title: Analysis of the ABCA4 gene by next-generation sequencing.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, Columbia University, New York, New York 10032, USA.
authors.forename: Jana
authors.initials: J
authors.lastname: Zernant
authors.forename: Carl
authors.initials: C
authors.lastname: Schubert
authors.forename: Kate M
authors.initials: KM
authors.lastname: Im
authors.forename: Tomas
authors.initials: T
authors.lastname: Burke
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Brown
authors.forename: Gerald A
authors.initials: GA
authors.lastname: Fishman
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Tsang
authors.forename: Peter
authors.initials: P
authors.lastname: Gouras
authors.forename: Michael
authors.initials: M
authors.lastname: Dean
authors.forename: Rando
authors.initials: R
authors.lastname: Allikmets
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics; D005784:Gene Amplification; D006801:Humans; D008268:Macular Degeneration / Q000151:congenital / Q000235:genetics; D008297:Male; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010375:Pedigree; D016133:Polymerase Chain Reaction; D058499:Retinal Dystrophies / Q000235:genetics; D012174:Retinitis Pigmentosa / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D000080362:Stargardt Disease; D041623:Tomography, Optical Coherence
chemical_list: C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters