The COBLL1 C allele is associated with lower serum insulin levels and lower insulin resistance in overweight and obese children.

BACKGROUND: Childhood obesity is a growing epidemic worldwide, and it is associated with metabolic complications, such as insulin resistance. Recently, a genetic variation (rs7607980) in the COBLL1 gene has been associated with lower insulin resistance in adults. The aim of the study was to investigate if the association between COBLL1 rs7607980 genetic variant and lower insulin resistance was present early in life. METHODS: This sequence variant was genotyped in 878 overweight and obese children (mean age: 10 years) from Sardinia, Italy, from the outpatient clinic of the Pediatric Endocrine Unit, at the Regional Hospital for Microcitaemia in Cagliari. Insulin resistance was assessed by measurement of fasting circulating insulin levels before and after an oral glucose tolerance test and by HOMA-IR. RESULTS: The COBLL1 rs7607980 C allele was associated with lower fasting insulin and HOMA-IR levels (p = 0.002 and p = 0.035, respectively) in overweight and obese children. Importantly, lower insulin levels were also observed 2 h after oral glucose tolerance test in C allele carriers (p = 0.009). CONCLUSIONS: The present study shows for the first time, the association between COBLL1 rs7607980 C allele, lower serum insulin levels and lower insulin resistance in overweight and obese children.

The role of FTO genotype on eating behavior in obese Sardinian children and adolescents.

AIM: We aimed to study the influence of the fat mass and obesity-associated (FTO) gene on eating behavior in 412 obese Sardinian children and adolescents. Genome-wide association studies (GWAS) have identified several susceptibility loci for obesity. Among these, the polymorphisms in the intron 1 of the FTO gene has been found associated to weight gain and obesity in various populations. METHODS: All obese patients were genotyped for the FTO single nucleotide polimorphysm (SNP) rs9939609. In all subjects we evaluated eating behavior using the Child Eating Behaviour Questionnaire (CEBQ). RESULTS: We found no differences in eating behavior according to the genotype, either in the entire cohort, or when subjects were subdivided into four different age groups. CONCLUSIONS: FTO genotype is associated with body mass index but does not influence eating behavior in a selected cohort of obese children from the isolated genetic population of Sardinia.

rs9939609 in the FTO gene is associated with obesity but not with several biochemical parameters in Sardinian obese children.

Several studies have reported an association of the intronic single nucleotide polymorphism (SNP) rs9939609 of the fat mass and obesity-associated (FTO) gene with obesity and with a number of obesity-related features. We studied the association of rs9939609 with obesity in 912 obese children and adolescents (426 males and 486 females, mean ± SD age 10.5 ± 3.3 years) and in 543 normal weight subjects. A number of biochemical and clinical parameters was also evaluated in 700 of these patients. In the obese group, mean body mass index standard deviation score (BMI-SDS) was similar between the three genotypes. The A allele was present in 55% of the patients' and in 43% of controls' chromosomes. The distribution of heterozygotes was similar between patients and controls (47%), while the distribution of AA homozygotes was significantly higher in patients (31% vs. 20%). Logistic regression analysis on the genotypes yielded a χ(2) of 35.5 with an odds ratio of 1.6 (CI = 1.3-1.8), P < 1 × 10(-5) . None of the clinical and metabolic parameters tested was associated with the genotype. In conclusion, we have confirmed the strong association between FTO and obesity, and shown that only AA homozygotes are predisposed to develop obesity while TT homozygotes might be protected. Finally, we found no association between rs9939609 and a number of obesity-related abnormalities.

Effect of body mass index on the growth hormone response to clonidine stimulation testing in children with short stature.

OBJECTIVES: An inverse relationship has been shown between body mass index (BMI) and the peak growth hormone (GH) response to stimulation in adults and in children with short stature. This relation is observed even within a normal range of BMI. The aim of this study was to investigate the effect of BMI on the GH response to clonidine in a large number of children with short stature. DESIGN: We conducted a retrospective study on the GH response to clonidine in a single centre. METHODS: We studied 202 children with short stature (135 M and 67 F) who underwent clonidine testing from 2007 to 2009. RESULTS: One hundred and twenty-eight patients had a GH peak >10 µg/l. In univariate regression analysis, the peak GH after clonidine was negatively correlated with BMI-standard deviation score (BMI-SDS) and positively correlated with height velocity-SDS and IGF-I-SDS. Only the relationship between peak GH and BMI-SDS remained significant in children with a BMI-SDS from -2 to +2. In the multivariate stepwise regression analysis, BMI-SDS and IGF-I-SDS were the only significant variables in the entire cohort, explaining 19·5% of the variance in peak GH. When only subjects with BMI-SDS between -2·0 and +2·0 were included in the analysis (n = 173), BMI-SDS alone explained 21·4% of the variability in peak GH. The number of patients who failed the clonidine test increased with increasing BMI-SDS. CONCLUSIONS: BMI affects the GH response to clonidine in children with short stature and should be considered when interpreting the results to the stimulation test.

An INSIG2 polymorphism affects glucose homeostasis in Sardinian obese children and adolescents.

Allelic variants of a single nucleotide polymorphism (SNP), rs7566605, located approximately 10 kb upstream of the INSIG2 gene have been found in association with body weight and with other clinical features related to obesity in some populations but not in others. Our objective was to test the association of this SNP in obese children and adolescents from the genetically isolated population of Sardinia. We tested the association of rs7566605 with body mass index (BMI) and with serum glucose and insulin concentrations and a surrogate measure of insulin resistance (HOMA-IR) in a cohort of 747 Sardinian obese children and adolescents. A case control analysis was performed using 548 ethnically-matched healthy controls. Allelic frequencies of the SNP were similar between patients and controls. Mean glucose and insulin concentration and mean HOMA-IR values were significantly higher in patients carrying the CC genotype than in the CG and GG carriers. In the patients with impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT), allele C was significantly more frequent than in controls. Although INSIG2 polymorphisms do not consistently associate with BMI, the observation of an association with glucose concentration would support a role for this gene in the metabolic complications of obesity.

The 148M allele of the PNPLA3 gene is associated with indices of liver damage early in life.

BACKGROUND & AIMS: Childhood obesity is a growing problem worldwide. Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity in children. Recently, the PNPLA3 gene I148M (rs738409) variant was demonstrated to be strongly associated with hepatic steatosis in obese adults. In this study we add further insight into the role of PNPLA3 by exploring whether this association begins early in life in obese children or becomes manifest only in adulthood. METHODS: Four hundred and seventy-five obese/overweight children and adolescents were genotyped for the I148M allele. Clinical and biochemical parameters were collected for all participants, including indices of hepatic injury, glucose tolerance and insulin resistance. Ultrasound imaging of the liver was obtained to assess the degree of steatosis in a subset of children. RESULTS: Carriers of two 148M alleles had a 52% increase in circulating ALT levels compared to carriers of two 148I alleles, with individuals with one 148M allele showing a 9.5% increase (p=0.001). AST concentration was also significantly higher in carriers of two and one M alleles (17.4% and 4%, respectively, p=0.022). A total of 36% of carries of two 148M alleles showed elevated ALT, defined as >30U/L, compared to only 10% of carriers of two 148I alleles (p<0.001). Liver steatosis was more prevalent in carriers of two 148M alleles. Glucose tolerance and insulin sensitivity were similar across all three genotypes. CONCLUSIONS: Our data show that the PNPLA3 gene I148M variant is associated with increased levels of ALT/AST in obese children and adolescents, suggesting that it confers genetic susceptibility to liver damage from a young age.

Characterization of thyroglobulin epitopes in Sardinian adults and juveniles with Hashimoto's thyroiditis: evidence against a major effect of age and genetic background on B-cell epitopes.

BACKGROUND: Using recombinant human monoclonal thyroglobulin antibodies expressed as Fab molecules (TgAb-Fab), we have recently confirmed the restriction of Tg epitopes in Hashimoto's thyroiditis (HT). OBJECTIVE: To investigate Tg epitopes of serum TgAb in HT adults and HT juveniles from a geographically isolated area (Sardinia). DESIGN AND PATIENTS: Serum TgAb of 39 Sardinian HT adults, 53 Sardinian HT juveniles and 45 non-Sardinian HT adults were evaluated. The binding of serum TgAb to Tg in ELISA was inhibited by four recombinant human TgAb-Fab, identifying Tg epitopic regions A-D. The percentage of Tg binding inhibition was calculated comparing the binding of serum TgAb in presence of each TgAb-Fab with that in its absence. RESULTS: In the whole cohort of 137 patients, A region TgAb-Fab induced the highest levels of inhibition (55.3 +/- 17.8%) (mean +/- SD). Lower levels of inhibition were induced by TgAb-Fab of regions B (27.8 +/- 25.8%), C (26.8 +/- 24.6%) and D (17.5 +/- 18.4%). In Sardinian HT adults inhibition by TgAb-Fab of regions A, B and C were comparable to Sardinian HT juveniles; the marginal D region TgAb-Fab induced a slightly higher inhibition (22.1 vs. 13.8%; P = 0.034) in the former than in the latter group. In Sardinian and non-Sardinian HT adults inhibitions by the four TgAb-Fab were similar. CONCLUSIONS: In HT, the Tg epitope pattern of serum TgAb was similar in juveniles and adults from a geographically restricted area and in two adult populations from different geographical areas. Thus, in HT, neither age nor genetic background appear to influence B-cell epitopes.

Oral glucose tolerance test in Italian overweight/obese children and adolescents results in a very high prevalence of impaired fasting glycaemia, but not of diabetes.

BACKGROUND: Very few studies on glucose abnormalities in European overweight/obese children and adolescents are available, and scientific evidence on the value of standard oral glucose tolerance test (OGTT) in childhood is lacking. We therefore aimed to establish prevalence and features of impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and type 2 diabetes (T2D) in a large cohort of Italian overweight/obese children and adolescents and to assess the validity of standard OGTT in the paediatric population. METHODS: This is a 1-year observational study conducted on 736 (535 overweight/obese and 201 normal weight) consecutive paediatric patients attending the outpatient clinic of Paediatric Endocrine Unit. Clinical and biochemical parameters were collected for all participants. All overweight/obese subjects underwent OGTT. RESULTS: We observed a high prevalence of IFG (7.66%), more than twice that observed in other European children, but a low prevalence of IGT (3.18%) and T2D (0.18%). IFG was useless to predict IGT, having very low predictive value (7.3%) and sensitivity (17.6%). Compared to normal weight children, overweight/obese subjects showed significant differences in most metabolic and clinical parameters. In the overweight/obese group, having hyperglycaemia was associated to significantly higher blood pressure, homeostasis model assessment for insulin resistance, insulin and triglycerides. CONCLUSIONS: In our children, the prevalence of IFG is higher than that reported in other European cohorts, whereas T2D is rare. IFG appears not useful to detect IGT in childhood. Paediatric diagnostic cut-points, glucose load and timing of sampling need to be further validated to define glucose abnormalities in obese children that, compared with normal weight subjects, already are characterised by a different metabolic phenotype.

Puberty is associated with a marked increase of the female sex predominance in chronic autoimmune thyroiditis.

BACKGROUND: Chronic autoimmune thyroiditis (CAT) displays a strong female predominance with female-to-male (F:M) ratios of 4-20:1 in adults and 2-9:1 in children and adolescents. Both genetic and hormonal factors are involved in this phenomenon. The relation between puberty and F:M ratio in CAT has never been evaluated. METHODS: The F:M ratio of 133 children with CAT (group A, age at diagnosis 2.4-17.7 years) was compared with that of 113 adult CAT patients (group B, age at diagnosis 21-79 years). Group A included 64 prepubertal (aged 2.4-13.2 years, group A1) and 69 pubertal (aged 9.2-17.4, group A2) children. RESULTS: The F:M ratio in group A was 3.0, which is significantly (p < 0.001) lower than that (10.3) found in group B patients. The F:M ratio of group A1 prepubertal children was lower (1.6) and significantly different from that of pubertal (6.7, p < 0.01) and adult patients (10.3, p < 0.0001). This phenomenon was more evident in hypothyroid as compared to euthyroid CAT. CONCLUSIONS: This study provides the first evidence that female predominance of CAT strongly increases during puberty, suggesting a major role for sex hormones in this phenomenon. Further studies are needed to clarify this point.

Cardiovascular Risk Factors in Children and Adolescents With Obesity: Sex-Related Differences and Effect of Puberty.

Objectives: To evaluate the effect of gender and puberty on cardiovascular risk factors (CVRF) in obese children and adolescents. Methods: One thousand four hundred and nine obese patients [age 9.7 (2.2-17.9) y; 646 Male] were studied. Subjects were stratified according to Tanner pubertal staging and age into prepubertal ≤ and >6 ys (G1 and G2), pubertal stage 2-3 (G3), and pubertal stage 4-5 (G4). Waist circumference (WC), systolic and diastolic blood pressure (SP, DP), fasting plasma glucose, insulin, post Oral Glucose Tolerance Test glucose and insulin, and lipids were evaluated. Insulin resistance was evaluated by HOMA index. Patients with no CVRF were considered metabolically healthy (MHO). Results: The percentage of MHO patients was 59.8% in G1 while was consistently around 30% in the other groups. WC was more frequently abnormal in G2 males. Pubertal progression was associated with a decrease in WC abnormalities. SP was more frequently abnormal in G4 males and pubertal progression was associated with higher prevalence of abnormal SP in males. Pubertal progression was associated with an increase in hypertension rate in both sexes. HOMA was more frequently abnormal in G2 and G3 females. HDL, LDL, and TG were more frequently abnormal in G2 females. Dyslipidemia rate was higher in G2 females. Pubertal progression was associated with higher prevalence of abnormal HDL in males. Conclusions: Sex and pubertal status influence the frequency of abnormalities of CVRF in obese children and adolescents. CVRF are already present in prepubertal age. Identifying patients with higher risk of metabolic complications is important to design targeted and effective prevention strategies.

Adult height in patients with permanent growth hormone deficiency with and without multiple pituitary hormone deficiencies.

CONTEXT: It has been reported that patients with multiple pituitary hormone deficiencies (MPHDs) achieve a greater final height, compared with patients with isolated GH deficiency (IGHD). However, the outcome of patients with permanent GH deficiency (GHD) has not yet been reported. OBJECTIVES: The objectives of the study were to evaluate and compare adult height data and the effect of spontaneous or induced puberty after long-term treatment with GH in young adults with either permanent IGHD or MPHD. DESIGN AND SETTING: This was a retrospective multicenter study conducted in university research hospitals and a tertiary referral endocrine unit. PATIENTS AND METHODS: Thirty-nine patients with IGHD (26 males, 13 females) and 49 with MPHD (31 males, 18 females), diagnosed at a median age of 7.7 and 6.9 yr, respectively, were reevaluated for GH secretion after adult height achievement (median age 17.6 and 19.8 yr). The diagnosis of permanent GHD was based on peak GH levels less than 3 microg/liter after an insulin tolerance test or peak GH levels less than 5 microg/liter after two different tests. Fifteen subjects had idiopathic GHD and seventy-three had magnetic resonance imaging evidence of congenital hypothalamic-pituitary abnormalities. Height sd score (SDS) was analyzed at diagnosis, the onset of puberty (either spontaneous or induced), and the time of GH withdrawal. RESULTS: The subjects with IGHD entered puberty at a median age of 12.6 yr (females) and 13.4 yr (males). Puberty was induced at a median age of 13.5 and 14.0 yr, respectively, in males and females with MPHD. Median height SDS at the beginning of puberty was similar in the IGHD and MPHD subjects. Total pubertal height gain was similar between patients with IGHD or MPHD. Median adult height was also not significantly different between IGHD and MPHD patients (males, 168.5 vs. 170.3 cm; females, 160.0 vs. 157.3 cm). The adult height SDS of the IGHD subjects was positively correlated with height at the time of diagnosis and with total pubertal height gain. Conversely, the adult height SDS of the MPHD subjects was positively correlated with both the duration of GH treatment and height SDS at the time of GHD diagnosis. CONCLUSIONS: Adult height in patients with permanent IGHD and spontaneous puberty is similar to adult height in patients with MPHD and induced puberty.

Cut-off limits of the peak GH response to stimulation tests for the diagnosis of GH deficiency in children and adolescents: study in patients with organic GHD.

OBJECTIVE: The diagnosis of GH deficiency (GHD) in children and adolescents is established when GH concentrations fail to reach an arbitrary cut-off level after at least two provocative tests. The objective of the study was to define the optimal GH cut-offs to provocative tests in children and adolescents. DESIGN: Retrospective study in 372 subjects who underwent evaluation of GH secretion. GH and IGF-I were measured by chemiluminescence assay in all samples. Receiver operating characteristic (ROC) analysis was used to evaluate the optimal GH cut-offs and the diagnostic accuracy of provocative tests. METHODS: Seventy four patients with organic GHD (GH peak <10µg/L after two provocative tests) and 298 control subjects (GH response >10µg/L to at least one test) were included in the study. The provocative tests used were arginine, insulin tolerance test (ITT) and clonidine. Diagnostic criteria based on cut-offs identified by ROC analysis (best pair of values for sensitivity and specificity) were evaluated for each test individually and for each test combined with IGF-I SDS. RESULTS: The optimal GH cut-off for arginine resulted 6.5µg/L, 5.1µg/L for ITT and 6.8µg/L for clonidine. IGF-I SDS has low accuracy in diagnosing GHD (AUC=0.85). The combination of the results of provocative tests with IGF-I concentrations increased the specificity. CONCLUSIONS: The results of the ROC analysis showed that the cut-off limits which discriminate between normal and GHD are lower than those commonly employed. IGF-I is characterized by low diagnostic accuracy.

Thyroid function in obese children and adolescents.

OBJECTIVE: Obesity is frequently associated with modifications of thyroid size and function. We evaluated the prevalence of thyroid function abnormalities and the effects of puberty and weight loss in obese children and adolescents. METHODS: We examined 468 obese children (255 girls and 213 boys aged 3.7-17.9 years) and 52 normal-weight age-matched children as controls. TSH, fT3, fT4, fasting serum insulin and glucose were measured at baseline. fT3, fT4 and TSH were also measured after 6 months of lifestyle intervention in a subset of 43 patients. RESULTS: 109 obese children showed abnormal circulating thyroid hormone concentrations (84 had elevated fT3 levels, 15 elevated TSH, 6 elevated fT4, 3 elevated fT3 and TSH, and 1 elevated fT3, fT4 and TSH levels). Serum TSH and fT3 concentrations were positively correlated with BMI-SDS. The prevalence of patients with abnormal thyroid hormone concentrations was similar between sexes and between prepubertal and pubertal subjects. After 6 months of lifestyle intervention, thyroid hormone concentrations normalized in 27 of the patients with decreased BMI-SDS, and in 2 patients in whom BMI-SDS increased. CONCLUSIONS: In obese children, an increased fT3 concentration is the most frequent thyroid function abnormality. Serum fT3 and TSH correlate with BMI. Moderate weight loss frequently restores these abnormalities.

Prevalence of type 1 diabetes autoantibodies (GADA, IA2, and IAA) in overweight and obese children.

OBJECTIVE: Little is known about the prevalence of beta-cell autoantibodies in children with excess body weight. The prevalence of type 1 diabetes autoantibodies and its relation with hyperglycemia was analyzed in 686 overweight/obese children and adolescents. RESEARCH DESIGN AND METHODS: All children underwent an oral glucose tolerance test, and anti-GAD, anti-IA2, and anti-IAA autoantibodies were measured. Autoantibody prevalence was evaluated in 107 normal-weight children for comparison. RESULTS: A single autoantibody was present in 2.18% of overweight/obese subjects and 1.86% normal-weight subjects (P = NS). Postload glycemia was significantly higher in antibody-positive children (133 +/- 69.9 vs. 105.4 +/- 17.7 mg/dl, P < 0.0001) compared with autoantibody-negative subjects. No difference in autoantibody distribution was seen when our cohort was stratified by age, sex, SDS-BMI, pubertal stage, and homeostasis model assessment-insulin resistance (HOMA-IR). CONCLUSIONS: The 2.18% prevalence of type 1 diabetes autoantibodies is similar to that reported in nonobese children. This study provided evidence that excess body weight and insulin resistance do not influence autoantibody frequency.

IRAK-M is involved in the pathogenesis of early-onset persistent asthma.

Asthma is a multifactorial disease influenced by genetic and environmental factors. In the past decade, several loci and >100 genes have been found to be associated with the disease in at least one population. Among these loci, region 12q13-24 has been implicated in asthma etiology in multiple populations, suggesting that it harbors one or more asthma susceptibility genes. We performed linkage and association analyses by transmission/disequilibrium test and case-control analysis in the candidate region 12q13-24, using the Sardinian founder population, in which limited heterogeneity of pathogenetic alleles for monogenic and complex disorders as well as of environmental conditions should facilitate the study of multifactorial traits. We analyzed our cohort, using a cutoff age of 13 years at asthma onset, and detected significant linkage to a portion of 12q13-24. We identified IRAK-M as the gene contributing to the linkage and showed that it is associated with early-onset persistent asthma. We defined protective and predisposing SNP haplotypes and replicated associations in an outbred Italian population. Sequence analysis in patients found mutations, including inactivating lesions, in the IRAK-M coding region. Immunohistochemistry of lung biopsies showed that IRAK-M is highly expressed in epithelial cells. We report that IRAK-M is involved in the pathogenesis of early-onset persistent asthma. IRAK-M, a negative regulator of the Toll-like receptor/IL-1R pathways, is a master regulator of NF- kappa B and inflammation. Our data suggest a mechanistic link between hyperactivation of the innate immune system and chronic airway inflammation and indicate IRAK-M as a potential target for therapeutic intervention against asthma.