Functional characterization of non-coding variants in disease-causing regions of benign choreoathetosis (CAHTP) and thyroid dysgenesis (CHTD)

转录因子NKX2-1不仅是甲状腺滤泡细胞的主要调控因子，也是腹侧端脑、间脑和肺泡上皮细胞中特定神经细胞类型的主要调控因子。NKX2-1基因突变导致良性舞蹈病和先天性甲状腺功能减退，伴或不伴肺功能障碍（CAHTP, OMIM #610978），这是一种常染色体显性遗传病，具有不同的外显率和甲状腺和肺表型的严重程度；正如我们小组在2002年所展示的那样。在第一个资助期，我们使用WGS鉴定了一组具有完整NKX2-1基因的CAHTP患者的NKX2-1基因位点的非编码变异。通过分析NKX2-1基因位点的表观基因组特征，我们在NKX2-1基因下游14q13处指定了一个假定的100 kb的疾病相关区域。在本项目的第一部分中，我们将以该区域为目标，采用不同的技术来研究该基因组区域的顺式调控作用，并分析WGS检测到的非编码变异体对25例CAHTP指数患者的影响。为了进一步了解甲状腺发育不良（CHTD）的遗传机制，我们与日本东京庆应义塾大学医学院的Satoshi Narumi进行了一项全基因组关联研究（GWAS），并在2q33.3的非编码基因组区域确定了CHTD的第一个疾病相关区域。根据表观基因组、转录组和染色质相互作用数据集推断，显著的顶部基因型SNP （rs9789446）位于一个72 kb的内含子区域，该区域包含甲状腺组织中FZD5和CCNYL1表达的顺式调控序列。为了开始正式证明该非编码区与甲状腺发育不良的疾病相关性，我们将在本项目的第二个资助期对2q33.3处假定的顺式调控元件的活性进行表征。

The transcription factor NKX2-1 is a master regulator not only of thyroid follicular cells, but also of specific neuronal cell types in the ventral telencephalon, the diencephalon, and of pulmonary alveolar epithelial cells. Mutations of the NKX2-1 gene cause benign choreoathetosis and congenital hypothyroidism with or without pulmonary dysfunction (CAHTP, OMIM #610978), an autosomal dominant disorder with variable penetrance and severity of thyroid and pulmonary phenotypes; as initially shown by our group already in 2002. During the first funding period we used WGS to identify non-coding variants in the NKX2-1 gene locus of a cohort of CAHTP patients with an intact NKX2-1 gene. By analyzing epigenomic signatures of the NKX2-1 gene locus, we assigned a putative 100 kb disease-associated region at 14q13 downstream of the NKX2-1 gene. In the first part of this project, we will target this region with different techniques to examine the cis-regulatory role of this genomic region and to analyze the impact of the non-coding variants detected by WGS in our cohort of 25 CAHTP index patients. To gain additional insights into the genetic mechanisms of thyroid dysgenesis (CHTD), we conducted a collaborative genome-wide association study (GWAS) with Satoshi Narumi from Keio University School of Medicine, Tokyo (Japan) and identified a first disease-associated region for CHTD in a non-coding genomic region at 2q33.3. The significant top genotyped SNP (rs9789446) was located in a 72 kb intronic region that contains cis-regulatory sequences for FZD5 and CCNYL1 expression in thyroid tissue as inferred from a combination of epigenome, transcriptome, and chromatin interaction data sets. To begin to formally demonstrate the disease relevance of this non-coding region for thyroid dysgenesis, we will characterize the activity of putative of cis-regulatory elements at 2q33.3 in the second funding period of this project.