Insulin-Like Growth Factor 2/H19 Methylation at Birth and Risk of Overweight and Obesity in Children

Article · February 2012with39 Reads
DOI: 10.1016/j.jpeds.2012.01.015 · Source: PubMed
Abstract
To determine whether aberrant DNA methylation at differentially methylated regions (DMRs) regulating insulin-like growth factor 2 (IGF2) expression in umbilical cord blood is associated with overweight or obesity in a multiethnic cohort. Umbilical cord blood leukocytes of 204 infants born between 2005 and 2009 in Durham, North Carolina, were analyzed for DNA methylation at two IGF2 DMRs by using pyrosequencing. Anthropometric and feeding data were collected at age 1 year. Methylation differences were compared between children >85th percentile of the Centers for Disease Control and Prevention growth charts weight-for-age (WFA) and children ≤ 85th percentile of WFA at 1 year by using generalized linear models, adjusting for post-natal caloric intake, maternal cigarette smoking, and race/ethnicity. The methylation percentages at the H19 imprint center DMR was higher in infants with WFA >85th percentile (62.7%; 95% CI, 59.9%-65.5%) than in infants with WFA ≤ 85th percentile (59.3%; 95% CI, 58.2%-60.3%; P = .02). At the intragenic IGF2 DMR, methylation levels were comparable between infants with WFA ≤ 85th percentile and infants with WFA >85th percentile. Our findings suggest that IGF2 plasticity may be mechanistically important in early childhood overweight or obese status. If confirmed in larger studies, these findings suggest aberrant DNA methylation at sequences regulating imprinted genes may be useful identifiers of children at risk for the development of early obesity.
Insulin-Like Growth Factor 2/H19 Methylation at Birth and Risk
of Overweight and Obesity in Children
Ellen Perkins, MD
1
, Susan K. Murphy, PhD
2,3
, Amy P. Murtha, MD
4
, Joellen Schildkraut, PhD
1,5
, Randy L. Jirtle, PhD
6
,
Wendy Demark-Wahnefried, PhD
8
, Michele R. Forman, PhD
9
, Joanne Kurtzberg, MD
7
, Francine Overcash, MPH
1,5
,
Zhiqing Huang, MD, PhD
2
, and Cathrine Hoyo, PhD
1
Objective To determine whether aberrant DNA methylation at differentially methylated regions (DMRs) regulating
insulin-like growth factor 2 (IGF2) expression in umbilical cord blood is associated with overweight or obesity in
a multiethnic cohort.
Study design Umbilical cord blood leukocytes of 204 infants born between 2005 and 2009 in Durham, North
Carolina, were analyzed for DNA methylation at two IGF2 DMRs by using pyrosequencing. Anthropometric and
feeding data were collected at age 1 year. Methylation differences were compared between children >85th percen-
tile of the Centers for Disease Control and Prevention growth charts weight-for-age (WFA) and children #85th per-
centile of WFA at 1 year by using generalized linear models, adjusting for post-natal caloric intake, maternal
cigarette smoking, and race/ethnicity.
Results The methylation percentages at the H19 imprint center DMR was higher in infants with WFA >85th per-
centile (62.7%; 95% CI, 59.9%-65.5%) than in infants with WFA #85th percentile (59.3%; 95% CI, 58.2%-
60.3%; P= .02). At the intragenic IGF2 DMR, methylation levels were comparable between infants with WFA
#85th percentile and infants with WFA >85th percentile.
Conclusions Our findings suggest that IGF2 plasticity may be mechanistically important in early childhood over-
weight or obese status. If confirmed in larger studies, these findings suggest aberrant DNA methylation at se-
quences regulating imprinted genes may be useful identifiers of children at risk for the development of early
obesity. (J Pediatr 2012;161:31-9).
The prevalence of obesity in children <5 years old has more than doubled since the 1990s, affecting 1 in 5 children in the
United States, with minority populations disproportionately affected.
1,2
Early childhood obesity and excessive infant
weight gain have been associated with higher blood pressure
3
and wheezing
4
in childhood and obesity and metabolic
and cardiovascular diseases in adulthood.
5
Childhood obesity may be an early adaptive response, hypothesized to be largely
driven by epigenetic mechanisms that guide expression of genes involved in energy balance, culminating in gene expression
profiles that predispose children to overweight and obesity.
A commonly studied epigenetic mechanism is DNA methylation, in part because of its stability in conditions in which hu-
man specimens are collected. Animal evidence from the last decade indicates that DNA methylation alterations at susceptible
loci link the early environment to obesity in later life. The monoallelic expression of imprinted genes-a class of genes that is
over-selected for growth effectors
6
is regulated (and dysregulated) by DNA methylation at differentially methylated regions
(DMRs). Aberrant methylation at these DMRs has been associated with aberrant changes in gene expression. Because imprinted
genes occur in clusters throughout the genome
7
and their regulation may be networked,
6
a single DMR can regulate the expres-
sion of several genes; suggesting aberrant methylation at a single DMR can affect the expression of several of these growth
effectors. The most studied imprinted gene is insulin-like growth factor 2 (IGF2). IGF2 is a paternally expressed imprinted
gene that encodes a potent mitogenic growth factor that plays a critical role in placental and fetal development. Aberrant
DNA methylation at the IGF2 DMRs has been associated with increased gene ex-
pression, and presumably, circulating IGF2 levels and risk of overweight status,
obesity, and overgrowth disorders.
8
Numerous epidemiological studies have reported a small but consistently
lower risk of rapid growth and obesity in breastfed children. Although epigenetic
mechanisms have been proposed,
9
the mechanism by which breastfeeding
From the
1
Department of Community and Family
Medicine,
2
Department of Obstetrics and Gynecology,
Division of Gynecologic Oncology,
3
Department of
Pathology,
4
Department of Obstetrics and Gynecology,
Division of Maternal-Fetal Medicine,
5
Program of Cancer
Detection, Prevention, and Control,
6
Department of
Radiation Oncology, and
7
Department of Pediatrics,
Duke University, Durham, NC;
8
Department of Nutrition
Sciences, University of Alabama at Birmingham,
Birmingham, AL; and
9
Department of Epidemiology, MD
Anderson Cancer Center, Houston, TX
Supported by the National Institutes of Health (grants
ES016772, R21ES014947, K01CA104517,
R01DK085173, and R01 ES015165). The authors declare
no conflicts of interest.
0022-3476/$ - see front matter. Copyright ª2012 Mosby Inc.
All rights reserved. 10.1016/j.jpeds.2012.01.015
BMI Body mass index
CpG Cytosine-phosphate-Guanine
DMR Differentially methylated region
IGF2 Insulin-like growth factor 2
NEST Newborn Epigenetics Study
WFA Weight-for-age
31
confers a lower risk of childhood obesity remains unknown.
Because breastfeeding varies by race/ethnicity, we evaluated
whether aberrant DNA methylation at two IGF2 DMRs at
birth increases the risk of overweight and obesity status in
early childhood, and this association may vary by ethnic
group and breastfeeding.
Methods
Study participants were children born to women who sought
obstetric care at Duke Obstetrics and Durham Regional Hos-
pital (Durham, North Carolina) between 2005 and 2009, as
part of the Newborn Epigenetics Study (NEST). NEST is
a prospective, perinatal epidemiologic study aimed at deter-
mining how the in utero environment influences epigenetic
profiles and phenotypes in children. Procedures for partici-
pant enrollment have been detailed elsewhere.
10
In brief, be-
tween 2005 and 2009, women who attended prenatal care at
Duke’s Maternal Fetal Medicine and one affiliated clinic and
intended to use Duke or Durham Regional Hospitals for
their obstetrics care were enrolled in the study. Inclusion cri-
teria were age $18 years and English speaking. We excluded
women who planned to give offspring up for adoption and
women who were human immunodeficiency virus-positive,
because the effect of anti-retroviral medications on the
methylation profile is still unknown. Most women (>50%)
were from Durham County, North Carolina, although the
catchment area also included contiguous counties.
Between 2005 and 2009, 940 of the 1101 women (85%) ap-
proached consented to participate and were enrolled in
NEST. Methylation analyses were performed on cord blood
leukocyte DNA of the first 438 neonates (46%). The final
sample includes the first 204 offspring who had methylation
data and had reached 1 year of age by August 2010 and whose
mothers completed a follow-up questionnaire. Follow-up is
ongoing. The distribution of factors that may affect over-
weight status or obesity, including maternal age (P= .54),
education (P= .94), race/ethnicity (P= .43), and sex of infant
(P= .80) were comparable between the 940 infant-mother
pairs enrolled and the 438 infant-mother pairs with DNA
methylation data. These factors were also comparable be-
tween the first 204 infants in whom follow-up data have
been collected to date and the 428 in whom methylation anal-
yses were conducted. Most questionnaires (78%) were com-
pleted via mailed survey, 16% were interviewer-administered
during a pediatric office visit, and 6% were telephone-admin-
istered.
To characterize the in utero environment, pregnant
women completed a questionnaire soliciting information
on sociodemographic characteristics, maternal lifestyle fac-
tors, and morbidity at recruitment. Women self-reported
maternal pre-pregnancy anthropometric measurements,
race, level of education, and cigarette smoking status. Self-
reported height and usual pre-pregnancy weight were used
to calculate maternal body mass index (BMI). At delivery,
medical records were abstracted to obtain maternal age at
delivery and parturition data, including morbidity during
pregnancy, mode of delivery, infection in labor, gestational
age at birth, and infant data including birth-weight, head
circumference, and Apgar score. Approximately 1 year after
the child’s birth, a 1-year questionnaire was completed to ob-
tain data on anthropometric measures, temperament, and
use of childcare. We also estimated the infant’s caloric intake
from a single 24-hour dietary recall by using the University of
Minnesota’s Nutrition Data System for Research (2008).
Although a single 24-hour recall cannot adequately reflect to-
tal energy intake in the first year of life, we used this informa-
tion as an indicator to adjust for, in the investigation of IGF2
DMR methylation at birth and overweight and obesity in
early childhood.
To estimate breastfeeding status, we used additional die-
tary information collected in the 1-year questionnaire, which
asked mothers to report—for each of the first 12 months of
life—whether their child was fed breast milk, cow’s milk for-
mula, and/or soy milk. Specifically, mothers were asked,
“How did you feed your baby during his/her first year?
(Please go month by month)”; for each month, mothers re-
sponded either “yes” or “no” individually to breast milk, for-
mula, and soy milk. Questionnaires were completed when the
child was between 12 and 29 months of age. The median age
of the child when the first follow-up questionnaire was
mailed was 14 months, and the median time to returning
the questionnaire was 1 month.
To verify the accuracy of the infant weights reported by
mothers in the questionnaire, anthropometric measurements
at child age 1 year were abstracted from the medical records
of 72 infants who had the data available within 1 month of
receipt of their 1-year questionnaire. The Pearson correlation
co-efficient between anthropometric measurements ab-
stracted from the medical record and weights reported by
mothers was 0.94 (P< .0001), suggesting that mothers accu-
rately reported their offspring’s weight.
At delivery, cord blood specimens were collected in ethyl-
enediaminetetraacetic acid-treated tubes within minutes of
delivery. Specimens were processed to obtain plasma and
buffy coat for DNA extraction (Qiagen, Valencia, California);
samples were stored at 80C until processed. DNA was ex-
tracted by using Puregene reagents according to the manufac-
turer’s protocol (Qiagen).
DNA methylation from leukocytes of umbilical cord blood
samples is generally used as a surrogate measure of genomic
stability in the study of prenatal exposures and epigenetic re-
sponse to these exposures, because they contribute to long-
term health in humans.
11-16
Genomic DNA was modified
by treatment with sodium bisulfite by using high throughput
methods as previously described.
17
Bisulfite treatment of de-
natured DNA converts unmethylated cytosines to uracils and
leaves methylated cytosines unchanged. Pyrosequencing was
performed by using a Biotage Pyromark MD pyrosequencing
instrument (Qiagen). We evaluated two regions, including 3
cytosine-phosphate-Guanine (CpG) sites comprising the in-
tragenic IGF2 DMR, upstream of exon 3 (chr11p15.5, site 1:
2 109 519; site 2: 2 109 516; and site 3: 2 109 500; NCBI
Human Genome Build 37.1; National Institutes of Health,
THE JOURNAL OF PEDIATRICS www.jpeds.com Vol. 161, No. 1
32 Perkins et al
Bethesda, Maryland) and 4 CpG sites, hereafter referred to as
the H19 DMR, within a sequence motif that binds the
CCCTC-binding factor zinc finger protein within the IGF2/
H19 imprint center (chr11p15.5, site 1: 1 964 261; site 2:
1 964 259; site 3: 1 964 257; and site 4: 1 964 254; NCBI Hu-
man Genome Build 37.1). With the same CpGs evaluated
here as estimates of DMR methylation, hypermethylation at
the intergenic DMR upstream of H19
18,19
(referred to as
the H19 DMR) and CpG hypomethylation at the intragenic
DMR upstream of IGF2 exon 3 (referred to as the IGF2
DMR)
19
have been associated with increased IGF2 transcrip-
tional activity and loss of imprinting and had been found
dysregulated in multiple obesity-related cancer types.
18,20-26
These sequences have been described extensively
27
and are
available at National Center for Biotechnology Information.
Pyrosequencing assays were designed with PSQ Assay Design
Software (Biotage AB, Uppsala, Sweden). Bisulfite conver-
sion efficiency for each specimen was confirmed to be
>95.5% through evaluation of non-CpG cytosines within
the region sequenced. Average methylation at each CpG
site was calculated from duplicate runs. Methylation data
were 98% complete. The study protocol was approved by
the institutional review boards of Duke University, Univer-
sity of Texas MD Anderson Cancer Center, and National
Cancer Institute.
Obesity and overweight status in early childhood are often
estimated with weight adjusted for length. In the absence of
length measurements, we estimated rapid early growth by ap-
proximately 1 year of age for each infant. We used children’s
weight at the most recent doctor’s visit (reported by mothers)
and age in months at date of return of the 1-year question-
naire to compute sex-specific weight-for-age (WFA) percen-
tile rank, on the basis of the Centers for Disease Control and
Prevention growth charts expected sex-specific smoothed for
WFA.
28
Because WFA was not normally distributed, infants
were dichotomized into overweight or obese status, when
WFA was >85th percentile or non-obese or overweight
when WFA was #85th percentile, although a cutoff point
at the WFA >95th percentile was also considered. Also con-
sidered was weight gain, defined as the difference between
birth weight and weight at follow-up interview, with cutoff
points at >85th and >95th percentiles, and weight for length
in the subset of infants in whom length data were available at
age approximately 1 year. To test for potential bias associated
with the length of time to return the questionnaire in infants
who were breastfed and infants who were not, we used ttests
to evaluate the association between time to return the ques-
tionnaire and both breastfeeding status (P= .4) and over-
weight or obesity status (P= .5).
For each of the 3 CpGs at the IGF2 DMR and 4 CpGs at the
H19 DMR, the distribution of methylation percentages ob-
tained from Pyrosequencing data were evaluated for normal-
ity by using Kolmogorov-Smirnov tests, and we found no
evidence to suggest the values were not normally distributed.
To determine whether a single mean could be used to esti-
mate methylation fractions, we computed correlation co-
efficients in CpG dinucleotide percents for each DMR. The
correlation co-efficient for the 4 CpG dinucleotides at the in-
tergenic H19 DMR ranged from 93% to 96%, and the corre-
lation co-efficients in the methylation fractions at the 3 CpG
dinucluiotides at the IGF2 DMR ranged from 81% to 83%.
On the basis of these high correlations, we used a single
(average) mean for each DMR, to compare DMR methyla-
tion fractions between infants with a WFA >85th percentile
and WFA #85th percentile in early childhood. Despite these
high correlations in CpGs at each DMRs, we also repeated
these analyses with mixed linear models to allow for unstruc-
tured model entry of individual CpGs.
We examined two measures of breastfeeding status. First,
we dichotomized all participants into “never” (“no” to breast
milk for all 12 months) versus “ever” breastfed (“yes” to
breast milk for any of the 12 months). We also evaluated
the effect of exclusive breastfeeding in the first 3 months by
categorizing children in 3 categories: “never breastfed”
(“no” breast milk for all 3 months), “mixed breastfeeding
and formula” (“yes” to both breast milk and formula in
any of the first 3 months), and “exclusively breastfed”
(“yes” to breast milk for all 3 months and “no” to formula
and soy milk for all 3 months). Self-reported maternal
race/ethnicity was used as a proxy for the race/ethnicity of
the infant.
Statistical Analyses
c
2
tests were used to compare maternal and offspring charac-
teristics in relation to overweight status or obesity (dichoto-
mized at WFA >85th percentile of the Centers for Disease
Control and Prevention growth charts). Least squares mean
DNA methylation percentages at the H19 and at the IGF2
DMRs were then compared between children with WFA
>85th percentile and children with WFA #85th percentile,
by using generalized linear models. Because early obesity
has been shown to differ by race/ethnicity and breastfeeding
status in the general population, we also explored potential
effect modification of the associations between methylation
percentages and obesity in early childhood with stratified
analyses and also by computing and including in final models
cross-product terms for methylation fraction and race/
ethnicity and for methylation fraction and breastfeeding.
A cross-product term with a Pvalue <.10 was considered
to be statistically significant. Factors found significantly
associated with overweight status or obesity in Table I
were evaluated for potential confounding in these models,
and those significantly associated were retained. Factors
considered for potential confounding were maternal
pre-pregnancy BMI, prenatal morbidity, mode of delivery,
race/ethnicity, education, cigarette smoking, birth weight,
sex, and caloric intake. To minimize residual confounding
by caloric input, birth weight, and maternal BMI, these
factors were entered in statistical models as continuous
variables. Maternal morbidity (any versus none), mode of
delivery (vaginal versus cesarean delivery), race/ethnicity
(African-American, white, and other), sex (male versus
female), cigarette smoking (never smoked, stopped during
pregnancy, or quit before knowledge of pregnancy),
July 2012 ORIGINAL ARTICLES
Insulin-Like Growth Factor 2/H19 Methylation at Birth and Risk of Overweight and Obesity in Children 33
education (up to some college versus college or higher) were
entered as categorical variables.
These analyses were repeated with multiple logistic regres-
sion models examining the association between aberrant
DNA methylation and obesity in early childhood, stratifying
by race/ethnicity and breastfeeding status, while adjusting for
the same potential confounding factors. For these models,
methylation fractions were dichotomized in aberrant and
normal categories by using a cutoff point $75th percentile
for the H19 DMR, because hypermethylation at this DMR
has most frequently been associated with IGF2 deregulation.
Aberrant methylation for the IGF2 DMR was defined as
#25th percentile, because hypomethylation at this DMR
has been associated with IGF2 deregulation.
19
All statistical
analyses were conducted with SAS software version 9.2
(SAS Institute, Cary, North Carolina).
Table I. Characteristics of mothers and children, by percentile of child’s weight at age 1 year
Characteristic £85th percentile weight for age (n = 172) >85th percentile weight for age (n = 26) Pvalue*
Maternal race .10
African-American 64 (37.7%) 15 (57.7%)
White 97 (57.1%) 11 (42.3%)
All others 9 (5.3%) 0
Missing 2 0
Maternal level of education .32
Up to some college 88 (51.2%) 16 (61.5%)
College graduate or graduate school 84 (48.8%) 10 (38.5%)
Maternal BMI
Mean, kg/m
2
26.4 27.2 .66
Range, kg/m
2
16.3-57.0 18.9-43.9
BMI <25 94 (58.0%) 11 (44.0%) .40
BMI 25-30 31 (19.1%) 7 (28.0%)
BMI $30 37 (22.8%) 7 (28.0%)
Missing 10 1
Maternal self-reported health status .74
Excellent 40 (23.3%) 6 (23.1%)
Very good or good 118 (68.6%) 19 (73.1%)
Fair or poor 14 (8.1%) 1 (3.9%)
Maternal smoking .24
Never smoked 107 (62.2%) 12 (46.2%)
Smoked during pregnancy 51 (29.7%) 10 (38.5%)
Quit before knowledge of pregnancy 14 (8.1%) 4 (15.4%)
Mean maternal age at delivery, years
Range, years 30 29 .32
Gestational age at birth 18-49 18-42
Mean, weeks 38 39 .24
Range, weeks 26-42 34-41
<35 weeks 13 (7.6%) 1 (3.9%) .49
$35 weeks 159 (92.4%) 25 (96.2%)
Infant sex .10
Male 96 (55.8%) 10 (38.5%)
Female 76 (44.2%) 16 (61.5%)
Birth weight .13
Mean, g 3135.6 3347.6
Range, g 760.0-5160.0 2070.0-4510.0
Use of any childcare .87
No 87 (51.8%) 13 (50.0%)
Yes 81 (48.2%) 13 (50.0%)
Missing 4 0
Weight gain, pounds 14.5 19.6 <.01
Range, pounds 8.7-22.1 9.9-28.9
Mean daily caloric intake, Kcal 1061.2 1369.8 .002
Range, Kcal 187.9-2938.3 304.6-3998.9
Missing
Breastfeeding: first 12 months of life .83
Never breastfed 58 (33.9%) 8 (30.8%)
Ever breastfed 113 (66.1) 18 (69.2%)
Breastfeeding: first 3 months of life .34
z
Never breastfed 58 (33.7%) 8 (30.8%)
Mixed breast milk and formula 46 (26.7%) 4 (15.4%)
Exclusively breastfed 68 (39.5%) 14 (53.9%)
Prenatal morbidity
None 82 (47.7%) 10 (38.5%)
Any chronic disease
x
90 (52.3%) 16 (61.5%) .38
*Pvalue for association between characteristic and child’s WFA percentile. Calculated with ttest for continuous variables and with c
2
for categorical variables.
†Calculated with Fisher exact test.
zCalculated with Cochran-Armitage trend test.
xAny chronic disease includes gestational diabetes, asthma, hypertension, depression, diabetes mellitus, heart diseases, allergies, migraine headaches, epilepsy, anxiety, treated for cancer.
THE JOURNAL OF PEDIATRICS www.jpeds.com Vol. 161, No. 1
34 Perkins et al
Results
Table I summarizes the distribution of sociodemographic
characteristics, anthropometric measures, and lifestyle factors
of the mother-infant pairs. Thirteen percent of infants were
categorized as overweight or obese, on the basis of the WFA
cutoff point >85th percentile. Compared with children with
WFA #85th percentile, children with WFA >85th percentile
were more likely to be female (P= .10) and African-
American (P= .10) and have a larger birth weight (P=.13),
alargerweightgain(P< .01), and a higher caloric intake
during the first year of life (P< .002). Obese and overweight
children, however, were comparable with non-obese children
in maternal age at delivery, self-reported health status,
chronic morbidity, gestational age at birth, educational level,
maternal BMI before pregnancy, infant breastfeeding, and
use of childcare outside the home.
Of the 204 mothers, 66% (n = 133) reported ever breast-
feeding their offspring during the first year of life; these pro-
portions were comparable in overweight or obese children
(69%) and non-obese or overweight children (66%). During
the follow-up period, 8.4% of infants (n = 17) had received
a diagnosis of conditions that included eczema, seizure disor-
ders, cerebral palsy, and tonsillitis. All the 17 children except
two were breastfed, and only one child was obese or over-
weight. A combination of breast- and formula-feeding was
reported in 27% of non-obese children and 15% of obese
or overweight children, and exclusive breastfeeding for at
least 3 months was reported by 54% of mothers of obese or
overweight children, compared with 40% of non-obese chil-
dren (P= .34). Never breastfeeding was comparable in obese
or overweight children (31%) and non-obese children (34%).
In infants who were ever breastfed, the average duration of
breastfeeding was 6.1 months (SD, 4.5 months; P= .95).
Table II summarizes unadjusted CpG-specific and average
DNA methylation levels at the H19 and IGF2 DMRs at birth,
by the child’s obesity and overweight status at approximately
age 1 year. In all infants, the average methylation fraction was
61% at the H19 DMR (SD, 8%; IQR, 56%-63%) and 47% at
the IGF2 DMR (SD, 7%; IQR, 43%-51%). Unadjusted
average methylation fraction at the H19 DMR was 3.4%
higher in overweight and obese children compared with
children who were neither obese nor overweight (P= .03).
This methylation difference was similar at all CpGs
evaluated. Adjusting for maternal cigarette smoking, race/
ethnicity, and postnatal caloric intake did not materially
alter this methylation difference (2.9% higher in obese or
overweight children). Although birth weight and age at
follow-up may be causally related to early obesity because
intrauterine growth restriction may result in “catch up”
growth during the early post-natal period, additionally
adjusting for these factors and maternal pre-pregnancy
BMI, age at follow-up, mode of delivery, education, and
sex did not alter these findings (data not shown).
We explored the possibility that DNA methylation differ-
ences may vary by race and by ever-breastfeeding status by re-
peating the analyses, stratified by these factors (Table III). We
found no differences in methylation fractions of overweight or
obese children (59.6%; 95% CI, 56.2%-63.1%) compared with
children who were neither overweight nor obese (59.2%;
95% CI, 57.9%-60.5%) who were ever breastfed (P= .82).
However, in infants who were never breastfed, we noted an
8.4% (P= .01) methylation difference between overweight
and obese children (68.3%; 95% CI, 62.3%-74.2%) and
children who were neither overweight nor obese (59.8%;
95% CI, 57.6%-61.9%). However, the cross-product terms
for breastfeeding and H19 methylation in the model
adjusted for cigarette smoking and postnatal caloric intake
were not statistically significant (P= .26), although further
adjusting for race/ethnicity reduced the cross-product term
Pvalue (P= .20). Repeating these analyses with linear mixed
models to allow simultaneous entry of individual CpG
dinucleotide methylation fractions in statistical models did
not alter our findings (data not shown). Neither modeling
these differences as continuous in linear regression models
dichotomized at WFA >85th percentile nor WFA >95th
percentile materially altered our findings, although estimates
were less stable. Repeating these analyses with weight gain
dichotomized at >85th percentile as the outcome, further
adjusted for sex and age of offspring, revealed a 2.7%
(P= .19) higher methylation fraction at birth in children
with higher weight gain at age 1 year (data not shown). In
the subset of children with length data at age 1 year,
methylation fraction differences of a similar magnitude were
also observed between children with weight-for-length
>85th percentile and children with weight-for-length #85th
percentile (P= .19). After race/ethnicity stratification, we
also found that methylation fractions of African-American
children with WFA #85th percentile were comparable with
African-American children with WFA >85th percentile (P=
.46); however, white children with WFA >85th percentile
had somewhat higher methylation fractions at birth than
white children with WFA #85th percentile (P= .09).
However, the cross-product term for methylation fraction
Table II. Methylation fraction at H19 and IGF2 DMRs
at birth by percentile of child’s weight at age
approximately 1 year
DMR
£85th percentile
of WFA (n = 172)
>85th percentile
of WFA (n = 26)
P
value*
Mean
methylation
fraction 95% CI
Mean
methylation
fraction 95% CI
H19 DMR
Mean 59.3 58.2-60.3 62.7 59.9-65.5 .03
CG1 61.6 60.3-62.9 65.6 62.2-69.0 .03
CG2 57.8 56.7-58.9 59.8 56.8-62.8 .22
CG3 58.9 57.7-60.0 62.3 59.2-65.4 .04
CG4 59.0 57.8-60.2 63.1 60.0-66.3 .02
IGF2 DMR
Mean 47.5 46.6-48.4 47.2 44.8-49.7 .84
CG1 41.7 40.3-43.1 39.1 35.5-42.8 .19
CG2 50.9 49.8-51.9 50.4 47.7-53.2 .78
CG3 51.2 49.9-52.6 49.7 46.1-53.3 .43
*Pvalue comparing unadjusted least square means.
July 2012 ORIGINAL ARTICLES
Insulin-Like Growth Factor 2/H19 Methylation at Birth and Risk of Overweight and Obesity in Children 35
and race/ethnicity was associated with a Pvalue of .58.
We found no significant differences in IGF2 DMR
methylation fractions between overweight children and
obese children during the first year of life.
Because continuous data such as methylation fraction are
sensitive to extreme values, we further explored the relation-
ship among breastfeeding and race, in the association be-
tween aberrant methylation and subsequent obesity or
overweight status by repeating these analyses with logistic re-
gression models. Hypermethylation was defined as DNA
methylation fractions >75th percentile, and normal methyl-
ation was defined otherwise, at the H19 locus (Table IV).
We found that in all children, after adjusting for caloric
intake and cigarette smoking, the OR for overweight or
obese status associated with hypermethylation at the H19
DMR was 3.7 (95% CI, 1.4-9.7; data not shown); further
adjusting for maternal BMI before pregnancy, education,
birth weight, and sex, did not alter this association.
However, further adjustment for race/ethnicity reduced this
association somewhat (OR, 3.1; 95% CI, 1.1-8.3; Table IV).
Race/ethnicity-stratified analyses suggested the association
between H19 DMR hypermethylation and obesity or
overweight status may not be more apparent in the 108
white children (OR, 4.4; 95% CI, 1.0-20.4) than in the 85
African-American children (OR, 2.3; 95% CI, 0.6-9.1). In
contrast, breastfeeding-stratified analyses after adjusting for
postnatal caloric intake, race, and maternal smoking
suggested the association between hypermethylation at the
H19 DMR and overweight or obesity status in early life was
most pronounced in children who were never breastfed
(OR, 22.3; 95% CI, 2.1-239.8) compared with children who
were ever breastfed (OR, 1.3; 95% CI, 0.3-4.7). The cross-
product term for breastfeeding and the H19 DMR
methylation fraction was statistically significant (P= .05).
Further adjusting for race/ethnicity reduced the Pvalue for
the cross-product term to .03 (Table IV). At the IGF2
Table IV. ORs and 95% CIs for the association between
DNA methylation at birth and obese or overweight
status in breastfed and non-breastfed African-American
and white children*
DMR OR 95% CI Pvalue
H19 DMR*
All participants (n = 204) 3.12 1.13-8.60 .03
Never breastfed (n = 70) 22.27 2.07-239.84 .01
Ever breastfed (n = 133) 1.25 0.34-4.67 .74
Pvalue for cross-product term for
breastfeeding and H19 DMR
methylation
.03
African-American (n = 85) 2.38 0.62-9.10 .21
White (n = 108) 4.40 0.95-20.37 .06
Pvalue for cross-product term for race
and H19 DMR methylation
.46
IGF2 DMR
All participants 1.19 0.36-3.93 .78
Never breastfed 1.91 0.22-16.69 .56
Ever breastfed 0.69 0.14-3.43 .65
Pvalue for cross-product term for
breastfeeding and IGF2 DMR
methylation
.25
African-American 2.33 0.48-11.34 .30
White 0.57 0.06-5.16 .62
Pvalue for cross-product term for race
and IGF2 DMR methylation
.23
Models for all participants adjusted for maternal cigarette smoking, breastfeeding, race, and
postnatal caloric intake.
Breastfeeding-restricted models adjusted for maternal cigarette smoking, race, and postnatal
caloric intake.
Race/ethnicity-restricted models adjusted for maternal cigarette smoking, breastfeeding, and
postnatal caloric intake.
*H19 DMR mean methylation fraction $75th percentile.
IGF2 DMR mean methylation fraction #25th percentile.
Table III. DNA methylation differences in obese or overweight status in breastfed and non-breastfed African-American
and white children
DMR
£85th percentile of WFA (n = 172) >85th percentile of WFA (n = 26)
PvalueMean methylation fraction 95% CI Mean methylation fraction 95% CI
H19 DMR
All participants (n = 204) 59.3 58.3-60.4 62.4 59.5-65.4 .05
Never breastfed (n = 70) 59.8 57.6-61.9 68.3 62.3-74.2 .01
Ever breastfed (n = 133) 59.2 57.9-60.5 59.6 56.2-63.1 .82
Pvalue for cross-product term for breastfeeding
and H19 DMR methylation
.20
African-American (n = 85) 61.5 59.4-63.6 63.4 58.9-68.0 .46
White (n = 108) 58.1 56.8-59.4 61.9 57.7-66.2 .09
Pvalue for cross-product term for race
and H19 DMR methylation
.58
IGF2 DMR
All participants 47.5 46.5-48.4 47.2 44.6-49.7 .83
Never breastfed 46.7 45.1-48.2 45.4 40.9-49.9 .59
Ever breastfed 47.8 46.6-49.0 48.2 44.9-51.4 .82
Pvalue for cross-product term for breastfeeding
and IGF2 DMR methylation
.71
African-American 47.5 45.9-49.0 46.0 42.5-49.4 .43
White 47.4 46.2-48.7 48.2 44.4-51.9 .71
Pvalue for cross-product term for race and
IGF2 DMR methylation
.87
Models for all participants adjusted for maternal cigarette smoking, breastfeeding, race, and postnatal caloric intake.
Breastfeeding-restricted models adjusted for maternal cigarette smoking, race, and postnatal caloric intake.
Race/ethnicity-restricted models adjusted for maternal cigarette smoking, breastfeeding, and postnatal calori c intake.
THE JOURNAL OF PEDIATRICS www.jpeds.com Vol. 161, No. 1
36 Perkins et al
DMR, we found no DNA methylation differences between
overweight or obese children and children who are neither
obese nor overweight.
Discussion
We found that children who were overweight or obese at age
1 year had higher methylation percentages at the H19 DMR at
birth compared with children who were neither overweight
nor obese. Methylation differences of strikingly similar mag-
nitude have been reported previously in relation to gene ex-
pression and several phenotypic differences.
18,20,23,24,29
DNA
methylation differences of a similar magnitude were found
between Dutch famine survivors and their same-sex sib-
lings
30
and in Gambians conceived in the nutritionally chal-
lenging rainy season compared with those conceived in the
dry season.
31
Our findings are consistent with the interpreta-
tion that the plasticity of IGF2 may be mechanistically impor-
tant in early childhood overweight status and obesity.
Although it has been hypothesized that epigenetic mecha-
nisms may drive obesity in early childhood,
9
our study offers
empirical evidence linking obesity and overweight status to
methylation patterns at a well-known DMR regulating
IGF2. DNA hypermethylation at the H19 DMR has been pre-
viously associated with deregulation of paternally expressed
IGF2. Through mechanisms that are still unclear, IGF2 dysre-
gulation relaxes imprint controls, resulting in aberrant bial-
lelic expression of a gene that is otherwise monoallelically
expressed from the paternally derived allele, thereby increas-
ing transcription activity and, presumably, IGF2 protein
levels. Higher circulating IGF2 protein levels have been asso-
ciated with obesity in adults.
32
If confirmed in larger studies
and hypothesized co-regulation of imprinted genes is fully
characterized, these findings would support the hypothesis
that aberrant DNA methylation at regulatory sequences of
imprinted genes may be useful biosensors or markers to iden-
tify newborns exposed to an intra-uterine environment that
increase risk of obesity in early childhood.
Some of our findings suggested that the magnitude of
methylation differences between overweight or obese chil-
dren and children whose weight was within reference range
was modified by breastfeeding status. Although cause-and-
effect cannot be established in this epidemiologic study, these
findings raise the possibility that lack of breastfeeding, a mod-
ifiable postnatal behavior, may interact with a prenatally ac-
quired aberrant DNA methylation profile to increase the risk
of obesity or overweight status in early life. This possibility
warrants further investigation because the potential public
health implications could be sizable. However, the mecha-
nisms are still unknown. The myriad of differences between
breastfeeding and formula-feeding have made it difficult to
elucidate the reasons for possible interaction of breastfeeding
and H19 DMR hypermethylation. It is possible that infants
with H19 DMR hypermethylation at birth also are more likely
to have metabolic dysregulation, which, with infant formula,
may increase the risk of obesity or overweight status. Also,
breast milk contains not only IGF2, but also IGF1 and IGF
binding proteins.
33
The early protein hypothesis posits that
the higher levels of protein in infant formula exceed meta-
bolic requirements and that the metabolic products of excess
protein may stimulate secretion of excess insulin and IGF1,
leading to increased weight gain in early life.
34
The results
of the European Childhood Obesity Project support the early
protein hypothesis; the study, which randomized infants to
higher or lower protein formula, revealed a lower prevalence
of obesity at age 2 years in the lower protein group.
35
These findings, however, do not exclude the possibility
that the effect of the interaction between H19 DMR methyl-
ation and breastfeeding we observed may not be epigeneti-
cally driven. Breastfeeding is also associated with other
psychosocial factors that are not adequately captured by so-
cioeconomic status (as measured with educational level),
raising the possibility of confounding by these unmeasured
factors. For example, breastfed infants take smaller and
more frequent meals than non-breastfed infants, which
may influence later eating habits.
36
In addition, day-to-day
variability in the taste and smell of human milk, as opposed
to the consistency of formula, may program infants to make
more varied food choices later in life.
37
Finally, breastfeeding
may enhance emotional bonding between mother and child,
establishing a psychological well being that subsequently
could influence health in general. Disentangling these effects
will require larger studies.
Although our study assessed for confounding maternal
education, cigarette smoking, mode of delivery, birth weight,
pre-pregnancy BMI, and race, there may still be residual con-
founding by maternal nutrition during pregnancy, differential
postnatal morbidity, and breastfeeding that could influence
methylation patterns and infant growth patterns. Only one
of the 17 children with postnatal morbidity was overweight
or obese. However, if confirmed in larger studies in which ma-
ternal nutrition and breastfeeding are assessed at shorter inter-
vals, our observation that the growth trajectory of infants with
DNA hypermethylation of the H19 DMR at birth depends on
breastfeeding status offers possibilities for early public health
interventions on childhood obesity and a means by which
such benefits can be monitored. Evidence from animal studies
involving Agouti mice demonstrated that hypomethylation
induced by in utero or neonatal exposure to bisphenol A
was negated by maternal dietary supplementation with methyl
group donor nutrients.
38
Such findings suggest that stable
methylation alterations are potentially reversible with nutri-
tion, presumably restoring normal gene function and offering
prospects for public health intervention.
Reasons for the lack of association between the intragenic
IGF2 DMR methylation profile and obesity in our study are
unclear. Adults exposed to severe caloric restriction peri-
conceptionally during the Dutch Hunger Winter had de-
creased methylation compared with unexposed same-sex
siblings at this DMR.
30
In general, adult survivors of the
Dutch Famine have a higher risk of obesity and obesity-
related chronic disease.
39
A strength of our study is the analysis of multiple, previ-
ously evaluated CpG dinucleotides at two DMRs regulating
July 2012 ORIGINAL ARTICLES
Insulin-Like Growth Factor 2/H19 Methylation at Birth and Risk of Overweight and Obesity in Children 37
a well-characterized imprinted gene that encodes a potent
growth factor that also has been associated with obesity in
children
40-42
and adults,
32
although inconsistently.
43,44
A po-
tential limitation is that we did not measure and control for
the potential confounding effect of white blood cell counts,
because their relative abundance in specimen may influence
methylation percentages. However, adjusting for infection
during parturition (a major cause of variation in white blood
cell counts) did not alter our findings. Furthermore, measur-
ing DNA methylation obtained from leukocytes of unfractio-
nated umbilical cord blood raises the possibility that DNA
methylation percentages may be dependent on the predom-
inant cell population in the specimen. However, we have pre-
viously shown that at the DMRs being evaluated, the
methylation profiles were similar between polymorphonu-
clear and mononuclear cells,
27
suggesting our findings may
not have been unduly influenced by differences in blood
composition. In addition, although we did not evaluate the
temporal stability of methylation marks at the DMRs being
studied, several other studies have shown the temporal stabil-
ity of methylation at one of these DMRs,
30,45
although at
older ages.
Although findings from our exploratory race-stratified
analyses were intriguing, disentangling potential epigenetic
effects of race/ethnicity from those of breastfeeding will re-
quire larger studies. A small sample size also limited our abil-
ity to identify factors associated with varying degrees of
childhood overweight and obesity status, including WFA
>95th or 99th percentiles. Our use of WFA unadjusted for
length, to estimate obesity, makes comparisons with many
childhood obesity studies difficult. However, repeating these
analyses with weight gain as an outcome and weight-for-
length in a subgroup of 72 infants in whom height data
were recorded within 1 month of questionnaire administra-
tion revealed DNA methylation differences of a strikingly
similar magnitude, although less stable. Further, mothers’ in-
ability to recall infant feeding practices in the first year of life
point to a need for a cautious interpretation of our findings,
although in an earlier study, maternal recall did not apprecia-
bly modify the magnitude of the OR for growth in infancy.
46
We did not collect information on maternal diet during ges-
tation and breastfeeding, nor did we assess the varying nutri-
ent and hormone composition of breast milk. We also did
not inquire about reasons for not breastfeeding; some of
which also may vary by breastfeeding status and race. Because
the effect of these factors on the methylation profile at the
IGF2/H19 region is unknown, we cannot predict possible
changes in the direction of the association between methyla-
tion profiles and obesity or overweight status according to
variations in breast milk composition. Although paternal
height has been associated with childhood obesity,
47
most
mothers in our study were unable to report paternal height.
Finally, we did not conduct subanalyses by weight-for-
gestational-age at birth; however, on the basis of the 12
small-for-gestational-age and 18 large-for-gestational-age in-
fants in our sample, we found no association between weight-
for-gestational age and breastfeeding status (P= .2) or
methylation fraction (P= .6-.7), suggesting that infants’
size at birth did not unduly influence the associations found.
Because methylation patterns can be evaluated at birth, our
findings offer the possibility to identify individuals at higher
risk of obesity before obesity becomes clinically evident, tar-
geting interventions at mothers of at-risk infants. Should sug-
gested breastfeeding differences be replicated in larger studies,
this insight may offer new avenues for public health interven-
tions aimed at decreasing or preventing early obesity. n
Submitted for publication Feb 15, 2011; last revision received Nov 28, 2011;
accepted Jan 6, 2012.
Reprint requests: Cathrine Hoyo, PhD, Department of Community and Family
Medicine,Duke University,PO Box 104006,Durham, NC 27710.E-mail: cathrine.
hoyo@duke.edu
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July 2012 ORIGINAL ARTICLES
Insulin-Like Growth Factor 2/H19 Methylation at Birth and Risk of Overweight and Obesity in Children 39
    • For imprinted IGF2, these regulatory methylation marks are established during spermatogenesis, while the same sequences in the oocyte are unmethylated. The multifaceted effects of IGF2 have led many, including our group, to study how the methylation status of this DMR, hereafter referred to as the IGF2 DMR, varies in disease states12131415, as well as how methylation of this region is influenced by the in utero environment1617181920212223. The first reports of methylation plasticity of the IGF2 DMR came from studies of individuals with colon cancer.
    [Show abstract] [Hide abstract] ABSTRACT: Paternally expressed Insulin-like Growth Factor II (IGF2) encodes a gene whose protein product functions as a potent growth mitogen. Overexpression of IGF2 has been implicated in a wide number of disorders and diseases. IGF2 is regulated in part by differential methylation of the two parentally derived alleles. The differentially methylated region (DMR) located upstream of the imprinted promoters of IGF2 exhibits plasticity under environmental stress and is hypomethylated in several types of cancer. Through bisulfite pyrosequencing and confirmation by nucleotide sequencing, we discovered a CpG to CpC transversion that results in hypomethylation of one of the three CpGs comprising this DMR. The presence of the polymorphism introduces a genetic rather than an environmentally-driven epigenetic source of hypomethylation that is additive to non-genetic sources.
    Full-text · Article · Sep 2015
    • Information on anthropometric measurements during pregnancy, birth outcomes , including birth weight and length, was abstracted from medical records. Umbilical cord blood specimens were also collected at birth, as previously described [36] [37].
    [Show abstract] [Hide abstract] ABSTRACT: At birth, elevated IGF-I levels have been linked to birth weight extremes; high birth weight and low birth weight are risk factors for adult-onset chronic diseases including obesity, cardiovascular disease, and type 2 diabetes. We examined associations between plasma IGF-I levels and birth weight among infants born to African American and White obese and nonobese women. Prepregnancy weight and height were assessed among 251 pregnant women and anthropometric measurements of full term infants (≥37 weeks of gestation) were taken at birth. Circulating IGF-I was measured by ELISA in umbilical cord blood plasma. Linear regression models were utilized to examine associations between birth weight and high IGF-I, using the bottom two tertiles as referents. Compared with infants with lower IGF-I levels (≤3rd tertile), those with higher IGF-I levels (>3rd tertile) were 130 g heavier at birth, (β-coefficient = 230, se = 58.0, P = 0.0001), after adjusting for gender, race/ethnicity, gestational age, delivery route, maternal BMI and smoking. Stratified analyses suggested that these associations are more pronounced in infants born to African American women and women with BMI ≥30 kg/m(2); the cross product term for IGF-I and maternal BMI was statistically significant (P ≤ 0.0004). Our findings suggest that the association between IGF-I levels and birth weight depends more on maternal obesity than African American race/ethnicity.
    Full-text · Article · May 2013
    • The connection of the H19 locus to growth disorders may suggest a possible involvement of the H19 locus in being overweight and obesity. Indeed, it was reported that one-year-old children who were overweight or obese had significantly higher methylation percentages at the H19 DMR at birth compared with children who were not overweight or obese [28]. Moreover, it seems that, as a response to starvation during periconception, ICR1 becomes hypermethylated, leading to restricted growth due to lower IGF2 expression in adult life.
    [Show abstract] [Hide abstract] ABSTRACT: The field of the long non-coding RNA (lncRNA) is advancing rapidly. Currently, it is one of the most popular fields in the biological and medical sciences. It is becoming increasingly obvious that the majority of the human transcriptome has little or no-protein coding capacity. Historically, H19 was the first imprinted non-coding RNA (ncRNA) transcript identified, and the H19/IGF2 locus has served as a paradigm for the study of genomic imprinting since its discovery. In recent years, we have extensively investigated the expression of the H19 gene in a number of human cancers and explored the role of H19 RNA in tumor development. Here, we discuss recently published data from our group and others that provide further support for a central role of H19 RNA in the process of tumorigenesis. Furthermore, we focus on major transcriptional modulators of the H19 gene and discuss them in the context of the tumor-promoting activity of the H19 RNA. Based on the pivotal role of the H19 gene in human cancers, we have developed a DNA-based therapeutic approach for the treatment of cancers that have upregulated levels of H19 expression. This approach uses a diphtheria toxin A (DTA) protein expressed under the regulation of the H19 promoter to treat tumors with significant expression of H19 RNA. In this review, we discuss the treatment of four cancer indications in human subjects using this approach, which is currently under development. This represents perhaps one of the very few examples of an existing DNA-based therapy centered on an lncRNA system. Apart from cancer, H19 expression has been reported also in other conditions, syndromes and diseases, where deregulated imprinting at the H19 locus was obvious in some cases and will be summarized below. Moreover, the H19 locus proved to be much more complicated than initially thought. It houses a genomic sequence that can transcribe, yielding various transcriptional outputs, both in sense and antisense directions. The major transcriptional outputs of the H19 locus are presented here.
    Full-text · Article · Feb 2013
    • It has been proposed that prenatal exposure to different compounds, among them xenoestrogens, might exert their effects by altering these tightly regulated epigenetic programs (Heindel and vom Saal, 2009). Interestingly, functional estrogen response elements (ERE), at least in rat testis, have been described in the IGF2-H19 locus, a key imprinted genomic region involved in the control of fetal growth and which is usually disregulated in cancer (Charalambous et al., 2007; Hamelers and Steenbergh, 2003; Koukoura et al., 2011; Nativio et al., 2011; Pathak et al., 2010; Perkins et al., 2012). In addition, the IGF2 gene is overexpressed in the hypothalamus of rats after estrogen treatment (Takeo et al., 2009).
    [Show abstract] [Hide abstract] ABSTRACT: BACKGROUND: Fetal exposure to endocrine disrupting chemicals may increase the risk for adverse health effects at birth or later in life. OBJECTIVES: The objective of this study is to analyze the combined effect of xenoestrogens on reproductive and perinatal growth outcomes (child birthweight, early rapid growth and body mass index (BMI) at 14months) using the biomarker total effective xenoestrogen burden (TEXB). METHODS: 490 placentas were randomly collected in the Spanish prospective birth cohort Environment and Childhood (INMA) project. TEXB was used to assess the estrogenicity of placental samples in two fractions: that largely attributable to environmental organohalogenated xenoestrogens (TEXB-alpha), and that mostly due to endogenous estrogens (TEXB-beta), both expressed in estrogen equivalent units (Eeq) per gram of tissue. Linear or logistic regression models were performed adjusting for cohort and confounders. Sex interactions were investigated. RESULTS: The median TEXB-alpha level was 0.76pMEeq/g (interquartile range (iqr): 1.14). In multivariate models, higher TEXB-alpha levels (third tertile, >1.22pMEeq/g; iqr: 1.73) were associated with increased birthweight in boys but not in girls (β=148.2g, 95% CI: 14.01, 282.53, p(int)=0.057). Additionally, higher TEXB-alpha values in boys were related with a lower risk of early rapid growth (OR=0.37; 95% CI: 0.15, 0.88) and with a non significant association with larger BMI z-scores at 14months of age (β=0.29; 95% CI: -0.11, 0.69). CONCLUSIONS: These findings suggest that prenatal exposure to xenoestrogens may increase birthweight in boys, which might have an impact on child obesity and other later health outcomes.
    Full-text · Article · Dec 2012
    • Our study found repeated positive associations of greater IGF2/H19 ICR methylation with greater subcutaneous adiposity measured by multiple methods, including direct ultrasound and by calliper assessed skin folds at multiple body sites. The positive association of IGF2/ H19 ICR methylation with subcutaneous adiposity in this study adds to the recent observations by Perkins et al. showing greater IGF/H19 ICR methylation in overweight or obese one-year-olds compared to normal weight counterparts [30]. The current study shows that an association between adiposity and methylation at this site persists to young adult life.
    [Show abstract] [Hide abstract] ABSTRACT: Background The insulin-like growth factor 2 (IGF2) and H19 imprinted genes control growth and body composition. Adverse in-utero environments have been associated with obesity-related diseases and linked with altered DNA methylation at the IGF2/H19 locus. Postnatally, methylation at the IGF2/H19 imprinting control region (ICR) has been linked with cerebellum weight. We aimed to investigate whether decreased IGF2/H19 ICR methylation is associated with decreased birth and childhood anthropometry and increased contemporaneous adiposity. DNA methylation in peripheral blood (n = 315) at 17 years old was measured at 12 cytosine-phosphate-guanine sites (CpGs), analysed as Sequenom MassARRAY EpiTYPER units within the IGF2/H19 ICR. Birth size, childhood head circumference (HC) at six time-points and anthropometry at age 17 years were measured. DNA methylation was investigated for its association with anthropometry using linear regression. Results The principal component of IGF2/H19 ICR DNA methylation (representing mean methylation across all CpG units) positively correlated with skin fold thickness (at four CpG units) (P-values between 0.04 to 0.001) and subcutaneous adiposity (P = 0.023) at age 17, but not with weight, height, BMI, waist circumference or visceral adiposity. IGF2/H19 methylation did not associate with birth weight, length or HC, but CpG unit 13 to 14 methylation was negatively associated with HC between 1 and 10 years. β-coefficients of four out of five remaining CpG units also estimated lower methylation with increasing childhood HC. Conclusions As greater IGF2/H19 methylation was associated with greater subcutaneous fat measures, but not overall, visceral or central adiposity, we hypothesize that obesogenic pressures in youth result in excess fat being preferentially stored in peripheral fat depots via the IGF2/H19 domain. Secondly, as IGF2/H19 methylation was not associated with birth size but negatively with early childhood HC, we hypothesize that the HC may be a more sensitive marker of early life programming of the IGF axis and of fetal physiology than birth size. To verify this, investigations of the dynamics of IGF2/H19 methylation and expression from birth to adolescence are required.
    Full-text · Article · Nov 2012
    • In humans, the degree of IGF2 methylation at birth has recently been related to the development of overweight or obesity in early childhood (Perkins et al., 2012 ). Interestingly, breastfeeding modified the magnitude of methylation differences between overweight or obese children and children whose weight was within reference range, thus suggesting an interplay between prenatal environment (nutrient transfer from mother to fetus) and early postnatal feeding behavior which could stabilize or change the epigenetic patterns acquired in utero (Perkins et al., 2012). A potential clinical implication of these findings is that IGF2 methylation may represent an easily assessable marker of intrauterine programming and long-term metabolic risk, thus driving the deprogramming strategies aimed at reducing the metabolic risk in subjects exposed to a suboptimal intrauterine environment.
    [Show abstract] [Hide abstract] ABSTRACT: Insulin-like growth factor-II (IGF-II), traditionally considered as a growth factor implicated in growth of fetal tissues and cancer cells, is now emerging as a potential metabolic regulator. The aim of this overview is to provide the available evidence, obtained in both experimental conditions and in humans, for a role of IGF-II in the fine-tuning of metabolism and body composition. The underlying mechanisms and the potential clinical implications are discussed.
    Full-text · Article · Oct 2012
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