Search

Background Lifestyle based weight loss interventions are hampered by long-term inefficacy. Prediction of individuals successfully reducing body weight would be highly desirable. Although sympathetic activity is known to contribute to energy homeostasis, its predictive role in body weight maintenance has not yet been addressed. Objectives We investigated, whether weight regain could be modified by a weight maintenance intervention and analyzed the predictive role of weight loss-induced changes of the sympathetic system on long-term weight regain. Design 156 subjects (age > 18; BMI ≥ 27 kg/m2) participated in a 12-week weight reduction program. After weight loss (T0), 143 subjects (weight loss > 8%) were randomized to a 12-month lifestyle intervention or a control group. After 12 months (T12) no further intervention was performed until month 18 (T18). Weight regain at T18 (regainBMI) was the primary outcome. Evaluation of systemic and tissue specific estimates of sympathetic system was a pre-defined secondary outcome. Results BMI was reduced by 4.67 ± 1.47 kg/m2 during the initial weight loss period. BMI maintained low in subjects of the intervention group until T12 (+0.07 ± 2.98 kg/m2; p = 0.58 compared to T0), while control subjects regained +0.98 ± 1.93 kg/m2 (p Conclusions Our lifestyle intervention successfully maintained body weight during the intervention period. However, no long-term effect could be observed beyond the intervention period. Predictive sympathetic activity was not persistently modified by the intervention, which may partially explain the lack of long-term success of such interventions.

Genetic defects underlying the melanocortin-4 receptor (MC4R) signaling pathway lead to severe obesity. Three severely obese LEPR-deficient individuals were administered the MC4R agonist setmelanotide, resulting in substantial and durable reductions in hyperphagia and body weight over an observation period of 45–61 weeks. Compared to formerly developed and tested MC4R agonists, setmelanotide has the unique capability of activating nuclear factor of activated T cell (NFAT) signaling and restoring function of this signaling pathway for selected MC4R variants. Our data demonstrate the potency of setmelanotide in treatment of individuals with diverse MC4R-related pathway deficiencies. Treatment with setmelanotide, a new-generation MC4R agonist, provides durable weight loss in hyperphagic, leptin receptor–deficient patients, suggesting a pharmacological avenue to treat patients with various MC4R pathway defects.

Patients with rare defects in the gene encoding proopiomelanocortin (POMC) have extreme early-onset obesity, hyperphagia, hypopigmentation, and hypocortisolism, resulting from the lack of the proopiomelanocortin-derived peptides melanocyte-stimulating hormone and corticotropin. In such patients, adrenal insufficiency must be treated with hydrocortisone early in life. No effective pharmacologic treatments have been available for the hyperphagia and obesity that characterize the condition. In this investigator-initiated, open-label study, two patients with proopiomelanocortin deficiency were treated with setmelanotide, a new melanocortin-4 receptor agonist. The patients had a sustainable reduction in hunger and substantial weight loss (51.0 kg after 42 weeks in Patient 1 and 20.5 kg after 12 weeks in Patient 2).

Naturally occurring thyrotropin (TSH) mutations are rare, which is also the case for the homologous heterodimeric glycoprotein hormones (GPHs) follitropin (FSH), lutropin (LH), and choriogonadotropin (CG). Patients with TSH-inactivating mutations present with central congenital hypothyroidism. Here, we summarize insights into the most frequent loss-of-function β-subunit of TSH mutation C105Vfs114X, which is associated with isolated TSH deficiency. This review will address the following question. What is currently known on the molecular background of this TSH variant on a protein level? It has not yet been clarified how C105Vfs114X causes early symptoms in affected patients, which are comparably severe to those observed in newborns lacking any functional thyroid tissue (athyreosis). To better understand the mechanisms of this mutant, we have summarized published reports and complemented this information with a structural perspective on GPHs. By including the ancestral TSH receptor agonist thyrostimulin and pathogenic mutations reported for FSH, LH, and choriogonadotropin in the analysis, insightful structure function and evolutionary restrictions become apparent. However, comparisons of immunogenicity and bioactivity of different GPH variants is hindered by a lack of consensus for functional analysis and the diversity of used GPH assays. Accordingly, relevant gaps of knowledge concerning details of GPH mutation-related effects are identified and highlighted in this review. These issues are of general importance as several previous and recent studies point towards the high impact of GPH variants in differential signaling regulation at GPH receptors (GPHRs), both endogenously and under diseased conditions. Further improvement in this area is of decisive importance for the development of novel targeted therapies.

Context The α subunit of the stimulatory G protein (Gαs) links numerous receptors to adenylyl cyclase. Gαs, encoded by GNAS, is expressed predominantly from the maternal allele in certain tissues. Thus, maternal heterozygous loss-of-function mutations cause hormonal resistance, as in pseudohypoparathyroidism type Ia, whereas somatic gain-of-function mutations cause hormone-independent endocrine stimulation, as in McCune-Albright syndrome. Objective We report two unrelated boys presenting with a new combination of clinical findings that suggest both gain and loss of Gαs function. Design and Setting Clinical features were studied and sequencing of GNAS was performed. Signaling capacities of wild-type and mutant Gαs were determined in the presence of different G protein–coupled receptors (GPCRs) under basal and agonist-stimulated conditions. Results Both unrelated patients presented with unexplained hyponatremia in infancy, followed by severe early onset gonadotrophin-independent precocious puberty and skeletal abnormalities. An identical heterozygous de novo variant (c.1136T>G; p.F376V) was found on the maternal GNAS allele in both patients; this resulted in a clinical phenotype that differed from known Gαs-related diseases and suggested gain of function at the vasopressin 2 receptor (V2R) and lutropin/choriogonadotropin receptor (LHCGR), yet increased serum PTH concentrations indicative of impaired proximal tubular PTH1 receptor (PTH1R) function. In vitro studies demonstrated that Gαs-F376V enhanced ligand-independent signaling at the PTH1R, LHCGR, and V2R and, at the same time, blunted ligand-dependent responses. Structural homology modeling suggested mutation-induced modifications at the C-terminal α5 helix of Gαs that are relevant for interaction with GPCRs and signal transduction. Conclusions The Gαs p.F376V mutation causes a previously unrecognized multisystem disorder.

Context The optimal levothyroxine (LT4) dose to treat congenital hypothyroidism (CH) remains unclear, with debate over whether higher starting doses (>10 µg/kg) are necessary and safe for a normal intelligence quotient (IQ). Objective To examine psychomotor, metabolic, and quality of life (QoL) outcomes in patients with CH treated with a mean high initial LT4 dose. Design, settings, participants A cross-sectional cohort study of patients with CH identified in the Berlin newborn screening program from 1979 to 2003; 76 patients with CH (mean age, 18 years; mean initial LT4 dose, 13.5 µg/kg) and 40 siblings completed the study. Main outcome measures Psychomotor (Wechsler Intelligence Test, CNS Vital Signs), QoL (short form-36 Health Survey), anthropometric (body mass index, height), and metabolic (intima media thickness, laboratory parameters) outcomes were compared with those of healthy siblings. Mean values and percentage of episodes of elevated thyroxine (T4) and tri-jod-thyronin (T3) and suppressed thyrotropin (TSH) before age 2 years were analyzed. A meta-analysis of CH treatment studies was performed. Results There were no significant differences in IQ, QoL, or other outcome measures in patients with CH compared with controls. Most T4 levels were high before age 2 years and during subsequent testing, but mean T3 and TSH levels remained normal. The meta-analysis showed a significant IQ difference in severe vs mild CH cases only when treatment started with an LT4 dose Conclusions High initial LT4 dosing was effective and safely achieved optimal cognitive development in patients with CH, including those severely affected. Supranormal T4 values during infancy were not associated with impaired IQ in adolescence.

Context Early-onset obesity, characteristic for disorders affecting the leptin–melanocortin pathway, is also observed in pseudohypoparathyroidism type 1A (PHP1A), a disorder caused by maternal GNAS mutations that disrupt expression or function of the stimulatory G protein α-subunit (Gsα). Mutations and/or epigenetic abnormalities at the same genetic locus are also the cause of pseudohypoparathyroidism type 1B (PHP1B). However, although equivalent biochemical and radiographic findings can be encountered in these related disorders caused by GNAS abnormalities, they are considered distinct clinical entities. Objectives To further emphasize the overlapping features between both disorders, we report the cases of several children, initially brought to medical attention because of unexplained early-onset obesity, in whom PHP1B or PHP1A was eventually diagnosed. Patients and Methods Search for GNAS methylation changes or mutations in cohorts of patients with early-onset obesity. Results Severe obesity had been noted in five infants, with a later diagnosis of PHP1B due to STX16 deletions and/or abnormal GNAS methylation. These findings prompted analysis of 24 unselected obese patients, leading to the discovery of inherited STX16 deletions in 2 individuals. Similarly, impressive early weight gains were noted in five patients, who initially lacked additional Albright hereditary osteodystrophy features but in whom PHP1A due to GNAS mutations involving exons encoding Gsα was diagnosed. Conclusions Obesity during the first year of life can be the first clinical evidence for PHP1B, expanding the spectrum of phenotypic overlap between PHP1A and PHP1B. Importantly, GNAS methylation abnormalities escape detection by targeted or genome-wide sequencing strategies, raising the question of whether epigenetic GNAS analyses should be considered for unexplained obesity.

The monocarboxylate transporter 8 (MCT8) is a member of the major facilitator superfamily (MFS). These membrane-spanning proteins facilitate translocation of a variety of substrates, MCT8 specifically transports iodothyronines. Mutations in MCT8 are the underlying cause of severe X-linked psychomotor retardation. At the molecular level, such mutations led to deficiencies in substrate translocation due to reduced cell-surface expression, impaired substrate binding, or decreased substrate translocation capabilities. However, the causal relationships between genotypes, molecular features of mutated MCT8, and patient characteristics have not yet been comprehensively deciphered. We investigated the relationship between pathogenic mutants of MCT8 and their capacity to form dimers (presumably oligomeric structures) as a potential regulatory parameter of the transport function of MCT8. Fourteen pathogenic variants of MCT8 were investigated in vitro with respect to their capacity to form oligomers. Particular mutations close to the substrate translocation channel (S194F, A224T, L434W, and R445C) were found to inhibit dimerization of MCT8. This finding is in contrast to those for other transporters or transmembrane proteins, in which substitutions predominantly at the outer-surface inhibit oligomerization. Moreover, specific mutations of MCT8 located in transmembrane helix 2 (del230F, V235M, and ins236V) increased the capacity of MCT8 variants to dimerize. We analyzed the localization of MCT8 dimers in a cellular context, demonstrating differences in MCT8 dimer formation and distribution. In summary, our results add a new link between the functions (substrate transport) and protein organization (dimerization) of MCT8, and might be of relevance for other members of the MFS. Finally, the findings are discussed in relationship to functional data combined with structural–mechanistical insights into MCT8.

MAMLD1 is suggested to play a role in the development of 46,XY disorders of sexual development (46,XY DSD). So far, mutations in this gene have been detected in several cases of hypospadias with normal testosterone levels at birth. From in vitro studies it was concluded that Mamld1 might transiently affect testosterone synthesis during genital development. We describe the first MAMLD1 mutation in a 46,XY patient with complete gonadal dysgenesis. The novel MAMLD1 missense mutation (p.P677L) results in a severely reduced transactivation in vitro of the promoter of the MAMLD1 target gene HES3/Hes3. However, as knowledge about the functional role of MAMLD1 in gonadal development is limited, we suggest that additional factors (digenic or oligogenic cause) play a role in the development of complete gonadal dysgenesis in this patient.

Activation of trace amine-associated receptor 1 (TAAR1) in endocrine pancreas is involved in weight regulation and glucose homeostasis. The purpose of this study was the identification and characterization of potential TAAR1 variants in patients with overweight/obesity and disturbed glucose homeostasis. Screening for TAAR1 variants was performed in 314 obese or overweight patients with impaired insulin secretion. The detected variants were functionally characterized concerning TAAR1 cell surface expression and signaling properties and their allele frequencies were determined in the population- based Study of Health in Pomerania (SHIP). Three heterozygous carriers of the single nucleotide missense variants p.Arg23Cys (R23C, rs8192618), p.Ser49Leu (S49L, rs140960896), and p.Ille171Leu (I171L, rs200795344) were detected in the patient cohort. While p.Ser49Leu and p.Ille171Leu were found in obese/overweight patients with slightly impaired glucose homeostasis, p.Arg23Cys was identified in a patient with a complete loss of insulin production. Functional in vitro characterization revealed a like wild-type function for I171L, partial loss of function for S49L and a complete loss of function for R23C. The frequency of the R23C variant in 2018 non-diabetic control individuals aged 60 years and older in the general population-based SHIP cohort was lower than in the analyzed patient sample. Both variants are rare in the general population indicating a recent origin in the general gene pool and/or the consequence of pronounced purifying selection, in line with the obvious detrimental effect of the mutations. In conclusion, our study provides hints for the existence of naturally occurring TAAR1 variants with potential relevance for weight regulation and glucose homeostasis.

Context: Congenital hypothyroidism, the most frequent endocrine congenital disease, can occur either based on a thyroid hormone biosynthesis defect or can predominantly be due to thyroid dysgenesis. However, a genetic cause could so far only be identified in less than 10% of patients with a thyroid dysgenesis. Objectives: Exome sequencing was used for the first time to find additional genetic defects in thyroid dysgenesis. Patients and Methods: In a consanguineous family with thyroid dysgenesis, exome sequencing was applied, and findings were further validated by Sanger sequencing in a cohort of 94 patients with thyroid dysgenesis. Results: By exome sequencing we identified a homozygous missense mutation (p.Leu597Ser) in the SLC26A4 gene of a patient with hypoplastic thyroid tissue, who was otherwise healthy. In the cohort of patients with thyroid dysgenesis, we observed a second case with a homozygous missense mutation (p.Gln413Arg) in the SLC26A4 gene, who was additionally affected by severe hearing problems. Both mutations were previously described as loss-of-function mutations in patients with Pendred syndrome and nonsyndromic enlarged vestibular aqueduct. Conclusion: We unexpectedly identified SLC26A4 mutations that were hitherto diagnosed in thyroid dyshormonogenesis patients, now for the first time in patients with structural thyroid defects. This result resembles the historic description of thyroid atrophy in patients with the so-called myxedematous form of cretinism after severe iodine deficiency. Most likely the thyroid defect of the two homozygous SLC26A4 gene mutation carriers represents a kind of secondary thyroid atrophy, rather than a primary defect of thyroid development in the sense of thyroid agenesis. Our study extends the variable clinical spectrum of patients with SLC26A4 mutations and points out the necessity to analyze the SLC26A4 gene in patients with apparent thyroid dysgenesis in addition to the known candidate genes TSHR, PAX8, NKX2.1, NKX2.5, and FOXE1.

Obesity is one of the most challenging global health problems. One key player in energy homeostasis is the melanocortin-4 receptor (MC4R), which is a family A G-protein-coupled receptor (GPCR). It has recently been shown that MC4R has the capacity to form homo- or heterodimers. Dimerization of GPCRs is of great importance for signaling regulation, with major pharmacological implications. Unfortunately, not enough is yet known about the detailed structural properties of MC4R dimers or the functional consequences of receptor dimerization. Our goal, therefore, was to explore specific properties related to MC4R dimerization. First, we aimed to induce the dissociation of dimers to monomers and to compare the functional parameters of wild-type and MC4R variants. To inhibit homodimerization, we designed MC4R chimeras with the cannabinoid-1 receptor, a receptor that does not interact with MC4R. Indeed, we identified several substitutions in the intracellular loop 2 (ICL2) and adjacent regions of transmembrane helix 3 (TMH3) and TMH4 that lead to partial dimer dissociation. Interestingly, the capacity for signaling activity was generally increased in these MC4R variants, although receptor expression remained unchanged. This increase in activity for dissociated receptors might indicate a link between receptor dimerization and signaling capacity. Moreover, dimer dissociation was also observed in a naturally occurring activating MC4R mutation in ICL2. Taken together, this study provides new information on the structural prerequisites for MC4R dimerization and identifies an approach to induce the dissociation of MC4R dimers. This might be useful for further investigation of pharmacological properties.

SummaryObjective Rare congenital conditions with incongruence of chromosomal, gonadal and phenotypic sex have been classified as differences/disorders of sex development (DSD). Included in DSD are conditions with diverse genetic aetiology, varying levels of prenatal androgen effects, phenotypes and, subsequently, different medical treatments. Quality of life (QoL) and psychological well-being are indicators of successful psychosocial adaptation to the conditions. We sought to investigate the HRQoL and psychological well-being in this population. Design This multicentre clinical evaluation study was part of a German network related to DSD funded by the German Ministry of Science and Education (BMBF 2003 to 2007). Methods To assess health-related quality of life (HRQoL), we used the Short Form Health Survey (SF-36), and for psychological well-being, the Brief Symptom Inventory (BSI). Participants were classified into five groups: females with CAH, females with XY DSD conditions where there is a partial androgen effect (partial androgen insensitivity, mixed/partial gonadal dysgenesis, disorders of androgen biosynthesis), females with XY DSD without androgen effect (complete androgen insensitivity, complete gonadal dysgenesis), males with XY DSD, and individuals with DSD conditions and other gender. Results Participants included 110 adults with DSD (age range 17–62). We found a trend of lowered mental HRQoL and significant higher physical HRQoL for participants as compared to the norm. The high physical HRQoL especially applied to females with androgen effect and XY karyotype. Participants reported significant higher psychological distress compared to the norm. Forty-seven participants (42·7%) reported distress in a clinically relevant range on the BSI. Conclusions Although we did not find significant impairments in overall HRQoL, participants reported significant impaired psychological well-being. Specialized interdisciplinary care should focus in particular on psychological issues to ensure good overall health and well-being.

Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside-mediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.

Context: In this paper we report two new TSH receptor (TSHR) mutations. One mutation (Pro6396.50Leu) was identified in two siblings with congenital hypothyroidism, and a second mutation (Cys6366.47Arg) was found in a patient suffering from nonautoimmune hyperthyroidism. Both mutations are located in transmembrane helix (TMH) 6 at the conserved Cys6.47-Trp(Met)6.48-Leu(Ala)6.49-Pro6.50 motif of family A G protein-coupled receptors (GPCR). Objective: To study the pathogenic mechanisms, we tested patients' mutations and further side chain variations regarding their effects on TSHR signaling. Results: Substitution Pro639Leu fully inactivates the promiscuous TSHR for cAMP (Gs) and IP (Gq) signaling. In contrast, Cys636Arg leads to constitutive activation of Gs. Organization of TSHR in oligomers was not modified by mutations at position 636. Interestingly, it is known from crystal structures of GPCR that Pro6.50 is located at a TMH6 kink-distortion, which is a pivot during activation-related helical movements. However, the cell surface expressions of all mutants at position 639 were comparable to wild type, indicating a helical conformation like wild type. Conclusion: Until now, only naturally occurring constitutively activating mutations in TSHR TMH6 have been reported, but here we present the first pathogenic inactivating mutation (Pro639Leu). Our data are indicative of differentiated regulation of Gs and Gq signaling at particular TMH6 positions, but without any effects on TSHR oligomer constellation. Details of signaling modulation by each mutant at positions 6366.47 and 6396.50 help us to understand high conservation of these amino acids in family A GPCR. Described molecular (pathogenic) mechanisms are likely not unique for TSHR.

The worldwide obesity epidemic is increasing, yet at this time, no long-acting and specific pharmaceutical therapies are available. Peripheral hormonal signals communicate metabolic status to the hypothalamus by activating their corresponding receptors in the arcuate nucleus (ARC). In this brain region, a variety of G protein-coupled receptors (GPCRs) are expressed that are potentially involved in weight regulation, but so far, the detailed function of most hypothalamic GPCRs is only partially understood. An important and underappreciated feature of GPCRs is the capacity for regulation via di- and heterodimerization. Increasing evidence implicates that heterodimerization of GPCRs results in profound functional consequences. Recently, we could demonstrate that interaction of the melanocortin 3 receptor (MC3R) and the growth hormone secretagogue receptor (GHSR)-1a results in a modulation of function in both receptors. Although the physiological role of GPCR-GPCR interaction in the hypothalamus is yet to be elucidated, this concept promises new avenues for investigation and understanding of hypothalamic functions dependent on GPCR signaling. Since GPCRs are important targets for drugs to combat many diseases, identification of heterodimers may be a prerequisite for highly specific drugs. Therefore, a detailed understanding of the mechanisms and their involvement in weight regulation is necessary. Fundamental to this understanding is the interplay of GPCR-GPCR in the hypothalamic nuclei in energy metabolism. In this review, we summarize the current knowledge on melanocortin receptors and GHSR-1a in hypothalamic weight regulation, especially as they pertain to possible drug targets. Furthermore, we include available evidence for the participation and significance of GPCR dimerization.

ObjectiveTo study whether metformin reduces obesity, homeostasis model assessment for insulin resistance index (HOMA-IR), and the metabolic syndrome (MtS) in obese European adolescents in addition to previous unsuccessful lifestyle intervention.Design and methodsAfter 6 months of multiprofessional lifestyle intervention, 70 out of 86 adolescents without improvement in body mass index (BMI) and HOMA-IR were randomized into either the placebo (n=34) or the metformin group (2×500 mg/day,n=36) in addition to ongoing lifestyle intervention for another 6 months.ResultsAge was 13.8 years, BMI was 33.1 kg/m2, 65% were female, and 89% were Caucasians. During lifestyle intervention alone, BMI and HOMA-IR deteriorated significantly. In the subsequent medication period, HOMA-IR and fasting insulin improved similarly in the placebo and metformin groups (HOMA-IR decreased 73 vs 54% respectively in metformin versus placebo;P=0.048), but BMI remained unchanged. The insulin sensitivity index, however, only improved in the metformin group. High fasting insulin is correlated with a subsequent BMI increase irrespective of the medication. MtS remained unchanged.ConclusionsObese European adolescents' insulin sensitivity improved without weight change during placebo or metformin intervention in addition to lifestyle intervention. Most differences did not reach statistical significance, probably due to improved compliance with lifestyle intervention as a placebo effect. In addition, the metformin dose may be too low.

ObjectiveHypospadias is a frequent congenital anomaly but in most cases an underlying cause is not found. Steroidogenic factor 1 (SF-1, NR5A1, Ad4BP) is a key regulator of human sex development and an increasing number of SF-1 (NR5A1) mutations are reported in 46,XY disorders of sex development (DSD). We hypothesized that NR5A1 mutations could be identified in boys with hypospadias.Design and methodsMutational analysis of NR5A1 in 60 individuals with varying degrees of hypospadias from the German DSD network.ResultsHeterozygous NR5A1 mutations were found in three out of 60 cases. These three individuals represented the most severe end of the spectrum studied as they presented with penoscrotal hypospadias, variable androgenization of the phallus and undescended testes (three out of 20 cases (15%) with this phenotype). Testosterone was low in all three patients and inhibin B/anti-Müllerian hormone (AMH) were low in two patients. Two patients had a clear male gender assignment. Gender re-assignment to male occurred in the third case. Two patients harbored heterozygous nonsense mutations (p.Q107X/WT, p.E11X/WT). One patient had a heterozygous splice site mutation in intron 2 (c.103-3A/WT) predicted to disrupt the main DNA-binding motif. Functional studies of the nonsense mutants showed impaired transcriptional activation of an SF-1-responsive promoter (Cyp11a). To date, adrenal insufficiency has not occurred in any of the patients.ConclusionsSF-1 (NR5A1) mutations should be considered in 46,XY individuals with severe (penoscrotal) hypospadias, especially if undescended testes, low testosterone, or low inhibin B/AMH levels are present. SF-1 mutations in milder forms of idiopathic hypospadias are unlikely to be common.

Thyroid hormone transport into cells requires plasma membrane transport proteins. Mutations in one of these, monocarboxylate transporter 8 (MCT8), have been identified as underlying cause for the Allan–Herndon–Dudley syndrome, an X-linked mental retardation in which the patients also present with abnormally high 3′,3,5-triiodothyronine (T3) plasma levels. Mice deficient inMct8replicate the thyroid hormone abnormalities observed in the human condition. However, no neurological deficits have been described in mice lackingMct8. Therefore, we subjectedMct8-deficient mice to a comprehensive immunohistochemical, neurological, and behavioral screen. Several behavioral abnormalities were found in the mutants. Interestingly, some of these behavioral changes are compatible with hypothyroidism, whereas others rather indicate hyperthyroidism. We thus hypothesized that neurons exclusively dependent onMct8are in a hypothyroid state, whereas neurons expressing other T3transporters become hyperthyroid, if they are exposed directly to the high plasma T3. The majority of T3uptake in primary cortical neurons is mediated by Mct8, but pharmacological inhibition suggested functional expression of additional T3transporter classes. mRNAs encoding six T3transporters, including L-type amino acid transporters (LATs), were coexpressed withMct8in isolated neurons. We then demonstratedLat2expression in cultured neurons and throughout murine brain development. In contrast,LAT2is expressed in microglia in the developing human brain during gestation, but not in neurons. We suggest that lack of functional complementation by alternative thyroid hormone transporters in developing human neurons precipitates the devastating neurodevelopmental phenotype inMCT8-deficient patients, whereasMct8-deficient mouse neurons are functionally complemented by other transporters, for possiblyLat2.

The challenge to eradicate iodine deficiency disorders (IDD) by the year 2000 worldwide will have positive consequences for more than 20 million people suffering from IDD. This is the main and common goal for the developing but also for a lot the developed countries. Sensitive tools like neonatal thyroid screening (NTS) can be used in order to make changes in iodine supplementation more transparent and to have a dynamic target oriented approach. The comparison between six districts in Bulgaria and Berlin revealed despite of some positive changes after the fortification of the iodine supplementation program still existing iodine deficiency in Bulgaria which is more profound than in Berlin in 1990. The shift to higher TSH-values in the Black-Sea region, classified by goitre-prevalence in 1956 as non-endemic for iodine deficiency, needs further investigation. Besides of iodine deficiency an influence of iodine-containing disinfectants and/or goitrogens is possible.

Screening for congenital hypothyroidism for all newborns of the former western parts of the city of Berlin was instituted in 1978 by determination of the TSH levels in dried filter paper blood spots of the 3rd to 5th day of life. Since 1991 the newborns of the former eastern parts have been also included in the same screening program. From 1978 to 1995 a total number of 104 newborns with permanent congenital hypothyroidism have been detected resulting in a prevalence of congenital hypothyroidism of 1:3800. The etiological diagnosis of hypothyroidism was made by imaging studies (ultrasonography or 99mTc scintigraphy) and assessment of serum thyroglobulin and thyroid hormone levels. Using this approach in 37 children (30 female, 7 male) the diagnosis of athyrosis, in 20 children (15 female, 5 male) the diagnosis of ectopy and in 21 children (18 female, 3 male) the diagnosis of thyroid hypoplasia was made, 16 children (8 female, 8 male) had a normally sized gland and 4 (1 female, 3 male) had congenital goiter. In 86% of all patients the age at onset of thyroxine (L-T4) replacement therapy was 8 or 9 days of life (3-42 days) and the median initial L-T4 dose was 14 micrograms/kg/day (10-16 micrograms/kg/day). The intellectual outcome of 77 children (2-16 years) was studied and normal scores for the intelligence (IQ) and developmental (DQ) quotients were found in 71 (92%). Outcome was not correlated to the age at onset of treatment, the initial dose and the severity of hypothyroidism, but there was a positive correlation of the socioeconomic status of the family and the IQ of the patients. The results of the screening program in Berlin document that an early and efficient thyroxine replacement can normalize the intellectual outcome of patients with congenital hypothyroidism independent of the severity of the disease as assessed by the residual thyroid function detectable at diagnosis.

HNF1B-maturity-onset diabetes of the young is caused by abnormalities in the HNF1B gene encoding the transcription factor HNF-1beta. We aimed to investigate detailed clinical features and the type of HNF1B gene anomaly in five pediatric cases with HNF1B-MODY.From a cohort of 995 children and adolescents with diabetes, we analyzed the most frequent maturity-onset diabetes of the young genes (GCK, HNF1A, HNF4A) including HNF1B sequencing and deletion analysis by quantitative Multiplex-PCR of Short Fluorescent Fragments (QMPSF) if patients were islet autoantibody-negative and had one parent with diabetes or associated extrapancreatic features or detectable C-peptide outside honeymoon phase. Presence and size of disease-causing chromosomal rearrangements detected by QMPSF were further analyzed by array comparative genomic hybridization.Overall, five patients had a heterozygous HNF1B deletion, presenting renal disease, elevated liver enzymes, and diabetes. Diabetes was characterized by insulin resistance and adolescent onset of hyperglycemia. Additionally, clinical features in some patients were pancreas dysplasia and exocrine insufficiency (two of five patients), genital defects (three of five), mental retardation (two of five), and eye abnormalities (coloboma, cataract in two of five). One case also had severe growth deficit combined with congenital cholestasis, and another case had common variable immune deficiency. All patients reported here had monoallelic loss of the entire HNF1B gene. Whole genome array comparative genomic hybridization confirmed a precurrent genomic deletion of approximately 1.3-1.7 Mb in size.The clinical data of our cases enlarge the wide spectrum of patients with HNF1B anomaly. The underlying molecular defect in all cases was a 1.3- to 1.7-Mb deletion, and paired, segmental duplications along with breakpoints were most likely involved in this recurrent chromosomal microdeletion.

ObjectiveMore than 30 years ago Frisch and Revelle proposed a body weight threshold for the onset of menarche. Based on this hypothesis, a further acceleration of age at menarche can be expected in times of childhood obesity.DesignA cross-sectional study of 1840 healthy school girls (Berlin school children's cohort, BSCOC) within the age groups 10–15 years was conducted in 2006–2007.MethodsMedian age of menarche was calculated by Kaplan–Meier survival analysis. Bi- and multivariate analyses were performed to analyze the associations between menarche age and weight status. A locally weighted regression was used to analyze the relationship respectively between height, weight, and body mass index (BMI)–SDS and age stratified by menarche status.ResultsNine hundred and thirty six (50.9%) girls had already experienced menarche at a median age of 12.8 years. Two hundred and thirty six of these girls reached their menarche recently. Obese/overweight girls reached menarche significantly earlier (12.5 years), than normal weight (12.9 years), and underweight girls (13.7 years). The mean total body weight was similar in all girls at menarche irrespective of age (mean 51.1 kg,s.d.8.1) and height. BMI–SDS remained the only significant factor for onset of menarche within a multiple regression model for early menarche (OR 2.1, 95% confidence interval 1.3–3.3,P=0.002).ConclusionsAge at onset of menarche did not accelerate even in a childhood population with more than 10% obesity prevalence. Nevertheless, a negative correlation of BMI–SDS with age at onset of menarche exists.

DEHAL1 has been identified as the gene encoding iodotyrosine deiodinase in the thyroid, where it controls the reuse of iodide for thyroid hormone synthesis. We screened patients with hypothyroidism who had features suggestive of an iodotyrosine deiodinase defect for mutations in DEHAL1. Two missense mutations and a deletion of three base pairs were identified in four patients from three unrelated families; all the patients had a dramatic reduction of in vitro activity of iodotyrosine deiodinase. Patients had severe goitrous hypothyroidism, which was evident in infancy and childhood. Two patients had cognitive deficits due to late diagnosis and treatment. Thus, mutations in DEHAL1 led to a deficiency in iodotyrosine deiodinase in these patients. Because infants with DEHAL1 defects may have normal thyroid function at birth, they may be missed by neonatal screening programs for congenital hypothyroidism.

Intact melanocortin signaling via the G protein-coupled receptors (GPCRs), melanocortin receptor 4 (MC4R), and melanocortin receptor 3 (MC3R) is crucial for body weight maintenance. So far, no connection between melanocortin signaling and hypothalamic inflammation has been reported. Using a bimolecular fluorescence complementation library screen, we identified a new interaction partner for these receptors, ring finger protein 11 (RNF11). RNF11 participates in the constitution of the A20 complex that is involved in reduction of tumor necrosis factor α (TNFα)-induced NFκB signaling, an important pathway in hypothalamic inflammation. Mice treated with high-fat diet (HFD) for 3 days demonstrated a trend toward an increase in hypothalamic Rnf11 expression, as shown for other inflammatory markers under HFD. Furthermore, Gs-mediated signaling of MC3/4R was demonstrated to be strongly reduced to 20-40% by co-expression of RNF11 despite unchanged total receptor expression. Cell surface expression was not affected for MC3R but resulted in a significant reduction of MC4R to 61% by co-expression with RNF11. Mechanisms linking HFD, inflammation, and metabolism remain partially understood. In this study, a new axis between signaling of specific body weight regulating GPCRs and factors involved in hypothalamic inflammation is suggested.

Endocrine-disrupting chemicals (EDCs) became the focus of both public and scientific interest when defects in sexual behavior and reproductive ability of wild-living animals were ascribed to their steroid-like or anti-steroid androgenic properties (Colborn et al. 1993). In the aftermath, these observations were reproduced in many laboratory animal models. Whether human reproduction is also affected by the action of EDCs is currently a topic of controversial discussion (Safe 2004; Toppari 2002; Waring and Harris 2005). Furthermore, there is growing evidence that, in addition to the reproductive, other endocrine systems such as the hypothalamus–pituitary–thyroid (HPT) axis may be targets of endocrine disruption. In particular, poly-halogenated phenolic compounds such as polychlorinated biphenyls (PCBs) and poly-brominated diphenyl ethers (PBDEs), probably because of their structural resemblance to thyroid hormones (Cody et al. 1986), may cause disturbance of thyroid hormone homeostasis, hypothyroidism, thyroid hyperplasia, and neoplasia (Hagmar 2003; Siddiqi et al. 2003), and developmental defects of the central nervous system (CNS) in experimental animals and humans (Koopman-Esseboom et al. 1994; Meerts et al. 2004). It now has been shown that there may be multiple targets for interference by various EDCs with the complex regulatory network of thyroid hormone synthesis, metabolism, distribution, and action on the various levels of endocrine regulation and feedback control. These targets include thyrotropin receptor (Auf’mkolk et al. 1985a, 1985b; Santini et al. 2003); iodide uptake by the sodium iodide symporter (NIS; Schroder-van der Elst et al. 2004); type I 5′-deiodinase (5′DI) (Auf’mkolk et al. 1986; Ferreira et al. 2002; Schmutzler et al. 2004); transthyretin (TTR) (Kohrle et al. 1988; van den Berg 1990; Yamauchi et al. 2003); thyroid hormone receptor (TR) (Bogazzi et al. 2003; Moriyama et al. 2002); and thyroid hormone-dependent growth of pituitary cells (Ghisari and Bonefeld-Jorgensen 2005). In the brain, PCBs may disrupt normal differentiation regulated by thyroid hormones, although they do not act as thyroid hormone-like ligands (Zoeller 2005). Moreover, complex biological developmental programs controlled by thyroid hormones may be disturbed by EDC action such as metamorphosis in the amphibian Xenopus laevis (Kloas 2002). Yet, data on EDCs affecting the HPT axis are comparatively scarce. Within the CREDO (cluster of research into endocrine disruption in Europe) Cluster, which is part of the Sixth European Union framework program, the EURISKED (multi-organic risk assessment of selected endocrine disruptors) consortium worked on a group of projects assessing multiorganic effects of selected endocrine disruptors. These projects included a focus on the thyroid gland and its target organs. In this article we summarize the first results. Chosen for analysis was an environmentally and nutritionally relevant collection of substances suspected to have endocrine-disrupting activity because of their steroid-like or anti-steroid effects. These substances included genistein as well as glycitein and daidzein (isoflavones from soybean); resveratrol (phytoalexin from grapes); silymarin (flavonol–lignane mixture from the milk thistle); xanthohumol (XN; prenylated chalcone from hops, Humulus lupulus L.); 8-prenylnaringenin (8-PN; prenylated flavonone from hops); benzophenone-2, ben-zophenone-3, 4-methylbenzylidene camphor, and octyl-methoxycinnamate [BP2, BP3, OMC, 4-MBC, respectively; ultraviolet (UV) filters]; F21388 (synthetic, halogenated flavonoid); 4-nonylphenol (4-NP; e.g., emulgator); bisphenol A (BPA; building block for, e.g., polycarbonate plastics); dibutylphthalate (e.g., plasticizer); linuron and procymidon (pesticides); 5α-androstane-3β,17β-diol [adiol; proposed to be an endogenous ligand of estrogen receptor β (ER-β)] and 17β-estradiol benzoate (E2). We show that several of these substances also interfere with multiple targets at various levels of the HPT axis in a tissue-specific manner. These targets included weight and morphology of the thyroid gland, iodide uptake by the NIS, iodide organification by thyroid peroxidase (TPO), binding of the thyroid hormone thyroxine (T4) to TTR, the metabolism of thyroid hormones by deiodinases, and the action of the biologically active thyroid hormone triiodothyronine (T3) mediated by TRs functioning as ligand-dependent transcription factors.

Steroidogenic factor 1 (SF1, NR5A1) is a nuclear receptor that regulates multiple genes involved in adrenal and gonadal development, steroidogenesis, and the reproductive axis. Human mutations in SF1 were initially found in two 46,XY female patients with severe gonadal dysgenesis and primary adrenal failure. However, more recent case reports have suggested that heterozygous mutations in SF1 may also be found in patients with 46,XY partial gonadal dysgenesis and underandrogenization but normal adrenal function. We have analyzed the gene encoding SF1 (NR5A1) in a cohort of 27 patients with 46,XY disorders of sex development (DSD) from the German network of DSD. Heterozygous SF1 mutations were found in 5 out of 27 (18.5%) of cases. Four patients with SF1 mutations presented with the similar phenotype of mild gonadal dysgenesis, severe underandrogenization, and absent Müllerian structures. Of these, two patients harbored missense mutations within the DNA-binding region of SF1 (p.C33S, p.R84H), one patient had a nonsense mutation (p.Y138X) and one patient had a frameshift mutation (c.1277dupT) predicted to disrupt RNA stability or protein function. One additional patient ([c.424_427dupCCCA]+[p.G146A]) displayed a more marked phenotype of severe gonadal dysgenesis, normal female external genitalia, and Müllerian structures. Functional studies of the missense mutants (p.C33S, p.R84H) and of one nonsense mutant (p.Y138X) revealed impaired transcriptional activation of SF1-responsive target genes. To date, adrenal insufficiency has not occurred in any of the patients. Thus, SF1 mutations are a relatively frequent cause of 46,XY DSD in humans.

Background: Congenital hypothyroidism (CH) is the most frequent congenital endocrine disorder. The diagnosis in industrialized countries is usually made with population-based newborn screening that measures thyroid-stimulating hormone (TSH) or TSH and total thyroxine (T4) in dried blood spots in the first 3 days of life. In newborns with a screening result suspicious for hypothyroidism, the diagnosis of primary CH is confirmed when serum TSH levels are above and T4 (free T4) levels are below the age-related reference ranges. Hypothalamic-pituitary hypothyroidism is more difficult to diagnose. Most infants with this diagnosis are missed in screening programs unless T4 (free T4)/TSH or TSH/T4/thyroxine binding globulin is simultaneously measured. If hypothyroidism is confirmed by laboratory analysis, imaging studies should be performed immediately; however, it is not acceptable to delay hormone replacement therapy if imaging studies are not readily available. Conclusions: The goal of treatment of CH is to avoid disturbed mental development, and initial treatment can be adjusted to physiological conditions. To match the higher thyroid hormone concentrations in the first weeks of life, substitution with l-thyroxine should aim to achieve serum T4/free T4 levels in the upper half of the normal age-related reference range. Some newborns and infants will have persistently high TSH levels despite normalized T4/free T4 serum concentrations.

Mostin vivoeffects of 3-iodothyronamine (3-T1AM) have been thus far thought to be mediated by binding at the trace amine-associated receptor 1 (TAAR1). Inconsistently, the 3-T1AM-induced hypothermic effect still persists inTaar1knockout mice, which suggests additional receptor targets. In support of this general assumption, it has previously been reported that 3-T1AM also binds to the α-2A-adrenergic receptor (ADRA2A), which modulates insulin secretion. However, the mechanism of this effect remains unclear. We tested two different scenarios that may explain the effect: the sole action of 3-T1AM at ADRA2A and a combined action of 3-T1AM at ADRA2A and TAAR1, which is also expressed in pancreatic islets. We first investigated a potential general signaling modification using the label-free EPIC technology and then specified changes in signaling by cAMP inhibition and MAPKs (ERK1/2) determination. We found that 3-T1AM induced Gi/oactivation at ADRA2A and reduced the norepinephrine (NorEpi)-induced MAPK activation. Interestingly, in ADRA2A/TAAR1 hetero-oligomers, application of NorEpi resulted in uncoupling of the Gi/osignaling pathway, but it did not affect MAPK activation. However, 3-T1AM application in mice over a period of 6 days at a daily dose of 5 mg/kg had no significant effects on glucose homeostasis. In summary, we report an agonistic effect of 3-T1AM on the ADRA2A-mediated Gi/opathway but an antagonistic effect on MAPK induced by NorEpi. Moreover, in ADRA2A/TAAR1 hetero-oligomers, the capacity of NorEpi to stimulate Gi/osignaling is reduced by co-stimulation with 3-T1AM. The present study therefore points to a complex spectrum of signaling modification mediated by 3-T1AM at different G protein-coupled receptors.

The symptoms of severe early-onset obesity, adrenal insufficiency, and red hair define the proopiomelanocortin (POMC) deficiency syndrome as described so far in two children with complete loss-of-function mutations of the human POMC gene. In POMC deficiency, obesity reflects the lack of POMC-derived peptides as ligands at the melanocortin (MC) MC4 and MC3 receptors, which are expressed in the hypothalamic leptin-melanocortin pathway of body weight regulation. Hypocortisolism and alteration of pigmentation are caused by the lack of POMC-derived peptides at the adrenal MC2 receptor and the skin MC1 receptor, respectively. Here we describe three new cases of complete loss-of-function mutations of the POMC gene. Patients were diagnosed based on the clinical trials of red hair, adrenal insufficiency, and early-onset severe obesity. One previously described translation initiation mutation (C3804A) as well as one new nonsense (A6851T) and two new frame-shift mutations (6996del and 7100 + 2G) were found in homozygosity or compound heterozygosity. The heterozygous parents were found to have high normal or mildly elevated body weight, suggesting a dosage effect of the POMC gene product on weight regulation. To compensate for the lack of hypothalamic melanocortin function, we initiated a trial in the two previously published patients with intranasal ACTH4-10, a melanocortin fragment for which an anorexic effect has been described recently. During 3 months with increasing doses of ACTH4-10, no change of body weight or metabolic rate was observed, suggesting that at least in these two POMC-deficient patients ACTH4-10 is without any compensatory effect. In the same two patients, further investigation revealed a mildly elevated TSH. However, a 1-yr treatment with thyroid hormone did not result in a significant reduction of body weight.

The melanocortin peptides and their receptors represent one of the most complex systems in human endocrinology. Hormonal regulation includes pigmentation, weight maintenance, adrenal function, and exocrine gland secretion via endocrine, paracrine, autocrine, and neurocrine action of melanocortin peptides at five different but homologous melanocortin receptors. Genetic relevance of the melanocortin system for human physiology was initially shown by mutations in the different melanocortin receptor genes, first described in the melanocortin-2 receptor gene in 1993 as one reason for congenital hypocortisolism. Because all ligands within the melanocortin systems are derived from one single precursor hormone, proopiomelanocortin (POMC), a genetic defect in the POMC gene could have been expected to affect all functional components of the melanocortin system. Accordingly, patients with a complete defect of the POMC gene product due to homozygous or compound heterozygous loss of function mutations were shown to be affected mainly by red hair, early-onset obesity, and congenital hypocortisolism. No further obvious clinical problems were described in these patients, suggesting that no additional function of the melanocortin system has escaped recognition. However, whether partial loss of function mutations in the POMC gene might lead to more circumscribed phenotypes, especially common obesity, remains an open question.

Prenatal deficit of androgens or androgen action results in atypical genitalia in individuals with XY disorders of sex development (XY,DSD). XY,DSD include mainly disorders of gonadal development and testosterone synthesis and action. Previously, most XY,DSD individuals were assigned to the female sex. Constructive genital surgery allowing heterosexual intercourse, gonadectomy, and hormone therapy for feminization were often performed. However, outcome studies are scarce.Our objective was evaluation of satisfaction with genital surgery and sexual life in adults with XY,DSD.We evaluated 57 individuals with XY,DSD from the German multicenter clinical evaluation study with a condition-specific questionnaire. The individuals were divided into subgroups reflecting the absence/presence of partial androgen effect or genital constructive surgery.Dissatisfaction with function of the surgical result (47.1%) and clitoral arousal (47.4%) was high in XY,DSD partially androgenized females after feminization surgery. Dissatisfaction with overall sex life (37.5%) and sexual anxieties (44.2%) were substantial in all XY,DSD individuals. Problems with desire (70.6%), arousal (52.9%), and dyspareunia (56.3%) were significant in XY,DSD complete females. 46,XY partially androgenized females reported significantly more often partners of female (9.1%) or both sexes (18.2%) and dyspareunia (56.5%) compared with controls. Individuals with complete androgen insensitivity syndrome stated significant problems with desire (81.8%), arousal (63.6%), and dyspareunia (70%).Care should be improved in XY,DSD patients. Constructive genital surgery should be minimized and performed mainly in adolescence or adulthood with the patients' consent. Individuals with DSD and their families should be informed with sensibility about the condition. Multidisciplinary care with psychological and nonprofessional support (parents, peers, and patients' support groups) is mandatory from child to adulthood.

Application of 3-iodothyronamine (3-T(1)AM) results in decreased body temperature and body weight in rodents. The trace amine-associated receptor (TAAR) 1, a family A G protein-coupled receptor, is a target of 3-T(1)AM. However, 3-T(1)AM effects still persist in mTaar1 knockout mice, which suggest so far unknown further receptor targets that are of physiological relevance. TAAR5 is a highly conserved TAAR subtype among mammals and we here tested TAAR5 as a potential 3-T(1)AM target. First, we investigated mouse Taar5 (mTaar5) expression in several brain regions of the mouse in comparison to mTaar1. Secondly, to unravel the full spectrum of signaling capacities, we examined the distinct G(s)-, G(i/o)-, G(12/13)-, G(q/11)- and MAP kinase-mediated signaling pathways of mouse and human TAAR5 under ligand-independent conditions and after application of 3-T(1)AM. We found overlapping localization of mTaar1 and mTaar5 in the amygdala and ventromedial hypothalamus of the mouse brain. Second, the murine and human TAAR5 (hTAAR5) display significant basal activity in the G(q/11) pathway but show differences in the basal activity in G(s) and MAP kinase signaling. In contrast to mTaar5, 3-T(1)AM application at hTAAR5 resulted in significant reduction in basal IP3 formation and MAP kinase signaling. In conclusion, our data suggest that the human TAAR5 is a target for 3-T(1)AM, exhibiting inhibitory effects on IP3 formation and MAP kinase signaling pathways, but does not mediate Gs signaling effects as observed for TAAR1. This study also indicates differences between TAAR5 orthologs with respect to their signaling profile. In consequence, 3-T(1)AM-mediated effects may differ between rodents and humans

By screening patients with X-linked nephrogenic diabetes insipidus (NDI) for mutations within the V(2) vasopressin receptor (AVPR2) gene, we have identified six novel and two recurrent mutations. Additionally, one patient revealed a genomic deletion of 3.2 kb encompassing most of the AVPR2 gene and the last exon/3'-region of C1 gene, which is in close proximity to the AVPR2 locus. In-depth characterization of the mutant AVPR2s by a combination of functional and immunological techniques allowed to gain further insight into molecular mechanisms leading to the receptor dysfunction. Aiming at the functional reconstitution of mutant G protein-coupled receptors, several strategies of potential therapeutic usefulness have been tested. Because the functional rescue of truncated receptors is most challenging, we addressed this issue by applying an aminoglycoside approach. Here, we demonstrate that the misreading capacity of the aminoglycoside antibiotic geneticin was sufficient to restore function of mutant AVPR2s harboring premature stop codons in an in vitro expression system.

Neonatal TSH screening has been a major achievement for the early detection and treatment of primary congenital hypothyroidism. It nevertheless fails to reveal cases of central hypothyroidism caused by TSH levels in the low normal range. In the last 10 yr, homozygous mutations in the TSHbeta-subunit gene have been recognized as a cause of central hypothyroidism with isolated TSH deficiency. The most frequent TSHbeta mutation 313DeltaT (C105V) has been described in six apparently unrelated families. We investigated the frequency and possible monophyletic origin of the different TSHbeta 313DeltaT alleles of the three affected families. Haplotype analysis of five polymorphic single-nucleotide polymorphism loci in the TSHbeta region revealed the presence of seven different haplotypes in the general population. In all six parental lines, the mutation occurred on the same haplotype. Extending the haplotype by two flanking microsatellite markers led to a mutational age estimate of about 150 generations. In 500 unrelated individuals from the general population, we did not detect any TSHbeta 313DeltaT allele, suggesting a population heterozygote carrier frequency less than 1:170 with more than 95% probability. Accordingly, the disease risk in the general population because of homozygosity is low. Our data suggest a monophyletic origin of the TSHbeta 313DeltaT mutation from a common ancestor and no significant population prevalence. Therefore, identification and genetic counseling of heterozygous carriers in affected families seems to be more advisable than population-wide neonatal T(4) screening programs for an early detection of this rare condition.

OBJECTIVE—To investigate thyroid autoimmunity in a very large nationwide cohort of children and adolescents with type 1 diabetes. RESEARCH DESIGN AND METHODS—Data were analyzed from 17,749 patients with type 1 diabetes aged 0.1–20 years who were treated in 118 pediatric diabetes centers in Germany and Austria. Antibodies to thyroglobulin (anti-TG) and thyroperoxidase (anti-TPO) were measured and documented at least once in 7,097 patients. A total of 49.5% of these patients were boys, the mean age was 12.4 years (range 0.3–20.0 years), and the mean duration of diabetes was 4.5 years (range 0.0–19.5 years). A titer exceeding 100 units/ml or 1:100 was considered significantly elevated. RESULTS—In 1,530 patients, thyroid antibody levels were elevated on at least one occasion, whereas 5,567 were antibody-negative during the observation period. Patients with thyroid antibodies were significantly older (P < 0.001), had a longer duration of diabetes (P < 0.001), and developed diabetes later in life (P < 0.001) than those without antibodies. A total of 63% of patients with positive antibodies were girls, compared with 45% of patients without antibodies (P < 0.001). The prevalence of significant thyroid antibody titers increased with increasing age; the highest prevalence was in the 15- to 20-year age group (anti-TPO: 16.9%, P < 0.001; anti-TG: 12.8%, P < 0.001). Thyroid-stimulating hormone (TSH) levels were higher in patients with thyroid autoimmunity (3.34 μU/ml, range 0.0–615.0 μU/ml) than in control subjects (1.84 μU/ml, range 0.0–149.0 μU/ml) (P < 0.001). Even higher TSH levels were observed in patients with both anti-TPO and anti-TG (4.55 μU/ml, range 0.0–197.0 μU/ml). CONCLUSIONS—Thyroid autoimmunity seems to be particularly common in girls with diabetes during the second decade of life and may be associated with elevated TSH levels, indicating subclinical hypothyroidism.

ObjectiveSteroidogenic factor 1 (SF1, NR5A1) is a key transcriptional regulator of genes involved in the hypothalamic–pituitary–gonadal axis. Recently, SF1 mutations were found to be a frequent cause of 46,XY disorders of sex development (DSD) in humans. We investigate the frequency of NR5A1 mutations in an Egyptian cohort of XY DSD.DesignClinical assessment, endocrine evaluation and genetic analysis of 50 Egyptian XY DSD patients (without adrenal insufficiency) with a wide phenotypic spectrum.MethodsMolecular analysis of NR5A1 gene by direct sequencing followed by in vitro functional analysis of the two novel missense mutations detected.ResultsThree novel heterozygous mutations of the coding region in patients with hypospadias were detected. p.Glu121AlafsX25 results in severely truncated protein, p.Arg62Cys lies in DNA-binding zinc finger, whereas p.Ala154Thr lies in the hinge region of SF1 protein. Transactivation assays using reporter constructs carrying promoters of anti-Müllerian hormone (AMH), CYP11A1 and TESCO core enhancer of Sox9 showed that p.Ala154Thr and p.Arg62Cys mutations result in aberrant biological activity of NR5A1. A total of 17 patients (34%) harboured the p.Gly146Ala polymorphism.ConclusionWe identified two novel NR5A1 mutations showing impaired function in 23 Egyptian XY DSD patients with hypospadias (8.5%). This is the first study searching for NR5A1 mutations in oriental patients from the Middle East and Arab region with XY DSD and no adrenal insufficiency, revealing a frequency similar to that in European patients (6.5–15%). We recommend screening of NR5A1 in patients with hypospadias and gonadal dysgenesis. Yearly follow-ups of gonadal function and early cryoconservation of sperms should be performed in XY DSD patients with NR5A1 mutations given the risk of future fertility problems due to early gonadal failure.

The thyroid hormone derivative 3-iodothyronamine (3T1AM) is an agonist for trace amine-associated receptor 1 (TAAR1). Application of 3T1AM in rodents results in a variety of effects, including decreased body temperature. Taar1-/- mice still show these effects, excluding Taar1 as primary mediator of 3T1AM induced action in vivo. Because of its role as potential treatment option for stroke patients, possible targets and effects of 3T1AM in humans are of medical interest. Here we investigated the human (hTAAR5) and mouse Taar5 (mTaar5) as potential target to explain 3T1AM effects in Taar1-/- mice. In situ hybridization for mTaar1 and mTaar5 revealed overlapping expression profiles in the amygdala and VMH. Next, we investigated several signaling pathways of mTaar5 and hTAAR5. We confirmed previously reported data that the volatile amine dimethylethylamine is an agonist for Gs activation by mTaar5, however, not for hTAAR5. Further investigations revealed a basal tone of ERK and Gq activation for hTAAR5 and mTaar5 and additional basal Gs activity for mTaar5 mediating cAMP accumulation. In response to 3T1AM, no signaling via Gs, Gq, Gi, G12/13 and ERK was observed for hTAAR5 and mTaar5, while 3T1AM acted as inverse agonist on hTAAR5 by decreasing the basal tone of IP3 accumulation and ERK. To elucidate the particular determinants involved in signaling differences between hTAAR5 and mTaar5 we created chimeric receptors, aiming to transfer murine signaling properties to hTAAR5. We identified 6 amino acids in the ligand binding and G protein-coupling region to be involved. Our data demonstrated that caution is needed when transferring signaling properties of mTaar5 to the human situation. While observed pharmacological effects of 3T1AM on Taar1-/- mice are likely not related to mTaar5, according to our in vitro data, hTAAR5 functions as a target for 3T1AM and exhibits inhibitory 3T1AM effects on Gq and ERK signaling. Supported by DFG-SPP1629 ThyroidTransAct

A case of nonclassic (NC) 21-hydroxylase deficiency, with a moderately elevated 17-hydroxyprogesterone level (145 nmol/L in filter paper blood spot), was detected in newborn screening. The newborn's phenotype was female, with no sign of virilization. Confirmatory diagnosis revealed elevated serum levels of 17-hydroxyprogesterone and of 21-desoxycortisol, whereas cortisol, PRA, and electrolytes were normal. Hydrocortisone substitution was considered at the age of 6 months, when virilization became obvious. For clinical reasons, this case had to be classified as late-onset congenital adrenal hyperplasia (CAH) with unusually early manifestation. However, the diagnosis of classic 21-hydroxylase deficiency was obtained by Southern blotting studies, showing that she was homozygous for the 30-kb deletion, including the 3' end of CYP21P pseudogene, the C4B gene, and the 5' end of the functional CYP21 gene. Further studies, using PCR and sequencing, were conducted to explain the discrepancy between this genotype, usually associated with a classic salt-wasting form, and the girl's phenotype. Typically, patients homozygous for the 30-kb deletion encoding classic CAH possess a unique CYP21P/21 hybrid gene with the junction site located after the third exon, yielding a nonfunctional pseudogene. The girl in question, however, was heterozygous for the 8-bp deletion, suggesting that the chimeric pseudogene on one allele had a junction site before the third exon. She was compound heterozygous for a 30-kb deletion encoding classic CAH on the paternal allele, and a 30-kb deletion encoding NC CAH on the maternal allele. This novel maternal CYP21P/21 hybrid gene is characterized by a junction site before intron 2 and differs from the normal CYP21 gene only by the P30L mutation in exon 1 and the promoter region of the CYP21P pseudogene. Because the P30L mutation has been described to result in an enzyme with 30-60% activity of the normal P450c21 enzyme, and the CYP21P promoter reduced the transcription to 20% of normal, this puzzling phenotype of a NC CAH with early onset may be fully explained by the genotype of the patient and considered as an intermediate form between the simple virilizing and NC form.

X-Linked nephrogenic diabetes insipidus (NDI) is a rare inherited disorder characterized by the excretion of abnormal large volumes of diluted urine mainly caused by mutations in the V2 vasopressin receptor (AVPR2) gene. By screening NDI patients for mutations within the AVPR2 gene we have identified three novel (I46K, F105V, I130F) and four recurrent (D85N, R106C, R113W, Q225X) mutations. In addition, a recurrent missense mutation (A147T) within the aquaporin-2 gene was identified in a female patient with autosomal recessive NDI associated with sensorineural deafness. Selected clinical data of the NDI patients were compared with the results from the in vitro studies. Functional analysis of I46K and I130F revealed reduced maximum agonist-induced cAMP responses as a result of an improper cell surface targeting. In contrast, the F105V mutation is delivered to the cell surface and displayed an unchanged maximum cAMP response, but impaired ligand binding abilities of F105V were reflected in a shifted concentration-response curve toward higher vasopressin concentrations. As the extracellularly located F105 is highly conserved among the vasopressin/oxytocin receptor family, functional analysis of this residue implicates an important role in high affinity agonist binding.

Isolated TSH deficiency leading to hypothyroidism seems to be a rare condition, escaping the diagnosis by neonatal screening programs, which are based on the primary determination of TSH. This is the first report of a case with an autosomal recessive TSH defect caused by a homozygous mutation of the betaTSH gene that was diagnosed in the early neonatal period. Hypothyroidism in the first child of apparently unrelated parents was suspected because of the classical symptoms of congenital hypothyroidism, which were fully expressed already on the 11th day of life. Routine neonatal TSH-screening on the 4th day of life had been normal, but subsequent determination of serum thyroid hormone levels revealed almost undetectable levels and thyroid hormone substitution was immediately started. Because there was no indication for other pituitary hormone deficiencies, sequence analysis of the betaTSH gene was initiated. A homozygous T deletion in codon 105 was found resulting in a change of a highly conserved cysteine to valine followed by eight altered amino acids and a premature stop codon due to the frame-shift. This altered betaTSH is a biologically inactive peptide. Because of the early development of severe symptoms, it is possible that this altered TSH suppresses the physiologic constitutive activity of the unliganded TSH receptor. Rapid molecular diagnosis in this patient clarified the diagnosis without additional endocrine and imaging studies and it is concluded, that symptoms of hypothyroidism in the neonatal period should result always in an immediate comprehensive work-up of thyroid function including molecular genetic studies irrespective of the screening result.

Gain of function mutations in the TSH receptor (TSHR) have been identified as the molecular basis for congenital and acquired forms of autonomous thyroid function. Herein, we report the molecular characterization of a case of severe congenital hyperthyroidism with a history of hyperthyroidism in the paternal aunt and the paternal grandmother, who were both found to be heterozygous for a mutation (R528H) located in exon 10 of the TSHR gene. Functional expression of the mutant TSHR-R528H in COS-7 cells, however, did not result in constitutive activity of the TSHR. Subsequent analysis of exons 1-9 led to the detection of an additional heterozygous mutation (S281N) in the patient, but not in other family members. Interestingly, the latter mutation is located in the extracellular domain of the TSHR, and functional studies revealed a marked increase in basal cAMP levels when the mutant receptor was expressed in COS-7 cells. To address the question of whether both mutations were present on the same allele, a double mutant TSHR (S281N/R528H) was generated and characterized. These functional studies in conjunction with RT-PCR analysis of thyroid tissue obtained from subtotal thyroidectomy performed at the age of 6 yr revealed that the patient bears two distinct mutations on different alleles: the familial paternal R528H mutation to be regarded as a polymorphism and a de novo mutation (S281N) on the maternal allele accounting for the clinical picture. Thus, the main conclusions to be drawn from this case are 1) a search for mutations in cases of congenital nonautoimmune hyperthyroidism should not remain restricted to exon 10 of the TSHR gene, because germ-line gain of function mutations of the TSH receptor can be located outside of the transmembrane core of the receptor; and 2) this case illustrates the necessity for careful functional characterization of any novel mutation before a causal relationship to hyperthyroidism can be established.

The pathogenesis of congenital hypothyroidism due to thyroid dysgenesis is still unknown. A point mutation in the TSH receptor (TSHR) of the hypothyroid hyt/hyt mouse invoked the TSHR as a candidate gene for congenital hypothyroidism. Therefore, we screened for mutations in the TSHR gene in patients with congenital hypothyroidism and hypoplasia of the gland. In one girl detected in neonatal screening with the confirmed diagnosis of permanent congenital hypothyroidism with reduced thyroid volume, two novel mutations in the TSHR gene were identified. Single strand conformational polymorphism and subsequent DNA sequencing studies of a fragment of the TSHR gene showed that the patient is a compound heterozygote for 2 loss of function mutations in exon 10 of the TSHR gene. In the mutant maternal allele, 18 nucleotides (positions 1217–1234) are deleted, and 4 novel bp are inserted, resulting in a frameshift and premature termination of the coding sequence. Transfection studies showed that this truncated TSHR wa...

The TSH receptor (TSHR) is a member of the glycoprotein hormone receptors, a subfamily of family A G protein-coupled receptors. The TSHR is of great importance for the growth and function of the thyroid gland. The TSHR and its endogenous ligand TSH are pivotal proteins with respect to a variety of physiological functions and malfunctions. The molecular events of TSHR regulation can be summarized as a process of signal transduction, including signal reception, conversion, and amplification. The steps during signal transduction from the extra- to the intracellular sites of the cell are not yet comprehensively understood. However, essential new insights have been achieved in recent years on the interrelated mechanisms at the extracellular region, the transmembrane domain, and intracellular components. This review contains a critical summary of available knowledge of the molecular mechanisms of signal transduction at the TSHR, for example, the key amino acids involved in hormone binding or in the structural conformational changes that lead to G protein activation or signaling regulation. Aspects of TSHR oligomerization, signaling promiscuity, signaling selectivity, phenotypes of genetic variations, and potential extrathyroidal receptor activity are also considered, because these are relevant to an understanding of the overall function of the TSHR, including physiological, pathophysiological, and pharmacological perspectives. Directions for future research are discussed.

Liesenkötter KP, Göpel W, Bogner U, Stach B, Grüters A. Earliest prevention of endemic goiter by iodine supplementation during pregnancy. Eur J Endocrinol 1996;134:443–8. ISSN 0804–4643 During pregnancy complex changes of maternal thyroid function occur and they are influenced by the maternal iodine supply. It has been demonstrated that with decreasing iodine supply maternal goiter and hypothyroxinemia as well as fetal and neonatal hypothyroidism become more prevalent. Therefore iodine supplementation during pregnancy is now strongly recommended also in areas of moderate iodine deficiency. To monitor the success of iodine supplementation and its theoretical risk of increasing the frequency of thyroid autoantibodies, we have investigated the thyroid volume, thyroid function, urinary iodine excretion and antibodies to thyroid peroxidase at 10–12 weeks of gestation and postpartum in 38 mothers receiving 300 μg potassium iodide/day and in 70 mothers without iodine supplementation. In all of their newborns thyroid volume was determined by ultrasound. The thyrotropin (TSH) levels and antibodies to thyroid peroxidase (TPO-ab) in the neonates were measured in dried blood spots on filter paper from their newborn screening. Urinary iodine excretion was increased significantly after iodine supplementation in mothers (p < 0.001) and their newborns ( Klaus Peter Liesenkötter, Kinderklinik Kaiserin Auguste Victoria Haus (KAVH), Virchow-Klinikum der Medizinische Fakultät der Humboldt-Universität zu Berlin, Heubnerweg 6, 14059 Berlin, Germany

In dizygotic premature twins delivered by Caesarean section after prolonged efforts to effect premature birth we found markedly enhanced thyroid stimulating hormone levels. The mother had been subjected to local polyvidone iodine therapy for seven weeks with PVP suppositories to prevent vaginal infections. Control checks revealed persistently elevated TSH levels, whereas T3/T4 levels were always reduced. Greatly pronounced iodine excretion was seen in the urine of both the babies. This raised the suspicion of iodine-induced hypothyroidism in both children. Substitution with 1-thyroxine was initiated and a withdrawal trial performed after 12 weeks. The patients remained euthyroid after the treatment had been discontinued. Due to possible transfer of iodide to the fetus associated with the risk of iodine-induced hypothyroidism, it is recommended to abstain from vaginal application of iodine during pregnancy.