Details

Autor(en) / Beteiligte

• Booij, Judith C., MD, PhD
• Bakker, Arne, BSc
• Kulumbetova, Jamilia, MD, PhD
• Moutaoukil, Youssef, BSc
• Smeets, Bert, PhD
• Verheij, Joke, MD, PhD
• Kroes, Hester Y., MD, PhD
• Klaver, Caroline C.W., MD, PhD
• van Schooneveld, Mary, MD, PhD
• Bergen, Arthur A.B., PhD
• Florijn, Ralph J., PhD

Titel

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

Ist Teil von

• Ophthalmology (Rochester, Minn.), 2011, Vol.118 (1), p.160-167.e3

Ort / Verlag

New York, NY: Elsevier Inc

Erscheinungsjahr

2011

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. Participants 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. 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.