Gene Mapping And Analysis Of Candidate Genes In Two Chinese Families With Retinitis Pigmentosa

ObjectiveTo determine the clinical phenotyps of two Chinese families with autosomal dominant retinitis pigmentosa (adRP), and to dissect their genetic basis by using genetic linkage analysis.1. To clinically characterize two families with adRP (kindred RPSZ and RPQY), and to investigate the relationship between gene mutation and clinical phenotype in kindred RPSZ.2. To identify the chromosomal locations of the disease loci in the two families with adRP.3. To sequence the candidate genes in the critical region in order to reveal mutations.4. To perform a restriction assay to confirm the mutation status in the family, and to exclude it as a polymorphism in a reference population.Methods:1. Clinical investigation 1.1 Routine ophthalmologic examinations Ophthalmologic examinations including disease history, best correct visualacuity, slit-lamp examinations, direct funduscopy and picture of fundus. 1.2 Electrophysiological examinationsHumphrey threshold perirnetry (Humphrey 750), full field electroretinography (ERG) and multifocal ERG (mfERG) was performed in all patients of the two families. 2. Molecular genetic study2.1 Human genomic DNA was extracted from peripheral blood leukocytes using DNA Isolation Kits for Mammalian Blood (Roche Biochemical, Inc.).2.2 Linkage analysisA genome-wide linkage screening was conducted by genotyping of 370 microsatellite markers representing all autosomes at an average resolution of approximately 10 cM (Weberset 6.0; Research Genetics). The polymerase chain reactions (PCR) were carried out to amplify all 370 markers. The PCR products were appropriately pooled according to allele sizes and labelling, and were run in ABI 377XL for fluorescent detection. The allele sizes were determined according to an internal size standard and the results were analyzed using Genescan 3.1 and Genotyper 2.0 software (Perkin Elmer). Linkage analyses were carried out by calculating multipoint LOD scores using the LINKAGE software package of SimWalk2, Version 3.35.2.3 Sequence analysisDirect genomic sequencing was used to evaluate all the exons and flanking intron sequences of the candidate genes. Once the probable mutation was identified, a restriction assay was used to confirm the mutation status in the family, and to exclude it as a polymorphism in a reference population.Results:1. Kindred RPSZBy linkage analysis the disease gene was mapped close to RP11 in chromosomal region 19ql3.4 defined by microsatellite markers D19S589 and D19S254. A novel single heterozygous base substitution (G>C) was detected at the beginning of intron 8 in the PRPF31 gene whereby the consensus GT dinucleotide of the intron 8 splice donor site was changed to CT. The mutation co-segregated completely with the disease phenotype, but was absent in the unaffected relatives and 100 reference subjects, thus supporting its pathogenic nature in the family. The clinical findings were consistent with a relatively severe and diffuse type of RP.2. Kindred RPQYThe disease locus was mapped to a minimum critical region (MCR) less than 600Kb between microsatellite markers D19S246 and D19S601 by linkage analysis. Sequencing of two candidate genes (CRX and PRPF31) near the MCR showed no mutation. There may be a new gene caused adRP in the MRC in chromosome 19 in that kindred.Conclusion:1. Pedigree analysis demonstrated that the RP conditions in kindred RPSZ and RPQY are both inherited as an autosomal dominant (adRP) trait.2. A novel splice site mutation (IVS8+1G>C) in the PRPF31 gene caused retinitis pigmentosa in the four-generation Chinese adRP family RPSZ studied. The relatively severe clinical findings in the RP patients and the high penetrance observed support the severity of the mutation detected in this family.