Welzel et al. 2008

J Clin Endocrinol Metab. 2008 Apr;93(4):1418-25. Epub 2008 Feb 5.
Carboxyl-terminal mutations in 3beta-hydroxysteroid dehydrogenase type II cause severe salt-wasting congenital adrenal hyperplasia.

Welzel M, Wüstemann N, Simic-Schleicher G, Dörr HG, Schulze E, Shaikh G, Clayton P, Grötzinger J, Holterhus PM, Riepe FG.
Division of Pediatric Endocrinology, Department of Pediatrics, Christian-Albrechts Universität zu Kiel, 24105 Kiel, Germany.

INTRODUCTION: 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) deficiency is a rare cause of congenital adrenal hyperplasia caused by inactivating mutations in the HSD3B2 gene. Most mutations are located within domains regarded crucial for enzyme function. The function of the C terminus of the 3beta-HSD protein is not known. OBJECTIVE: We studied the functional consequences of three novel C-terminal mutations in the 3beta-HSD protein (p.P341L, p.R335X and p.W355X), detected in unrelated 46,XY neonates with classical 3beta-HSD type II deficiency showing different degrees of under-virilization. METHODS AND RESULTS: In vitro expression of the two truncated mutant proteins yielded absent conversion of pregnenolone and dehydroepiandrosterone (DHEA), whereas the missense mutation p.P341L showed a residual DHEA conversion of 6% of wild-type activity. Additional analysis of p.P341L, including three-dimensional protein modeling, revealed that the mutant's inactivity predominantly originates from a putative structural alteration of the 3beta-HSD protein and is further aggravated by increased protein degradation. The stop mutations cause truncated proteins missing the final G-helix that abolishes enzymatic activity irrespective of an augmented protein degradation. Genital appearance did not correlate with the mutants' residual in vitro activity. CONCLUSIONS: Three novel C-terminal mutants of the HSD3B2 gene are responsible for classical 3beta-HSD deficiency. The C terminus is essential for the enzymatic activity. However, more studies are needed to clarify the exact function of this part of the protein. Our results indicate that the genital phenotype in 3beta-HSD deficiency cannot be predicted from in vitro 3beta-HSD function alone.

Kulle et al. 2010

J Clin Endocrinol Metab. 2010 Mar 3. [Epub ahead of print]
A Novel Ultrapressure Liquid Chromatography Tandem Mass Spectrometry Method for the Simultaneous Determination of Androstenedione, Testosterone, and Dihydrotestosterone in Pediatric Blood Samples: Age- and Sex-Specific Reference Data.

Kulle AE, Riepe FG, Melchior D, Hiort O, Holterhus PM.
Department of Pediatrics (A.E.K., F.G.R., P.M.H.), Division of Pediatric Endocrinology and Diabetes, and Department of Urology and Pediatric Urology (D.M.), Christian-Albrechts University of Kiel, 24105 Kiel, Germany; and Department of Pediatrics (O.H.), University of Lübeck, 23538 Lübeck, Germany.

Context: Current immunoassays for analysis of plasma androgens in children have several limitations due to antibody-specific variations of data and normal ranges. Mass spectrometry-based methods are available for individual steroids but need complex sample preparation and report only fragmentary reference data for the pediatric population. Objective: Our objective was to develop a state of the art sensitive and specific tandem mass spectrometry method for high-throughput simultaneous determination of plasma concentrations of androstenedione (A), testosterone (T), and dihydrotestosterone (DHT) and to report age-, sex-, and pubertal stage-specific reference levels for these steroids in children aged 0-18 yr. Subjects and Methods: Plasma (100 mul) was mixed with internal standard and extracted by solid-phase extraction. Androgens were measured by ultrapressure liquid chromatography tandem mass spectrometry. Samples of 138 boys and 131 girls with neither signs of endocrine nor systemic disease were considered for the generation of reference data. The following age groups were used: less than 1 wk, 2 wk to 2 months, 3-5 months, 6-11 months, 1-3 yr, 4-6 yr, 7-9 yr, 10-12 yr, 13-15 yr, and over 16 yr. Results: Lower quantification limit was 2.9 ng/dl (0.1 nmol/liter) for A, T, and DHT. No relevant interference with other steroids was detected. Reference data for A, T, and DHT are reported as functions of age, sex, pubertal maturation, and testicular volume. Conclusion: Simplicity, velocity, sensitivity, specificity, and the availability of pediatric reference data allow application of our new method in clinical routine as well as in research settings.

Bens et al. 2010

J Clin Endocrinol Metab. 2010 Mar;95(3):1301-8. Epub 2010 Jan 15.
Congenital Lipoid Adrenal Hyperplasia: Functional Characterization of Three Novel Mutations in the STAR Gene.

Bens S, Mohn A, Yüksel B, Kulle AE, Michalek M, Chiarelli F, Nuri Ozbek M, Leuschner I, Grötzinger J, Holterhus PM, Riepe FG.
Division of Pediatric Endocrinology, Department of Pediatrics, University Hospital Schleswig- Holstein, Schwanenweg 20, D-24105 Kiel, Germany.

Context: The steroidogenic acute regulatory protein (StAR) has been shown to be essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause the typical clinical picture of congenital lipoid adrenal hyperplasia. Objective: The objective of the investigation was to study the functional and structural consequences of three novel StAR mutations (p.N148K in an Italian patient; p.P129fs and p.Q128R in a Turkish patient). Methods and Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.N148K mutant, the combined p.P129fs and p.Q128R mutant, and the p.P129fs mutant by itself. The p.Q128R mutant led to a higher cholesterol conversion than the wild-type StAR protein. As derived from three-dimensional protein modeling, the residue N148 is lining the ligand cavity of StAR. A positively charged lysine residue at position 148 disturbs the hydrophobic cluster formed by the alpha4-helix and the sterol binding pocket. The frame shift mutation p.P129fs truncates the StAR protein. Residue p.Q128 is situated at the surface of the molecule and is not part of any functionally characterized region of the protein. Conclusion: The mutations p.N148K and p.P129fs cause adrenal insufficiency in both cases and lead to a disorder of sex development with complete sex reversal in the 46, XY case. The mutation p.Q128R, which is not relevant for the patient's phenotype, is the first reported variant showing a gain of function. We speculate that the substitution of hydrophilic glutamine with basic arginine at the surface of the molecule may accelerate cholesterol transfer.

Appari et al. 2009

J Mol Med. 2009 Jun;87(6):623-32. Epub 2009 Mar 30.
Apolipoprotein D (APOD) is a putative biomarker of androgen receptor function in androgen insensitivity syndrome.

Appari M, Werner R, Wünsch L, Cario G, Demeter J, Hiort O, Riepe F, Brooks JD, Holterhus PM.
Department of Pediatrics, University-Hospital Schleswig-Holstein, Christian Albrechts University of Kiel, Germany.

Androgen insensitivity syndrome (AIS) is the most common cause of disorders of sex development usually caused by mutations in the androgen receptor (AR) gene. AIS is characterized by a poor genotype-phenotype correlation, and many patients with clinically presumed AIS do not seem to have mutations in the AR gene. We therefore aimed at identifying a biomarker enabling the assessment of the cellular function of the AR as a transcriptional activator. In the first step, we used complementary DNA (cDNA) microarrays for a genome-wide screen for androgen-regulated genes in two normal male primary scrotal skin fibroblast strains compared to two labia majora fibroblast strains from 46,XY females with complete AIS (CAIS). Apolipoprotein D (APOD) and two further transcripts were significantly upregulated by dihydrotestosterone (DHT) in scrotum fibroblasts, while CAIS labia majora cells were unresponsive. Microarray data were well correlated with quantitative real-time polymerase chain reaction (qRT-PCR; R = 0.93). Subsequently, we used qRT-PCR in independent new cell cultures and confirmed the significant DHT-dependent upregulation of APOD in five normal scrotum strains [13.5 +/- 8.2 (SD)-fold] compared with three CAIS strains (1.2 +/- 0.7-fold, p = 0.028; t test) and six partial androgen insensitivity syndrome strains (2 +/- 1.3-fold, p = 0.034; t test). Moreover, two different 17ss-hydroxysteroid dehydrogenase III deficiency labia majora strains showed APOD induction in the range of normal scrotum (9.96 +/- 1.4-fold), supporting AR specificity. Therefore, qRT-PCR of APOD messenger RNA transcription in primary cultures of labioscrotal skin fibroblasts is a promising tool for assessing AR function, potentially allowing a function-based diagnostic evaluation of AIS in the future.

Holterhus et al. 2007

BMC Genomics. 2007 Oct 18;8:376.
Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome.

Holterhus PM, Deppe U, Werner R, Richter-Unruh A, Bebermeier JH, Wünsch L, Krege S, Schweikert HU, Demeter J, Riepe F, Hiort O, Brooks JD.
Department of Pediatrics, University-Hospital Schleswig-Holstein, Campus Kiel, Schwanenweg 20, Kiel, Germany.

BACKGROUND: To better understand the molecular programs of normal and abnormal genital development, clear-cut definition of androgen-dependent gene expression patterns, without the influence of genotype (46, XX vs. 46, XY), is warranted. Previously, we have identified global gene expression profiles in genital-derived fibroblasts that differ between 46, XY males and 46, XY females with complete androgen insensitivity syndrome (CAIS) due to inactivating mutations of the androgen receptor (AR). While these differences could be due to cell autonomous changes in gene expression induced by androgen programming, recent work suggests they could also be influenced by the location from which the fibroblasts were harvested (topology). To minimize the influence of topology, we compared gene expression patterns of fibroblasts derived from identical urogenital anlagen: the scrotum in normally virilized 46, XY males and the labia majora from completely feminized 46, XY individuals with CAIS. RESULTS: 612 transcripts representing 440 unique genes differed significantly in expression levels between scrotum and CAIS labia majora, suggesting the effects of androgen programming. While some genes coincided with those we had identified previously (TBX3, IGFBP5, EGFR, CSPG2), a significant number did not, implying that topology had influenced gene expression in our previous experiments. Supervised clustering of gene expression data derived from a large set of fibroblast cultures from individuals with partial AIS revealed that the new, topology controlled data set better classified the specimens. CONCLUSION: Inactivating mutations of the AR, in themselves, appear to induce lasting changes in gene expression in cultured fibroblasts, independent of topology and genotype. Genes identified are likely to be relevant candidates to decipher androgen-dependent normal and abnormal genital development.

Holterhus et al. 2009

BMC Genomics. 2009 Jul 1;10:292.
Disorders of sex development expose transcriptional autonomy of genetic sex and androgen-programmed hormonal sex in human blood leukocytes.

Holterhus PM, Bebermeier JH, Werner R, Demeter J, Richter-Unruh A, Cario G, Appari M, Siebert R, Riepe F, Brooks JD, Hiort O.
Department of Pediatrics, Christian Albrechts University of Kiel, Germany.

BACKGROUND: Gender appears to be determined by independent programs controlled by the sex-chromosomes and by androgen-dependent programming during embryonic development. To enable experimental dissection of these components in the human, we performed genome-wide profiling of the transcriptomes of peripheral blood mononuclear cells (PBMC) in patients with rare defined "disorders of sex development" (DSD, e.g., 46, XY-females due to defective androgen biosynthesis) compared to normal 46, XY-males and 46, XX-females. RESULTS: A discrete set of transcripts was directly correlated with XY or XX genotypes in all individuals independent of male or female phenotype of the external genitalia. However, a significantly larger gene set in the PBMC only reflected the degree of external genital masculinization independent of the sex chromosomes and independent of concurrent post-natal sex steroid hormone levels. Consequently, the architecture of the transcriptional PBMC-"sexes" was either male, female or even "intersex" with a discordant alignment of the DSD individuals' genetic and hormonal sex signatures. CONCLUSION: A significant fraction of gene expression differences between males and females in the human appears to have its roots in early embryogenesis and is not only caused by sex chromosomes but also by long-term sex-specific hormonal programming due to presence or absence of androgen during the time of external genital masculinization. Genetic sex and the androgen milieu during embryonic development might therefore independently modulate functional traits, phenotype and diseases associated with male or female gender as well as with DSD conditions.

Riepe et al. 2009

Clin Endocrinol (Oxf). 2009 Feb;70(2):252-8.
Revealing a subclinical salt-losing phenotype in heterozygous carriers of the novel S562P mutation in the alpha subunit of the epithelial sodium channel.

Riepe FG, van Bemmelen MX, Cachat F, Plendl H, Gautschi I, Krone N, Holterhus PM, Theintz G, Schild L.
Division of Paediatric Endocrinology, Department of Paediatrics, Christian Albrechts Universität zu Kiel, Kiel, Germany.

OBJECTIVE: Pseudohypoaldosteronism type I (PHA1) is a rare inborn disease causing severe salt loss. Mutations in the three coding genes of the epithelial sodium channel (ENaC) are responsible for the systemic autosomal recessive form. So far, no phenotype has been reported in heterozygous carriers. PATIENTS: A consanguineous family from Somalia giving birth to a neonate suffering from PHA1 was studied including clinical and hormonal characteristics of the family, mutational analysis of the SCNN1A, SCNN1B, SCNN1G and CFTR genes and in vitro analysis of the functional consequences of a mutant ENaC channel. RESULTS: CFTR mutations have been excluded. SCNN1A gene analysis revealed a novel homozygous c.1684T > C mutation resulting in a S562P substitution in the alphaENaC protein of the patient. Functional analysis showed a significantly reduced S562P channel function compared to ENaC wild type. Protein synthesis and channel subunit assembly were not altered by the S562P mutation. Co-expression of mutant and wild-type channels revealed a dominant negative effect. In heterozygote carriers, sweat sodium and chloride concentrations were increased without additional hormonal or clinical phenotypes. CONCLUSION: Hence, the novel mutation S562P is causing systemic PHA1 in the homozygous state. A thorough clinical investigation of the heterozygote SCNN1A mutation carriers revealed increased sweat sodium and chloride levels consistent with a dominant effect of the mutant S562P allele. Whether this subclinical phenotype is of any consequence for the otherwise asymptomatic heterozygous carriers has to be elucidated