Simultaneous mutation detection in 90 retinal disease genes in multiple patients using a custom-designed 300-kb retinal resequencing chip.
OBJECTIVE
To develop a high-throughput, cost-effective diagnostic strategy for the identification of known and new mutations in 90 retinal disease genes.


METHODS
Evidence-based study.


METHODS
Sixty patients with a variety of retinal disorders, including Leber's congenital amaurosis, ocular albinism, pseudoxanthoma elasticum, retinitis pigmentosa, and Stargardt's disease.


METHODS
We designed a custom 300-kb resequencing chip. Polymerase chain reaction (PCR) amplification, DNA fragmentation, and chip hybridization were performed according to Affymetrix recommendations. Hybridization signals were analyzed using Sequence pilot module seq-C mutation detection software (2009). This resequencing approach was validated by Sanger sequence technology.


METHODS
Disease-causing sequence changes.


RESULTS
We developed a retinal resequencing chip that covers all exons of 90 retinal disease genes. We developed and tested multiplex primer sets for 1445 amplicons representing the genes included on the chip. We validated our approach by screening 87 exons from 25 retinal disease genes containing 87 known sequence changes previously identified in our patient group using Sanger sequencing. Call rates for successfully hybridized amplicons were 98% to 100%. Of the known single nucleotide changes, 99% could be detected on the chip. As expected, deletions could not be detected reliably.


CONCLUSIONS
We designed a custom resequencing chip that can detect known and new sequence changes in 90 retinal disease genes using a new high-throughput strategy with a high sensitivity and specificity for one tenth of the cost of conventional direct sequencing. 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.
Booij
Judith C
JC
Department of Clinical and Molecular Ophthalmogenetics, Netherlands Institute for Neuroscience, an institute of the Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands.
Bakker
Arne
A
Kulumbetova
Jamilia
J
Moutaoukil
Youssef
Y
Smeets
Bert
B
Verheij
Joke
J
Kroes
Hester Y
HY
Klaver
Caroline C W
CC
van Schooneveld
Mary
M
Bergen
Arthur A B
AA
Florijn
Ralph J
RJ
D003362:Cost-Benefit Analysis; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D005136:Eye Proteins / Q000235:genetics*; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000379:methods*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012164:Retinal Diseases / Q000235:genetics*; D012680:Sensitivity and Specificity
D005136:Eye Proteins