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

该研究项目的假设假设是，生殖器雄激素靶组织中雄激素受体(AR)启动子的甲基化增加可导致雄激素不敏感综合征(AIS)，该综合征通常源于AR基因突变的失活。在这个研究项目的背景下，我们可以在两个指标患者的AR上游区域中发现一个迄今未被描述的顺式活性元件，我们使用多种复杂的分子生物学方法对其进行了结构和功能分析。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。对总共96名指标性患者的焦磷酸测序显示，在18%的病例中，分析区域的单个CPGS有超过60%的甲基化，这突显了该区域的相关性。在更新建议的背景下，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联。同时，该研究项目导致了非常罕见的雄激素不敏感患者生殖器皮肤成纤维细胞的全球最大生物库的建立，该生物库将在分子水平上进行广泛的分析。我们计划在基尔汇集、培养和存储总共385个雄激素不敏感生物库的细胞培养物，并通过以下方式对所有385个细胞培养物进行全面的功能鉴定：(1)通过APOD分析依赖双氢睾酮(DHT)的AR靶基因载脂蛋白D(APOD)的反式激活，包括计算该方法对AIS的特异性和敏感性，(2)转录(AR-mRNA)和蛋白质(AR-蛋白质)水平的AR-表达分析，(3)通过亚硫酸氢盐测序对AR上游区域进行序列分析，包括识别AR突变阴性的AIS-AR上游区域甲基化和AR转录减少的患者。根据DFG审查委员会关于促进最初提案和我们迄今未被接受的更新提案的建议，我们在此提交一份大幅修订的更新提案，供DFG考虑资助。

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.