Array comparative genomic hybridisation analysis of boys with X linked hypopituitarism identifies a 3.9 Mb duplicated critical region at Xq27 containing SOX3.

INTRODUCTION: Array comparative genomic hybridisation (array CGH) is a powerful method that detects alteration of gene copy number with greater resolution and efficiency than traditional methods. However, its ability to detect disease causing duplications in constitutional genomic DNA has not been shown. We developed an array CGH assay for X linked hypopituitarism, which is associated with duplication of Xq26-q27. METHODS: We generated custom BAC/PAC arrays that spanned the 7.3 Mb critical region at Xq26.1-q27.3, and used them to search for duplications in three previously uncharacterised families with X linked hypopituitarism. RESULTS: Validation experiments clearly identified Xq26-q27 duplications that we had previously mapped by fluorescence in situ hybridisation. Array CGH analysis of novel XH families identified three different Xq26-q27 duplications, which together refine the critical region to a 3.9 Mb interval at Xq27.2-q27.3. Expression analysis of six orthologous mouse genes from this region revealed that the transcription factor Sox3 is expressed at 11.5 and 12.5 days after conception in the infundibulum of the developing pituitary and the presumptive hypothalamus. DISCUSSION: Array CGH is a robust and sensitive method for identifying X chromosome duplications. The existence of different, overlapping Xq duplications in five kindreds indicates that X linked hypopituitarism is caused by increased gene dosage. Interestingly, all X linked hypopituitarism duplications contain SOX3. As mutation of this gene in human beings and mice results in hypopituitarism, we hypothesise that increased dosage of Sox3 causes perturbation of pituitary and hypothalamic development and may be the causative mechanism for X linked hypopituitarism.

"Giving body" to embryos. Modeling, mechanism, and the microtome in late nineteenth-century anatomy.

Reinvestigating the work of the anatomist Wilhelm His (1831-1904) shows how engaging with models in three dimensions can revise our accounts of scientific change. His is known to historians of biology for articulating a mechanical approach to embryology and for inventing a section cutter, or microtome. Focusing on the wax models that he also made in the late 1860s shows how the other two innovations were linked; reconstructing embryos from the sections, His claimed, provided compelling evidence for mechanical views. The next generation of embryologists appropriated His's work selectively. In the 1880s anatomists took up "plastic reconstruction" to visualize the complex forms of higher vertebrate, especially human, embryos. An increasingly dominant experimental embryology, by contrast, drew on His's mechanical approach but had little use for the waxes and effaced them from the history of his work. Recovering these models offers a fresh perspective on the transformation of a central science of animal life and enriches our understanding of the relations between representation in two dimensions and three.

Hyperinsulinism and hyperammonemia in infants with regulatory mutations of the glutamate dehydrogenase gene.

BACKGROUND: A new form of congenital hyperinsulinism characterized by hypoglycemia and hyperammonemia was described recently. We hypothesized that this syndrome of hyperinsulinism and hyperammonemia was caused by excessive activity of glutamate dehydrogenase, which oxidizes glutamate to alpha-ketoglutarate and which is a potential regulator of insulin secretion in pancreatic beta cells and of ureagenesis in the liver. METHODS: We measured glutamate dehydrogenase activity in lymphoblasts from eight unrelated children with the hyperinsulinism-hyperammonemia syndrome: six with sporadic cases and two with familial cases. We identified mutations in the glutamate dehydrogenase gene by sequencing glutamate dehydrogenase complementary DNA prepared from lymphoblast messenger RNA. Site-directed mutagenesis was used to express the mutations in COS-7 cells. RESULTS: The sensitivity of glutamate dehydrogenase to inhibition by guanosine 5'-triphosphate was a quarter of the normal level in the patients with sporadic hyperinsulinism-hyperammonemia syndrome and half the normal level in patients with familial cases and their affected relatives, findings consistent with overactivity of the enzyme. These differences in enzyme insensitivity correlated with differences in the severity of hypoglycemia in the two groups. All eight children were heterozygous for the wild-type allele and had a mutation in the proposed allosteric domain of the enzyme. Four different mutations were identified in the six patients with sporadic cases; the two patients with familial cases shared a fifth mutation. In two clones of COS-7 cells transfected with the mutant sequence from one patient, the sensitivity of the enzyme to guanosine 5'-triphosphate was reduced, findings similar to those in the child's lymphoblasts. CONCLUSIONS: The hyperinsulinism-hyperammonemia syndrome is caused by mutations in the glutamate dehydrogenase gene that impair the control of enzyme activity.

Partial growth-hormone insensitivity: the role of growth-hormone receptor mutations in idiopathic short stature.

Mutations in the GHR locus may play a role in the cause of idiopathic short stature (ISS) by impairing growth-hormone (GH) receptor (GHR) function. At one extreme, mutations that nullify the function of the GH receptor are linked to complete GH insensitivity syndrome, or Laron syndrome, and we hypothesized that less-disruptive mutations could contribute to partial GH insensitivity syndrome. Low levels of GH binding protein may indicate mutations in the extracellular domain of the receptor, and by focusing on 14 children with ISS who had low GH binding protein and insulin-like growth factor I levels, we found three heterozygotes and one compound heterozygote for mutations in the extracellular domain of the receptor. We have since extended our study to a broader spectrum of patients, adding 76 patients with ISS who were treated with GH in a phase II study of the safety and efficacy of recombinant human GH in ISS and also adding 10 patients who were ascertained as having ISS by pediatric endocrinologists in private practice. The GHR gene has thus been analyzed in 100 patients with ISS, eight of whom were found to carry mutations: four in our original study and four with normal or elevated levels of GH binding protein. The latter group consists of three carriers of heterozygous extracellular domain mutations and one carrier of a heterozygous intracellular domain mutation. Family data suggest that the carriers of these mutations have a range of phenotypes, supporting our hypothesis that the expression of these heterozygous mutations as partial GH insensitivity syndrome depends on the genetic makeup of the person.

Growth hormone bioactivity in girls with Turner's syndrome: correlation with insulin-like growth factor I.

We have recently developed a new bioassay for growth hormone (GH) in serum, which is based on the ability of GH to suppress glucose use in cultured murine adipocytes. We tested the hypothesis that bioactive GH (B-GH) concentrations would correlate better with the GH-dependent peptides, IGF-I, and IGF-binding protein-3 (IGFBP-3) than would GH determined by conventional RIA (RIA-GH). Twenty-five girls with Turner's syndrome were studied. The subjects had ages ranging from 4.8 to 15.9 y and height SD from the mean (SD score) ranging from -0.77 to -5.67. Blood samples were obtained every 15 or 20 min for 12 h overnight. For each girl, an equal aliquot of each overnight sample was pooled for determination of B-GH, RIA-GH, IGF-I, IGFBP-3, LH, FSH, and estradiol. Measurable estradiol concentrations were present in six girls and were sufficient to suppress gonadotropin concentrations in two girls, but they did not alter B-GH, RIA-GH, IGF-I, and IGFBP-3 concentrations compared with the age-matched girls without measurable estradiol concentrations. Hence, data for all girls were combined for subsequent regression analyses. RIA-GH did not correlate significantly with B-GH, IGF-I, or IGFBP-3. B-GH exhibited a significant correlation with IGF-I (r = 0.407, p < 0.05), and the correlation with IGFBP-3 was better than that for RIA-GH (r = 0.355 versus 0.064, B-GH and RIA-GH, respectively). None of the B-GH, RIA-GH, IGF-I, or IGFBP-3 concentrations had a significant correlation with height SD score or height velocity SD score.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid masses: approach to diagnosis and management in childhood and adolescence.

The approach to the evaluation of a neck mass requires careful history and physical examination to determine if the mass is thyroidal or non-thyroidal. Thyromegaly can be classified as diffuse or nodular, painless or painful, or associated with a solitary or multiple nodules. While the most common cause of diffuse enlargement is chronic lymphocytic thyroiditis, the presence of nodularity should prompt consideration of cancer. Results of a radionuclide scan, ultrasonogram, and/or a fine-needle aspiration of a cystic nodule should help guide the physician to those patients in need of an open thyroid biopsy.

Somatostatin withdrawal alone is an ineffective generator of pulsatile growth hormone release in man.

To assess the relative roles of growth hormone-releasing hormone (GHRH) pulse and somatostatin withdrawal as potential generators of pulsatile growth hormone (GH) release in humans, we studied GH responses to iv bolus GHRH (1 microgram/kg) and to termination of a 4 h iv somatostatin infusion (7.2 micrograms.kg-1.h-1) in five normal young men, and in five men with previously diagnosed isolated GH deficiency. The patients were diagnosed 8-15 years previously on the basis of typical auxological and hormonal criteria, were treated with exogenous GH and were off GH therapy for 1.5-8.9 years prior to this study. Growth hormone rises to a bolus GHRH were similar between the controls and the patients (maximum GH 27.3 +/- 15.3 vs 8.0 +/- 4.0 micrograms/l). The controls exhibited only a small GH rise to somatostatin withdrawal (maximum GH 2.9 +/- 1.2 micrograms/l), while the patients did not (maximum GH 0.7 +/- 0.1 micrograms/l; p < 0.05). We conclude that somatostatin withdrawal by itself is an ineffective promoter of GH pulsatility. Periodic quiescence of somatostatinergic neurons must be associated with a concomitant GHRH pulse in order to result in a robust GH pulse.

Growth response of children with non-growth-hormone deficiency and marked short stature during three years of growth hormone therapy.

Short-term administration of human growth hormone to children with idiopathic short stature can improve mean growth rate and predicted adult height. It is yet unknown whether therapy would alter pubertal development or affect final height. Three-year treatment results in a group of children with idiopathic short stature are reported. For year 1 of the study, 121 prepubertal children were randomly selected to receive somatotropin, 0.3 mg/kg per week, administered subcutaneously three times weekly (n = 63), or to be nontreatment control subjects (n = 58). After 1 year, all subjects were again randomly selected to receive either three-times-weekly or daily dosing at the same total dose. For the 92 subjects who completed 36 months of treatment, mean growth rate increased from a mean of 4.6 cm/yr before treatment to a mean of 8.0 cm/yr in the first year of treatment. Daily dosing resulted in a significantly faster mean growth rate (9.0 cm/yr) than three-times-weekly dosing (7.8 cm/yr) (p = 0.0005). Mean growth rates were 7.6 and 7.2 cm/yr during years 2 and 3, respectively, and did not differ by dosing group. Mean standardized height for all subjects improved from -2.7 to -1.6 after 3 years. When the growth rate was standardized for bone age, however, subjects who remained prepubertal had a significantly greater gain in mean height SD score than subjects who became pubertal during that 3-year period (p < 0.02). Mean standardized Bayley-Pinneau predicted adult height SD score increased from -2.7 to -1.6 and was independent of the timing of pubertal onset, but for individuals this score was more variable. Year-1 growth response, expressed as growth rate or change in height SD score, was the best predictor of growth in subsequent years. Responses to therapy could not be reliably predicted from baseline anthropometric variables, plasma insulin-like growth factor I SD score, growth hormone levels. Final height assessment will be needed to determine the ultimate benefit of therapy.

Bioactivity of human growth hormone in serum: validation of an in vitro bioassay.

GH, in clinical practice, is determined by RIA, but RIA estimates may not accurately reflect serum GH bioactivity. The available measures of GH bioactivity lack either sensitivity, specificity, or a physiologically relevant end point. The objective of this research was to develop a physiologically relevant GH bioassay which would not only measure the bioactivity of purified GH preparations, but would also have sufficient sensitivity to measure GH bioactivity in human serum. The method consisted of incubating murine 3T3-F442A adipocytes in serum-free medium containing BSA, 14C-glucose, and increasing concentrations of GH or test materials for 24 h, followed by measurement of conversion of glucose to lipid. Interference by nonspecific serum factors was reduced by the addition of 10 micrograms/liter insulin, 25 nM dexamethasone, and 37 nM estradiol to the medium. In the presence of 10 micrograms/liter insulin, 50 micrograms/liter insulin-like growth factor-1 did not alter the ability of GH to suppress lipid accumulation. Epinephrine and glucagon could suppress lipid accumulation but only at concentrations greatly in excess of the physiological range in serum. Twenty two thousand dalton hGH produced dose-dependent suppression of lipid accumulation which was linear between 0.625 and 10 micrograms/liter (r = 0.926; P = 0.0001) with a half-maximal response of 3.0 +/- 0.2 micrograms/liter (n = six experiments). The intra- and interassay coefficients of variation were 7% and 19%, respectively. The assay was specific for GH since addition of human PRL produced suppression of lipid accumulation only at concentrations where contamination of the preparation by GH became a significant factor. ACTH also suppressed lipid accumulation but only at doses of 1000 micrograms/liter or greater. Human placental lactogen and hLH, hFSH, and hTSH did not cross-react with GH in this assay. Addition of human serum did not alter the slope of ED50 of the GH dose-response curve. Pools of serum from prepubertal and pubertal boys and girls, subjects treated with arginine or insulin, a diabetic girl, and a boy with gigantism who had a serum GH content of 80 micrograms/liter by RIA and 40 micrograms/liter by bioassay, produced dose response curves parallel to that of the GH standard curve. Serum from patients with hypopituitarism did not produce significant suppression of lipid accumulation in any assay. Recovery of 5 micrograms/liter GH added to human serum was 94%. Twenty thousand dalton GH also suppressed lipid accumulation in this assay, but was 2-fold less potent than 22,000 dalton GH.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of growth hormone (rhGH) therapy on tooth formation in idiopathic short statured children.

The purpose of this preliminary study was to evaluate tooth formation in children with idiopathic short stature, before and during treatment with recombinant growth hormone (rhGH). Twenty-nine short-statured children ages 6 to 13 years were assigned into two treatment groups, an "experimental" group (n = 18), which received rhGH, and a "control" group (n = 11), which was observed for 1 year before commencing rhGH treatment. Clinical and radiographic records were obtained at the initial, year 1, and year 2 visits. Tooth formation and stature were assessed by calculating Z-scores, appropriate for the age and gender of each child. Delta-Z scores, which measure the change in Z-score over time, were also calculated between annual visits. Height was measured and recorded every 3 months, and Z-score statural norms for age and gender were derived from the 1977 National Center for Health Services national probability sampling. Tooth formation standards were derived from Moorrees et al. A matched control sample for tooth development was derived from untreated children. Tooth formation was initially delayed although the degree of reduction in stature exceeded the initial degree of delay in tooth formation. During this 2-year study, rhGH therapy had a significant influence on acceleration or gain in stature, but did not have a significant influence on tooth formation.

Evaluation of gonadotropin responses to synthetic gonadotropin-releasing hormone in girls with idiopathic hypopituitarism.

We hypothesized that prepubertal girls with gonadotropin deficiency would produce less follicle-stimulating hormone (FSH) in response to synthetic gonadotropin-releasing hormone (GnRH) than would gonadotropin-sufficient children. To test this hypothesis, we performed 103 GnRH tests serially in 21 children who had idiopathic hypopituitarism with growth hormone deficiency. We tried to predict whether puberty would occur in the 17 girls with bone ages of 8 years or less. Of these 17 girls, 4 failed to have spontaneous secondary sexual characteristics by age 16 1/2 years, and 12 had spontaneous complete pubertal development. One girl had incomplete pubertal maturation with partial gonadotropin deficiency; her results were combined with those of the girls who had no spontaneous pubertal development. With increasing bone age, the girls with complete pubertal development had a decrease in the increment of FSH released in response to GnRH, although basal gonadotropin concentrations did not change. For GnRH tests performed at bone ages of 8 years or less, basal luteinizing hormone (LH) values did not differ between girls with complete puberty and those with absent or incomplete puberty. However, basal FSH and the incremental response of LH and FSH to GnRH were greater in those with complete puberty. Only two girls with prepubertal bone ages at the time of testing, who subsequently had complete puberty, had incremental FSH responses to GnRH that were less than 5 IU/L. Individual incremental LH responses to GnRH did not discriminate well between groups. None of the girls with adrenocorticotropic hormone deficiency, either originally or subsequently, had spontaneous puberty, but 4 of 12 girls with thyrotropin deficiency, either originally or subsequently, had complete puberty. We conclude that a significant increase in GnRH-stimulated FSH suggests that spontaneous pubertal development will occur in girls with idiopathic hypopituitarism. However, a low FSH response to GnRH may not be diagnostic of gonadotropin deficiency.

Expression of XMyoD protein in early Xenopus laevis embryos.

A monoclonal antibody specific for Xenopus MyoD (XMyoD) has been characterized and used to describe the pattern of expression of this myogenic factor in early frog development. The antibody recognizes an epitope close to the N terminus of the products of both XMyoD genes, but does not bind XMyf5 or XMRF4, the other two myogenic factors that have been described in Xenopus. It reacts in embryo extracts only with XMyoD, which is extensively phosphorylated in the embryo. The distribution of XMyoD protein, seen in sections and whole-mounts, and by immunoblotting, closely follows that of XMyoD mRNA. XMyoD protein accumulates in nuclei of the future somitic mesoderm from the middle of gastrulation. In neurulae and tailbud embryos it is expressed specifically in the myotomal cells of the somites. XMyoD is in the nucleus of apparently every cell in the myotomes. It accumulates first in the anterior somitic mesoderm, and its concentration then declines in anterior somites from the tailbud stage onwards.

Muscle gene activation in Xenopus requires intercellular communication during gastrula as well as blastula stages.

In Xenopus an early morphological marker of mesodermal induction is the elongation of the mesoderm at the early gastrula stage (Symes and Smith, 1987). We show here that the elongation of equatorial (marginal) tissue is dependent on protein synthesis in a mid blastula, but has become independent of it by the late blastula stage. In animal caps induced to become mesoderm, the time when protein synthesis is required for subsequent elongation immediately follows the time of induction, and is not related to developmental stage. For elongation, intercellular communication during the blastula stage is of primary importance. Current experiments involving cell transplantation indicate a need for further cell:cell interactions during gastrulation, and therefore after the vegetal-animal induction during blastula stages. These secondary cell interactions are believed to take place among cells that have already received a vegetal induction, and may facilitate some of the later intracellular events known to accompany muscle gene activation.

Xenopus embryos contain a somite-specific, MyoD-like protein that binds to a promoter site required for muscle actin expression.

We identify the "M region" of the muscle-specific Xenopus cardiac actin gene promoter from -282 to -348 as necessary for the embryonic expression of a cardiac actin-beta-globin reporter gene injected into fertilized eggs. Four DNA-binding activities in embryo extracts, embryonic M-region factors 1-4 (EMF1-4), are described that interact specifically with this region. One of these, EMF1, is detected in extracts from microdissected somites, which differentiate into muscle, but not in extracts from the adjacent neurectoderm, which differentiates into a variety of other cell types. Moreover, EMF1 is detected in embryo animal caps induced to form mesoderm, which includes muscle, and in which the cardiac actin gene is activated, but not in uninduced animal caps. EMF1 is also first detectable when cardiac actin transcripts begin to accumulate; therefore, both its temporal and spatial distributions during Xenopus development are consistent with a role in activating cardiac actin expression. Two lines of evidence suggest that EMF1 contains the myogenic factor Xenopus MyoD (XMyoD): (1) XMyoD synthesized in vitro can bind specifically to the same site as EMF1; and (2) antibodies raised against XMyoD bind to EMF1. DNA-binding studies indicate that EMF1 may be a complex between XMyoD and proteins found in muscle and other tissues. Our results suggest that the myogenic factor XMyoD, as a component of somite EMF1, regulates the activation of the cardiac actin gene in developing embryonic muscle by binding directly to a necessary region of the promoter.