Schwarz Jana Marie, Becker Lena-Luise, Wahle Monika, Faßbender Jessica, Thomale Ulrich-Wilhelm, Tietze Anna, Morales-Gonzalez Susanne, Knierim Ellen, Schuelke Markus, Kaindl Angela M
Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, 13353 Berlin, Germany.
Center for Chronically Sick Children, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Augustenburger Platz 1, 13353 Berlin, Germany.
Int J Mol Sci. 2025 Jan 19;26(2):815. doi: 10.3390/ijms26020815.
Epilepsy affects 50 million people worldwide and is drug-resistant in approximately one-third of cases. Even when a structural lesion is identified as the epileptogenic focus, understanding the underlying genetic causes is crucial to guide both counseling and treatment decisions. Both somatic and germline DNA variants may contribute to the lesion itself and/or influence the severity of symptoms. We therefore used whole exome sequencing (WES) to search for potentially pathogenic somatic DNA variants in brain samples from children with lesional epilepsy who underwent epilepsy surgery. WES was performed on 20 paired DNA samples extracted from both lesional brain tissue and reference tissue from the same patient, such as leukocytes or fibroblasts. The paired WES data were jointly analyzed using GATK Mutect2 to identify somatic single nucleotide variants (SNVs) or insertions/deletions (InDels), which were subsequently evaluated in silico for their disease-causing potential using MutationTaster2021. We identified known pathogenic somatic variants in five patients (25%) with variant allele frequencies (VAF) ranging from 3-35% in the genes , , , , and as potential causes of cortical malformations or central nervous system (CNS) tumors. Depending on the VAF, we used different methods such as Sanger sequencing, allele-specific qPCR, or targeted ultra-deep sequencing (amplicon sequencing) to confirm the variant. In contrast to the usually straightforward confirmation of germline variants, the validation of somatic variants is more challenging because current methods have limitations in sensitivity, specificity, and cost-effectiveness. In our study, WES identified additional somatic variant candidates in additional genes with VAFs ranging from 0.7-7.0% that could not be validated by an orthogonal method. This highlights the importance of variant validation, especially for those with very low allele frequencies.
癫痫在全球影响着5000万人，约三分之一的病例对药物耐药。即使确定结构性病变为致痫灶，了解潜在的遗传原因对于指导咨询和治疗决策也至关重要。体细胞和生殖系DNA变异都可能导致病变本身和/或影响症状的严重程度。因此，我们使用全外显子组测序（WES）在接受癫痫手术的患有病灶性癫痫的儿童脑样本中寻找潜在的致病性体细胞DNA变异。对从同一患者的病灶脑组织和参考组织（如白细胞或成纤维细胞）中提取的20对DNA样本进行WES。使用GATK Mutect2对配对的WES数据进行联合分析，以识别体细胞单核苷酸变异（SNV）或插入/缺失（InDel），随后使用MutationTaster2021在计算机上评估它们的致病潜力。我们在5名患者（25%）中鉴定出已知的致病性体细胞变异，其变异等位基因频率（VAF）在基因 、 、 、 和 中为3%-35%，作为皮质畸形或中枢神经系统（CNS）肿瘤的潜在原因。根据VAF，我们使用不同的方法，如桑格测序、等位基因特异性定量PCR或靶向超深度测序（扩增子测序）来确认变异。与通常直接确认生殖系变异不同，体细胞变异的验证更具挑战性，因为目前的方法在灵敏度、特异性和成本效益方面存在局限性。在我们的研究中，WES在其他基因中鉴定出额外的体细胞变异候选者，其VAF范围为0.7%-7.0%，无法通过正交方法进行验证。这突出了变异验证的重要性，特别是对于那些等位基因频率非常低的变异。
