Sabine Hannema

Regulation of wolffian duct development

Wolffian ducts (WDs) are the embryonic structures that form the male internal genitalia. These ducts develop in both the male and female embryo. However, in the female they subsequently regress, whereas in the male they are stabilised by testosterone. The WDs then develop into separate but contiguous organs, the epididymis, vas deferens and seminal vesicles. Recently, considerable progress has been made in identifying genes that are involved in these different stages of development which is described in this review. In addition, WD development in (atypical forms of) cystic fibrosis and intersex disorders, such as the complete androgen insensitivity syndrome, 17β-hydroxysteroid dehydrogenase deficiency and LH-receptor defects, is discussed. The apparent increase in male reproductive tract disorders is briefly discussed from the perspective of the potential endocrine-disrupting effects of the numerous chemicals in the environment to which the developing male foetus can be exposed.

An activating mutation in the kinase homology domain of the natriuretic peptide receptor-2 causes extremely tall stature without skeletal deformities

BACKGROUND C-type natriuretic peptide (CNP)/natriuretic peptide receptor 2 (NPR2) signaling is essential for long bone growth. Enhanced CNP production caused by chromosomal translocations results in tall stature, a Marfanoid phenotype, and skeletal abnormalities. A similar phenotype was described in a family with an activating NPR2 mutation within the guanylyl cyclase domain. CASE Here we describe an extremely tall male without skeletal deformities, with a novel NPR2 mutation (p.Arg655Cys) located in the kinase homology domain. OBJECTIVES The objective of the study was to investigate the functional and structural effects of the NPR2 mutation. METHODS Guanylyl cyclase activities of wild-type vs mutant NPR2 were analyzed in transfected human embryonic kidney 293 cells and in skin fibroblasts. The former were also used to study possible interactions between both isoforms. Homology modeling was performed to understand the molecular impact of the mutation. RESULTS CNP-stimulated cGMP production by the mutant NPR2 was markedly increased in patient skin fibroblasts and transfected human embryonic kidney 293 cells. The stimulatory effects of ATP on CNP-dependent guanylyl cyclase activity were augmented, suggesting that this novel mutation enhances both the responsiveness of NPR2 to CNP and its allosteric modulation/stabilization by ATP. Coimmunoprecipitation showed that wild-type and mutant NPR2 can form stable heterodimers, suggesting a dominant-positive effect. In accordance with augmented endogenous receptor activity, plasma N-terminal pro-CNP (a marker of CNP production in tissues) was reduced in the proband. CONCLUSIONS We report the first activating mutation within the kinase homology domain of NPR2, resulting in extremely tall stature. Our observations emphasize the important role of this domain in the regulation of guanylyl cyclase activity and bone growth in response to CNP.

Novel Leptin Receptor Mutations Identified in Two Girls with Severe Obesity Are Associated with Increased Bone Mineral Density.

Background: Recessive mutations in the leptin receptor (LEPR) are a rare cause of hyperphagia and severe early-onset obesity. To date, the phenotype has only been described in 25 obese children, some of whom also had altered immune function, hypogonadotropic hypogonadism, reduced growth hormone secretion, hypothalamic hypothyroidism or reduced adult height. We provide a detailed description of the phenotype of 2 affected girls to add to this knowledge. Methods: Whole-exome sequencing and targeted sequencing were used to detect the LEPR mutations. RNA analysis was performed to assess the effect of splice-site mutations. Results: In 2 unrelated girls with severe obesity, three novel LEPR mutations were detected. Longitudinal growth data show normal childhood growth, and in the older girl, a normal adult height despite hypogonadotropic hypogonadism and the lack of an obvious pubertal growth spurt. Bone age is remarkably advanced in the younger (prepubertal) girl, and bone mineral density (BMD) is high in both girls, which might be directly or indirectly related to leptin resistance. Conclusion: The spectrum of clinical features of LEPR deficiency may be expanded with increased BMD. Future observations in LEPR-deficient subjects should help further unravel the role of leptin in human bone biology.

Androgen receptor gene mutations in androgen insensitivity syndrome cause distinct patterns of reduced activation of androgen-responsive promoter constructs

Assessment of quantitative impairment of reporter gene activation is an important strategy proving pathogenetic relevance of androgen receptor (AR)-gene mutations in androgen insensitivity syndrome (AIS). We hypothesized the additional existence of mutation-specific patterns of reduced target gene activation. Four AR-gene mutations causing AIS, L712F, M780I, R855H, and V866M, respectively, were recreated in an AR-expression plasmid. Activation of three structurally different androgen-dependent promoters (MMTV, (ARE)2TATA, and GRE-OCT) was measured in transfected CHO-cells in response to dihydrotestosterone (DHT), testosterone, androstenedione and stanozolol (S). V866M showed the lowest activity across all conditions. R855H exhibited strikingly high activation of MMTV in response to DHT. M780I showed markedly low activation of (ARE)2TATA by S. L712F demonstrated high activation of GRE-OCT. In essence, each mutation was characterized in this model by a specific pattern of reduced reporter gene activation. Our AR crystal structure analyses showed that L712 and M780 may cause distinct alterations of AR-ligand- and AR-coregulator interaction interfaces supporting the experimental observations. Our data support the hypothesis that mutations of the AR-gene in AIS induce mutation-specific patterns of reduced promoter activation in vitro. Considering the diversity of natural androgen-regulated promoters, mutation-specific differences of androgen response patterns may be of relevance in vivo and consequently may influence the AIS-phenotype. Assessment of transactivation patterns in vitro may be an interesting concept to extend functional description of AR-gene mutations in AIS.

Changes in gene expression during wolffian duct development

Background: Wolffian ducts (WDs) are the embryonic precursors of the male reproductive tract. Their development is induced by testosterone, which interacts with the androgen receptor (AR). The molecular pathways underlying androgen-dependent WD development are largely unknown. We aimed to identify AR target genes important in this process. Methods: RNA was isolated from rat WDs at E17.5 and E20.5. Affymetrix GeneChip expression arrays were used to identify transcripts up- or downregulated more than 2-fold. Regulation of seven transcripts was confirmed using quantitative PCR. Results: Transcripts from 76 known genes were regulated, including modulators of insulin-like growth factor and transforming growth factor-βsignalling. By controlling these modulators, androgens may indirectly affect growth factor signalling pathways important in epithelial–mesenchymal interactions and organ development. Caveolin-1, also upregulated, may play a role in modifying as well as mediating AR signalling. Differentiation of WD epithelium and smooth muscle, innervation and extracellular matrix synthesis were reflected in regulation of other transcripts. Several genes were previously suggested to be regulated by androgens or contained functional or putative androgen/glucocorticoid response elements, indicating they may be direct targets of androgen signalling. Conclusion: Our results suggest novel cohorts of signals that may contribute to androgen-dependent WD development and provide hypotheses that can be tested by future studies.

The Evaluation and Management of Tall Stature

Tall stature is a common reason for consultation of a paediatric endocrinologist. It is important to always consider underlying pathology. We propose a diagnostic flowchart based on five questions. (1) Does the child have tall stature? (2) Is there evidence of a syndrome? (3) Has there been growth acceleration? (4) Are there signs of puberty? (5) Does the child grow within the target height range? Diagnostic tests can then be ordered targeted to the suspected disorder. The Bayley-Pinneau and Tanner-Whitehouse methods are reasonably accurate in predicting adult height based on bone age in girls, but neither method performs well in boys. Tall stature is not a pathological condition and generally does not need treatment. However, adolescents with a strong treatment wish and their parents should be counselled on the effectivity and safety of available treatments including surgery and high-dose sex steroids. Surgical epiphysiodesis has the advantage that a reasonable height reduction can be achieved at a more advanced bone age, allowing a more accurate adult height prediction to base any treatment decision on. We feel that high-dose oestrogen treatment should no longer be used because of its association with reduced fecundity and imminent ovarian failure.

Management of Gonads in Adults with Androgen Insensitivity: An International Survey

Background: Complete and partial androgen insensitivity syndrome (CAIS, PAIS) are associated with an increased risk of gonadal germ cell cancer (GGCC). Recent guidelines recommend gonadectomy in women with CAIS in late adolescence. Nevertheless, many adult women prefer to retain their gonads. Aims: This study aims to explore attitudes towards gonadectomy in AIS in centres around the world, estimate the proportion of adults with retained gonads and/or who developed GGCC, and explore reasons for declining gonadectomy. Methods: A survey was performed among health care professionals who use the International DSD Registry (I-DSD). Results: Data were provided from 22 centres in 16 countries on 166 women (CAIS) and 26 men (PAIS). In CAIS, gonadectomy was recommended in early adulthood in 67% of centres; 19/166 (11.4%) women refused gonadectomy. Among 142 women who had gonadectomy, evidence of germ cell neoplasm in situ (GCNIS), the precursor of GGCC, was reported in 2 (1.4%) out of 8 from whom pathology results were formally provided. Nine out of 26 men with PAIS (34.6%) had retained gonads; 11% of centres recommended routine gonadectomy in PAIS. Conclusion: Although development of GGCC seems rare, gonadectomy after puberty is broadly recommended in CAIS; in PAIS this is more variable. Overall, our data reflect the need for evidence-based guidelines regarding prophylactic gonadectomy in AIS.