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Genotyping Microarray (Disease Chip) for Leber Congenital Amaurosis

¹ Ophthalmology, Columbia University, New York, NY, United States
² Asper Biotech, Tartu, Estonia
³ Hopital Des Enfants Malades, Paris, France
⁴ University Medical Center, Nijmegen, Netherlands
⁵ Johns Hopkins University, Baltimore, MD, United States
⁶ Montreal Children's Hospital, Montreal, PQ, Canada

Commercial Relationships: R.L. Allikmets, None; J. Zernant, None; M. Külm, Asper Biotech E; I. Perrault, None; A. den Hollander, None; S. Dharmaraj, None; R. Koenekoop, None; J. Kaplan, None; F. Cremers, None; I. Maumenee, None.

Grant Identification: Support: NIH Grant EY13435, RPB, FFB

Abstract

Purpose: Sequence variation in at least eight genes (RPE65, GUCY2D, CRB1, RPGRIP1, CRX, AIPL1, MERTK, and LRAT) has been associated with diagnosis consistent with Leber congenital amaurosis (LCA). Genetically heterogeneous (multi-genic) inheritance of this congenital retinal blindness substantially complicates genetic analyses of LCA cases, especially sporadic, where conventional screening methods are of limited or no value. To overcome these limitations, we designed a genotyping microarray (disease chip) for LCA, which includes all >250 variants currently described in all eight genes, allowing for the detection of all known LCA-associated variants (mutations) in a DNA sample in one simple reaction. Methods: The LCA genotyping microarray was constructed by the allele-specific primer extension (APEX) technology (described at: http://www.asperbio.com). Every known disease-associated sequence change described in LCA genes, and a selection of common polymorphisms for haplotype analysis, was included on the chip via allele-specific oligonucleotides. Results: Screening of ~100 LCA patients previously analyzed by SSCP technology was performed to validate the chip. The microarray yielded >98% effective in determining the existing genetic variation, yielding many sequence changes undetected by SSCP. More than the expected two mutant alleles were discovered in a substantial fraction of patients, suggesting multi-allelic inheritance or a modifier effect from more than one gene. The latter finding and the overall efficiency of the array are currently being tested on a large (>200) cohort of previously not screened sporadic and familial LCA cases. Conclusions: The LCA genotyping microarray offers a next generation tool for molecular diagnostics, representing the first "disease chip" for genotyping of patients with a genetically heterogeneous disorder. The chip is available for ophthalmologists and geneticists for effective, high-throughput, accurate, and affordable screening of patient populations.

Keywords: gene microarray • genetics • retinal degenerations: hereditary

© 2003, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any part of this abstract, contact the ARVO Office at arvo{at}arvo.org.