RP33, A Novel Locus Of Autosomal Dominant Retinitis Pigmentosa; And A Complete Mutation Screening Of PRPF31 Gene In RP11 Linked Family

RP33. a novel locus of autosomal dominant retinitis pigmentosa；and a complete mutation screening of PRPF31 gene in RP11 linked familyObjectiveTo study the clinical manifestations of two Chinese families with autosomal dominant retinitis pigmentosa(adRP)and investigate their genetic pathogenesis．A genome wide linkage analysis was applied and followed by a complete mutation screening of candidate genes．1. To clinically characterize two families with adRP(kindred AA and AB)．2. To identify the chromosomal locations of the disease loci in the two adRP families by genome-wide linkage screening．3．To search the Causative mutation of cardidate genes within linked regions in the two families respectively．4．To screen any substantial big deletion mutation in PRPF31 gene in family AB by mutiplex ligation-dependent probe amplication (MLPA) reaction and single nucleotide polymorphism(SNPs)testing．Methods:1. Clinical investigation1.1 Routine ophthalmologic examinationsOphthalmologic examinations including disease history, best correct visual acuity, slit-lamp examinations, direct funduscopy and photographphic fundus．1.2 Electrophysiological examinations Humphrey threshold perimetry (Humphrey 750), full field electroretinogram (ERG) and multifocal ERG (mfERG) were 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 analysis2.2.1 Genome-wide linkage analysisA genome-wide linkage screening was conducted by genotyping 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. Linkage analyses were carried out by calculating multipoint LOD scores using the LINKAGE software package of SimWalk2 2.89, Version 3.35.2.2.2 Fine mappingBased upon the genome-wide linkage analysis, more fluorescent microsatellite markers located within the initial linked regions were genotyped to refine the critical interval.2.3 Sequence analysisCandidate genes were selected from linked regions of the two families respectively and direct sequencing was performed using ABI 3730 Genetic Analyzer to evaluate all the exons and flanking introns sequences of the candidate genes.2.4 Detection for big deletion in PRPF31 geneSNPs testing and MLPA reaction were applied in family AB to detect any substantial big deletions (hemizygousity) ofPRPF31 gene.Result: 1. AA familyClinically, AA family presented relatively late onset of night blindness, gradually decreased visual acuity and progressive loss of peripheral visual field. Variable clinical features and incomplete penetrance were found in the affected members of AA family.After excluding all known adRP loci by genome-wide linkage analysis, a novel disease locus RP33 (OMIM %610395) was assigned to the long arm of chromosome2. From meiotic recombinations in two unaffected members, RP33 was further refined to a 4.8 cM (9.5 Mb) interval flanked by D2S2159 and D2S1343 in chromosomal region 2cen-q12.1.No disease-associated mutations were detected in the candidate genes SEMA4C,CNGA3 or HNKlST from within RP33 region. MERTK, a known disease gene for autosomal recessive RP located close to RP33 was similarly excluded.2. AB familyAB family presented a form of diffuse adRP phenotype with high penetrance. In addition, cortex cataract was also observed in 13 affected members with adRP phenotype.AdRP in family AB was mapped to chromosomal region 19q13.4 (RP11 locus, OMIM #600138) , a 5.71 cM interval flanked by microsatellite markers D19S924 and D19S880. A maximum multi-point LOD score of 7.46 was reached at marker D19S927.After a relatively complete mutation screening throughout the open reading frame of PRPF31 gene, it has been excluded that the PRPF31 gene is the causative gene of AB family. No disease-associated mutations were detected in other candidate genes PRKCG, TFPT, TSEN34 or RDH13 from within the linked region. U2AF2, an essential pre-mRNA splicing factor coding gene located close to the critical interval was similar excluded.Conclusions: 1. Pedigrees analysis demonstrated that the RP conditions in kindred AA and AB are both inherited as an autosomal dominant (adRP) trait.2. A novel adRP locus RP33 (OMIM %610395) was assigned to chromosomal region 2cen-q12.1 in AA family. It's the first novel RP locus reported by China. SEMA4C, CNGA3, HNK1ST and MERTK genes are not the causative gene in AA family. There is a new gene in RP33 region which is responsible for adRP phenotype.3. Family AB was linked to chromosomal region 19q13.4 (RP11 locus). PRPF31 gene is not the causative gene for the adRP phynotype in AB family. In addition to PRPF31, there may be a new adRP related gene in RP11 locus, which caused the diffuse form of adRP phenotype in AB family, or might be also causative for the cataract phenotype.