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Sequencing arrays for screening multiple genes associated with early-onset human retinal degenerations on a high-throughput platform.

Abstract：

PURPOSE:To develop and apply microarray-based resequencing technology to detect sequence alterations in multiple autosomal recessive retinal disease genes on a single high-throughput platform. METHODS:Oligonucleotides corresponding to both strands of the target exons and the flanking intron sequences of 29,214 bp from 11 genes associated with autosomal recessive retinitis pigmentosa (arRP) were tiled on 20 x 25-microm microarrays (arRP-I arrays). A total of 155 exons were amplified from 35 arRP patient DNA samples, with each sample being sequenced on an arRP-I chip by hybridization. RESULTS:With the arRP-I arrays, 97.6% of the tiled sequence were determined with more than 99% accuracy and reproducibility. Of the 2.4% unread sequence, 89.5% involved stretches of G or C. In analyzing the 903,140-bp sequence from the 35 patient samples, 506 sequence changes have been detected in which 386 are previously reported alterations, and 120 are novel. In addition to four known causative mutations, six novel sequence changes that are potentially pathogenic were observed. Additional analysis is needed to determine whether these changes are responsible for arRP in these patients. CONCLUSIONS:The use of microarray for sequencing is a novel approach, and the arRP-I chip is the first successful application of this technology for determining sequence alteration in multiple disease-related genes. These arrays can be used for high-throughput genotyping of patients with relevant retinal conditions. In addition, these arrays offer a unique opportunity to interrogate complex patterns of inheritance due to the involvement of more than one gene by screening multiple genes on a single platform.

journal_name

Invest Ophthalmol Vis Sci

journal_title

Investigative ophthalmology & visual science

authors

Mandal MN,Heckenlively JR,Burch T,Chen L,Vasireddy V,Koenekoop RK,Sieving PA,Ayyagari R

doi

10.1167/iovs.05-0007

keywords:

subject

Has Abstract

pub_date

2005-09-01 00:00:00

pages

3355-62

issue

eissn

0146-0404

issn

1552-5783

pii

46/9/3355

journal_volume

pub_type

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Sequencing arrays for screening multiple genes associated with early-onset human retinal degenerations on a high-throughput platform.