Oxygen-sensitive regulation and neuroprotective effects of growth hormone-dependent growth factors during early postnatal development.

Perinatal hypoxia severely disrupts metabolic and somatotrophic development, as well as cerebral maturational programs. Hypoxia-inducible transcription factors (HIFs) represent the most important endogenous adaptive mechanisms to hypoxia, activating a broad spectrum of growth factors that contribute to cell survival and energy homeostasis. To analyze effects of systemic hypoxia and growth hormone (GH) therapy (rhGH) on HIF-dependent growth factors during early postnatal development, we compared protein (using ELISA) and mRNA (using quantitative RT PCR) levels of growth factors in plasma and brain between normoxic and hypoxic mice (8% O2, 6 h; postnatal day 7, P7) at P14. Exposure to hypoxia led to reduced body weight (P < 0.001) and length (P < 0.04) compared with controls and was associated with significantly reduced plasma levels of mouse GH (P < 0.01) and IGF-1 (P < 0.01). RhGH abrogated these hypoxia-induced changes of the GH/IGF-1 axis associated with normalization of weight and length gain until P14 compared with controls. In addition, rhGH treatment increased cerebral IGF-1, IGF-2, IGFBP-2, and erythropoietin mRNA levels, resulting in significantly reduced apoptotic cell death in the hypoxic, developing mouse brain. These data indicate that rhGH may functionally restore hypoxia-induced systemic dysregulation of the GH/IGF-1 axis and induce upregulation of neuroprotective, HIF-dependent growth factors in the hypoxic developing brain.

Rare copy number variants are a common cause of short stature.

Human growth has an estimated heritability of about 80%-90%. Nevertheless, the underlying cause of shortness of stature remains unknown in the majority of individuals. Genome-wide association studies (GWAS) showed that both common single nucleotide polymorphisms and copy number variants (CNVs) contribute to height variation under a polygenic model, although explaining only a small fraction of overall genetic variability in the general population. Under the hypothesis that severe forms of growth retardation might also be caused by major gene effects, we searched for rare CNVs in 200 families, 92 sporadic and 108 familial, with idiopathic short stature compared to 820 control individuals. Although similar in number, patients had overall significantly larger CNVs (p-value<1×10(-7)). In a gene-based analysis of all non-polymorphic CNVs>50 kb for gene function, tissue expression, and murine knock-out phenotypes, we identified 10 duplications and 10 deletions ranging in size from 109 kb to 14 Mb, of which 7 were de novo (p<0.03) and 13 inherited from the likewise affected parent but absent in controls. Patients with these likely disease causing 20 CNVs were smaller than the remaining group (p<0.01). Eleven (55%) of these CNVs either overlapped with known microaberration syndromes associated with short stature or contained GWAS loci for height. Haploinsufficiency (HI) score and further expression profiling suggested dosage sensitivity of major growth-related genes at these loci. Overall 10% of patients carried a disease-causing CNV indicating that, like in neurodevelopmental disorders, rare CNVs are a frequent cause of severe growth retardation.

DNA methylation of the p66Shc promoter is decreased in placental tissue from women delivering intrauterine growth restricted neonates.

OBJECTIVE: The adaptor protein p66Shc generates mitochondrial reactive oxygen species and translates oxidative signals into apoptosis. We aimed to analyze potential alterations in total methylation and in p66Shc activation in placental tissues from women delivering intrauterine growth restricted neonates (IUGR) versus appropriate for gestational age (AGA) and small for gestational age (SGA) neonates. METHOD: DNA methylation of the p66Shc promoter and of long interspersed nuclear elements (LINE-1), as a marker for total methylation, was quantified by automatic pyrosequencing in 15 IUGR, 25 AGA and 15 SGA placentas. Placental gene expression of p66Shc was determined by TaqMan real-time polymerase chain reaction. RESULTS: No significant difference was found for LINE-1 methylation between IUGR, AGA and SGA newborns. DNA methylation of the p66Shc promoter was significantly decreased in the IUGR compared with the AGA group (p < 0.0001) and the SGA group (p < 0.0001). However, analysis of placental p66Shc gene expression did not show a significant difference between the three groups. CONCLUSION: It remains speculative if the decreased p66Shc promoter methylation might play a role in the pathophysiology of endothelial dysfunction and cardiovascular disease after IUGR.

Sex chromosome DSD individuals with mosaic 45,X0 and aberrant Y chromosomes in 46,XY cells: distinct gender phenotypes and germ cell tumour risks§.

"Differences of Sexual Development (DSD)," individuals with rearranged Y chromosome breaks in their 46,XY cells are reported with male and female gender phenotypes and differences in germ cell tumour (GCT) risk. This raised the question of whether male or female gender and GCT risk depends on the site of the break and/or rearrangement of the individual´s Y chromosome. In this paper, we report molecular mapping of the breakpoint on the aberrant Y chromosome of 22 DSD individuals with a 45,X/46,XY karyotype reared with a different gender. Their Y chromosome breaks are found at different sites on the long and short Y arms. Our data indicate that gender rearing is, neither dependent on the site of Y breakage, nor on the amount of 45,X0 cells in the individuals' leukocytes. Most prominent are secondary rearrangements of the Y chromosome breaks forming di-centric Y-structures ("dic-Y"). Duplications of the short Y arm and the proximal part of the long Y arm are the results. A putative GCT risk has been analysed with immunohistochemical experiments on some dysgenetic gonadal tissue sections. With specific antibodies for OCT3/4 expression, we marked the pluripotent germ cell fraction being potential tumour precursor cells. With specific antibodies for DDX3Y, TSPY, and UTY we analyzed their putative Gonadoblastoma Y (GBY) tumour susceptibility function in the same specimen. We conclude GBY expression is only diagnostic for GCT development in the aberrant germ cells of these DSD individuals when strong OCT3/4 expression has marked their pluripotency.

Evolutionary conserved networks of human height identify multiple Mendelian causes of short stature.

Height is a heritable and highly heterogeneous trait. Short stature affects 3% of the population and in most cases is genetic in origin. After excluding known causes, 67% of affected individuals remain without diagnosis. To identify novel candidate genes for short stature, we performed exome sequencing in 254 unrelated families with short stature of unknown cause and identified variants in 63 candidate genes in 92 (36%) independent families. Based on systematic characterization of variants and functional analysis including expression in chondrocytes, we classified 13 genes as strong candidates. Whereas variants in at least two families were detected for all 13 candidates, two genes had variants in 6 (UBR4) and 8 (LAMA5) families, respectively. To facilitate their characterization, we established a clustered network of 1025 known growth and short stature genes, which yielded 29 significantly enriched clusters, including skeletal system development, appendage development, metabolic processes, and ciliopathy. Eleven of the candidate genes mapped to 21 of these clusters, including CPZ, EDEM3, FBRS, IFT81, KCND1, PLXNA3, RASA3, SLC7A8, UBR4, USP45, and ZFHX3. Fifty additional growth-related candidates we identified await confirmation in other affected families. Our study identifies Mendelian forms of growth retardation as an important component of idiopathic short stature.

SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syndrome.

BACKGROUND: Heterozygous gain-of-function mutations in various genes encoding proteins of the Ras-MAPK signalling cascade have been identified as the genetic basis of Noonan syndrome (NS) and cardio-facio-cutaneous syndrome (CFCS). Mutations of SOS1, the gene encoding a guanine nucleotide exchange factor for Ras, have been the most recent discoveries in patients with NS, but this gene has not been studied in patients with CFCS. METHODS AND RESULTS: We investigated SOS1 in a large cohort of patients with disorders of the NS-CFCS spectrum, who had previously tested negative for mutations in PTPN11, KRAS, BRAF, MEK1 and MEK2. Missense mutations of SOS1 were discovered in 28% of patients with NS. In contrast, none of the patients classified as having CFCS was found to carry a pathogenic sequence change in this gene. CONCLUSION: We have confirmed SOS1 as the second major gene for NS. Patients carrying mutations in this gene have a distinctive phenotype with frequent ectodermal anomalies such as keratosis pilaris and curly hair. However, the clinical picture associated with SOS1 mutations is different from that of CFCS. These findings corroborate that, despite being caused by gain-of-function mutations in molecules belonging to the same pathway, NS and CFCS scarcely overlap genotypically.

Individualised growth response optimisation (iGRO) tool: an accessible and easy-to-use growth prediction system to enable treatment optimisation for children treated with growth hormone.

BACKGROUND: Growth prediction models (GPMs) exist to support clinical management of children treated with growth hormone (GH) for growth hormone deficiency (GHD), Turner syndrome (TS) and for short children born small for gestational age (SGA). Currently, no prediction system has been widely adopted. CONTENT: The objective was to develop a stand-alone web-based system to enable the widespread use of an 'individualised growth response optimisation' (iGRO) tool across European endocrinology clinics. A modern platform was developed to ensure compatibility with IT systems and web browsers. Seventeen GPMs derived from the KIGS database were included and tested for accuracy. SUMMARY: The iGRO system demonstrated prediction accuracy and IT compatibility. The observed discrepancies between actual and predicted height may support clinicians in investigating the reasons for deviations around the expected growth and optimise treatment. CONCLUSIONS: This system has the potential for wide access in endocrinology clinics to support the clinical management of children treated with GH for these three indications.

Prevalence of autoantibodies associated with thyroid and celiac disease in Ullrich-Turner syndrome in relation to adult height after growth hormone treatment.

A prospective, multicenter study of patients with Ullrich-Turner syndrome (UTS) was conducted to estimate the prevalence of autoantibodies to tissue transglutaminase (tTg), thyroid stimulating hormone receptor (TSH-R), thyroglobulin (TG) and thyroid peroxidase (TPO) in relation to adult height after long-term growth hormone (GH) treatment. Out of 347 near-adult (> 16 years) patients with UTS from 96 German centers, whose longitudinal growth was documented within the Pharmacia International Growth Study (KIGS), 188 returned for a standardized follow-up visit at a median chronological age of 18.7 (16.0-23.6) years (bone age > 15 years). Serum samples of 120 patients were obtained for central measurements of TSH, thyroxine (T4) and free T4 and autoantibodies by standard immunoassays. Information regarding thyroid disease, karyotype and anthropometric data was extracted from the KIGS database. Thirty-six percent of the patients with UTS had positive TG and/or TPO autoantibodies and 4% had positive tTg autoantibodies, whereas 2% had positive TG and/or TPO autoantibodies as well as positive tTg autoantibodies. TSH-R autoantibodies were undetectable in all patients. The detection of autoantibodies was unrelated to a specific karyotype. Median height standard deviation scores (SDS, UTS) at start of GH treatment (0.43; -1.07, 1.85) and at follow-up (1.36; -0.11, 2.57) were comparable in all patients independent of their antibody status. The total deltaheight SDS, however, was higher in patients with negative autoantibody titers (1.08; -0.03, 2.25) compared to those with positive antibody titers (0.68; -0.44, 1.82; p < 0.01). Our study confirms the high prevalence of autoantibodies in patients with UTS predisposing them to autoimmune thyroid disease and celiac disease, and indicates for the first time that autoimmune pathologies may interfere with GH therapy and thus compromise final height. Therefore, medical care for patients with UTS should routinely include screening for these autoimmune disorders in order to assure early detection and appropriate treatment.

Measurement of amniotic fluid steroids of midgestation via LC-MS/MS.

INTRODUCTION: Analysis of steroids by mass spectrometry (MS) has evolved into a reliable tool for the simultaneous detection of multiple steroids. As amniotic fluid (AF) and fetal serum composition of early pregnancy are closely related, the analysis of AF can yield information on the physiological status of the developing fetus. We evaluated the use of liquid-chromatography tandem mass spectrometry (LC-MS/MS) for AF steroid analysis, including the analysis of its sensitivity and accuracy for gender verification in healthy subjects. MATERIALS AND METHODS: AF of 78 male and 94 female healthy newborns was analyzed by LC-MS/MS at 16 weeks of gestation. The levels of androstenedione, corticosterone, cortisol, cortisone, deoxycorticosterone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S), 17-hydroxyprogesterone, progesterone (17-OHP) and testosterone were measured. Steroid levels were compared to RIA and GC-MS levels of midgestation from the literature. Cross-validated logistic regression was used to obtain statistical predictions of gender at birth from testosterone and the above steroids. RESULTS: LC-MS/MS analysis of AF steroids yielded comparable results with published GC-MS data. Gender specific differences were found for androstenedione and testosterone concentrations with higher levels in the male fetus. In contrast to published RIA data no gender specific differences were observed for 17-hydroxyprogesterone and dehydroepiandrosterone AF concentrations. Testosterone concentrations yielded highly accurate predictions for male gender at birth. Additional analysis of further steroids did neither increase the accuracy, sensitivity nor specificity of this prediction. The estimated optimal cut-off value for amniotic testosterone level was 0.074 µg/L for healthy male newborns. CONCLUSIONS: LC-MS/MS is a reliable method for the determination of steroids in amniotic fluid. The determination of testosterone in amniotic fluid by LC-MS/MS in early pregnancy of healthy subjects can be used to offer a reliable prediction of fetal gender at birth.

Detection of expressional changes induced by intrauterine growth restriction in the developing rat mammary gland via exploratory pathways analysis.

BACKGROUND: Intrauterine growth restriction (IUGR) is thought to lead to fetal programming that in turn contributes to developmental changes of many organs postnatally. There is evidence that IUGR is a risk factor for the development of metabolic and cardiovascular disease later in life. A higher incidence of breast cancer was also observed after IUGR. This could be due to changes in mammary gland developmental pathways. We sought to characterise IUGR-induced alterations of the complex pathways of mammary development at the level of the transcriptome in a rat model of IUGR, using pathways analysis bioinformatics. METHODOLOGY/PRINCIPAL FINDINGS: We analysed the mammary glands of Wistar rats with IUGR induced by maternal low protein (LP) diet at the beginning (d21) and the end (d28) of pubertal ductal morphogenesis. Mammary glands of the LP group were smaller in size at d28, however did not show morphologic changes. We identified multiple differentially expressed genes in the mammary gland using Agilent SurePrint arrays at d21 and d28. In silico analysis was carried out using Ingenuity Pathways Analysis. In mammary gland tissue of LP rats at d21 of life a prominent upregulation of WT1 and CDKN1A (p21) expression was observed. Differentially regulated genes were associated with the extracellular regulated kinase (ERK)-1/-2 pathway. Western Blot analysis showed reduced levels of phosphorylated ERK-1/-2 in the mammary glands of the LP group at d21. To identify possible changes in circulating steroid levels, serum LC-Tandem mass-spectrometry was performed. LP rats showed higher serum progesterone levels and an increased corticosterone/dehydrocorticosterone-ratio at d28. CONCLUSIONS/SIGNIFICANCE: Our data obtained from gene array analysis support the hypothesis that IUGR influences pubertal development of the rat mammary gland. We identified prominent differential regulation of genes and pathways for factors regulating cell cycle and growth. Moreover, we detected new pathways which appear to be programmed by IUGR.

Height gain in Ullrich-Turner syndrome after early and late growth hormone treatment start: results from a large retrospective German study and potential basis for an individualized treatment approach.

BACKGROUND: Ullrich-Turner syndrome (UTS) girls often present with short stature in adolescence to the endocrinologist when the efficacy of growth hormone (GH) to improve growth remains unknown and parameters to estimate individual GH responsiveness have yet to be determined. OBJECTIVE: Retrospective evaluation of adult height (AH) and predicted adult height at GH start (descriptive model of Ranke, Model PredAH) in early and late GH-treated German UTS patients. SUBJECTS/METHODS: 313 patients treated with GH, early [chronological age (CA) at GH start <12 years, n = 259] or late (CA at GH start ≥12 years, n = 54) who reached AH were selected from KIGS (Pfizer International Growth Database). RESULTS: AH (152.5 ± 5.9 vs. 151.1 ± 5.4 cm, p = n.s.) after GH treatment for 7.5 ± 2.12 years (GH start early) and for 5.2 ± 1.2 years (GH start late) were similar (p = n.s.) as Model PredAH (155.7 ± 4.8 vs. 154.7 ± 4.8 cm; p = n.s.) but higher (p < 0.001) than projected adult height (Ranke, ProjAH; 148.2 ± 5.5 vs. 145.2 ± 6.7 cm; p = 0.001). Total height gain over ProjAH was 4.3 ± 4.6 cm (GH start early) and 5.8 ± 4.7 cm (GH start late, p = 0.021), respectively. CONCLUSIONS: GH may improve AH in UTS patients even when started late. The individual growth response could be estimated by the descriptive Model PredAH independent of age at treatment start.

Growth in pre-pubertal children with myelomeningocele (MMC) on growth hormone (GH): the KIGS experience.

PURPOSE: To analyse the auxological data of children with myelomeningocele (MMC) on growth hormone (GH) therapy whose growth data was documented within KIGS (Pfizer International Growth Database). Longitudinal growth data of a sub-group of pre-pubertal children were studied after a treatment period of 3 years. PATIENTS AND METHODS: Eighty patients (38 m, 42 f) with MMC with a median chronological age (CA) of 11.6 years (at latest visit) on GH were registered in the KIGS database. In 52 patients, GH deficiency was documented. GH therapy started with a median dose of 0.23 mg kg(-1) per week. The 3-year longitudinal growth was analysed in 21 patients (13 m, 8 f; median CA 9.2 years, latest visit), all of whom were pre-pubertal at start and during GH therapy. RESULTS: GH therapy started at 7.5 years with a dose of 0.23 mg kg(-1) per week. Birth length SDS (-0.51) and mid-parental height SDS (+0.07) were in the normal range. BMI SDS at start was +0.24, at latest visit -0.03. After a median treatment duration of 3.0 years (latest visit), height SDS improved from -2.97 (start of GH) to -2.01. The sub-group of pre-pubertal MMC patients started GH therapy (dose 0.22 mg kg(-1) per week) at 6.2 years. Growth velocity (GV) SDS increased significantly (at start: -1.77; 1 year: +2.60, 2 years: +2.25, 3 years: +1.24), thus height SDS improved from -3.25 at start to -1.87 at 36 months. BMI SDS was in the normal range and remained unchanged during GH therapy. No major side effects of GH were recorded. CONCLUSION: GH had positive effects on height SDS in MMC patients. The analysis of the longitudinal growth data of pre-pubertal MMC patients showed a significant increase in GV SDS and improvement of height SDS.

Uterine size in women with Turner syndrome after induction of puberty with estrogens and long-term growth hormone therapy: results of the German IGLU Follow-up Study 2001.

BACKGROUND: To evaluate the factors influencing uterine size in young adult women with Turner syndrome (TS) after long-term growth hormone (GH) treatment. METHODS: Cross-sectional study. Out of 188 women with TS from 96 German centres, whose longitudinal growth was documented within KIGS (Pfizer International Growth Database), data on uterine size were collected voluntarily at a standardized follow-up visit: 75 TS women (ages: 15.8-30.8 years) with complete data were included. Classification according to karyotype: 45,X (78.6%), 45,X/46,XX (5.4%), 45,X/46,iXq (8%), 45,X/46,XY (8%). Puberty was induced with estrogens in all women. At follow-up, 66 were on cyclic estrogens and progestins. RESULTS: 13/66 (19.6%) TS women who received estrogens had a reduced uterine length <5 cm. Calculating the data in standard deviation scores (SDS), only women with 45,X/46,XX karyotype had normal median uterine length and volume of 0.6 and 1.59 SDS respectively. An incomplete breast development (Tanner stage B 3) was found in women with 45,X karyotype (n = 11; 18.6%) and with 45,X/46,XY (n = 2). CONCLUSIONS: Only TS women with karyotype 45,X/46,XX had normal uterine sizes, whereas 26% of the TS women with karyotype 45,X had a uterine length <-2 SDS, and 18% a volume <-2 SDS.

Genotype-phenotype correlations in Noonan syndrome.

OBJECTIVE: To study genotype-phenotype correlations in a cohort of clinically well-characterized pediatric patients with Noonan syndrome (NS). Study design Fifty-seven unrelated patients with the clinical diagnosis of NS ascertained according to standardized inclusion criteria were prospectively enrolled. Mutational analysis was performed by direct sequencing of the entire coding sequence of the PTPN11 gene. RESULTS: Sixteen known and 3 novel PTPN11 mutations could be detected in 60% of index patients, in all familial and in 52% of the sporadic cases. Presence of pulmonic stenosis, short stature, easy bruising, and thorax deformities was significantly associated with a PTPN11 mutation, whereas cardiomyopathy was more common in patients without a mutation. On average, PTPN11 mutation-negative probands fulfilled fewer clinical criteria of NS, but more than half-among them all with cardiomyopathy-had the full clinical picture of NS indistinguishable from typical cases with PTPN11 mutation. CONCLUSIONS: The phenotype of NS due to PTPN11 mutations is clinically unambiguous in the majority of patients and represents a highly penetrant trait. Individuals with the clinical diagnosis of NS but without a PTPN11 mutation presumably represent a heterogeneous group in which patients with cardiomyopathy appear to constitute an interesting subgroup for future research.