Identification of mutations in HPRP3, the Gene for Autosomal Dominant Retinitis Pigmentosa on Chromosome 1 and Functional Analysis of Mutations

Abstract

Abstract: : Purpose: To undertake functional characterisation of HPRP3 mutations, the gene responsible for the RP18 locus on chromosome 1p13-q21. We recently identified HPRP3 as a third splicing factor gene to be implicated in adRP along with PRPC8 and PRPF31, the adRP genes on chromosome 17p and 19q respectively. . Methods: PCR, Denaturing High Performance Liquid Chromatography (DHPLC) and direct sequencing was used for mutation detection. Mutagenesis was performed using the QuikChange site-directed mutagenesis method using the pTriEx-1 vector. To test for splicing defects the HPRP3 mutants were co-transfected with a splicing construct that contain a recombinant bovine rhodopsin transcript inclusive of intron 4 Results: We have identified two different missense mutations within HPRP3 in two English families, a Danish family and in three individuals with RP. Both mutations (T494M and P493S) are clustered within a two-codon stretch in the eleventh exon of the gene. In order to identify the functional consequences of the two mutations we created mutant constructs of HPRP3 in a vector allowing for expression in E. coli and mammalian cells. The His-tagged mutant protein also allows for immuno-localisation studies within the mammalian cells. Conclusion: The identification of mutations in a third pre-mRNA splicing factor gene further highlights a novel mechanism of photoreceptor degeneration due to defects in the splicing process. Preliminary functional data indicate that the pathophysiological basis of adRP at this locus is due to functional loss of one allele resulting in haploinsuffiency.

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