Molecular Diagnostics of Stargardt Disease by Genotyping Patients on the ABCR (ABCA4) Microarray

Abstract

Abstract: : Purpose: We had designed the genotyping microarray (gene chip) for the ABCR gene to enable comprehensive screening of populations with ABCR-associated retinal pathology, e.g., Stargardt disease (STGD). The chip currently includes all >400 sequence variants described in this gene, allowing the detection of all known ABCR variants in a DNA sample in one reaction. Here, we further evaluated the efficiency of the array on a series of STGD patient cohorts derived from multiple independent eye clinics around the world. Methods: The ABCR400 array was constructed by the allele-specific primer extension (APEX) technology (described at: http://www.asperbio.com). Every sequence change described in the ABCR gene, including all mutations and a selection of common polymorphisms, was included on the chip by synthesis and application of allele-specific oligonucleotides. STGD patient cohorts were ascertained at three centers in the US and at three centers in Europe. Results: First, the efficiency of the array was determined by screening two large cohorts of STGD patients from the US and The Netherlands whom we had analyzed previously by SSCP technology. The combination of the ABCR400 chip with SSCP detected 70-78% of all possible disease-associated alleles. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded an additional 15-20% disease-associated alleles not found by SSCP. Genetic analysis of STGD patients of European descent, collected at five independent Centers, by screening with the array alone detected 54-60% of alleles across all cohorts. Chip analysis of 100 healthy control subjects suggested a high carrier frequency (up to 10%) of ABCR variants in the general population. Conclusions: In tested STGD patient cohorts, the array detected between 54% and 78% of all possible disease-associated alleles. The efficiency depended on the ethnic composition and the degree of clinical and molecular characterization of a cohort. The ABCR400 genotyping microarray is robust, cost-effective, and comprehensive screening tool for genetic variation in STGD patients.

Keywords: gene microarray • genetics • retinal degenerations: hereditary