Department of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, PO Box 2040, 3000 Rotterdam, the Netherlands.
Department of Pediatrics, Erasmus MC, University Medical Center Rotterdam, PO Box 2040, 3000 Rotterdam, the Netherlands.
Am J Hum Genet. 2023 Feb 2;110(2):251-272. doi: 10.1016/j.ajhg.2022.12.015. Epub 2023 Jan 19.
For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.
对于神经发育障碍（NDD），分子诊断是管理、预测结果和咨询的关键。通常，常规的基于 DNA 的测试无法在 NDD 中建立遗传诊断。转录组分析（RNA 测序 [RNA-seq]）有望提高诊断产量，但尚未在 NDD 的常规诊断中应用。在这里，我们在包括 67 名未确诊的 NDD 个体在内的 96 个人中探索了 RNA-seq 的诊断潜力。我们对单个个体的培养皮肤成纤维细胞进行了 RNA-seq，同时进行了和未进行环已酰亚胺处理，并使用改良的 OUTRIDER Z 分数来检测基因表达异常和外显子和内含子异常剪接。通过用户友好的网络应用程序进行分析，并通过二次检测来确认候选致病性转录事件。我们确定了基因内缺失、单等位基因表达和假外显子插入，但也确定了对转录有有害影响的同义和非同义变体，从而将 NDD 的诊断产量提高了 13%。我们发现环已酰亚胺处理和外显子/内含子 Z 分数分析增加了异常剪接的检测和分辨率。重要的是，在一个个体中，候选基因中的剪接错误与个体的特定表型几乎匹配。然而，另一个神经元表达的基因中发生了致病性剪接，并提供了分子诊断，强调了定制 RNA-seq 的必要性。最后，我们的网络浏览器应用程序允许自定义分析设置，以促进诊断应用，并将致病性转录本列为顶级候选者。我们的结果表明，RNA-seq 是 NDD 基因组诊断的一种补充方法，通过提供具有更高敏感性的可访问分析，我们的转录组分析方法促进了 RNA-seq 在常规基因组诊断中的更广泛应用。
