Androgen receptor genotyping in a large Australasian cohort with androgen insensitivity syndrome; identification of four novel mutations

We genotyped the androgen receptor (AR) gene in 31 Australasian patients with androgen insensitivity syndrome (AIS). The entire coding region of AR was examined including analysis of polymorphic CAG and GGN repeats in all patients. AR defects were found in 66.7% (6/9) of patients with complete AIS (CAIS) and 13.6% (3/22) of patients with partial AIS (PAIS). A novel deletion (N858delG) leading to a premature stop codon was found in CAIS patient P1. CAIS patient P2 has a novel deletion (N2676delGAGT) resulting in a stop at codon 787. These mutations would result in inactivation of AR protein. A novel insertion of a cysteine residue in the first zinc finger of the AR DNA-binding domain (N2045_2047dupCTG) was found in CAIS patient P3. PAIS patient P4 has a novel amino acid substitution (Arg760Ser) in the AR ligand binding domain, which may impair ligand binding. Five patients were found to have previously reported AR mutations and no mutations were identified in the remaining patients

Substitution of Ala564 in the first zinc cluster of the deoxyribonucleic acid (DNA)-binding domain of the androgen receptor by Asp, Asn, or Leu exerts differential effects on DNA binding

In the androgen receptor of a patient with androgen insensitivity, the alanine residue at position 564 in the first zinc cluster of the DNA-binding domain was substituted by aspartic acid. In other members of the steroid receptor family, either valine or alanine is present at the corresponding position, suggesting the importance of a neutral amino acid residue at this site. The mutant receptor was transcriptionally inactive, which corresponded to the absence of specific DNA binding in gel retardation assays, and its inactivity in a promoter interference assay. Two other receptor mutants with a mutation at this same position were created to study the role of position 564 in the human androgen receptor on DNA binding in more detail. Introduction of asparagine at position 564 resulted in transcription activation of a mouse mammary tumor virus promoter, although at a lower level compared with the wild-type receptor. Transcription activation of an (ARE)2-TATA promoter was low, and binding to different hormone response elements could not be visualized. The receptor with a leucine residue at position 564 was as active as the wild-type receptor on a mouse mammary tumor virus promoter and an (ARE)2-TATA promoter, but interacted differentially with several hormone response elements in a gel retardation assay. The results of the transcription activation and DNA binding studies could partially be predicted from three-dimensional modeling data. The phenotype of the patient was explained by the negative charge, introduced at position 564

The Human Glucocorticoid Receptor Isoform: EXPRESSION, BIOCHEMICAL PROPERTIES, AND PUTATIVE FUNCTION

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, hGRalpha and hGRbeta, which differ at their carboxyl termini. The hGRalpha isoform conveys endocrine information to target tissues by altering patterns of gene expression in a hormone-dependent fashion. In contrast to hGRalpha, very little is known about the hGRbeta splice variant. Using hGRalpha- and hGRbeta-specific riboprobes on human multiple tissue Northern blots, we show that the hGRbeta message has a widespread tissue distribution. We also prove by reverse transcriptase-polymerase chain reaction that the alternative splicing event underlying the formation of the hGRbeta message occurs in these tissues. Because the hGRbeta protein differs from hGRalpha at the extreme COOH terminus, we investigated several of the biochemical properties of hGRbeta expressed in transfected cells. hGRbeta does not bind the glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist RU38486 in vivo. Moreover, in contrast to hGRalpha, hGRbeta is located primarily in the nucleus of transfected cells independent of hormone administration. Finally, in the absence of hGRalpha, hGRbeta is transcriptionally inactive on a glucocorticoid-responsive enhancer. However, when both isoforms are expressed in the same cell, hGRbeta inhibits the hormone-induced, hGRalpha-mediated stimulation of gene expression. Thus, hGRbeta potentially functions as a dominant negative inhibitor of hGRalpha activity

Predicting outcome of acute kidney transplant rejection using

Acute kidney transplant rejection is an important risk factors for adverse graft outcome. Once diagnosed, it remains difficult to predict the risk of graft loss and the response to anti-rejection treatment. The aim of this thesis was to identify biomarkers during acute rejection, which predict the response to corticosteroid therapy and renal allograft survival. We demonstrated that steroid resistance is a multifactorial condition, in which both immunological and non-immunological factors are involved. Response to steroid therapy correlates with the expression level and characteristics of allograft infiltrating T cells and macrophages, indicating that steroid resistance resides in specific cell populations and is not a feature of all lymphocytes. In addition, zinc regulation plays a role in the response to corticosteroids. Increased expression of zinc-regulating molecules may diminish the zinc-requiring anti-inflammatory effects of corticosteroids. Therefore, kidney transplant recipients may benefit from additional zinc intake to optimize steroid signaling. Furthermore, we demonstrated that a multivariate prediction model, containing biomarkers related to different aspects of corticosteroid signaling, offers the best prognostic value for assessing steroid response. Finally, we demonstrated that determination of S100A8 and S100A9 expression levels in renal allograft tissue can be used for assessing the risk of renal allograft loss over time.UBL - phd migration 201

Modelling altered Glucocorticoid Sensitivity: From HPA axis to metabolic abnormalities in mice and humans

The primary determinants of tissue glucocorticoid action are glucocorticoid receptor (GR) density and intracellular levels of ligand, the latter determined both by activity of the hypothalamic-pituitary-adrenal (HPA) axis and cellular activity of 11beta- hydroxysteroid dehydrogenase (11beta-HSD) enzymes that interconvert active 11- hydroxy (corticosterone, cortisol) and inactive 11-keto (11-dehydrocorticosterone, cortisone) glucocorticoids. Here, the contribution of GR density and ligand levels in determining body composition and metabolic phenotype have been investigated in mice and in humans. Genetic evidence in humans implicates variations in the GR gene in the regulation of the HPA axis as well as the control of body fat distribution, metabolic parameters and blood pressure. Although GR deficient mouse models have been previously generated (with homozygous nulls dying at birth), the effects of altered GR density upon fat distribution and blood pressure have not been described. This study addresses the relationship between GR density and metabolic parameters, including body fat distribution, insulin resistance and hypertension. A novel line of mice harbouring a null mutation in the GR gene (GR+/-) was generated from an ES cell line in which a beta-galactosidase-neomycin phosphotransferase (beta geo) reporter cassette was fused with GR. The resulting fusion protein lacks part of the DNA binding domain and the entire ligand binding domain and is transcriptionally inactive. In addition, the beta-galactosidase enzyme activity “reports” activity of the GR gene promoter. GR-/- mice are present in a normal Mendelian ratio before birth. Intriguingly, 1 (of 36/146 expected if null allele not lethal) survived to adulthood suggesting this might be a hypomorphic rather than a null allele. Heterozygous 15 (GR+/-) mice showed 40-45% reductions in GR mRNA levels in the hippocampus, paraventricular nucleus of the hypothalamus, pituitary gland and adipose tissue, 30% in liver, 56% in muscle and 67% in adrenals. X-gal staining of GR+/- brain sections showed that GR-beta gal is present throughout, mirroring GR mRNA expression. Adult GR+/- mice had larger adrenals, higher evening plasma corticosterone levels and greater corticosterone responses following 10 minute restraint suggesting a hyperactive HPA axis. Compared to GR+/+ littermates, GR+/- mice had similar body weight gain on normal chow or high fat diet, with unaltered fat depot (inguinal, epididymal, mesenteric) weights and similar glucose and insulin tolerance. However, GR+/ - mice had higher (10%) systolic blood pressure, associated with activation of the renin-angiotensin system. Thus GR haploinsufficiency in mice causes increased blood pressure and accords with data associating GR polymorphisms with hypertension in humans. The role of altered GC sensitivity was also investigated in a mouse model of HPA axis hypoactivity pro-opiomelanocortin null (POMC) mice. POMC-null mice are obese due to central melanocortin deficiency. In contrast to most rodent models of obesity, POMC-null mice are also glucocorticoid deficient due to ACTH deficiency. Previous data have shown that glucocorticoid replacement in POMC-null mice exaggerated hyperphagia, obesity and insulin resistance and caused hypertension. Here, the contribution of peripheral glucocorticoid sensitivity was investigated. POMC-null mice have increased liver and retroperitoneal fat GR mRNA levels but, specifically in adipose tissue, decreased levels of mRNA encoding 11beta-HSD1, a reductase which regenerates active glucocorticoids, thus amplifying their action