Abstract
Purpose: :
Abnormal retinal vasculature is present in patients with various pediatric vitreoretinal diseases such as FEVR, Norrie’s, PFVS and ROP. We collected genomic DNA from 160 patients with pediatric retinal diseases(FEVR;n=32, Norries;n=3, ROP;n=96). Three genes (NDP, Fz4 and LRP–5) of the Wnt ß–cantenin signaling pathway have been implicated in vascularization of the retina. A panel of known mutations was selected for these three genes. Appropriate primers were designed and a protocol was optimized to efficiently and accurately screen the selected panel of mutations.
Methods: :
Single base extension technology on a capillary electrophoreses platform was used to screen mutations using genomic DNA. Initially, single mutations were tested individually. PCR and interrogation primers were then designed to construct a multiplex panel. Primer/template concentrations, thermocycling and cleanup conditions were optimized to obtain well separated products. The reactions were separated and analyzed using the Beckman CEQ 8000 genetic analysis system.
Results: :
The mutation panel was successfully developed. This assay yields reproducible and accurate results.
Conclusions: :
This Beckman Coulter SNP kit provides and easy and efficient means for screening three genes of the Wnt ß–cantenin signaling pathway.
Keywords: mutations • retinal neovascularization • gene screening