THE ANDROGEN RECEPTOR GGN POLYMORPHISM. Genetic and functional analyses

Sammanfattning: Male sex development and reproductive function is regulated by androgens acting via the androgen receptor (AR). The AR harbours two polymorphic repeats of CAG and GGN triplets, encoding glutamines and glycines, respectively. Both polymorphisms affect the AR transactivation ability, although the function of the GGN repeat was virtually unknown at the start of this project. Thus, the main purpose with this thesis was to study the in vivo and in vitro impact of GGN repeat variation on AR function. A reliable method for studying the segment was established, after which the GGN repeat was studied in normal men, and in men with androgen-related disorders such as prostate cancer, testicular cancer, infertility, cryptorchidism, and hypospadias. Two predominant alleles of 23 and 24 GGN repeats, respectively, were found in all subject categories studied. GGN23 was the most common allele in all men except cryptorchid men and boys with penile hypospadias, in whom GGN24 was predominant. Point mutations within the GGN repeat were present in ~1% of the Swedish population, and were not associated with severe genital malformations. In normal men, GGN<23 was associated with a decrease in semen volume compared to GGN?23. Expression vectors harbouring the AR cDNA with GGN10, 23, 24, or 27, in combination with the median CAG repeat length, were constructed and tested in an in vitro expression system. Cos-1 cells were transiently transfected with vectors containing the GGN constructs, the prostate specific antigen promoter in combination with the Luciferase reporter gene, and ?-galactosidase gene. Cells were grown in the presence of 0, 0.1, 1.0, 10 and 100 nM of 5?-dihydrotestosterone or R1881. Western blot analysis was performed to assess the amount of androgen receptor protein. The in vitro results indicated a functional difference between the two predominant alleles, with GGN23 being superior in function. A non-linear association between GGN repeat length and AR transcriptional function was seen. No differences in AR protein levels for the different GGN repeat lengths were seen. The mode of inheritance for the CAG and GGN repeats was assessed by single nucleotide polymorphism analysis. Linkage disequilibrium was observed between the CAG and GGN repeats, with long GGN repeats being inherited together with short CAG repeats, and vice versa. Thus, in this thesis I have shown that the AR gene GGN repeat plays an important role in normal male sex development and male reproductive function.