Mutational analysis of RHO, RDS and PRPF31 genes in Chinese patients with retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) is a group of genetically heterogeneous eye diseases. A characteristic feature of RP is the progressive degeneration of photoreceptors in the retina and eventual blindness. It is one of the most common genetic eye diseases with a prevalence of about 1 in 4000. More than one thousand people are estimated to have RP in Hong Kong. Genetic mutations play a definitive role in RP. In order to identify the genetic alterations underlying RP in Hong Kong Chinese, DMA samples from 79 RP subjects were screened for mutations by single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) amplified fragments (PCR-SSCP). Seventy five samples from anonymous healthy blood donors were used for determining the frequencies of base changes in normal subjects. Three genes were investigated in this study: rhodopsin (RHO), retinal degeneration, slow (RDS) and PRP31 pre-mRNA processing factor 31 homolog (yeast) (PRPF31). All exons and their flanking regions of these three genes were screened and all sequence variations were characterized by cycle sequencing. A putative promoter region of RHO was also investigated. In total, 37 sequence variations were found in this study. Of these variations, 14 have been reported previously while 23 were novel. Sequence variations were scattered throughout the genes. Nine sequence variations were identified in RHO, 10 in RDS and 18 in PRPF31. Five significant findings can be summarized as follows. First, a 3-T deletion of RHO located in the 5' negative regulatory element was identified in a subject with Usher syndrome (RP plus inherited hearing impairment). In sequence comparison between organisms, 3-T was found conserved among human, cow, mouse and rat. The consequences of this variation should be further confirmed by functional assays. Second, the well-known RHO missense mutation P347L and a novel +6307delT (also denoted as F262/sX320) frameshift mutation of PRPF31 were identified in an autosomal dominant RP (adRP) family. Affected subjects were found to carry either one or both of these two mutations: homozygous P347L RHO mutation with wild type PRPF31 (P347L +/+; F262feX320 -/-), wild type RHO mutation with heterozygous F262feX320 PRPF31 mutation (P347L -/-; F262feX320 +/-), and homozygous P347L RHO mutation with heterozygous F262feX320 PRPF31 mutation (P347L +/+; F262feX320 +/-). Third, a novel S2C missense of PRPF31 was also identified in two adRP families. Incomplete penetrance was suspected in one of the families that had an asymptomatic carrier of this mutation. Fourth, a novel complex RDS mutation, +232G>C;+232_+233insT, was found in an RP subject. These base changes caused a frameshift (A78feX176) and were predicted to produce a truncated protein. Finally, the frequencies of haplotypes consisting of three polymorphism alleles in RDS, E304Q, K310R and G338D, showed a statistical significant difference (p=0.0131) between the RP and the control groups. This indicated that there might be an association between the haplotype and RP.