The human androgen receptor (hAR) is a ligand-activated transcription factor, and like other nuclear receptors, consists of a N-terminal modulatory domain, a central DNA-binding domain, and a C-terminal ligand-binding domain (LBD). Several missense mutations in the LBD cause androgen insensitivity syndrome (AI), a condition in XY individuals with absent or subnormal male primary and secondary sexual characteristics. On the other hand, abnormal expansion of a polyglutamine tract in the N-terminal domain of the hAR causes spinal and bulbar muscular atrophy (SBMA) which also affects males and causes milder forms of AI, in addition to adult-onset motor neuron degeneration and gradual wasting and weakening of the muscles of the limbs, face, throat, and tongue. However, it was not clear how and to what extent these mutations contribute to the clinical phenotype of the affected individuals. In order to investigate this matter, I used PCR site-directed mutagenesis to create plasmids expressing hARs with two pairs of missense mutations in the LBD (Val865Leu and Val865Met, and Arg839His and Arg839Cys), discovered in AI individuals with varying severity of the phenotype, and two abnormal expansions of the polyglutamine repeat discovered in SBMA patients (40 and 50 glutamines). I also synthesized plasmids expressing no glutamines (0 glutamines), 12 glutamines, or 20 glutamines in the same N-terminal region of the hAR. These plasmids were transiently expressed in heterologous cells (COS-1 and PC-3), and the mutant hARs were assayed for ligand binding, stability, and transactivational capacity. / In contrast to the findings by others (McPhaul et al., 1992; Marcelli et al., 1994), in some instances involving identical mutations, I consistently observed a correlation between the biochemical phenotype of the mutant hARs and the clinical phenotype of AI individuals; that is, the more severe receptor phenotype was associated with the more severe AI. These results support the hypothesis that hAR phenotype is the dominant factor in the development of the secondary sexual characteristics in normal and affected individuals. / I also observed a tight negative correlation between polyglutamine tract length and transactivational capacity. This suggests that polyglutamine modulates the activity of the hAR, and that hAR activity might be suppressed in various androgen-sensitive tissues (including motor neurons) in SBMA individuals, thereby contributing to the age of onset and/or progression of the disease, even if it cannot be the primary pathogenic agent of the disease.

The major objective of this thesis was to determine the molecular basis of a "ligand-selective" mutant androgen receptor (AR) phenotype. Methyltrienelone (MT), a synthetic androgen, dissociates normally from this receptor but mibolerone (MB), another synthetic androgen, dissociates from it two-fold faster than normal. This mutant receptor was identified within genital skin fibroblasts (GSF) from two unrelated individuals with different degrees of androgen insensitivity (AI). Sequence analysis of the AR gene from both subjects revealed a G to A transition at nt 2969 in exon 6 that alters codon 813 from serine to asparagine (S813N). Transiently expressed hAR.S813N did not reproduce the mutant phenotype in several heterologous cells: COS-1, BHK, CHO or HeLa cells. In contrast, when AR free (R$ sp-$) GSF were used as host cells, MB-R.S813N complexes dissociated almost two fold faster than the controls (n = 4) while MT-R.S813N complexes dissociated normally. These results establish the G to A transition at nt 2969 as the cause of the ligand-selective phenotype. Such host-cell restricted expression of the mutant dissociation rate points to cell-specific factors that can suppress abnormal dissociation of A-R complexes. Host cell-restricted expression of the abnormal dissociation rates has also been observed for two other transiently expressed mutant AR, hAR.V865L and hAR.R839H (n = 3). / Expansion of the glutamine (gln) tract within the N-terminus of the AR causes spinal bulbar muscular atrophy (SBMA), a disease of motor neurons, but the mechanism of this neuropathology is unknown. To determine the effect of gln-tract expansion upon AR function, SBMA-associated mutant AR was transiently expressed and characterized in COS-1 cells. The androgen-binding parameters of the mutant receptor were normal, but it had decreased transactivation competence (50-66% of normal; n = 3). This abnormal transregulatory function may account for the expression of traits associated with minimal androgen insensitivity (MAI) that are variably expressed in the SBMA patients.

Two previously identified sequence alterations in the androgen receptor gene of patients with complete androgen resistance are studied to prove their pathogenicity. Family studies confirm that the mutation segregates with the phenotype and that the mothers are heterozygous carriers. In one family a sibling of the patient is identified as a heterozygous carrier. Mutant cDNAs encoding the mutant receptors are constructed and expressed in COS-1 cells. The resulting mutant receptors show a decreased apparent equilibrium constant for androgens, faster dissociation rates and impaired transactivation. Further studies reveal that both mutant receptors were either inactivated or destroyed in the presence of hormone, while the normal receptor is stabilized and up-regulated by incubation with ligand. These results prove that the sequence alterations thus identified are pathogenic and illustrate a dual mechanism of pathogenicity: an affinity defect combined with a loss of binding activity in the presence of hormone, resulting in receptors incapable of supporting normal male sexual development.

This work proves the pathogenicity of four substitution mutations in the androgen-binding domain of the human androgen receptor (hAR) gene of four subjects with varying degrees of androgen insensitivity syndrome (AIS): complete (CAIS), partial (PAIS), or mild (MAIS). Of three unrelated CAIS subjects, two have Arg830Leu, the third has Arg830Gln. Their genital skin fibroblasts (GSF) have negligible androgen binding, but in overexpressing transfectants, the mutant androgen-binding activities have increased dissociation rates and decreased affinity for androgen. Owing to the instability of AR-androgen complexes, both mutants fail to transactivate a reporter gene. Glu771Ala and Arg870Gly caused PAIS and MAIS, respectively. Their normal levels of GSF androgen-binding activity have normal androgen affinity but increased dissociation rates. In transfectants, rates of dissociation resemble those in GSF, but the androgen affinities are questionably abnormal. Instability of Glu771Ala and Arg870Gly AR-androgen complexes caused subnormal transactivation of a reporter gene.

Sry on the Y-chromosome triggers the fetal gonad to begin differentiation into testis in mammals. Mutation or absence of Sry results in development of ovaries and the female phenotype. However, XY sex reversal in the presence of wild-type Sry exists in mice and man. One such example is the B6-YTIR mouse, whose autosomes and X-chromosome are of the C57BL6/J mouse (Mus musculus molossinus) whereas the Y-chromosome is from a mouse originating in Tirano, Italy (Mus musculus domesticus). B6-YTIR mice develop only ovaries or ovotestes in fetal life. The objective of my thesis was to identify the mechanism of sex reversal in the B6-YTIR mouse. The results indicate that onset of Sry transcription in B6-YTIR gonads is comparable to control B6 XY gonads. On the other hand, onset of Mis, 17alpha-HA, 3beta-HSD (testicular cell products), p450arom as well as inactivation of Sry transcription are delayed or absent in the sex reversed gonads. It has been suggested that low levels of Sry transcription may account for aberrant testis differentiation in B6-YTIR mice. We observed relatively low levels of Sry transcripts not only in B6-YTIR but also in B6 mice. However, levels in normal B6-YSJL mice were significantly greater. On the SJLB6F1 background, where no sex reversal occurs, Sry transcript levels of the TIR allele increased while those of B6 and SJL alleles remained the same as in the B6 background. Thus, low levels of Sry transcript from the B6 allele are sufficient whereas the levels from TIR and SJL alleles (both DOM type) appear to be critical for testis determination. We then compared the levels of endogenous Sry proteins. A combination of immunoprecipitation and immunoblotting succeeded in detecting a protein band whose expression profile and molecular size are consistent with those of the predicted Sry. Sry protein levels in B6-Y TIR gonads were roughly two fold greater than in B6 XY gonads. We hypothesize that the Sry protein of the TIR/SJL alleles is less efficient

Inaug.-Diss.--Heidelberg. / Lebenslauf. "Benutzte literatur": p. [21]-22.

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