Studies of androgen receptor gene mutations in patients phenotypically ranging from complete androgen insensitivity to men with preserved fertility

University dissertation from Stockholm : Karolinska Institutet, Department of Molecular Medicine

Abstract: The androgen insensitivity syndrome (AIS) is the single most common cause of male pseudohermaphroditism, i. e., deficient masculinization which is not explained by disturbed testis development. In its most severe form it affects at least 1/20 000 newborn 46, XY males. This X-linked recessive disorder is caused by mutations in the androgen receptor (AR) gene, and has classically been divided into two subgroups according to severity; complete (CAIS) and partial androgen insensitivity syndrome (PAIS). In the complete form, the 46, XY individual presents as a phenotypically normal girl, except for absence of sexual hair. These patients have intraabdominal testes and, due to regression of Müllerian ducts, a short vagina, no uterus and lack oviducts. Partial forms of AIS present as varying degrees of undermasculinization, ranging from a predominantly female phenotype to boys with genital malformations, such as hypospadias or cryptorchidism. It has also been speculated, that subtle androgen receptor defects could cause impaired spermatogenesis without genital malformations. In the present work 13 missense mutations are described, identified in the AR gene of patients phenotypically ranging from complete androgen insensitivity to men with preserved fertility at the other end of the spectrum. The functional properties of 10 mutations have been characterized, using the approaches of site-directed mutagenesis, transient expression in COS-1 cells, and transactivation assays using an androgen sensitive reporter gene. Hormone binding assays in transfected COS-1 cells and genital skin fibroblasts from some patients were also performed. With a few exceptions, the degrees of impairment of mutant ARs in vitro were roughly in agreement with the severity of symptoms seen in the patients. Mutation A596T was an exception. A596T was functionally normal at high concentrations of androgens in vitro, although it was found in two newborns with PAIS. In accordance with this finding, treatment of the two boys with high doses of androgens resulted in a positive response. When this study was initiated, there was no molecular evidence for involvement of the AR in infertility. However, an elongated CAG repeat in exon I of the AR was known to be associated with dysfunctional sperm production in Kennedy's disease. Therefore, the length of the CAG-repeat of 33 infertile men was determined and compared to the CAG-repeats of 294 normal men. We found no difference in repeat lengths between the two groups. On the other hand, two missense mutations, N233K and N756S, were identified in two out of ten cases of infertility, selected due to elevated levels of LH and testosterone as well as azoo- or oligozoospermia. In both men, in vitro studies showed reduced transactivational capacity as compared to wild type AR. The patient carrying the N233K mutation displayed additional symptoms not generally seen in patients with AIS; he suffered from musculoskeletal and urogenital pain. He reported a remarkable relief upon high-dose androgen treatment. We speculate, that these symptoms result from abnormal protein- protein interactions arising as a consequence of the mutation, which is located in the transactivating domain of the AR where very few mutations previously have been found. Mutations in the AR gene have not been considered to be compatible with fertility. The Q824K mutation was found in three individuals of a family who complained of gynecomastia and showed hormonal levels indicating AIS. The mutant AR showed slight functional impairment in vitro. The patients had inherited the mutation from their grandfathers through their mothers, and one of them has fathered a daughter. The E653K mutation was found in a father of two daughters, who were affected with congenital adrenal hyperplasia due to 2 1 -hydroxylase deficiency. The daughters, who were heterozygous for the mutation, showed unusually mild signs of androgen excess, but in vitro assays of the E653K mutant failed to detect any functional abnormality. In conclusion, mutations in the AR gene have been found in patients covering the whole range of clinical phenotypes of androgen insensitivity. We confirm that the syndrome can be classified into three entities; CAIS, PAIS, and minimal androgen insensitivity (MAIS), where MAIS is defined as biochemical signs of AIS, gynecomastia and / or infertility, but without genital malformations.

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