New liquid chromatography tandem mass spectrometry reference data for estradiol show mini-puberty in both sexes and typical pre-pubertal and pubertal patterns.

CONTEXT: Reliable estradiol (E2) reference intervals (RIs) are crucial in pediatric endocrinology. OBJECTIVES: This study aims to develop a sensitive ultra-performance liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method for E2 in serum, to establish graphically represented RI percentiles and annual RIs for both sexes, and to perform a systematic literature comparison. METHODS: First, a UPLC-MS/MS method for E2 was developed. Second, graphically represented RI percentiles and annual RIs covering 0-18 years were computed (cohort of healthy children [1181 girls and 543 boys]). Subsequently, RIs were compared with published data by systematic searches. RESULTS: Lower limit of quantification was 11 pmol/L, indicating high sensitivity. Estradiol first peaked during mini-puberty in both sexes (girls up to 192 pmol/L; boys up to 225 pmol/L). As could be expected, girls showed higher pubertal E2 (up to 638 pmol/L). However, boys' RIs (up to 259 pmol/L) overlapped considerably. We found 4 studies in the literature that also used LC-MS/MS to determine E2 and published RIs for the complete pediatric age range. Reference intervals varied considerably. Pre-pubertal and pubertal phases were present in all studies. Higher E2 during the time of mini-puberty in both sexes was documented in 3 studies including ours. CONCLUSIONS: Variability of RIs for E2 between studies illustrates the importance of laboratory-specific RIs despite using a LC-MS/MS reference method. In boys, the striking E2 peak during mini-puberty as well as high pubertal E2 without phenotypic estrogenization in regular male puberty indicates that the role of E2 in children and, especially in boys, requires better functional understanding.

Mini-Puberty, Physiological and Disordered: Consequences, and Potential for Therapeutic Replacement.

There are 3 physiological waves of central hypothalamic-pituitary-gonadal (HPG) axis activity over the lifetime. The first occurs during fetal life, the second-termed "mini-puberty"-in the first months after birth, and the third at puberty. After adolescence, the axis remains active all through adulthood. Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by a deficiency in hypothalamic gonadotropin-releasing hormone (GnRH) secretion or action. In cases of severe CHH, all 3 waves of GnRH pulsatility are absent. The absence of fetal HPG axis activation manifests in around 50% of male newborns with micropenis and/or undescended testes (cryptorchidism). In these boys, the lack of the mini-puberty phase accentuates testicular immaturity. This is characterized by a low number of Sertoli cells, which are important for future reproductive capacity. Thus, absent mini-puberty will have detrimental effects on later fertility in these males. The diagnosis of CHH is often missed in infants, and even if recognized, there is no consensus on optimal therapeutic management. Here we review physiological mini-puberty and consequences of central HPG axis disorders; provide a diagnostic approach to allow for early identification of these conditions; and review current treatment options for replacement of mini-puberty in male infants with CHH. There is evidence from small case series that replacement with gonadotropins to mimic "mini-puberty" in males could have beneficial outcomes not only regarding testis descent, but also normalization of testis and penile sizes. Moreover, such therapeutic replacement regimens in disordered mini-puberty could address both reproductive and nonreproductive implications.

Effects of Burosumab Treatment on Mineral Metabolism in Children and Adolescents With X-linked Hypophosphatemia.

CONTEXT: Burosumab has been approved for the treatment of children and adults with X-linked hypophosphatemia (XLH). Real-world data and evidence for its efficacy in adolescents are lacking. OBJECTIVE: To assess the effects of 12 months of burosumab treatment on mineral metabolism in children (aged <12 years) and adolescents (aged 12-18 years) with XLH. DESIGN: Prospective national registry. SETTING: Hospital clinics. PATIENTS: A total of 93 patients with XLH (65 children, 28 adolescents). MAIN OUTCOME MEASURES: Z scores for serum phosphate, alkaline phosphatase (ALP), and renal tubular reabsorption of phosphate per glomerular filtration rate (TmP/GFR) at 12 months. RESULTS: At baseline, patients showed hypophosphatemia (-4.4 SD), reduced TmP/GFR (-6.5 SD), and elevated ALP (2.7 SD, each P < .001 vs healthy children) irrespective of age, suggesting active rickets despite prior therapy with oral phosphate and active vitamin D in 88% of patients. Burosumab treatment resulted in comparable increases in serum phosphate and TmP/GFR in children and adolescents with XLH and a steady decline in serum ALP (each P < .001 vs baseline). At 12 months, serum phosphate, TmP/GFR, and ALP levels were within the age-related normal range in approximately 42%, 27%, and 80% of patients in both groups, respectively, with a lower, weight-based final burosumab dose in adolescents compared with children (0.72 vs 1.06 mg/kg, P < .01). CONCLUSIONS: In this real-world setting, 12 months of burosumab treatment was equally effective in normalizing serum ALP in adolescents and children, despite persistent mild hypophosphatemia in one-half of patients, suggesting that complete normalization of serum phosphate is not mandatory for substantial improvement of rickets in these patients. Adolescents appear to require lower weight-based burosumab dosage than children.

Genetic Obesity in Children: Overview of Possible Diagnoses with a Focus on SH2B1 Deletion.

INTRODUCTION: Genetic obesity is rare and quite challenging for pediatricians in terms of early identification. Src-homology-2 (SH2) B adapter protein 1 (SH2B1) is an important component in the leptin-melanocortin pathway and is found to play an important role in leptin and insulin signaling and therefore in the pathogenesis of obesity and diabetes. Microdeletions in chromosome 16p11.2, encompassing the SH2B1 gene, are known to be associated with obesity, insulin resistance, hyperphagia, and developmental delay. The aim of our study is to report on a case series of young individuals with 16p11.2 microdeletions, including the SH2B1 gene, and provide detailed information on body mass index (BMI) development and obesity-associated comorbidities. In this way, we want to raise awareness of this syndromic form of obesity as a differential diagnosis of genetic obesity. METHODS: We describe the phenotype of 7 children (3 male; age range: 2.8-18.0 years) with 16p11.2 microdeletions, encompassing the SH2B1 gene, and present their BMI trajectories from birth onward. Screening for obesity-associated comorbidities was performed at the time of genetic diagnosis. RESULTS: All children presented with severe, early-onset obesity already at the age of 5 years combined with variable developmental delay. Five patients presented with elevated fasting insulin levels, 1 patient developed diabetes mellitus type 2, 4 patients had dyslipidemia, and 4 developed nonalcoholic fatty-liver disease. DISCUSSION/CONCLUSION: Chromosomal microdeletions in 16p11.2, including the SH2B1 gene, in children are associated with severe, early-onset obesity and comorbidities associated with insulin resistance. Early genetic testing in suspicious patients and early screening for comorbidities are recommended.

Endocrine-disrupting chemicals and their effects on puberty.

Sexual maturation in humans is characterized by a unique individual variability. Pubertal onset is a highly heritable polygenic trait but it is also affected by environmental factors such as obesity or endocrine disrupting chemicals. The last 30 years have been marked by a constant secular trend toward earlier age at onset of puberty in girls and boys around the world. More recent data, although more disputed, suggest an increased incidence in idiopathic central precocious puberty. Such trends point to a role for environmental factors in pubertal changes. Animal data suggest that the GnRH-neuronal network is highly sensitive to endocrine disruption during development. This review focuses on the most recent data regarding secular trend in pubertal timing as well as potential new epigenetic mechanisms explaining the developmental and transgenerational effects of endocrine disrupting chemicals on pubertal timing.

EAP1 regulation of GnRH promoter activity is important for human pubertal timing.

The initiation of puberty is orchestrated by an augmentation of gonadotropin-releasing hormone (GnRH) secretion from a few thousand hypothalamic neurons. Recent findings have indicated that the neuroendocrine control of puberty may be regulated by a hierarchically organized network of transcriptional factors acting upstream of GnRH. These include enhanced at puberty 1 (EAP1), which contributes to the initiation of female puberty through transactivation of the GnRH promoter. However, no EAP1 mutations have been found in humans with disorders of pubertal timing. We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited delayed puberty (DP). Variants were analyzed for rare, potentially pathogenic variants enriched in case versus controls and relevant to the biological control of puberty. We identified one in-frame deletion (Ala221del) and one rare missense variant (Asn770His) in EAP1 in two unrelated families; these variants were highly conserved and potentially pathogenic. Expression studies revealed Eap1 mRNA abundance in peri-pubertal mouse hypothalamus. EAP1 binding to the GnRH1 promoter increased in monkey hypothalamus at the onset of puberty as determined by chromatin immunoprecipitation. Using a luciferase reporter assay, EAP1 mutants showed a reduced ability to trans-activate the GnRH promoter compared to wild-type EAP1, due to reduced protein levels caused by the Ala221del mutation and subcellular mislocation caused by the Asn770His mutation, as revealed by western blot and immunofluorescence, respectively. In conclusion, we have identified the first EAP1 mutations leading to reduced GnRH transcriptional activity resulting in a phenotype of self-limited DP.

ErbB4 point mutation in CU3 inbred rats affects gonadotropin-releasing-hormone neuronal function via compromised neuregulin-stimulated prostaglandin E2 release from astrocytes.

Gonadotropin releasing hormone (GnRH)-secretion is not only regulated by neuronal factors but also by astroglia cells via growth factors and ErbB receptors of the epidermal growth factor family. Studies in transgenic mice carrying mutations in the ErbB receptor system experience impaired reproductive capacity. In addition, some of these animals show a typical skin phenotype with wavy hair and curly whiskers. The rat strain SPRD-CU3 (CU3), examined in this study, displays a similar skin phenotype and a significant impairment of the timing of puberty onset and reproductive performance, suggesting a disruption in the astrocytic to GnRH neuronal communication. To address this issue, we analyzed astrocytic prostaglandin E2 (PGE2 ) release from primary hypothalamic astrocytic cell cultures after stimulation with transforming growth factor α (TGFα), ligand for ErbB1/ErbB2, or Neuregulin 1 beta 2 (NRG1ß2 ), ligand for ErbB4/ErbB2 signaling pathway. Compared to cultures from wild type animals, astrocytic cultures from CU3 rats were unable to respond to NRG stimulation, suggesting a disruption of the ErbB4/ErbB2 signaling pathway. This is confirmed by mutational analysis of ErbB4 that revealed a single point mutation at 3125 bp resulting in an amino acid change from proline to glutamine located at the carboxy-terminal region. As a consequence, substantial conformational changes occur in the transmembrane and intracellular domain of the protein, affecting the ability to form a receptor dimer with a partner and the ability to function as a transcriptional regulator. Thus, astroglia to GnRH neuronal signaling via ErbB4 is essential of timely onset of puberty and reproductive function.

A mathematical model for predicting the adult height of girls with idiopathic central precocious puberty: A European validation.

BACKGROUND: A previous single-center study established a mathematical model for predicting the adult height (AH) in girls with idiopathic central precocious puberty (CPP). OBJECTIVE: To perform internal and external validations by comparing the actual AH to the calculated AH established by this model and to update it. METHODS: The original formula, calculated AH (cm) = 2.21 (height at initial evaluation, SD) + 2.32 (target height, SD) - 1.83 (luteinizing hormone/follicle-stimulating hormone peaks ratio) + 159.68, was established in a sample of 134 girls (group 4) and was applied to additional girls with CPP seen in the same center (group 1, n = 35), in Germany (group 2, n = 43) and in the Netherlands (group 3, n = 72). This formula has been updated based on these extended data, and both versions are available at the following location: http://www.kamick.org/lemaire/med/girls-cpp15.html. RESULTS: Despite the differences among the 4 groups in terms of their characteristics at the initial evaluation and the percentages of patients treated with the gonadotropin-releasing hormone analogue, they have similar calculated and actual AHs. The actual AHs are 162.2±7.0, 163.0±7.6, 162.4±7.7 and 162.1±5.6 cm in groups 1 to 4, respectively. They are highly correlated with the AHs calculated by the formula established in the original group (group 4), with R at 0.84, 0.67 and 0.69 in groups 1 to 3, respectively. When the actual AHs and the AHs predicted by the Bayley and Pinneau method are compared, the R is 0.76, 0.51 and 0.64 in groups 1 to 3, respectively. The absolute differences between actual AHs and the calculated AHs are greater than 1 SD (5.6 cm) in 15%, 35% and 28% of the patients in groups 1 to 3, respectively. CONCLUSION: This study validates and updates the previously established formula for predicting AH in girls with CPP. This updated formula can help clinicians to make treatment decisions.

Multiples of Median-Transformed, Normalized Reference Ranges of Steroid Profiling Data Independent of Age, Sex, and Units.

BACKGROUND/AIMS: The high complexity of pediatric reference ranges across age, sex, and units impairs clinical application and comparability of steroid hormone data, e.g., in congenital adrenal hyperplasia (CAH). We developed a multiples-of-median (MoM) normalization tool to overcome this major drawback in pediatric endocrinology. METHODS: Liquid chromatography tandem mass spectrometry data comprising 10 steroid hormones representing 905 controls (555 males, 350 females, 0 to > 16 years) from 2 previous datasets were MoM transformed across age and sex. Twenty-three genetically proven CAH patients were included (21-hydroxylase deficiency [21OHD], n = 19; 11ß-hydroxylase deficiency [11OHD], n = 4). MoM cutoffs for single steroids predicting 21OHD and 11OHD were computed and validated through new, independent patients (21OHD, n = 8; adrenal cortical carcinoma, n = 6; obesity, n = 40). RESULTS: 21OHD and 11OHD patients showed disease-typical, easily recognizable MoM patterns independent of age, sex, and concentration units. Two single-steroid cutoffs indicated 21OHD: 3.87 MoM for 17-hydroxyprogesterone (100% sensitivity and 98.83% specificity) and 12.28 MoM for 21-deoxycortisol (94.74% sensitivity and 100% specificity). A cutoff of 13.18 MoM for 11-deoxycortisol indicated 11OHD (100% sensitivity and 100% specificity). CONCLUSIONS: Age- and sex-independent MoMs are straightforward for a clinically relevant display of multi-steroid patterns. In addition, defined single-steroid MoMs can serve alone as predictors of 21OHD and 11OHD. Finally, MoM transformation offers substantial enhancement of routine and scientific steroid hormone data exchange due to improved comparability.

Psychological well-being and independent living of young adults with childhood-onset craniopharyngioma.

AIM: To assess the psychological well-being and social integration of adults with craniopharyngioma diagnosed in childhood. METHOD: A cross-sectional study of a nationwide cohort of young adults with craniopharyngioma in Germany was performed. A structured questionnaire covered the sociodemographic, clinical data, and subjective effects of the condition on social integration. Psychological well-being was assessed using the Hospital Anxiety and Depression Scale (HADS). Results were compared to young adults with type 1 diabetes mellitus (T1DM). RESULTS: The study included 59 participants (29 females, 30 males; mean age 25y 2mo [SD 5y 10mo]), mean age at first surgery 10y 2mo [SD 3y 7mo]. Compared to the T1DM group, significantly more young people with craniopharyngioma aged 25 to 35 years lived at their parents' homes (craniopharyngioma 43.34%; T1DM 13.7%; χ2 =4.14, p=0.049), and fewer lived in a relationship (craniopharyngioma 8.69%; T1DM 54.7%; χ2 =15.74, p<0.001). The HADS revealed a score for depression above the cut-off in 20.69 per cent of young adults with craniopharyngioma and in 6 per cent of young adults with T1DM (χ2 =13.42, p<0.001). INTERPRETATION: Young adults with craniopharyngioma reported subjective disadvantages in professional and social integration. Further, they presented with reduced well-being and increased depression rates. Better psychosocial support and self-management education might reduce the long-term burden of the disease.

17α-hydroxylase deficiency diagnosed in early infancy caused by a novel mutation of the CYP17A1 gene.

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.

Pubertal timing after neonatal diethylstilbestrol exposure in female rats: neuroendocrine vs peripheral effects and additive role of prenatal food restriction.

We studied the effects of neonatal exposure to diethylstilbestrol (DES) on pubertal timing in female rats. We examined associated neuroendocrine changes and effects of prenatal food restriction. Age at vaginal opening was advanced after exposure to 10 µg/kg/d of DES and delayed after 1 µg/kg/d (subcutaneous injections). Using this lower dose, pulsatile GnRH secretion was slower at 25 days of age. Both doses reduced KiSS1 mRNA levels at 15 days of age. Using functional Kisspeptin promoter assay, 1 or 10 µM DES reduced or increased KISS1 transcription, respectively. Leptin stimulatory effect on GnRH secretion in vitro (15 days of age) was reduced after prenatal food restriction and neonatal DES exposure (higher dose), both effects being cumulative. Thus, alterations in pubertal timing by DES neonatally are not unequivocally toward precocity, the level of exposure being critical. We provide evidence of neuroendocrine disruption and interaction with prenatal food availability.

Endocrine disrupting chemicals affect the gonadotropin releasing hormone neuronal network.

Endocrine disrupting chemicals have been shown to alter the pubertal process. The controlling levels of the Gonadotropin releasing hormone (GnRH) network involve GnRH itself, KiSS1, and the transcriptional regulators enhanced at puberty 1 (EAP1), Thyroid Transcription Factor 1 (TTF1), and Yin Yang 1 (YY1). While Genistein and Bisphenol A (BPA) have been shown to advance the advent of puberty, exposure to Dioxin delayed pubertal onset. Utilizing in vitro approaches, we observed that Genistein and BPA suppress inhibitory and activate stimulatory components of the GnRH network, while Dioxin exhibit an inhibitory effect at all regulatory hierarchical levels of the GnRH network. It repressed KiSS1, Gnrh, Ttf1 and Yy1 transcription via the xenobiotic response element (XRE), while EAP1 was not affected. Therefore, EDCs alter the neuroendocrine GnRH regulatory network at all hierarchical levels.

Molecular and gene network analysis of thyroid transcription factor 1 (TTF1) and enhanced at puberty (EAP1) genes in patients with GnRH-dependent pubertal disorders.

BACKGROUND/AIM: TTF1 and EAP1 are transcription factors that modulate gonadotropin-releasing hormone expression. We investigated the contribution of TTF1 and EAP1 genes to central pubertal disorders. PATIENTS AND METHODS: 133 patients with central pubertal disorders were studied: 86 with central precocious puberty and 47 with normosmic isolated hypogonadotropic hypogonadism. The coding region of TTF1 and EAP1 were sequenced. Variations of polyglutamine and polyalanine repeats in EAP1 were analyzed by GeneScan software. Association of TTF1 and EAP1 to genes implicated in timing of puberty was investigated by meta-network framework GeneMANIA and Cytoscape software. RESULTS: Direct sequencing of the TTF1 did not reveal any mutation or polymorphisms. Four EAP1 synonymous variants were identified with similar frequencies among groups. The most common EAP1 5'-distal polyalanine genotype was the homozygous 12/12, but the genotype 12/9 was identified in 2 central precocious puberty sisters without functional alteration in EAP1 transcriptional activity. TTF1 and EAP1 were connected, via genetic networks, to genes implicated in the control of menarche. CONCLUSION: No TTF1 or EAP1 germline mutations were associated with central pubertal disorders. TTF1 and EAP1 may affect puberty by changing expression in response to other members of puberty-associated gene networks, or by differentially affecting the expression of gene components of these networks.

Neonatal screening: identification of children with 11ß-hydroxylase deficiency by second-tier testing.

BACKGROUND: 21-Hydroxylase deficiency (21-OHD) is the target disease of newborn screening for congenital adrenal hyperplasia (CAH). We describe the additional detection of patients suffering from 11ß-hydroxylase deficiency (11-OHD) by second-tier testing. METHOD: Over a period of 5 years, screening for CAH was done in a total of 986,098 newborns by time-resolved immunoassay (DELFIA®) for 17α-hydroxyprogesterone (17-OHP). Positive samples were subsequently analyzed in an LC-MS/MS second-tier test including 17-OHP, cortisol, 11-deoxycortisol, 4-androstenedione and 21-deoxycortisol. RESULTS: In addition to 78 cases of 21-OHD, 5 patients with 11-OHD were identified. Diagnostic parameters were a markedly elevated concentration of 11-deoxycortisol in the presence of a low level of cortisol. Androstenedione was also increased. In contrast to 21-OHD, concentrations of 21-deoxycortisol were normal. CONCLUSION: Steroid profiling in newborn blood samples showing positive results in immunoassays for 17-OHP allows for differentiating 21-OHD from 11-OHD. This procedure may not detect all cases of 11-OHD in the newborn population because there may be samples of affected newborns with negative results for 17-OHP in the immunoassay.

Transcription of the human EAP1 gene is regulated by upstream components of a puberty-controlling Tumor Suppressor Gene network.

Mammalian puberty is initiated by an increased pulsatile release of gonadotropin-releasing hormone (GnRH) from specialized neurons located in the hypothalamus. GnRH secretion is controlled by neuronal and glial networks, whose activity appears to be coordinated via transcriptional regulation. One of the transcription factors involved in this process is thought to be the recently described gene Enhanced at Puberty 1 (EAP1), which encodes a protein with dual transcriptional activity. In this study we used gene reporter and chromatin immunoprecipitation (ChIP) assays to examine the hypothesis that EAP1 expression is controlled by transcriptional regulators earlier postulated to serve as central nodes of a gene network involved in the neuroendocrine control of puberty. These regulators include Thyroid Transcription Factor 1 (TTF1), Yin Yang 1 (YY1), and CUX1, in addition to EAP1 itself. While TTF1 has been shown to facilitate the advent of puberty, YY1 (a zinc finger protein component of the Polycomb silencing complex) may play a repressive role. The precise role of CUX1 in this context is not known, but like EAP1, CUX1 can either activate or repress gene transcription. We observed that DNA segments of two different lengths (998 and 2744bp) derived from the 5'-flanking region of the human EAP1 gene display similar transcriptional activity. TTF1 stimulates transcription from both DNA segments with equal potency, whereas YY1, CUX1, and EAP1 itself, behave as transcriptional repressors. All four proteins are recruited in vivo to the EAP1 5'-flanking region. These observations suggest that EAP1 gene expression is under dual transcriptional regulation imposed by a trans-activator (TTF1) and two repressors (YY1 and CUX1) previously postulated to be upstream components of a puberty-controlling gene network. In addition, EAP1 itself appears to control its own expression via a negative auto-feedback loop mechanism. Further studies are needed to determine if the occupancy of the EAP1 promoter by these regulatory factors changes at the time of puberty.

Hypothalamic EAP1 (enhanced at puberty 1) is required for menstrual cyclicity in nonhuman primates.

Mammalian reproductive cyclicity requires the periodic discharge of GnRH from hypothalamic neurons into the portal vessels connecting the neuroendocrine brain to the pituitary gland. GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The transcriptional control of this process is not well understood, but it appears to involve several genes. One of them, termed enhanced at puberty 1 (EAP1), has been postulated to function in the female hypothalamus as an upstream regulator of neuroendocrine reproductive function. RNA interference-mediated inhibition of EAP1 expression, targeted to the preoptic region, delays puberty and disrupts estrous cyclicity in rodents, suggesting that EAP1 is required for the normalcy of these events. Here, we show that knocking down EAP1 expression in a region of the medial basal hypothalamus that includes the arcuate nucleus, via lentiviral-mediated delivery of RNA interference, results in cessation of menstrual cyclicity in female rhesus monkeys undergoing regular menstrual cycles. Neither lentiviruses encoding an unrelated small interfering RNA nor the placement of viral particles carrying EAP1 small interfering RNA outside the medial basal hypothalamus-arcuate nucleus region affected menstrual cycles, indicating that region-specific expression of EAP1 in the hypothalamus is required for menstrual cyclicity in higher primates. The cellular mechanism by which EAP1 exerts this function is unknown, but the recent finding that EAP1 is an integral component of a powerful transcriptional-repressive complex suggests that EAP1 may control reproductive cyclicity by inhibiting downstream repressor genes involved in the neuroendocrine control of reproductive function.

Transcriptional regulation of the human KiSS1 gene.

Kisspeptin, the product of the KiSS1 gene, has emerged as a key component of the mechanism by which the hypothalamus controls puberty and reproductive development. It does so by stimulating the secretion of gonadotropin releasing hormone (GnRH). Little is known about the transcriptional control of the KiSS1 gene. Here we show that a set of proteins postulated to be upstream components of a hypothalamic network involved in controlling female puberty regulates KiSS1 transcriptional activity. Using RACE-PCR we determined that transcription of KiSS1 mRNA is initiated at a single transcription start site (TSS) located 153-156bp upstream of the ATG translation initiation codon. Promoter assays performed using 293 MSR cells showed that the KiSS1 promoter is activated by TTF1 and CUX1-p200, and repressed by EAP1, YY1, and CUX1-p110. EAP1 and CUX-110 were also repressive in GT1-7 cells. All four TFs are recruited in vivo to the KiSS1 promoter and are expressed in kisspeptin neurons. These results suggest that expression of the KiSS1 gene is regulated by trans-activators and repressors involved in the system-wide control of mammalian puberty.

A single copy of the recently identified dual oxidase maturation factor (DUOXA) 1 gene produces only mild transient hypothyroidism in a patient with a novel biallelic DUOXA2 mutation and monoallelic DUOXA1 deletion.

CONTEXT: Dual oxidases (DUOX1 and DUOX2) play a crucial role in the generation of hydrogen peroxide required in the oxidation of iodide and the synthesis of thyroid hormone. Heterodimerization with specific maturation factors (DUOXA1 and DUOXA2) is essential for the maturation and function of the DUOX enzyme complexes. Biallelic loss-of-function mutations of DUOX2 result in congenital hypothyroidism (CH), whereas a single reported case of homozygous DUOXA2 mutation (Y246X) has been associated with mild CH. OBJECTIVE: We now report an infant with transient CH due to a complex genetic alteration of the DUOX/DUOXA system. RESULTS: Our patient was born to euthyroid nonconsanguineous parents and presented with an elevated TSH and enlarged thyroid gland at neonatal screening. Genetic analysis revealed a missense mutation (C189R) on the maternal DUOXA2 allele. The mutant DUOXA2 protein showed complete loss-of-function in reconstituting DUOX2 in vitro. The apparent C189R homozygosity of the proband in the absence of the same mutation in the father led to detailed gene mapping, revealing an approximately 43-kb pair deletion encompassing DUOX2, DUOXA1, and DUOXA2. Thus, in addition to being deficient in DUOXA2, the proband lacks one allele of DUOX2 and DUOXA1 but has two functioning DUOX1 alleles. CONCLUSION: The transient CH in the presence of only one functional maturation factor allele indicates a high level of functional redundancy in the DUOX/DUOXA system.