Grazia Cirillo

Hepcidin in obese children as a potential mediator of the association between obesity and iron deficiency.

CONTEXT Obesity and iron deficiency are two of the most common nutritional disorders worldwide. Several studies found higher rates of iron deficiency in obese than in normal-weight children. Hepcidin represents the main inhibitor of intestinal iron absorption, and its expression is increased in adipose tissue of obese patients. Leptin is able, in vitro, to raise hepcidin expression. OBJECTIVES Aims of this work were 1) to assess the association between poor iron status and obesity, 2) to investigate whether iron homeostasis of obese children may be modulated by serum hepcidin variations, and 3) to assess the potential correlation between leptin and serum hepcidin variations. METHODS Iron status and absorption as well as hepcidin, leptin, and IL-6 levels were studied in 60 obese children and in 50 controls. RESULTS Obese children showed lower iron and transferrin saturation (both P < 0.05) and higher hepcidin levels (P = 0.004) compared with controls. A direct correlation between hepcidin and obesity degree (P = 0.0015), and inverse correlations between hepcidin and iron (P = 0.04), hepcidin and transferrin saturation (P = 0.005), and hepcidin and iron absorption (P = 0.003) were observed. A correlation between leptin and hepcidin (P = 0.006) has been found. The correlation remained significant when adjusted for body mass index, sex, pubertal stage, and IL-6 values. CONCLUSIONS We propose that in obese patients, increased hepcidin production, at least partly leptin mediated, represents the missing link between obesity and disrupted iron metabolism.

Low frequency of melanocortin-4 receptor (MC4R) mutations in a Mediterranean population with early-onset obesity.

Background: Melanocortin-4 receptor (MC4R) mutations have been reported as the most common single genetic cause of obesity in some populations and it has been suggested that they may be responsible for more than 4% of early-onset obesity.Objectives: To verify the presence of mutations of the MC4R coding region in children from southern Italy with early-onset obesity.Subjects and Methods: Two-hundred and eight unrelated obese children and adolescents were included in the study. The average age at obesity onset was 4.5±2.6 y. MC4R coding region was screened using both single-strand conformation polymorphism (SSCP) analysis and denaturing high-performance liquid chromatography (DHPLC). Automatic sequencing of PCR products of all individuals that showed an aberrant SSCP and/or DHPLC pattern was performed.Results: One novel missense mutation and one previously described polymorphism (Vall03Ile) were identified. The missense mutation C142T, resulting in the substitution of proline with serine at codon 48, within the first MC4R transmembrane domain, was detected at the heterozygous state in a 15-y-old obese girl (body mass index (BMI)=35 kg/m2) who has been obese since she was 8 y old. The mutation co-segregated with the obesity phenotype for over three generations and was not found in the control population.Conclusions: Our data show MC4R obesity causing mutations in less than 0.5% of the patients (ie 1 out of 208 patients) and therefore indicate a low prevalence of MC4R variants in the obese population from southern Italy. The specific genetic background of the Mediterranean population could make it difficult for MC4R mutations to produce an essentially polygenic trait such as common obesity, at least during childhood.

Molecular screening of the ghrelin gene in Italian obese children: the Leu72Met variant is associated with an earlier onset of obesity

OBJECTIVE: To test whether ghrelin variants could play a role in modulating some aspects of the obese phenotype during childhood.DESIGN: We screened the ghrelin gene in 300 Italian obese children and adolescents (mean age 10.5±3.2 y; range 4–19 y) and 200 controls by using the single-strand conformation polymorphism and the restriction fragment length polymoprhism analysis.RESULTS: No mutations were detected with the exception of two previously described polymorphisms, Arg51Gln and Leu72Met. For both variations, allelic frequencies were similar between patients and controls. Interestingly, we showed that the Leu72Met polymorphism was associated with differences in the age at obesity onset. Patients with the Met72 allele became obese earlier than homozygous patients for the wild Leu72 allele. The logrank test comparing the plots of the complement of Kaplan–Meier estimates between the two groups of patients was statistically significant (P<0.0001).CONCLUSION: It is unlikely that ghrelin variations cause the obesity due to single-gene mutations. The Leu72Met polymorphism of the ghrelin gene seems to play a role in anticipating the onset of obesity among children suggesting, therefore, that ghrelin may be involved in the pathophysiology of human adiposity.

The association of PNPLA3 variants with liver enzymes in childhood obesity is driven by the interaction with abdominal fat.

Background and Aims A polymorphism in adiponutrin/patatin-like phospholipase-3 gene (PNPLA3), rs738409 C->G, encoding for the I148M variant, is the strongest genetic determinant of liver fat and ALT levels in adulthood and childhood obesity. Aims of this study were i) to analyse in a large group of obese children the role of the interaction of not-genetic factors such as BMI, waist circumference (W/Hr) and insulin resistance (HOMA-IR) in exposing the association between the I148M polymorphism and ALT levels and ii) to stratify the individual risk of these children to have liver injury on the basis of this gene-environment interaction. Methods 1048 Italian obese children were investigated. Anthropometric, clinical and metabolic data were collected and the PNPLA3 I148M variant genotyped. Results Children carrying the 148M allele showed higher ALT and AST levels (p = 0.000006 and p = 0.0002, respectively). Relationships between BMI-SDS, HOMA-IR and W/Hr with ALT were analysed in function of the different PNPLA3 genotypes. Children 148M homozygous showed a stronger correlation between ALT and W/Hr than those carrying the other genotypes (p: 0.0045) and, therefore, 148M homozygotes with high extent of abdominal fat (W/Hr above 0.62) had the highest OR (4.9, 95% C. I. 3.2–7.8, p = 0.00001) to develop pathologic ALT. Conclusions We have i) showed for the first time that the magnitude of the association of PNPLA3 with liver enzymes is driven by the size of abdominal fat and ii) stratified the individual risk to develop liver damage on the basis of the interaction between the PNPLA3 genotype and abdominal fat.

Molecular screening of the proopiomelanocortin ( POMC ) gene in Italian obese children: report of three new mutations

BACKGROUND: Although linkage studies strongly suggest that proopiomelanocortin (POMC) alterations could play a role in the genetic predisposition to obesity, systematic POMC mutational analysis did not completely confirm this hypothesis.OBJECTIVES: To verify the presence of mutations of the POMC coding region in Italian children with very early onset obesity.SUBJECTS AND METHODS: Eighty seven unrelated Italian obese children and adolescents were studied. Mean age at obesity onset was 4.7±2.5 y. The POMC gene coding region was screened using single-strand conformation polymorphism (SSCP) analysis. Bi-directional automatic sequencing of PCR products was performed for all individuals who showed an aberrant SSCP pattern.RESULTS: Three new mutations have been identified in the heterozygous state in three patients: (a) G3834C, resulting in the substitution of Ser with Thr at codon 7 within the POMC signal peptide; (b) C3840T, resulting in the substitution of Ser with Leu at codon 9 of the pre-proopiomelanocortin signal peptide; and (c) C7406G, producing the substitution of Arg with Gly at codon 236 within the β-endorphin peptide. A polymorphism consisting of a 9 bp insertion, AGC AGC CGC, between position 6997 and 6998 has been found at the heterozygous state in nine patients. They showed leptin levels adjusted for BMI, gender and pubertal stage significantly higher than obese subjects homozyous for the POMC wild-type allele.CONCLUSIONS: Mutations in codons 7 and 9 of the signal peptide may alter the translocation of the pre-proopiomelanocortin into the endoplasmic reticulum and, therefore, can be implicated in obesity. Although further studies are required, the polymorphism between position 6997 and 6998 may represent one of the genetic variations that explain the linkage between obesity and POMC.

Abdominal fat interacts with PNPLA3 I148M, but not with the APOC3 variant in the pathogenesis of liver steatosis in chronic hepatitis C

The patatin‐like phospholipase domain‐containing 3 gene (PNPLA3) and the apolipoprotein C3 gene (APOC3) have been studied in relation to liver steatosis and liver disease outcome. The aim of this study was to evaluate the influence of PNPLA3 p.I148M and APOC3 rs2854116 and rs2854117 polymorphisms on the clinical and histological presentation of chronic hepatitis C in an Italian population and their relationship with viral and anthropometric parameters. Patients with hepatitis C (n = 166) entered the study receiving a clinical, histological, virological and biochemical evaluation. APOC3 (rs2854116 and rs2854117) and PNPLA3 (p.I148M) variants were genotyped. PNPLA3 polymorphisms were associated with liver steatosis, which was significantly higher in patients with p.148I/M (P = 0.034) and p.148M/M (P = 0.004) variants than those homozygous for the PNPLA3 wild type. Excluding patients with HCV genotype 3, the association with liver steatosis and PNPLA3 variants was more marked (p.148I/I genotype vs p.148I/M, P = 0.02, and vs p.148M/M, P = 0.005). The APOC3 polymorphism was not associated with any of the evaluated parameters. Among the interacting factors, BMI and waist circumference correlated with liver steatosis (P = 0.008 and 0.004, respectively). Relationship between waist circumference and liver steatosis was analysed for the different PNPLA3 genotypes. Homozygous 148M patients showed a stronger correlation between waist circumference and steatosis than those carrying the other genotypes (P = 0.0047). In our hepatitis C‐infected population, the PNPLA3 polymorphism influenced the development of liver steatosis, but not fibrosis progression. APOC3 polymorphisms had no effect on the development of steatosis and no influence on the PNPLA3 polymorphism. The amount of abdominal fat can increase the association of PNPLA3 p.I148M with liver steatosis.

Benign familial neonatal convulsions (BFNC) resulting from mutation of the KCNQ2 voltage sensor.

Benign familial neonatal convulsions (BFNC) is a rare autosomal inherited epilepsy. We studied the KCNQ2 coding region in a large, four-generation, Italian family with BFNC. A missense mutation C686T predicting the change of one of the innermost arginine (R214W) of the key functional voltage sensor (S4 helix), has been found in all affected members. This substitution probably reduces the movement of the voltage sensor that precedes channel opening during voltage-dependent activation. Several mutations affecting the trans-membrane domain and the pore region of the K+ channels belonging to the KQT-like family have been described in some human diseases associated with altered regulation of cellular excitability (ie BFNC, some LQT syndromes and DFNA2). R214W represents the first mutation involving the region of the voltage sensor.

TM6SF2 E167K variant is associated with severe steatosis in chronic hepatitis C, regardless of PNPLA3 polymorphism

A common non‐synonymous polymorphism, E167K, in transmembrane six superfamily member 2 (TM6SF2) gene has been recently associated with an increased hepatic triglyceride content, dyslipidemia and liver fibrosis in NAFLD patients. We investigated possible associations between the TM6SF2 variants and liver lesions in chronic hepatitis C.

TM6SF2 Glu167Lys polymorphism is associated with low levels of LDL-cholesterol and increased liver injury in obese children.

The Glu167Lys (E167K) transmembrane 6 superfamily member 2 (TM6SF2) variant has been associated with liver steatosis, high alanine transaminase (ALT) levels and reduced plasma levels of liver‐derived triglyceride‐rich lipoproteins.

Variations of retinol binding protein 4 levels are not associated with changes in insulin resistance during puberty.

Retinol binding protein 4 (RBP4) is an adipokine involved in the pathogenesis of insulin resistance in obese adults and children. Since insulin resistance occurs during puberty, independently of adiposity, a role for RBP4 in the onset of this phenomenon may be hypothesized. In order to verify our hypothesis, we studied 90 subjects (45 obese and 45 lean controls). A complete physical examination was assessed, the z-score body mass index (BMI) was calculated, fat mass was assessed by bioelectric impedance analysis, and pubertal stage was assessed according to Tanner. Serum insulin and serum RBP4 levels were assayed. Obese and lean children differed for z-score BMI, fat mass, homeostasis model assessment of insulin resistance (HOMA-IR) and RBP4 levels. z-score BMI and HOMA-IR showed a direct correlation with RBP4 in the total population. When the subjects were divided in lean and obese, this correlation was evident only in obese (r2: 0.2; p=0.009 and r2: 0.2; p=0.01), but not in lean subjects (r2: 0.09; p=0.1 and r2: 0.03; p=0.4). Both in obese and lean HOMA-IR values were higher in pubertal subjects than in pre-pubertal (p<0.001), while serum RBP4 levels were similar in pubertal and in pre-pubertal subjects (>0.1). We conclude that RBP4 is correlated with adiposity and insulin resistance in obese children, but it is not involved in the insulin resistance occurring during puberty.