Resequencing chip enables rapid detection of mutations in retinal disease genes

Ophthalmology. 2011;118(1):160-167.

A custom resequencing chip accurately and efficiently identified novel and known mutations in retinal disease genes in multiple patients, a study conducted in the Netherlands found.

The chip offers rapid analysis with high sensitivity and specificity, at about one-tenth the cost of conventional direct sequencing.

Genetic heterogeneity and the complexity of retinal phenotypes hinder the efficient identification of mutations with conventional methods, the study authors said.

"The developed amplification strategy allows for the pooling of multiple patients with non-overlapping phenotypes, enabling many patients to be analyzed simultaneously in a fast and cost-effective manner," they said.

The investigators designed a custom 300-kb Affymetrix resequencing chip that affords direct sequencing of 265,000 double-stranded bases of retinal disease genes.

The evidence-based study included 60 patients with retinal disorders such as Leber's congenital amaurosis, ocular albinism, optic atrophy, glaucoma, pseudoxanthoma elasticum, retinitis pigmentosa and Stargardt's disease.

Direct sequencing of forward and reverse strands in relevant disease genes was performed to verify the presence of mutations in the DNA of the pooled patient sample.

The investigators identified 87 known pathogenic and polymorphic sequence changes from 25 retinal disease genes. They developed primer sets for 1,445 amplicons representing genes tested on the chip.

Study results showed a detection rate of 99% and reproducibility rate of 100%, with no false positives.

The chip is unable to detect deletions of genetic material. As a result, it should be used to prescreen patients to improve the efficiency of subsequent formal DNA testing, the authors said.