Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease
Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease
复制标题
DOI：
10.1002/humu.23787
复制
发表时间：
2019-10-01
期刊：
影响因子：
3.9
通讯作者：
Cremers, Frans P. M.
中科院分区：
文献类型：
--
作者：
Khan, Mubeen;Cornelis, Stephanie S.;Cremers, Frans P. M.
目的Stargardt病（STGD 1）是由ABCA 4基因的双等位基因突变引起的，但由于突变扫描方法不敏感，许多患者的遗传学未得到解决。我们的目的是开发一种成本效益的测序方法ABCA 4外显子和区域携带已知的因果深内含子变异。方法采用基于smMIP的新一代测序技术，对ABCA 4基因50个外显子和12个区域（含14个深度内含子变异体）进行测序。使用483个smMIP对16个携带29个ABCA 4等位基因的STGD 1病例和4个健康人的DNA进行测序。此后，对411例有一个或没有ABCA 4变异的STGD 1病例的DNA进行测序。使用体外剪接测定分析新的非编码变体对剪接的影响。结果在16例STGD 1患者中发现了34个ABCA 4变异。在155/411先证者（38%）中，确定了两个因果变异。我们确定了11个深内含子变异存在于62个等位基因。两个已知的和两个新的非典型剪接位点变异体显示剪接缺陷，一个新的深内含子变异体（c.4539+ 2065 C>G）导致170-nt mRNA假外显子插入（p. [Arg1514Lysfs*35，=]）。结论：基于smMIP的ABCA 4编码区和非编码区序列分析能够在以前未解决的病例中进行STGD 1病例的成本效益突变检测。
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.