An ABCA4 genomic deletion in patients with Stargardt disease

Stargardt disease (STGD1) segregates with mutations in the ABCA4 (ABCR) locus. However, mutations of the ABCA4 coding region detected by sequencing account for only 66-80% of disease chromosomes. We hypothesized a potential contribution of otherwise undetected genomic rearrangements of the ABCA4 region. To investigate this hypothesis, we performed genomic Southern analysis on samples from 96 STGD families in which we had identified either one or no ABCA4 mutations by conventional methods. Among 192 chromosomes evaluated, we found one deletion (0.52%), IVS17-905_IVS18+35del, that spans 1,030 bp and eliminates exon 18 of ABCA4. By conceptual translation, this alteration creates an in-frame deletion of 30 amino acids, G885_H915del, and cosegregates with the disease in this family, implying a disease-associated allele. STGD subjects with this deletion were found to have a second mutant ABCA4 allele, 2588G>C. DNA sequence analysis of the deletion junction revealed consensus DNA topoisomerase I sites at both breakpoints that may predispose to nonhomologous recombination. Using deletion-specific PCR, we found the same allele in 2 of 308 STGD subjects (0.32%), in 1 of 96 age-related macular degeneration (AMD) subjects (0.52%), and in 2 of 480 (0.2%) individuals with no known eye diseases, but it was absent in a control group consisting of 96 individuals over age 60 and with normal eye examinations. In vitro biochemical studies of the cloned G885_H915del mutation revealed diminished expression, suggesting that partial deletion of the putative nucleotide-binding domain I leads to either misfolding or defective membrane interactions and eventually reduces the protein function in the retinopathy-affected subjects. Our experiments suggest that genomic alterations contribute to only a small fraction of retinopathy-associated alleles.