Analysis of the ABCA4 gene by next-generation sequencing

Abstract

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 prescreened 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 (~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.

Figure 1.

Fundus autofluorescence image of the left eye of patient 3032 harboring ABCA4 variants c.2300T>A (p.V767D) and c.735T>G (p.Y245*). This combination of ABCA4 mutations resulted in early disease onset at 5 years of age. At 16 years of age, the patient was found to have extensive hypoautofluorescence, indicative of atrophy of the retinal pigment epithelium throughout the macula with patchy extension of hypoautofluorescence into the extramacular retina. Note the relative “sparing” of uniform hyperautofluorescence in the peripapillary region.

Figure 2.

Pedigrees segregating Stargardt disease. (A) An example of a pedigree with pseudodominant inheritance. Father and son are both affected with arSTGD. Mother is a carrier of a frequent c.3322C>T (p.R1108C) mutation. The new c.3522+5delG variant affecting splicing (Table 1) was detected by NGS. (B) An example of a pedigree segregating a frequent complex ABCA4 allele [c.1622T>C; 3113C>T] (p.L541P;A1038V), in which either mutation separately can cause the disease. The c.3543delT frameshift variant was detected by NGS. (C) An example of a pedigree segregating a complex allele in which one variant (c.2894A>G, p.N965S) causes disease and the other, c.4283C>T, p.T1428M, is a benign polymorphism, although it was originally described as a rare mutation in patients of European descent. The new c.3655G>C, p.A1219P variant was detected by NGS.