Trial to Introduce an Economic and Efficient System for Genetic Diagnosis in Patients with Retinitis Pigmentosa in Kyoto University Hospital

Abstract

Abstract: : Purpose: Retinitis Pigmentosa (RP) could develop from a mutation in number of photoreceptor or retinal pigment epithelium (RPE) related genes with a similar pathological phenotype. Diagnosis of causal gene will become more crucial with the future introduction of gene therapy or transplantation trials of RPE or neural cells. The goal of this study was to test a new genetic diagnostic screening system in our clinic in terms of efficiency and practicality. Methods: PCR primers were designed to amplify all the genomic fragments that include causal mutation for autosomal dominant (AD), autosomal recessive (AR) and x–linked (XR) RP listed on RetNet web sight (http://www.sph.uth.tmc.edu/Retnet/). In order to detect a mutation in amplified products, denaturing HPLC (d–HPLC) was performed in which the double stranded PCR products from a single (AD) or a number of patients (AR and XR) were subjected to a hetero–duplex reaction and the resultant samples were analyzed on HPLC (Wave system; Transgenomic Inc. USA). Results: All the amplification reactions (113 fragments from 29 cloned genes) could be performed under one PCR condition except for one fragment in RPGR. The use of d–HPLC enabled the detection of all the 25 artificially introduced mutations tested. The estimated total screening time for HPLC analysis of all the genes (AD, AR and XR) was approximately 60 hours per patient. The evaluation by HPLC pattern change was efficient and reproducible. Among 25 patients screened so far, an average of 18% of screened fragments showed some HPLC pattern change and were followed by sequence analysis. Half of the positives were false positive by sequencing confirmation. Most positives contained single nucleotide polymorphism and mutations were detected in 2 patients. This system reduced the total cost of diagnosis per patient by 70% of that done by full sequencing. Conclusions: The use of d–HPLC could allow an efficient and economic screening system for genetic diagnosis in RP patients

Keywords: retinal degenerations: hereditary • clinical research methodology • genetics