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Abstract：
Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands.
Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays.
In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband.
Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.
Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays.
In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband.
Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.
摘要：
人类疾病的遗传力缺失是一个重大挑战，对于ABCA4相关的Stargardt病（STGD1）尤其如此。我们旨在阐明1054个未解决的STGD和STGD样先证中的基因组和转录组变异。
使用单分子分子倒置探针（smMIPs）对完整的128kbABCA4基因进行测序，基于半自动化和经济有效的方法。使用相对读段覆盖分析鉴定结构变体(SV),并使用体外测定研究推定的剪接缺陷。
在448个双等位基因先证中发现了14个已知的和13个新的深内含子变体，导致105个等位基因的假外显子（PE）插入或外显子延伸。有趣的是，内含子13变体c.1938-621G>A和c.1938-514G>A导致由相同上游组成的双PE插入，但下游PE不同。内含子44变体c.6148-84A>T导致两个PE插入和侧翼外显子缺失。发现了11个明显的大缺失，其中两个包含小的倒置段。在一个先证中鉴定出1号染色体的单亲等异体性。
ABCA4的深度测序和基于midigene的剪接分析允许在25％的双等位基因STGD1病例中鉴定SVs和致病性深内含子变异，这代表了可以应用于其他遗传性疾病的模型研究。
使用单分子分子倒置探针（smMIPs）对完整的128kbABCA4基因进行测序，基于半自动化和经济有效的方法。使用相对读段覆盖分析鉴定结构变体(SV),并使用体外测定研究推定的剪接缺陷。
在448个双等位基因先证中发现了14个已知的和13个新的深内含子变体，导致105个等位基因的假外显子（PE）插入或外显子延伸。有趣的是，内含子13变体c.1938-621G>A和c.1938-514G>A导致由相同上游组成的双PE插入，但下游PE不同。内含子44变体c.6148-84A>T导致两个PE插入和侧翼外显子缺失。发现了11个明显的大缺失，其中两个包含小的倒置段。在一个先证中鉴定出1号染色体的单亲等异体性。
ABCA4的深度测序和基于midigene的剪接分析允许在25％的双等位基因STGD1病例中鉴定SVs和致病性深内含子变异，这代表了可以应用于其他遗传性疾病的模型研究。
