PURPOSE: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. METHODS: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. RESULTS: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). CONCLUSIONS: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.
PURPOSE:Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. METHODS: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. RESULTS: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). CONCLUSIONS: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.
Authors: Esmee H Runhart; Mubeen Khan; Stéphanie S Cornelis; Susanne Roosing; Marta Del Pozo-Valero; Tina M Lamey; Petra Liskova; Lisa Roberts; Heidi Stöhr; Caroline C W Klaver; Carel B Hoyng; Frans P M Cremers; Claire-Marie Dhaenens Journal: JAMA Ophthalmol Date: 2020-10-01 Impact factor: 7.389
Authors: Tomasz Z Tomkiewicz; Nuria Suárez-Herrera; Frans P M Cremers; Rob W J Collin; Alejandro Garanto Journal: Int J Mol Sci Date: 2021-04-28 Impact factor: 5.923
Authors: Zeinab Fadaie; Mubeen Khan; Marta Del Pozo-Valero; Stéphanie S Cornelis; Carmen Ayuso; Frans P M Cremers; Susanne Roosing Journal: Hum Mutat Date: 2019-09-03 Impact factor: 4.878
Authors: Gang Zou; Tao Zhang; Xuesen Cheng; Austin D Igelman; Jun Wang; Xinye Qian; Shangyi Fu; Keqing Wang; Robert K Koenekoop; Gerald A Fishman; Paul Yang; Yumei Li; Mark E Pennesi; Rui Chen Journal: Mol Vis Date: 2021-03-18 Impact factor: 2.367