Smal Noor, Millevert Charissa, De Wachter Matthias, De Vriendt Els, Eddafir Zakaria, Schoonjans An-Sofie, Bayat Allan, Møller Rikke Steensbjerre, Mei Davide, Balestrini Simona, Guerrini Renzo, Meeuwissen Marije E C, Jansen Anna C, Weckhuysen Sarah
Applied Translational Neurogenomics Group, Vlaams Instituut voor Biotechnology (VIB) Center for Molecular Neurology, VIB, Antwerp, Belgium.
Applied Translational Neurogenomics Group, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
Epilepsia. 2025 May;66(5):1613-1627. doi: 10.1111/epi.18279. Epub 2025 Jan 29.
This study aims to improve genetic diagnosis in childhood onset epilepsy with neurodevelopmental problems by utilizing RNA sequencing of fibroblasts to identify pathogenic variants that may be missed by exome sequencing and copy number variation analysis.
We enrolled 41 individuals with childhood onset epilepsy and neurodevelopmental problems who previously had inconclusive genetic testing. Fibroblast samples were cultured and analyzed using RNA sequencing to detect aberrant expression, aberrant splicing, and monoallelic expression using the Detection of RNA Outlier Pipeline (DROP) pipeline. Detected events were correlated with phenotypes, and long-read genome sequencing was performed on individuals with strong candidate events to identify the causal genomic variant. A systematic literature review on RNA sequencing in rare disorders was conducted to contextualize our findings.
RNA sequencing identified five strong candidate events in four individuals, affecting the genes QRICH1, TSC1, SMARCA1, GNAI1, and PTEN. (Likely) pathogenic genomic variants affecting expression of QRICH1, TSC1, and SMARCA1 were detected, resulting in a diagnostic yield of 7% (3/41). Two variants were not covered in the initial exome sequencing data but were revealed through long-read sequencing. The identification of a pathogenic TSC1 variant led to a previously unrecognized diagnosis of tuberous sclerosis complex. This prompted guideline-based screening, which revealed tuberous sclerosis lesions in the brain and lung, directly impacting clinical care. Notably, two of the three pathogenic events would not have been detected using whole blood due to the lack of expression of the involved genes. The lower yield of this study compared to studies in other rare disorders reflects the genetic heterogeneity of epilepsy and neurodevelopmental disorders, and the inaccessibility of affected tissue.
This research underscores RNA sequencing of cultured fibroblasts as a valuable tool in genetic diagnostics for childhood onset epilepsy, particularly when conventional methods fail. Expanding the control dataset with age-matched samples and incorporating RNA sequencing with nonsense-mediated decay inhibition could further enhance diagnostic yield.
本研究旨在通过利用成纤维细胞的RNA测序来识别可能被外显子组测序和拷贝数变异分析遗漏的致病变异，从而改善伴有神经发育问题的儿童期起病癫痫的基因诊断。
我们招募了41名患有儿童期起病癫痫和神经发育问题且先前基因检测结果不明确的个体。培养成纤维细胞样本，并使用RNA测序进行分析，通过RNA异常值检测流程（DROP）检测异常表达、异常剪接和单等位基因表达。将检测到的事件与表型相关联，并对具有强烈候选事件的个体进行长读长基因组测序，以识别因果基因组变异。对罕见疾病中的RNA测序进行了系统的文献综述，以将我们的发现置于背景中。
RNA测序在4名个体中识别出5个强烈候选事件，影响QRICH1、TSC1、SMARCA1、GNAI1和PTEN基因。检测到影响QRICH1、TSC1和SMARCA1表达的（可能）致病变异，诊断率为7%（3/41）。两个变异在初始外显子组测序数据中未涵盖，但通过长读长测序得以揭示。致病性TSC1变异的鉴定导致了先前未被识别的结节性硬化症复合体诊断。这促使了基于指南的筛查，发现了脑和肺中的结节性硬化症病变，直接影响了临床护理。值得注意的是，由于相关基因缺乏表达，使用全血无法检测到这三个致病变异中的两个。与其他罕见疾病的研究相比，本研究的诊断率较低，这反映了癫痫和神经发育障碍的基因异质性以及受影响组织难以获取。
本研究强调了培养成纤维细胞的RNA测序作为儿童期起病癫痫基因诊断的有价值工具，特别是在传统方法失败时。用年龄匹配的样本扩展对照数据集，并将RNA测序与无义介导的衰变抑制相结合，可进一步提高诊断率。
