ABSTRACT:
Setting: DNA methylation is an epigenetic mechanism through which environmental
factors including obesity influence health. Obesity is a major modifiable risk factor for
many common diseases including cardiovascular diseases and cancer. Obesity-
5 induced metabolic stress and inflammation are key mechanisms that affect disease
risk and which may result from changes in methylation of metabolic and
inflammatory genes.
Objectives: This review aims to report the effects of weight loss induced by bariatric
surgery (BS) on DNA methylation in adults with obesity focusing on changes in
10 metabolic and inflammatory genes.
Methods: A systematic review was performed using Medline, EMBASE and Scopus,
to identify studies in adult humans that reported DNA methylation following BS.
Results: Out of 15996 screened titles, 15 intervention studies were identified, all of
which reported significantly lower body mass index (BMI) post-surgery. DNA
15 methylation was assessed in five different tissues (blood=7 studies, adipose tissues
=4, skeletal muscle =2, liver and spermatozoa). Twelve studies reported significant
changes in DNA methylation after BS. Meta-analysis showed that BS increased
methylation of PDK4 loci in skeletal muscle and blood in two studies while the effects
of BS on IL6 methylation levels in blood were inconsistent. BS had no overall effect
20 on LINE1 or PPARGC1 methylation.
Conclusion: The current evidence supports the reversibility of DNA methylation at
specific loci in response to BS-induced weight loss. These changes are consistent
with improved metabolic and inflammatory profiles of patients after BS. However, the
1
evidence regarding the effects of BS on DNA methylation in humans is limited and
25 inconsistent, which makes it difficult to combine and compare data across studies.
Key words: Bariatric surgery, DNA methylation, obesity, inflammation
30
35
40
2
Introduction:
45 Obesity is a major modifiable, and preventable, risk factor for many common
diseases including cardiovascular diseases and cancer1. Obesity increases disease
risk by multiple mechanisms, including increased metabolic stress and chronic
inflammation. Obesity-induced inflammation is orchestrated by metabolic cells,
results in local expression of inflammatory mediators and creates a proinflammatory
50 tissue environment that is maintained in the long-term.2 This dysregulated
metabolism is characterised by abnormal glucose metabolism, dyslipidemia and
insulin resistance which subsequently increase the inflammatory response3. These
effects lead to endothelial dysfunction and atherosclerosis, increasing the risk of
cardiovascular diseases4. In addition, these mechanisms may underpin the greater
55 cancer risk in those with obesity.3
DNA methylation is an epigenetic mechanism through which obesity may influence
disease risk. In humans, DNA is methylated by the addition of a methyl group to the
5’ position on cytosine (C) residues in CpG dinucleotides and is a key element in the
regulation of gene expression5. Abnormal patterns of DNA methylation result in
60 reduced DNA integrity, changes in gene expression and mutations6. Patterns of DNA
methylation respond to many environmental factors, including dietary interventions
and weight loss7.
Bariatric surgery (BS) is an effective therapy which induces long-term weight loss
and improves comorbidities in obese patients.8 BS induces remission from type 2
65 diabetes (T2D) in a large proportion of initially obese patients, lowers risk of
cardiometabolic disease9 and lowers incident cancer risk including breast,
endometrial and colorectal cancers10. However, whilst these changes are associated
3
with decreased systemic and adipose tissue inflammation11, the underlying
molecular mechanisms remain unresolved.
70 This systematic review reports the effects of weight loss induced by BS on DNA
methylation in adults with obesity, aiming to: i) synthesize the evidence for the
relationships between weight loss and corresponding changes in DNA methylation
and ii) establish the links between these changes and specific metabolic and
inflammatory genetic loci.
75
Methods:
The systematic review is reported following the PRISMA checklist and flowchart12
(Supplementary Figure1). The systematic review was registered with PROSPERO
(CRD42018112261).
80 Search strategy and screening:
The databases, Embase, Scopus and Medline, were searched from inception until
January 2019 by using the following search terms: ( ( (methylat*) OR methylation
[Mesh] OR dna methylation [Mesh] ) AND ( ( Surg*) OR Surgery [Mesh] OR
Bariatric Surgery [Mesh] ) ). Other databases that were searched included: Prospero,
85 Cochrane library, ClinicalTrial.gov and International clinical trials registry platform
(WHO) for relevant protocols of clinical trials and systematic reviews that addressed
DNA methylation and bariatric surgery.
Articles were screened against the pre-set inclusion criteria (PICOS): a) Population:
adult human beings (≥16 years old); b) Intervention: bariatric surgical interventions or
90 procedures; c) Comparator: healthy control group, other bariatric interventions, and
4
other interventions aiming for weight loss including dietary and physical exercise; d)
Outcome: DNA methylation measured using any technique (global or locus specific)
as a primary or secondary outcome, assessed before and after the intervention; e)
Study design: any observational or intervention study, randomized or non-
95 randomized. Studies that recruited patients who had a history of, or were undergoing
active treatment for, specific diseases (e.g. cancer) or patients with hereditary
genetic disorders were excluded because of the likelihood that such conditions or
therapies would confound the intervention effects.
Titles and abstracts were screened by two independent reviewers (KE and FCM).
100 Neither of the reviewers was blind to the journal titles or to the study authors or
institutions. Following screening of the titles and abstracts, full texts were reviewed to
ensure eligibility for inclusion. Comparisons were made between the results of the
two reviewers. Any discrepancy between their decisions regarding inclusion in the
study was resolved by a third reviewer (JCM).
105
Data extraction, narrative synthesis and meta-analysis:
The following data were collected using a pre-tested standard form: year of
publication; study design; health or disease status of participants; number of
participants; BMI of participants before and after intervention; nature of bariatric
110 intervention; duration of follow up; any other pre-procedure intervention; nature of
other interventions; sample site; DNA methylation assessment method (including
genomic loci, where appropriate), and DNA methylation levels of participants pre-
and post- intervention, with measures of variance and level of significance. These
5
data were recorded using Microsoft® Excel 2017 which was used to synthesize
115 descriptive statistics and summary tables to support the narrative synthesis.
Eligible studies were included in a meta-analysis conducted using the Review
Manager software (v5.3, The Cochrane Collaboration, 2014) and intervention effects
were quantified using a random effects model (due to heterogeneity) and
standardized mean difference (due to the different methods used to quantify DNA
120 methylation). The quality of the included studies was assessed using the Newcastle-
Ottawa Scale (NOS). Heterogeneity between studies was assessed using the Chi2
statistic (expressed as p value) and I2 statistics (expressed as percentage) using
Review Manager v5.3.
125 Results:
The PRISMA flowchart12 (Supplementary Figure 1) summarizes the outcomes of the
search strategy. Out of 15996 screened titles, 15 studies were included. Of these
studies, two were cross sectional and eight were cohort, three of which did not
include a control group. None of the studies was a randomized controlled trial (RCT)
130 (Table 1).
Two BS procedures were applied in the included studies: Roux-en-Y Gastric Bypass
(RYGB, n=15) and Sleeve Gastrectomy (SG, n=3). In the fifteen studies, 312 obese
patients underwent BS (range 6 – 120 patients, median =11) with an average follow
up of 10.1 months (range 6 – 24 months). Mean BMI dropped from 45.9 kg/m2 (42.1-
135 50.9) to 32.8 kg/m2 (25.7-36.4) after BS. Only three studies13–15 reported mean BMI
below the obesity cutoff (30) at ≥ 12-month follow-up. DNA methylation was
6
assessed in five different tissues: blood (n=7), adipose tissues (n=4), skeletal
muscles (n=2), liver (n=1) and spermatozoa (n=1) (Table1).
140 Effects of bariatric surgery on DNA methylation in blood:
Five studies investigated the effects of BS on DNA methylation at specific genomic
loci in blood. Kirchner et al.13 followed up 7 patients who had undergone RYGB and
for whom mean BMI was 27.3 kg/m2 after 12-month follow-up. Methylation of PDK4
(Pyruvate Dehydrogenase Kinase 4) (involved in metabolic homeostasis), IL1-B
145 (Interleukin 1 beta), IL6 (Interleukin 6) and TNF (Tumor Necrosis Factor)
(inflammatory genes) in whole blood was significantly higher at the end of the 12-
month follow-up compared with pre-surgery levels. In addition, methylation of IL1-B,
IL6 and TNF and PPARGC1A (Peroxisome proliferator-activated receptor gamma
coactivator 1-alpha gene) was lower immediately after surgery (two days), indicating
150 that the effects of acute stress by BS on the inflammatory process may mediated
through these hypomethylated inflammatory genes.
Nicoletti et al16 investigated the effects of BS on the methylation of IL6 and
SERPINE1 (Serpin Family E Member 1). After 6- month follow-up, there was no
change in the methylation of SERPINE1 but IL6 methylation decreased. In a larger
155 study of 120 participants with obesity, Morcillo et al. 17 found increased SCD
(Stearoyl-CoA Desaturase) methylation in peripheral blood mononuclear cells
(PBMC) at 6 months after BS. In a more recent study, Gonzales et al.18 found no
significant change in methylation of IL6, SLC19A1 (Solute Carrier Family 19 Member
1) or PPARγ (Peroxisome Proliferator-Activated Receptor Gamma) at 6 months after
160 the surgery. However, methylation of NFkB1 (Nuclear Factor Kappa B Subunit 1)
7
was increased and correlated significantly with the decrease in blood pressure in
patients after BS.
Three studies13,16,19 investigated global DNA methylation and none found significant
effects of BS at 6 or 12 months after surgery. Furthermore, Martín-Núñez et al.19
165 found no significant differences in global DNA methylation (assessed as LINE1
methylation20- Long interspersed nuclear elements) levels in either diabetic or non-
diabetic patients or when stratified according to the bariatric procedure (RYGB vs
SG).
Two studies investigated genome-wide DNA methylation using the Infinium
170 HumanMethylation450k Bead Chip technology21,22. In 11 patients, Nilsson et al.21
identified 51 regions with significantly altered methylation at 6 months after BS.
Importantly, after BS, methylation at these loci was similar to the methylation levels
in a healthy control group. These changes included decreased methylation of INCA1
(Inhibitor Of CDK, Cyclin A1 Interacting Protein 1), a gene that has anti-cancer and
175 anti-proliferative properties23 and decreased methylation of ADK (Adenosine
Kinase), a gene that increases extracellular concentrations of adenosine, improving
insulin and glucagon secretion.24
Effects of bariatric surgery on DNA methylation in adipose tissues:
180 Four studies assessed DNA methylation in abdominal subcutaneous tissue, with one
study15 including additional samples from omental fat. All studies investigated
genome-wide methylation except one study25 that investigated methylation of the
LEP gene. The latter study reported no significant difference in the LEP methylation
8
in DNA from adipose tissue of 8 females with obesity before, and at 24 months after,
185 RYGB.
The remaining three studies reported significant effects of BS on genome-wide
methylation. Both Benton et al.15 and Dahlman et al. 14 reported lower overall
methylation levels after RYGB and Multhaup et al.26 reported significant changes in
227 differentially methylated regions (DMRs) at 6 months after BS. Importantly, for
190 105 of those DMRs, methylation levels after BS were similar to those in healthy
controls. In addition, Dahlman et al.14 found over-representation of DMRs related to
cellular differentiation pathways and adipogenesis in post-obese females. Overall
methylation levels of the DNA extracted from omental fat cells were lower after BS
and only 15 differentially methylated CpG sites were identified in DNA from omental
195 samples compared with 3601 in the SC samples15.
Effects of bariatric surgery on DNA methylation in other tissues:
Two cohort studies27,28 assessed methylation in biopsies from the vastus lateralis.
Barres et al.27 followed up eight patients at 6 months after BS and observed that
200 methylation of 11 out of the 14 studied genetic loci were similar to those of normal
healthy controls. Day et al.28 reported lower methylation at 29 cytosine residues in
SORBS3 (Sorbin And SH3 Domain Containing 3) in skeletal muscle DNA from 7
obese female patients 3 months after BS.
In liver biopsies from patients at multiple stages of non-alcoholic fatty liver disease
205 (NAFLD) who underwent RYGB, Ahrens et al.29 examined genome wide methylation
patterns using the Illumina HumanMethylation450k Bead Chip approach. Before
surgery, the authors found 273 CpG sites that showed phenotypic progression
9
(changed methylation) from normal controls, to healthy obese, to steatosis and to
non-alcoholic steatohepatitis (NASH). After RYGB, a total of 113 CpGs were
210 identified in which those CpGs that had lower methylation after bariatric surgery
typically had higher methylation during progression from normal liver to NASH29.
Donkin et al.30 assessed methylation levels in the spermatozoa of obese men at one
week and at one year after BS. More than 1500 unique genes were differently
methylated at one week compared with pre-surgery, indicating that such changes
215 can occur up to the last stages of sperm maturation. In addition, 3910 genes were
differentially methylated at one-year follow-up. Of these genes, 2681 were
differentially methylated when compared with samples from lean men.
Correlation between methylation and gene expression after bariatric surgery:
220 Seven studies investigated expression of the corresponding genes as functional
consequences of the observed methylation changes (see Supplementary Table 2).
Two studies found no correlation between methylation and expression of SCD in
blood17 or LEP in adipose tissues25. Five studies found inverse correlations between
methylation and expression i.e. decreased methylation was associated with
225 increased expression and vice versa at specific genomic loci e.g. PTPE (protein-
tyrosine phosphatase epsilon) in liver29, SORBS3 in skeletal muscles28 and CETP
(Cholesteryl ester transfer protein) in adipose tissues15. However, Benton et al. and
Barres et al. found positive correlations between methylation and expression of
CTGF15 (connective tissue growth factor) in adipose tissues and EXOC5 (Exocyst
230 Complex Component 5), ACOX1 (Acyl-CoA Oxidase 1) and ACACB (Acetyl-CoA
Carboxylase Beta) in skeletal muscle27 respectively (see Supplementary Table 2).
10
Meta-analysis of effects of bariatric surgery on DNA methylation on specific
genetic loci:
Meta-analysis was undertaken for four genetic loci with five studies included in total
235 (Figure 1). Martín-Núñez et al.19 and Nicoletti et al. 16 quantified LINE1 methylation
as a surrogate marker for global methylation20 in whole blood and buffy coats
respectively at 6 months after BS. There was no significant heterogeneity between
the studies (p=0.61, I2=0%) and no evidence for an effect of BS on LINE1
methylation (p=0.94).
240 Six months after BS, Nicolletti et al.16 found significantly lower IL6 methylation in
blood, whereas Gonzales et al.18 did not find any significant effect. In contrast,
Kirchner et al.13 reported significantly higher IL6 methylation in blood one year after
BS. There was significant heterogeneity between the three studies (p<0.01,
I2=90%), with no significant overall effect of BS on IL6 methylation levels in blood
245 (p=0.25).
In both blood and skeletal muscle, methylation of the PDK4 gene was greater after
BS in studies by Kirchner et al13 and Barres et al27 . There was no heterogeneity
between the two studies (p=0.8, I2=0%) with evidence of a highly significant (p<0.01)
hypermethylation of PDK4 after BS (Fig. 1).
250 Methylation of PPARGC1A was also quantified in blood 13,18 and skeletal muscles
27. At 6 months after BS, PPARGC1A methylation in skeletal muscles was decreased
significantly27, but there was no effect in blood18. In a separate study, methylation of
PPARGC1A had increased in blood at one year post BS13. There was no overall
effect of BS on methylation of PPARGC1A (p=0.7) with no significant heterogeneity
255 between the three studies (p=0.13, I2=51%).
11
Quality assessment:
Use of the Newcastle-Ottawa Scale (NOS) for study quality assessment showed that
all five studies included in the meta-analyses had similar scores for the “selection”
(2/4) and “outcome” (3/3) criteria but they scored differently in the comparability
260 section (0-2/2) according to the NOS (Supplementary Table 1). All the studies
included selected groups of patients who underwent intervention, with inadequate
description of the derivation of the non-exposed group. However, all studies had an
appropriate assessment of the outcomes and adequate follow-up. The studies
differed in respect of matching of the exposed and control groups and in adjustment
265 for confounders. For example, Martín-Núñez et al.19 controlled for diabetic status and
adjusted the analysis for age and gender while Nicoletti et al.16 recruited patients for
the control group who were not age or gender- matched to the intervention group.
Discussion:
270 Principal findings:
Twelve of the fifteen studies included in this systematic review reported significant
effects of BS on patterns of DNA methylation. Four studies reported that weight loss
following BS resulted in methylation levels (gene-specific, or genome-wide) that were
similar to those of the (non-obese) control group, despite the post-BS mean BMI
275 remaining in the obese range (31.2 to 36.4 kg/m2).
Seven inflammation-related genes were investigated in five studies. Although
changes in IL6 gene methylation were seen after BS in individual studies 13,16, meta-
analysis indicated no overall effect (Figure 1). Differences in participant
characteristics, in the follow-up duration and in the degree of weight change
12
280 achieved after BS are likely contributing factors to the differential effects on IL6 gene
methylation. After 12 months, levels of methylation of IL1B and TNF were increased
in response to BS-induced weight loss13. In a more recent study by Gonzales et al.18,
methylation of NF-kB was significantly higher 6 months after BS. In addition,
Gonzales et al.18 reported a significant correlation between high sensitivity CRP (hs-
285 CRP) as an indicator of inflammation and methylation of SLC19A1, a gene that link
inflammation and insulin resistance.31
SCD, encoding Stearoyl-CoA Desaturase, is part of a regulatory mechanism for
cellular inflammation and has been implicated in severe inflammatory disorders
including dermatitis and colitis32. In addition, increased expression of SCD has been
290 associated with insulin resistance and obesity. Morcillo et al.17 reported increased
methylation levels of SCD within the promotor region 6 months after BS which were
similar to the levels observed in the control group. Increased SCD methylation was
also observed following weight loss through dietary intervention by Martín-Núñez et
al.33, which suggests that the methylation response observed after BS may be due to
295 the resulting weight loss.
SORBS3 is a tumor suppressor gene and inhibitor of cell growth34. Lower SORBS3
gene expression can lead to mitochondrial dysfunction, which can be induced by
chronic inflammation and obesity35. Day et al.28 reported a reduction in SORBS3
methylation post-BS that was accompanied by increased expression, suggesting that
300 SORBS3 expression changes induced by DNA methylation may contribute to the
anti-cancer effect of weight loss after BS.
Strikingly, after BS, methylation of PDK4 increased to a similar extent in both blood
and skeletal muscles13,27 which was associated with improved insulin sensitivity and
13
glucose metabolism. This finding also suggests that methylation of PDK4 in blood
305 may be a potential surrogate for changes in PDK4 methylation in skeletal muscles
but there was no direct comparison between tissues in either study.
Study limitations:
This review summarizes the available evidence for the impact of weight loss induced
by BS on DNA methylation in several tissues in humans. More than two thirds
310 (71.4%) of the studies assessed methylation in samples from blood or adipose
tissues. Little is known about the effect of BS on DNA methylation in other tissues
and none of the included studies correlated methylation levels between target
tissues and other surrogate tissues. The availability of such data could promote
validation of assays of DNA methylation in more accessible surrogate tissues,
315 facilitating larger population-based studies. In addition, most studies did not
investigate the functional consequences of changes in gene methylation.
There are many sources of heterogeneity in this systematic review. DNA
methylation is gene and site-specific 5 and the effects of interventions are
dependent on the target tissues and the duration of follow-up7. In addition, there
320 were multiple methods and approaches used for assessing DNA methylation. Few
studies were suitable for inclusion in meta-analyses and statistical heterogeneity was
apparent. Furthermore, publication bias could not be assessed due to the small
numbers of studies included for each genetic locus studied. In addition, most of the
studies included a small number of participants, which affects the overall strength of
325 the evidence. Finally, none of the included studies was a randomized controlled trial
which affects the quality of the evidence with a higher risk of bias.
Conclusion:
14
The current evidence supports the hypothesis that obesity-related aberrant patterns
of DNA methylation at specific loci may be mitigated following BS-induced weight
330 loss. These changes are suggestive of improved metabolic and inflammatory profiles
of patients after BS. However, the evidence for the effects of BS on the methylation
at all genomic loci is limited or inconsistent. Little is known about the effects of BS on
tissues other than blood and adipose tissue. In addition, multiple assays and
different genomic loci have been used in investigations of the effects of BS on DNA
335 methylation, which makes it challenging to compare or combine data across studies.
Standardization of outcome measurements would facilitate future research.
Financial Support:
340 This research received no specific grant from any funding agency, or from the commercial or not-for-profit sectors.
Conflict of Interest:
None
Authorship:
345 Khalil ElGendy: formulating the research question, designing the study, carrying out the study, analyzing the data and writing the manuscript
Fiona C. Malcomson: second independent screener of the titles, review of the manuscript
Michael D. Bradburn: critical review of the manuscript and final approval
350 John C Mathers: formulating the research question, designing the study, writing up and critical review of the manuscript, final approval
15
Tables:
Table 1: Effects of bariatric surgery (BS) on DNA methylation in different tissues: 355 Summary of findings
Supplementary Table 1: Newcastle-Ottawa Quality Assessment of the studies included in the meta-analysis
Supplementary Table 2: Correlation between DNA methylation and gene expression: summary of findings
360
Figures:
Figure 1: Forest plot of non-randomized studies investigating the effects of BS on the methylation levels of specific genetic loci using different techniques of quantification of DNA methylation using the Review Manager (v5.3)
365 Supplementary Figure 1: PRISMA flowchart summarizing the results of the search strategy
370
375
16
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