title: ABCA4 gene screening by next-generation sequencing in a British cohort.
abstract: OBJECTIVE
  We applied a recently reported next-generation sequencing (NGS) strategy for screening the ABCA4 gene in a British cohort with ABCA4-associated disease and report novel mutations.
  
  
  METHODS
  We identified 79 patients with a clinical diagnosis of ABCA4-associated disease who had a single variant identified by the ABCA4 microarray. Comprehensive phenotypic data were obtained, and the NGS strategy was applied to identify the second allele by means of sequencing the entire coding region and adjacent intronic sequences of the ABCA4 gene. Identified variants were confirmed by Sanger sequencing and assessed for pathogenicity by in silico analysis.
  
  
  RESULTS
  Of the 42 variants detected by prescreening with the microarray, in silico analysis suggested that 34, found in 66 subjects, were disease-causing and 8, found in 13 subjects, were benign variants. We detected 42 variants by NGS, of which 39 were classified as disease-causing. Of these 39 variants, 31 were novel, including 16 missense, 7 splice-site-altering, 4 nonsense, 1 in-frame deletion, and 3 frameshift variants. Two or more disease-causing variants were confirmed in 37 (47%) of 79 patients, one disease-causing variant in 36 (46%) subjects, and no disease-causing variant in 6 (7%) individuals.
  
  
  CONCLUSIONS
  Application of the NGS platform for ABCA4 screening enabled detection of the second disease-associated allele in approximately half of the patients in a British cohort where one mutation had been detected with the arrayed primer extension (APEX) array. The time- and cost-efficient NGS strategy is useful in screening large cohorts, which will be increasingly valuable with the advent of ABCA4-directed therapies.
authors: Laboratory of Visual Physiology, National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan.
authors: Kaoru
authors: K
authors: Fujinami
authors: Jana
authors: J
authors: Zernant
authors: Ravinder K
authors: RK
authors: Chana
authors: Genevieve A
authors: GA
authors: Wright
authors: Kazushige
authors: K
authors: Tsunoda
authors: Yoko
authors: Y
authors: Ozawa
authors: Kazuo
authors: K
authors: Tsubota
authors: Andrew R
authors: AR
authors: Webster
authors: Anthony T
authors: AT
authors: Moore
authors: Rando
authors: R
authors: Allikmets
authors: Michel
authors: M
authors: Michaelides
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000293:Adolescent; D000328:Adult; D000368:Aged; D000483:Alleles; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000235:genetics*; D004596:Electroretinography; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D015994:Incidence; D008268:Macular Degeneration / Q000453:epidemiology / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010375:Pedigree; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012160:Retina / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D012374:Rod Cell Outer Segment; D000080362:Stargardt Disease; D041623:Tomography, Optical Coherence; D006113:United Kingdom / Q000453:epidemiology; D055815:Young Adult
chemical_list: C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters; D004247:DNA
keywords: ABCA4; Stargardt disease; next generation sequencing
references: Doc Ophthalmol. 2009 Feb;118(1):69-77
references: 19030905
references: Hum Genet. 2001 Sep;109(3):326-38
references: 11702214
references: Hum Mutat. 2000;15(1):7-12
references: 10612815
references: Hum Mol Genet. 2001 Nov 1;10(23):2671-8
references: 11726554
references: Ophthalmology. 2012 Mar;119(3):596-605
references: 22182799
references: Hum Genet. 2001 Apr;108(4):346-55
references: 11379881
references: Mol Vis. 2009;15:638-45
references: 19352439
references: Mol Vis. 2009 Dec 20;15:2841-7
references: 20029649
references: Eur J Hum Genet. 2004 Dec;12(12):1024-32
references: 15494742
references: Invest Ophthalmol Vis Sci. 2002 Jun;43(6):1980-5
references: 12037008
references: Acta Ophthalmol. 2011 May;89(3):e297-8
references: 20163366
references: Am J Hum Genet. 1999 Feb;64(2):422-34
references: 9973280
references: Invest Ophthalmol Vis Sci. 2000 Jan;41(1):16-9
references: 10634594
references: Am J Hum Genet. 1999 Apr;64(4):1024-35
references: 10090887
references: Nucleic Acids Res. 2003 Jul 1;31(13):3812-4
references: 12824425
references: Hum Mol Genet. 1998 Mar;7(3):355-62
references: 9466990
references: Invest Ophthalmol Vis Sci. 2010 Nov;51(11):5943-51
references: 20554613
references: Am J Hum Genet. 2000 Oct;67(4):800-13
references: 10958763
references: Am J Ophthalmol. 2013 Jun;155(6):1075-1088.e13
references: 23499370
references: Exp Eye Res. 2010 Nov;91(5):592-600
references: 20696155
references: Exp Eye Res. 2001 Dec;73(6):877-86
references: 11846518
references: Hum Mutat. 2003 Nov;22(5):395-403
references: 14517951
references: Ophthalmology. 2013 Nov;120(11):2324-31
references: 23769331
references: Arch Ophthalmol. 2001 Mar;119(3):359-69
references: 11231769
references: Hum Mol Genet. 2013 Dec 20;22(25):5136-45
references: 23918662
references: J Med Genet. 1999 Jun;36(6):447-51
references: 10874631
references: Nat Methods. 2010 Apr;7(4):248-9
references: 20354512
references: Br J Ophthalmol. 2007 Dec;91(12):1650-5
references: 18024811
references: Clin Chem. 2004 Aug;50(8):1336-43
references: 15192030
references: Invest Ophthalmol Vis Sci. 2001 Sep;42(10):2229-36
references: 11527935
references: Arch Ophthalmol. 1999 Apr;117(4):504-10
references: 10206579
references: Ophthalmic Genet. 2011 Sep;32(3):165-74
references: 21510770
references: Invest Ophthalmol Vis Sci. 2012 Apr 30;53(4):2112-8
references: 22427542
references: Eur J Hum Genet. 1998 May-Jun;6(3):291-5
references: 9781034
references: Nat Genet. 1997 Mar;15(3):236-46
references: 9054934
references: Invest Ophthalmol Vis Sci. 2011 Oct 31;52(11):8479-87
references: 21911583
references: Arch Ophthalmol. 2007 Feb;125(2):252-8
references: 17296903
references: Hum Mutat. 2002 Mar;19(3):189-208
references: 11857735
references: Invest Ophthalmol Vis Sci. 2001 May;42(6):1179-89
references: 11328725
references: Nat Genet. 1998 Jan;18(1):11-2
references: 9425888
references: Doc Ophthalmol. 2013 Feb;126(1):1-7
references: 23073702