Economic and Efficient System for Genetic Diagnosis in Reitnitis Pigmentosa Patients Using Denaturing HPLC

Abstract

Abstract: : Purpose:The hereditary pattern of many of the retinitis pigmentosa patients are difficult to determine despite that such information is one of patients' most concerns. For such needs genetic diagnosis is the decisive way, but considering the diversity of causal genes of the disease, the introduction of an efficient system of gene diagnosis is mandatory. Here we report our newly established genetic diagnostic system using denaturing HPLC (d–HPLC: WAVE system). Methods: DNA was extracted from the peripheral blood leukocytes of typical retinitis pigmentosa patients who gave their informed consent. PCR was performed under one condition for all the genomic exons that include previously reported causal mutations for autosomal dominant (AD), autosomal recessive (AR) and x–linked (XR) RP (29 genes and 110 exons). The double stranded PCR products of each exon from a single (AD) or a number of patients (AR and XR) were then subjected to a hetero–duplex reaction and the resultant samples were analyzed on HPLC (Wave system; Transgenomic Inc. USA). Results: Most of the positive samples by Wave analysis contained a single nucleotide polymorphism and possible causal heterozygous mutations were detected in 9 patients. Four patients had mutations in RDS, 3 in rhodopsin and 2 in FSCN2 genes. Among the 9 patients, hereditary patterns of 5 patients had not been identified by their family history. Conclusions: The use of d–HPLC may allow an efficient and economic screening system for genetic diagnosis in RP patients and help identifying hereditary patterns of pigmentosa patients.

Keywords: gene screening • retinal degenerations: hereditary • genetics