Abu Safieh, Leen; (2003) Molecular genetic study of autosomal dominant retinitis pigmentosa on chromosome 19q13.4. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Retinitis Pigmentosa (RP) describes a clinically and genetically heterogeneous group of inherited retinal disorders characterized by progressive loss of peripheral vision and night blindness. RP can be inherited as an autosomal dominant, autosomal recessive or X-linked trait. The autosomal dominant form of RP (adRP) can be caused by mutations in 11 genes and one other locus, for which the gene remains to be identified. The work presented in this thesis relates to the adRP locus that maps to chromosome 19q13.4 (designated RP11). Affected members from RP11-linked pedigrees have type II, the regional form of RP. Uniquely all RP11 linked families also exhibit an incomplete penetrance phenotype defined by the presence of completely asymptomatic disease gene carriers. The aim of this work was to identify and characterize the RP11 gene and to determine the molecular basis of the partial penetrance phenotype. At the time this work was initiated the RP11 locus was confined to a ~ 500kb genomic region, DNA of which was represented by several BAC clones. As the sequence of these BAC clones was emerging due to the efforts of the Human Genome Mapping Consortium, a systematic analysis of this sequence was carried out to identify positional candidates for RP11. This thesis specifically describes the characterization of genes located in BAC clone 3093M3. In addition to the sequence analysis, ESTs mapping to the RP11 interval were also characterized. The screening and exclusion of one such EST i.e. WI-17997, encoding a hypothetical gene LOC911663, is described in chapter 3. This EST was one of the few ESTs that mapped to the interval. Hence it was fully characterized and screened as a candidate for RP11. Screening of insilico-predicted genes located in BAC clone 3093 M3 led to the identification of pathogenic mutations in a novel gene, PRPF31 subsequently found to encode a pre-mRNA splicing factor. PRPF31 protein is an integral component of the human U4.U6 snRNP particle and therefore essential to the spliceosome complex, which mediates pre-mRNA splicing. Identification of the RP11 gene is described in chapter 4. To investigate the possibility that the partial penetrance of disease is due to the differential expression of wild type PRPF31 allele, the level of PRPF31 expression in symptomatic and asymptomatic individuals both at the mRNA and the protein levels was investigated (chapter 5). Real-time quantitative RT-PCR was performed on RNA from lymphoblastoid cell lines derived from the adRP family, ADRP5 that segregates an 11bp deletion in exon 11 of PRPF31. The mRNA levels from only the wild-type allele of PRPF31 were assayed using a probe designed across the deletion. The PRPF31 protein levels from symptomatic and asymptomatic individuals were also assayed by Western blot analysis using an antibody specific to the wild-type PRPF31 protein. The use of cell lines was validated by the observation that cell transformation did not alter PRPF31 expression in the cell lines as compared with nucleated blood cells and donor retinas. A significant difference in wild-type PRPF31 mRNA levels was observed between symptomatic and asymptomatic individuals (p<0.001). This data were supported by Western blot analysis of the PRPF31 protein. Therefore, partial penetrance in RP11 could be due to the co-inheritance of a PRPF31 gene defect and a low expressed wild type allele. This study revealed a potential avenue for future therapy, as it appears that moderate over expression of wild type PRPF31 may prevent clinical manifestation of the disease.

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