splicing in different tissues. The most important transcript of this gene is RPGRORF15, which shows highest expression in photoreceptors and is the only transcript known to be involved in retinal disease. Human RPGRORF15 contains exons 1-14 of RPGRex1-19 plus a large alternatively spliced C-terminal exon, ORF15. Recent studies on population from England, North America, Germany and Japan all indicate that exon ORF15 is a mutational hot spot in XLRP. About 60-80% of XLRP patients have pathogenic mutations in exon ORF15. The highest frequency of mutations is associated with a central repetitive, purine rich region of ORF15 in which pyrimidines account only for 2-3%. ObjectiveIn our study we employed genetic linkage analysis and haplotype analysis to identify potential loci responsible for this disease in a newly collected X-linked RP (XLRP) family and investigate the X-linked mode of the family. This enabled us to apply genetic methodology to detect carrier of female and antenatal diagnosis of fetus.After that, we sequenced the candidate genes in the critical region in order to reveal mutations and get some genetic background information about the Chinese XLRP patients and also to gain more insights into genotype-phenotype correlation of XLRP. The data we get from these efforts are beneficial for such future endeavors as genetic consultation, clinical diagnosis and therapy.MethodsIn the former work of our group, we have localized the disease loci in 4 doubtful XLRP families and there are 3 families linked to RP2 or RP3 loci.On this research, we first localized the disease loci in a newly doubtful XLRP family. 12 microsatellite markers were selected from the known gene loci such as RP2, RP3, RP6, RP23 and RP24. Linkage analysis was performed using the GENE HUNTER program. Haplotype analysis for the familiy was performed to determine the critical region.Secondly, we sequenced the candidate genes of RP2 and RPGRORF15 especially the mutational hot spot exon ORF15 of the 3 former families which were linked to RP2 or RP3 loci and the other 10 doubtful XLRP sporadic cases. Once the probable mutation was identified, the sequencing of all the members of the families was performed to confirm the mutation status in the family, and to exclude it as a polymorphism in a reference population.ResultsBy linkage analysis and haplotype analysis, the disease gene of ZSC pedigree was mapped close to RP23 or RP6 loci. 6 reported polymorphisms have been detected during the sequencing of the exon1-14 and the flanking intron sequences of RPGR gene. 2 pathogenic mutations, 5 reported polymorphisms, 5 novel SNPs and 1 novel 21bp-duplication were identified in ORF15 of RPGR gene. In the two pathogenic mutations, g.ORF15+483₄84delGA, which is detected in the ZLK family, has been reported in several pieces of research and g.ORF15+821₈22delGG, which is detected in the XY family, has never been reported till now. These two mutations co-segregated completely with the disease phenotype, but was absent in the unaffected relatives, thus supporting their pathogenic nature in the families.ConclusionsWe have identified the disease loci in a doubtful X-linked RP (XLRP) family by using microsatellite markers. Through genetic linkage analysis and haplotype analysis, the disease gene was mapped close to RP23 or RP6 loci. It is important to detect carrier of female and make antenatal diagnosis of fetus.By sequencing the candidate genes of RP2 and RPGRORF15 especially the mutational hot spot exon ORF15, we have discovered two pathogenic mutations which were predicted to result in the early termination of the proteins, and 17 polymophisms. In our result there is one ORF15 mutation--g.ORF15+821delGG and 6 polymophisms being novel. This study has expanded the XLRP genotypic and phenotypic spectrum of RPGR mutations. Besides, both of the two pathogenic mutations and 11 of 17 polymophisms have also been detected in ORF15, which indicates that ORF15 is the mutaional hot spot not only in families from England, North America, Germany and Japan but also in Chinese families.In this study, we have successfully established the strategy to screen exon ORF15 for mutations, and provided the technical platform to detect RPGR mutations in Chinese RP patients.