Identifying the causal genetic variants in congenital brain disorders by integrating genome and transcrip-tome sequencing

罕见的孟德尔疾病主要是单基因的，由8000多个不同的实体组成。先天性脑疾病，包括小头畸形、皮质畸形、后脑畸形和胼胝体畸形，属于单基因疾病。全外显子组测序(WES)是疾病基因发现的主要驱动力，使人们对基因、相关临床表型和分子机制之间的关系有了更全面的了解(S)。全基因组测序(WGS)是识别WES未检测到的致病变异的合乎逻辑的下一步，例如RNA基因和非编码区的变异以及结构变异。作为一种伴随的方法，转录组测序有助于寻找表达水平异常、剪接异常和单等位基因表达异常的基因，并有能力将研究范围缩小到WGS确定的候选基因的子集。要分析任何潜在的致病变异的影响，疾病基因表达的细胞环境是重要的。然而，在大多数情况下，受疾病影响的组织标本是无法获取的。人诱导多能干细胞(HiPSCs)可以从患者来源的细胞中建立，并在体外分化为感兴趣的细胞类型，是适合于转录组测序和研究细胞水平表型的模型系统。在这个项目中，我们的目标是确定25例WES阴性的先天性脑疾病患者的遗传原因：7例患者有连合异常，13例后脑畸形，3例皮质发育畸形，均伴有或不伴有小头畸形，2例小头畸形合并宫内生长迟缓。我们将首先使用更新的生物信息学工作流程重新分析现有的TRIO-WES数据，以在25名患者中最多20%的患者中识别疾病或候选基因中的潜在致病变异。作为下一步，我们将在20名遗传未解决的患者中进行Trio-WGS，以确定其中一部分患者的基因或非编码区中的潜在致病变异。最多5名TRIO-WGS后出现候选非编码变异或没有分子诊断的患者及其健康父母将被选为获得原代尿路细胞，将其重新编程为HiPSCs，将其分化为诱导的人诱导神经干细胞(HiNSCs)，并在hiNSCs来源的RNA中进行转录组测序。WGS和转录组数据的整合将可能导致发现或确认患者的遗传原因。其中一个或最多两个患者-父母三人组中的hiNSCs将被分化为人类诱导的皮质神经元(HiNeurons)。将研究患者和父母来源的海马神经元的形态发生表型，以深入了解潜在的病理生理学。

Rare Mendelian disorders are largely monogenic and comprise over 8,000 distinct entities. Congenital brain disorders, including microcephaly, malformations of the cortex, hindbrain, and corpus callosum, belong to monogenic diseases. Whole-exome sequencing (WES), mainly WES in patient-parents trios, was the main driver in disease gene discovery and led to a comprehensive understanding of the relationship between genes, associated clinical phenotype(s), and molecular mechanisms. Whole-genome sequencing (WGS) is the logical next step in identifying disease-causing variants not detected by WES, such as variants in RNA genes and non-coding regions and structural variants. As a concomitant approach, transcriptome sequencing is useful to search for genes with aberrant expression levels, aberrant splicing, and monoallelic expression and has the power to narrow down the investigation to a subset of candidate genes identified by WGS. To analyze the impact of any potential pathogenic variant, the cellular context in which a disease gene is expressed is important. In most cases, however, tissue specimen affected by the disease are not accessible. Human induced pluripotent stem cells (hiPSCs) that can be established from patient-derived cells and in vitro differentiation of hiPSCs into the cell type of interest are suitable model systems for transcriptome sequencing and studying the cellular-level phenotypes. In this project, we aim to identify the genetic cause in 25 WES-negative individuals with a congenital brain disorder: 7 patients have commissural abnormalities, 13 hindbrain malformations, 3 malformations of cortical development, all with or without microcephaly, and 2 have microcephaly with intrauterine growth retardation. We will first re-analyze existing trio-WES data using an updated bioinformatics workflow to identify a potentially pathogenic variant in a disease or candidate gene in up to 20% of the 25 patients. As a next step, we will perform trio-WGS in 20 genetically unsolved patients to identify a potentially pathogenic variant in a gene or non-coding region in a portion of these patients. A maximum of 5 patients with a candidate non-coding variant or without molecular diagnosis after trio-WGS and their healthy parents will be selected for obtaining primary urinary cells to reprogram them into hiPSCs, differentiate hiPSCs into induced human induced neural stem cells (hiNSCs), and perform transcriptome sequencing in hiNSCs-derived RNA. Integration of WGS and transcriptome data will potentially lead to the discovery or confirmation of the genetic cause in the patients. hiNSCs of one or a maximum of two patient-parent trios in which we have identified the pathogenic variant will be differentiated into human induced cortical neurons (hiNeurons). The morphogenetic phenotype of patient- and parent-derived hiNeurons will be studied to gain insight into the underlying pathophysiology.