Molecular analysis of candidate genes in retinitis pigmentosa
Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal degenerations which are one of the most common causes of inherited visual handicap. The aims of this study were three fold. Firstly, samples from 81 patients with RP and various other retinal degenerations were screened for mutations in four of the RP candidate genes (rhodopsin, ROM1 peripherin/RDS and CRBP1) by SSCP analysis followed by direct sequencing. Two heterozygous sequence changes, GCC299TCC (ala299ser) and G4084A, were identified in the rhodopsin gene, both of them in isolated cases of RP. Three alterations were found in the ROM1 gene. A heterozygous CGC16CAA (arg16gln) alteration was disease specific in available members of an autosomal dominant RP family and a heterozygous GGG298AGG was found in an isolated case of RP. The third change, CGG223CGC (arg223arg), has been previously reported as a benign polymorphism. Four sequence changes were identified in peripherin/RDS exon 3 (glu304gln, lys310arg, gly338asp and C1302T) all of which have been previously reported as benign polymorphisms. No sequence changes were identified in the CRBP1 gene. Secondly, the RP candidate genes rhodopsin, peripherin/RDS and ROM1 were shown to be illegitimately transcribed in human peripheral blood lymphocytes (PBLs). This allowed an analysis of the effect of three rhodopsin splice site sequence changes on mRNA processing. One of these changes, a heterozygous G5167A change in the 3' acceptor splice site of intron 4 and disease specific in an adRP family was found to abolish normal splicing. The second two changes (GGC120GGT and G4084A) were both found in isolated cases of RP and neither of them is thought to affect mRNA processing. aFinally, RNA arbitrarily primed PCR (RAP-PCR) of PBL RNA was investigated as a means to identify novel RP candidate genes.