| Background Primary retinitis pigmentosa (RP) is among the most common cause of inheritedblindness, which affects about 1 in every 3500 people worldwide. Most patients with RP typically experience night blindness, a gradual loss of peripheral visual field and eventuallyloss of central vision due to degeneration of rod photoreceptor cells followed by cone ceils. RP is very heterogeneous, both phenotypically and genetically. With the widespread application of linkage analysis and mutation detection techniques, a complex underlying etiology has now been revealed. So far, at least 31 distinct genetic loci have been implicatedin RP, with mutations identified in at least 15 genes that cause various forms of primary RP, including adRP (autosomal dominant RP), arRP (autosomal recessive RP) and XLRP(X-linked RP). Rhodopsin (RHO) on chromosome 3q represent the most common known RPgene, whose mutations contribute to about 10% of all RP cases or about 25% of adRP inNorth America. However the frequency is lower elsewhere in the world due to the absence of RHO P23H, which is only found in America and causes about 10% of adRP. Excluding P23H, the most common RP mutation is RHO P347L, accounting for 3-5% of adRP patients or 0.5-1.0% of all unrelated RP patients in Europe, Asia and Africa. Recently, family linkage analysis identified a novel photoreceptor-specific gene, RP1, on chromosome 8q11-13, a locus of adRP. Truncating mutations in the RP1 gene cause about 7% of adRP, and approximately 3% of all RP cases in North America, appearing to be a second predominant cause of RP after RHO. However, the function of RP 1 is yet unclear at present. So far, more than 20 truncating mutations in RP1 causative of RP have been identified in Caucasians, among which R677X is responsible for about 3% of adRP, making it the third most common adRP mutation in Caucasians after the RHO mutations, Pro23His (10%) and Pro347Leu (5%) To date, no study about screening the entire RHO or RP 1 gene for sequence alterations among Chinese individuals has been reported.Purpose In order to evaluate the role of RHO/RP1 mutations in RP and also to learn the patterns and frequency of RHO/KP 1 mutations in Chinese RP patients, we screened the entire coding regions as well as the adjacent flanking splice sites of RHO and RP1 genes in 100 unrelated Chinese RP patients and 190 normal controls. Additionally, based on the mutation analysis of RP genes, we attempt to establish a simple, fast and accurate mutation detection system applied in molecular diagnosis of RP.Methods Leukocyte DNA was isolated from peripheral blood samples of 100 unrelated Chinesepatients diagnosed with RP and 190 unrelated individuals without RP or other known eyedisease. The conformation sensitive gel electrophoresis (t2SGE) technique and direct DNA sequencing were used to evaluate 7 DNA fragments of RHO and 26 of RP1, amplified by PCR from the DNA samples, that in total encompassed the entire coding regions and the flanking splice sites of RHO and RP1. genes respectively. When a suspecteddisease-associated mutation was detected in one index patient of RP, his/her first-degree and/or second-degree relatives were also recruited and genotyped correspondingly to determine whether the identified mutation cosegregated with RP in their family. In addition we made some modifications with the original procedure of CSGE, including decreasing the gel thickness by half, using two half-width double free sharkstooth combs for 96 wells, increasing the number of loadings per lane and using SYBR Gold for DNA staining in stead of EB (ethidium bromide), to increase the number of PCR samples analyzed on one gel without decreasing the resolution of the bands.Results For RHO we have totally identified six nucleotide changes, among which three are silent mutations, two are missense mutations and one is deletion mutation. P347L, the most common RHO mutation causative of RP worldwide, was found in one RP proband as well as three children of h...
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