Fracture rate associated with quality metric-based anti-osteoporosis treatment in glucocorticoid-induced osteoporosis.

UNLABELLED: Anti-osteoporosis medication (AOM) use in patients exposed to glucocorticoids is thought to reduce fractures. We found post-menopausal women using glucocorticoids for at least 90 days who also used an AOM within 90 days had 48 % fewer fractures by 1 year and 32 % fewer fractures by 3 years compared to non-AOM users. INTRODUCTION: The purpose of this study is to explore the effectiveness of adherence to quality measures by estimating the effect of anti-osteoporosis medication (AOM) initiation within 90 days after chronic (≥90 days) glucocorticoid (GC) therapy on osteoporotic fracture. METHODS: A new-user cohort was assembled using the MarketScan databases between 2000 and 2012. Included patients were female, age ≥50 at GC initiation, had a first GC fill daily dose ≥10 mg and persisted for at least 90 days. During a 365-day baseline period, patients were excluded for prior GC or AOM (bisphosphonate, denosumab, teriparatide) use, fracture, or cancer diagnosis. Initiators of an AOM in the 14 days pre- or 90 days post-GC fill were characterized as AOM users; those without, AOM non-users. Follow-up began 91 days after GC fill with patients followed until fracture, loss of continuous enrollment, initiation of AOM by AOM non-users, or end of study period. A propensity score was estimated for AOM receipt using all measured covariates and converted to a stabilized inverse probability of treatment weights (IPTW). Weighted hazard ratios (HR) and associated 95% confidence intervals (95% CI) were estimated using weighted Cox proportional hazard models. RESULTS: Of the 7885 women eligible for the study, 12.1% were AOM users. AOM use was associated with lower fracture incidence: weighted HR of 0.52 (95% CI 0.29, 0.94) at 1 year and weighted HR of 0.68 (95% CI 0.47, 0.99) at 3 years. CONCLUSIONS: AOM initiation within 90 days of chronic GC use was associated with a fracture reduction of 48% at 1 year and 32% at 3 years.

Parental genomic imprinting of the human IGF2 gene.

The mouse igf2 gene, coding for the insulin-like growth factor II (IGF-II) is parentally imprinted, only the gene copy derived from the father is expressed. To know whether IGF2, the human homologue, is also imprinted, we used an ApaI polymorphism at the 3' untranslated region in order to distinguish between mRNA derived from each copy of the gene in placentae from heterozygote human fetuses, studied after careful removal of the decidua. Six term and two pre-term placentae of heterozygotes were studied, and in each case the cDNA contained only one of the two alleles present in the genomic DNA. In three cases the mother was homozygous for the non-expressed allele, allowing assignment of paternal origin to the transcribed gene copy. We conclude that, as in the mouse, human IGF2 is parentally imprinted.

46, XY gonadal dysgenesis: new SRY point mutation in two siblings with paternal germ line mosaicism.

Familial recurrence risks are poorly understood in cases of de novo mutations. In the event of parental germ line mosaicism, recurrence risks can be higher than generally appreciated, with implications for genetic counseling and clinical practice. In the course of treating a female with pubertal delay and hypergonadotropic hypogonadism, we identified a new missense mutation in the SRY gene, leading to somatic feminization of this karyotypically normal XY individual. We tested a younger sister despite a normal onset of puberty, who also possessed an XY karyotype and the same SRY mutation. Imaging studies in the sister revealed an ovarian tumor, which was removed. DNA from the father's blood possessed the wild type SRY sequence, and paternity testing was consistent with the given family structure. A brother was 46, XY with a wild type SRY sequence strongly suggesting paternal Y-chromosome germline mosaicism for the mutation. In disorders of sexual development (DSDs), early diagnosis is critical for optimal psychological development of the affected patients. In this case, preventive karyotypic screening allowed early diagnosis of a gonadal tumor in the sibling prior to the age of normal puberty. Our results suggest that cytological or molecular diagnosis should be applied for siblings of an affected DSD individual.

Pituitary blastoma: a unique embryonal tumor.

Pituitary blastoma, a recently described tumor of the neonatal pituitary, exhibits differentiation to Rathke epithelium and adenohypophysial cells of folliculostellate and secretory type, a reflection of arrested pituitary development and unchecked proliferation (Scheithauer et al. in Acta Neuropathol 116(6):657-666, 2008). Herein, we report the pathologic features of three additional cases, all ACTH-producing. One involved a 9-month-old male presenting with progressive right ophthalmoplegia, MRI findings of a large suprasellar mass with cavernous sinus invasion, and elevated plasma ACTH levels. The second was nonfunctioning and occurred in a 13-month-old female with right third nerve palsy. The third had been previously published as a "pituitary adenoma" in a 2-year-old female (Min et al. in Pathol Int 57(9):600-605, 2007). The subtotally resected tumors were subject to histochemical, immunohistochemical and, in two cases, ultrastructural study. Histologically, the complex tumors consisted of glands of varying from rosettes to glandular structures resembling Rathke epithelium, small undifferentiated-appearing cells (blastema), and large secretory cells. Mucin-producing goblet cells were noted in case 3. Cell proliferation was high in two cases and low in case 3. Immunoreactivity of the secretory cells included synaptophysin, chromogranin, various keratins and, to a lesser extent, ACTH and beta endorphin. MGMT immunolabeling was 40-60%. Mitotic activity was moderate to high in cases 1 and 2 and was low in case 3. The same was true for MIB-1 labeling. Germ cell markers were lacking in all cases. One tumor ultrastructurally consisted of three cell populations including (a) small, polyhedral, primitive-appearing cells (blastema) with scant cytoplasm, abundant glycogen and few organelles, (b) folliculostellate cells and (c) large corticotroph cells containing rough endoplasmic reticulum, golgi membranes, spherical, 150-400 nm secretory granules and occasional perinuclear, intermediate filament bundles. A second example (case 3) lacked a blastema and glandular component. The clinical and morphologic features of our three cases were those of pituitary blastoma. The finding of cellular elements of adenohypophysial development is consistent with a diagnosis of pituitary blastoma and aligns it with blastomas of other organs. It also suggests an underlying specific genetic abnormality. Marked variations in cellular proliferative activity suggest blastomas occur in low- and higher-grade form. Variable MGMT reactivity suggests an incomplete response to temozolomide therapy. Literature regarding similar morphologically complex, infantile, Cushing disease-associated lesions is briefly reviewed.

Risk of autoimmune diabetes in APECED: association with short alleles of the 5'insulin VNTR.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autoimmune disease causing a wide spectrum of autoimmune dysfunction potentially including diabetes of an autoimmune etiology. We have previously described a pair of discordant APECED siblings and pointed to a possible role of 5'insulin variable number of tandem repeats (VNTR) locus IDDM2 in the appearance of diabetes within this disease. In vitro studies have previously suggested that class I VNTR alleles were associated with decreased fetal thymic insulin expression. We genotyped the 5'INS VNTR locus and several flanking 11p15.5 markers in 50 Finnish APECED subjects and explored the possible contribution of IDDM2 in the development of diabetes. The shorter 5'INS VNTR class I alleles (<35 repeats) were more prevalent in the diabetic Finnish APECED subjects than in non-diabetic APECED subjects. Logistic regression analysis revealed that having 1 short (<35) VNTR allele did not increase the risk of developing diabetes (95% CI 0.6-27.0), whereas having 2 short alleles conferred a 43.5-fold increased risk (95% CI 3.0-634.6). We conclude that short 5'INS VNTR class I alleles play a role in susceptibility to autoimmune diabetes in the context of APECED.

Molecular studies of a patient with complete androgen insensitivity and a 47,XXY karyotype.

A phenotypic female with complete androgen insensitivity from a maternally inherited mutation in the androgen receptor had a 47,XXY karyotype. Partial maternal X isodisomy explained the expression of androgen insensitivity despite the presence of 2 X chromosomes.

[Pattern and evolution of the prescription of olanzapine during one year: Results of the cohort study ECOL]. / Modalités et évolution de la prescription d'olanzapine : résultats de l'étude de cohorte ECOL.

INTRODUCTION: The necessary evidence of new therapies of clinical interest extends beyond clinical trials in a less controlled population and closer to clinical practice justified since several years the need of conducting observational, noninterventional studies. Observational studies must include epidemiological (quantitative observational) data to define prevalence and natural history of the target conditions. Moreover, pharmacological interventions in "naturalistic" patients populations, selected by clinicians as per clinical judgment within the scope of the target disease will allow to generate data to complement clinical trials. Clinical trials designed to show robust data on efficacy and tolerability particularly during registration trials must be complemented by robust observational research to confirm and better describe clinical effectiveness in the target population. A noninterventional, observational trial is a study where the medicinal product(s) is (are) prescribed in the usual manner in accordance with the terms of the marketing authorization. The assignment of the patient to a particular therapeutic strategy is not decided in advance by a trial protocol but falls within current practice and the prescription of the medicine is clearly separated from the decision to include the patient in the study. No additional diagnosis or monitoring procedures shall be applied to the patients and epidemiological methods shall be used for the analysis of collected data. Olanzapine is a new antipsychotic therapy registered in Europe for the treatment of schizophrenia since 1996. AIMS OF THE STUDY: The primary objective of this observational research was to study the evolution of the olanzapine dosage under naturalistic settings. Secondary objectives included patients characteristics, severity of disease, therapeutic evolution and coprescriptions, in a patient's cohort, suffering from schizophrenia, adult patients, diagnosis based on ICD-10; patients were followed during a total of 12 months. DESIGN OF THE STUDY: The cohort study was conducted in France. Between the period of June 2000 and February 2001, 407 psychiatrics randomized to participate in the study had consolidated the patient's cohort. RESULTS: A total of 1810 patients were included, 1093 (60, 4%) male, 717 (39, 6%) females. Age was recorded for a total of 1802 (99, 6%) patients, mean age was 37.8 years as per inclusion criteria and patients consent according to current regulations. Patients entered in the cohort as per clinicians decision underwent a treatment with olanzapine during an outpatient's consultation or at hospitalization. More than two thirds of the patients were followed up during 12 months after onset of this treatment. Clinical outcome was assessed at three, six, nine and 12 months following cohort inclusion using the following tools: CGI, PANSS, Calgary and GAF; as per CGI 78% of the patients cohort were severely ill, the mean PANSS score was 94.1. At second month of treatment clinicians were requested to very well document any changes in olanzapine dosage as well as reasons for the dosage modifications and potential coprescriptions. DISCUSSION: The daily mean dosage of olanzapine was 9.5mg at initiation of treatment, 10.5mg after one month and 11.2mg after 12 months of follow-up. The increase of the dosage after one month was associated with factors such as younger age, schizophrenia diagnosis and severity of the symptoms as measured by CGI and PANSS scores evolution, low initial dosage and hospitalization at treatment initiation. Within the 1810 participants included in the cohort, 1383 (76.5%) received a coprescrition of a psychotropic, for example, 811 (44.8%) a benzodiazepine, 506 (28.0%) an antidepressant. Among the patients cohort that were followed during 12 months, all the clinical and patient-functioning indicators progressed in the direction of a significant improvement.

Consensus statement on the diagnosis and treatment of children with idiopathic short stature: a summary of the Growth Hormone Research Society, the Lawson Wilkins Pediatric Endocrine Society, and the European Society for Paediatric Endocrinology Workshop.

OBJECTIVE: Our objective was to summarize important advances in the management of children with idiopathic short stature (ISS). PARTICIPANTS: Participants were 32 invited leaders in the field. EVIDENCE: Evidence was obtained by extensive literature review and from clinical experience. CONSENSUS: Participants reviewed discussion summaries, voted, and reached a majority decision on each document section. CONCLUSIONS: ISS is defined auxologically by a height below -2 sd score (SDS) without findings of disease as evident by a complete evaluation by a pediatric endocrinologist including stimulated GH levels. Magnetic resonance imaging is not necessary in patients with ISS. ISS may be a risk factor for psychosocial problems, but true psychopathology is rare. In the United States and seven other countries, the regulatory authorities approved GH treatment (at doses up to 53 microg/kg.d) for children shorter than -2.25 SDS, whereas in other countries, lower cutoffs are proposed. Aromatase inhibition increases predicted adult height in males with ISS, but adult-height data are not available. Psychological counseling is worthwhile to consider instead of or as an adjunct to hormone treatment. The predicted height may be inaccurate and is not an absolute criterion for GH treatment decisions. The shorter the child, the more consideration should be given to GH. Successful first-year response to GH treatment includes an increase in height SDS of more than 0.3-0.5. The mean increase in adult height in children with ISS attributable to GH therapy (average duration of 4-7 yr) is 3.5-7.5 cm. Responses are highly variable. IGF-I levels may be helpful in assessing compliance and GH sensitivity; levels that are consistently elevated (>2.5 SDS) should prompt consideration of GH dose reduction. GH therapy for children with ISS has a similar safety profile to other GH indications.

Corticosterone and related receptor expression are associated with increased beta-amyloid plaques in isolated Tg2576 mice.

Previously, we reported that the stress associated with chronic isolation was associated with increased beta-amyloid (Abeta) plaque deposition and memory deficits in the Tg2576 transgenic animal model of Alzheimer's disease (AD) [Dong H, Goico B, Martin M, Csernansky CA, Bertchume A, Csernansky JG (2004) Effects of isolation stress on hippocampal neurogenesis, memory, and amyloid plaque deposition in APP (Tg2576) mutant mice. Neuroscience 127:601-609]. In this study, we investigated the potential mechanisms of stress-accelerated Abeta plaque deposition in this Tg2576 mice by examining the relationship between plasma corticosterone levels, expression of glucocorticoid receptor (GR) and corticotropin-releasing factor receptor-1 (CRFR1) in the brain, brain tissue Abeta levels and Abeta plaque deposition during isolation or group housing from weaning (i.e. 3 weeks of age) until 27 weeks of age. We found that isolation housing significantly increased plasma corticosterone levels as compared with group-housing in both Tg+ mice (which contain and overexpress human amyloid precursor protein (hAPP) gene) and Tg- mice (which do not contain hAPP gene as control). Also, isolated, but not group-housed animals showed increases in the expression of GR in the cortex. Furthermore, the expression of CRFR1 was increased in isolated Tg+ mice, but decreased in isolated Tg- mice in both cortex and hippocampus. Changes in the components of hypothalamic-pituitary-adrenal (HPA) axis were accompanied by increases in brain tissue Abeta levels and Abeta plaque deposition in the hippocampus and overlying cortex in isolated Tg+ mice. These results suggest that isolation stress increases corticosterone levels and GR and CRFR1 expression in conjunction with increases in brain tissue Abeta levels and Abeta plaque deposition in the Tg2576 mouse model of AD.

Tracing the origins of "fetal origins" of adult diseases: programming by oxidative stress?

Too small size at birth (due to poor fetal growth and/or preterm delivery) has been associated with substantially elevated risks of the metabolic syndrome (dislipidemia, insulin resistance, hypertension), type 2 diabetes and cardiovascular disease in adulthood. The mechanisms of such "fetal origins" or "programming" of disease phenomenon remain unresolved. Too large size at birth seems also associated with an increased risk. Many known or suspected causes of or conditions associated with adverse (poor or excessive) fetal growth or preterm birth have been associated with oxidative stress. Plausibly, oxidative stress may be a common link underlying the superficial "programming" associations between adverse fetal growth or preterm birth and elevated risks of certain chronic diseases. The mechanisms of oxidative stress programming may be through directly modulating gene expression or indirectly through the effects of certain oxidized molecules. Experimental investigations have well demonstrated the role of redox balance in modulating gene expression, and recent studies indicate that both the insulin functional axis and blood pressure could be sensitive targets to oxidative stress programming. Adverse programming may occur without affecting fetal growth, but more frequently among low birth weight infants merely because they more frequently experienced known or unknown conditions with oxidative insults. As oxidative stress levels are easily modifiable during pregnancy and early postnatal periods (which are plausible critical windows), the hypothesis, if proved valid, will suggest new measures that could be very helpful on fighting the increasing epidemic of the metabolic syndrome, type 2 diabetes and cardiovascular disease. Currently, there are several ongoing large randomized trials of antioxidant supplementation to counter oxidative stress during pregnancy for the prevention of preeclampsia. It would be invaluable if long-term follow-ups of infants born to women in such trials could be realized to test the oxidative stress programming hypothesis in such experimental trial settings.

GH safety workshop position paper: a critical appraisal of recombinant human GH therapy in children and adults.

Recombinant human GH (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that 'for approved indications, GH is safe'; however, the statement highlighted a number of areas for on-going surveillance of long-term safety, including cancer risk, impact on glucose homeostasis, and use of high dose pharmacological rhGH treatment. Over the intervening years, there have been a number of publications addressing the safety of rhGH with regard to mortality, cancer and cardiovascular risk, and the need for long-term surveillance of the increasing number of adults who were treated with rhGH in childhood. Against this backdrop of interest in safety, the European Society of Paediatric Endocrinology (ESPE), the GRS, and the Pediatric Endocrine Society (PES) convened a meeting to reappraise the safety of rhGH. The ouput of the meeting is a concise position statement.

Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2.

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in mammalian cell membranes and is essential for cell viability. The levels of this lipid must be tightly controlled to maintain homeostasis. Therefore, changes in the rate of PtdCho synthesis are generally balanced by changes in PtdCho catabolism and vice versa. It is commonly accepted that the rate of PtdCho synthesis is regulated by CTP:phosphocholine cytidylyltransferase (CT). However, it is not certain if PtdCho mass is regulated by specific catabolic enzyme(s). Our goal is to determine if PtdCho homeostasis is regulated by a phospholipase A2 (PLA2). To this end, we have prepared Chinese hamster ovary (CHO) cell lines that overexpress CT. CT activity is 7-10-fold higher in the transfected cells than in parental CHO cells. This increase in CT activity is associated with increases in both PtdCho synthesis and PtdCho catabolism. Glycerophosphocholine is the PtdCho catabolite that accumulates in the transfected cells, which suggests that PtdCho turnover is mediated by a phospholipase A2 (PLA2). Indeed, higher levels of calcium-independent PLA2 activity are measured in the cytosols of the CHO cells that overexpress CT, compared to parental CHO cells. The elevated calcium-independent PLA2 activity is associated with increases in the expression of the 80-kDa calcium-independent PLA2 (iPLA2). Together, these data suggest that the 80-kDa iPLA2 may be modulated in response to changes in PtdCho levels and therefore is involved in the regulation of PtdCho homeostasis in CHO cells.

Affinity purification and subcellular localization of kinesin in human neutrophils.

Studies of granule-microtubule interactions in human neutrophils have suggested that mechanochemical ATPases such as kinesin or dynein may play a role in granule mobilization during neutrophil activation by inflammatory signals. In this study we show that proteins extracted from the surface of neutrophil granules, found previously to contain microtubule-dependent ATPase activity, caused microtubules polymerized from phosphocellulose-purified rat brain tubulin to move across glass slides. Antibodies were generated against peptides based on two regions of the amino acid sequence of Drosophila kinesin: the ATPase active site (amino acids 86-99) in the head of the kinesin heavy chain and the tail of the heavy chain (residues 913-933). These antibodies were found to recognize kinesin in rat brain extracts as well as kinesin-like polypeptides in extracts of human neutrophils. Furthermore, when used in immunoaffinity chromatography, these antibodies permitted the isolation of a protein from neutrophil granule extracts that was recognized by Drosophila kinesin antibodies. Subcellular localization by immunofluorescence microscopy showed this protein to be associated principally with the cytoplasmic granules of neutrophils.

Effects of 14-day infusions of growth hormone and/or insulin-like growth factor I on the obesity of growing Zucker rats.

The response of fat tissue to GH or insulin-like growth factor I (IGF-I) differs between humans with hypopituitarism and those with exogenous obesity; the effects of combined GH and IGF-I administration have not been compared in these two situations. In GH-deficient dwarf rats (who have a primary GH deficiency), the excessive fat deposition induced by a high fat diet is completely reversed by combined infusion of GH and IGF-I. Whether the same phenomenon would be observed in genetically obese Zucker rats (in whom, as in obese humans, the decrease in GH secretion is secondary to the obese state) remained to be determined. Growing (6-week-old) female obese Zucker rats received a continuous sc infusion of vehicle, recombinant human GH, recombinant human IGF-I, or GH plus IGF-I for 14 days (3 mg/kg x day for both GH and IGF-I). Combined GH and IGF-I stimulated body weight gain and in naso-anal length to the same extent as IGF-I alone, whereas GH alone was less potent. Because all treatments stimulated weight linear growth proportionately, the progression of obesity was similar in treated and control animals. However, GH plus IGF-I (but not either agent alone) induced a 25% decrease in the relative weight of inguinal fat. GH and IGF-I exerted distinct effects on the relative weights of liver, kidney, and spleen and on the circulating levels of IGF-I and IGF-binding protein-3. Circulating glucose and insulin levels did not change in any group. In summary, GH plus IGF-I infusions decrease the relative weight of inguinal fat in Zucker rats as in obese GH-deficient dwarf rats; however, this effect is of more modest magnitude despite the use of a 2- to 3-fold higher dose and is limited to the inguinal site. Thus, GH plus IGF-I infusions did not influence the obesity index in Zucker rats. Inasmuch as Zucker rats are a better model of childhood-onset obesity than dwarf rats fed a high fat diet, the present results do not appear promising for extrapolation to clinical studies in children. The mechanisms by which the primary vs. secondary nature of the decreased GH secretion influences the effect of GH plus IGF-I on obesity remain to be determined.

Hormonal control of growth in the genetically obese Zucker rat. I. Linear growth, plasma insulin-like growth factor-I (IGF-I) and IGF-binding proteins.

The genetically obese Zucker rat is a widely used model of early-onset obesity. Like obese children, these obese rats are hyperinsulinemic and have low GH secretion. However, data on linear growth and insulin-like growth factor-I (IGF-I) levels in this model are scanty and contradictory. In the present study, we investigated linear growth and its hormonal control in Zucker rats (male and female) from 4-20 weeks of age. In the obese animals, compared to their lean littermates, the naso-anal length was normal or slightly greater, whereas the tails and femurs were shorter. The plasma concentration of IGF-I increased between 4-20 weeks of age, and IGF-I levels were normal or slightly higher in the obese animals. The serum level of IGF-binding protein-3 (IGFBP-3) measured by Western ligand blotting was not significantly different in lean vs. obese rats. To assess the IGF-I response to GH, bovine GH was administered (250 micrograms/100 g BW, ip, daily for 3 days) to 16- to 20-week-old female Zucker rats; plasma IGF-I concentrations increased more in the obese (percent increase over baseline, 347 +/- 44% vs. 194 +/- 31%; P < 0.01). These results show that despite low GH secretion, genetically obese Zucker rats have 1) normal linear (nasoanal) growth, 2) normal or increased circulating levels of IGF-I and IGFBP-3, and 3) increased plasma IGF-I responses to exogenous GH. These results suggest that the GH-independent growth in this model could result from direct effects of hyperinsulinism on circulating IGF-I and IGFBP-3 levels and/or indirect effects through increased GH receptor function.

Human placental trophoblasts secrete a disintegrin metalloproteinase very similar to the insulin-like growth factor binding protein-3 protease in human pregnancy serum.

During the course of human pregnancy, there is a marked increase in insulin-like growth factor (IGF) binding protein (IGFBP)-3 protease activity in maternal serum that is first evident at 6 weeks of gestation, persists through term, and returns to nonpregnancy levels by day 5 postpartum. This protease activity cleaves IGFBP-3 into smaller fragments that have markedly reduced affinity for the IGFs. To date, the precise identity and cellular origin of the pregnancy-associated serum IGFBP-3 protease have not been established. To investigate whether placental and/or decidual tissues, which uniquely develop during pregnancy, may be sources of the pregnancy-associated serum IGFBP protease, we examined the secretion of IGFBP-3 protease in vitro by isolated human cytotrophoblasts or fibroblasts from second trimester placentae and by in vitro decidualized human endometrial stromal cells. Cytotrophoblasts were either cultured alone, which favors aggregation and fusion, or cocultured with decidualized endometrial stromal cells, which favors differentiation to an invasive phenotype. IGFBP-3 protease activity was detected in trophoblast, but not in placental fibroblast or decidualized endometrial cultures, and was also present in trophoblast-endometrial cocultures. Western ligand blot and Western immunoblot analyses showed that most of the endogenous IGFBP-3 in trophoblast cultures was in the form of low molecular weight fragments with reduced IGF binding affinity. The substrate specificity of the trophoblast-derived protease was identical to that in pregnancy serum, showing activity against IGFBP-2, -3, and -4, but being inactive against IGFBP-1. IGFBP-3 proteolysis by both pregnancy serum and trophoblast conditioned medium showed a major peak of activity at neutral pH. The trophoblast-derived activity caused time-and temperature-dependent proteolysis of IGFBP-3 into fragments of identical size as those produced by pregnancy serum, and also shared its sensitivity to protease inhibitors: highly sensitive to EDTA and o-phenanthroline, partially sensitive to the serine protease inhibitors AEBSF and aprotinin, and insensitive to alpha2-antiplasmin, and to aspartic and cysteine protease inhibitors. IGFBP-3 proteolysis by both pregnancy serum and trophoblast conditioned medium was also insensitive to tissue inhibitor of metalloproteinase-1, precluding the involvement of the matrix metalloproteinases. In contrast, both the pregnancy serum- and trophoblast-derived proteases were preferentially inhibited by a hydroxamic acid derivative with selective activity against the disintegrin-metalloproteinase tumor necrosis factor-alpha converting enzyme. This study shows that placental trophoblasts produce an IGFBP-3 protease with characteristics very similar to the activity found in pregnancy serum and indicates these cells at the maternal-fetal interface are a potential source of the pregnancy-associated serum IGFBP-3 protease. The findings further suggest that the main IGFBP-3 protease activity in both pregnancy serum and trophoblast conditioned medium may correspond to a disintegrin-metalloproteinase type enzyme.

An autosomal dominant form of familial persistent hyperinsulinemic hypoglycemia of infancy, not linked to the sulfonylurea receptor locus.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a rare disorder due to defective negative feedback regulation of insulin secretion by low glucose levels, is often familial. Most cases are recessively inherited, and mutations of the sulfonylurea receptor gene (SUR) or the closely linked KIR6.2 gene have been found in several families. Both of these genes encode components of the potassium channels responsible for glucose-regulated insulin release. However, in some families recessive PHHI is not linked to the SUR-KIR6.2 locus, suggesting genetic heterogeneity. We report here a French Canadian kindred with hypoglycemia in five first cousins. All five patients had documented hypoglycemia, and all responded well to diazoxide. In two, inappropriately elevated insulin levels during hypoglycemia were documented. This familial clustering strongly suggests the existence of an autosomal dominant form of PHHI. By preliminary linkage analysis, we tested the possibility of a dominant negative SUR or KIR6.2 mutant. The insulin (INS) and glucokinase (GCK) genes were also tested as additional candidates. Microsatellite markers closely linked to each gene were used, and large negative Lod scores were obtained at the known recombination fractions between all three genes studied and the corresponding marker. We conclude that mutation of a gene other than SUR or KIR6.2 is responsible for the dominant PHHI in this family, and this gene cannot be INS or GCK. We propose that a genome-wide search for this gene is important for elucidating this rare disorder and, more importantly, for determining its potential impact on understanding noninsulin-dependent diabetes mellitus and on the effort to develop bioengineered beta-cells for transplantation.

Pediatric adrenocortical tumors: molecular events leading to insulin-like growth factor II gene overexpression.

It has been previously shown that adrenocortical tumors (ACT) in adults exhibit structural abnormalities in tumor DNA in approximately 30% of cases. These abnormalities involve chromosome 11p15 and include loss of heterozygosity, paternal isodisomy, and overexpression of the gene for insulin-like growth factor II (IGF2), correlating with DNA demethylation at this locus. It has been hypothesized that these events occur late in the tumorigenic process in adults and seem to correlate with a worse prognosis. We present 4 pediatric cases of ACT diagnosed at 2.5 yr, 10 months, 12 yr, and 2.2 yr. All 4 patients presented with virilization, and 1 patient also showed signs and symptoms of glucocorticoid excess. The youngest patient's maternal aunt had surgical excision of a more than 15-cm ACT 18 yr previously, but the aunt is doing well at age 23 yr. They all had surgical removal of their tumors. The 2.5-yr-old child also received chemotherapy and radiotherapy because of capsular rupture and, after 3 local recurrences, died 3.3 yr after initial presentation. We investigated all 4 tumors for chromosome 11 structural abnormalities (11p15.5 to 11q23), IGF2 and H19 expression by competitive RT-PCR analysis, and IGF2 methylation patterns by Southern analysis. All 4 tumors (100%) showed a combination of structural abnormalities at the 11p15 locus with mosaic loss of heterozygosity involving 11p. All tumors also had significantly increased IGF2 messenger ribonucleic acid levels relative to normal adrenal (up to 36-fold) and significant IGF2 demethylation (mean, 87%). H19 messenger ribonucleic acid levels were undetectable in 3 of 4 tumors, explained in part by mosaic loss of the actively expressed maternal allele for this imprinted gene. By immunohistochemistry we were able to confirm increased IGF-II peptide levels within the tumor tissue in 10 pediatric patients, including the 4 patients described above. Concomitantly, we also observed nuclear accumulation of p53, suggesting somatic mutations. For the 10-month-old patient, sequencing revealed a p53 germline mutation. We therefore conclude that in pediatric ACT, structural abnormalities of tumor DNA and IGF2 overexpression as well as p53 mutations are very common and are therefore less useful for prognosis than in adults. Our findings support the theory that pediatric ACT, whose IGF2 expression and steroidogenesis evoke the phenotype of the fetal adrenal cortex, may arise because of defective apoptosis.

Apparent congenital athyreosis contrasting with normal plasma thyroglobulin levels and associated with inactivating mutations in the thyrotropin receptor gene: are athyreosis and ectopic thyroid distinct entities?

Loss-of-function mutations in the TSH receptor gene (TSH-R), usually leading to asymptomatic hyperthyrotropinemia, have been reported since 1995 in a total of eight pedigrees, with a pattern of transmission suggesting autosomal recessive inheritance. Although normal TSH secretion and action are not necessary for normal migration of the thyroid analage, they are essential for normal thyroid growth and function. In keeping with this concept, we report a severely hypothyroid boy with a normally located but very hypoplastic and hypofunctional thyroid caused by TSH-R loss-of-function mutations. The propositus' maternal great aunt also had apparent athyreosis. The propositus had undetectable uptake on 99mpertechnetate scintigraphy but normal plasma thyroglobulin at 15 days of age. He was found to be a compound heterozygote for TSH-R mutations, with the maternal allele carrying a splicing mutation (G to C transversion at position +3 of the donor site of intron 6) and the other allele a deletion of two nucleotides (2 bases of codon 655 in exon 10). The great aunt's TSH-R was normal. We also report the sex ratio of hypothyroid newborns referred to our center since 1989 with apparent athyreosis (5 girls, 7 boys) and with ectopic thyroid tissue (41 girls, 15 boys). We conclude that different genetic and nongenetic mechanisms for athyreosis and ectopic thyroid are likely, and that these two distinct entities are themselves heterogeneous. Our results further show that inactivating mutations in TSH-R may account for some cases of apparent congenital athyreosis and should be suspected, especially if plasma thyroglobulin levels are normal.

Outcome of severe congenital hypothyroidism: closing the developmental gap with early high dose levothyroxine treatment.

We have previously reported that despite neonatal screening, children with severe congenital hypothyroidism treated at 5 weeks of age with 6 micrograms/kg.day levothyroxine have clinically significant intellectual impairment, whereas those with the moderate form of the disease are indistinguishable from controls. The developmental outcome of children with severe congenital hypothyroidism treated earlier with higher initial doses of levothyroxine remained to be determined. In the present study, 45 infants with permanent congenital hypothyroidism detected by neonatal screening are described. For the group, the median age at starting treatment was 14 days, and the median initial dose of levothyroxine was 11.6 micrograms/kg.day. Based on the area of their knee epiphyses at diagnosis, the patients were divided into 2 subgroups: severe (< 0.05 cm2; n = 10) and moderate (> or = 0.05 cm2; n = 35). The psychomotor development of 8 patients in each subgroup, matched for the socioeducational level of their families, was assessed at 18 months. Mean plasma free T4 levels were supraphysiological during the first few months of life, but mean plasma T3 levels remained within the normal range, and there were no signs or symptoms of hyperthyroidism. The mean plasma TSH concentration was less than 4.5 mIU/L 4 weeks after starting treatment. Bone maturation remained delayed at 12 months in the severe cases and was not unduly advanced in the moderate cases. The mean (+/- SD) developmental quotients at 18 months were similar in severe and moderate cases (107 +/- 10 and 110 +/- 5, respectively). We conclude that with earlier treatment and a higher initial dose of levothyroxine, the early developmental outcome of infants with severe congenital hypothyroidism is now indistinguishable from that of infants with the moderate form of the disease who were used as controls.