The neuronotoxicity of genes with expanded CAG repeats is most likely mediated by their respective polyglutamine (Gln)-expanded gene products. Gln- expanded portions of these products may be sufficient, or necessary, for pathogenesis. We tested whether a Gln-expanded human androgen receptor (AR) is structurally altered, so that it allows for the proteolytic generation of a potentially pathogenic portion that may be resistant to further degradation. We found, in vitro , that a Gln-expanded AR is more proteolytically resistant than normal, and that it yields a distinct set of Gln-expanded fragments even after extended proteolysis in the presence of 2 M urea. Furthermore, COS cells transfected with CAG-expanded AR cDNA generate an aberrant, nuclear-associated 75 kDa derivative containing the Gln-expanded tract. They are also twice as likely to die by 24 h apoptotically than those transfected with normal AR cDNA. Our data support the notion that an unconventional derivative of the Gln- expanded AR is a component of the proximate motor neuronopathic agent in spinobulbar muscular atrophy. They also focus attention on two ways in which neuronotoxic derivatives may originate from various Gln-expanded proteins: (i) generation of an unusual derivative that is pathogenic de novo ; and (ii) the toxic accumulation of a normal derivative because of an inability to dispose of it.

Animals↗

PubMed1997

Poland sequence with dextrocardia: which comes first?

We report on two cases of Poland sequence (defect of the pectoralis major and hand on the same side) with dextrocardia, and review the literature on such patients. In all 16 reported cases, the Poland defect was on the left side, and associated with a rib defect, whereas most cases of Poland sequence involve the right side, and few have a rib defect. The dextrocardia appeared to be a dextroposition, and was not associated with other cardiac defects, whereas isolated dextrocardias (without situs inversus) frequently are. These observations suggest that the dextrocardia associated with Poland sequence is usually secondary to it.

Dextrocardia↗

PubMed1997

The androgen receptor gene mutations database.

The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 212 to 272. We have expanded the database: (i) by adding a large amount of new data on somatic mutations in prostatic cancer tissue; (ii) by defining a new constitutional phenotype, mild androgen insensitivity (MAI); (iii) by placing additional relevant information on an internet site (http://www.mcgill.ca/androgendb/ ). The database has allowed us to examine the contribution of CpG sites to the multiplicity of reports of the same mutation in different families. The database is also available from EMBL (ftp.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker Pro or Word file (MC33@musica,mcgill.ca)

Computer Communication Networks↗

PubMed1997

Codon-usage variants in the polymorphic (GGN)n trinucleotide repeat of the human androgen receptor gene.

The human androgen receptor gene (hAR) has a long, polymorphic trinucleotide (GGN; glycine)n repeat in the 3' portion of its first exon, with n = 10-31. Owing to technical difficulties that have precluded routine sequencing of this region, it is widely unknown that N represents T, G or C, and that the usual sense codon sequence of the GGN tract is (GGT)3GGG(GGT)2(GGC)4-25. Furthermore, on 4 of 61 X chromosomes, we observed that the internal GGT sequence was present three or four times instead of twice. Strikingly, each of the three alleles with an internal (GGT)3, and only these three, also had a (GGC)20 repeat. The size or composition of a (GGN)n repeat was not correlated with the length of the accompanying (CAG)nCAA repeat in the 5' portion of exon one. Hence, codon-usage variants of the GGN tract may be used to seek associations with particular diseases, as diagnostic aids in families with androgen insensitivity whose AR mutations have not yet been identified, or as internal controls for observations on intergenerational contractions or expansions of the (CAG)nCAA tract in a given hAR allele.

Alleles↗

PubMed1997

Androgen insensitivity with mental retardation: a contiguous gene syndrome?

We present data to suggest the existence of a mental retardation (MR) locus at Xq11.2-q12 between DXS1 and DXS905, identified in two subjects with complete androgen insensitivity syndrome (CAIS) and MR. Androgen insensitivity syndrome is a disorder of male sexual differentiation caused by a defect in the androgen receptor (AR) gene (Xq11-q12). Two subjects with CAIS resulting from a complete deletion of the AR gene have previously been reported, one of whom also has MR. We have identified another mentally retarded person with a complete deletion of the AR gene. The deletion in the two patients with CAIS and MR extends past the AR gene and includes several marker loci both proximal and distal to the AR gene, the limits of the deletions being DXS1 and DXS905. The deletions in the CAIS patients who do not have MR do not include any of the markers outside the AR gene itself. These data suggest that located close to the AR gene is a gene which is implicated in non-specific MR.

Androgens↗

PubMed1997

Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation, impaired sperm production, and male infertility.

The X-linked androgen receptor (AR) gene contains two polymorphic trinucleotide repeat segments that code for polyglutamine and polyglycine tracts in the N-terminal trans-activation domain of the AR protein. Changes in the lengths of these polymorphic repeat segments have been associated with increased risk of prostate cancer, an androgen-dependent tumor. Expansion of the polyglutamine tract causes a rare neuromuscular disease, spinal bulbar muscular atrophy, that is associated with low virilization, reduced sperm production, testicular atrophy, and infertility. As spermatogenesis is exquisitely androgen dependent, it is plausible that changes in these two repeat segments could have a role in some cases of male infertility associated with impaired spermatogenesis. To test this hypothesis, we examined the lengths of the polyglutamine and polyglycine repeats in 153 patients with defective sperm production and compared them to 72 normal controls of proven fertility. There was no significant association between the polyglycine tract and infertility. However, patients with 28 or more glutamines (Gln) in their AR had more than 4-fold (95% confidence interval, 4.9-3.2) increased risk of impaired spermatogenesis, and the more severe the spermatogenic defect, the higher the proportion of patients with a longer Gln repeat. Concordantly, the risk of defective spermatogenesis was halved when the polyglutamine tract was short (< or = 23 Gln). Whole cell transfection experiments using AR constructs harboring 15, 20, and 31 Gln repeats and a luciferase reporter gene with an androgen response element promoter confirmed an inverse relationship between Gln number and trans-regulatory activity. Immunoblot analyses indicated that the reduced androgenicity of the AR was unlikely to be due to a change in AR protein content. The data indicate a direct relation between length of the AR polyglutamine tract and the risk of defective spermatogenesis that is attributable to the decreased functional competence of AR with longer glutamine tracts.

Blotting, Western↗

PubMed1997

Mutant and wild-type androgen receptors exhibit cross-talk on androgen-, glucocorticoid-, and progesterone-mediated transcription.

Androgen, glucocorticoid, and progesterone receptors (ARs, GRs, and PRs) often can regulate transcription via composite hormone response elements in target genes. We have used artificial and natural mutant ARs from patients with androgen resistance to study their effects on dominant negative activity on wild type AR, GR, and PR function on mouse mammary tumor virus (MMTV) and tyrosine aminotransferase (TAT) promoters. Artificial ARs that contained internal deletions within the amino-terminal region had minimal transcriptional activity but blocked ligand-mediated transcription by wild type AR. Mutants containing deletions of the DNA-binding and ligand-binding domains had minimal or weak dominant negative activity. We then tested the ability of wild type and mutant ARs to modulate GR- and PR-mediated transcriptional activity. The amino-terminal deletion mutants exerted dominant negative effects on GR- and PR-mediated activity, both in the absence and presence of testosterone. Surprisingly, wild type AR, which had approximately 20% of the maximal transcriptional activity of GR on the MMTV promoter, also had dominant negative activity on dexamethasone-regulated transcription mediated by GR. This dominant negative activity likely involves DNA binding because a point mutation in the DNA-binding domain abrogated such activity of an amino-terminal deletion mutant. Additionally, natural human AR mutants from patients with androgen resistance, which do not bind either DNA or ligand, did not block dexamethasone-mediated transcription. In summary, these studies suggest that mutant and wild type ARs can display dominant negative activity on other steroid hormone receptors that bind to a composite hormone response element This cross-regulation may be important in regulating maximal transcriptional activity in tissues where these receptors are coexpressed and may contribute to the phenotype of patients with steroid hormone resistance.

Androgens↗

PubMed1996

The androgen receptor gene mutations database.

The current version of the androgen receptor (AR) gene mutations database is described. We have added (if available) data on the androgen binding phenotype of the mutant AR, the clinical phenotype of the affected persons, the family history and whether the pathogenicity of a mutation has been proven. Exonic mutations are now listed in 5'-->3' sequence regardless of type and single base pair changes are presented in codon context. Splice site and intronic mutations are listed separately. The database has allowed us to substantiate and amplify the observation of mutational hot spots within exons encoding the AR androgen binding domain. The database is available from EML (ftp://www.ebi.ac.uk/pub/databases/androgen) or as a Macintosh Filemaker file (MC33@musica.mcgill.ca).

Androgens↗

PubMed1995

Characterization of alternative amino acid substitutions at arginine 830 of the androgen receptor that cause complete androgen insensitivity in three families.

We have studied two different missense mutations at arginine-830 in exon 7 of the human androgen receptor (hAR) gene that cause complete androgen insensitivity (CAIS) in three families. These substitutions result from point mutations at nucleotide 2489: a G-->T transversion causes Arg830Leu and a G-->A transition causes Arg830Gln. Genital skin fibroblasts of the patients have negligible androgen-binding capacity. The mutations were recreated in an hAR cDNA expression vector that was transiently transfected into COS-1 cells. Both mutant androgen receptors have increased dissociation rate constants and apparent equilibrium rate constants when measured with 5 alpha-dihydrotestosterone or the synthetic, nonmetabolizable androgens, mibolerone or methyltrienolone. The mutant androgen-binding activities share a distinctive thermal misbehavior. At 37 degrees C R830Q and R830L are about 40% and 10% of normal, respectively. At 22 degrees C both mutants gain androgen binding while the normal decreases by 20%; for R830Q the augmented value approaches 60% of the normal. During prolonged 18 h incubation at 37 degrees C, androgen binding of the normal AR is stable while that of both mutants decreases by at least 85%. Both mutants have a very reduced ability to transactivate a cotransfected androgen-responsive reporter gene, but R830Q is better than R830L. We conclude that arginine-830 is important for A-R complex stability, and that its replacement by glutamine or leucine yields distinctive functional aberrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Evidence for a repressive function of the long polyglutamine tract in the human androgen receptor: possible pathogenetic relevance for the (CAG)n-expanded neuronopathies.

We have reported that polyglutamine (polyGln)-expanded human androgen receptors (hAR) have reduced transactivational competence in transfected cells. We presumed that maximal hAR transactivation requires a normal-size polyGln tract. Here we report, however, that hAR transactivity and polyGln-tract length are related inversely: n = 0 > 12 > 20 > 40 > 50. Thus, a normal-size polyGln tract represses the transactivational competence of a polyGln-free hAR, and polyGln expansion increases that negative effect. This observation has pathogenetic implications for X-linked spinobular muscular atrophy (Kennedy syndrome), and possibly for the autosomal dominant central neuronopathies associated with (CAG)n expansion in the translated portion of four different genes.

Base Sequence↗

PubMed1995

Characterization of normal and point-mutated human androgen receptors expressed in the baculovirus system.

The baculovirus system is able to generate large amounts of a protein, permitting detailed analysis of structure-function relations. We have used this system to overexpress and characterize normal human androgen receptors (hAR) and mutant hARs from humans with complete or partial androgen insensitivity. Maximum specific binding of [3H]mibolerone (MB) in recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells varied from 15 to 40 pmol/mg protein, about 1000-fold higher than in genital skin fibroblasts, and peaked 48-72 h after infection. In contrast, Coomassie blue staining and Western blotting revealed maximum accumulation of 100-120 kDa hAR proteins 96 h post-infection. Normal and mutant hARs were specifically photo-affinity-labeled with [3H]methyltrienolone (MT), and had normal steroid-binding selectivity: the order of competition was androgen > estrogen > progestin > glucocorticoid. Normal hAR was phosphorylated in Sf9 cells, reacted with antibodies against phosphoserine and phosphothreonine after purification using testosterone-biotin, and transactivated a transfected androgen response element-luciferase reporter in infected Sf9 cells. Two mutant hARs had increased rates of dissociation from MB and MT that were in accord with the associated degree of clinical androgen insensitivity: complete, Pro903Ser > partial, Leu820Val; the third, Ile663Asn, was not abnormal. Our data extend the characterization of normal hAR produced by baculovirus-infected Sf9 cells, and demonstrate, for the first time, that point-mutated hARs so produced can display distinctive biochemical phenotypes.

Amino Acid Sequence↗

PubMed1994

Complete androgen insensitivity due to mutations in the probable alpha-helical segments of the DNA-binding domain in the human androgen receptor.

We describe different single-amino acid aberrations in the DNA-binding domain (DBD) of the human androgen receptor (hAR) in three families with complete androgen insensitivity. No additional alteration was found in the translated portion of each mutant gene. In one family, an in-frame 3 nt deletion removes codon 581-(or 582) and, thereby, one of two phenylalanines that invariably occupy adjacent positions in the N-terminal alpha-helical region of the DBD in the steroid/thyroid/vitamin D receptor superfamily. In the second, an in-frame 3 nt loss deletes Arg614, an invariant residue in the C-terminal alpha-helix of the DBD. In the third, a G-->A transition causes Arg614His. Following transient transfection of COS cells with each mutant AR plasmid, there is a normal concentration of specific androgen-binding activity that has a reduced ability to bind two types of androgen response element (ARE), and to transregulate an androgen-responsive human growth hormone reporter gene. In genital skin fibroblasts with delta Phe581 or Arg614His, androgen-binding, AR protein and AR mRNA are markedly reduced; in gonadal fibroblasts with delta Arg614, AR mRNA may be reduced. Our data substantiate the primary contributions of Phe581 and Arg614 to normal hAR-ARE binding, and expose important secondary effects of the mutations affecting each residue.

Amino Acid Sequence↗

PubMed1994

Substitution of arginine-839 by cysteine or histidine in the androgen receptor causes different receptor phenotypes in cultured cells and coordinate degrees of clinical androgen resistance.

We aim to correlate point mutations in the androgen receptor gene with receptor phenotypes and with clinical phenotypes of androgen resistance. In two families, the external genitalia were predominantly female at birth, and sex-of-rearing has been female. Their androgen receptor mutation changed arginine-839 to histidine. In a third family, the external genitalia were predominantly male at birth, and sex-of-rearing has been male: their codon 839 has mutated to cysteine. In genital skin fibroblasts, both mutant receptors have a normal androgen-binding capacity, but they differ in selected indices of decreased affinity for 5 alpha-dihydrotestosterone or two synthetic androgens. In transiently cotransfected androgen-treated COS-1 cells, both mutant receptors transactivate a reporter gene subnormally. The His-839 mutant is less active than its partner, primarily because its androgen-binding activity is more unstable during prolonged exposure to androgen. Adoption of a nonbinding state explains a part of this instability. In four other steroid receptors, another dibasic amino acid, lysine, occupies the position of arginine-839 in the androgen receptor. Androgen receptors with histidine or cysteine at position 839 are distinctively dysfunctional and appear to cause different clinical degrees of androgen resistance.

Adult↗

PubMed1994

The androgen receptor gene mutations database.

The androgen receptor gene mutations database is a comprehensive listing of mutations published in journals and meetings proceedings. The majority of mutations are point mutations identified in patients with androgen insensitivity syndrome. Information is included regarding the phenotype, the nature and location of the mutations, as well as the effects of the mutations on the androgen binding activity of the receptor. The current version of the database contains 149 entries, of which 114 are unique mutations. The database is available from EMBL (NetServ@EMBL-Heidelberg.DE) or as a Macintosh Filemaker file (mc33001@musica.mcgill.ca).

Databases, Factual↗