Modifiers of penetrance and expressivity in monogenic dystonia: Insights from systems biology

一些基因的突变，包括THAP1（含THAP结构域的凋亡相关蛋白1），SGCE（肌聚糖，epsilon）和GCH1 （GTP环水解酶1）已被证实是肌张力障碍的遗传原因。这些基因的突变外显率约为50%，并且在受影响的携带者之间存在巨大的可变表达性。在第一个资助期，我们专注于THAP1外显率的遗传修饰因子。为此，我们从5个受影响的THAP1突变携带者和5个未受影响的THAP1突变携带者的成纤维细胞系中产生了20个iPSC克隆。这些克隆和14个匹配的对照系已分化为皮质神经元系，并用于转录组分析。进一步，对13个受影响和未受影响的THAP1突变携带者进行全基因组测序，并生成无THAP1突变的皮质神经元系的染色体构象芯片捕获（4C）数据。利用这些全面的数据资源，我们证明了与不同途径相关的几个基因的差异表达，包括对I型干扰素的反应、膜融合和线粒体裂变。对于其中的一些基因，THAP1转录因子的结合已经显示在各自的启动子区域内（ENCODE数据）。此外，基因组数据排除了受影响携带者中其他肌张力障碍基因中简单的编码序列变异。最后，基于与THAP1基因的3D相互作用和家族分离，KAT6A（赖氨酸乙酰转移酶6A）启动子内的一个常见变异被提名为THAP1外显率的潜在修饰因子。在这一提议中，总体假设是THAP1、SGCE或GCH1突变的外显率和表达性被进一步的遗传变异和/或影响基因表达和/或代谢物的表观遗传标记所修饰。为了证实这一假设，我们将重新分析THAP1的现有数据，重点关注分子功能，结合转录组学、基因组和规则组数据，采用反式组学多变量建模方法，阐明与THAP1突变外显率相关的协同作用（目标1）。我们将把重点扩大到SGCE和GCH1。虽然SGCE的外显率降低主要是由母体印迹造成的，但正如我们之前所证明的那样，差异甲基化和基因表达对可变表达率的影响在很大程度上是难以捉摸的，将通过转录组、基因组和甲基组研究作为目标（目的2）。最后，将通过转录组学、基因组测序和代谢组学研究高度降低的外显率，特别是男性GCH1突变携带者（目的3）。该方法将有助于更好地了解THAP1、SGCE和GCH1突变携带者的分子变异，并有可能阐明影响这些基因外显率降低和突变表达性变化的因素。P4将在肌张力障碍方面与P5、P8和P9密切合作，在方法上与P10和Z2、P1和P3的核心密切合作。

Mutations in several genes, including THAP1 (THAP domain-containing apoptosis-associated protein 1), SGCE (sarcoglycan, epsilon), and GCH1 (GTP cyclohydrolase 1) have been confirmed as inherited cause of dystonia. Penetrance of mutations in these genes is about 50%, and there is enormous variable expressivity among the affected carriers. In the first funding period, we focused on genetic modifiers of THAP1 penetrance. For this, we generated 20 iPSC clones from fibroblast lines of 5 affected and 5 unaffected THAP1 mutation carriers. These clones and 14 matched control lines have been differentiated into cortical neuronal lines and used for transcriptome analysis. Further, 13 affected and unaffected THAP1 mutation carriers were whole genome- sequenced and Chromosome Conformation Capture-on-Chip (4C) data of a cortical neuronal line without THAP1 mutation were generated. Using these comprehensive data resources, we demonstrated differential expression of several genes linked to diverse pathways including response to type I interferon, membrane fusion, and mitochondrial fission. For several of those genes, binding of the THAP1 transcription factor has been shown within the respective promoter regions (ENCODE data). Further, the genome data ruled out simple, coding sequence variants in other dystonia genes in the affected carriers. Finally, a common variant within the promoter of KAT6A (lysine acetyltransferase 6A) has been nominated as a potential modifier of THAP1 penetrance based on 3D interaction with the THAP1 gene and segregation in a family.Within this proposal, the overall hypothesis is that penetrance and expressivity of mutations in THAP1, SGCE, or GCH1 are modified by further genetic variants and/or epigenetic marks affecting gene expression and/or metabolites. To substantiate this hypothesis, we will re-analyze existing data for THAP1 with a focus on molecular function combining transcriptome, genome, and regulome data in a trans-Omics multi-variate modeling approach to elucidate synergistic actions relevant to the penetrance of THAP1 mutations (Objective 1). We will broaden our focus to SGCE and GCH1. While the reduced penetrance in SGCE is mainly explained by maternal imprinting, as we demonstrated earlier, the impact of differential methylation and gene expression on the variable expressivity is largely elusive and will be targeted by transcriptome, genome, and methylome studies (Objective 2). Finally, the highly reduced penetrance in, especially, male GCH1 mutation carriers will be investigated by transcriptomics, genome sequencing, and metabolomics (Objective 3). This approach will contribute to a better understanding of molecular variations in THAP1, SGCE, and GCH1 mutation carriers and potentially elucidate factors influencing reduced penetrance and variable expressivity of mutations in these genes.P4 will closly collaborate with P5, P8, and P9 in terms of dystonia, and methodologically with P10 and the Cores in Z2, P1 and P3.