Sequencing arrays for screening multiple genes associated with early-onset human retinal degenerations on a high-throughput platform.
OBJECTIVE
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.
Mandal
Md Nawajes A
MN
Department of Ophthalmology and Visual Sciences, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105, USA.
Heckenlively
John R
JR
Burch
Tracy
T
Chen
Lianchun
L
Vasireddy
Vidyullatha
V
Koenekoop
Robert K
RK
Sieving
Paul A
PA
Ayyagari
Radha
R
D001483:Base Sequence; D053938:DNA Fragmentation; D004252:DNA Mutational Analysis; D005136:Eye Proteins / Q000235:genetics; D020869:Gene Expression Profiling; D005808:Genes, Recessive; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D006801:Humans; D008869:Microtubule-Associated Proteins; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012174:Retinitis Pigmentosa / Q000235:genetics*
D005136:Eye Proteins; D008869:Microtubule-Associated Proteins; C120042:RP1 protein, human