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The Association between the FTO Gene and Fat Mass in Humans Develops by the Postnatal Age of Two Weeks

Diabetes, Endocrinology and Nutrition Unit (A.L.-B.), Dr. Josep Trueta Hospital, 17007 Girona, Spain; Department of Paediatrics (C.J.P., D.B.D.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; Endocrinology Unit (M.D., G.S., L.I.), Sant Joan de Déu Children’s Hospital, University of Barcelona, 08950 Esplugues, Barcelona, Spain; and Department of Woman and Child (F.d.Z.), University of Leuven, 3000 Leuven, Belgium

Address all correspondence and requests for reprints to: Lourdes Ibáñez, M.D., Ph.D., Endocrinology Unit, Hospital Sant Joan de Déu, University of Barcelona, Passeig de Sant Joan de Déu, 2, 08950 Esplugues, Barcelona, Spain. E-mail: libanez{at}hsjdbcn.org.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Objective: Little is known about the genetic determinants of fat mass around birth. We hypothesized that the common rs9939609 single-nucleotide polymorphism (SNP) in FTO is associated with fat mass and metabolic parameters in neonates.

Design: We conducted a cross-sectional, hospital-based study.

Patients: Patients included 234 full-term, healthy newborns [122 girls and 112 boys; gestational age (mean, range), 39.0 (37.0–42.0) wk; birth weight, 3.2 (1.9–4.2) kg].

Methods: Cord-blood insulin, IGF-I, IGF-binding protein-1, adiponectin, and visfatin were measured by specific immunoassays. Body composition was assessed by dual-energy x-ray absorptiometry at about 13 d (range, 9–20 d). Genotyping of rs9939609 was achieved by restriction fragment length polymorphism analysis.

Results: The rs9939609 SNP in FTO was not associated with birth weight; however, it was associated with serum visfatin (P < 0.001), with weight and ponderal index at age 2 wk (P < 0.05), and with total, truncal, and abdominal fat (P < 0.05 to P = 0.01), so that AA homozygotes had 37% higher plasma visfatin concentration and 17, 20, and 17% higher total, truncal, and abdominal fat mass, respectively, than T-carrier neonates.

Conclusion: Our findings support a role of the common rs9939609 SNP in FTO gene in the early stages of fat accretion in humans and disclose novel associations between this SNP and both serum visfatin and abdominal fat mass in neonates.

	Introduction

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The phenotype of metabolic syndrome is known to be modulated by prenatal and postnatal growth patterns (1). Among the latter, rapid increase in weight during the first months of life has been related to increased risk for metabolic syndrome (2). Genetics may influence the rate of fat accretion early in life (3). However, little is known about the genetic determinants of body adiposity around birth.

Common single-nucleotide polymorphisms (SNPs) in the recently described fat mass- and obesity-associated (FTO) gene (4) have been related to both body weight and fat mass in humans (5, 6, 7). FTO is highly expressed in human hypothalamus and in pituitary and adrenal glands, suggesting a potential role for this gene in the regulation of body weight (4, 5). In mice, the FTO gene is expressed in the arcuate nucleus where it can regulate energy balance (8). In population-based studies, homozygotes for the rs9939609 variant show increased body weight and fat mass already in prepuberty (5).

We tested the hypothesis that the rs9939609 SNP in FTO is associated with fat mass and metabolic parameters in neonates. Our findings disclose an association between this common SNP in FTO, serum visfatin, and both total fat mass and abdominal fat in infants as early as at age 2 wk.

	Subjects and Methods

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Subjects

As shown in Fig. 1, the study population consisted of 234 full-term, healthy newborns [122 girls and 112 boys; gestational age (mean, range), 39.0 (37.0–42.0) wk; birth weight, 3.2 (1.9–4.2) kg; birth length, 49.0 (43.0–54.0) cm], recruited at the Neonatal Unit of the Obstetrics and Gynecology Department of the Hospital Sant Joan de Déu, Barcelona, among those consecutively born between May 2006 and February 2007.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Flow chart for recruitment and selection of the study population. The availability of cord blood samples was exclusively dependent on study logistics (personnel availability).

Exclusion criteria were 1) maternal use of alcohol, drugs, or illicit substances; 2) prematurity; 3) major congenital or chromosomal anomalies; 4) arterial blood pH less than 7; and 5) 10-min Apgar score of less than 7.

The newborns included in the study did not differ in terms of clinical or pregnancy outcomes from those whose families refused to participate in the study.

Gestational age at delivery was calculated according to the last menstrual period and confirmed by ultrasound examination during the second trimester (20 wk of gestation). Cigarette consumption during pregnancy was categorized as nonsmokers during pregnancy (n = 209) or active smokers (n = 25).

Study protocol and measurements

Venous cord blood (10 ml) was collected and processed as described (9).

All children had weight and supine length measured at birth, by the same physician. Length (centimeters) was measured with a standardized plastic length board. The mean of three measurements was used in the analysis. Weight (grams) was measured to the nearest 10 g using a standard beam balance (Seca, Hamburg, Germany).

Body composition was assessed by dual-energy x-ray absorptiometry at approximately 13 d (range, 9–20 d), with a Lunar Prodigy coupled to specific pediatric Lunar software (version 3.4/3.5; Lunar Corp., Madison, WI), as described (10), appropriately adapted by the manufacturer for measurements in newborns and babies. The instrument underwent daily quality assessment and was calibrated against a water phantom weekly. The measurements were obtained within 30 min before the next feeding, without sedation, and after allowing for spontaneous sleep. The baby was undressed completely, swaddled in a standard towel, placed on a waterproof sheet in supine position on the scanner, and kept in position using thin strips to gently hold the legs and buttocks. The room temperature was between 24 and 25 C, and a radiant source of heat was placed near the child. The average duration of each measurement was about 10 min; all scans were performed and processed by the same operator.

Absolute (grams) whole-body fat and lean mass were assessed as well as fat content in the abdominal region, which was defined as the area between the dome of the diaphragm (cephalad limit) and the top of the great trochanter (caudal limit). Total irradiation dose per assessment was 0.1 mSievert (about 1/10 of the usual radiation received with a regular x-ray of the thorax). Coefficients of variation for scanning precision were less than 3% for fat and lean body mass (10, 11).

Genetic analysis

Genomic DNA was purified from cord blood leukocytes using QIAGEN QIAmpBlood kits. Successful genotyping was achieved in 96% of subjects. Recall rate was higher than 99%.

The FTO SNP (rs9939609) was genotyped by mismatch PCR (12) and restriction fragment length polymorphism analyses. Genomic DNA (20 ng) was incubated in a 10-µl solution containing 1x NH₄ buffer, 2.5 mmol/liter magnesium, 200 µmol/liter each dNTP, 20 pmol forward (5'-AACTGGCTCTTGAATGAAATAGGATTCAGA-3') and reverse (5'-AGAGTAACAGAGACTATCCAAGTGCAGTAC-3') oligonucleotide primers, and 0.5 U Taq DNA polymerase (Bioline Ltd., London, UK). The PCR mix was incubated at 94 C for 5 min followed by 20 cycles of 94 C for 45 sec, 61 C for 45 sec (dropping 0.5 C per cycle), and 72 C for 45 sec. After this, the PCR mix was incubated for 15 cycles of 94 C for 45 sec, 51 C for 45 sec, and 72 C for 45 sec, followed by a final incubation at 72 C for 10 min. This was then incubated at 37 C for 16 h with 2 U ScaI (New England Biolabs, Hitchin, UK). Upon running the final products on a 3% agarose gel, the T allele produced a 182-bp band and the A allele produced 154- and 28-bp bands.

Hormone assays

Serum glucose, insulin, IGF-I, IGF-binding protein-1 (IGFBP-1), total adiponectin, and visfatin were measured as described (9).

Statistics and ethics

The protocol and collection of normal blood was approved by the Institutional Review Board of Barcelona University, Hospital of Sant Joan de Déu, and informed consent was obtained from parents.

Statistical analyses were performed using SPSS for Windows (version 12.0; SPSS Inc., Chicago, IL). Continuous data are expressed as geometric mean ± 95% confidence interval. Quantitative phenotypic data were compared across genotypes by one-way ANOVA followed by general linear models to adjust for possible effect modifiers, such as body weight, gender, parity, and age at assessment of body composition. P < 0.05 was considered statistically significant throughout. In this study, there was 80% statistical power to be able to detect a difference of 0.26 SD of the dependent variable across genotypes.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Genotype frequencies for the rs9939609 SNP in FTO were 85 (37.8%), 103 (45.8%), and 37 (16.4%), for T/T, T/A, and A/A genotypes, respectively. These frequencies were in Hardy-Weinberg equilibrium (P = 0.82).

The common rs9939609 SNP in FTO was associated with cord blood visfatin, with weight and ponderal index at age 2 wk, with weight change over the first 2 wk of life, and with total, truncal, and abdominal fat also at age 2 wk, so that homozygous newborns for the at-risk allele (AA) had 37% higher cord blood visfatin concentration and 17, 20, and 17% higher total, truncal, and abdominal fat mass at age 2 wk, respectively, than newborns carrying the T allele (Table 1). These results remained unchanged when further adjusting for the type of feeding [breastfed (n = 143; 64%) vs. bottle-fed (n = 82; 36%); data not shown].

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The FTO gene has been recently reported to be a major candidate for human obesity (5). Although the mechanisms by which variants in FTO lead to obesity are unknown, three independent studies have shown strong associations between a cluster of SNPs in the first intron of FTO, where the rs9939609 SNP lies, and obesity-related traits (5, 6, 7). This effect is seen as early as in children aged 7–9 yr, in whom the minor allele was associated with both body weight and fat mass, as measured by dual-energy x-ray absorptiometry scan (5). Additionally, in healthy women carrying the obesity-protective allele in homozygosis (TT), adipocyte lipolytic activity was increased both in vivo and in vitro, independently of the body mass index, suggesting that, at least in part, the FTO gene may regulate body fat through lipolysis (13).

Our findings not only extend the association with body weight to the neonatal period but also indicate, for the first time, an association between the rs9939609 SNP and both serum visfatin and abdominal fat mass at age approximately 13 d. In line with the study by Frayling et al. (5), however, we failed to show an association between rs9939609 SNP and birth weight. Taken together, these findings suggest an early postnatal effect of the FTO gene on fat mass and abdominal fat in humans that is associated with increased weight gain over the first 2 wk of life.

The potential interaction between the rs9939609 SNP and cord visfatin is intriguing. This adipocytokine is highly, but not exclusively, expressed in visceral fat mass, promotes adipogenesis, and has insulinomimetic properties; however, its role in human physiology remains largely unknown (14). For example, cord visfatin has been found to have no influence or to be related to birth size in uncomplicated pregnancies (9, 15). Recent observations indicate that the FTO gene is expressed in both human sc and omental fat tissue, with increased expression in obesity (13). The protein is also localized to the cell nucleus where it can demethylate DNA and consequently regulate gene expression (8). It is thus tempting to speculate that FTO might regulate visfatin gene expression in human adipose tissue.

We acknowledge the small sample size and the fact that genetic associations are shown without correction for multiple comparisons. Our results, however, agree with both the direction and the extent of the association of the rs9939609 in FTO with fat mass in children aged 9 yr (5), which makes it unlikely for our results to be consistent with a type I error.

In conclusion, our findings disclose an association between a common SNP in FTO and serum visfatin and both fat mass and abdominal fat in neonates. These observations may have implications in the early stages of fat mass accretion in humans.

	Acknowledgments

 
We thank Dr. Lola Gómez-Roig for compiling pregnancy data and Montserrat Visa for performing the DNA extraction and hormone measurements.

	Footnotes

 
Supported by the Social Security Research Fund, Health Institute Carlos III, Spain (PI05/2405). L.I. is a Clinical Investigator of CIBERDEM CB07/08/0044 (FIS, Instituto de Salud Carlos III, Madrid, Spain). A.L.-B. is an Investigator of the Fund for Scientific Research "Ramon y Cajal" (Ministry of Education and Science, Spain). F.d.Z. is a Clinical Investigator of the Fund for Scientific Research (Flanders, Belgium).

Disclosure Statement: A.L.-B., C.J. P., M.D., G.S., F.d.Z., D.B.D, and L.I. have nothing to declare.

First Published Online February 5, 2008

Abbreviations: FTO, Fat mass- and obesity-associated; IGFBP-1, IGF-binding protein-1; SNP, single-nucleotide polymorphism.

Received October 22, 2007.

Accepted January 25, 2008.

	References

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