Verónica Mericq

Regulation of Growth Plate Chondrogenesis by Bone Morphogenetic Protein-21

Bone morphogenetic proteins (BMPs) regulate embryonic skeletal development. We hypothesized that BMP-2, which is expressed in the growth plate, also regulates growth plate chondrogenesis and longitudinal bone growth. To test this hypothesis, fetal rat metatarsal bones were cultured for 3 days in the presence of recombinant human BMP-2. The addition of BMP-2 caused a concentration-dependent acceleration of metatarsal longitudinal growth. As the rate of longitudinal bone growth depends primarily on the rate of growth plate chondrogenesis, we studied each of its three major components. BMP-2 stimulated chondrocyte proliferation in the epiphyseal zone of the growth plate, as assessed by [H]thymidine incorporation. BMP-2 also caused an increase in chondrocyte hypertrophy, as assessed by quantitative histology and enzyme histochemistry. A stimulatory effect on cartilage matrix synthesis, assessed by SO4 incorporation into glycosaminoglycans, was produced only by the highest concentration of BMP-2. These BMP-2-mediated stimulatory effects were reversed by recombinant human Noggin, a glycoprotein that blocks BMP-2 action. In the absence of exogenous BMP-2, Noggin inhibited metatarsal longitudinal growth, chondrocyte proliferation, and chondrocyte hypertrophy, which suggests that endogenous BMPs stimulate longitudinal bone growth and chondrogenesis. We conclude that BMP-2 accelerates longitudinal bone growth by stimulating growth plate chondrocyte proliferation and chondrocyte hypertrophy. (Endocrinology 142: 430–436, 2001) B MORPHOGENETIC proteins (BMPs) appear to play an important role in skeletal development (1–5). These glycoproteins were initially characterized by their ability to induce ectopic cartilage and bone formation in soft tissues (6). Expression of BMP-2 and BMP-4 in specific areas of the limb bud suggests a regulatory role for these factors during early skeletal development (7). Consistent with this concept, overexpression of BMP-2 in embryonic limbs causes dysplastic changes in the cartilaginous structures (8). Because BMP-2 is also expressed in the growth plates of long bones (9, 10), we hypothesized that this growth factor also plays a role at a later stage of skeletal development, after morphogenesis is completed. In long bones, morphogenesis is followed by an extended period of growth. Longitudinal growth occurs at the growth plate by endochondral ossification (11, 12), a process in which cartilage is formed and then remodeled into bone. Growth plate chondrocyte proliferation, hypertrophy, and extracellular matrix secretion lead to the formation of new cartilage, chondrogenesis (13). Simultaneously, the growth plate is invaded from the metaphysis by blood vessels and bone cell precursors, which remodel the cartilage into bone tissue (14). The rate of longitudinal bone growth depends primarily on the rate of growth plate chondrogenesis. To study the role of BMP-2 in growth plate chondrogenesis, we assessed the effects of recombinant human BMP-2 (rhBMP-2) on cultured fetal rat metatarsal bone rudiments. Unlike isolated cells in culture, this organ culture system preserves the histological architecture of the growth plate and thus the intercellular interactions and local microenvironments found in vivo. Materials and Methods

Estrogen levels in girls with premature thelarche compared with normal prepubertal girls as determined by an ultrasensitive recombinant cell bioassay

OBJECTIVE Estradiol levels in girls with premature thelarche have not previously been well defined because of the lack of adequate sensitivity of previously available estradiol assays. The ultrasensitive recombinant cell bioassay for estradiol has made the study of estradiol levels in premature thelarche possible. We hypothesized that girls with premature thelarche have higher estradiol levels than normal prepubertal girls. STUDY DESIGN We used an ultrasensitive recombinant cell bioassay to study estradiol levels in 20 girls with premature thelarche and 15 normal prepubertal girls less than 3 years of age. The 2 groups were compared by Student t test. RESULTS Estradiol levels were significantly greater in the girls with premature thelarche (8.4 4. 5 pmol/L estradiol equivalents) than in the normal prepubertal girls (3.3 3.5 pmol/L estradiol equivalents; P <.01). The estradiol level was not significantly correlated with age, height, weight, body mass index, age at onset of thelarche, or the presence or absence of ovarian cysts. CONCLUSION Girls with premature thelarche have significantly higher estradiol levels than normal prepubertal girls. This is consistent with the hypothesis that the mechanism of premature thelarche involves increased estradiol levels rather than increased sensitivity of breast tissue to normal estradiol levels.

Expanding the Phenotype and Genotype of Female GnRH Deficiency

CONTEXT GnRH deficiency is a rare genetic disorder of absent or partial pubertal development. The clinical and genetic characteristics of GnRH-deficient women have not been well-described. OBJECTIVE To determine the phenotypic and genotypic spectrum of a large series of GnRH-deficient women. DESIGN, SETTING, AND SUBJECTS Retrospective study of 248 females with GnRH deficiency evaluated at an academic medical center between 1980 and 2010. MAIN OUTCOME MEASURES Clinical presentation, baseline endogenous GnRH secretory activity, and DNA sequence variants in 11 genes associated with GnRH deficiency. RESULTS Eighty-eight percent had undergone pubarche, 51% had spontaneous thelarche, and 10% had 1-2 menses. Women with spontaneous thelarche were more likely to demonstrate normal pubarche (P = 0.04). In 27% of women, neuroendocrine studies demonstrated evidence of some endogenous GnRH secretory activity. Thirty-six percent (a large excess relative to controls) harbored a rare sequence variant in a gene associated with GnRH deficiency (87% heterozygous and 13% biallelic), with variants in FGFR1 (15%), GNRHR (6.6%), and PROKR2 (6.6%) being most prevalent. One woman had a biallelic variant in the X-linked gene, KAL1, and nine women had heterozygous variants. CONCLUSIONS The clinical presentation of female GnRH deficiency varies from primary amenorrhea and absence of any secondary sexual characteristics to spontaneous breast development and occasional menses. In this cohort, rare sequence variants were present in all of the known genes associated with GnRH deficiency, including the novel identification of GnRH-deficient women with KAL1 variants. The pathogenic mechanism through which KAL1 variants disrupt female reproductive development requires further investigation.

Insulin resistance markers in children.

The prevalence of obesity among children and adolescents is progressively increasing around the world. One of the important consequences of obesity is the development of insulin resistance (IR). This condition has a multifactorial pathogenesis and is associated with cardiovascular risk, diabetes, hypertension, polycystic-ovary syndrome and a shorter lifespan. IR during childhood may be diagnosed by physical examination or there may be clues in the histories of the patient and his/her family. When IR is suspected, tests on a blood sample (which are more reliable) are recommended. Most of the biochemical markers have been well defined in adults, but appropriate reference data for children are still lacking. Here we discuss the usefulness of various currently known biochemical markers to evaluate insulin sensitivity (homeostatic model assessment, the quantitative insulin sensitivity check index, the oral glucose tolerance test, Matsuda method and the whole-body insulin resistance index), hormones (leptin, adiponectin, resistin, glucocorticoids, the insulin-like growth factor-1-binding protein/growth hormone axis, ghrelin, sex hormone-binding globulin and retinol-binding protein-4) and inflammatory markers (C-reactive protein, IL-6, intercellular adhesion molecule-1, vascular adhesion molecule-1 and E-selectin), which can be used in the diagnosis of IR in children.

Copy number variants in patients with short stature

Height is a highly heritable and classic polygenic trait. Recent genome-wide association studies (GWAS) have revealed that at least 180 genetic variants influence adult height. However, these variants explain only about 10% of the phenotypic variation in height. Genetic analysis of short individuals can lead to the discovery of novel rare gene defects with a large effect on growth. In an effort to identify novel genes associated with short stature, genome-wide analysis for copy number variants (CNVs), using single-nucleotide polymorphism arrays, in 162 patients (149 families) with short stature was performed. Segregation analysis was performed if possible, and genes in CNVs were compared with information from GWAS, gene expression in rodents’ growth plates and published information. CNVs were detected in 40 families. In six families, a known cause of short stature was found (SHOX deletion or duplication, IGF1R deletion), in two combined with a de novo potentially pathogenic CNV. Thirty-three families had one or more potentially pathogenic CNVs (n=40). In 24 of these families, segregation analysis could be performed, identifying three de novo CNVs and nine CNVs segregating with short stature. Four were located near loci associated with height in GWAS (ADAMTS17, TULP4, PRKG2/BMP3 and PAPPA). Besides six CNVs known to be causative for short stature, 40 CNVs with possible pathogenicity were identified. Segregation studies and bioinformatics analysis suggested various potential candidate genes.

Metabolic Effects of Oral Versus Transdermal 17β-Estradiol (E2): A Randomized Clinical Trial in Girls With Turner Syndrome

CONTEXT The long-term effects of pure 17β-estradiol (E₂) depending on route of administration have not been well characterized. OBJECTIVE Our objective was to assess metabolic effects of oral vs transdermal (TD) 17β-E₂ replacement using estrogen concentration-based dosing in girls with Turner syndrome (TS). PATIENTS Forty girls with TS, mean age 16.7 ± 1.7 years, were recruited. DESIGN Subjects were randomized to 17β-E₂ orally or TD. Doses were titrated using mean E₂ concentrations of normally menstruating girls as therapeutic target. E₂, estrone (E₁), and E₁ sulfate (E₁S) were measured by liquid chromatography tandem mass spectrometry and a recombinant cell bioassay; metabolites were measured, and dual-energy x-ray absorptiometry scan and indirect calorimetry were performed. MAIN OUTCOME Changes in body composition and lipid oxidation were evaluated. RESULTS E₂ concentrations were titrated to normal range in both groups; mean oral dose was 2 mg, and TD dose was 0.1 mg. After 6 and 12 months, fat-free mass and percent fat mass, bone mineral density accrual, lipid oxidation, and resting energy expenditure rates were similar between groups. IGF-1 concentrations were lower on oral 17β-E₂, but suppression of gonadotropins was comparable with no significant changes in lipids, glucose, osteocalcin, or highly sensitive C-reactive protein between groups. However, E₁, E₁S, SHBG, and bioestrogen concentrations were significantly higher in the oral group. CONCLUSIONS When E₂ concentrations are titrated to the normal range, the route of delivery of 17β-E₂ does not affect differentially body composition, lipid oxidation, and lipid concentrations in hypogonadal girls with TS. However, total estrogen exposure (E₁, E₁S, and total bioestrogen) is significantly higher after oral 17β-E₂. TD 17β-E₂ results in a more physiological estrogen milieu than oral 17β-E₂ administration in girls with TS.

Two different 5′ splice site mutations in the growth hormone gene causing autosomal dominant growth hormone deficiency

Abstract Four distinct types of isolated growth hormone deficiency (IGHD) have been described to date. Of these IGHD type II has been defined as having a dominant mode of inheritance. We performed a molecular genetic analysis of two patients clinically characterized as IGHD type II. One of the patients and her father shared a heterozygous G–A transition in the first 5′ donor splice site of intron III. The second father and daughter studied also showed a heterozygous G–A transition in the fifth base from the 5′ donor splice site in the same intron. Both mutations altered the correct splicing of the growth hormone pre-mRNA when the corresponding genes were expressed in COS-7 cells. We propose that both inherited mutations are responsible for IGHD type II in these patients.

Familial Tumoral Calcinosis Caused by a Novel FGF23 Mutation: Response to Induction of Tubular Renal Acidosis with Acetazolamide and the Non-Calcium Phosphate Binder Sevelamer

Hyperphosphatemic familial tumoral calcinosis (HFTC) is an uncommon disease characterized by periarticular calcifications produced by the deposition of amorphous extraosseous calcifications of hydroxyapatite. It is associated with hyperphosphatemia due to increased tubular phosphate reabsorption, despite normal renal function and normal plasma PTH levels. The disease can be caused by inactivating mutations in either the fibroblast growth factor 23 (FGF23) gene, the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene or in human KLOTHO (KL) gene. Herein, we describe a Caucasian 3-year-old girl with tumoral calcinosis who presented with elevated serum phosphorus levels and a large calcified mass at her left elbow which led to ulceration of the skin. Treatment with the phosphate binder sevelamer and the carbonic anhydrase inhibitor acetazolamide successfully reduced the serum phosphate levels and led to a reduction of the calcified mass. This medical management has not been described previously. Her 7-month-old sister also had elevated serum phosphate levels, but did not have ectopic calcifications. Sequencing analysis revealed a novel homozygous FGF23 missense mutation (c.367G>T, p.Gly123Trp) in both siblings while the parents were carriers of the mutation.

Obesity is positively associated with dehydroepiandrosterone sulfate concentrations at 7 y in Chilean children of normal birth weight

Background: In low-birth-weight girls, obesity increases the risk of premature adrenarche and metabolic complications. However, the consistency of this association in normal-birth-weight children and its potential mediators remain unknown. Objectives: The objectives were to assess the associations between obesity indicators and dehydroepiandrosterone sulfate (DHEAS) at 7 y of age and to evaluate the role of hormonal markers on these associations. Design: We assessed in 969 participants (6.9 y; 48% girls; all Tanner I) in the Growth and Obesity Chilean Cohort Study the associations between DHEAS and weight, BMI, waist circumference (WC), waist-to-height ratio, skinfold thickness, and percentage total fat (bioimpedance) and determined whether these associations were related to insulin, insulin-like growth factor I (IGF-I), and leptin. We also compared BMI and height growth from 0 to 7 y of age in nonobese and obese children with normal and high DHEAS (≥75th percentile) at 7 y. Results: DHEAS concentrations were similar between girls (30.3 ±1.86 μg/dL) and boys (29.4 ±1.73 μg/dL) (P > 0.05); 17.3% of children were obese (BMI-for-age z score ≥2 SD). Adiposity indicators were positively and similarly associated with DHEAS [ie, BMI, β standardized regression coefficient: 0.23 (95% CI: 0.17, 0.29); WC, β standardized regression coefficient: 0.23 (95% CI: 0.16, 0.30)]; these associations were only partially related to IGF-I and leptin. Obese children had twice the risk of high DHEAS (OR: 2.16; 95% CI: 1.51, 3.09); at 7 y, obese children with high DHEAS were fatter and more centrally obese than their counterparts (P < 0.05), although their previous growth was similar (P > 0.05). None of the results differed by sex (P > 0.05). Conclusion: In children of normal birth weight, obesity is positively associated with DHEAS at 7 y of age.

Latin American Consensus: Children Born Small for Gestational Age

BackgroundChildren born small for gestational age (SGA) experience higher rates of morbidity and mortality than those born appropriate for gestational age. In Latin America, identification and optimal management of children born SGA is a critical issue. Leading experts in pediatric endocrinology throughout Latin America established working groups in order to discuss key challenges regarding the evaluation and management of children born SGA and ultimately develop a consensus statement.DiscussionSGA is defined as a birth weight and/or birth length greater than 2 standard deviations (SD) below the population reference mean for gestational age. SGA refers to body size and implies length-weight reference data in a geographical population whose ethnicity is known and specific to this group. Ideally, each country/region within Latin America should establish its own standards and make relevant updates. SGA children should be evaluated with standardized measures by trained personnel every 3 months during year 1 and every 6 months during year 2. Those without catch-up growth within the first 6 months of life need further evaluation, as do children whose weight is ≤ -2 SD at age 2 years. Growth hormone treatment can begin in SGA children > 2 years with short stature (< -2.0 SD) and a growth velocity < 25th percentile for their age, and should continue until final height (a growth velocity below 2 cm/year or a bone age of > 14 years for girls and > 16 years for boys) is reached. Blood glucose, thyroid function, HbA1c, and insulin-like growth factor-1 (IGF-1) should be monitored once a year. Monitoring insulin changes from baseline and surrogates of insulin sensitivity is essential. Reduced fetal growth followed by excessive postnatal catch-up in height, and particularly in weight, should be closely monitored. In both sexes, gonadal function should be monitored especially during puberty.SummaryChildren born SGA should be carefully followed by a multidisciplinary group that includes perinatologists, pediatricians, nutritionists, and pediatric endocrinologists since 10% to 15% will continue to have weight and height deficiency through development and may benefit from growth hormone treatment. Standards/guidelines should be developed on a country/region basis throughout Latin America.