Knowledge associating AR mutations to PAIS is often unclear. Patient gender assignment maybe problematic, pubertal progression unknown and the mechanisms that underlie genital phenotype diversity are only now being revealed. In association with other members of the consortium, WP03 will obtain pubertal information for PAIS patients throughout Europe. This clinical data will be correlated to the functional activity of PAIS AR mutations (Lübeck and Cambridge). WP03 also aims to understand the molecular mechanisms of the wild-type and mutant AR action during early genital development. AR binding partners expressed in the mouse genital tubercle will be identified (Lübeck). The second approach develops RCME technology for the analysis of mutant AR function (Cambridge).
The outcome of this European collaboration will establish the relationship between AR mutations, genital phenotype and pubertal outcome in PAIS patients. The first phase of the study uses the EuroDSD database to identify PAIS phenotypes of post-pubertal age with AR mutations; this will allow identification of a critical number of patients required for correlation of mutation with pubertal outcome. The second phase will quantify mutant AR function using established in vitro assays. Functional data will be correlated with clinical outcome data and the findings published. It is anticipated that this will provide clinicians with valuable information for patients born with PAIS and a known mutation that has been documented by the study. The cohort of patients will be of particular value in understanding the molecular function of the AR during both early and later stages of sexual development. The study will highlight patient mutations for extended investigation in task 2 and 3 of WP03.
AR function is required for the development of the male phenotype during early development and later during puberty. We will use yeast 2 hybrid system to define AR binding protein partners expressed during early genital development. Central to this approach will be construction of the mouse genital tubercle cDNA ‘PREY’ library. This will be screened for interactive partners to the ligand binding region of the AR. The screen should identify novel AR co-regulators and their expression in the mouse embryo at defined stages of development will be examined. A validated library of AR binding partners present in the genital tubercle would be a valuable resource to all members of WP03 and other groups investigating AIS. The second stage will examine how PAIS AR mutations perturb interaction with co-regulators, focusing on human orthologues of the genes identified in the genital tubercle screen.
This task will define mutant AR function in relation to the role of androgens during Wolffian duct development. Murine Wolffian duct cell lines will be modified to allow efficient recombinase mediated introduction of mutations into AR locus. RMCE technology results in the stable expression of mutant AR only. These lines are then compatible with a range of molecular and cellular approaches to define mutant AR function.