The Role of Obesity-associated Loci Identified in Genome-wide Association Studies in the Determination of Pediatric BMI

Authors

  • Jianhua Zhao,

    1. Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Jonathan P. Bradfield,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Mingyao Li,

    1. Department of Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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  • Kai Wang,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Haitao Zhang,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Cecilia E. Kim,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Kiran Annaiah,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Joseph T. Glessner,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Kelly Thomas,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Maria Garris,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Edward C. Frackelton,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • F. George Otieno,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Julie L. Shaner,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Ryan M. Smith,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Rosetta M. Chiavacci,

    1. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Robert I. Berkowitz,

    1. Behavioral Health Center and Department of Child and Adolescent Psychiatry, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
    2. Center for Weight and Eating Disorders, Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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  • Hakon Hakonarson,

    Corresponding author
    1. Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
    2. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
    3. Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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  • Struan F.A. Grant

    Corresponding author
    1. Division of Human Genetics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
    2. Center for Applied Genomics, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
    3. Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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  • The first two authors contributed equally to this work.

(grants@chop.edu)

(hakonarson@chop.edu)

Abstract

The prevalence of obesity in children and adults in the United States has increased dramatically over the past decade. Besides environmental factors, genetic factors are known to play an important role in the pathogenesis of obesity. A number of genetic determinants of adult BMI have already been established through genome-wide association (GWA) studies. In this study, we examined 25 single-nucleotide polymorphisms (SNPs) corresponding to 13 previously reported genomic loci in 6,078 children with measures of BMI. Fifteen of these SNPs yielded at least nominally significant association to BMI, representing nine different loci including INSIG2, FTO, MC4R, TMEM18, GNPDA2, NEGR1, BDNF, KCTD15, and 1q25. Other loci revealed no evidence for association, namely at MTCH2, SH2B1, 12q13, and 3q27. For the 15 associated variants, the genotype score explained 1.12% of the total variation for BMI z-score. We conclude that among 13 loci that have been reported to associate with adult BMI, at least nine also contribute to the determination of BMI in childhood as demonstrated by their associations in our pediatric cohort.

In the past 3 years, 13 genetic loci have been implicated for BMI from the outcomes of genome-wide association (GWA) studies primarily in adults. Insulin-induced gene 2 (INSIG2) was the first locus to be reported by this method to have a role in obesity (1) but replication attempts have yielded inconsistent outcomes (2,3,4,5,6). The second reported locus, the fat mass- and obesity-associated gene (FTO) (7), has been more robustly observed by others (8,9,10,11). Subsequent larger studies have uncovered 11 additional genes (12,13,14), first melanocortin 4 receptor (MC4R) from a multicenter meta-analysis (12), then the GIANT consortium revealed six more genes (transmembrane protein 18 (TMEM18), potassium channel tetramerization domain containing 15 (KCTD15), glucosamine-6-phosphate deaminase 2 (GNPDA2), SH2B adaptor protein 1 (SH2B1), mitochondrial carrier 2 (MTCH2), and neuronal growth regulator 1 (NEGR1)) (14), five of which were confirmed in the GWA study reported from Iceland (but not GNPDA2 due to an unavailable proxy single-nucleotide polymorphism (SNP)), who also uncovered and reported loci on 1q25, 3q27, and 12q13 (ref. 13) and verified association with the brain-derived neurotrophic factor (BDNF) gene (15).

In this study, we aimed at examining these findings in a large pediatric cohort with BMI measures and to determine the relative impact of these variants in childhood. For this purpose, we leveraged genotyping data from our ongoing GWA study of BMI variation in children. The 25 SNPs corresponding to the 13 previously reported obesity loci were investigated with respect to their association to normalized pediatric BMI (Table 1; also Supplementary Table S1 for analyses by age categories).

Table 1.  Quantitative association results for the candidate loci in the European-American BMI cohort (n = 6,078), sorted by chromosomal location
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In summary, 15 of these SNPs yielded at least nominally significant association to BMI (P < 0.05), representing nine different loci with the same direction of effect as previously reported. Of these nine loci, variants at the FTO locus yielded the strongest association with P < 10−4, namely rs8044769 and rs3751812 (P = 7.26 × 10−5 and 9.68 × 10−5, respectively); in addition, this locus also yielded association with rs8050136 and rs7190492 (P = 1.40 × 10−4 and 0.021, respectively) but not with rs6499640.

With a similar magnitude of association to FTO was TMEM18, with rs2867125 yielding a P = 9.72 × 10−5, together with almost as strongly associated SNPs, rs7561317 and rs4854344 (P = 1.02 × 10−4 and 1.52 × 10−4, respectively). Indeed, it is interesting to note that the two adult cohorts that uncovered TMEM18 in obesity also showed it to be second only in significance to FTO (13,14). The third most significantly associated locus was at GNPDA2, (rs13130484; P = 1.32 × 10−4).

Overall, in addition to FTO, TMEM18, and GNPDA2, we found evidence for association at the INSIG2, MC4R, NEGR1, 1q25, BDNF, and KCTD15 loci. One could argue that we have carried out multiple testing in our BMI cohort for these previously reported SNPs, albeit at a number of magnitudes less than for a full GWA study. If we were to apply the strictest correction, i.e., the Bonferroni correction based on 25 SNPs, then FTO, TMEM18, GNPDA2, and MC4R would still be considered significant. This is very much in line with the observations made with the pediatric cohort utilized by Willer et al. (14); however the one exception is that we do not observe strong association with KCTD15.

It was also observed that SNPs residing at the 12q13, 3q27, MTCH2, and SH2B1 loci did not reveal any evidence of association with BMI in our pediatric cohort.

The positive results for FTO and MC4R come as no surprise as we have previously reported their association with the Centers for Disease Control and Prevention–defined 95th percentile of BMI, i.e., obesity, in our pediatric cohort, but limited to ages 2–18 years old (8,16). In this study, where we utilized BMI z-score on all children 0–18 years old, it is satisfying that they continue to show association at a similar magnitude.

One of the more notable results is the positive association with INSIG2. This association with pediatric BMI, albeit at just the nominal level, will further add to the debate on the relevance of INSIG2 in BMI determination.

For the loci we did not observe any evidence for association for at all may be due to power issues, but could also indicate that they have a less pronounced role in a pediatric setting. Indeed, many of the newly uncovered genes have been implicated in neurological functions, genes which may be more important in BMI determination in adults rather than in children, where other more direct metabolic genes could play a more important role.

Finally, we investigated the 15 significant SNPs further by testing for association between BMI z-score and the genotype score by summing the number of BMI increasing alleles across all these SNPs. The resulting P value for the genotype score was 2.53 × 10−16 (Figure 1). The genotype score explains 1.12% of the total variation for BMI z-score. We also tested pair-wise interactions between the 15 significant SNPs, but none of the interaction effects were significant (Supplementary Table S2), suggesting that these 15 SNPs act additively on the pediatric BMI z-score. As such, we did observe a cumulative effect but not as striking as reported by the GIANT consortium in their adult cohorts (14).

Figure 1.

Scatter plot for association between BMI z-score and the genotype score by summing the number of BMI increasing alleles across all 15 BMI-associated SNPs.

From this analysis, it is clear that a number of loci previously reported from GWA analyses of adult BMI and/or obesity also play a role in our phenotype of interest. Although these recently discovered loci unveil several new biomolecular pathways not previously associated with obesity, it is important to note that these well-established genetic associations with obesity explain very little of the genetic risk for this pediatric phenotype, suggesting the existence of additional loci whose number and effect size remain unknown. Once our GWA study is complete, we will have the opportunity to look for other variants in the genome that are associated with BMI in childhood.

Research Methods and Procedures

Study Subjects

All subjects were consecutively recruited from the Greater Philadelphia area from 2006 to 2008 at the Children's Hospital of Philadelphia. Our study cohort consisted of 6,078 children of European ancestry with BMI information. All subjects were biologically unrelated and were aged between 0 and 18 years. This study was approved by the Institutional Review Board of the Children's Hospital of Philadelphia. Parental informed consent was given for each study participant for both the blood collection and subsequent genotyping.

Genotyping

We performed high throughput genome-wide SNP genotyping using either the Illumina Infinium II HumanHap550 or Human 610 BeadChip (Illumina, San Diego, CA) technology in the same manner as our center has reported previously (17). The SNPs analyzed survived the filtering of the genome-wide data set for SNPs with call rates <95%, minor allele frequency <1%, missing rate per person <2%, and Hardy–Weinberg equilibrium P < 10−5.

Loci described from GWA studies published to date have been found using either the Affymetrix or Illumina platform. In the event, a locus was reported using both the Illumina and Affymetrix arrays, we used the SNPs present on the Illumina array. In the event of a signal only being described on the Affymetrix array, we either already had that SNP on our Illumina array or we identified and used the best surrogate SNP available. As such, as rs7566605 at INSIG2 and rs10938397 at GNPDA2 were not available on the BeadChip, we employed perfect surrogates for the association analysis, i.e., rs17047697 and rs13130484, respectively (r2 = 1). With rs7498665 at the SH2B1 locus, rs8049439 and rs4788102 are already in strong linkage disequilibrium with this SNP so no additional surrogate was selected.

Analysis

From our database of heights and weights for our multidimensional scaling determined whites, we eliminated BMI outliers using 2% cutoff for each age category in order to remove measurement error and syndromic forms of obesity. As BMI values vary widely across pediatric age groups, each BMI value was adjusted for age and sex then expressed as a z-score.

We queried the data for the indicated SNPs in our pediatric samples. All statistical analyses were carried out using the software package plink (18). By treating BMI as a quantitative trait, association analysis for each SNP was carried out using linear regression with the SNP included as an independent variable (coded as 0, 1, and 2).

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at http:www.nature.comoby

Acknowledgments

We thank all participating subjects and families. Hope Thomas, Kisha Harden, Andrew Hill, Kenya Fain, Crystal Johnson-Honesty, Cynthia Drummond, Shanell Harrison, and Sarah Wildrick provided expert assistance with genotyping or data collection and management. We also thank Smari Kristinsson, Larus Arni Hermannsson, and Asbjörn Krisbjörnsson of Raförninn ehf for their extensive software design and contribution. This research was financially supported by the Children's Hospital of Philadelphia. The study is supported in part by a Research Development Award from the Cotswold Foundation (H.H. and S.F.A.G.) and National Institutes of Health grant 1R01HD056465-01A1.

Disclosure

The authors declared no conflict of interest.

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