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Objective: The Anti-Muellerian Hormone (AMH) has been reported as surrogate marker of antral follicles, which are the origins of hyperandrogenism in polycystic ovarian syndrome (PCOS). Therefore, AMH may be useful for the diagnosis of PCOS. The objective was to study the longitudinal changes in AMH concentrations in girls with and without PCOS., Design: This is a longitudinal study of obese girls participating in a 1-year lifestyle intervention., Patients: Forty obese girls aged 13-16 years (50% with PCOS) were included in the study. Girls with and without PCOS were matched to age, BMI and change in weight status., Measurements: AMH, gonadotropins, androstenedione, testosterone, oestradiol and sex hormone-binding globulin (SHBG) were determined., Results: Obese girls with PCOS demonstrated significantly (P<.001) higher AMH concentrations (5.8±3.1 ng/mL) compared to obese girls without PCOS (2.4±1.4 ng/mL). None of the girls without PCOS had AMH concentrations ≥6 ng/mL and none of the PCOS girls showed AMH concentrations ≤3 ng/mL. Weight loss in girls with PCOS was associated with a significant drop in AMH concentrations (-1.4±1.8 ng/mL, P=.045). AMH was significantly related to testosterone (cross-sectional: b-coefficient 3.7±1.7, P=.001, longitudinal: b-coefficient 0.54±0.47, P=.026) and luteinizing hormone (LH) (cross-sectional: b-coefficient 0.05±0.04, P=.039, longitudinal: b-coefficient 0.005±0.004, P=.039), but not to any other analysed parameter in multiple linear regression analyses adjusted to multiple confounders., Conclusions: AMH was increased in adolescent girls with PCOS and normalized with weight loss. AMH was cross-sectionally and longitudinally related to hyperandrogenism., (© 2017 John Wiley & Sons Ltd.)

According to the concept of fetal programming, prenatal distress has long-lasting consequences on the offspring's health later in life. The hypothalamic-pituitary-adrenal axis is considered a mediating system by which maternal distress is transferred to the fetus in intrauterine environment. In this longitudinal explorative study on 30 mother-child-dyads, reported maternal distress and salivary cortisol were collected during pregnancy. Infant temperament, motor and cognitive development were assessed at 16 months. Additionally, infant cortisol levels throughout the day and in reaction to a psycho-social stressor where measured. As expected, infants whose mothers had experienced higher prenatal distress expressed a more difficult temperament and lower fine motor development at 16 months. No association could be shown between prenatal maternal distress and infant salivary cortisol.

Disorders or differences in sex development (DSD) comprise a heterogeneous group of conditions with an atypical sex development. For optimal diagnosis, highly specialised laboratory analyses are required across European countries. Working group 3 of EU COST (European Cooperation in Science and Technology) Action BM 1303 'DSDnet' 'Harmonisation of Laboratory Assessment' has developed recommendations on laboratory assessment for DSD regarding the use of technologies and analytes to be investigated. This position paper on steroid hormone analysis in diagnosis and treatment of DSD was compiled by a group of specialists in DSD and/or hormonal analysis, either from participating European countries or international partner countries. The topics discussed comprised analytical methods (immunoassay/mass spectrometry-based methods), matrices (urine/serum/saliva) and harmonisation of laboratory tests. The following positions were agreed upon: support of the appropriate use of immunoassay- and mass spectrometry-based methods for diagnosis and monitoring of DSD. Serum/plasma and urine are established matrices for analysis. Laboratories performing analyses for DSD need to operate within a quality framework and actively engage in harmonisation processes so that results and their interpretation are the same irrespective of the laboratory they are performed in. Participation in activities of peer comparison such as sample exchange or when available subscribing to a relevant external quality assurance program should be achieved. The ultimate aim of the guidelines is the implementation of clinical standards for diagnosis and appropriate treatment of DSD to achieve the best outcome for patients, no matter where patients are investigated or managed., (© 2017 The authors.)

Objective: The underlying mechanisms of polycystic ovarian syndrome (PCOS) are not fully understood yet. The aim of the study was to get functional insights into the regulation of steroid hormones in PCOS by steroid metabolomics., Design: This is a longitudinal study of changes of steroid hormones in 40 obese girls aged 13-16 years (50% with PCOS) participating in a 1-year lifestyle intervention. Girls with and without PCOS were matched to age, BMI and change of weight status., Methods: We measured progesterone, 17-hydroxyprogesterone, 17-hydroxyprogenolon, 11-deoxycorticosterone, 21-deoxycorticosterone, deoxycorticosterone, corticosterone, 11-deoxycortisol, cortisol, cortisone, androstenedione, testosterone, dehydroepiandrostendione-sulfate (DHEA-S), estrone and estradiol by LC-MS/MS steroid profiling at baseline and one year later., Results: At baseline, obese PCOS girls demonstrated significantly higher androstenedione and testosterone concentrations compared to obese girls without PCOS, whereas the other steroid hormones including glucocorticoids, mineralocorticoids, estrogens and precursors of androgens did not differ significantly. Weight loss in obese PCOS girls was associated with a significant decrease of testosterone, androstenedione, DHEA-S, cortisol and corticosterone concentrations. Weight loss in obese non-PCOS girls was associated with a significant decrease of DHEA-S, cortisol and corticosterone concentrations, whereas no significant changes of testosterone and androstenedione concentrations could be observed. Without weight loss, no significant changes of steroid hormones were measured except an increase of estradiol in obese PCOS girls without weight loss., Conclusions: The key steroid hormones in obese adolescents with PCOS are androstenedione and testosterone, whereas glucocorticoids, mineralocorticoids, estrogens and precursors of androgens did not differ between obese girls with and without PCOS., (© 2017 The authors.)

Objective: Childhood and Juvenile Absence Epilepsy account for 30% of all genetic generalized epilepsies with a strong genetic contribution. At the current state the genetic background remains to be resolved. The aim of this study was to identify disease associated transcripts pinpointing potential underlying disease mechanisms in patients with CAE and JAE., Methods: We performed gene expression analysis from peripheral blood mononuclear cells (PBMCs) in 30 patients with newly-diagnosed absence epilepsy prior to initiating treatment and 30 healthy age - and gender-matched pediatric controls. In a first group (group 1), 10 patients and controls we performed genome-wide transcriptome analysis using the Affymetrix HG U133 2.0+ microarray. 75 differentially expressed genes were followed up by qRT-PCR in two independent groups of 10 patients and controls (group 2 and 3). Furthermore, we analyzed 18 candidate genes by qRT-PCR in groups 2 and 3, which had previously been considered strong candidates for genetic epilepsies., Results: Genome-wide gene expression analysis in group 1 revealed 601 differentially regulated genes. Independent validation of 75 group 1-derived genes by qRT-PCR in groups 2 and 3 confirmed candidate genes with a consistent, but non-significant pattern of up- or down-regulation across all groups (ATP1B3, CAND1, PRPF6, TRIM8). Previously known genes including GABRA1, GABRB3, GABRG2, and RCN2 showed evidence for up- or down-regulation in individual experiments, but were not reliable across groups either., Discussion: Gene expression analysis in absence epilepsy from PMBCs displayed a high degree of heterogeneity between different patient groups. Our study provides several potentially interesting candidate genes, while demonstrating the limits of using gene expression analysis from blood in the identification of novel pathogenic mechanisms. In particular, we found that gene expression levels vary in response to altered experimental conditions, representing a substantial challenge for the identification of disease-related gene expression signatures for neurological diseases from whole blood., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mutations in NR5A1 have been reported as a frequent cause of 46,XY disorders of sex development (DSD) associated to a broad phenotypic spectrum ranging from infertility, ambiguous genitalia, anorchia to gonadal dygenesis and female genitalia. Here we present the clinical follow up of four 46,XY DSD patients with three novel heterozygous mutations in the NR5A1 gene leading to a p.T40P missense mutation and a p.18DKVSG22del nonframeshift deletion in the DNA-binding domain and a familiar p.Y211Tfs*83 frameshift mutation. Functional analysis of the missense and nonframeshift mutation revealed a deleterious character with loss of DNA-binding and transactivation capacity. Both, the mutations in the DNA-binding domain, as well as the familiar frameshift mutation are associated with highly variable endocrine values and phenotypic appearance. Phenotypes vary from males with spontaneous puberty, substantial testosterone production and possible fertility to females with and without Müllerian structures and primary amenorrhea. Exome sequencing of the sibling's family revealed TBX2 as a possible modifier of gonadal development in patients with NR5A1 mutations.

Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1 , a gene encoding 11β-hydroxylase, represents a rare autosomal recessive Mendelian disorder of aberrant sex steroid production. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where consanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not frequently measured, is the most robust biochemical marker for diagnosing 11β-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11β-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, genetic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11β-hydroxylase deficiency CAH.

Context: It is well established that boys are heavier than girls at birth. Although the cause of birth weight (BW) difference is unknown, it has been proposed that it could be generated from prenatal androgen action., Objective: The aim of the current study was to determine the BW of children with disorders of sex development (DSD) of different etiologies and to evaluate the effects of androgen action on BW., Methods: Data regarding diagnosis, BW, gestational age, karyotype, and concomitant conditions were collected from the International Disorders of Sex Development (I-DSD) Registry (www.i-dsd). BW standard deviation score was calculated according to gestational age. Cases were evaluated according to disorder classification in I-DSD (i.e., disorders of gonadal development, androgen excess, androgen synthesis, androgen action, nonspecific disorder of undermasculinization groups, and Leydig cell defect)., Results: A total of 533 cases were available; 400 (75%) cases were 46,XY, and 133 (25%) cases were 46,XX. Eighty cases (15%) were born small for gestational age (SGA). Frequency of SGA was higher in the 46,XY group (17.8%) than in the 46,XX (6.7%) group (P = 0.001). Mean BW standard deviation scores of cases with androgen excess and androgen deficiency [in disorders of gonadal development, androgen synthesis, and Leydig cell defect groups and androgen receptor gene (AR) mutation-positive cases in disorders of androgen action groups] were similar to normal children with the same karyotype. SGA birth frequency was higher in the AR mutation-negative cases in disorders of androgen action group and in the nonspecific disorders of the undermasculinization group., Conclusions: BW dimorphism is unlikely to be explained by fetal androgen action per se. 46,XY DSDs due to nonspecific disorders of undermasculinization are more frequently associated with fetal growth restriction, SGA, and concomitant conditions., (Copyright © 2017 by the Endocrine Society)

Background: Information sharing in chronic conditions such as disorders of/differences in sex development (DSD) is essential for a comprehensive understanding by parents and patients. We report on a qualitative analysis of communication skills of fellows undergoing training in paediatric endocrinology. Guidelines are created for the assessment of communication between health professionals and individuals with DSD and their parents., Methods: Paediatric endocrinology fellows worldwide were invited to study two interactive online cases (www.espe-elearning.org) and to describe a best practice communication with (i) the parents of a newborn with congenital adrenal hyperplasia and (ii) a young woman with 46,XY gonadal dysgenesis. The replies were analysed regarding completeness, quality, and evidence of empathy. Guidelines for structured assessment of responses were developed by 22 senior paediatric endocrinologists worldwide who assessed 10 selected replies. Consensus of assessors was established and the evaluation guidelines were created., Results: The replies of the fellows showed considerable variation in completeness, quality of wording, and evidence of empathy. Many relevant aspects of competent clinical communication were not mentioned; 15% (case 1) and 17% (case 2) of the replies were considered poor/insufficient. There was also marked variation between 17 senior experts in the application of the guidelines to assess communication skills. The guidelines were then adjusted to a 3-level assessment with empathy as a separate key item to better reflect the qualitative differences in the replies and for simplicity of use by evaluators., Conclusions: E-learning can play an important role in assessing communication skills. A practical tool is provided to assess how information is shared with patients with DSD and their families and should be refined by all stakeholders, notably interdisciplinary health professionals and patient representatives., (© 2017 S. Karger AG, Basel.)

Background: 17β-hydroxysteroid dehydrogenase (17β-HSD) type 3 deficiency is an autosomal recessive disorder with diminished testosterone synthesis and consequently underandrogenisation. 46,XY patients with 17β-HSD type 3 deficiency are often assigned a female sex at birth but have a high virilisation potential at the time of puberty., Methods: We studied four 46,XY patients with 17β-HSD type 3 deficiency at puberty with regard to the underlying mutations, the hormone values, and the clinical findings., Results: Three patients were initially assigned a female sex and 1 was assigned a male sex. All had relevant mutations in the HSD17B3 gene. The 2 patients with deleterious mutations had lower testosterone values at the time of puberty than the patients with possible residual activity of 17β-HSD type 3. One of the latter patients changed to male gender., Conclusion: All 4 patients with 17β-HSD type 3 deficiency synthesized relevant amounts (>0.7 µg/L) of testosterone at puberty, which lead to variable androgenisation. In patients with presumable residual activity of the mutated enzyme, testosterone values in the male reference range can be achieved, thereby inducing male pubertal development. These patients should possibly be assigned a male sex. Any surgical intervention should be avoided until the patients are old enough to consider their options of medical and surgical intervention.
., (© 2016 S. Karger AG, Basel.)

Background: Dehydroepiandrosterone sulfate (DHEAS) and 17-hydroxypregnenolone (17OHPreg) are important for understanding the Δ5 pathway (e.g., in adrenarche and obesity). Although mass spectrometry has become the state-of-the-art method for quantifying steroids, there are few comprehensive age-, sex-, and pubertal stage-specific reference ranges for children., Aims: To develop a sensitive and reliable ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of DHEAS and 17OHPreg and to establish entire age-, sex- and pubertal stage-specific reference ranges in children., Methods: A total of 684 children, 453 (243 female, 210 male) with normal body mass index (BMI; <90th) and 231 (132 female, 99 male) obese subjects (>97th), were categorized into 11 age groups, and age- and Tanner stage (PH)-specific reference ranges were determined., Results: The limit of detection was 0.05 nmol/L for 17OHPreg and 0.5 nmol/L for DHEAS. Levels of both steroids declined after the neonatal period. Comparisons with RIA assays (Siemens, Munich, Germany) (DHEAS) and an in-house kit (17OHPreg) revealed 0.95 and 0.93, respectively, as coefficients of determination. Although DHEAS-generally higher in boys-increased continuously starting at 3 to 6 years, 17OHPreg remained largely constant. In obese patients, both were significantly elevated, also in part after alignment to Tanner stages (PH)., Conclusions: UPLC-MS/MS is sensitive and reliable for quantifying DHEAS and 17OHPreg. Our data support differential maturation of CYP17 during adrenarche with successively increasing 17,20-lyase activity but largely constant 17α-hydroxylation activity. Endocrine interpretation of 17OHPreg and DHEAS must consider differential patterns for age, sex, pubertal stage, and BMI., (Copyright © 2017 by the Endocrine Society)

Background/aims: To elucidate the genetic causes of severe primary insulin-like growth factor-I deficiency (SPIGFD) by systematic, targeted, next-generation sequencing (NGS)-based resequencing of growth-related genes., Methods: Clinical phenotyping followed by NGS in 17 families including 6 affected sib pairs., Results: We identified disease-causing, heterozygous, de novo variants in HRAS (p.Gly13Cys) and FAM111A (p.Arg569His) in 2 male patients with syndromic SPIGFD. A previously described homozygous GHR nonsense variant was detected in 2 siblings of a consanguineous family (p.Glu198*). Furthermore, we identified an inherited novel variant in the IGF2 gene (p.Arg156Cys) of a maternally imprinted gene in a less severely affected father and his affected daughter. We detected 2 other novel missense variants in SH2B1 and SOCS2, both were inherited from an unaffected parent., Conclusions: Screening of growth-related genes using NGS-based, large-scale, targeted resequencing identified disease-causing variants in HRAS, FAM111A, and GHR. Considering the increased risk of subjects with HRAS mutations for neoplasms, close clinical monitoring and a thorough discussion of the risk/benefit ratio of the treatment with recombinant IGF-I is mandatory. Segregation analysis proved to be critical in the interpretation of potential SPIGFD-associated gene variations., (© 2017 S. Karger AG, Basel.)

Objective: Copeptin has been reported to be associated with stress, obesity and the metabolic syndrome (MetS) in adults. However, data in childhood are scarce. Therefore, we studied the relationships between copeptin, cortisol, puberty and parameters of the MetS in children., Design: Cross-sectional study., Patients: A total of 51 obese children (10·8 ± 3·2 years, 39% male, 45% prepubertal, body mass index standard deviation score (BMI-SDS) 2·77 ± 0·56) and 24 lean children of similar age, gender and pubertal stage., Measurements: Copeptin, serum cortisol, 24-h urinary free cortisol, BMI-SDS and, as parameters of the MetS, insulin resistance index (HOMA), HbA1c, uric acids, blood pressure and lipids., Results: Copeptin levels were significantly (P = 0·047) higher in obese children (5·8 ± 2·8pmol/l) compared to lean children (4·6 ± 2·2pmol/l). BMI-SDS (β-coefficient 0·38 ± 0·35, P =0·033), but not any parameter of the MetS, was significantly related to copeptin in multiple linear regression analyses adjusted for age, gender and pubertal stage. A 24-h urinary free cortisol (β-coefficient 0·13 ± 0·06, P < 0·001), but not serum cortisol, was significantly related to copeptin in multiple linear regression analyses adjusted for age, gender, pubertal stage and BMI-SDS. Pubertal boys (6·6 ± 2·8pmol/l) demonstrated significantly (P = 0·042) higher copeptin levels compared to pubertal girls (4·8 ± 2·6pmol/l), while copeptin concentrations did not differ between prepubertal girls and boys., Conclusions: Copeptin levels are related to 24-h urinary free cortisol in obese children. Pubertal boys, but not prepubertal boys, demonstrated higher copeptin levels than girls, suggesting that sex hormones are involved in the regulation of copeptin levels. Further studies are necessary to understand the relationship between obesity, cortisol, gender, pubertal stage and copeptin levels., (© 2016 John Wiley & Sons Ltd.)

Background: In boys with suspected partial androgen insensitivity syndrome (PAIS), systematic evidence that supports the long-term prognostic value of identifying a mutation in the androgen receptor gene (AR) is lacking., Objective: To assess the clinical characteristics and long-term outcomes in young men with suspected PAIS in relation to the results of AR analysis., Methods: Through the International Disorders of Sex Development Registry, clinical information was gathered on young men suspected of having PAIS (n = 52) who presented before the age of 16 years and had genetic analysis of AR., Results: The median ages at presentation and at the time of the study were 1 month (range, 1 day to 16 years) and 22 years (range, 16 to 52 years), respectively. Of the cohort, 29 men (56%) had 20 different AR mutations reported. At diagnosis, the median external masculinization scores were 7 and 6 in cases with and without AR mutation, respectively (P = .9), and median current external masculinization scores were 9 and 10, respectively (P = .28). Thirty-five men (67%) required at least one surgical procedure, and those with a mutation were more likely to require multiple surgeries for hypospadias (P = .004). All cases with an AR mutation had gynecomastia, compared to 9% of those without an AR mutation. Of the six men who had a mastectomy, five (83%) had an AR mutation., Conclusions: Boys with genetically confirmed PAIS are likely to have a poorer clinical outcome than those with XY DSD, with normal T synthesis, and without an identifiable AR mutation. Routine genetic analysis of AR to confirm PAIS informs long-term prognosis and management.

Context: Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene., Objective: The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls., Design: Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus., Setting: The study was conducted at a university hospital endocrine research laboratory., Patients: GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46)., Intervention(s): There were no interventions., Main Outcome Measure(s): DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured., Results: The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling., Conclusions: AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high clinical relevance.

Unlabelled: The aim of this study was to characterize the phenotype and treatment of young patients (manifestation <30 years) with diabetes of mitochondrial origin (DMO), based on the German/Austrian DPV (Diabetes Patienten Verlaufsdokumentation) registry. Only 13 (0.02 %) of all patients with diabetes in this cohort were identified with DMO, mainly due to the Kearns-Sayre (n = 5), Pearson (n = 3), or mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome (n = 2). The onset of DMO (14.2, interquartile range (IQR) 7.1-16 years) was later than diabetes onset in individuals with T1D but earlier than in T2D. At manifestation, patients exhibited a mild elevation of blood glucose concentrations (251, IQR 178-299 mg/dl) without ketoacidosis. They had lower body mass index (BMI) values (-1.39 ± 0.28 kg/m(2)) than peers with T1D or T2D (p < 0.0001) and higher triglycerides (211, IQR 134-574 mg/dl) than in T1D (p = 0.04) while there was a high rate of dyslipidemia (86 %). Insulin requirements (0.58, IQR 0.37-0.90 U/kg/d) were between T1D and T2D while glucometabolic control (glycated hemoglobin A1c (HbA1c) 7.4 ± 0.52 %) in DMO was comparable to age-matched T2D and stable over a 5-year follow-up., Conclusion: Primary mitochondrial disorders are a rare cause of juvenile diabetes and likely to be underdiagnosed. As there is clinical overlap with T1D and T2D, dyslipidemia and low body weight may help to identify further DMO cases., What Is Known: • In adults diabetes of mitochondrial origin (DMO) is a rare cause of non-autoimmune diabetes, affecting about 0.8 % of diabetes cases. • Common features are a maternal family history of diabetes, hearing loss and neurological abnormalities. What is New: • In our juvenile cohort 0.02 % of diabetes patients (age < 30 years) were affected by DMO, while Kearns Sayre, MELAS and Pearson syndrome were the most frequent entities. • Juvenile DMO patients exhibited dyslipidemia, higher triglycerides and a lower BMI than peers with T1D or T2D, while some patients also showed retinal changes.

A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5' untranslated region (5'-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5'UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5'UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general.

Background: Data on regional differences in the quality of medical care in Germany are scarce. This study aimed to compare outcome quality and medical treatment of pediatric patients with type 1 diabetes between the federal states of Germany., Methods: 24,928 patients (< 18 years of age) with type 1 diabetes and German residence were selected from the Diabetes-Patienten-Verlaufsdokumentation database. Indicators of outcome quality were HbA1C, overweight prevalence, and rate of severe hypoglycemia. To reflect medical treatment, use of insulin pumps and use of rapid-acting or long-acting insulin analogues were analyzed. Logistic regression models were created for binary variables with federal state as independent predictor. Linear regression was applied for HbA1C and Poisson regression for rate of severe hypoglycemia. Confounders: Sex, age, diabetes duration, migratory background., Results: Disparity was observed for indicators of outcome quality between the 16 federal states of Germany (all p<0.05). After adjustment, HbA1C varied between 55.8 mmol/mol and 67.3 mmol/mol, overweight prevalence between 10.0 and 15.3%, severe hypoglycemia ranged from 0.06 events/PY to 0.21 events/PY. Overall, the best outcome quality appeared to be present in Saxony. Medical treatment also differed. The percentage of pediatrics on insulin pumps varied between 26.3 and 51.8%. The use of rapid-acting analogues ranged from 56.6 to 96.2% and the use of long-acting analogues varied between 41.9 and 96.9% (all p<0.0001)., Conclusions: Medical treatment and outcome quality in pediatrics with type 1 diabetes differed within Germany. Disparities in individual socioeconomic status, regional deprivation, or differences in medical reimbursement decisions might have contributed to the patterns observed., (© Georg Thieme Verlag KG Stuttgart · New York.)

Objective: Heterozygosity in 21-hydroxylase deficiency (21OHD) has been associated with hyperandrogenemic symptoms in children and adults. Moreover, the carrier status is mandatory for genetic counseling. We aimed at defining a hormonal parameter for carrier detection by mass spectrometry., Design: Eleven basal and ACTH-stimulated steroid hormones of heterozygous carriers of CYP21A2 mutations and control individuals were compared., Method: Hormones were determined in plasma samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 58 carriers (35 males, 23 females, age range 6-78 years) and 44 random controls (25 males, 19 females, age range 8-58 years)., Results: Heterozygotes could be identified best applying the 17-hydroxyprogesterone+21-deoxycortisol/cortisol×1000 ((17OHP+21S)/F×1000) equation 30  min after ACTH injection. An optimal cut-off value of 8.4 provided 89% sensitivity and specificity. Considering this data and a published frequency of heterozygotes of 1/50 to 1/61, the positive predictive value (PPV) of this cut-off is 12%. Of note, the negative predictive value (NPV) excluding heterozygosity in a given patient is 99.8%., Conclusion: Considering only marginal biochemical effects anticipated from heterozygosity, the stimulated ((17OHP+21S)/F×1000) identifies and excludes heterozygotes remarkably well. Nevertheless, LC-MS/MS cannot replace genetic testing, since sensitivity and specificity did not reach 100%. However, due to the considerably high NPV of the optimal cut-off and to a specificity of even 100% applying a cut-off higher than 14.7, hormonal assessment of heterozygosity can be of significant aid in conditions with limited access to genetic testing, as in some health care systems. The ((17OHP+21S)/F×1000) equation can guide diagnostic considerations in the differential diagnosis of hyperandrogenism., (© 2015 European Society of Endocrinology.)

Objective: To investigate rates and risk factors of hospital admission for diabetic ketoacidosis (DKA) or severe hypoglycemia in young patients with established type 1 diabetes., Design: In total, 31,330 patients with type 1 diabetes (median age 12.7 years) from the Diabetes Patienten Verlaufsdokumentation (DPV) Prospective Diabetes Registry treated between 2011 and 2013 in Germany were included., Methods: Admission rates for DKA (pH < 7.3 or bicarbonate <15 mmol/l) and severe hypoglycemia (requiring assistance from another person) were calculated by negative binomial regression analysis. Associations of DKA or hypoglycemia with patient and treatment characteristics were assessed by multivariable regression analysis., Results: The mean admission rate for DKA was 4.81/100 patient-years (95% CI, 4.51-5.14). The highest DKA rates were observed in patients with HbA1c ≥ 9.0% (15.83 (14.44-17.36)), age 15-20 years (6.21 (5.61-6.88)) and diabetes duration of 2-4.9 years (5.60 (5.00-6.27)). DKA rate was higher in girls than in boys (5.35 (4.88-5.86) vs 4.34 (3.95-4.77), P = 0.002), and more frequent in migrants than in non-migrants (5.65 (4.92-6.49) vs 4.57 (4.23-4.93), P = 0.008). The mean admission rate for severe hypoglycemia was 1.45/100 patient-years (1.30-1.61). Rates were higher in migrants compared to non-migrants (2.13 (1.72-2.65) vs 1.28 (1.12-1.47), P < 0.001), and highest in individuals with severe hypoglycemia within the preceding year (17.69 (15.63-20.03) vs patients without preceding hypoglycemia 0.42 (0.35-0.52), P < 0.001). Differences remained significant after multivariable adjustment., Conclusions: The identification of at-risk individuals for DKA (patients with high HbA1c, longer diabetes duration, adolescents, girls) and for severe hypoglycemia (patients with preceding severe hypoglycemia, migrants) may facilitate targeted diabetes counselling in order to prevent these complications., (© 2015 European Society of Endocrinology.)

Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualized evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analyzed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analyzed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.

The differential diagnosis of 46,XY disorders of sex development (DSD) is based on the distinction between forms of gonadal dysgenesis and disorders of androgen biosynthesis and action. However, clinical and endocrine evaluations are often not conclusive. Here, we describe an adolescent female with hirsutism and hyperandrogenization at puberty. Her karyotype was 46,XY, and clinical investigation demonstrated clitoromegaly, but no uterine remnants were detected. Histology of the gonads revealed a testicular structure with a Sertoli-cell-only pattern. Endocrine evaluation showed hypergonadotropic hypogonadism, and the Sertoli cell markers inhibin B and anti-Müllerian hormone were also low. Several molecular genetic studies were initiated. While analyses of the androgen receptor gene, the SRD5A2 gene and HSD17B3 gene were uninformative, a novel p.L230R mutation was found in the NR5A1 gene. A mutant construct proved a severe dysfunction of this variant in functional analysis after recreation and transfection into HeLa cells. We conclude that the NR5A1 p.L230R mutation most likely leads to a spatial and time-dependent Leydig cell and Sertoli cell dysfunction during development not causing the classical gonadal dysgenesis phenotype. This case demonstrates that the current classification should be updated to encompass the overlapping phenotypes of some genetic conditions within 46,XY DSD., (© 2015 S. Karger AG, Basel.)

Background/aims: Primary hypoaldosteronism is a rare inborn disorder with life-threatening symptoms in newborns and infants due to an aldosterone synthase defect. Diagnosis is often difficult as the plasma aldosterone concentration (PAC) can remain within the normal range and thus lead to misinterpretation and delayed initiation of life-saving therapy. We aimed to test the eligibility of the PAC/plasma renin concentration (PRC) ratio as a tool for the diagnosis of primary hypoaldosteronism in newborns and infants. Meth ods: Data of 9 patients aged 15 days to 12 months at the time of diagnosis were collected. The diagnosis of primary hypoaldosteronism was based on clinical and laboratory findings over a period of 12 years in 3 different centers in Switzerland. To enable a valid comparison, the values of PAC and PRC were correlated to reference methods., Results: In 6 patients, the PAC/PRC ratio could be determined and showed constantly decreased values <1 (pmol/l)/(mU/l). In 2 patients, renin was noted as plasma renin activity (PRA). PAC/PRA ratios were also clearly decreased. The diagnosis was subsequently genetically confirmed in 8 patients., Conclusion: A PAC/PRC ratio <1 pmol/mU and a PAC/PRA ratio <28 (pmol/l)/(ng/ml × h) are reliable tools to identify primary hypoaldosteronism in newborns and infants and help to diagnose this life-threatening disease faster., (© 2015 S. Karger AG, Basel.)

Background and Objective: It is unclear whether the proportion of infants with a disorder of sex development who are raised as male or female has changed over time. The temporal trends in sex assignment of affected cases entered in the International Disorder of Sex Development (I-DSD) Registry were studied., Methods: Cases of disorders of sex development reported as partial androgen insensitivity syndrome (PAIS; n = 118), disorder of gonadal development (DGD; n = 232), and disorder of androgen synthesis (DAS; n = 104) were divided into those who were born before 1990, 1990-1999, and after 1999. External appearance of the genitalia was described by the external masculinization score., Results: The median (5th-95th percentile) external masculinization scores of those infants with PAIS, DGD, and DAS who were raised as boys were 6 (2-9), 6 (3-9), and 6 (1-12), respectively, and were significantly higher than in those raised as girls (2 [0-6], 2 [0-7], and 0 [0-5], respectively); this difference was maintained in the 3 temporal birth cohorts (P < .01). Of the 118 cases in the pre-1990 cohort, 41 (35%) were raised as boys; of the 148 cases in the 1990-1999 cohort, 60 (41%) were raised as boys; and of the 188 cases in the post-1999 cohort, 128 (68%) were raised as boys., Conclusions: Although there is an association between the external appearance of the genitalia and the choice of sex assignment, there are clear temporal trends in this practice pointing toward an increased likelihood of affected infants being raised as boys. The impact of this change in practice on long-term health outcomes requires additional focus., (Copyright © 2014 by the American Academy of Pediatrics.)

The medical term disorders of sex development (DSDs) is used to describe individuals with an atypical composition of chromosomal, gonadal and phenotypic sex, which leads to differences in the development of the urogenital tract and reproductive system. A variety of genetic factors have been identified that affect sex development during gonadal differentiation or in specific disorders associated with altered androgen biosynthesis or action. The diagnosis of DSDs in individuals and the subsequent management of patients and their families requires a targeted and structured approach, involving a multidisciplinary team with effective communication between the disciplines. This approach includes distinct clinical, imaging, laboratory and genetic evaluations of patients with DSDs. Although treatment of patients with DSDs can include endocrine and surgical options, many patients have concerns that arise from past incorrect treatments that were founded on the traditional binary concept of the sexes. To dispel these concerns, it is necessary to create centres of expertise for DSDs that include physicians, surgeons, psychologists and specialists in diagnostic procedures to manage patients and their families. Additionally, the inclusion of trained peer support in the multidisciplinary DSD team seems to be integral to the supportive management of patients with DSDs. Most importantly, dealing with DSDs requires acceptance of the fact that deviation from the traditional definitions of gender is not necessarily pathologic.

Context: Clinical features of Metabolic Syndrome (MetS) and Cushing's Syndrome are similar, suggesting a pathogenetic role of hypothalamus-pituitary-adrenal axis in MetS., Objective: The aim of the study was to determine whether MetS diagnosis and specific clusters of MetS components (waist circumference, dyslipidemia, hypertension, and impaired glucose metabolism) are associated with serum cortisol (SC) or 24-h urinary free cortisol (UFC) levels., Design and Setting: We conducted cross-sectional analyses of data from our obesity cohort. We studied 264 obese children (age, 11.0 ± 2.8 years; male, 48%; BMI, 28.2 ± 5.4 kg/m(2)). We examined UFC, SC, homeostasis model assessment (HOMA), and features of MetS (waist circumference, blood pressure, fasting lipids, and glucose)., Results: Slightly increased UFC concentrations were measured in 30.7% of the children. Obese children with MetS had significantly (P = .003) higher UFC levels compared with obese children without MetS. Girls demonstrated significantly higher UFC concentrations compared with boys independent of pubertal stage. UFC and SC levels were significantly related to features of MetS, but the associations were stronger for UFC. In multivariate analyses adjusted for age, sex, and body mass index, none of the features of MetS but HOMA index was correlated with UFC, whereas SC demonstrated no significant association to any parameter of MetS or HOMA., Conclusions: Our findings support the hypothesis that changes in the hypothalamus-pituitary-adrenal axis are related to MetS in obesity. UFC seems to be a suitable marker for this relationship. Norm values for UFC adapted to obese children may help to avoid unnecessary dexamethasone suppression tests.

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling.

Background: Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine diseases. Steroid 11β-hydroxylase (P450c11) deficiency (11OHD) is the second most common form of CAH., Aim: The aim of the study was to study the functional consequences of three novel CYP11B1 gene mutations (p.His125Thrfs*8, p.Leu463&#95;Leu464dup and p.Ser150Leu) detected in patients suffering from 11OHD and to correlate this data with the clinical phenotype., Methods: Functional analyses were done by using a HEK293 cell in vitro expression system comparing WT with mutant P450c11 activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein., Results: Two mutations (p.His125Thrfs*8 and p.Leu463&#95;Leu464dup) detected in patients with classic 11OHD showed a complete loss of P450c11 activity. The mutation (p.Ser150Leu) detected in a patient with non-classic 11OHD showed partial functional impairment with 19% of WT activity., Conclusion: Functional mutation analysis enables the correlation of novel CYP11B1 mutations to the classic and non-classic 11OHD phenotype respectively. Mutations causing a non-classic phenotype show typically partial impairment due to reduced maximum reaction velocity comparable with non-classic mutations in 21-hydroxylase deficiency. The increasing number of mutations associated with non-classic 11OHD illustrate that this disease should be considered as diagnosis in patients with otherwise unexplained hyperandrogenism.

Context: The focus of care in disorders of sex development (DSD) is often directed to issues related to sex and gender development. In addition, the molecular etiology remains unclear in the majority of cases., Objective: To report the range of associated conditions identified in the international DSD (I-DSD) Registry., Design, Setting, and Patients: Anonymized data were extracted from the I-DSD Registry for diagnosis, karyotype, sex of rearing, genetic investigations, and associated anomalies. If necessary, clarification was sought from the reporting clinician., Results: Of 649 accessible cases, associated conditions occurred in 168 (26%); 103 (61%) cases had one condition, 31 (18%) had two conditions, 20 (12%) had three conditions, and 14 (8%) had four or more conditions. Karyotypes with most frequently reported associations included 45,X with 6 of 8 affected cases (75%), 45,X/46,XY with 19 of 42 cases (45%), 46,XY with 112 of 460 cases (24%), and 46,XX with 27 of 121 cases (22%). In the 112 cases of 46,XY DSD, the commonest conditions included small for gestational age in 26 (23%), cardiac anomalies in 22 (20%), and central nervous system disorders in 22 (20%), whereas in the 27 cases of 46,XX DSD, skeletal and renal anomalies were commonest at 12 (44%) and 8 (30%), respectively. Of 170 cases of suspected androgen insensitivity syndrome, 19 (11%) had reported anomalies and 9 of these had confirmed androgen receptor mutations., Conclusions: Over a quarter of the cases in the I-DSD Registry have an additional condition. These associations can direct investigators toward novel genetic etiology and also highlight the need for more holistic care of the affected person.

In this study, we present a Sudanese 46,XY patient raised as a female and diagnosed at the age of 20 years with having 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency. She presented with primary amenorrhea, undeveloped breasts and a male pattern of secondary sexual characteristics. Examination of her external genitalia showed type IV genital circumcision. Steroid measurements both in urine and serum pointed to 17β-HSD3 deficiency. A novel homozygous splice-site mutation [c.524 + 2T>A] was detected in intron 7 of the HSD17B3 gene. In this patient, steroid concentration clearly supported both the clinical diagnosis of 17β-HSD3 deficiency and the functional relevance of the mutation. Interestingly, despite of the type IV genital circumcision, the patient expressed her interest in reassigning her sex from female to male., (© 2014 S. Karger AG, Basel.)

Androgens are important for male sex development and physiology. Their actions are mediated by the androgen receptor (AR), a ligand-dependent nuclear transcription factor. The activity of the AR is controlled at multiple stages due to ligand binding and induced structural changes assisted by the foldosome, compartmentalization, recruitment of coregulators, posttranslational modifications and chromatin remodeling, leading to subsequent transcription of androgen-responsive target genes. Beside these short-term androgen actions, there is phenomenological and experimental evidence of long-term androgen programming in mammals and in the human during sensitive programming time windows, both pre- and postnatally. At the molecular level, research into androgen insensitivity syndrome has unmasked androgen programming at the transcriptome level, in genital fibroblasts and peripheral blood mononuclear cells, and at the epigenome level. Androgens are crucial for male sex development and physiology during embryogenesis, at puberty and in adult life. Testosterone and its more potent metabolite, dihydrotestosterone, which is converted from testosterone within the target cell by 5α-reductase II, are the main androgens involved in male sex differentiation. Androgen action is mediated by a single AR. The AR belongs to the nuclear receptor 3 group C, composed of the glucocorticoid receptor (NR3C1), mineralocorticoid receptor (NR3C2), progesterone receptor (NR3C3) and AR (NR3C4), and acts as a ligand-dependent transcription factor., (© 2014 S. Karger AG, Basel.)

Background: Mutations of the CYP17A1 gene cause 17α-hydroxylase deficiency (17OHD) resulting in 46,XY disorder of sex development, hypertension, hypokalemia and absent pubertal development. It is a rare, autosomal recessive form of congenital adrenal hyperplasia (CAH)., Patient: We report on a neonate with prenatally determined 46,XY karyotype. At 20 weeks of gestation, lack of development of male external genitalia was noticed. A phenotypically female child was born at 41 weeks of gestation., Results: Postnatal ultrasound revealed testes in both labia majora, an absence of uterus and normal adrenal glands. Steroid hormone analysis in serum revealed low basal levels of cortisol, testosterone and androstenedione in the presence of massively elevated corticosterone at the age of 2 weeks. The urinary steroid profile from spot urine showed excessive excretion of 17-desoxysteroids, decreased glucocorticoid metabolites and absent C19 steroids, thus proving 17OHD. Molecular analysis identified a novel mutation of the CYP17A1 gene: c.896T>A (p.I299N) in exon 5. Substitution with hydrocortisone was started. The child is raised as a girl and is developing well so far., Conclusion: Herein, we report the unusually early diagnosis of a newborn with the rare CAH form of 17OHD allowing an early start of treatment., (© 2014 S. Karger AG, Basel.)

Intersex is an inherited incongruence of chromosomal, gonadal, and genital sexual characteristics. A typical clinical situation of intersex is the ambiguous genitalia in the newborn. Diagnostics, counseling, and therapy should be offered by specialized multidisciplinary health-care teams. The focus is not only on medical issues but also on psychological, social, and ethical aspects. In the international literature, intersex is now termed "disorders of sex development" (DSD). Alternatively, some authors use "differences of sex development" to underline that patients do not necessarily feel they have a "disorder" but rather a "difference" of sex development compared with normal sex development.

Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS) due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

Context: Little information is available on the steroid hormone profiles in obese children and their changes after weight loss., Objective: We compared liquid chromatography-tandem mass spectrometry of serum steroid hormone profiles between obese and normal-weight children and studied the differential effects of weight loss on these hormones., Design: This study was a cross-sectional comparison between obese and normal-weight children and a longitudinal 1-year follow-up study during lifestyle intervention in obese children., Setting: The setting of the study was primary care., Patients: Forty obese prepubertal (mean age 8.5 ± 2.1 years, 48% female, mean body mass index 24.8 ± 3.5 kg/m(2)) and 40 normal-weight children matched for gender, age, and pubertal stage., Intervention: The study consisted of an outpatient 1-year intervention program based on exercise, behavior, and nutrition therapy., Main Outcomes Measures: Progesterone, 17-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione, T, dihydrotestosterone, insulin resistance index of the homeostasis model assessment, and blood pressure were measured., Results: Prepubertal obese children showed significantly increased androgens (DHEAS, androstenedione, T), mineralocorticoid precursor corticosterone, and glucocorticoids (11-deoxycortisol, cortisol, cortisone) compared with normal-weight children. In contrast to 20 obese children without weight loss, the 20 obese children with substantial weight loss demonstrated a significant decrease of cortisol, cortisone, and corticosterone. Androstenedione and T decreased but DHEAS remained elevated. Changes of the homeostasis model assessment correlated significantly positively with changes of cortisol (r = 0.38) and cortisone (r = 0.43) in partial regression analyses adjusted to changes of weight status., Conclusions: In obese prepubertal children, the increased androgens, mineralocorticoid precursors, and glucocorticoids were responsive to weight loss in contrast to DHEAS, suggesting that DHEAS does not seem to be regulated by changes in body mass index.

Objective: We aimed at developing and cross-validating a mathematical prediction model for an optimal basal insulin infusion pattern for children with type 1 diabetes on continuous subcutaneous insulin infusion therapy (CSII)., Research Design and Methods: We used the German/Austrian DPV-Wiss database for quality control and scientific surveys in pediatric diabetology and retrieved all CSII patients <20 years of age (November 2009). A total of 1,248 individuals from our previous study were excluded (dataset 1), resulting in 6,063 CSII patients (dataset 2) (mean age 10.6 ± 4.3 years). Only the most recent basal insulin infusion rates (BRs) were considered. BR patterns were identified and corresponding patients sorted by unsupervised clustering. Logistic regression analysis was applied to calculate the probabilities for each BR pattern. Equations were based on both independent datasets separately, and probabilities for BR patterns were cross-validated using typical test patients., Results: Of the 6,063 children, 5,903 clustered in one of four major circadian BR patterns, confirming our previous study. The oldest age-group (mean age 12.8 years) was represented by 2,490 patients (42.18%) with a biphasic dawn-dusk pattern (BC). A broad single insulin maximum at 9-10 p.m. (F) was unveiled by 853 patients (14.45%) (mean age 6.3 years). Logistic regression analysis revealed that age, to a lesser extent duration of diabetes, and partly sex predicted BR patterns. Cross-validation revealed almost identical probabilities for BR patterns BC and F in the two datasets but some variation in the remaining two BR patterns., Conclusions: Reconfirmation of four key BR patterns in two very large independent cohorts supports that these patterns are realistic approximations of the circadian distribution of insulin needs in children with type 1 diabetes. Prediction of an optimal pattern a priori can improve initiation and clinical follow-up of CSII in children and adolescents. In addition, these BR patterns represent valuable information for insulin-infusion algorithms in closed-loop CSII.

Background: Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disease caused by mutations in the genes encoding the human mineralocorticoid receptor (MR) or the α (SCNN1A), β (SCNN1B) or γ (SCNN1G) subunit of the epithelial Na(+) channel (ENaC). While autosomal dominant mutation of the MR cause renal PHA1, autosomal recessive mutations of the ENaC lead to systemic PHA1. In the latter, affected children suffer from neonatal onset of multi-organ salt loss and often exhibit cystic fibrosis-like pulmonary symptoms., Objective: We searched for underlying mutations in seven unrelated children with systemic PHA1, all offsprings of healthy consanguineous parents., Methods and Results: Amplification of the SCNN1A gene and sequencing of all 13 coding exons unraveled mutations in all of our patients. We found five novel homozygous mutations (c.587&#95;588insC in two patients, c.1342&#95;1343insTACA, c.742delG, c.189C>A, c.1361-2A>G) and one known mutation (c.1474C>T) leading to truncation of the αENaC protein. All parents were asymptomatic heterozygous carriers of the respective mutations, confirming the autosomal recessive mode of inheritance. Five out of seven patients exhibited pulmonary symptoms in the neonatal period., Conclusion: The α subunit is essential for ENaC function and mutations truncating the pore-forming part of the protein leading to systemic PHA1. Based on current knowledge, the pulmonary phenotype cannot be satisfactorily predicted.

Background: The steroidogenic acute regulatory protein (StAR) is essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause lipoid congenital adrenal hyperplasia., Objective and Methods: To identify causative mutations in a patient presenting with adrenal failure during early infancy. The objective was to study the functional and structural consequences of the novel StAR mutation p.Trp147Arg in a Turkish patient detected in compound heterozygosity with the p.Glu169Lys mutation., 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.Trp147Arg mutant. The previously described p.Glu169Lys mutant led to significantly lower cholesterol conversion than wild-type StAR protein. As derived from three-dimensional protein modeling, the residue W147 is stabilizing the C-terminal helix in a closed conformation hereby acting as gatekeeper of the ligand cavity of StAR., Conclusions: The novel mutation p.Trp147Arg causes primary adrenal insufficiency and complete sex reversal in the 46,XY patient. Clinical disease, in vitro studies and three-dimensional protein modeling of the mutation p.Trp147Arg underscore the relevance of this highly conserved residue for StAR protein function., (Copyright © 2013 S. Karger AG, Basel.)

Background: Sensitive and accurate determination of steroids is essential for diagnosing congenital and acquired adrenal diseases. Since plasma concentrations change during childhood, age-specific reference ranges are the prerequisite for clinical interpretation. The objectives of this study were to develop a sensitive and reliable method for simultaneous detection and quantification of progesterone, 17-hydroxyprogesterone, deoxycorticosterone (DOC), 11-deoxycortisol, 21-deoxycortisol, corticosterone, cortisol (F) and cortisone (E) by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and to establish age- and sex-specific reference ranges from birth to adulthood., Methods: All eight steroids were measured simultaneously in 0.1 ml plasma by UPLC-MS/MS. Samples of 905 children were measured and grouped in five age groups., Results: The assay was linear up to 70 ng/ml (700 ng/ml for F; r(2) > 0.992). The limit of detection ranged between 0.01 ng/ml for DOC and 0.07 ng/ml for E. Correlations with radioimmunoassays yielded a coefficient of determination between 0.82 and 0.99. Reference data are reported as a function of age and sex., Conclusions: The UPLC-MS/MS method presented here for the simultaneous detection of eight C-21 adrenal hormones together with the detailed reference ranges for children provides a valuable methodology for assessing adrenal steroids in clinical routine and research., (Copyright © 2013 S. Karger AG, Basel.)

Unlabelled: Study Type--Therapy (case series) Level of Evidence 4. What's known on the subject? and What does the study add? In some individuals with disorders of sex development (DSD), gonadal tumour risk is increased. The individual risk is estimated based on the molecular diagnosis and the age and approaches 30% in the high-risk group. In the past, early gonadectomy has been advised for all individuals with 46XY DSD. Gonadectomy clearly represents an overtreatment for many individuals with 46XY DSD. Thus, further clinical indicators of individual tumour risk are urgently needed. The present study provides a comprehensive description of gonadal morphology, as seen during laparoscopy. For the first time, laparoscopic features, molecular diagnosis and histopathological findings are presented in a comprehensive context. The present study adds a detailed morphological description of the variability found in different subgroups of 46XY DSD. As three of four detected tumours were microscopic, early diagnosis by inspection appears unfeasible. Biopsy, gonadopexy and precise localisation of the gonad will potentially allow for gonadal preservation in well-defined clinical situations., Objective: • To investigate the role of laparoscopy for the early detection of gonadal tumours, with emphasis on gonadal preservation, in patients with 46XY disorders of sex development (DSD). In patients with DSD, gonadectomy is frequently recommended and depending on the age and the molecular diagnosis, an increased gonadal tumour risk exists and undesired hormone effects may arise. However, gonadectomy is irreversible and impacts considerably on body image. It represents an overtreatment for some patients and should be considered after a comprehensive diagnostic evaluation. Laparoscopy is an important technique, because it is able to retrieve small gonads and allows guided biopsies., Patients and Methods: • We performed laparoscopic assessment of the gonads in 40 patients with various 46XY DSD. • In all, 77 gonads were evaluated, images were analysed and compared with histological findings. • Laparoscopic procedures included gonadectomy, biopsy, laparoscopic orchidolysis or the Fowler-Stephens procedure as well as the removal or splitting of uterine remnants., Results: • In all, 19 patients underwent gonadectomy and tumours were discovered in four. • Three patients had only microscopic evidence of tumour, in one the tumour was diagnosed intraoperatively. • In 21 patients, biopsies were taken and the gonads preserved. • Laparoscopic biopsy and gonadopexy was performed in six patients with complete androgen insensitivity syndrome (CAIS)., Conclusion: • Laparoscopy and biopsy detected three microscopic tumours, one tumour was macroscopically evident. • In CAIS, gonadopexy improved the visibility of the gonads on postoperative ultrasonography. This procedure facilitated the examination of the gonad at follow-up. • In complete gonadal dysgenesis, a highly variable morphology of the gonads was found. Laparoscopy improved exposure of gonads and Müllerian structures, and facilitated biopsies and organ-preserving procedures., (© 2012 BJU INTERNATIONAL.)

Context: Acrodysostosis is a rare skeletal dysplasia that is associated with multiple resistance to G protein-coupled receptor (GPCR) signaling hormones in a subset of patients. Acrodysostosis is genetically heterogeneous because it results from heterozygous mutations in PRKAR1A or PDE4D, two key actors in the GPCR-cAMP-protein kinase A pathway., Objective: Our objective was to identify the phenotypic features that distinguish the two genotypes causing acrodysostosis., Patients and Methods: Sixteen unrelated patients with acrodysostosis underwent a candidate-gene approach and were investigated for phenotypic features., Results: All patients had heterozygous de novo mutations. Fourteen patients carried a PRKAR1A mutation (PRKAR1A patients), five each a novel PRKAR1A mutation (p.Q285R, p.G289E, p.A328V, p.R335L, or p.Q372X), nine the reported PRKAR1A p.R368X mutation; two patients harbored a mutation in PDE4D (PDE4D patients) (one novel mutation, p.A227S; one reported, p.E590A). All PRKAR1A, but none of the PDE4D mutated patients were resistant to PTH and TSH. Two PRKAR1A patients each with a novel mutation presented a specific pattern of brachydactyly. One PDE4D patient presented with acroskyphodysplasia. Additional phenotypic differences included mental retardation in PDE4D patients. In addition, we report the presence of pigmented skin lesions in PRKAR1A and PDE4D patients, a feature not yet described in the acrodysostosis entity., Conclusions: All PRKAR1A and PDE4D patients present similar bone dysplasia characterizing acrodysostosis. Phenotypic differences, including the presence of resistance to GPCR-cAMP signaling hormones in PRKAR1A but not PDE4D patients, indicate phenotype-genotype correlations and highlight the specific contributions of PRKAR1A and PDE4D in cAMP signaling in different tissues.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease of steroid biosynthesis in humans. More than 90% of all CAH cases are caused by mutations of the 21-hydroxylase gene (CYP21A2), and approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. In this study, the CYP21A2 gene was genotyped in 50 patients in Tunisia with the clinical diagnosis of 21-hydroxylase deficiency. CYP21A2 mutations were identified in 87% of the alleles. The most common point mutation in our population was the pseudogene specific variant p.Q318X (26%). Three novel single nucleotide polymorphism (SNP) loci were identified in the CYP21A2 gene which seems to be specific for the Tunisian population. The overall concordance between genotype and phenotype was 98%. With this study the molecular basis of CAH has been characterized, providing useful results for clinicians in terms of prediction of disease severity, genetic and prenatal counseling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Steroid 11β hydroxylase deficiency (11β-OHD) (OMIM # 202010) is the second most common form of congenital adrenal hyperplasia (CAH), accounting for 5-8% of all cases. It is an autosomal recessive enzyme defect impairing the biosynthesis of cortisol. The CYP11B1 gene encoding this enzyme is located on chromosome 8q22, approximately 40kb from the highly homologous CYP11B2 gene encoding for the aldosterone synthase. Virilization and hypertension are the main clinical characteristics of this disease. In Tunisia, the incidence of 11β-OHD appears higher due to a high rate of consanguinity (17.5% of congenital adrenal hyperplasia). The identical presentation of genital ambiguity (females) and pseudo-precocious puberty (males) can lead to misdiagnosis with 21 hydroxylase deficiency. The clinical hallmark of 11β hydroxylase deficiency is variable, and biochemical identification of elevated precursor metabolites is not usually available. In order to clarify the underlying mechanism causing 11β-OHD, we performed the molecular genetic analysis of the CYP11B1 gene in a female patient diagnosed as classical 11β-OHD. The nucleotide sequence of the patient's CYP11B1 revealed two novel mutations in exon 4: a missense mutation that converts codon AGT (serine) to ATT (isoleucine) (c.650G>T; p.S217I) combined with an insertion of a thymine at the c.652-653 position (c.652&#95;653insT). This insertion leads to a reading frame shift, multiple incorrect codons, and a premature stop in codon 258, that drastically affects normal protein function leading to a severe phenotype with ambiguous genitalia of congenital adrenal hyperplasia due to 11β hydroxylase deficiency., (Copyright © 2011 Elsevier Inc. All rights reserved.)

17β-hydroxysteroid dehydrogenase 3 (17β-HSD 3) deficiency is a rare cause of 46,XY disorders of sex development (DSD). At puberty, these patients experience a surge of androstenedione and also testosterone, leading to substantial virilization. The origin of testosterone synthesis in these patients remains elusive. We investigated the expression of the isoenzyme AKR1C3 (17β-HSD 5) in the testis and patient-derived genital skin fibroblasts (GSF) as well as the ability of GSF to synthesize testosterone. Supernatants of GSF cultures and serum samples of one patient before and after gonadectomy were analyzed by liquid and gas chromatography/mass spectrometry. The androgenic potential of GSF-derived supernatants was also assessed by androgen receptor-mediated transactivation of a reporter gene in transiently transfected Chinese hamster ovary cells. Although AKR1C3 is expressed both in the testes and in GSF, androstenedione is rapidly metabolized and is not synthesized to testosterone. The transactivation potential of GSF supernatants towards the androgen receptor is declining within 48 h. However, under testis-equivalent androstenedione concentration, testosterone can be synthesized in 17β-HSD 3-negative GSF. After gonadectomy, both androstenedione and testosterone decline rapidly in vivo. In 17β-HSD 3 deficiency, relevant amounts of testosterone are synthesized most probably through AKR1C3 in the testis and not peripherally in GSF., (Copyright © 2012 S. Karger AG, Basel.)

Background: Non-syndromic clefts of the orofacial region occur in approximately 1 per 500 to 2,500 live births, depending on geographical area and ethnicity. It can be supposed that the disruption of the normal facial structure and the long-standing pressure of treatment from birth to adulthood bring about a range of life stressors which may lead to a long-lasting impact on affected subjects throughout their lives. Therefore, the present study aimed to assess different aspects of psychosocial stress in affected individuals., Methods: The study was divided into two parts: first, the Trier Social Stress Test which involves uncontrollability and high levels of social-evaluative stress under real conditions and second, the query of various aspects of coping with psychosocial stress. The test group consisted of 30 affected adult subjects, and an equally sized control group of unaffected volunteers. Cortisol dysregulation was determined by saliva samples before and after stress induction. Meanwhile, participants were asked to complete the SVF 120 stress-coping questionnaire., Results: The analysis of saliva samples showed a similar baseline concentration as well as a similar increase in cortisol levels after stress induction for both groups. Subsequently, the decline in cortisol concentrations was significantly faster in the CLP group (course: p<0.001; groups: p = 0.102; interaction: p = 0.167). The evaluation of the stress-coping questionnaire revealed a significantly shorter rumination about a stressful event in individuals with CLP-related malformations (p = 0.03)., Conclusion: We conclude that adults with CLP have significantly better stress-coping strategies than their healthy peers., Trial Registration: German Clinical Trials Organization DRKS00003466.

Hormone concentrations decline with aging. Up to now it was not clear, whether the decrease of hormone concentrations in blood samples are also present in cutaneous suction blister fluids, and whether skin from different anatomical sites shows different hormone concentrations.Analysis of suction blister fluids and paired blood samples from young (mean 27.8 y) and old (mean 62.6 y) male subjects by UPLC-MS/MS showed that DHEA concentration in blood samples was age-dependently significantly reduced, but increased in suction blister fluids, while androstenedione behaved in an opposite manner to DHEA. Testosterone decreased age-dependently in blood samples and in suction blister fluids. Regarding skin sites, DHEA was lower in samples from upper back compared with samples from the forearm. In contrast, the concentrations of androstenedione and testosterone were higher in samples from upper back.In vitro analyses showed that SZ95 sebocytes, but neither primary fibroblasts nor keratinocytes, were able to use DHEA as precursor for testosterone biosynthesis, which was confirmed by expression analysis of 3β-hydroxysteroiddehydrogenase in skin biopsies.In conclusion, we show an inverse pattern of DHEA and androstenedione concentrations in blood vs. suction blister fluids, highlighting age-dependent changes of dermal testosterone biosynthesis, and a stronger metabolism in young skin. Furthermore, sebocytes play a central role in cutaneous androgen metabolism.

Background/aim: X-linked adrenal hypoplasia congenita (AHC) is a rare disorder characterized by primary adrenal insufficiency and hypogonadic hypogonadism. It is caused by deletions or point mutations of the NR0B1 gene, on Xp21. AHC can be associated with glycerol kinase deficiency, Duchenne muscular dystrophy and mental retardation (MR), as part of a contiguous gene deletion syndrome. A synthetic probe set for multiplex ligation-dependent probe amplification analysis was developed to confirm and characterize NR0B1 deletions in patients with AHC and to correlate their genotypes with their divergent phenotypes., Results: In 2 patients, isolated AHC was confirmed, while a patient at risk for metabolic crisis was revealed as the deletion extends to the GK gene. A deletion extending to IL1RAPL1 was confirmed in both patients showing MR. Thus, a good genotype-phenotype correlation was confirmed., Conclusions: Multiplex ligation-dependent probe amplification analysis is a valuable tool to detect NR0B1 and contiguous gene deletions in patients with AHC. It is especially helpful for IL1RAPL1 deletion detection as no clinical markers for MR are available. Furthermore, multiplex ligation-dependent probe amplification has the advantage to identify female carriers that, depending on the deletion extension, have a high risk of giving birth to children with MR, AHC, glycerol kinase deficiency and Duchenne muscular dystrophy., (Copyright © 2012 S. Karger AG, Basel.)

Background: There are very few laboratory markers which reflect the biological sensitivity of children to recombinant human growth hormone (rhGH) treatment. Genome-wide transcriptional changes in peripheral blood mononuclear cells (PBMC) have been widely used as functional readout for different pharmacological stimuli., Objective: To characterize transcriptional changes in PBMC induced by rhGH during a routine short-term IGF-I generation test (IGFGT) in children with growth disorders., Materials and Methods: Blood was obtained for IGF-I determination and RNA-preparation from PBMC of 12 children before and after 4days treatment with 30μgrhGH/kg body weight/day s.c. Transcriptional changes were assessed by cDNA-microarrays in the first six children. Selected genes were validated in all 12 cases by RT-qPCR., Results: Serum IGF-I rose in all patients except one (p<0.0001), confirming biological response to rhGH. Unsupervised microarray data analysis in the first six children revealed 313 transcripts with abundant transcriptional changes but considerable inter-individual variability of response patterns. Many patients showed a large cluster of up-regulated genes, including EGR1, EGR2, FOS and to a lesser extent STAT2 and 5b. Exemplarily, EGR1, EGR2 and FOS data were independently reproduced by RT-qPCR. Gene ontology analysis revealed that pathways involved in cell proliferation and immune functions were significantly over represented., Conclusion: The IGFGT is a suitable method for measuring reproducible and biologically conclusive transcriptional changes in PBMC. As our unsupervised data analysis strategy exposed a considerable inter-individual variability of response profiles a search for molecules of diagnostic and even prognostic value needs to be based on large long-term studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)