Gene Mapping In A Chinese Bai Pedigree With Autosomal Dominant Retinitis Pigmentosa

BackgroundAutosomal dominant retinitis pigmentosa (adRP) is a clinically and genetically heterogeneous disorder. It can be transmitted as autosomal dominant RP (adRP), autosomal recessive RP (arRP), X-link RP (XLRP) and some with digenic or mitochondrial inheritance. RP is the most frequent form of inherited retinopathy, with an approximate incidence of 1 in 3500 individuals worldwide and over one million being affected. But to this day, there is no prevention or treatment available for it. Identification of additional loci as well as of the causative genes is the first step toward understanding the molecular basis of RP, and subsequently, toward the genetic counseling, prenatal diagnosis or treatment of this sight threatening problem. Studies show that over 70 loci were relative to the disfounction of human retinal photoreceptor, and now 53 loci have been identified. Therefore gene mapping of RP is still hot research in molecular genetics.Of the 53 loci,19 have been determined in adRP, and rhodopsin (RHO),PRPF31,RP1,RDS and IMPDH1 are more frequently reported as adRP gene in many populations. Although RHO and PRPF31 gene are common in Han Chinese, it has not been detected in patients from the Bai nationality, one of minority ethnic groups of southwest China. Up to now, there are 120 different mutations reported in RHOand many of which shows a point mutation. Studies have discovered that these mutations displayed different distribution in different population, such as in the west it accounts for 25%and of that 5.9%in Japan,7.7%in china. Pro23His was the most common mutation of RHO in South American while in Asian, all of the RHO mutation located in c-terminal and Pro347Lue was most commonly reported in the globe. PRPF31 gene, second only to see frequently except RHO gene, was firstly found in a large English adRP pedigree and mapped in chromosome by linkage analysis. Nowadays over 30 different mutations have been reported in PRPF3 gene that include missense, deletion and insertion and splice site alterations. The most frequent mutational site of PRPF31 gene was locating in exon5, exon6, exon7, exon8, exon11 and intron splice sites. Shown in data that RP1 gene was common in the west but rare in china, its mutation causing adRP accounted for 7%and exon4 (R677X) of this gene was the most frequent mutational site. Mutation in RDS gene may result many of retinal degeneration and today over 70 mutants of RDS were associated with adRP and autosomal dominant macula degeneration, of which accounting for 5%in adRP. RDS gene's mutations, including the most frequent mutational site (exonl) also characterized by territory disparation. The majority of these mutations were found in American or Norther Europe whereas rare in Asian. Mutations of IMPDH1 gene mapping in chromosome 7q32.1 was seldom in our country and its frequent mutational site was exon7, but the clinical symptom suffered from these mutants were early-onset and severiou.As the reported from literatures, the mapping of RP disease-causing mutation was largely carried out through linkage analysis. Science in 1990s, Inglehearn and Greenberg have used the second generation genetic marks, short tandem repeat (STR) to perform linkage analysis then they found two novel loci in chromosome 7p and chromosome 17p. With the help of generation genetic marks, linkage analysis could make a fair decision in whether there would be a linkage relation between markers and disease locus and finally confirm a candidate for the studied pedigree.Inherited disease holds a genetic predisposition in a pedigree, and it was a characteristic in genetic research to perform gene mapping by taking advantage of pedigrees especially from a big Chinese family. RP was charactered by genetic heterogeneity, affected individuals, even coming from a pedigree maybe harbor different disease-causing gene. The Bai (Pai) nationality, one of minority ethnic groups of southwest China shares its own culture feature and rarely contacts with others beyond toward to this region, of which the background was beneficial for linkage analysis. To hunt for the mutant gene in the Bai family will enrich the mutation spectrum of human RP gene in different ethnic groups and supply more information for the establishment of Chinese RP gene map and system of gene order structure or function.ObjectiveTo study the relationship between causative genes of a pedigree with adRP and RHO, PRPF31, RP1,RDS and IMPDH1 genesMethods1. Microsatellite markers D3S3606 and D3S1292, which closed to the RHO locus and with a distance approximately one-centimorgan (cM) on chromosome 3 were selected to perform genotyping. The size of allele was determined on the basis of an internal size standard, and data were analyzed using the Gene Mapper version 3.5 software package.2. LOD scores for two-point linkage analysis between the disease locus and the genetic markers was performed with LINKAGE package 5.1. And the judgement of linkage degree according to the following criteria:if LOD score is large than or equal to 3, marker linkage to disease locus is certain; if LOD score less than negative or equal to 2, it dose not support linkage; if LOD score belongs to the others, it should be made a larger samples. In addition, Haplotype analysis was completed by using Cyrillic 2.1 software.3. Primers were designed with Primer premier version 5.0 to amplify all coding region and intron splice sites of RHO and the hot mutation spot of the PFPR31,IMPDH1 and RDS gene. The PCR products were sequenced directly by ABI PRISM(?)3100 Genetic Analyze and the results were Blast with the sequence from GenBank database for mutation analysis.4. Reaction of restriction enzyme was carried out for 391bp PCR products of exon5 of RHO amplified from 13 affected and 9 unaffected members withl U of NlaIV to confirm the results of sequencing.5. Statistical analysis was not performed with SPSS13.0 software to test chi-square for SNPs rs7984, rs2269736, rs57960425 and rs2303557 because the sample size collected were too small and the related species were not independent.Results1. We identified and characterized a Bai adRP pedigree, of which 13 individuals were affected and 9 were unaffected members, However, during to younger ofⅣ4,Ⅳ5,Ⅳ6andⅤ1, there were no changes in their ERG or fundus examination, but a nyctalopia had been represented. The inheritance pattern in the pedigree is autosomal dominant, all of the affected individuals have a similar juvenile-onset night blindness, visual deterioration with growths and posterior subcapsular opacity in advanced stage, but no others syndrome were found except for these.2. The maximum two-point LOD score obtained was 3.61 and 4.52 at a recombination fraction (0) of zero with markers D3S3606 and D3S1292, respectively. Haplotype analysis showed cosegregation one-centimorgan region harboring the RHO gene between the two markers with the disease, which indicates that the disease-caused gene locus is located in this region.3. Direct sequencing of RHO revealed a heterozygous nucleotide substitution (c.1040C＞T) in exon 5, which was carried by all the affected individuals. Two single nucleotide polymorphisms rs7984 and rs2269736 were found in exon 1, and higher frequencies (61.54%) of genotypes AA of rs7984 and GG of rs2269736 were found in affected members of this pedigree. A four-base deletion at intron 6 of PRPF31 (IVS6-78_IVS6-75del4CACA) was observed in eleven members (50%), including seven (53.8%) adRP patients and four (44.4%) unaffected individuals. A variation IVS6-31 C/T was also found at intron 6 of PRPF31 in eight affectedmembers (61.5%) and three unaffected members (33.3%). No variations were found in IMPDH1 and RDS genes.4. With digested by the restriction enzyme NlaIV, All affected individuals examined in the pedigree are heterozygous carriers who have all five bands at 307 bp,177 bp,130 bp,50 bp and 43 bp. The unaffected individuals are homozygous and have only the four lower bands (177 bp,130 bp,50 bp and 43 bp), which supported that c.1040C>T mutation was co-segregated with this disease.Conclusion1. The hot mutation spot of PRPF31,IIMPDH1 and RDS gene were not relative to the adRP in this Bai pedigree.2. A c.1040C＞T (p.Pro3471eu) mutation in exon 5 of RHO was cosegregated with disease and responsible for the molecule-based pathogenesis in a Chinese Bai pedigree with adRP. 3. The co-expression of homozygotic genotype of single nucleotide polymorphisms rs7984 (AA) and rs2269736 (GG) may be associated with the onset of adRP in the Bai pedigree.