Three point mutations have been found in the hormone-binding domain (HBD) of the human androgen receptor (hAR): one in the N-terminal end (Ile663Asn in a family with partial androgen insensitivity syndrome (PAIS)); one in the middle, (Leu820Val in a family with PAIS); and one in the C-terminal end (Pro903Ser, in a family with complete AIS). The positions 663 and 903 were the most terminal mutation sites in the HBD found to date. The three mutant hARs have been previously characterized biochemically in genital skin fibroblasts. In the family with the Leu820Val substitution, the mother and the grandmother were found to be carriers for the same mutation. To prove their pathogenicity, each of the three mutations has been reproduced in an hAR expression vector that was transfected into COS-1 cells. In COS-1 cells, the complexes from Pro903Ser and Leu820Val had: increased thermolability; increased dissociation rates; decreased affinity; and abnormal transactivation. There was a hierarchy in the severity of the mutations expressed in kinetic and transactivation assays that correlated with the severity of the clinical phenotype. The pathogenicity of the Pro903Ser and the Leu820Val mutations was thereby confirmed. In COS-1 cells, the AR with Ile663Asn had normal thermolability, normal dissociation rates, and normal transactivation, but a decreased affinity. Although this sequence alteration has only been found in a PAIS patient, its pathogenicity is not considered to be proven. More sensitive assays are needed for this purpose.

The human androgen receptor (hAR) is a ligand-activated, nuclear transcription factor. Mutations affecting the formation and/or action of the hAR cause androgen insensitivity syndrome (AIS). The majority of mutations identified to date are within the DNA- and hormone-binding domains; very few have been identified in the transactivational modulatory domain, encoded by exon 1. This work presents an analysis of exon 1 and the 5$ sp prime$-flanking region of the hAR in a set of subjects whose AIS was believed to be caused by a mutation within these regions. Six of twelve strains had a nonsense or frameshift mutation in exon 1; a seventh strain had two missense and one silent substitution; no mutations were identified in the remaining subjects. The two missense mutations were recreated, individually and together, in an hAR complementary DNA (cDNA) expression vector and expressed in heterologous COS-1 cells. Their pathogenicity could not be proven with the system and assays used. In addition, mRNA and protein levels were analyzed and correlated with the identified mutations and the subjects' phenotype.

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.

Because of its perceived ability to predict future health and its relevance for family members, genetic information challenges the traditional justifications for medical confidentiality. This thesis examines the question whether a health care professional should have the discretion or a duty to breach confidentiality in order to inform a patient's relatives of their increased genetic risk. There is currently no exception to the statutory, common law and ethical duties of confidentiality for the non-consensual disclosure of genetic information to relatives. Precedents developed in the context of threats of harm and communicable diseases are of limited value. The law should not recognise the existence of a duty to warn in the context of genetics. As a last resort, health care professionals should be authorized, but not required, to disclose genetic risk information when there is a serious risk of preventable harm and when the potential harm of non-disclosure outweighs that of disclosure.

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.

This thesis analyzes the rationale, appropriateness and value of the available legal and regulatory means to deal with genetic discrimination in the context of insurance. Insurance is used as a paradigm case for discussing the legal means to address the concerns related to the impact of new medical technologies. A new framework is proposed for evaluating the potential impact of such new technologies on people's ability to participate fully in social life and to have access to important social goods without unfair discrimination based on certain inherited traits. / A "thick" contextual method is used, which involves a detailed description of the medical, social, and legal context of the debate. The approach is based on Michael Walzer's theory of justice, which posits that in assessing the fairness of the distribution of a particular good, one must take into account the nature of the good as determined by the specific socio-historical context in which it obtains its shared meaning. Walzer's theory is used in the thesis to critically analyze the regulatory and legislative means introduced in several countries to curb genetic discrimination. It is further argued that Walzer's contextual analysis resembles the approach taken by the Canadian Supreme Court in the context of anti-discrimination law. Canadian human rights law is analyzed in detail to describe how genetic discrimination could be dealt with under the current provisions and how human rights law can be used to create conditions of substantive equality. The thesis concludes with an analysis of various legal and regulatory options to deal with genetic discrimination and its impact on human rights in the Canadian context. The establishment of a regulatory body is proposed, with the mandate to review the appropriateness of the use of new tests in the context of insurance. I argue that this review process, and the contextual analysis that should be involved in this process, would constitute a useful step towards creating conditions for substantive equality, not only for those who are genetically disabled, but for all those who are affected by real or perceived disabling conditions and stigmatizing traits.

Thesis (Ph. D.)--Dept. of Paediatrics, Faculty of Medicine, University of Adelaide, 1991. / Typescript (Photocopy). Includes published papers co-authored by the author at the end of volume 2. Includes bibliographical references (leaves 195-237 of vol. 1).

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.