Analysis of the ABCA4 Gene by Next-Generation Sequencing

PURPOSE. To find all possible disease-associated variants in coding sequences of the ABCA4 gene in a large cohort of patients diagnosed with ABCA4-associated diseases. METHODS. One hundred sixty-eight patients who had been clinically diagnosed with Stargardt disease, cone-rod dystrophy, and other ABCA4-associated phenotypes were pre-screened for mutations in ABCA4 with the ABCA4 microarray, resulting in finding 1 of 2 expected mutations in 111 patients and 0 of 2 mutations in 57 patients. The next-generation sequencing (NGS) strategy was applied to these patients to sequence the entire coding region and the splice sites of the ABCA4 gene. Identified new variants were confirmed or rejected by Sanger sequencing and analyzed for possible pathogenicity by in silico programs and, where possible, by segregation analyses. RESULTS. Sequencing was successful in 159 of 168 patients and identified the second disease-associated allele in 49 of 103 (similar to 48%) of patients with one previously identified mutation. Among those with no mutations, both disease-associated alleles were detected in 4 of 56 patients, and one mutation was detected in 10 of 56 patients. The authors detected a total of 57 previously unknown, possibly pathogenic, variants: 29 missense, 4 nonsense, 9 small deletions and 15 splice-site-altering variants. Of these, 55 variants were deemed pathogenic by a combination of predictive methods and segregation analyses. CONCLUSIONS. Many mutations in the coding sequences of the ABCA4 gene are still unknown, and many possibly reside in noncoding regions of the ABCA4 locus. Although the ABCA4 array remains a good first-pass screening option, the NGS platform is a time-and cost-efficient tool for screening large cohorts. (Invest Ophthalmol Vis Sci. 2011;52:8479-8487) DOI: 10.1167/iovs.11-8182