Androgen Insensitivity Without Androgenreceptor Mutations: from functional studies to the epigenotype

该研究项目的假设假定生殖器雄激素靶组织中雄激素受体（AR）启动子甲基化增加可导致雄激素不敏感综合征（AIS），其通常起源于失活的AR基因突变。在本研究项目的背景下，我们可以确定一个迄今尚未描述的顺式活性元件在AR上游区域的两个索引患者，我们分析了结构和功能，使用多种和复杂的分子生物学方法。AR-上游区域的功能相关性的确认是通过实验建立的，并将在更新提案中进行分析。共96名索引患者的焦磷酸测序显示，在18%的病例中，分析区域中的单个CpG甲基化超过60%，强调了该区域的相关性。在更新提案的背景下，这种甲基化将与生殖器皮肤成纤维细胞中AR的功能分析和表达数据相关。同时，该研究项目导致建立世界上最大的生殖器皮肤成纤维细胞生物库之一，该生物库将在分子水平上进行广泛分析。我们计划把，培养并储存基尔雄激素不敏感性生物库的共385个细胞培养物，并通过（1）AR靶基因载脂蛋白D（APOD）的双氢睾酮（DHT）依赖性反式激活的功能分析，全面表征所有385个细胞培养物的功能通过APOD测定，包括计算该测定在AIS方面的特异性和灵敏度，（2）转录（AR-mRNA）和蛋白质的AR表达分析，（AR蛋白）水平，（3）通过亚硫酸氢盐测序进行AR上游区的序列分析，包括鉴定具有AR上游区高甲基化和减少的AR-突变阴性AIS患者。转录。根据DFG审查委员会关于促进初步建议以及我们迄今尚未接受的续约的建议，根据这一建议，我们在此提交一份经过大幅修订的更新建议，供您考虑DFG的资助。

The hypothesis of the research project postulates that increased methylation of the androgen receptor (AR) promoter in the genital androgen target tissue can cause androgen insensitivity syndrome (AIS) which usually originates from inactivating AR-gene mutations. In the context of this research project we could identify a so far not described cis-active element in the AR upstream region of two index patients that we analyzed structurally and functionally using manifold and complex molecular biology methods. The confirmation of the functional relevance of the AR-upstream region is experimentally established and will be analyzed in the renewal proposal. Pyrosequencing of a total of 96 index patients showed an over 60% methylation at individual CpGs in the analyzed region in 18% of the cases underlining the relevance of this region. In the context of the renewal proposal this methylation will be correlated with the functional analyses and expression data of the AR in gential skin fibroblasts.At the same time this research project leads to the buildup of one of the worldwide largest biobanks of genital skin fibroblasts of very rare androgen-insensitivity patients that will be extensively analyzed on a molecular level. We are planning to bring together, to culture and to store a total of 385 cell cultures of the androgen insensitivity biobank in Kiel as well as to comprehensively functionally characterize all 385 cell cultures through (1) functional analysis of the dihydrotestosterone (DHT)-dependent transactivation of the AR-target gene ApolipoproteinD (APOD) through the APODassay including the calculation of the specificity and sensitivity of this assay in regard of AIS, (2) AR-expression analysis on the transcriptional (AR-mRNA) and protein (AR-protein) level ,(3) sequence analysis of the AR upstream region through bisulfite sequencing including the identification of AR-mutation negative AIS-patients with hypemethylation of the AR upstream region and reduced AR-transcription.Following the recommendations of the DFG review board concerning the promotion of the initial proposal as well as of our so far not accepted renewal proposal, we here submit a substantially revised renewal proposal for your consideration for funding by the DFG.