Elsevier

Ophthalmology

Volume 118, Issue 1, January 2011, Pages 160-167.e3
Ophthalmology

Original article
Simultaneous Mutation Detection in 90 Retinal Disease Genes in Multiple Patients Using a Custom-designed 300-kb Retinal Resequencing Chip

https://doi.org/10.1016/j.ophtha.2010.04.022Get rights and content

Purpose

To develop a high-throughput, cost-effective diagnostic strategy for the identification of known and new mutations in 90 retinal disease genes.

Design

Evidence-based study.

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.

Main Outcome Measures

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.

Financial Disclosure(s)

The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Section snippets

Materials and Methods

For this study, we designed a custom 300-kb Affymetrix resequencing chip8(http://media.affymetrix.com/support/technical/other/customseq_design_manual.pdf, accessed April 10, 2010) capable of sequencing both forward and reverse strands of 90 retinal disease genes. For validation of the chip, we included DNA from patients with retinal diseases containing 87 known sequence changes in 25 of the genes on the chip. The study was performed in agreement with the Declaration of Helsinki. Approval of the

Custom Resequencing Chip Design

We designed a custom 300-kb Affymetrix resequencing chip, allowing for the direct sequencing of 265 000 double-stranded bases of retinal disease genes. For 90 retinal disease genes, the array covers all coding exons and 12 bases of flanking intronic sequence on either side of the exons. For 7 genes, several single nucleotide polymorphisms (SNPs) are included (Table 1). Array specifications required that 12 additional bases are added on the 5' and 3' side of each amplicon; these 24 bases are not

Discussion

This study presents a high-throughput diagnostic tool for the detection of known and new mutations in patients with inherited retinal diseases. We describe the design, validation, and use of a new 300-kb retinal resequencing chip that allows for the direct sequencing of all coding exons and flanking introns of 90 retinal disease genes. We devised a unique pooling strategy that allows for the screening of multiple patients or genes per chip, which will reduce the costs per screened individual

Acknowledgments

The authors thank Drs. M. H. Breuning, A. Plomp, L. Prick, F. Meire, and M. van Genderen for sending in DNA of patients.

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    Manuscript no. 2009-1455.

    Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

    Funded by the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Landelijke Stichting voor Blinden en Slechtzienden, Rotterdamse Vereniging Blindenbelangen, Stichting Blindenpenning, Stichting Oogfonds Nederland, and Gelderse Blindenstichting.

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