局灶性皮质发育不良中体细胞变异的挑战。

Q3 Medicine
Innovations in clinical neuroscience Pub Date : 2023-12-01 eCollection Date: 2023-10-01
Joana Jesus-Ribeiro, Luís Miguel Pires, Ilda Patrícia Ribeiro, Olinda Rebelo, Ricardo Pereira, Francisco Sales, Isabel Santana, António Freire, Joana Barbosa Melo
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引用次数: 0

摘要

目的:下一代测序技术(NGS)的出现使得在局灶性皮质发育不良(FCD)术后组织中检测低水平脑体细胞变异成为可能。FCD I 型的遗传背景仍然难以捉摸,而哺乳动物雷帕霉素靶标(mTOR)通路似乎在 FCD II 型的发病机制中起着相关作用。我们的目标是通过对手术后组织进行全基因组测序(WGS)来发现候选脑特异性体细胞变异,并评估其临床意义,从而揭示 FCD 的分子基础信息:设计:使用配对的外周静脉血和手术后病理脑脱氧核糖核酸(DNA)样本进行全基因组测序。使用罗氏 KAPA HyperPrep 聚合酶链反应(PCR)免费文库制备试剂盒制备文库。在Illumina NovaSeq平台上生成了150bp的成对端读数。使用 nf-core sarek 管道(3.0 版)处理 FASTQ 文件以调用体细胞变异,然后使用 ANNOVAR 对其进行注释。采用筛选策略获得相关变异:纳入了两名接受手术治疗的 FCD 引起的女性耐药性癫痫患者。在神经病理学诊断方面,一名患者为 FCD Ia 型,另一名患者为 FCD IIa 型。利用WGS检测到了五个体细胞非同义单核苷酸变异(SNV),其中三个在FCD Ia型组织中(WDR24 p.Trp259Gly;MICAL1 p.Lys1036Arg;KATNB1 p.Leu566Ile),两个在FCD IIa型组织中(MATN4 p.Phe91Val和ANKRD6 p.His386Gln)。所有变异都被至少两种不同的工具预测为潜在致病变异。然而,根据美国医学遗传学和基因组学学院(ACMG)的标准,它们被归类为意义不确定的变异(VUS):结论:利用手术后 FCD 组织,通过 NGS 在新的候选基因(WDR24、MICAL1、KATNB1、MATN4 和 ANKRD6)中发现了脑特异性体细胞错义变异,这可能有助于进一步了解 FCD 的遗传背景。所有报告的基因以前都与癫痫和/或中枢神经系统(CNS)和皮层发育畸形有关。然而,这些变异基因的致病性评估及其对临床实践的影响仍然是一个重要的挑战。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
The Challenge of Somatic Variants in Focal Cortical Dysplasia.

Objective: The advent of next-generation sequencing (NGS) enabled the detection of low-level brain somatic variants in postsurgical tissue of focal cortical dysplasia (FCD). The genetic background of FCD Type I remains elusive, while the mammalian target of rapamycin (mTOR) pathway seems to have a relevant role in the pathogenesis of FCD Type II. Our goal was to uncover information on the molecular basis of FCD, performing whole genome sequencing (WGS) in postsurgical tissue to detect candidate brain-specific somatic variants, and evaluate their clinical significance.

Design: WGS was performed using paired peripheral venous blood and postsurgical pathological brain deoxyribonucleic acid (DNA) samples. Libraries were prepared using the Roche KAPA HyperPrep polymerase chain reaction (PCR) free library preparation kit. Paired-end 150bp reads were generated on the Illumina NovaSeq platform. The FASTQ files were processed using the nf-core sarek pipeline (version 3.0) to call somatic variants, which were then annotated with ANNOVAR. A screening strategy was applied to obtain relevant variants.

Results: Two female patients with drug-resistant epilepsy due to FCD who underwent surgical treatment were included. Regarding neuropathological diagnosis, one patient had FCD Type Ia and the other had FCD Type IIa. Five somatic nonsynonymous single nucleotide variants (SNVs) were detected using WGS, three in FCD Ia tissue (WDR24 p.Trp259Gly; MICAL1 p.Lys1036Arg; and KATNB1 p.Leu566Ile) and two in FCD IIa tissue (MATN4 p.Phe91Val and ANKRD6 p.His386Gln). All variants were predicted to be potentially pathogenic by at least two different tools. However, they were classified as variants of uncertain significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG) criteria.

Conclusion: Brain-specific somatic missense variants were identified by NGS in new candidate genes (WDR24, MICAL1, KATNB1, MATN4, and ANKRD6) using postsurgical FCD tissue, which may contribute to further understanding of the genetic background of FCD. All the reported genes were previously related to epilepsy and/or malformations of central nervous system (CNS) and cortical development. However, the pathogenicity assessment of these variants and, consequently, their impact on clinical practice still poses an important challenge.

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Innovations in clinical neuroscience
Innovations in clinical neuroscience Medicine-Psychiatry and Mental Health
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