Whole genome and transcriptome sequencing for gene discovery in dystonia

全基因组和转录组测序用于肌张力障碍的基因发现

• 批准号: 458949627
• 负责人: Dr. Barbara Schormair
• 金额: --
• 依托单位: Klinikum rechts der Isar der Technischen Universität München (MRI)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/458949627?language=en
• 关键词: Whole; genome; transcriptome; sequencing; gene

Dystonias are hyperkinetic movement disorders, often imposing severe limitations on patients’ health and quality of life. They are rare diseases and show extensive heterogeneity in their etiology and phenotypic presentation. Their molecular etiology is poorly understood, limiting the availability of effective therapies. Many forms of dystonia have a genetic basis; therefore identification of the causal genes is an excellent starting point for the urgently needed progress in diagnostics and treatment options. At present, whole exome sequencing (WES) is the main driving force for gene discovery in monogenic disorders. We have sequenced more than 700 index cases and have compiled one of the largest WES cohorts for dystonia in Europe. These efforts have led to the discovery of novel disease genes and have suggested new pathophysiological concepts for dystonia. However, WES has a limited detection scope, leaving the majority of cases unsolved at present.Therefore, we want to combine whole genome sequencing (WGS) and transcriptome sequencing (RNA-seq) to identify the genetic causes of dystonia in WES-negative individuals.WGS has uniform coverage of all genes, detects structural variants, and enables the analysis of non-coding regulatory variants, which also contribute to monogenic disorders, but are challenging to annotate. We address this by combining WGS with RNA-seq to aid interpretation of the consequences of variation detected by WGS. Using WGS and/or RNA-seq has already proven successful for disease gene discovery in other types of monogenic disorders.We will apply WGS and RNA-seq in a total of 300 individuals selected from our cohort: 76 trios, 25 families with an autosomal-dominant, and 11 families with an autosomal-recessive mode of inheritance. Samples are selected using a prioritization score which we have developed based on our WES results. It integrates several phenotype-based clinical predictors and was shown to identify individuals with a high likelihood of a genetic cause of their dystonia. We will call genetic variants from WGS and filter these based on frequency, function, and mode of inheritance. Function refers to both coding variants (e.g. LoF) and non-coding variants (e.g. regulatory variant in an enhancer). For trios, variants have to be consistent with a de novo model (sequencing parents and affected offspring), whereas in families they have to be consistent with the observed mode of inheritance and between two sequenced family members. We will integrate these variants with genes prioritized by three different readouts from RNA-seq (gene expression, monoallelic expression, splicing patterns) to prioritize genes as candidate causal genes for dystonia. We expect to identify at least four novel disease genes with compelling evidence in our cohort. These genes will then be taken forward to validation in functional follow-up studies. In addition, we will seek additional confirmation in independent dystonia cohorts.

肌张力障碍是一种多动性运动障碍，通常对患者的健康和生活质量造成严重限制。它们是罕见的疾病，在病因和表型表现上表现出广泛的异质性。其分子病因尚不清楚，限制了有效治疗的可用性。许多形式的肌张力障碍都有遗传基础；因此，确定致病基因是诊断和治疗方案迫切需要的进展的一个很好的起点。目前，全外显子组测序（WES）是单基因疾病基因发现的主要推动力。我们对700多个索引病例进行了测序，并编制了欧洲最大的肌张力障碍WES队列之一。这些努力导致了新的疾病基因的发现，并提出了肌张力障碍的新的病理生理学概念。然而，WES的检测范围有限，目前大多数病例尚未解决。因此，我们希望结合全基因组测序（WGS）和转录组测序（RNA-seq）来确定wes阴性个体肌张力障碍的遗传原因。WGS具有对所有基因的统一覆盖，检测结构变异，并能够分析非编码调节变异，这些变异也有助于单基因疾病，但具有挑战性。我们通过将WGS与RNA-seq结合来解决这个问题，以帮助解释WGS检测到的变异的后果。使用WGS和/或RNA-seq在其他类型的单基因疾病的疾病基因发现中已经被证明是成功的。我们将对从我们的队列中选择的总共300个个体进行WGS和RNA-seq分析：76个三人组，25个常染色体显性遗传模式家族，11个常染色体隐性遗传模式家族。使用我们根据WES结果开发的优先级评分来选择样本。它整合了几种基于表型的临床预测因素，并被证明可以识别出具有高可能性的肌张力障碍遗传原因的个体。我们将从WGS中调用遗传变异，并根据频率、功能和遗传模式对其进行过滤。功能是指编码变体（如LoF）和非编码变体（如增强子中的调控变体）。对于三人组，变异必须与从头模型（对父母和受影响的后代进行测序）一致，而在家庭中，它们必须与观察到的遗传模式和两个测序的家庭成员之间的遗传模式一致。我们将通过RNA-seq的三种不同读数（基因表达、单等位基因表达、剪接模式）将这些变异与优先排序的基因结合起来，优先排序基因作为肌张力障碍的候选致病基因。我们期望在我们的队列中确定至少四种具有令人信服的证据的新型疾病基因。这些基因将在功能性后续研究中进行验证。此外，我们将在独立的肌张力障碍队列中寻求额外的证实。