The molecular basis of androgen insensitivity.

Androgen action is mediated in the peripheral target cell via the androgen receptor (AR). The AR is a nuclear transcription factor, combining a DNA-binding and a hormone-binding domain with a large transactivation unit. Androgen insensitivity syndrome (AIS) as the clinical entity of defective androgen action with variable phenotypes in 46,XY patients is caused by mutations of the X-chromosomal AR gene. Most variations in the AR gene are point mutations inhibiting either hormone or DNA binding. However, even within the same family, the phenotype for a given mutation can vary widely. Only few influential factors have been identified for the phenotypic diversity. For mutations affecting hormone binding, ligand concentration variability during fetal life may be an important influence on residual androgen action. A second factor is the occurrence of postzygotic de novo mutations, which are present at a high rate in single-case families. These somatic mutations lead to expression of both mutant and wild-type AR in a single patient and thus allow androgen action despite a deleterious mutation of the AR gene. Third, residual androgen response may be mediated by additional transcripts of the AR gene which are present in several cell types and can be affected in a different pattern by splice-site mutations. Whether differential expression of AR-interacting proteins has an influence on phenotype has not yet been proven. Moreover, little is known about the regulation of AR-dependent genes. Their identification is needed to understand post-AR action and, hence, androgenic control of sexual differentiation and maturation.

Oxygenation and lung morphology in a rabbit pediatric ARDS- model under high peak pressure ventilation plus nitric oxide and surfactant compared with veno-venous ECMO.

UNLABELLED: The aim of the study is to investigate which of two treatment options of saline lavage induced ARDS in rabbits is better in terms of oxygenation and prevention of barotrauma: combined high peak pressure ventilation with surfactant administration and inhaled nitric oxide or veno-venous ECMO combined with low peak inspiratory pressure ventilation. MATERIALS AND METHODS: After saline lavage (10 cc/kg repeated as long as foamy retrieval was observed) two combined therapeutic strategies were examined: ventilation with high inspiratory pressures (35 cm H2O) with additional exogenous surfactant administration (100 mg/kg) and inhaled nitric oxide (10 PPM) (n=5, group 1) and low inspiratory pressure (20 cm H2O) ventilation under veno-venous ECMO support (n=5, group 2). The FiO2 was maintained at 1.0 in both groups. The paO2/FiO2 ratio was calculated in 30 minute intervals for 4 hours. After that the animals were sacrificed and the lungs examined macro- and microscopically. Aeration was described in a semiquantitative method using the alveolar expansion index. Oxygenation in group 1 was significantly better than in group 2, it increased significantly after surfactant but not after additional nitric oxide administration. However, the lungs in group 1 showed severe signs of baro/ergotrauma (Hyaline membranes, air leaks, infiltration of polymorphonuclear (PMN) granulocytes and macrophages, break down of alveolar capillary membranes) after 4 hrs of combined therapy, whereas the lungs in group 2 appeared normal. Adding surfactant and NO to a high tidal volume ventilation improved oxygenation, but did not prevent baro/ergotrauma. Ventilation with low inspiratory pressures combined with ECMO caused little baro/ergotrauma but adequate oxygenation could not be achieved, probably due to anatomical features of the rabbit which do not allow appropriate blood flow within the ECMO-circuit.

Inherited and de novo androgen receptor gene mutations: investigation of single-case families.

OBJECTIVE: The objective of this study was to assess somatic and inherited androgen receptor gene mutations in families with only one affected individual. METHODS: Molecular genetic analysis of the androgen receptor gene in DNA derived from blood leukocytes from 30 families with single-strand conformation analysis, direct sequencing, and restriction fragment analysis was performed. RESULTS: In 22 families the mothers and all investigated grandmothers were heterozygous carriers. However, within the sisters and aunts, both heterozygous carriers and noncarriers were present. In eight families a de novo mutation was characterized. In three of these patients indication for somatic mosaicism was found. CONCLUSIONS: De novo mutations occur at a high rate within the androgen receptor gene (8 of 30 = 26.7%); a high proportion (3 of 8) arise after the zygote stage. Thus only direct analysis of the underlying mutation of the androgen receptor gene in the proband and his or her family can provide the basis for genetic counseling.

Physiology and pathophysiology of androgen action.

Knowledge of the physiology of male sexual differentiation and the clinical presentation of androgen insensitivity syndromes (AIS) has led to an increasing understanding of the mechanisms of androgen action. Androgens induce their specific response via the androgen receptor (AR), which in turn regulates the transcription of androgen-responsive target genes. The androgen-dependent development of male genital structures and the induction of the normal male phenotype depends on the presence of an intact AR. Structural alterations leading to malfunction of the AR are associated with variable inhibition of virilization despite normal or even supranormal serum levels of androgens. The mapping, cloning and sequencing of the AR gene have facilitated new insights into the study of androgen action. Functional investigation of the normal and the mutant AR in vivo as well as in vitro has led to the characterization of the distinct molecular steps involved in the normal androgen action pathways that are inhibited in the androgen insensitivity syndrome.

Functional assessment and clinical classification of androgen sensitivity in patients with mutations of the androgen receptor gene. German Collaborative Intersex Study Group.

UNLABELLED: In the genetic male, mutations of the androgen receptor (AR) gene cause phenotypes ranging from female to subfertile male. Binding assays on genital skin fibroblasts and DNA analysis alone provide incomplete information about receptor function. We used the sex hormone-binding globulin (SHBG) response to stanozolol as a measure of AR function and correlated the results with phenotypes which were classified according to the degree of defective masculinization. Of the 34 patients investigated, 9 had complete, and 14 had partial androgen insensitivity syndrome (AIS) with predominantly female, ambiguous, or predominantly male phenotype. Eleven subjects served as controls. Mutations were characterized using polymerase chain reaction-single strand conformation polymorphism analysis and direct DNA sequencing. DNA analysis revealed two major deletions, two minor defects leading to premature stop codons in exon 1, and 19 point mutations in the DNA- and hormone-binding domains of the AR gene. After stanozolol, SHBG remained unchanged in patients with complete AIS (102.0 +/- 3.8 [SE]%; range 92.4%-129% of the initial value). The SHBG decrease was diminished in partial AIS with predominantly female (83.8% +/- 1.7%; range 81.3%-87.0%), ambiguous (80.4% +/- 4.4%, range 68.4%-89.1%), and predominantly male (mean 65.9% +/- 4.9%, range 48.6%-80.8%) phenotypes, and normal in controls (51.4% +/- 2.1%, range 35.6%-62.1%). Differences between controls and each AIS group were statistically significant (P < 0.05 - < 0.0001). A close correlation was found between the degree of undermasculinization (AIS phenotype) and the SHBG response. CONCLUSIONS: The SHBG test provides functional information about the severity of the receptor defect in vivo and hence adds to the structural information provided by DNA analysis. It detects receptor defects due to mutations within the entire gene, including the DNA-binding domain, and is a rapid, simple, and cost effective procedure. It may provide useful information for the diagnosis and management of affected children.

The clinical and molecular spectrum of androgen insensitivity syndromes.

Androgen insensitivity syndromes (AIS) are due to end-organ resistance to androgenic steroids in males leading to defective virilization of the external genitalia. The phenotype encompasses a wide array of genital ambiguity and may range from completely female to undervirilized but unequivocally male with infertility. This disorder is caused by mutations of the androgen receptor and is an X-linked recessive trait. We have studied 47 patients with AIS and have characterized the underlying molecular abnormality in the androgen receptor gene. Twenty patients had complete AIS and twenty-seven had partial AIS. Of the latter, 11 were of predominantly female phenotypic appearance and gender was assigned accordingly, while 16 were raised as males. Within the group of complete AIS, two patients had gross deletions within the gene, one had a small deletion, and one had an insertion. In the other patients with complete AIS, as well as all individuals with partial AIS, single nucleotide substitutions within the coding region were detected, each leading to an amino acid alteration. Seven codons were involved in more than one mutation in different cases. In addition, in one patient with spinal and bulbar muscular atrophy, an elongation of a glutamine-repeat was characterized. We conclude that mutations in the androgen receptor gene may be present throughout the whole coding region. However, our study provides evidence that several mutational hot spots exist.

Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression.

Male sexual differentiation is a process that involves androgen action via the androgen receptor. Defects in the androgen receptor, many resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA from human neonatal genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequences were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP-2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Four of these clones again are similar to ETS from unknown genes. Three differential expressed sequences that appear to be upregulated in the presence of testosterone show significant homology to the cDNAs of L-plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression.

Serum interferon level and (2'-5')oligoadenylate synthetase activity in lymphocytes during clinical interferon application.

Determinations of (2'-5')oligoadenylate synthetase (OAS) in extracts of peripheral mononuclear cells were used to monitor clinical treatment by human leukocyte-derived alpha-interferon (IFN-alpha). The maximum activity of this enzyme was detected about 6 h after the maximum IFN activity in the patient's serum, its half-life being severalfold longer than that of circulating IFN. Thus, even some days after its clearance, IFN can be detected by means of this enzyme. Sixfold increases of the IFN dose were not able to further increase OAS activity induced by doses of about 5 X 10(4) units/kg body weight in adult persons. Intravenous administration of IFN seems not to be superior to its i.m. injection.