MUTATIONS AND POLYMORPHISMS OF THE ANDROGEN RECEPTOR GENE CAUSING PARTIAL AND MINIMAL ANDROGEN INSENSITIVITY SYNDROMES

Abstract

Genetic defects of the human androgen receptor (AR) cause a wide spectrum of androgen insensitivity syndromes (AIS) ranging from phenotypic females in those with complete AIS; ambiguous genitalia in partial AIS; to male infertility in minimal AIS. The defects in complete AIS are well characterized and are usually due to mutations that totally disrupt the function of the AR protein. However, the molecular pathogenesis of partial and minimal AIS is less well understood. This thesis investigates the molecular mechanisms whereby, firstly a point mutation in the AR gene causes partial AIS, and secondly how variations in the length of a polymorphic polyglutamine tract in the AR protein results in minimal AIS, defective spermatogenesis and idiopathic male infertility. ln the first part of this study, investigation of a patient with ambiguous genitalia and partial AIS revealed a point mutation affecting codon 758 of the AR ligand-binding domain (LBD) that changed the sense of the codon from asparagine to threonine (N758T). The mutant receptor displayed normal binding affinity but abnormal dissociation kinetics with androgens both in patient's fibroblasts and transfected COS-7 cells. The mutant AR was thermolabile, and resulted in -50% reduction in receptor transactivation capacity when examined with a receptor gene incorporating an androgen-response-element. This indicates that the mutant (N758T) AR is the cause of ambiguous genitalia in that patient. Amino acid 758 lies in the predicted linker region between the 5th ?-helix (H5) and the first ?-strand (S1) of the AR. Our analyses show that mutations leading to partial AIS tend to cluster in the predicted linker regions located between the structural helixes of the AR LBD. Most strikingly, the predicted linker regions contain over 70% of the mutant ARs associated with prostate cancer in the LBD. The occurrence of mutations associated with both partial AIS and prostate cancer in the same predicted linker regions, suggests that this clustering is not coincidental and that the predicted linker regions are likely to have important, but subtle, roles in defining androgen binding and ligand specificity. In the second part of this study, two polymorphic trinucleotide repeat segments, coding for polyglutamine and polyglycine tracts in the N-terminal transactivation domain of the AR protein, were investigated for their roles in the etiology of male infertility due to defective spematogenesis. Changes in the lengths of these polymorphic repeat segments have been associated with increased risk of prostate cancer, an androgen-dependent tumor. Expansion of the polyglutamine tracts causes a rare neuromuscular disease, spinal bulbar muscular atrophy, that is associated with low virilization, reduced sperm production, testicular atrophy, and infertility. As spermatogenesis is exquisitely androgen dependent, it is plausible that changes in these two repeat segments could have a role in some cases of male infertility associated with impaired spermatogenesis. To test this hypothesis, the lengths of the polyglutamine and polyglycine repeats were examined in 153 patients with defective sperm production and compared them to 72 normal controls of proven fertility. There was no significant association between the polyglycine tracts and infertility. However, patients with 28 or more glutamines (Gln) in their AR had more than 4-fold (95% confidence interval, 4.9-3.2) increased risk of impaired spermatogenesis, and the more severe the spermatogenic defect, the higher the proportion of patients with a longer Gln repeat. Concordantly, the risk of defective spermatogenesis was halved when the polyglutamine tract was short (? 23 Gln). Whole cell transfection experiments using AR constructs harboring 15, 20, and 31 Gln repeats and a luciferase reporter gene with an androgen response element promoter confirmed an inverse relationship between Gln number and trans-regulatory activity. lmmunoblot analyses indicated that the reduced androgenicity of the AR was unlikely to be due to a change in AR protein content. The data indicate a direct relation between length of the AR polyglutamine tract and the risk of defective spermatogenesis that is attributable to the decreased functional competence of AR with longer polyglutamine tracts. The results of these two studies demonstrate the subtle molecular mechanisms whereby mutations and polymorphisms of the AR protein can modulate receptor function in health and disease.