Paul-Martin Holterhus

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.

Deciding on Gender in Children with Intersex Conditions

Biologic factors such as genetic and hormonal influences contribute to gender identity, gender role behavior, and sexual orientation in humans, but this relationship is considerably modified by psychologic, social, and cultural factors. The recognition of biologically determined conditions leading to incongruity of genetically determined sex, somatic phenotype, and gender identity has led to growing interest in gender role development and gender identity in individuals with intersex conditions. Sex assignment of children with ambiguous genitalia remains a difficult decision for the families involved and subject to controversial discussion among professionals and self-help groups. Although systematic empirical data on outcomes of functioning and health-related quality of life are sparse, anecdotal evidence from case series and individual patients about their experiences in healthcare suggests traumatic experiences in some. This article reviews the earlier ‘optimal gender policy’ as well as the more recent ‘full consent policy’ and reviews published data on both surgical and psychosocial outcomes. The professional debate on deciding on sex assignment in children with intersex conditions is embedded in a much wider public discourse on gender as a social construction. Given that the empirical basis of our knowledge of the causes, treatment options, long-term outcomes, and patient preferences is insufficient, we suggest preliminary recommendations based on clinical experience, study of the literature, and interviews with affected individuals.

Anabolic steroids, testosterone-precursors and virilizing androgens induce distinct activation profiles of androgen responsive promoter constructs☆

Different androgens, e.g. virilizing androgens such as testosterone and its precursors as well as synthetic anabolic steroids, respectively, induce diverse biological effects. The molecular basis for this variety in biological actions, however, is not well understood. We hypothesized that this variability of actions may be due to steroid-specific target gene expression profiles following androgen receptor (AR)-activation. Therefore, we investigated androgen receptor dependent transactivation of three structurally different androgen responsive promoter constructs ((ARE)(2)TATA-luc, MMTV-luc, GRE-OCT-luc) in co-transfected Chinese hamster ovary (CHO)-cells as an artificial model simulating different natural target genes. Three virilizing androgens (dihydrotestosterone, testosterone, methyltrienolone), three anabolic steroids (oxandrolone, stanozolol, nandrolone) and two testosterone-precursors of gonadal and adrenal origin (dehydroepiandrosterone, androstenedione) were used as ligands (0.001-100 nM). All steroids proved to be potent activators of the AR. Remarkably, anabolic steroids and testosterone-precursors showed characteristic promoter activation profiles distinct from virilizing androgens with significantly lower (ARE)(2)TATA-luc activation. Hierarchical clustering based on similarity of activation profiles lead to a dendrogram with two major branches: first virilizing androgens, and second anabolics/testosterone-precursors. We conclude that steroid-specific differences in gene transcription profiles due to androgen receptor activation could contribute to differences in biological actions of androgens.

Clinical, Endocrine, and Molecular Genetic Findings in Patients with 17β-Hydroxysteroid Dehydrogenase Deficiency

Mutations in the 17β-hydroxysteroid dehydrogenase (17β-HSD) type 3 gene are associated with the clinical findings of 17β-HSD deficiency. We investigated 5 patients of German descent with 46,XY karyotype and predominantly female phenotype. Androstenedione (A) and testosterone (T) levels in serum were determined before and after stimulation with human chorionic gonadotropin. DNA analysis of the whole coding region of the 17β-HSD type 3 gene was performed by PCR, single-strand conformation analysis, and direct sequencing. In all patients we found highly variable A and T levels before and after stimulation. However, the A-to-T ratio was abnormal in all cases suggestive of 17β-HSD deficiency. Molecular genetic analysis revealed mutations in all patients. We conclude that A and T levels may be highly variable in patients with 17β-HSD deficiency. Molecular genetic analysis of the 17β-HSD gene may support the diagnosis of this disorder.

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.

Complete androgen insensitivity caused by a splice donor site mutation in intron 2 of the human androgen receptor gene resulting in an exon 2-lacking transcript with premature stop-codon and reduced expression

Various mutations within the human androgen receptor gene have been documented to cause defective sexual differentiation in karyotypic male individuals. In this study, we report a previously undescribed point mutation at the donor splice-site of the second intron of the androgen receptor gene in a patient with a completely female phenotype. The sequence alteration was detected by single-strand-conformation-analysis-PCR and genomic sequencing. Applying competitive reverse transcribed PCR, cDNA sequencing and Western blotting, we could demonstrate considerable aberrations of structure and concentration of the transcript and its translation product in the patients fibroblasts from the genital region. (1) In the transcript, exon 1 and 3 are directly linked to each other, the complete second exon is skipped. The mRNA predictively suffers a codon frame-shift in exon 3 associated with a premature termination between codons 598 and 599, leading to a truncated androgen receptor protein lacking any in vivo function. (2) Steady-state concentration levels of transcript and protein are abnormally low. Our observations highlight the influence of exon-flanking intron sequences on proper expression and function of gene products.

The Basis of Gender Assignment in Disorders of Somatosexual Differentiation

Sex assignment of patients with disorders of somatosexual differentiation is a controversial topic. The aim is to enable the patient to develop a stable gender identity during childhood, adolescence and adulthood. Enormous advances have recently been made in our knowledge of the molecular mechanisms of sexual differentiation and it is understood that long-term outcome may depend on the underlying diagnosis. There is increasing evidence that genital development is dependent on the action of androgenic steroids; moreover, both androgens and oestrogens may have an impact on other developing organs including neuronal structures such as the brain. Long-term outcome studies on the various intersexuality disorders are desperately needed in order to establish a basis for evidence-based medicine regarding sex assignment and treatment options. Premature decisions leading to irreversible interventions before an accurate diagnosis has been established must be avoided.

Expression of two functionally different androgen receptors in a patient with androgen insensitivity

Abstract Recently, we demonstrated a previously unknown high rate of de novo mutations of the androgen receptor (AR) gene in androgen insensitivity syndrome (AIS) with some resulting in somatic mosaicism of mutant and wild type AR alleles. However, data on the genotype-phenotype relationship in the latter patients are sparse. We present here a 46,XY newborn with ambiguous genitalia carrying a mosaic of an 866 GTG (Val) → ATG (Met) mutation with the wild type AR gene. This mutation has usually been associated with complete AIS. Accordingly, we found markedly impaired transactivation due to the mutant Met866 AR. Essential information arose from Scatchard analysis of methyltrienolone binding on cultured genital skin fibroblasts. We demonstrated for the first time the expression of two functionally different ARs (Kd1: 5.58 nMu2009=u2009mutant, Kd2: 0.06u2009nMu2009=u2009wild type) in one AIS individual. This finding not only represents an important confirmation for the presence of the somatic mosaicism in the patient, it also indicates the most likely molecular mechanism responsible for the unexpectedly strong virilization of the patient: Androgen action through the wild type AR expressed by part of the somatic cells.nConclusions The present case clearly demonstrates the molecular mechanism by which somatic mosaicism of the androgen receptor gene can modulate in vivo androgen action. It underlines the importance of particular notice on somatic mosaicism in all androgen insensitivity syndrome patients carrying de novo mutations of the androgen receptor gene.