metribolone and Androgen-Insensitivity-Syndrome

The clinical phenotype of complete androgen insensitivity (CAIS) was associated with a mutation in the human androgen receptor (hAR) gene encoding the amino acid substitution, M745I, in the hAR protein. Transcriptional activation of hAR(M745I) by the synthetic androgen, methyltrienolone (R1881), was reduced compared to wild-type (wt) hAR. The transcriptional co-activator, androgen receptor associated protein 70 (ARA70), failed to enhance transactivation of hAR(M745I) at lower concentrations of R1881 (0.01-0.1 nM), whereas the p160 co-activators, SRC-1 and TIF2, stimulated activity. Transcriptional activity of hAR(M745I) was stimulated by 1 or 10 nM R1881 and activity was further enhanced by co-expression of ARA70 similar to that of the hAR(wt). Transcriptional activity of hAR(wt) was minimally stimulated by estradiol (E2) without or with co-expression of ARA70, whereas 10 or 100 nM E2 increased transactivation by hAR(M745I) of the androgen-responsive MMTV-luciferase reporter gene by 10-fold and activity was further enhanced by ARA70. Increasing concentrations of E2 competed more effectively for binding of R1881 to hAR(M745I) than to hAR(wt), indicative of the preferential binding of E2 to the mutant hAR. Partial tryptic digestion of hAR wt and M745I revealed that activation of the mutant protein was reduced in the presence of R1881. By contrast, tryptic digestion showed that the mutant hAR was activated by the binding of E2. In conclusion, the clinical phenotype of CAIS resulted from a hAR gene mutation encoding hAR(M745I) with reduced binding and transactivation by androgens, but the novel properties of enhanced affinity for and increased transactivation by estradiol.

Topics: Amino Acid Substitution; Androgen-Insensitivity Syndrome; Androgens; Animals; Brain; Cells, Cultured; Chlorocebus aethiops; COS Cells; Estradiol; Gene Library; HeLa Cells; Histone Acetyltransferases; Humans; Male; Metribolone; Mutation; Nuclear Receptor Coactivator 1; Nuclear Receptor Coactivator 2; Nuclear Receptor Coactivators; Oncogene Proteins; Receptors, Androgen; Transcription Factors; Transcriptional Activation; Two-Hybrid System Techniques

Superior androgen receptor (AR) function in subjects carrying a GGN repeat length of 23 (GGN23) has been indicated in vivo. Therefore, the activity of the AR carrying GGN23 combined with CAG22 was compared to the AR with GGN10, 24 and 27, respectively, in the presence of 0.1-100 nM testosterone or DHT. At 100 nM DHT, GGN24 showed 35% lower transactivating activity (95% [CI]: 20-50%) than GGN23. GGN10 and GGN27 also showed significantly less AR activity than GGN23 (mean differences [95% CI]: 54% [40-68%] and 58% [39-78%], respectively). The same trend was also observed at lower DHT concentrations. In response to R1881, GGN23 activity was significantly higher than for other lengths. ARs with other glutamine numbers than 23 have lower transactivating capacity in response to both testosterone and DHT. Congenital malformations and other signs of hypoandrogenism in subjects with AR gene GGN lengths other than 23 could, hence, be related to a lower AR activity.

Topics: Androgen-Insensitivity Syndrome; Cell Line; Humans; Male; Metribolone; Polymorphism, Genetic; Receptors, Androgen; Transcriptional Activation; Trinucleotide Repeat Expansion; Trinucleotide Repeats

The androgen insensitivity syndrome is a disorder caused by deficient function of the androgen receptor, characterized by varying degrees of undermasculinization in karyotypic males. We have identified four mutations in the androgen receptor gene, in the region encoding the DNA-binding domain of the protein. Two mutations, R607X and R615G, were found in patients with complete insensitivity to androgens, whereas the other two, S578T and A596T, were found in patients with partial insensitivity. The functional consequences of the three missense mutations were assayed in vitro after transient expression of the receptors in COS cells. All mutants showed normal androgen binding but abnormal abilities to stimulate transcription of an androgen-responsive reporter gene. R615G abolished transactivation whereas S578T and A596T were partially malfunctional. The function of A596T, but not of S578T, was normalized at high androgen concentrations in vitro, reflecting the in vivo situation. Thus, patients with specific mutations in the DNA-binding domain of the androgen receptor may benefit from androgen treatment.

Topics: Amino Acid Sequence; Androgen-Insensitivity Syndrome; Animals; Binding Sites; COS Cells; DNA; Exons; Humans; Male; Metribolone; Molecular Sequence Data; Mutation; Mutation, Missense; Receptors, Androgen; Testosterone; Testosterone Congeners; Transcriptional Activation; Transfection

We have characterized two different mutations of the human androgen receptor (hAR) found in two unrelated subjects with androgen insensitivity syndrome (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitutions have been found in both individuals: E772A in the PAIS subject, and R871G in the MAIS patient. In COS-1 cells transfected with the E772A and R871G hARs, the apparent equilibrium dissociation constants (Kd) for mibolerone (MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the PAIS subject (E772A) has elevated nonequilibrium dissociation rate constants (k(diss)) for both androgens. In contrast, the MAIS subject's hAR (R871G) has k(diss) values that are apparently normal for MB and methyltrienolone; in addition, the R871G hAR's ability to bind MB resists thermal stress better than the hAR from the PAIS subject. The E772A and R871G hARs, therefore, confer the same pattern of discordant androgen-binding parameters in transfected COS-1 cells as observed previously in the subjects' genital skin fibroblasts. This proves their pathogenicity and correlates with the relative severity of the clinical phenotype. In COS-1 cells transfected with an androgen-responsive reporter gene, trans-activation was 50% of normal in cells containing either mutant hAR. However, mutant hAR-MB binding is unstable during prolonged incubation with MB, whereas normal hAR-MB binding increases. Thus, normal equilibrium dissociation constants alone, as determined by Scatchard analysis, may not be indicative of normal hAR function. An increased k(diss) despite a normal Kd for a given androgen suggests that it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in terms of the three-dimensional model of the ligand-binding domain of the nuclear receptor superfamily.

Topics: Amino Acid Sequence; Androgen-Insensitivity Syndrome; Androgens; Animals; COS Cells; Drug Stability; Female; Hot Temperature; Humans; Male; Metribolone; Molecular Sequence Data; Mutagenesis, Site-Directed; Nandrolone; Point Mutation; Receptors, Androgen; Testosterone Congeners; Transcriptional Activation; Transfection

In the coding part and the intron-exon boundaries of the androgen-receptor gene of a patient with partial androgen insensitivity, no mutation was found. The androgen receptor of this patient displayed normal ligand-binding parameters and migrated as a 110-112-kD doublet on SDS-PAGE in the absence of hormone. However, after culturing of the patient's genital skin fibroblasts in the presence of hormone, the slower-migrating 114-kD protein, which reflects hormone-dependent phosphorylation, was hardly detectable. Furthermore, receptor protein was undetectable in the nuclear fraction of the fibroblasts, after treatment with hormone, which is indicative of defective DNA binding. By sequencing part of intron 2, a T-->A mutation was found 11 bp upstream of exon 3. In our screening of 102 chromosomes from unrelated individuals, this base-pair substitution was not found, indicating that it was not a polymorphism. mRNA analysis revealed that splicing involved a cryptic splice site, located 71/70 bp upstream of exon 3, resulting in generation of mRNA with an insert of 69 nucleotides. In addition, a small amount of a transcript with a deleted exon 3 and a very low level of wild-type transcript were detected. Translation of the extended transcript resulted in an androgen-receptor protein with 23 amino acid residues inserted between the two zinc clusters, displaying defective DNA binding and defective transcription activation.

Topics: Androgen-Insensitivity Syndrome; Animals; Blotting, Western; Cells, Cultured; DNA; DNA Mutational Analysis; Electrophoresis, Polyacrylamide Gel; Genes, Reporter; Humans; Introns; Male; Metribolone; Mutation; Nucleic Acid Hybridization; Pedigree; Phosphorylation; Receptors, Androgen; RNA Splicing; RNA, Messenger; Testosterone Congeners; Transcriptional Activation; Transfection

Premature stop codons of the human androgen receptor (AR) gene are usually associated with a complete androgen insensitivity syndrome. We, however, identified an adult patient with a 46,XY karyotype carrying a premature stop codon in exon 1 of the AR gene presenting with signs of partial virilization: pubic hair Tanner stage 4 and clitoral enlargement. No other family members were affected. A point mutation at codon position 172 of the AR gene was detected that replaced the original TTA (Leu) with a premature stop codon TGA (opal). Careful examination of the sequencing gel, however, also identified a wild-type allele, indicating a mosaicism. In addition, elimination of the unique AflII recognition site induced by the mutation was incomplete, thus confirming the coexistence of mutant and wild-type AR alleles in the patient. Normal R1881 binding and a normal 110/112-kDa AR doublet in Western immunoblots consolidated the molecular genetic data by demonstrating the expression of the wild-type AR in the patient's genital skin fibroblasts. Transfection analysis revealed that only relatively high plasmid concentrations carrying the mutated AR complementary DNA lead to expression of a shortened AR due to downstream reinitiation at methionine 189. Thus, reinitiation does not play a role in the presentation of the phenotype; rather, the partial virilization is caused by the expression of the wild-type AR due to a somatic mosaic. We conclude that somatic mosaicism of the AR gene can represent a substantial factor for the individual phenotype by shifting it to a higher degree of virilization than expected from the genotype of the mutant allele alone.

Topics: Adult; Androgen-Insensitivity Syndrome; Base Sequence; Blotting, Western; DNA; Gene Expression; Humans; Male; Metribolone; Mosaicism; Mutation; Phenotype; Polymerase Chain Reaction; Receptors, Androgen; Transcriptional Activation; Transfection

A wide spectrum of androgen receptor (AR) gene mutations has been reported in complete androgen insensitivity syndromes. The molecular basis of androgen resistance was investigated in a female newborn with complete testicular feminization. Sequencing identified a point mutation in exon 4 responsible for a leucine (CTG) to arginine (CGG) replacement at codon 707. This novel mutation is located in the amino-terminal part of the ligand-binding domain of the AR. To determine the functional properties of the mutated AR and to establish the correlation with the clinical phenotype of androgen resistance, the mutation was reproduced in AR wild-type complementary DNA, and the plasmid was transfected into AR-free mammalian cells. In vitro studies showed that the mutant AR was functionally defective as an androgen-binding molecule. Electrophoretic mobility shift assay revealed that the binding of mutated AR to DNA was reduced. Finally, the mutant was unable to induce the transcriptional activation of androgen-responsive reporter gene. This amino acid defect in the primary sequence probably involves the rupture of hydropathicity in a region that is conserved among members of the steroid receptor subfamily. Our data substantiate the major contribution of leucine 707 to normal AR function and demonstrate that its substitution by an arginine caused the complete androgen insensitivity in this patient. Our findings also contribute to the elaboration of the structure-function map of the AR based on naturally occurring mutations.

Topics: Amino Acid Sequence; Androgen-Insensitivity Syndrome; Androgens; Base Sequence; DNA; Drug Resistance; Exons; Humans; Infant, Newborn; Luciferases; Male; Metribolone; Molecular Sequence Data; Receptors, Androgen

The reproducible photolabeling of the androgen receptor from human skin fibroblasts, using [3H]methyltrienolone (R-1881) as ligand is described. Crude nuclei were irradiated for 2 min using a UV lamp with an emission line at 352 nm and a CuSO4 filter. After KCl extraction, proteins were precipitated with trichloroacetic acid, washed with ether and assayed for radioactivity. Specific binding was determined as the difference in bound radioactivity between cells incubated with [3H]R-1881 +/- a 200-fold excess of unlabeled dihydrotestosterone (DHT). The photolabeled proteins were analyzed on SDS-polyacrylamide gel electrophoresis yielding one peak of 90 kDa and in several cases, one of 43 kDa. These peaks comprised 60 +/- 20% of the saturable binding recovered on the gels. The overall efficiency of photolabeling was between 1 and 5%. The amount of covalently bound radioactivity was proportional to the number of cells used. The labeling was inhibited by R-1881, DHT, the anti-androgens hydroxyflutamide and cyproterone acetate and to a lesser extent by estradiol and progesterone. No covalent attachment of R-1881 to any protein was observed when nuclei from patients with androgen insensitivity were irradiated, whether or not the cells were receptor positive or negative. In conclusion the androgen receptor from human skin fibroblast can be efficiently photolabeled and could be used as a marker to follow receptor purification. The absence of photolabeling of nuclear extracts from receptor-positive androgen-insensitive patients may reflect some abnormality of the receptor.

Topics: Affinity Labels; Androgen-Insensitivity Syndrome; Dihydrotestosterone; Electrophoresis, Polyacrylamide Gel; Estrenes; Fibroblasts; Humans; Metribolone; Photochemistry; Receptors, Androgen; Skin

We have studied a kindred in which two parts of siblings, maternal first cousins, have a form of "minimal" androgen insensitivity that permits morphogenesis of unambiguous male external genitalia, but interferes with normal virilization at puberty. All four had gynecomastia that required reductive surgery. Apart from this common phenotype, they varied considerably in the temporal and regional aspects of their subvirilization and appreciably in their androgenic responsiveness to pharmacological doses of testosterone. The cultured genital skin fibroblasts from one set of siblings have an androgen-receptor activity with the following properties: (1) a normal maximum-binding capacity (Bmax) with 5 alpha-dihydrotestosterone (DHT), or the synthetic androgen, methyltrienolone (MT; R1881) as ligand; (2) a higher than normal apparent equilibrium dissociation constant (Kd) for DHT (0.77 nM) but not for MT; and (3) an elevated dissociation rate (k) of DHT-receptor (0.013 min-1, 37 degrees C), but not MT-receptor, complexes within intact cells. In addition, prolonged incubation with MT, but not DHT, augments the specific androgen-binding activity of the mutant cells as much as that of the controls. Normal cells yield lower values of apparent Kd for DHT (0.1-0.3 nM) after 2- than after 0.5-hr incubation (0.3-1.8 nM), and 1-hr values are intermediate. This occurs despite concurrent catabolic consumption of DHT from the medium and is considered to reflect transformation of initial, low-affinity DHT-receptor complexes to subsequent, higher-affinity states by a process that depends on time and initial ligand concentration. The mutant complexes described here can readily attain the highest state of affinity with MT, but have an impeded, variably expressed ability to do so with DHT. These findings suggest that a structural mutation at the X-linked locus that encodes the androgen-receptor protein is responsible for its androgen-selective dysfunction. Synthetic, nonhepatotoxic androgens, with corrective effects in vitro comparable to those of MT, may be therapeutically useful for these subjects.

Topics: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase; Adolescent; Adult; Androgen-Insensitivity Syndrome; Androgens; Cells, Cultured; Dihydrotestosterone; Disorders of Sex Development; Estrenes; Fibroblasts; Humans; Kinetics; Male; Metribolone; Mutation; Pedigree; Receptors, Androgen; Receptors, Steroid; Scrotum; Skin

To determine whether abnormalities of the androgen receptor previously observed in skin fibroblasts from patients with androgen insensitivity syndrome also occur in the gonads of affected individuals, androgen receptor activity in the gonads of a patient with testicular feminization syndrome was investigated. Using conditions for optimal recovery of androgen receptor from human testes established by previous studies, we detected the presence of a high-affinity (dissociation constant = 3.2 X 10(-10) mol/L), low-capacity (4.2 X 10(-12) mol/mg DNA), androgen-binding protein when tritium-labeled R1881 was incubated at 4 degrees C with nuclear extracts from the gonads of control patients or from a patient with testicular feminization syndrome but not when incubated at 37 degrees C. Thus this patient has an androgen receptor with a temperature lability similar to that of receptors from normal persons.

Topics: Adolescent; Aged; Androgen-Binding Protein; Androgen-Insensitivity Syndrome; Binding Sites; Cytosol; Estrenes; Humans; Male; Metribolone; Middle Aged; Receptors, Androgen; Receptors, Steroid; Temperature; Testis; Tritium

Enzyme kinetics of 5 alpha-reductase were compared in cultured genital skin fibroblasts taken from 6 control subjects and from an affected subject with 5 alpha-reductase deficiency in whom the diagnosis was established on hormonal grounds. The Km value for testosterone in the mutant enzyme was extremely high (1,427 vs. 185-417 nmol/l in controls). in the mutant enzyme was extremely high (1,427 vs 185-417 nmol/l in controls). A A mutant but stable enzyme with reduced affinity to steroid substrate is reported.

Topics: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase; Androgen-Insensitivity Syndrome; Cells, Cultured; Child, Preschool; Chorionic Gonadotropin; Disorders of Sex Development; Estrenes; Female; Fibroblasts; Genitalia; Hot Temperature; Humans; Hydrogen-Ion Concentration; Male; Metribolone; Oxidoreductases; Testosterone

Thermolability of androgen binding was compared in fibroblasts cloned from normal female skin, skin from a subject with testicular feminization whose mutation is known to be associated with a thermolabile androgen receptor, and from the mother of the subject with testicular feminization. Seven of 28 clones studied from the mother exhibited thermolability of binding, indicating that the mutant gene that causes thermolability of binding, like the gene responsible for the normal androgen receptor, is X-linked.

Topics: Adolescent; Adult; Androgen-Insensitivity Syndrome; Child; Clone Cells; Estrenes; Female; Fibroblasts; Genes; Heterozygote; Hot Temperature; Humans; Infant; Male; Metribolone; Mutation; Receptors, Androgen; Receptors, Steroid; X Chromosome

Patterns of protein synthesis by genital skin fibroblasts from three unrelated normal individuals and three unrelated patients with complete testicular feminization were compared to two-dimensional gel electrophoresis. cell lines were maintained in monolayer culture and pulse labeled with [35S]methionine. Cells were lysed in 9 M urea, and aliquots of 20 microliters subjected to isoelectric focussing and polyacrylamide gel electrophoresis followed by autoradiography. Gels of control fibroblasts showed two proteins (mol wt approximately 45,000, approximately 85,000; pKi approximately 5.0) markedly more prominent than on gels from affected fibroblasts. This pattern was unaltered by prior exposure to dihydrotestosterone, suggesting differences in constitutive proteins of the fibroblast cells. Parallel studies demonstrated a marked reduction in the ability of fibroblasts from patients with complete testicular feminization to bind androgens in vitro compared with those of normal individuals. The relationship between these proteins, androgen receptors, and androgen insensitivity requires further investigation.

Topics: Androgen-Insensitivity Syndrome; Cells, Cultured; Dihydrotestosterone; Estrenes; Fibroblasts; Humans; Male; Metribolone; Protein Biosynthesis; Receptors, Androgen; Testosterone Congeners

Human skin, an accessible tissue, is an androgen target organ. We have measured the androgen-binding capacity of human skin cytosol using either 5 alpha[3H]dihydrotestosterone ([3H]DHT) or [3H]methyltrienolone ([3H]R-1881) as ligand. Samples were incubated for 20 h at 0 C, and dextran-coated charcoal was used to separate bound from free steroids. The androgen receptor has a high affinity for both ligands (0.23 +/- 0.04 nM for [3H]DHT; 0.32 +/- 0.16 nM for [3H]R-1881). Testosterone, cyproterone acetate, and, to a lesser extent, estradiol also bind this protein. Progesterone displaces R-1881 from its binding sites, whereas its 5 alpha-reduced metabolite somewhat inhibits DHT binding. The highest binding capacity is measured in cytosol of skin from external genitalia (129.14 +/- 58.0 fmol/g skin; n = 34); it is lower in pubic skin (21.8 +/- 13 fmol/g skin; n = 6). There is no variation as a function of age or sex in genital skin; the higher concentrations observed in the cytosol of pubic skin of women compared to that of men are probably related to lower levels of endogenous steroids. Whereas most patients with the complete form of the testicular feminization syndrome do not have detectable concentrations of androgen receptor, one patient with apparent complete clinical androgen insensitivity had a normal androgen-binding capacity. The parity of values in genital skin from men and women, the absence of variation with age, and the presence of a cytosolic androgen receptor in some androgen-insensitive patients suggest that the androgen receptor in human skin cytosol is not regulated by androgens.

Topics: Adolescent; Adult; Aged; Androgen-Insensitivity Syndrome; Child; Child, Preschool; Cytosol; Dihydrotestosterone; Estrenes; Female; Genitalia; Hirsutism; Hot Temperature; Humans; Infant; Male; Metribolone; Middle Aged; Progesterone; Radioligand Assay; Receptors, Androgen; Receptors, Steroid; Skin

Topics: Androgen-Insensitivity Syndrome; Animals; Binding, Competitive; Cytosol; DNA; Estrenes; Hormones; Male; Metribolone; Muscles; Rats; Receptors, Androgen; Receptors, Steroid