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

• Applicants: Professor Dr. Ole Ammerpohl; Professor Dr. Paul-Martin Holterhus
• Subject Area: Pediatric and Adolescent Medicine ; Endocrinology, Diabetology, Metabolism ; Human Genetics
• Term: from 2012 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 208642470

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.

• DFG Programme: Research Grants