Investigating the molecular basis of partial penetrance with splicing factor gene, PRPF31, implicated in retinitis pigmentosa

Retinitis Pigmentosa (RP, MIM#268000) is a significant cause of blindness in the Western world with an incidence of 1 in 3500. The mode of inheritance in RP may be autosomal recessive (ar), autosomal dominant (ad), X-linked recessive or digenic. To date, 39 loci have been implicated in RP, of which 30 genes are known (http://www.sph.uth.tmc.edu/ Retnet/home.htm). Three recently identified pre-mRNA splicing factors, PRPF31, PRPFB, and PRPF3 correspond to RP11, 13, and 18, respectively. Our ongoing research on the genetic basis of RP resulted in the mapping of an autosomal dominant RP (adRP) locus on chromosome 19q13.4 (RP11). The genetic interval was refined to a 520kb region flanked by markers 019S927 and 019S781.2. Mutation screening led to the exciting discovery of splicing factor gene, PRPF31, implicated in RP11. The unique feature associated with this locus has been the consistent finding of partial penetrance phenotype in most RP11 families worldwide. The molecular mechanism responsible for this feature still remains unClear. We speculated that the reason for partial penetrance is due to the different levels of expression of the normal copy 9f the gene that is able to compensate for the mutant allele in asymptomatic individuals (or unable to in the symptqmatic individuals). Partial penetrance can be viewed as nature's way of curing the disease. Through our recent studies we demonstrated that symptomatic and asymptomatic individuals in a given sib-ship consistently inherited different wild-type (WT) alleles of PRPF31 from their unaffected parent. This resulted in the asymptomatics having higher levels of WT PRPF31 mRNA compared to symptomatic individuals. We feel this might explain the partial penetrance phenotype observed in most RP11 families. The aim of the thesis is to explore the molecular mechanism that underlies the partial penetrance phenotype associated with the PRPF31 gene, causing adRP. For this reason, the variation in the wild-type PRPF31 expression level was assessed in random unrelated individuals, at the mRNA level and protein level. From the data generated, it showed that in the general population there are individuals with two high expressing alleles (HH), individuals with one high and one low (HL) and others who have two low expressing alleles (LL), suggesting a continuing spectrum with three overlapping (high, intermediate and low) levels of PRPF31 expression. In order to identify the novel sequence change in PRPF31 gene, responsible for the differential expression of PAPF31 alleles, the -520kb region spanning PRPF31 gene has been targetted to be amplified and sequenced, where the genetic change might exist. A SNP rs460824 located -93 kb (based on the standard ENSEMBL database) upstream of PAPF31 showed significant association (chi square 7.5, P