BackgroundFor a proportion of individuals judged clinically to have a recessive Mendelian disease, only one heterozygous pathogenic variant can be found from clinical whole exome sequencing (WES), posing a challenge to genetic diagnosis and genetic counseling. One possible reason is the limited ability to detect disease causal structural variants (SVs) from short reads sequencing technologies. Long reads sequencing can produce longer reads (typically 1000 bp or longer), therefore offering greatly improved ability to detect SVs that may be missed by short-read sequencing.ResultsHere we describe a case study, where WES identified only one heterozygous pathogenic variant for an individual suspected to have glycogen storage disease type Ia (GSD-Ia), which is an autosomal recessive disease caused by bi-allelic mutations in the G6PC gene. Through Nanopore long-read whole-genome sequencing, we identified a 7.1 kb deletion covering two exons on the other allele, suggesting that complex structural variants (SVs) may explain a fraction of cases when the second pathogenic allele is missing from WES on recessive diseases. Both breakpoints of the deletion are within Alu elements, and we designed Sanger sequencing and quantitative PCR assays based on the breakpoints for preimplantation genetic diagnosis (PGD) for the family planning on another child. Four embryos were obtained after in vitro fertilization (IVF), and an embryo without deletion in G6PC was transplanted after PGD and was confirmed by prenatal diagnosis, postnatal diagnosis, and subsequent lack of disease symptoms after birth.ConclusionsIn summary, we present one of the first examples of using long-read sequencing to identify causal yet complex SVs in exome-negative patients, which subsequently enabled successful personalized PGD.

中文翻译：

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长读长测序在外显子组阴性病例中发现了因果结构变异，并实现了植入前遗传学诊断

背景对于临床判断为隐性孟德尔病的一部分个体，临床全外显子组测序（WES）只能发现一种杂合致病变异，这给遗传诊断和遗传咨询带来了挑战。一个可能的原因是通过短读长测序技术检测疾病因果结构变异（SV）的能力有限。长读长测序可以产生更长的读长（通常为 1000 bp 或更长），因此大大提高了检测短读长测序可能漏掉的 SV 的能力。结果在这里，我们描述了一个案例研究，其中 WES 仅识别出一个杂合致病性变异疑似患有 Ia 型糖原累积病 (GSD-Ia) 的个体，这是一种由 G6PC 基因双等位基因突变引起的常染色体隐性遗传疾病。通过 Nanopore 长读长全基因组测序，我们发现了覆盖另一个等位基因上两个外显子的 7.1 kb 缺失，这表明复杂结构变异 (SV) 可以解释隐性疾病 WES 中第二个致病等位基因缺失的一小部分病例。两个缺失断点都在 Alu 元件内，我们根据断点设计了桑格测序和定量 PCR 检测，用于另一个孩子的计划生育的植入前遗传学诊断 (PGD)。体外受精（IVF）后获得四个胚胎，PGD后移植一个G6PC无缺失的胚胎，并通过产前诊断、产后诊断以及随后出生后无疾病症状得到证实。结论综上所述，我们提出其中一项使用长读长测序来识别外显子组阴性患者中因果但复杂的 SV 的第一个例子，这随后实现了成功的个性化 PGD。