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Liquid-chromatography - tandem mass spectrometry (LC-MS/MS) is becoming the method of choice for clinical steroid analysis. In most instances, it has the advantage of higher sensitivity, better reproducibility and greater specificity than commercial immunoassay techniques. The method requires only minimal sample preparation and a small sample volume. Furthermore, it has the potential to analyze multiple steroids simultaneously. Modern instruments guarantee high throughput, allowing an affordable price for the individual assay. All this makes LC-MS/MS an attractive method for use in a clinical setting. Reliable reference ranges for the detected analytes are the pre-requisite for their clinical use. If these are available, LC-MS/MS can find application in congenital disorders of steroid metabolism, such as congenital adrenal hyperplasia, disorders of sex development and disorders of salt homeostasis, as well as in acquired disorders of steroid metabolism, such as primary aldosteronism, Cushing's disease, Addison's disease, and hyperandrogenemia, as well as in psychiatric disease states such as depression or anxiety disorders. The principles of LC-MS/MS for steroid measurement, the pros and cons of LC-MS/MS compared with conventional immunoassays and the possible applications in clinical routine, with a special focus on pediatric endocrinology needs, are discussed here.

Sex specific development in the human comprises irreversible sexual differentiation of the external genitalia during embryogenesis, sexual maturation of secondary sex characteristics during puberty (e.g., sex specific body proportions, pubertal voice change) and eventually sex specific development of extragenital tissues and organs, including the brain. The presence or absence of androgens acting via the androgen receptor plays a key role therein. At the single cell level, androgens cause reversible short term changes of gene transcription by activating the androgen receptor. From a developmental perspective, this often leads to irreversible long-term changes of anatomy (e.g., sex-specific differentiation of the external genitalia) and function (e.g., sex-specific play behavior in children). These observations are discussed in the context of recent genome-wide gene expression studies and first published experimental data at the epigenome level. In essence, there is evidence for a molecular androgen memory at both the transcriptome and the epigenome level.

We describe a 3.5 year old girl presenting with short stature, developmental delay, marked muscular hypotonia with ataxia, premature pubarche, and dysmorphic features. A 1.07-1.12Mb-sized de novo microdeletion of chromosome 19p13.11 is most likely the cause for the clinical phenotype. The patient did not show any abnormalities of the extremities which contrasts with the finding of one previously reported patient with an overlapping deletion presenting with split hand and foot malformation (SHFM). The remarkable difference is that in the previously described patient but not in the patient reported herein the genes EPS15L1 and CALR3 were deleted. As EPS15L1 has been associated with limb development previously, the presented case provides indirect evidence that this may be a new candidate gene for SHFM. A possible genotype-phenotype correlation is provided based on literature review and comparison of our patient to the previously reported patients with overlapping or partly overlapping copy number variations in 19p13.11., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Women and girls with complete androgen insensitivity syndrome (CAIS) invariably have a female typical core gender identity. In this case report, we describe the first case of male gender identity in a CAIS individual raised female leading to complete sex reassignment involving both androgen treatment and phalloplasty. CAIS was diagnosed at age 17, based on an unambiguously female phenotype, a 46,XY karyotype, and a 2660delT androgen receptor (AR) gene mutation, leading to a premature stop in codon 807. Bilateral gonadectomy was performed but a short period of estrogen treatment induced a negative emotional reaction and treatment was stopped. Since the age of 3, childhood-onset cross gender behavior had been noticed. After a period of psychotherapy, persisting male gender identity was confirmed. There was no psychiatric co-morbidity and there was an excellent real life experience. Testosterone substitution was started, however without inducing any of the desired secondary male characteristics. A subcutaneous mastectomy was performed and the patient received phalloplasty by left forearm free flap and scrotoplasty. Testosterone treatment was continued, without inducing virilization, and bone density remained normal. The patient qualifies as female-to-male transsexual and was treated according to the Standards of Care by the World Professional Association for Transgender Health with good outcome. However, we do not believe that female sex of rearing as a standard procedure should be questioned in CAIS. Our case challenges the role of a functional AR pathway in the development of male gender identity.

Male external genital differentiation is accompanied by implementation of a long-term, male-specific gene expression pattern indicating androgen programming in cultured genital fibroblasts. We hypothesized the existence of an epigenetic background contributing to this phenomenon. DNA methylation levels in 2 normal scrotal fibroblast strains from 46,XY males compared to 2 labia majora fibroblast strains from 46,XY females with complete androgen insensitivity syndrome (AIS) due to androgen receptor (AR) mutations were analyzed by Illumina GoldenGate methylation arrays®. Results were validated with pyrosequencing in labia majora fibroblast strains from fifteen 46,XY patients and compared to nine normal male scrotal fibroblast strains. HOXA5 showed a significantly higher methylation level in complete AIS. This finding was confirmed by bisulfite pyrosequencing of 14 CpG positions within the HOXA5 promoter in the same strains. Extension of the 2 groups revealed a constant low HOXA5 methylation pattern in the controls in contrast to a highly variable methylation pattern in the AIS patients. HOXA5 represents a candidate gene of androgen-mediated promoter methylation. The constantly low HOXA5 DNA methylation level of normal male scrotal fibroblast strains and the frequently high methylation levels in labia majora fibroblast strains in AIS indicate for the first time that androgen programming in sexual differentiation is not restricted to global gene transcription but also occurs at the epigenetic level., (2011 S. Karger AG, Basel.)

17α-Hydroxylase/17, 20-lyase deficiency (17OHD) is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90%) of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination.

Context: In 21-hydroxylase (CYP21A2) deficiency (21OHD), the level of in vitro enzymatic function allows for classification of mutation groups (null, A, B, C) and prediction of disease severity. However, genital virilization in affected females correlates only weakly with CYP21A2 mutation groups, suggesting the influence of genetic modifiers., Objective: The objective of the study was to investigate the influence of the polymorphic CAG and GGn repeats of the androgen receptor (AR) gene on the degree of genital virilization in 21OHD females., Design and Patients: Design of the study was the determination of CYP21A2 genotype, degree of genital virilization (Prader stage), and X-weighted biallelic mean of AR CAG and GGn repeat length in 205 females with 21OHD., Outcome Measurements: Correlation of AR CAG and GGn repeat lengths with Prader stages using nested stepwise logistic regression analysis was measured., Results: CYP21A2 mutation groups null and A showed significantly higher levels of genital virilization than groups B and C (P < 0.01). However, Prader stages varied considerably within mutation groups: null, Prader I-V (median IV); A, Prader I-V (median IV); B, Prader I-V (median III); C, 0-III (median I). Mean GGn repeat length of patients was not significantly associated with Prader stages, classified as low (0-I), intermediate (II-III), or severe (IV-V) (odds ratio per repeat: 0.98, 95% confidence interval 0.71-1.35). In contrast, patients with Prader 0-I showed a trend toward longer CAG repeats without reaching statistical significance (P = 0.07, odds ratio per repeat: 0.82, 95% confidence interval 0.65-1.02)., Conclusion: Neither CAG nor GGn repeat lengths are statistically significant modifiers of genital virilization in females with 21OHD.

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 microl) 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.

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.

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.

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.

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.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by mutations in SLC34A3, the gene encoding the renal sodium-phosphate co-transporter NaPi-IIc. Despite increased urinary calcium excretion, HHRH is typically not associated with kidney stones prior to treatment. However, here we describe two sisters, who displayed nephrolithiasis or nephrocalcinosis upon presentation. The index patient, II-4, presented with short stature, bone pain, and knee X-rays suggestive of mild rickets at age 8.5 years. Laboratory evaluation showed hypophosphatemia, elevated 1,25(OH) (2) vitamin D levels, and hypercalciuria, later also developing vitamin D deficiency. Her sister, II-6, had a low normal serum phosphorous level, biochemically vitamin D deficiency and no evidence for osteomalacia, but had undergone left nephro-ureterectomy at age 17 because of ureteral stricture secondary to renal calculi. Nucleotide sequence analysis of DNA from II-4 and II-6 revealed a homozygous missense mutation c.586G>A (p.G196R) in SLC34A3/NaPi-IIc. Ultrasonographic examinations prior to treatment showed grade I nephrocalcinosis for II-4, while II-6 had grade I-II nephrocalcinosis in her remaining kidney. Four siblings and the mother were heterozygous carriers of the mutation, but showed no biochemical abnormalities. With oral phosphate supplements, hypophosphatemia and hypercalciuria improved in both homozygous individuals. Renal calcifications that are presumably due to increased urinary calcium excretion can be the presenting finding in homozygous carriers of G196R in SLC34A3/NaPi-IIc, and some or all laboratory features of HHRH may be masked by vitamin D deficiency.

Background: Steroid 11-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Herein, we describe two novel CYP11B1 mutations (g659&#95;660dupTG, p.M92X; g.4817G>A, p.R453Q) found in a patient diagnosed with classic 11OHD, after presenting with borderline elevated 17-hydroxyprogesterone concentrations in CAH newborn screening., Methods: A novel CYP11B1 variant (p.R453Q) identified in a patient with classic 11OHD was characterized employing a COS7 cell assay and a computational three-dimensional CYP11B1 model., Results: The in vitro expression analysis revealed an almost complete loss of function of p.R453Q. This finding was consistent with the clinical presentation of classic 11OHD as the patient was compound heterozygous for p.R453Q and a nonsense mutation (p.M92X) on the other allele. Inserting the p.R453Q mutation into our CYP11B1 model provided two potential explanations for the almost complete loss of enzyme activity. Firstly, the heme coordination is most likely disturbed. A second possibility could be an altered interaction with the redox partner adrenodoxin., Conclusion: Results indicate that both novel mutations are disease-causing mutations. Proving the pathogenic effect of a missense sequence variation is of particular importance for clinical genetic counseling as this provides essential information on the prediction of recurrence risk and disease severity., (Copyright 2009 S. Karger AG, Basel.)

Background: Congenital adrenal hyperplasia is caused by insufficient adrenal steroid biosynthesis due to impaired steroidogenic enzymes. The majority of patients suffer from deficiency of 21-hydroxylase (CYP21) coded by the CYP21A2 gene., Objective: Our objective was to study the functional and structural consequences of the novel CYP21A2 missense mutation c.364A > C (K121Q) detected in a female patient with nonclassical 21-hydroxylase deficiency. The patient was compound heterozygous for the novel K121Q mutation and the mild P453S mutation., Results: In vitro expression analysis of the mutant K121Q enzyme in transiently transfected COS-7 cells revealed reduced CYP21 activity of 14.0 +/- 5% for the conversion of 17-hydroxyprogesterone and 19.5 +/- 4% for the conversion of progesterone. K121 is located on helix C in the CYP21 protein, which is part of the heme coordinating system. In addition, helix C is involved in the interaction with the electron-providing enzyme P450 oxidoreductase. Protein modeling revealed that the substitution of glutamine for the basic amino acid lysine introduces an electrostatic change on the surface of CYP21 and may additionally change heme coordination. We hypothesize that the electron flux between P450 oxidoreductase and CYP21 is impaired and, moreover, that substrate affinity is altered due to heme dislocation with K121Q., Conclusion: Both the interaction of P450 oxidoreductase and CYP21 as well as heme coordination are likely to be disturbed due to the K121Q mutation. Our data exemplify how the combination of in vitro expression and structural protein analysis provide novel insights into molecular mechanisms of reduced CYP21 activity, eventually explaining the patient's phenotype.

Background: Findings have been inconsistent regarding the effect of T1DM (type 1 diabetes) on age at menarche., Objective: The purpose was to investigate in young German women with T1DM menarcheal age and factors potentially affecting menarche, including glycemic control, BMI (body mass index), relative T1DM duration (proportion of life with diabetes), insulin dose, and insulin therapy intensity. Initiated in 1990, the DPV program is an ongoing, prospective long-term longitudinal follow-up study to benchmark the quality of care provided to pediatric and, more recently, adult diabetes patients. Two hundered two German diabetes centers participated in nationwide data collection. Based on ethnicity and the availability of menarche and T1DM onset data as the main inclusion criteria, 643 young German women were selected from 11,629 female T1DM patients aged <20 years, recruited by referral, clinic or hospital ascertainment, or self report. Mean age at menarche (+/-SD) was 13.22 +/- 1.31 years, representing a delay of 0.52 years (p < 0.001) relative to the general population. Significant delay (p < 0.05) was also found for relative T1DM duration, BMI SD score, insulin dose, and HbA1c level, with a 1% increase in HbA1c resulting in a delay in menarche by 0.07 years., Conclusions: Age at menarche is delayed in type 1 diabetes mellitus. The delay increases with relative T1DM duration and poor quality of glycemic control.

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.

In contrast to disorders of sexual differentiation caused by lack of androgen production or inhibited androgen action, defects affecting development of the bipotent genital anlagen have rarely been investigated in humans. We have previously documented that the transcription factor FOXF2 is highly expressed in human foreskin. Moreover, Foxf2 knockout mice present with cleft palate in combination with hypoplasia of the genital tubercle. We hypothesized that humans with disorders of sex development (DSD) in combination with cleft palate could have mutations in the FOXF2 gene. Eighteen children with DSD and cleft palate were identified in the Lübeck DSD database (about 1,500 entries). Genomic DNA sequence analysis of the FOXF2 gene was performed and compared with 10 normal female and 10 normal male controls, respectively. Two heterozygous DNA sequence variations were solely present in one single patient each but in none of the 20 normal controls: a duplication of GCC (c.97GCC[9]+[10]) resulting in an extra alanine within exon 1 and a 25*G>A substitution in the 3'-untranslated region. Two patients carried a c.262G>A sequence variation predicting for an Ala88Thr exchange which was also detected in 2 normal controls. Two silent mutations, c.1272C>T (Ser424Ser) and c.1284T>C (Tyr428Tyr), respectively, occurred in the coding region of exon 2, again in both patients and normal controls. In conclusion, the majority of the detected sequence alterations were polymorphisms without obvious functional relevance. However, it cannot be excluded that the 2 unique DNA sequence alterations could have affected FOXF2 on the mRNA or protein level thus contributing to the observed disturbances in genital and palate development.

Aims: To conduct a multicentre, matched-pair cohort analysis comparing glycaemic control and adverse events of continuous subcutaneous insulin infusion (CSII) with multiple daily injections (MDI) in paediatric patients., Methods: Using standardized computer-based prospective documentation, HbA(1c), insulin dose, body mass index-standard deviation score (BMI-SDS), rate of hypoglycaemia, rate of diabetic ketoacidosis (DKA) and intensity of care were analysed in 434 matched pairs during a follow-up period of 3 years after initiation of MDI or CSII., Results: HbA(1c) was significantly lower in the CSII group during the first year of new regimen (CSII 7.5 +/- 0.05 vs. MDI 7.7 +/- 0.06; P < 0.05), but rose to the same level as in the MDI group during year 3. Insulin requirement remained significantly lower in the CSII group. The BMI-SDS increased in both study groups, with no significant difference. The rate of severe hypoglycaemia decreased significantly after the change of regimen (CSII 17.87 +/- 2.85 vs. MDI 25.14 +/- 3.79; P < 0.05) and during year 3 of the regimen, particularly when compared with baseline (-21% vs. -16%). The rate of DKA was lower at baseline in the CSII group and remained significantly lower over all 3 years. Intensity of care was the same in both subsets., Conclusions: Employing a large cohort, this matched-pair analysis has demonstrated over a 3-year study period that CSII is a safe form of intensive insulin therapy with similar glycaemic effects, but with significantly reduced rates of hypoglycaemia and DKA and a lower insulin requirement when compared with MDI.

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.

Children exhibit gender-typical preferences in play, toys, activities and interests, and playmates. Several studies suggest that high concentrations of pre- and postnatal androgens contribute to male-typical behavior development, whereas female-typical behavior develops in the absence of high androgens levels. This study aims to explore the consequences of hypoandrogenization on gender-typical behavior in children who have an XY karyotype and disorder of sex development (DSD). Participants included 33 children (ages 2-12 years) with an XY karyotype and DSD; 21 reared as girls and 12 reared as boys. Children's preferred activities and interests and playmate preferences were assessed with parent report questionnaires, a structured free-play task, and choice of a toy to keep as a gift. Participant's responses were compared to those of children recruited in a pre-school and elementary school survey (N=166). In this study, the degree of hypoandrogenization as indicated by genital stage and diagnosis showed a significant relationship to nearly all of the gender-related behaviors assessed, supporting the hypothesis that masculinization of gender role behavior is a function of prenatal androgen exposure. Despite the fact that children with partial androgen effects reared as girls showed increased "boyish" behaviors, they did not show increased signs of gender identity confusion or instability on a group level. We conclude that androgen exposure plays a decisive role in the development of gender-typical behavior in children with XY karyotype and DSD conditions.

Objective: We hypothesized systematic differences in the patterns of programmed basal insulin infusion rates in children and adolescents with type 1 diabetes on continuous subcutaneous insulin infusion (CSII). We aimed at classification of basal insulin infusion rate regimens and comparing patients' underlying clinical characteristics., Research Design and Methods: The German/Austrian diabetes data acquisition system for prospective surveillance database for quality control and scientific surveys in pediatric diabetology served as the primary data source. Latest (September 2004) basal insulin infusion rates of all 1,248 patients with type 1 diabetes on CSII (0.38-18 years) were analyzed (dataset 1). Basal insulin infusion rates per hour were expressed relative to mean basal insulin infusion rates per 24 h. Unsupervised clustering was used to classify basal insulin infusion rate patterns. Clinical characteristics of patients falling into distinct basal insulin infusion rate clusters were compared by Kruskal-Wallis test. Changes of basal insulin infusion rates in 64 patients were followed from initial settings before CSII to latest programming in an independent dataset 2., Results: Seven different basal insulin infusion rate patterns occurred in dataset 1. A dawn-dusk pattern was used in 708 patients (14.9 +/- 2.4 years) with the peak basal insulin infusion rate at 5 a.m. Additional patterns showed only one basal insulin infusion rate oscillation per 24 h with a backshift of peak basal insulin infusion rates in younger children (P < 0.000001) (1 A.M.: n = 152, 12.4 years and 9 P.M.: n = 117, 8.9 years). All but two patients in dataset 2 were initially set on dawn-dusk patterns but showed a comparable diversification of basal insulin infusion rates during follow-up with backshift of peak basal insulin infusion rates in younger children (P < 0.01)., Conclusions: Pediatric diabetologists shape distinct basal insulin infusion rate profiles during treatment of CSII patients, mainly reflecting differences in age. Our data strongly suggest that age-dependent endocrine changes during childhood (e.g., puberty) affect circadian distribution of insulin needs in CSII, which should be kept in mind when considering basal insulin infusion rate strategies in children and adolescents.

Objective: Autosomal-dominant pseudohypoaldosteronism type 1 (PHA1) is mostly caused by mutations in the mineralocorticoid receptor (NR3C2) gene. However, several kindreds with clinical signs of PHA1 but without NR3C2 gene mutations or deletions are reported. Serum- and glucocorticoid-induced kinase 1 (Sgk1) is involved in epithelial sodium reabsorption by facilitating the accumulation of the epithelial sodium channel in the plasma membrane. Therefore variations in the SGK1 gene may aggravate renal salt loss or cause PHA1., Methods: The SGK1 genewas sequenced in 10 patients with the typical clinical signs of PHA1 but without NR3C2 or SCNN1A, SCNN1B and SCNN1C gene mutation., Results: No disease-causing SGK1 gene mutation was detected in the studied PHA1 patient group. Two novel intronic SNPs which were also present in the normal population were detected in 2 patients., Conclusion: Our data do not support that SGK1 gene variations are disease-causing factors in genetically unexplained PHA1. Therefore, systematic investigation of other factors downstream of the MR involved in epithelial sodium reabsorption is warranted in patients with autosomal-dominant PHA1., (Copyright 2007 S. Karger AG, Basel.)

Normal synthesis and action of androgens is essential for normal male sex differentiation. 17Beta-hydroxysteroid dehydrogenase (17beta-HSD) and 5alpha-reductase isoenzymes play essential roles in normal androgen biosynthesis. We hypothesized that differences in expression of these enzymes in genital skin could contribute to the pathogenesis of 46,XY disorders of sex development (DSD). We investigated the mRNA transcription patterns of 17beta-hydroxysteroid dehydrogenase-isoenzymes type 1, 2, 3, 4, 5, 7, and 10, 5alpha-reductase type 1 and 2 and the androgen receptor in genital skin fibroblasts from foreskin and scrotal skin obtained from healthy males and patients with unclassified 46,XY DSD. mRNA expression was semi-quantified by real-time PCR. Although no systematic differences of gene expression of any enzyme between normal controls and hypospadias patients could be detected, we found in nearly half of all investigated patients' samples noticeable differences in the transcription profiles of 17beta-hydroxysteroid dehydrogenase type 5. In scrotal skin samples of patients a significantly higher transcription of the androgen receptor was detected. A role for an altered expression pattern of different enzymes of steroidogenesis in the etiology of genital malformations in some patients may be postulated., (2007 S. Karger AG, Basel)

Normal genital skin fibroblasts (GSF) and the human prostate carcinoma cell line LNCaP have been used widely as cell culture models of genital origin to study androgen receptor (AR) signaling. We demonstrate that LNCaP shows a reproducible response to androgens as assessed using cDNA-microarrays representing approximately 32,000 unique human genes, whereas several independent GSF strains are virtually unresponsive. We show that LNCaP cells express markedly higher AR protein levels likely contributing to the observed differences of androgen responsiveness. However, previous data suggested that AR-expression levels alone do not determine androgen responsiveness of human GSF compared to LNCaP. We hypothesized that cell-specific differences in expression levels of AR coregulators might contribute to differences in androgen responsiveness and might be found by comparing LNCaP and GSFs. Using the Canadian McGill-database of AR coregulators ( http://www.mcgill.ca/androgendb ), we identified 61 AR-coregulator genes represented by 282 transcripts on our microarray platform that was used to measure transcript profiles of LNCaP and GSF cells. Baseline expression levels of 48 AR-coregulator transcripts representing 33 distinct genes showed significant differences between GSF and LNCaP, four of which we confirmed by reverse transcriptase polymerase chain reaction. Compared to LNCaP, GSFs displayed significant upregulation of AR coregulators that can function as repressors of AR-transactivation, such as caveolin 1. Analysis of a recently published comprehensive dataset of 115 microarrays representing 35 different human tissues revealed tissue-specific signatures of AR coregulators that segregated with ontogenetically related groups of tissues (e.g., lymphatic system and genital tissues, brain). Our data demonstrate the existence of cell-line and tissue-specific expression patterns of molecules with documented AR coregulatory functions. Therefore, differential expression patterns of AR coregulators could modify tissue-specificity and diversity of androgen actions in development, physiology, and disease.

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.

Background: Sufficient androgen receptor (AR) activity is crucial for normal male sexual differentiation. Here we report on two unrelated 46, XY patients suffering from undervirilization and genital malformations. Both patients had a short polyglycine (polyG) repeat of 10 residues and a relatively long polyglutamine (polyQ) repeat of 28 and 30 residues within the transactivation domain of the AR. In addition, they also harbor a rare A645D substitution., Objective: We made a set of AR expression plasmid constructs with varying polyQ and polyG tract sizes in context with or without the A645D substitution and analyzed their in vitro transactivation capacity in transfected CHO cells., Results: We found that a short polyG repeat downmodulated AR activity to approximately 60-65% of the wild-type receptor. This effect was aggravated by A645D in context of a long polyQ repeat to less than 50% activity. In contrast, in the context of a short polyQ and a short polyG repeat, the A645D mutation rescues AR activity to almost wild-type levels, demonstrating a contradictory effect of this mutation, depending on the size of the polymorphic repeats., Conclusions: A combination of a short polyG repeat with a long polyQ repeat and an A645D substitution might contribute to the development of virilization disorders and explain the observed phenotypes of our patients as a form of androgen insensitivity. The whole recreation of AR sequence variations including individual polymorphic repeat sizes could unravel possible interference of mutations and variations on AR activity by in vitro transfection.

17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 5alpha-reductase isoenzymes play a crucial role in the formation and metabolism of sex steroids. Not only the key androgens testosterone and dihydrotestosterone but also their precursors are potent activators of the androgen receptor and are, therefore, likely to act as determinants of male sexual differentiation and maturation in a differentially regulated way. The aim of the present study was to relatively quantify the expression of the mRNA of 17beta-HSD isoenzymes, namely, type 1, 2, 3, 4, 5, 7, and 10, together with the 5alpha-reductase type 1 and 2, and the androgen receptor in normal human males and females. RNA was isolated from peripheral blood cells of both sexes and from genital skin fibroblasts (GSFs) of two different localizations (foreskin and scrotal skin) obtained from phenotypically normal males. mRNA expression was semi-quantified by quantitative reverse-transcriptase polymerase chain reaction with the LightCycler Instrument (Roche). The examined enzymes show statistically significant differences in their transcription pattern between the blood and the GSF RNA samples. Within the GSF samples, there are also significant variations between the two examined localizations in the transcription of 17beta-HSD type 1, 2, 4, and 5 as well as for the androgen receptor. We found large interindividual variation of enzyme transcription patterns in all investigated tissues. In peripheral blood cells, no sex-specific differences were seen. We conclude that sex steroid enzymes are expressed not only in genital primary target tissues but also in peripheral blood. The expression in different target tissues may contribute to both the individual sexual and tissue-specific phenotype in humans.

Context: Congenital adrenal hyperplasia is a group of autosomal recessive inherited disorders of steroidogenesis. The deficiency of steroid 11-hydroxylase (CYP11B1) resulting from mutations in the CYP11B1 gene is the second most frequent cause., Objective: We studied the functional and structural consequences of two CYP11B1 missense mutations, which were detected in a 1.8-yr-old boy with acne and precocious pseudopuberty, to prove their clinical relevance and study their impact on CYP11B1 function., Results: The in vitro expression studies in COS-7 cells revealed an almost complete absence of CYP11B1 activity for the P94L mutant to 0.05% for the conversion of 11-deoxycortisol to cortisol. The A368D mutant severely reduced the CYP11B1 enzymatic activity to 1.17%. Intracellular localization studies by immunofluorescence revealed that the mutants were correctly localized. Introducing these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the effects measured in vitro. We hypothesize that the A368D mutation interferes with structures important for substrate specificity and heme iron binding, thus explaining its major functional impact. However, according to structural analysis, we would expect only a minor effect of the P94L mutant on 11-hydroxylase activity, which contrasts with the observed major effect of this mutation both in vitro and in vivo., Conclusion: Analyzing the in vitro enzyme function is a complementary procedure to genotyping and a valuable tool for understanding the clinical phenotype of 11-hydroxylase deficiency. This is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides valuable insights in cytochrome P450 structural-functional relationships, although one must be aware of the limitations of in silico-based methods.

Background: In this epidemiological study, we sought to capture the incidence of ambiguous genitalia in neonates and to describe initial management strategies., Methods: We used the registry for rare diseases in pediatrics in Germany to ascertain cases and asked reporting institutions for information on phenotype, laboratory tests, imaging results, diagnosis, initial management and sex assignment., Results: We identified 80 cases within a 2-year study period and calculated an incidence of 2 per 10,000 births with ambiguous genitalia per year in Germany. Prevalence was higher in infants from non-German family background. In more than 50% of all infants a definite diagnosis was lacking even at the age of 6 months. In those cases where the etiology was confirmed, the most common diagnosis was congenital adrenal hyperplasia, followed by androgen insensitivity syndrome and mixed gonadal dysgenesis. Associated malformations were very common, affecting 37.5% of all infants. Sex assignment was female in 46,XY infants with predominately female phenotype and all 46,XX infants. Early surgery was performed in many cases irrespective whether a definite diagnosis had been established or not. Integrated psychosocial care was the exception rather than the rule., Conclusions: Classification and management of ambiguous genitalia at birth remain a challenge for all professionals involved. National and international registries may help to provide a better understanding of the incidence and clinical course of such disorders., (Copyright (c) 2006 S. Karger AG, Basel.)

Androgen insensitivity syndrome (AIS) is characterized by deficient or absent virilization in 46,XY individuals despite normal or even elevated androgen levels. AIS is usually caused by mutations in the androgen receptor (AR) gene. We aimed at contrasting clinical, biochemical, and molecular genetic characteristics of three patients (P1-P3) with clinically evident partial (P1) and complete (P2, P3) AIS with and without AR gene mutations. AR expression was studied in cultured genital skin fibroblasts (GSF) by Western immunoblotting, ligand binding analyses, Northern blotting, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), and RT-PCR spanning exons 1-8. AR gene DNA sequence was analyzed by single-strand conformation analysis (SSCA), and DNA sequencing. GSF revealed reduced (P1) or absent (P2, P3) ligand binding. Northern blots showed either slightly reduced hybridization of the 10.5-kb AR transcript (P3) or no hybridization (P1, P2), as confirmed by semiquantitative RT-PCR. RT-PCR spanning exons 1-8 detected single AR mRNA bands in P1-P3 excluding splicing errors. Western analyses showed either low (P1) or no (P2, P3) AR protein. While SSCA initially did not reveal any molecular abnormality, sequencing showed a novel CAG (Gln) to TAG (stop) mutation at codon 59 (P3) and a previously described 2-bp deletion at codon 472, leading to a frameshift and premature stop in codon 499 (P2). Intriguingly, P1 showed an unaltered DNA sequence of the coding region of the AR gene including all intron-exon boundaries. In conclusion, patients with clinically evident complete AIS are likely to harbor an AR gene mutation, demanding that the two polymorphic regions must always be included in molecular analyses of the AR gene. Moreover, our data support the concept that in a subset of AIS patients, particularly those with partial AIS, molecular alterations outside the coding region of the AR gene must be presumed.

The majority of genetic variations in the androgen receptor (AR) gene are point mutations leading to impairment of the DNA- or hormone-binding domains. The N-terminus encoded by the first exon of the AR-gene usually harbors disruptive mutations associated with complete androgen insensitivity syndrome (CAIS) while missense mutations related with partial androgen insensitivity syndrome (PAIS) are seemingly rare. We present a 46,XY male with scrotal hypospadias in whom we detected a S432 F point mutation within the N-terminus. Transient transfections of an AR expression plasmid carrying the S432 F mutation using Chinese Hamster Ovary (CHO) cells revealed a significant partial reduction in transactivation of the co-transfected androgen responsive (ARE) (2)TATA luciferase reporter gene thus confirming PAIS. In two further 46, XY patients with slight to moderate virilization defects, we detected an S411 N mutation, and a 9 base pair deletion leading to the loss of amino acids 409 to 411 (L-A-S), respectively. These mutations did not compromise AR-function under the chosen experimental settings. The S432 F-patient supports particular significance of the AR-N-terminus for mild forms of AIS while the functional role of the two further mutations remains unclear. The N-terminus is a species-specific AR-domain possibly also involved in contributing to target tissue selectivity of AR-actions via mediating co-regulator interactions. Therefore, mild molecular defects of the AR-N-terminus may not necessarily inhibit general transactivation properties using currently established reporter gene models.

Objective: 5alpha-reductase enzymes reduce testosterone (T) to the most potent androgen dihydrotestosterone (DHT). Two isoenzymes are known to day. While the type 2-enzyme (5RII) is predominantly expressed in male genital tissues and mutations are known to cause a severe virilization disorder in genetic males, the role of the type 1-enzyme (5RI) in normal male androgen physiology is unclear. We investigated whether 5RI is transcribed in normal male genital skin fibroblasts (GSFs) and if the transcription is regulated by age or by androgens themselves., Methods: GSF from 14 normally virilized males of different ages, ranging from 8 months to 72 years, obtained at circumcision were cultured. Total RNA was isolated after incubation for 48 h with 100 nM T or without androgens. Each sample was amplified in triplicate by real-time PCR with porphobilinogen desaminase as a housekeeping gene used for semiquantification. Selected cultures were analyzed after incubation with 10 and 100 nM T and 1 and 100 nM DHT for 24, 48 and 120 h., Results: 5RI was transcribed in all investigated samples with a 4.5-fold variability in the mRNA concentration of different individuals. However, neither age-related regulation nor significant influence of T or DHT on the transcription rate was discovered., Conclusion: Since 5RI is abundantly transcribed in GSFs, we hypothesize that this isoenzyme may play important roles in the androgen physiology of normally virilized males and may contribute to masculinization in 5RII-deficient males at the time of puberty.

Leydig cell hypoplasia (LCH) is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals and is caused by inactivating mutations of the LH receptor gene. The clinical and biochemical diagnostic parameters of LCH are not always specific and may therefore show significant overlap with other causes of insufficient testicular steroid biosynthesis. We have studied a 46,XY newborn with completely female external genitalia and palpable testes. Due to an increased basal serum ratio of androstenedione/testosterone, 17 beta-hydroxysteroid dehydrogenase type 3 (17 beta-HSD 3) deficiency was initially suspected. DNA analysis of the corresponding HSD17B3 gene, however, showed no abnormalities in the entire coding region. In contrast, direct sequencing of the LH receptor gene revealed a novel homozygous single nucleotide insertion in exon 11 (codon A589fs) producing a frame shift in the open reading frame predicting for premature termination of translation 17 amino acids downstream. From the genetic perspective, this mutation represents the first frame shift mutation in the LH receptor gene ever reported to date. From the clinical standpoint, LCH should always be considered in the differential diagnosis as steroid profiles may not be informative. Therefore, molecular genetic analysis should be warranted for androgen biosynthesis defects in all cases.

Disruption of the P450 side-chain cleavage cytochrome (P450scc) enzyme due to deleterious mutations of the CYP11A1 gene is thought to be incompatible with fetal survival because of impaired progesterone production by the fetoplacental unit. We present a 46,XY patient with a homozygous disruption of CYP11A1. The child was born prematurely with complete sex reversal and severe adrenal insufficiency. Laboratory data showed diminished or absent steroidogenesis in all pathways. Molecular genetic analysis of the CYP11A1 gene revealed a homozygous single nucleotide deletion leading to a premature termination at codon position 288. This mutation will delete highly conserved regions of the P450scc enzyme and thus is predicted to lead to a nonfunctional protein. Both healthy parents were heterozygous for this mutation. Our report demonstrates that severe disruption of P450scc can be compatible with survival in rare instances. Furthermore, defects in this enzyme are inherited in an autosomal-recessive fashion, and heterozygote carriers can be healthy and fertile. The possibility of P450scc-independent pathways of steroid synthesis in addition to the current concept of luteoplacental shift of progesterone synthesis in humans has to be questioned.

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., ((c) 2005 S. Karger AG, Basel.)

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.

Background: Stromal cells play key roles during androgen-mediated male sexual differentiation. Our objective was to establish a transient transfection method for primary human fibroblasts enabling functional characterization of wild-type (wt) and mutant androgen receptor (AR) plasmid constructs, corresponding to partial and complete androgen insensitivity syndrome (PAIS/CAIS)., Methods: An AR-negative fibroblast strain (ARD842) was established from the gonads of a CAIS patient. Wt-AR or either mutants L712F (PAIS), R774C or V866M (CAIS) were transfected using a polyamine-based procedure. Alternatively, two AR-positive male foreskin fibroblast strains were investigated. Androgen-induced activation of two co-transfected reporter plasmids ((ARE)(2)TATA-, MMTV-luciferase) was measured., Results: All three fibroblast strains showed a ligand-dependent rise of luciferase activity after transfection of wt-AR. Mutant plasmids were assessed in AR-negative ARD842 cells. While L712F showed high partial activity, R774C and V866M were nearly inactive. The intrinsic AR of normal foreskin fibroblasts revealed no measurable ligand-inducible reporter gene activity., Conclusions: Polyamine-based transfection of AR plasmids into cultured fibroblasts provides a promising tool for analysis of AR transactivation, thereby considering a stromal cellular background. This is supported by the mutant ARs which showed the expected levels of impaired transactivation with respect to the corresponding AIS phenotypes. The role of the intrinsic AR in normal male human foreskin fibroblasts needs further exploration., (Copyright 2005 S. Karger AG, Basel.)

Despite enormous advances in the understanding of the molecular mechanisms of sexual differentiation, the medical decisions made in patients with disorders of somatosexual differentiation are mostly lacking evidence-based principles. Recent critical discussions have focused on approaches to gender assignment in infants with ambiguous genitalia. These decisions must be based on the correct diagnosis to be able to predict the development during puberty and adulthood in the affected individual. Puberty in this process plays a pivotal role not only because of the physical changes induced by endogenous or supplementary sex steroids to enhance the sex of rearing, but it is also the time for sexual orientation and promotion of gender identity. In this review we focus not only on the molecular impact of sex steroid action, but also view current opinions and studies on the gender development of the intersex patient during puberty.

Abnormal human spermatogenesis can be caused by defects in androgen action because of androgen insensitivity. A variety of mutations have been described in the human androgen receptor gene associated with male infertility. These can be attributed to two molecular mechanisms. First, point mutations in the androgen receptor gene cause alterations in the amino acid sequence and, hence, lead to apparently slight changes in the androgen receptor effector mechanisms and mild androgen insensitivity. Secondly, variations in the polymorphic poly glutamine segment within the N-terminal end of the androgen receptor have been ascribed to correlate with fertility aspects possibly because of modifications of transcriptional regulatory mechanisms. It has been postulated that longer poly glutamine segments are associated with decreased sperm counts. However, the molecular mechanisms that lead to inhibition of spermatogenesis because of a mutated androgen receptor are poorly understood and will need more focus in the future.

Background: Androgen insensitivity syndrome (AIS) comprises a range of phenotypes from male infertility to complete feminization. Most individuals with AIS carry germline mutations of the androgen receptor (AR) that interfere with or ablate its function. As genital fibroblasts retain expression of the AR in vitro, we used genital skin fibroblasts from normal males and 46,XY females with complete AIS due to known AR mutations to gain insights into the role of the AR in human genital differentiation., Results: Using DNA microarrays representing 32,968 different genes, we identified 404 transcripts with significant differences in transcription levels between genital skin fibroblasts cultured from normal and AIS-affected individuals. Gene-cluster analyses uncovered coordinated expression of genes involved in key processes of morphogenesis. On the basis of animal studies and human genetic syndromes, several of these genes are known to have specific roles in genital differentiation. Remarkably, genital fibroblasts from both normal and AIS-affected individuals showed no transcriptional response to dihydrotestosterone treatment despite expression of the AR., Conclusions: The results suggest that in addition to differences in the anatomic origin of the cells, androgen signaling during prenatal development contributes to setting long-lasting, androgen-independent transcriptional programs in genital fibroblasts. Our findings have broad implications in understanding the establishment and the stability of sexual dimorphism in human genital development.

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.

Background: 17beta hydroxysteroid dehydrogenase deficiency is a presumable rare cause for a severe virilization disorder in children with 46,XY karyotype due to a defect in the testicular testosterone biosynthesis from androstenedione., Patient: We report on a 14 year old child with 46,XY karyotype with a predominantly female phenotype., Results: Hormonal analysis showed low values for androstenedione and testosterone before and after stimulation with human chorionic gonadotropin, however, the androstenedione/testosterone ratio was elevated. Molecular genetic analysis proved the diagnosis of 17beta-hydroxysteroid dehydrogenase deficiency due to a homozygous mutation (325+4 A-T) in the HSD17B3-gene, which leads to an aberrant splicing process., Conclusions: This case demonstrates that in addition to a meticulous steroid analysis a direct molecular genetic analysis can be helpful in the diagnosis of 17beta-hydroxysteroid dehydrogenase deficiency.

Steroid 5alpha-reductase deficiency is a rare autosomal recessive disorder caused by mutations in the SRD5A2-gene, resulting in diminished dihydrotestosterone (DHT) formation and, hence, in a severe virilization deficit of the external genitalia in patients with 46,XY karyotype. The phenotype of affected individuals is variable and has been reported to range from completely female over genital ambiguity to normal male, depending on the type of mutation and its effect on enzyme activity. Here we report an adolescent 46,XY patient with predominantly female appearance, who had been gonadectomized in early infancy. Genital status revealed a urogenital sinus equivalent to Prader stage III. Molecular genetic analysis demonstrated a homozygous point mutation in exon 2 of the SRD5A2-gene, leading to a premature termination in codon position 111 of the 5alpha-reductase 2 enzyme, and not allowing formation of a functional 5alpha-reductase type 2 enzyme. This case demonstrates that even despite a complete loss of function of 5alpha-reductase type 2, marked virilization is possible, most likely the result of a testosterone (T) effect during foetal life.

In a patient with partial androgen insensitivity syndrome (AIS), we identified a single inherited presumably silent nucleotide variation (AGC -> AGT) in exon 8 (codon 888) of the AR gene. However, in the patient's genital skin fibroblasts, a considerably shortened transcript of 5.5 kb (normal: 10.5 kb) was detected, which misses a part of exon 8 and a prominent portion of the 3'-untranslated region. The translation product includes eight missense amino acids from codon 886 onward followed by a premature stop codon. As shown by in vitro expression analysis, the mutant protein lacks any residual function. However, reverse transcribed PCRs and sequence data indicate the existence of two additional splicing variants of 6.4 kb and 7.8-kb length both in patient and normal control genital skin fibroblasts. These splicing variants comprise the complete coding region but a shortened 3'-untranslated region. Thus, a distinct alternative pre-messenger RNA-processing event leading to two additional transcripts occurs generally in genital skin fibroblasts. In addition, this process partially prevents aberrant splicing in the patient and produces a small fraction of normal, functionally intact AR-protein that could explain the partial masculinization in this patient. This first report of an exonic splicing mutation in the AR-gene indicates a physiologic relevance of the regular AR-messenger RNA variants with shortened 3'-untranslated regions and their functional translation products in human genital development.

Molecular causes of phenotypic diversity in androgen insensitivity syndrome, occurring even in the same family, have rarely been identified. We report on a family with four affected individuals, three brothers (B1-3) and their uncle, displaying strikingly different external genitalia: B1, ambiguous; B2, severe micropenis; B3, slight micropenis; and uncle, micropenis and penoscrotal hypospadias. All had been assigned a male gender. We detected the same L712F mutation of the androgen receptor (AR) gene in each subject. Methyltrienolone binding on cultured genital skin fibroblasts of B2 suggested moderate impairment of the ligand-binding domain [maximal binding capacity, 38.2 fmol/mg protein (normal); Kd, 0.21 nmol/L; normal range, 0.03-0.13 nmol/L]. In trans-activation assays, the mutant 712F-AR showed considerable deficiency at low concentrations of testosterone (0.01-0.1 nmol/L) or dihydrotestosterone (0.01 nmol/L). Remarkably, this could be fully neutralized by testosterone concentrations greater than 1.0 nmol/L. Hence, the 712F-AR could switch its function from subnormal to normal within the physiological concentration range of testosterone. This was reflected by an excellent response to testosterone therapy in B1, B2, and the uncle. Taking into account the well documented individual and time-dependent variation in testosterone concentration in early fetal development, our observations clearly illustrate the potential impact of varying ligand concentrations for distinct cases of phenotypic variability in androgen insensitivity syndrome.

Abnormal human spermatogenesis is caused by a variety of genetic and acquired conditions. Because spermatogenesis is dependent on androgens, some males may have a minimal form of androgen insensitivity that does not inhibit virilization but impairs fertility. This has lead us to investigate the possibility of abnormalities in the androgen receptor (AR) gene in a large cohort of males suffering from infertility of unknown cause. We studied 180 males with variable impairment of spermatogenesis. In all patients, serum levels of testosterone and gonadotropins were analyzed to define an androgen sensitivity index (ASI). Single-strand conformation analysis and direct DNA sequencing of PCR-amplified blood leukocyte DNA were used to identify mutations within the whole coding region of the AR-gene. Endocrine and molecular investigations were compared with 53 normal males with proven fertility. In three infertile males, mutations in the AR were identified. Two unrelated males had the same variation within the first exon encoding for the transactivation domain of the receptor (Pro390Ser), whereas, in the third, a mutation in the hormone-binding region was characterized (Gln798Glu). All identified mutation carriers had a significantly elevated ASI. A proportion of males with idiopathic infertility carry relevant variations within the AR-gene. These males may be distinguished on the basis of hormone levels, calculating the ASI, although this index lacks specificity.

We recently found that postzygotic de novo mutations occur at the expected high rate of an X-linked recessive mutation in androgen insensitivity syndrome. The resulting somatic mosaicism can be an important molecular determinant of in vivo androgen action caused by expression of the wild-type androgen receptor (AR). However, the clinical relevance of this previously underestimated genetic condition in androgen insensitivity syndrome has not been investigated in detail as yet. Here, we present the clinical and molecular spectrum of somatic mosaicism considering all five patients with mosaic androgen insensitivity syndrome, whom we have identified since 1993: Patient 1 (predominantly female, clitoromegaly), 172 TTA(Leu)/TGA(Stop); patient 2 (ambiguous), 596 GCC(Ala)/ACC(Thr); patient 3 (ambiguous), 733 CAG(Gln)/ CAT(His); patient 4 (completely female), 774 CGC(Arg)/TGC (Cys); and patient 5 (ambiguous), 866 GTG(Val)/ATG(Met). Serum sex hormone binding globulin response to stanozolol, usually correlating well with in vivo AR function, was inconclusive for assessment of the phenotypes in all tested mosaic individuals. An unexpectedly strong virilization occurred in patients 1, 3, and 5 compared with phenotypes as published with corresponding inherited mutations and compared with the markedly impaired transactivation caused by the mutant ARs in cotransfection experiments. Only the prepubertal virilization of patients 2 and 4 matched appropriately with transactivation studies (patient 4) or the literature (patients 2 and 4). However, partial pubertal virilization in patient 4 caused by increasing serum androgens and subsequent activation of the wild-type AR could not be excluded. We conclude that somatic mosaicism is of particular clinical relevance in androgen insensitivity syndrome. The possibility of functionally relevant expression of the wild-type AR needs to be considered in all mosaic individuals, and treatment should be adjusted accordingly.