October 2014

Epigenetics Studies Uncover Obesity-Driven
Methylation Signatures
Researchers use the Infinium® HumanMethylation450 BeadChip to identify cell signaling disruptionthat potentially contributes to the negative downstream effects of high BMI.

Introduction
As he was finishing his research degree at the Universityof Leicester, Nilesh Samani made an important decision.
While his goal was to become a cardiologist, he hadenjoyed conducting research into the molecular biologyof hypertension. He decided to chart a new course,
combining his clinical practice with research into the geneticunderpinnings of cardiovascular disease. Over the next 30years, he produced groundbreaking research, publishing
more than 400 scientific papers including many of the key
genome-wide association studies (GWAS) in coronary
artery disease.
Professor Samani and his team took this work a step further
in 2013, conducting an epigenetic-wide association study(EWAS) to see if DNA methylation changes could be linked
to cardiovascular traits. The study leveraged blood andtissue samples from a number of well-known cardiovascularstudies, for assessment with the HumanMethylation450
BeadChip. GWAS and EWAS data analysis identified a DNA
methylation change in the hypoxia inducible transcription
factor (HIF) in patients with high body mass index (BMI).1
HIF is best known for its key role in hypoxia-sensing.
However, emerging evidence has also demonstrated its
involvement in metabolism, specifically the cellular response
to glucose and insulin, and as an accelerator of adipocyte
differentiation. Previously, the HIF system had been linked
with obesity in animal models, but this was the first timethat HIF signaling disruption was shown to potentiallycontribute to the deleterious downstream effects of highBMI in humans.
iCommunity spoke with Professor Samani to learn more
about this research and the role EWAS studies will play in
his future cardiovascular research.
Q: How has your research into the genetics of
cardiovascular disease evolved over the years?
Nilesh Samani (NS): I used experimental models in most of
my early hypertension research, including research performed
with spontaneously hypertensive rats. Once I completed
my clinical training, I began focusing on the genetics ofhypertension and coronary disease in humans, which both

Nilesh Samani, M.D., is British Heart Foundation Professor of Cardiology
and Head of the Department of Cardiovascular Sciences at the University ofLeicester, and Director of the NIHR Leicester Biomedical Research Unit inCardiovascular Disease.

have a strong genetic determination. I started with candidate
gene studies and linkage analysis using microsatellite markers,
moving into GWAS studies as part of the initial Wellcome Trust
Case Control Consortium (WTCCC). I’ve been privileged tobe involved in many of the GWAS studies performed in thecardiovascular disease area.
Q: What caused you to look for a link between epigenetic
changes and cardiovascular diseases?
NS: We became interested in epigenetic changes as they
could reflect the most proximate change to explain someof the GWAS signals we had identified. Techniques tolook systematically for epigenetic changes have trailed
behind those for GWAS and gene expression, but theHumanMethylation450 BeadChip has changed the picture.
Our initial hypothesis was that we would find methylation
changes linked to genotype. We thought that the process
would flow forward, with the genotype causing a methylation
change that in turn caused the disease in some way. As it
turned out, it is much more complex.
Q: What epigenetic and phenotype associations did you
investigate?
NS: We initially focused on BMI as this is an important risk
factor for cardiovascular disease. The association between
specific epigenetic signals and BMI appeared to be quite

October 2014
striking in our discovery cohort so we decided to see if we
could replicate those signals in other cohorts.
Q: What samples did you use for this study?
NS: The discovery cohort was made up of healthy blood
donors and individuals with a history of myocardial infarctionthat were recruited for the Cardiogenics Consortium.
Blood samples from the MARseille Thrombosis Association(MARTHA) and Cooperative Health Research in the Region ofAugsburg (KORA) studies made up our primary and secondary
replication cohorts, respectively. We also analyzed EWAS inskin and adipose tissues from the Multiple Tissue HumanExpression Resource (MuTHER) study.
Q: What did the EWAS results show in these cohorts?
NS: The methylation values of the discovery cohort wereadjusted for age, sex, smoking, and case-control status.
We identified five probes, three of which were in HIF3A on
chromosome 19. The three HIF3A probes—cg22891070,cg27146050, and cg16672562—reside alongside each otherin intron 1 of the gene encoding HIF.
We took the three HIF3A probes forward for analysis in the
primary replication cohort (MARTHA), and found all threesignificantly associated with BMI. The same was true inour secondary replication cohort (KORA), although the BMI
association was weaker.
Q: What is HIF and what does it regulate?
NS: HIF is an oxygen sensing system consisting of a numberof different proteins that mediate the cellular response tooxygen deprivation. Researchers have investigated the roleof HIF signaling in ischemia in general. In the past few years,
they’ve begun to study the role HIF plays in cancer, because
many cancers are ischemic.
Only recently have we begun to consider that this transcription
factor may modulate other genes as well. Its link with obesity
may not actually be driven by the hypoxia signal. There arestudies that show that knocking out certain HIF signaling
proteins can cause changes in metabolic parameters. We’rejust beginning to understand the multiple roles HIF plays inthe body.

“Techniques to look systematicallyfor epigenetic changes have
trailed behind those for GWAS
and gene expression, but theHumanMethylation450 BeadChip
has changed the picture.”

Q: Were you concerned that the epigenetic signature youwere seeing might represent variations in the proportionof different blood cells in the whole blood samples
you assayed?
NS: Yes, we recognized that the DNA used in our methylation
analysis was derived from a mixture of different white bloodcell types. Each of those cell types might have slightly
different epigenetic levels, causing us to mistake differencesin epigenetic signature for disease changes rather thandifferences in cell type proportion.
To clarify what we were seeing, we decided to determine if
there were epigenetic differences between each cell type.
We used one of the probes and examined the association ofmethylation and cell type, adjusting for lymphocyte, monocyte,
and neutrophil counts. We found that the association
between probe methylation and BMI was not attenuated afterthis adjustment.

“In this case, blood DNA acted
as a reasonable surrogate for
epigenetic changes present in abiologically relevant tissue.”
Q: What was the association between the HIF3A probes
and BMI?
NS: Using the cg22891070 as an exemplar, every 0.1 increase
in methylation ß value for the probe was associated with a
3.6% higher BMI in the discovery cohort. The increase in BMI
was greater in participants who had a myocardial infarction
than in healthy blood donors. We also investigated theassociation of DNA methylation at HIF3A with two componentsof BMI, height and weight. We found the methylation signaturesignificantly associated with weight, but not with height.
Q: How did the blood sample HIF3A methylation signaturescompare with those you found in skin and adipose tissue?
NS: We wondered if the epigenetic signature that we saw
in blood was indicative of changes in relevant tissues. Inthe MuTHER cohort, we assessed the methylation level at
the three HIF3A sites in skin and adipose tissue. We werepleased and surprised to see that the data showed a very
specific epigenetic change in adipose tissue that was strongly
associated with BMI. We did not see a similar association inskin. In this case, blood DNA acted as a reasonable surrogate
for epigenetic changes present in a biologically relevant tissue.
The results provide the epigenetic community with a measureof confidence that assessing epigenetic signatures in blood
has value.

October 2014
Q: Is the methylation at HIF3A responsible for high BMI?
NS: GWAS signals by definition are causal. One cannot makethe same interpretation for EWAS signals as they could bealtered by environmental factors. Indeed when we probed
into the association of methylation at HIF3A with BMI further,
we found evidence that indeed it may be BMI that is driving
the change in methylation at HIF3A. Specifically, we foundsome methylation QTLs for HIF3A (ie genetic variants affecting
methylation at this locus) but these did not associate with BMI
directly. That suggests that the epigenetic present at the HIF
locus represented obesity driving methylation.
Obesity causes all sorts of deleterious things downstream
and researchers have long wondered how obesity causesthese changes. I think the epigenetic signatures we uncovered
may provide important clues in this area. When we started
this study, we hadn’t appreciated that it could provide us
with important biological information about the downstream
mechanisms of obesity.
Q: Do your research results suggest a new approach to
reducing the health impact of obesity?
NS: Preventing obesity is the focus of most therapeutic
approaches. There’s been much less emphasis on thedownstream impact of obesity, including its links with cancer
and cardiovascular disease. There are few studies focusedon the biology behind why obesity increases the risk ofthese diseases.
What our study highlights is that we may have the opportunity
to influence the relationship between obesity and downstream
diseases. Methylation signatures in HIF3A may act as one
mediator of that link, providing us with therapeutic targets.
Q: Why did you choose the HumanMethylation450
BeadChip for this study?
NS: I think the HumanMethylation450 array is ideal for EWAS
studies. It is the best and most comprehensive array out there.
Q: How do you foresee EWAS studies contributing to ourunderstanding of complex disease?
NS: One of the things that our paper shows is that theinterpretation of EWAS studies is going to be much more
complicated. Apart from issues of tissue and cellular specificity,
the main challenge is to determine directionality. With GWAS,
the findings indicate causality: this gene caused this effect.
EWAS results can be bidirectional, with epigenetics being
either the cause or the consequence. We need to be aware ofthat. Provided the findings are interpreted carefully, they cannonetheless inform biology.
I think over the next two to three years we’re going to see
a fairly large increase in EWAS studies and more definitive
EWAS studies being published. I think we’ll see EWAS studies
evolving, maybe not quite to the scale of GWAS studies,but becoming substantially larger. I suspect that the sort ofepigenetic changes we’ll be looking for in EWAS studies are
not going to require 100,000 sample cohorts to generate
data. Data analysis will be more complicated because ofthe complexity of merging EWAS data sets due to cell type,etc. I think EWAS is going to require a higher degree ofsophistication that hasn’t been necessary for GWAS studies.

“Interpretation of EWAS studies
is going to be much more
complicated than GWAS studies.”
Q: Do you see array-based methods like the
HumanMethylation450 BeadChip and sequencing
complementing each other moving forward?
NS: I think we will see methylation array and sequencing usedin tandem, but it may require new sequencing approaches tobe developed. We saw half a dozen or more epigenetic signals
linked with BMI in our study. To study those signals, we’dneed a sequencing approach that doesn’t differentially amplify
methylated or non-methylated alleles.
Q: What are the next steps in your research?
NS: The HIF3A study was a valuable one and I think ithighlighted the opportunity to use the HumanMethylation450
array and EWAS approach to pursue the role of epigenetics
in cardiovascular disease. We’ll continue to use GWAS
and EWAS to pursue our study of coronary and othercardiovascular diseases.

References
1.

Dick KJ, Nelson CP, Tsaprouni L, et al. DNA Methylation and Body-Mass
Index: a Genome-Wide Analysis. Lancet 2014: 383: 1990-1998.

October 2014

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