Screening for genetic variants in BDNF that contribute to childhood obesity.

UNLABELLED: What is already known about this subject BDNF is involved in the regulation of food intake and body weight. BDNF deficient animal models are obese. Chromosomal abnormalities cause obesity in humans. What this study adds Evaluation of point mutations in BDNF. Identification of BDNF mutations in obese children. Point mutations in BDNF are not a common cause of childhood obesity. INTRODUCTION: There is ample evidence that BDNF has a role in the regulation of food intake and body weight. Study of various mouse models gave a clear indication that BDNF deficiency leads to the development of obesity. Functional loss of one copy of the BDNF gene, due to chromosomal rearrangements or microdeletions, can cause an obesity phenotype in humans. Therefore, we wanted to investigate whether point mutations in the gene also result in a comparable phenotype. METHODS: We screened 554 severely overweight and obese children and adolescents and 565 lean adults for mutations in the coding region of BDNF. Mutation screening was performed by high-resolution melting curve analysis and direct sequencing. RESULTS: Screening of obese patients led to the identification of two synonymous variations (V37V and H65H) and two non-synonymous coding mutations (T2I and V46M) in the BDNF gene. When we subsequently screened our control population, we found T2I with comparable frequency and confirmed that this is a rare and non-pathogenic variant. In addition, we found another non-synonymous mutation (N187S) in the control population. CONCLUSIONS: In silico analysis of the V46M variant did not support a clear disease-causing effect and no family data were available in order to determine whether the mutation segregates with obesity. However, we cannot rule out a possible pathogenic effect for this variant. In general, we tend to conclude that mutations in the coding region of BDNF are uncommon in obese patients and are therefore not likely to play an essential role in the pathogenesis of childhood obesity.

Mutation screen of the SIM1 gene in pediatric patients with early-onset obesity.

BACKGROUND: The transcription factor SIM1 (Single-minded 1) is involved in the control of food intake and in the pathogenesis of obesity. In mice, Sim1 is involved in the development of the paraventricular nucleus, and Sim1 deficiency leads to severe obesity and hyperphagia. In humans, chromosomal abnormalities in the SIM1 gene region have been reported in obese individuals. Furthermore, recent data also suggest that loss-of-function point mutations in SIM1 are responsible for SIM1 haplo-insufficiency that is involved in causing human obesity. In this study, we therefore wanted to expand the evidence regarding the involvement of SIM1 mutations in the pathogenesis of severe early-onset obesity. METHODS: We screened 561 severely overweight and obese children and adolescents and 453 lean adults for mutations in the coding region of the SIM1 gene. Mutation screening in all patients and lean individuals was performed by high-resolution melting curve analysis combined with direct sequencing. To evaluate the effect of the mutations on SIM1 transcriptional activity, luciferase reporter assays were performed. RESULTS: Mutation analysis identified four novel nonsynonymous coding variants in SIM1 in four unrelated obese individuals: p.L242V, p.T481K, p.A517V and p.D590E. Five synonymous variants, p.P57P, p.F93F, p.I183I, p.V208V and p.T653T, were also identified. Screening of the lean control population revealed the occurrence of four other rare SIM1 variants: p.G408R, p.R471P, p.S492P and p.S622F. For variants p.T481K and p.A517V, which were found in obese individuals, a decrease in SIM1 transcriptional activity was observed, whereas the transcriptional activity of all variants found in lean individuals resembled wild type. CONCLUSIONS: In this study, we have demonstrated the presence of rare SIM1 variants in both an obese pediatric population and a population of lean adult controls. Further, we have shown that functional in vitro analysis of SIM1 variants may help in distinguishing benign variants of no pathogenic significance from variants which contribute to the obesity phenotype.

Sleep-disordered breathing and systemic inflammation in overweight children and adolescents.

OBJECTIVE: To assess if the severity of sleep-disordered breathing (SDB) and mainly intermittent hypoxia is associated with increased peripheral leukocytes in overweight children and adolescents, controlling for adiposity and obesity-related metabolic abnormalities. METHODS: Consecutive subjects were recruited at a pediatric obesity clinic. All subjects underwent polysomnography and a fasting blood sample. RESULTS: In total, 95 subjects were included ( =11.1+/-2.6, 43 boys, body mass index, =2.3+/-0.5, 29 subjects were overweight and 66 obese). Total white blood cell count increased significantly by worsening of intermittent hypoxia. Total white blood cell count was correlated with the maximal degree of desaturation, independent of puberty, HOMA and HDL-cholesterol. Neutrophil levels were associated with the degree of desaturation, while controlling for puberty and HOMA. CONCLUSION: This study supports the hypothesis of an independent interaction between intermittent hypoxia and nocturnal desaturation during sleep, and increased white blood cell and neutrophil levels in overweight and obese children and adolescents. This finding may contribute to the mechanisms linking SDB with increased cardiovascular morbidity.

Sleep-disordered breathing and the metabolic syndrome in overweight and obese children and adolescents.

OBJECTIVE: To assess whether sleep-disordered breathing (SDB) is a risk factor of the metabolic syndrome (MS) in children and adolescents who are overweight and to examine whether the severity of SDB was independently associated with glucose intolerance, insulin resistance, and/or dyslipidemia. STUDY DESIGN: Consecutive subjects who were overweight or obese underwent polysomnography, fasting blood sample, and oral glucose tolerance test (for calculation of area under the curve [AUC]). SDB was defined as a respiratory disturbance index > or = 2. MS was present when > or = 3 of these factors were present: waist circumference > or = 90th percentile; fasting glucose level > or = 110 mg/dL; triglyceride level > or = 110 mg/dL; high-density lipoprotein cholesterol level < or = 40 mg/dL; blood pressure > or = 90th percentile. RESULTS: A total of 104 subjects were included in the study (44% boys; 58% prepubertal; mean age, 11.1 +/- 2.6 years; 69% obese). Mean SaO2 (odds ratio, 0.54) and SaO2nadir (odds ratio, 0.89) were independent, significant predictors of the presence of MS. Multiple regression showed significant associations between SaO2nadir and high-density lipoprotein cholesterol level, mean SaO2 and both AUC glucose and triglyceride levels, and between the percentage of total sleep time with SaO2 > or = 95% and cholesterol level, while controlling for adiposity and sex, puberty, or both. CONCLUSION: This study supports the hypothesis of an interaction between SDB and metabolic abnormalities, independent of estimates of body fat distribution, in children and adolescents who are overweight and obese.

Seasonality in clinical onset of type 1 diabetes in belgian patients above the age of 10 is restricted to HLA-DQ2/DQ8-negative males, which explains the male to female excess in incidence.

AIMS/HYPOTHESIS: Type 1 diabetes arises from an interplay between environmental and genetic factors. The reported seasonality at diagnosis supports the hypothesis that currently unknown external triggers play a role in the onset of the disease. We investigated whether a seasonal pattern is observed at diagnosis in Belgian Type 1 diabetic patients, and if so whether seasonality varies according to age, sex and genetic risk, all known to affect the incidence of Type 1 diabetes. METHODS: The seasonal pattern at clinical diagnosis was assessed in 2176 islet antibody-positive diabetic patients aged 0 to 39 years diagnosed between 1989 and 2000. Additional stratification was performed for age, sex and HLA-DQ genotype. RESULTS: Overall, a significant seasonal pattern at clinical diagnosis of diabetes was observed (p<0.001). More subjects were diagnosed in the period of November to February (n=829) than during the period of June to September (n=619) characterised by higher averages of maximal daily temperature and daily hours of sunshine. However, the seasonal pattern was restricted to patients diagnosed above the age of 10 (0-9 years: p=0.398; 10-19 years: p<0.001; 20-29 years: p=0.003; 30-39 years: p=0.015). Since older age at diagnosis is associated with a male to female excess and a lower prevalence of the genetic accelerator HLA-DQ2/DQ8, we further stratified the patients aged 10 to 39 years (n=1675) according to HLA-DQ genotype and sex, and we found that the seasonal pattern was largely restricted to male subjects lacking DQ2/DQ8 (n=748; p<0.00 vs all others: n=927; p=0.031). CONCLUSIONS/INTERPRETATION: In a subgroup of male patients diagnosed over the age of 10, the later stages of the subclinical disease process may be more driven by sex- and season-dependent external factors than in younger, female and genetically more susceptible subjects. These factors may explain the male to female excess in diabetes diagnosed in early adulthood.

Beta-cell, thyroid, gastric, adrenal and coeliac autoimmunity and HLA-DQ types in type 1 diabetes.

The autoimmune attack in type 1 diabetes is not only targeted to beta cells. We assessed the prevalence of thyroid peroxidase (aTPO), parietal cell (PCA), antiadrenal (AAA) and endomysial antibodies (EmA-IgA), and of overt autoimmune disease in type 1 diabetes, in relation to gender, age, duration of disease, age at onset, beta-cell antibody status (ICA, GADA, IA2A) and HLA-DQ type. Sera from 399 type 1 diabetic patients (M/F: 188/211; mean age: 26 +/- 16 years; duration: 9 +/- 8 years) were tested for ICA, PCA, AAA and EmA-IgA by indirect immunofluorescence, and for IA2A (tyrosine phosphatase antibodies), GADA (glutamic acid decarboxylase-65 antibodies) and aTPO by radiobinding assays. The prevalence rates were: GADA 70%; IA2A, 44%; ICA, 39%; aTPO, 22%; PCA, 18%; EmA-IgA, 2%; and AAA, 1%. aTPO status was determined by female gender (beta = - 1.15, P = 0.002), age (beta = 0.02, P = 0.01) and GADA + (beta = 1.06, P = 0.02), but not by HLA-DQ type or IA2A status. Dysthyroidism (P < 0.0001) was more frequent in aTPO + subjects. PCA status was determined by age (beta = 0.03, P = 0.002). We also observed an association between PCA + and GADA + (OR = 1.9, P = 0.049), aTPO + (OR = 1.9, P = 0.04) and HLA DQA1*0501-DQB1*0301 status (OR = 2.4, P = 0.045). Iron deficiency anaemia (OR = 3.0, P = 0.003) and pernicious anaemia (OR = 40, P < 0.0001) were more frequent in PCA + subjects. EmA-IgA + was linked to HLA DQA1*0501-DQB1*0201 + (OR = 7.5, P = 0.039), and coeliac disease was found in three patients. No patient had Addison's disease. In conclusion, GADA but not IA2A indicate the presence of thyrogastric autoimmunity in type 1 diabetes. aTPO have a female preponderance, PCA are weakly associated with HLA DQA1*0501-DQB1*0301 and EmA-IgA + with HLA DQA1*0501-DQB1*0201.

Gastric parietal cell antibodies are associated with glutamic acid decarboxylase-65 antibodies and the HLA DQA1*0501-DQB1*0301 haplotype in Type 1 diabetes mellitus. Belgian Diabetes Registry.

AIMS: To assess the prevalence of thyrogastric autoimmunity in relation to age, sex, beta-cell antibody status and HLA DQ haplotypes in Type 1 diabetes mellitus. METHODS: One hundred and seventy-one patients with Type 1 diabetes mellitus were studied (male/female 86/85; mean age 19 +/- 11 years; duration of diabetes 5 +/- 4 years). Islet cell antibodies (ICA) and parietal cell antibodies (PCA) were measured using indirect immunofluorescence; glutamic acid decarboxylase-65 antibodies (GADA) by radiobinding assay and thyroid peroxidase antibodies (TPO) with an immunoradiometric assay (IRMA). RESULTS: The majority of subjects (81.3%) showed one or more autoantibodies. The prevalence rates were: GADA 64.9%, ICA 46.2%, PCA 19.9% and TPO 19.3%. Patients with ICA+ > or = 3 years after diagnosis had a higher prevalence of GADA (P = 0.03, odds ratio (OR) 2.66) and thyrogastric antibodies (P = 0.05, OR 2.23) than subjects ICA- after 3 years. PCA+ patients were older (P = 0.04), had a higher prevalence of GADA (P = 0.005, OR 3.89) and TPO (P = 0.05, OR 2.50) than PCA- subjects. Logistic regression analysis showed that PCA status was determined by the HLA DQA1*0501-DQB1*0301 haplotype (beta = 2.94, P = 0.04) and GADA status (beta = 2.44, P = 0.041). CONCLUSIONS: Thyrogastric antibodies are highly prevalent in Type 1 diabetes mellitus, especially in patients with persisting ICA. Screening for gastric autoimmunity is particularly advised in patients who are positive for GADA and for the HLA DQA1*0501-DQB1*0301 haplotype.

Progressive deceleration in growth as an early sign of delayed puberty in boys.

OBJECTIVE: To describe the prepubertal growth pattern in boys with delayed puberty. METHODS: Growth curves for height and height velocity covering the age range 4-14 years were constructed on the basis of retrospectively obtained data in 85 boys with delayed puberty, who attained a normal final height. RESULTS: Between the age of 4 and 14 years the height in this cohort progressively deviated from the normal reference. At the age of 4 years, the height SDS was already significantly lower (median -0.8; p < 0.001) and progressively diminished during childhood, resulting in a median height SDS of -1.1 at the age of 12 years (p < 0.001). The median final height of this cohort (-0.4) was not different from their target height (-0.2). The degree of deceleration in growth during childhood was not determined by birth weight or birth height and did not influence final height. The decline of the height velocity with age in this group of boys with delayed puberty was significantly smaller (p < 0.001) than predicted by the model of Rikken and Wit. CONCLUSION: Late-maturing boys often show a prepubertal deceleration in growth that starts at an early age but that does not affect final height.

Hormonal changes during the first year of oestrogen treatment in constitutionally tall girls.

OBJECTIVE: Oestrogens are used to inhibit growth in girls with constitutionally tall stature. We studied the changes in different hormones that accompany such therapy. SUBJECTS AND METHODS: In this longitudinal study we examined the levels of total insulin-like growth factor-I (IGF-I), free thyroxine (FT(4)), thyrotrophin (TSH), testosterone, dehydroepiandrosterone sulphate (DHEA-S), cortisol and prolactin in two groups of girls receiving ethinyloestradiol at a dose of either 0.1mg daily (group A, n=22) or 0.2mg daily (group B, n=36). Hormonal measurements were performed at start of therapy and after 3, 6 and 12 months. RESULTS: In both groups the levels of IGF-I, testosterone and DHEA-S were reduced while the concentrations of cortisol and prolactin were increased. The pituitary-thyroid axis was not significantly affected by this therapy. The girls receiving 0.2mg ethinyloestradiol daily had lower IGF-I levels after 12 months of therapy and had higher serum prolactin concentrations than the girls treated with 0.1mg daily. The reduction in predicted height and the advancement in bone age were similar in both groups. CONCLUSIONS: Therapy with pharmacological doses of ethinyloestradiol changes the levels of several hormones including IGF-I, testosterone, DHEA-S, prolactin and cortisol but the role of the respective changes in the inhibition of growth is not clear. The suppression of DHEA-S levels by 40% suggests that the ovaries contribute significantly to the production of this hormone in pubertal girls.

Low thyroxinaemia occurs in the majority of very preterm newborns.

UNLABELLED: Transient hypothyroxinaemia with normal thyroid stimulating hormone (TSH) levels is a well-known condition in preterm neonates and is generally assumed to be a harmless epiphenomenon of prematurity. This assumption is, however, based on studies that included very few neonates with a gestational age (GA) below 30 weeks. We therefore measured serum free thyroxine (FT4) and serum TSH on days 1 and 14 in 263 neonates with a GA between 26 and 41 weeks. In 13 infants (5%), transient hypothyroidism (low FT4 and TSH >20 mU/l on day 14) was found. In the remaining 250 patients FT4 on days 1 and 14 but not TSH correlated positively with GA. In neonates with a GA of 35-41 weeks, FT4 increased postnatally to levels within or above the normal adult range. In contrast, in the very preterm group (26-31 weeks) the already low FT4 levels declined to values significantly below the range observed in term neonates. A significant proportion of these neonates had FT4 levels within the hypothyroid range. There was no difference in thyroid function between neonates treated with povidone-iodine or chlorhexidine. CONCLUSION: Very preterm neonates have FT4 levels on day 14 that are much lower than is generally assumed while TSH remains in the normal range. We therefore propose to measure FT4 in all preterms with a GA below 33 weeks, during the 2nd week of life.

Longitudinal data on growth and final height in diabetic children.

The available data on growth in children with insulin-dependent diabetes mellitus are conflicting and are mainly derived from cross-sectional studies. In this longitudinal study, height, weight, skeletal age, and pubertal development were recorded until final height was attained in 46 children (22 girls and 24 boys) with onset of diabetes before the age of 10 y. At the onset of diabetes, height SD score (SDS) averaged 0.41 in girls and 0.56 in boys, which was normal when corrected for the secular trend. Prepubertal growth was unaffected in both sexes. Diabetic boys had a marked delay in onset of puberty (mean age at genitalia stage 2: 13.7 y) but attained a normal final height (final height SDS: 0.48 +/- 0.89). In girls final height was slightly reduced (height SDS 0.27 +/- 0.97) due to a suboptimal pubertal growth spurt. Mean pubertal height gain in girls was 16.6 cm and mean age at breast stage 2 was 11.6 y. Diabetic girls also tended to become obese during puberty. Skeletal maturation was normal at all ages. These data suggest that conventional therapy does not guarantee optimal growth, especially in girls.

Imidazoline intoxication in children.

A 15-month-old girl with rhinopharyngitis was treated with a nasal solution containing the imidazoline derivative naphazoline. She rapidly developed profound CNS depression with stupor, hypothermia, hypoventilation and bradycardia. All symptoms disappeared within 24 h. The symptomatology of 18 other paediatric cases of naphthylimidazoline exposure reported to the Belgian National Poison Centre, is also discussed. Imidazoline intoxication due to overdose or accidental ingestion but also after normal therapeutic usage is frequent in children. It can cause severe CNS depression, especially in very young children. For these reasons vasoconstrictor imidazoline containing solutions should be prescribed with caution and kept out of reach of children.

Isolation of several fractions from human serum, inhibiting rabbit cartilage metabolism in vitro.

In contrast to the well characterized serum stimulators of cartilage metabolism, information is scarce on the nature of circulating inhibitors. Human serum was fractionated by molecular sieving chromatography on Sephadex G-200, G-75, G-50 and Biogel P-4 gels. Six fractions with molecular weights of 150, 45, 30, 16, 9 and 1.2 kD inhibited [35S]sulphate incorporation into rabbit cartilage segments. All fractions but the smallest one exhibited their inhibitory effect only in the serum-stimulated cartilage. The 1.2 kD fraction impaired [35S]sulphate and [3H]methyl-thymidine incorporation into the cartilage segments in both stimulated and basal conditions. A seventh inhibitory fraction corresponded to the serum salt peak.

Serum and serotonin induce retraction of calf aortic smooth muscle (CASM) cells in vitro: inhibition by ketanserin, a 5-HT2 receptor antagonist.

Calf aortic smooth muscle (CASM) cells cultured in vitro at high cell density (4 x 10(4) cells/cm2) on bacteriological petri dishes in the presence of serum pile up in clusters and create open spaces in the monolayer. This phenomenon is clearly visible 6 days after plating and is markedly enhanced by the addition of fetal calf serum. Serotonin is essential for the serum-induced retraction since (1) dialyzed serum has no effect, (2) of all the vasoactive agents we tested, only serotonin induced a similar degree of retraction, and (3) the serum-induced retraction was completely blocked by preincubating the cells with serotonin 5-HT2 receptor blockers such as ketanserin and ritanserin but not by preincubation with adrenergic-alpha 1 blockers or histamine antagonists. Serotonin caused CASM cell retraction in a dose-dependent way, with a maximum effect at 10(-6) M. The serotonin-induced retraction was reversible in time and was effectively blocked by ketanserin (IC50 = 1.2 x 10(-9) M). It is therefore concluded that serotonin induces retraction of CASM cells, mediated by the serotonin 5-HT2 receptor.

Somatomedin inhibitors from human serum produce abnormalities in mouse embryos in culture.

Two human serum fractions, one from normal individuals (Mr 1,150-1,310 daltons) and the other (Mr 800-1,100 daltons) from patients suffering with uremia (renal failure, azotemia), were added to the medium used to grow embryos in whole-embryo culture (WEC) beginning at the 3-5 (day 9) or 18-21 (day 10) somite stage. Both of these fractions possessed somatomedin (insulin-like growth factor) inhibitory activity. Day 9 embryos exposed to either of the serum fractions for 24 hr exhibited incomplete rotation and neural tube closure defects and were smaller than control embryos (decreased total protein content). Developmental abnormalities induced in day 10 embryos following 24 hr in culture included a marked decrease in expansion of the brain regions, hypoplasia of the first two branchial arches, and decreased amounts of total protein compared to controls. The visceral yolk sacs (VYSs) of somatomedin inhibitor (SI)-exposed conceptuses were opaque, and those from day 10 conceptuses contained significantly more protein than controls. Morphologically, the VYS endoderm cells from SI-exposed embryos contained a much higher density of "vacuoles" than controls. These results mimic those produced by exposure of conceptuses to an SI of Mr800-1,100 obtained from the serum of diabetic rats and suggest that similar substances and mechanisms are involved.

Effect of steroids on cartilage metabolism "in vitro".

Conditions characterized by high levels of glucocorticoids are associated with poor growth. Serum somatomedin or insulin-like growth factor activity measured by cartilage bioassay systems is low, but is generally not accompanied by a fall in somatomedin concentration. Hydrocortisone and a synthetic analogue, dexamethasone, impaired the serum stimulated "in vitro" 35S sulphate and 3H-thymidine incorporation in porcine rib cartilage at physiological concentrations. Hydrocortisone added at a concentration of 0.1 micrograms/ml decreased the potency of normal serum to 50% of controls. Dexamethasone was at least 10 times more potent. Removal of "in vitro" or "in vivo" administered hydrocortisone with dextran-coated charcoal restored the sulphate and thymidine activity to normal. We conclude that physiological amounts of glucocorticoids inhibit the "in vitro" porcine cartilage metabolism. Glucocorticoid administration "in vivo" does not abolish the activity of the cartilage stimulating effect of serum but affects cartilage metabolism directly or by the induction of locally produced inhibitors of cartilage metabolism.