Long-read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel-Gruber syndrome
Long-read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel-Gruber syndrome
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DOI：
10.1002/humu.23940
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发表时间：
2019-11-11
期刊：
影响因子：
3.9
通讯作者：
Bonthron, David T.
中科院分区：
文献类型：
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作者：
Watson, Christopher M.;Dean, Philip;Bonthron, David T.
大规模并行短读下一代测序（NGS）的诊断部署大大提高了基因检测的可用性、速度和诊断率，特别是对于罕见的遗传性疾病。尽管如此，基于短读段测序的诊断方法准确检测基因转换事件的能力很差。我们报告了一个家族的遗传分析，其中3个胎儿的临床特征与常染色体隐性遗传病Meckel-Gruber综合征（MKS）一致。29个已知MKS相关基因的靶向NGS揭示了杂合TMEM 231剪接供体变体c.929+1A>G。进行比较读取深度分析以鉴定第二致病等位基因，揭示了TMEM 231外显子4的明显杂合缺失。为了验证这一结果，我们对跨越该位点的长距离聚合酶链反应产物进行了单分子长读序测序。我们鉴定了四种错义变体，由于含有变体的读数优先映射到下游TMEM 231假基因，这些错义变体不存在于短读数数据集中。与亲本分离分析一致，我们证明单分子长读段可用于显示变体以反式排列。我们的经验表明，对明显剂量变体的稳健验证对于避免短读段测序的陷阱仍然至关重要，并且新的第三代长读段测序技术已经可以帮助常规临床护理。
The diagnostic deployment of massively parallel short-read next-generation sequencing (NGS) has greatly improved genetic test availability, speed, and diagnostic yield, particularly for rare inherited disorders. Nonetheless, diagnostic approaches based on short-read sequencing have a poor ability to accurately detect gene conversion events. We report on the genetic analysis of a family in which 3 fetuses had clinical features consistent with the autosomal recessive disorder Meckel-Gruber syndrome (MKS). Targeted NGS of 29 known MKS-associated genes revealed a heterozygous TMEM231 splice donor variant c.929+1A>G. Comparative read-depth analysis, performed to identify a second pathogenic allele, revealed an apparent heterozygous deletion of TMEM231 exon 4. To verify this result we performed single-molecule long-read sequencing of a long-range polymerase chain reaction product spanning this locus. We identified four missense variants that were absent from the short-read dataset due to the preferential mapping of variant-containing reads to a downstream TMEM231 pseudogene. Consistent with the parental segregation analysis, we demonstrate that the single-molecule long reads could be used to show that the variants are arranged in trans. Our experience shows that robust validation of apparent dosage variants remains essential to avoid the pitfalls of short-read sequencing and that new third-generation long-read sequencing technologies can already aid routine clinical care.