缺乏功能证据阻碍了变体解释,在全基因组或全外显子组测序(WES)后,大部分疑似孟德尔疾病的个体没有基因诊断。研究主张进一步对转录组进行测序,以直接和系统地探测基因表达缺陷。然而,收集额外的活检和建立实验室工作流程,分析管道,在常规诊断中采用RNA测序(RNA-seq)仍然需要在异常基因表达的临床解释中定义的概念。
我们实施了一个自动化的RNA-seq协议和一个计算工作流程,我们用它分析了303名以前经历过WES的疑似线粒体疾病的个体的皮肤成纤维细胞。我们还通过模拟评估了异常表达和单等位基因表达测试如何依赖于RNA-seq覆盖。
我们平均每个样本检测到12,500个基因,包括所有疾病基因的约60%-覆盖率大大高于全血,支持皮肤活检的使用。我们优先考虑表现出异常表达的基因,异常剪接,或单等位基因表达。从样品制备到结果报告,管道需要不到1周的时间,并为每位患者提供了8个疾病相关基因的中位数进行检查。在205例WES不确定的病例中,有16%进行了遗传诊断。异常表达的检测是诊断的主要原因,包括减少50%的情况。which,连同单等位基因表达,允许诊断由单倍体功能不全引起的显性疾病。此外,从RNA-seq数据中调用异常剪接和变体,能够检测和验证剪接破坏变体,其中大多数落在WES覆盖的地区之外。
一起,这些结果表明,简化的实验和计算过程可以加速RNA-seq在常规诊断中的实现.
Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics.
We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage.
We detected on average 12,500 genes per sample including around 60% of all disease genes-a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions.
Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.