Biochemical and molecular genetic analysis of mutant androgen receptors in humans

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

Human Nonsyndromic Hereditary Deafness DFNA17 Is Due to a Mutation in Nonmuscle Myosin MYH9

The authors had previously mapped a new locus—DFNA17, for nonsyndromic hereditary hearing impairment—to chromosome 22q12.2-q13.3. DFNA17 spans a 17- to 23-cM region, and MYH9, a nonmuscle–myosin heavy-chain gene, is located within the linked region. Because of the importance of myosins in hearing, MYH9 was tested as a candidate gene for DFNA17. Expression of MYH9 in the rat cochlea was confirmed using reverse transcriptase–PCR and immunohistochemistry. MYH9 was immunolocalized in the organ of Corti, the subcentral region of the spiral ligament, and the Reissner membrane. Sequence analysis of MYH9 in a family with DFNA17 identified, at nucleotide 2114, a G→A transposition that cosegregated with the inherited autosomal dominant hearing impairment. This missense mutation changes codon 705 from an invariant arginine (R) to histidine (H), R705H, within a highly conserved SH1 linker region. Previous studies have shown that modification of amino acid residues within the SH1 helix causes dysfunction of the ATPase activity of the motor domain in myosin II. Both the precise role of MYH9 in the cochlea and the mechanism by which the R705H mutation leads to the DFNA17 phenotype (progressive hearing impairment and cochleosaccular degeneration) remain to be elucidated