Strategic validation of variants of uncertain significance in ECHS1 genetic testing
Strategic validation of variants of uncertain significance in ECHS1 genetic testing
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DOI：
10.1136/jmg-2022-109027
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发表时间：
2023-04-13
影响因子：
4
通讯作者：
Okazaki，Yasushi
中科院分区：
文献类型：
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作者：
Kishita，Yoshihito;Sugiura，Ayumu;Okazaki，Yasushi
背景烯酰辅酶A水合酶短链1（ECHS1）是一种参与支链氨基酸和脂肪酸代谢的酶。 ECHS1基因突变导致线粒体短链烯酰辅酶A水合酶1缺乏，导致缬氨酸中间体的积累。这是线粒体疾病中最常见的致病基因之一。虽然基因分析研究已诊断出许多具有ECHS1变异的病例，但基因诊断中意义不确定的变异（VUS）数量的增加是一个主要问题。方法在这里，我们构建了一个检测系统来验证ECHS1基因的VUS功能。使用 ECHS1 敲除细胞进行高通量测定，通过表达含有 VUS 的 cDNA 来索引这些表型。与 VUS 验证系统并行，对线粒体疾病患者的样本进行了遗传分析。通过 RNA-seq 和蛋白质组分析验证了对病例中基因表达的影响。结果 VUS 的功能验证发现了导致 ECHS1 功能丧失的新变异。 VUS验证系统还揭示了VUS在复合杂合状态下的影响，并为变异解释提供了新的方法。此外，我们进行了多组学分析并鉴定了导致剪接异常的同义替换 p.P163=。多组学分析补充了一些VUS验证系统无法诊断的病例的诊断。结论综上所述，本研究基于VUS验证和组学分析发现了新的ECHS1病例；这些分析适用于与线粒体疾病相关的其他基因的功能评估。
BackgroundEnoyl-CoA hydratase short-chain 1 (ECHS1) is an enzyme involved in the metabolism of branched chain amino acids and fatty acids. Mutations in theECHS1gene lead to mitochondrial short-chain enoyl-CoA hydratase 1 deficiency, resulting in the accumulation of intermediates of valine. This is one of the most common causative genes in mitochondrial diseases. While genetic analysis studies have diagnosed numerous cases withECHS1variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem.MethodsHere, we constructed an assay system to verify VUS function forECHS1gene. A high-throughput assay usingECHS1knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. In parallel with the VUS validation system, a genetic analysis of samples from patients with mitochondrial disease was performed. The effect on gene expression in cases was verified by RNA-seq and proteome analysis.ResultsThe functional validation of VUS identified novel variants causing loss ofECHS1function. The VUS validation system also revealed the effect of the VUS in the compound heterozygous state and provided a new methodology for variant interpretation. Moreover, we performed multiomics analysis and identified a synonymous substitution p.P163= that results in splicing abnormality. The multiomics analysis complemented the diagnosis of some cases that could not be diagnosed by the VUS validation system.ConclusionsIn summary, this study uncovered newECHS1cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease.