Gene Promoter Methylation Is Associated With Lung Function In The Elderly: The Normative Aging Study

Abstract

Lung function is a strong predictor of mortality. While inflammatory markers have been associated with lung function decrease, pathways are still poorly understood and epigenetic changes may participate in lung function decline mechanisms. We studied the cross-sectional association between DNA methylation in nine inflammatory genes and lung function in a cohort of 756 elderly men living in the metropolitan area of Boston. Participants donated a blood sample for DNA methylation analysis and underwent spirometry at each visit every 3 to 5 y from 1999-2006. We used separate multivariate mixed effects regression models to study the association between each lung function measurement and DNA methylation within each gene. Decreased CRAT, F3 and TLR2 methylation was significantly associated with lower lung function. One interquartile range (IQR) decrease in DNA methylation was associated with lower forced vital capacity (FVC) and forced expiratory volume in one second (FEV 1), respectively by 2.94% (p < 10 (-4)) and 2.47% (p < 10 (-3)) for F3, and by 2.10% (p < 10 (-2)) and 2.42% (p < 10 (-3)) for TLR2. Decreased IFNγ and IL6 methylation was significantly associated with better lung function. One IQR decrease in DNA methylation was associated with higher FEV 1 by 1.75% (p = 0.02) and 1.67% (p = 0.05) for IFNγ and IL6, respectively. These data demonstrate that DNA methylation may be part of the biological processes underlying the lung function decline and that IFNγ and IL6 may have ambivalent roles through activation of negative feedback.

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www.landesbioscience.com Epigenetics 261
Epigenetics 7:3, 261-269; March 2012; © 2012 Landes Bioscience
RESEARCH PAPER RESEARCH PAPER
*Correspondence to: Johanna Lepeule; Email: jlepeule@hsph.harvard.edu
Submitted: 10/12/11; Revised: 12/28/11; Accepted: 12/29/11
http://dx.doi.org/10.4161/epi.7.3.19216
Introduction
Epigenetics describes both modifiable and stable changes in
gene expression control that do not depend on the underly-
ing nuclear sequence.1 The best understood of the epigenetic
mechanisms is DNA methylation, which involves the addition
of methyl groups to cytosine to form 5-methylcytosine. Low
methylation in regulatory sequences, such as regions dense in
CpG dinucleotides named CpG islands and in neighboring
sequences named CpG island shores, has been shown to be
associated with active genes or with genes that are poised to
Lung function is a strong predictor of mortality. While inammatory markers have been associated with lung function
decrease, pathways are still poorly understood and epigenetic changes may participate in lung function decline
mechanisms. We studied the cross-sectional association between DNA methylation in nine inammatory genes and lung
function in a cohort of 756 elderly men living in the metropolitan area of Boston. Participants donated a blood sample
for DNA methylation analysis and underwent spirometry at each visit every 3 to 5 y from 1999–2006. We used separate
multivariate mixed eects regression models to study the association between each lung function measurement and
DNA methylation within each gene. Decreased CRAT, F3 and TLR2 methylation was signicantly associated with lower
lung function. One interquartile range (IQR) decrease in DNA methylation was associated with lower forced vital capacity
(FVC) and forced expirator y volume in one second (FEV1), respectively by 2.94% (p < 10-4) and 2.47% (p < 10-3) for F3 and by
2.10% (p < 10-2) and 2.42% (p < 10-3) for TLR2. Decreased IFNγ and IL6 methylation was signicantly associated with better
lung function. One IQR decrease in DNA methylation was associated with higher FEV1 by 1.75% (p = 0.02) and 1.67%
(p = 0.05) for IFNγ and IL6, respectively. These data demonstrate that DNA methylation may be part of the biological
processes underlying the lung function decline and that IFNγ and IL6 may have ambivalent roles through activation of
negative feedback.
Gene promoter methylation is associated with
lung function in the elderly
The normative aging study
Johanna Lepeule,1,* Andrea Baccarelli,1 Letizia Tarantini,2 Valeria Motta,1,2 Laura Cantone,2 Augusto A. Litonjua,3, 4
David Sparrow,5 Pantel S. Vokonas5 and Joel Schwartz1,3
1Exposure, Ep idemiology and Risk Pr ogram; Departmen t of Environmental Health; H arvard School of Pub lic Health; Boston, MA USA; 2Center of Molecular and G enetic
Epidemiology; Department of Environmental and Occupational Health; Ca’ Granda Ospedale Maggiore Policlinico IRCCS Foundation; Università degli Studi di Milano;
Milan, Italy; 3Channing Laboratory; Brigham and Women’s Hospital; Harvard Medical School; Boston, MA USA; 4Division of Pulmonary and Critical Care Medicine; Brigham and
Women’s Hospita l; Harvard Medical S chool; Boston, MA USA; 5VA Norm ative Aging Study; Veterans Aa irs Boston Healthcar e System and the D epartment of Medi cine; Boston
Universit y School of Medicine; B oston, MA USA
Key words: DNA methylation, genes, spirometry, FEV1, lungs, TLR2, F3, INOS, GCR, OGG1
Abbreviations: %5mC, percentage of 5-methylcytosine; CI, confidence interval; COPD, chronic obstructive pulmonary disease;
CRAT, carnitine O-acetyltransferase; F3, coagulation factor-3, tissue factor; FEV1, forced expiratory volume in one second;
FVC, forced vital capacity; GCR, glucocorticoid receptor; CAM, intercellular adhesion molecule; IFNγ, interferongamma; IL6,
interleukin-6; iNOS, inducible nitric oxide synthase; IQR, interquartile range; MMEF, maximum midexpiratory flow;
mRNA, messenger RNA; NAS, Normative Aging Study; NFκB, nuclear factorkappa-light-chain-enhancer of activated B cells;
NO, nitric oxide; OGG1, 8-oxoguanine DNA glycosylase 1; RNS, reactive nitrogen species; ROS, reactive oxygen species; SD,
standard deviation; TFBS, transcription factor binding sites; TLR, toll-like receptor; UCSC, University of California Santa Cruz
be activated.2-4 Conversely, hypermethylation usually results in
lower gene expression. DNA methylation is known to change
through aging5 and has been associated with age-related dis-
eases including cancer,6,7 atherosclerosis8 and cardiovascular
diseases.9,10
Lung function is tightly related to aging, and starts to decline
in the third decade of life,11 but with different rates of decline
across individuals.12 Lung function is one of the strongest pre-
dictors of cardiorespiratory and cardiovascular health13 and

262 Epigenetics Volume 7 Issue 3
Association between white blood cells type and DNA meth-
ylation. DNA methylation may vary by white cell type, and
hence variations from sample to sample in e.g., the fraction of
cells that are lymphocytes may create differences in methylation
levels (measured in mixed cells) that do not reflect differences in
underlying levels of methylation in the participants. Upon exam-
ination, all genes except CR AT and ICAM exhibited associations
with the proportion of one or more cell types in the blood count
(Tabl e 3). GCR, IL6 and TLR2 were negatively associated with
percent neutrophils and positively associated with percent lym-
phocytes. Conversely, IFNγ and iNOS methylation were posi-
tively associated with percent neutrophils and monocytes (only
iNOS), and negatively associated with percent lymphocytes. F3
methylation was positively associated with percent lymphocytes
and negatively with percent monocytes. OGG1 methylation
was positively associated with percent basophils. Results for the
individual positions within each gene were similar (Ta b le S3).
However, further associations with individual positions were
observed, including negative association of F3 and ICAM meth-
ylation with percent eosinophils, and of IFNγ and OGG1 with
percent monocytes. Hence, for analyses with lung function,
methylation levels were standardized for cell type.
Cross-sectional association between DNA methylation and
lung function. Because lung function decrease with age was very
slow and most participants had only 1 or 2 measurements in the
study period, we were only able to examine the cross-sectional
association of methylation with lung function. Because persons
with a second visit may have represented a healthier subset of
the overall population, we used inverse probability weighting to
adjust for the potential of survival bias. These analyses excluded
persons with chronic lung disease. Decreased DNA methyla-
tion in the mean of all positions for CR AT, F3, iNOS, OGG1
and TLR 2 was associated with lower lung function (Table 4 ).
An IQR %5mC decrease in the mean of all positions tested for
F3 and TLR2 was associated with 2.94% lower FVC (p < 10-4)
and 2.47% lower FEV1 (p < 10-3), and with 2.10% lower FVC
(p < 10-2) and 2.42% lower FEV1 (p < 10-3), respectively.
Decreased DNA methylation of TLR2 was also associated with
2.86% lower MMEF (p = 0.02). Similarly for CR AT and OGG1,
decreased DNA methylation was significantly associated with
lower FEV1 and FEV1/FVC and with lower FVC, respectively.
Associations between iNOS and FVC and FEV1 were borderline
significant (p = 0.07). For ICAM, IFNγ and IL6, associations
with lung function were in the opposite direction: decreased
DNA methylation was associated with better lung function.
Especially, an IQR %5mC decrease in the mean of all positions
tested for IFNγ and IL6 was associated with 1.75% and 1.67%
higher FEV1 (p < 0.05), respectively. Decreased DNA methyl-
ation of IFNγ was also associated with 3.72% higher MMEF
(p < 10-2). Associations between ICAM and FEV1/FVC and
MMEF were borderline significant (p = 0.07). We did not
observe any significant association between DNA methylation of
GCR and lung function.
Similar associations were found in the secondary analyses
looking at individual positions within each gene (Table S 4),
but only some of the positions showed significant associations:
morta l ity,1 3- 15 though the causal pathways are poorly under-
stood.13,16 Faster lung function decline has been associated with
increased risks of hospitalizations related to chronic obstructive
pulmonary disease (COPD),17 which is a leading cause of mortal-
ity in all countries.18 By the year 2020, the prevalence of COPD
is expected to be the third leading cause of worldwide mortality
and the fifth leading cause of morbidity.19
Persons with impaired lung function have been found to
have higher levels of inflammatory markers such as fibrino-
gen,20 C-reactive protein21 and IL6,22 as well as oxidative stress
markers.23 Although inflammation is considered central to the
pathogenesis of airways diseases, the mechanisms responsible for
accelerated lung function decline remain unclear. DNA meth-
ylation can be meta-stable, and can be propagated through cell
division. This can represent a form of cellular memory that deter-
mines the levels of inflammation and oxidative stress. However,
whether changes in DNA methylation are associated with lower
lung function or accelerated lung function decline has not been
investigated.
We leveraged the prospective collection of data and biospeci-
mens in the Normative Aging Study (NAS), in which partici-
pants’ lung function was regularly monitored over a 7 year span,
to examine the relationships between DNA methylation in nine
genes related to inflammation and oxidative metabolism, and
lung function. We hypothesized that decreased methylation
of genes related to inflammation and oxidative stress would be
associated with lower lung function. Given that age is a major
risk-factor for decreased lung function24 and for changes in DNA
methylation,5 we further hypothesized that age could modify the
relationship between DNA methylation and lung function.
Results
Participants were 73.3 ± 6.7 y-old on average. From the 756 par-
ticipants, 368 (49%) had one visit, 290 (38%) had two visits,
97 (13%) had three visits and 1 had four visits (0.13%). Most
of the participants were former smokers (67.2%) or never smok-
ers (28.6%). Mean levels ± SD of forced vital capacity (FVC),
forced expiratory volume in one second (FEV1), FEV1/FVC and
maximum mid-expiratory flow (MMEF) are shown in Table 1.
From the 388 participants with more than one visit, the aver-
age time between the first and last visit was 4 y and 7 mo and
the average change in lung function during this time was 0.007
L for FEV1, 0.08 L for FVC, -1.5 for FEV1/FVC and -33.1 L/
min for MMEF. The distribution of blood DNA methylation for
each of the nine genes (Carnitine O-acetyltransferase (CR AT),
coagulation factor-3 (F3), glucocorticoid receptor (GCR), inter-
cellular adhesion molecule (ICAM), interferon-gamma (IFNγ),
interleukin-6 (IL6), inducible nitric oxide synthase (iNOS),
8-oxoguanine DNA glycosylase 1 (OGG1) and toll-like recep-
tor-2 (TLR2)), expressed as the percentage of 5-methylcytosine
(%5mC), is described in Table 2 and in Tabl e S1 in the online
Supplement. In general, the correlations between the individual
positions within each gene were low (<0.46), except between
position 1 and 2 within CRAT, ICAM, IFNγ and IL6 where the
correlation was higher than 0.7 (Table S2).

www.landesbioscience.com Epigenetics 263
Table1. Lung function and characteristics of the 756 men participating to the Normative Aging Study, 1999–2006, Boston
All visits (n = 1243) 1st visit (n = 756) 2nd visit (n = 388) 3rd visit (n = 98)
Outcomes
FVC ± SD, liters 3.3 ± 0.7 3.3 ± 0.7 3.4 ± 0.7 3.5 ± 0.7
FEV1 ± SD, liters 1st sec 2.5 ± 0.6 2.5 ± 0.6 2.5 ± 0.6 2.6 ± 0.6
FEV1/FVC ± SD 74.9 ± 8.0 75.4 ± 8.0 74.0 ± 8.3 75.1 ± 6.6
MMEF ± SD, liters min 239.7 ± 107.6 246.9 ± 112.2 228.7 ± 101.7 228.6 ± 88.1
Adjustment factors
Age ± SD (p25, p75), yr 73. 3 ± 6.7 (68, 78) 72.2 ± 6.8 74.8 ± 6.4 75.9 ± 5.4
Race, n (%)
Black 23 (1.9) 14 (1. 8) 6 (1.6) 3 (3 .1)
White 120 7 ( 97.1) 7 34 (9 7.1) 378 (97. 4) 94 (95.9)
missing 13 (1.0 ) 8 (1.1) 4 (1.0) 1 (1.0)
BMI ± SD, kg/m228.0 ± 4.1 28.2 ± 4.1 27.7 ± 4.1 27.4 ± 3.7
Height ± SD, cm 173.3 ± 7.2 173.5 ± 6.9 173.3 ± 7.5 172.4 ± 7.9
Weight ± SD, kg 84.3 ± 14.2 85.1 ± 14.2 83.5 ± 14.3 81.7 ± 19.7
Education, n (%), y
<12 46 (3.7) 30 (4.0) 13 (3.4) 3 (3 .1)
12 304 (24.5) 183 (24. 2) 96 (24.7) 25 (25.5)
13–15 3 61 ( 29.0) 214 (28.3) 112 (28.9) 34 (34.7)
>15 525 (42.2) 322 (426) 167 (43.0) 36 (36.7)
missing 7 (0.6) 7 (0.9) 0 (0.0) 0 (0.0)
Smoking status, n (%)
Never 366 (29.4) 216 (28.6) 115 (29. 6) 35 (35.7)
Current 50 (4.0) 32 (4.2) 16 (4 .1) 2 (2.0)
Former 827 (6 6.5) 508 (6 7. 2) 257 (66.2) 61 (62.2 )
Packs years* ± SD 20.5 ± 25.7 21.7 ± 26.9 19.8 ± 24 .6 14.4 ± 19.7
Season, n (%)
Spring (March–May) 290 (23.3) 181 (23.9) 95 (24.5) 14 (14.3)
Summer (June–Aug) 351 (28. 2) 213 (2 8.2 ) 105 (27.1) 32 (32.7)
Fall (Sep t-N ov) 407 (32.7) 234 (30.9) 134 (34.5) 39 (39.8)
Winter (Dec-Feb) 195 (15.7) 128 (16. 9) 5 4 (13.9) 13 (13 . 2)
Day of the week, n (%)
Tuesday 71 (5.7) 71 (9.4) 0 (0.0) 0 (0.0)
Wednesday 333 (26.8) 167 (2 2.1) 12 0 (30. 9) 0 (0.0)
Thursday 617 (49. 6) 329 (43.5) 235 (60.6) 45 (45.9)
Friday 222 (17.9) 189 (25.0) 33 (8.5) 53 (5 4.1)
Blood count
Neutrophils ± SD, % 62.2 ± 8.6 62.0 ± 8.6 62.6 ± 8.6 61.6 ± 8.0
Lymphocytes ± SD, % 25.5 ± 7.9 25.6 ± 7.8 25.0 ± 7.9 26.4 ± 7.7
Asthma, n (%) 83 (6.7) 45 (6.0) 31 (8 .0) 7 (7.1)
Chronic bronchitis, n (%) 81 (6.5) 52 (6.9) 24 (6.2) 5 (5.1)
Emphysema, n (%) 42 (3.4) 29 (3.8) 11 (2.8) 2 (2.0)
Positive methacholine challenge test, n (%) 115 (1 0 .9) 7 3 (11. 5) 36 (10.9 ) 6 (6.7)
Missing 184 (14.8) 119 (15.7) 57 (14.7) 8 (8.2)
Corticosteroids, n (%) 90 (7.2) 50 (6.6) 33 (8.5) 7 (7.1)
Sympathomimetic α and β, n (%) 95 (7.6) 54 (7.1) 37 (9.5) 4 (4.1)
Anticholinergic, n (%) 30 (2.4) 14 (1.9 ) 14 (3.6) 2 (2.0)
*Among current or former smokers.

264 Epigenetics Volume 7 Issue 3
genes or across positions within the genes, such that the asso-
ciation between DNA methylation and lung function was
sometimes more deleterious for younger and sometimes more
deleterious for older participants.
Sensitivity analyses. The sensitivity analyses including par-
ticipants with chronic lung diseases showed similar associations
between lung function and DNA methylation as the main analy-
ses, with only slight variations in significance (Table S5). The
association with lung function was no longer significant for the
mean of OGG1 and decreased DNA methylation in the mean of
ICAM became significantly associated with better lung function.
For the individual positions, the associations were consistent with
those observed in the main analysis (Ta ble S6).
When adjusting for the proportions of different types of
white blood cells that were associated with gene’s methylation
(Tabl e 3), the associations between the mean of ICAM methyla-
tion and lung function were no longer borderline significant (p =
0.15 with FEV1/FVC and p = 0.12 with MMEF). Furthermore,
the individual positions of ICAM that had been significantly
associated with lung function in the main analysis appear to be
borderline significant in the sensitivity analysis (p = 0.07 for pos
1 and FEV1/FVC, p = 0.05 for pos 3 and FEV1 and MMEF).
position 2 in CRAT, positions 3–5 in F3, position 1 in IL6 and
iNOS, positions 1, 2 and 4 in OGG1, position 1 and 3–5 in
TLR2. The only case where a significant association was seen
with an individual position that was not apparent using the mean
of all positions was for ICAM, where decreased methylation at
positions 1 and 3 was significantly associated with higher FEV1,
FEV1/FVC and MMEF.
Modification by age of the association between DNA
methylation and lung function. Of the genes with significant
main effects the following associations were modified by age
(p ≤ 0.05): mean TLR2 and FVC, position 5 in TLR2 and
FVC and FEV1, position 4 in OGG1 and FEV1, mean IFNγ
and FEV1, position 4 in F3 and FEV1/F VC (Table S5). For
these genes and positions, the association was stronger in older
people. Hence decreased methylation in TLR2 and OGG1 was
associated with a greater decrement in lung function in older
people than in younger ones, while decreased methylation in
IFNγ and F3 was associated with a greater increment in lung
function in older people than in younger ones. There were some
genes/positions for which there was no main effect, but where
a significant effect modification was seen with age. However,
in these cases the effect of age was not consistent either across
Table2. Descriptive statistics of DNA methylation (percentage of 5-methylcytosine) by visit for 756 men participating to the Normative Aging Study,
Boston, 1999–2006
Gene All visits (n = 1243) 1st visit (n = 756) 2nd visit (n = 388) 3rd visit (n = 98)
nmean ± SD (p25–p75) IQR mean ± SD mean ± SD mean ± SD
CR AT 115 0 3.2 ± 1.1 (2.4–3.9) 1.5 3.1 ± 1.0 3.3 ± 1.2 3.8 ± 0.9
F3 1093 2.4 ± 1.2 (1. 6 –3.0) 1. 4 2.2 ± 1.2 2.6 ± 1.2 3.2 ± 1.0
GCR 1034 47.2 ± 5.8 (44.0 –50.2) 6.2 47.0 ± 5.7 47.4 ± 6.4 48.1 ± 3.2
ICAM 926 4.3 ± 1.9 (3.1–5.2) 2.1 4.4 ± 1.9 4.3 ± 2.0 4.2 ± 1.0
IFNγ119 6 84.7 ± 5.3 (82.0–88.2) 6.2 84.4 ± 5.5 85.1 ± 5.0 85.2 ± 4.2
IL6 1202 43.4 ± 10.4 (3 7.1 – 49 . 3) 12. 2 43.7 ± 10.7 42.7 ± 10.1 43.1 ± 9.4
iNOS 792 69.7 ± 6.4 (65 .9–74.1) 8.2 70.1 ± 6.6 68.6 ± 6.1 71.4 ± 5.3
OGG1 745 2.3 ± 1.2 (1.4 –2. 9) 1.5 2.1 ± 1.1 2.3 ± 1.3 3.4 ± 1.2
TLR2 996 3.1 ± 1.3 (2.1–3.8) 1.7 3.1 ± 1.2 3.0 ± 1.4 3.1 ± 1.4
Table3. Unadjusted association between white blood cells counts (%) and DNA methylation (percentage of 5-methylcytosine) in 1243 visits under-
went by 756 men participating to the Normative Aging Study, Boston, 1999–2006
Gene White blood cell (%)
Neutrophils Lymphocytes Basophils Monocytes Eosinophils
βp value βp value βp value βp value βp value
CR AT -0.03 0.43 0.06 0 .14 -0.75 0.23 -0.08 0.63 -0.25 0 .11
F3 -0.06 0.20 0.11 0.03 0.32 0.66 -0.57 <10-2 - 0 .11 0.53
GCR -0.60 <10-2 0.98 <10-4 -0. 31 0.93 -1.48 0 .10 -1. 63 0.07
ICAM 0.06 0.40 -0.004 0.96 0.91 0.43 -0.28 0.36 - 0.53 0.07
IFNγ2.64 <10-4 -3.10 <10-4 -0.63 0.82 -1.2 8 0.10 0.54 0.48
IL6 -0.75 0.03 1.12 <10-2 -1. 59 0.73 0.003 1.0 0 -0.83 0.54
iNOS 0.68 0.02 -1.02 <10-3 -2.0 6 0.62 2.54 0.02 0.61 0.57
OGG1 -0. 02 0.68 0.03 0.62 2.16 0.02 -0.25 0.26 0.22 0.30
TLR2 -0.16 <10-2 0.18 <10-3 0.46 0.58 0 .18 0.38 - 0.06 0.78
β is expressed for a 10% increase in percent of white blood cells type.

www.landesbioscience.com Epigenetics 265
coagulation-inflammation cycle;30 hypomethylation of F3 there-
fore constitutes a prothrombic and proinflammatory state that
may also increase the risk for cardiovascular events, which are a
major cause of mortality in COPD.
TLR2 and iNOS, which are also related to inflammation,
were associated with lower FVC and FEV1 in the present study.
The TLR2 gene encodes a membrane protein that regulates the
activation of innate immunity. The highest constitutive expres-
sion of TLR2 is observed in peripheral blood leukocytes,31 but
TLR2 is also strongly expressed in the lungs. Consistent with our
results, other studies have reported higher expression of TLR2
in monocytes32 and neutrophils33 in COPD patients compared
with healthy patients, and a positive association between TLR2
expression and inflammatory lung diseases has been demon-
strated in mice.34 Decreased lung function is characterized by
airflow obstruction and inflammatory responses involving neu-
trophils production through the activation of TLR2, TLR4 and
TLR9. TLRs detect pathogen- or damage-associated molecular
patterns and trigger the production of NFκB cells which then
stimulate the production of inflammatory chemokines and cyto-
kines.33 These events also stimulate the production of iNOS, the
inducible form of nitric oxide (NO) synthase, which releases NO.
iNOS methylation has been associated with gene silencing.35
Increased iNOS production is seen in inflammation as well as in
the bronchi36 and in induced sputum37 of COPD patients com-
pared with healthy subjects, consistent with our finding of lower
iNOS methylation in subjects with lower lung function. Brindicci
et al. showed increased iNOS protein expression at all stages of
COPD, whereas iNOS mRNA was increased at earlier stages of
COPD, but decreased in patients with severe disease. NO is a free
radical that can interact with Reactive Oxygen Species (ROS) to
form Reactive Nitrogen Species (RNS), which may both have
deleterious effects for cell components and enhance inflamma-
tion, and lead to lung disorders such as COPD.38,39
We also found that lower CRAT and OGG1 (mean and posi-
tions 2 and 4) methylation was associated with lower lung func-
tion. Oxidative stress ha s been identified as one of the mechanisms
Also, the mean of OGG1 was no longer associated with FVC
(p = 0.08).
When further adjusting for cardiovascular diseases, diabetes
and hypertension, the mean of OGG1 was no longer associated
with FVC (p = 0.08) and the mean of IL6 was no longer associ-
ated with FEV1 (p = 0.07). The associations between the mean
of iNOS and FVC and FEV1 were a bit stronger (p = 0.04 and
p = 0.05 respectively). Results for the individual positions were
consistent with the main analysis.
Discussion
In the present cohort of elderly men, we found that decreased
DNA methylation in the mean of all positions tested for CR AT,
F3 and TLR2 was associated with lower lung function metrics,
especially FVC and FEV1, which are related to large airways.
We also observed borderline-significant associations in the same
direction for mean iNOS and OGG1 methylation. In contrast,
decreased DNA methylation in the mean of all positions tested
for IFNγ and IL6 was associated with better lung function in
both large and small airway markers (MMEF). We also observed
borderline-significant associations in the same direction for mean
ICAM.
We found that decreased F3 methylation was associated with
lower lung function. The F3 gene encodes coagulation factor III,
a cell surface glycoprotein with major roles in initiating the blood
coagulation cascade, chemokine production,25 pro-inflammatory
effects and innate immunity.25,26 There is no experimental data
to support the assumption that methylation in F3 promoter regu-
lates the gene. However, the sequence we analyzed was previously
suggested to have that role based on sequence characteristics.27
F3 is expressed in monocytes and neutrophils, which are both
involved in adhesion, migration and spreading and tend to accu-
mulate at inflammatory sites.28,29 Consistent with our results,
higher F3 levels in whole blood have been shown in patients
with COPD compared with healthy subjects.26 Moreover,
inflammation also promotes coagulation which accentuates the
Table4. Percent change in lung function associated* with an interquartile range decrease in gene-specific DNA methylation (percentage of 5-methyl-
cytosine) in 510 men without any chronic respirator y condition† participating to the Normative Aging Study, Boston, 1999–2006
Gene n observations/n subjects FVC FEV1FEV1/FVC‡MMEF
%p value %p value change p value %p value
CR AT 755/479 -0.99 0.21 -1.68 0.04 -0.51 0.04 -1. 51 0. 33
F3 716/4 66 -2.94 <10-4 -2.47 <10-3 0.34 0.13 1 .11 0 .41
GCR 68 9/4 41 - 0.78 0.22 -1. 2 2 0.07 - 0.31 0 .14 -1. 01 0.42
ICAM 621/413 0. 31 0.68 0.88 0.29 0.45 0.07 2.94 0.07
IFNγ784/489 1.32 0.07 1.75 0.02 0.36 0.12 3.72 <10-2
IL6 793/4 86 1. 09 0 .17 1.67 0.05 0.38 0.15 2.90 0.07
iNOS 517/ 363 -1. 83 0.07 -1.93 0.07 - 0.03 0.93 - 0 .31 0.87
OGG1 513/ 367 -1.37 0.05 -1.36 0.08 0.02 0.92 0.14 0.92
TLR2 66 2/431 -2.10 <10-2 -2.42 <10-3 -0.24 0.23 -2.86 0.02
*Results were adjusted for age (continuous), race (white/black), log(height), standardized weight (linear and quadratic term), % neutrophils, % leu-
cocytes, education level (<12, 12, 13–15 and >15 y), smoking status (former/current/never), cumulative smoking (continuous), season of the medical
exam (indicator variable), day of the week, corticosteroids (Y/N), sympathomimetic α and β (Y/N), anticholinergics (Y/N). †Subjects with asthma, chronic
bronchitis, emphysema or positive methacholine test were excluded. ‡Absolute change in FEV1/FVC.

266 Epigenetics Volume 7 Issue 3
SP1, Inil, EBF1, HEY1, BATF, BAF155, TAF1 and STS2). This
might help to explain the differences we saw between position 3
and positions 1–2 (Table S 4 ).
We found that age significantly modified the associations
between lung function and some of the genes. The associations
between DNA methylation and lung function were somewhat
stronger for older people for genes with main effects. For genes
without main effects, the patterns were not consistent either
across genes or across positions within genes (Table S5). This
suggests the association of methylation of some genes with lung
function may get stronger with age. Few studies have described
intra-individual changes over time in DNA methylation.50,51
Interestingly, Madrigano et al. found that longitudinal change
in age was associated with decreased methylation in GCR and
INOS and with increased methylation in CR AT, F3, IFNγ and
OGG1 in the same cohort as we investigated here.50 They also
showed that the cross-sectional effect of age (between individu-
als) was associated with increased TLR2 methylation, whereas
the longitudinal effect of age (within individuals) was associated
with decreased TLR2 methylation, which means that the effect of
age on DNA methylation might be difficult to disentangle. The
changes they observed in DNA methylation were quite small,
and given this and the small changes in lung function between
visits, we did not think it feasible to examine the association of
changes in methylation and changes in lung function.
While our study cannot draw conclusions on how gene
expression relates to lung function, our results in an aging healthy
population confirm that inflammation and oxidative stress are
key features that drive lung function decline and that meth-
ylation of inflammatory genes is part of these processes, which
involve a complex cascade of interactions between signaling cells.
However, the present study has a number of limitations. While
our results demonstrate that methylation patterns were associ-
ated with lung function characteristics, whether methylation
patterns is a cause or a consequence of the changes in lung func-
tion cannot be determined in our study design. We currently do
not have enough years of follow-up to examine methylation and
change in lung function. DNA methylation may be a biomarker
of lung function or may mediate the effects of environmental
factors such as smoking or air pollution that are associated with
lung function decline. How methylation patterns of these genes
relate to changes in lung function throughout the life course and
how the DNA methylation relates to expression for these genes
requires further investigation.
In this study, DNA methylation was measured in white blood
cells, and while these cells infiltrate the lungs, it is not clear to
what extent the observed changes in white blood cells DNA
methylation reflect similar changes in lung tissues and airways.
Several studies have shown elevated levels of inflammatory cyto-
kines in circulating blood associated with COPD, suggesting an
overspill of inflammatory mediators from peripheral lung tis-
sues,52 but this might also be due to other inflammatory diseases
commonly seen with aging and in subjects with impaired lung
function. However, a recent study found elevated circulating lev-
els of surfactant protein D, which is specific from lung tissues and
therefore provides more evidence for the overspill hypothesis.53,54
responsible for decreased lung function, including in persons free
of COPD. Previous work in this cohort has shown that statin use,
which in addition to lowering cholesterol has antioxidant activity,
was associated with higher than predicted lung function, inde-
pendent of chronic respiratory disease.40 OGG1 encodes a DNA
glycosylase enzyme expressed in the lungs and involved in exci-
sion of 8-oxoguanine resulting from ROS exposure. Free radicals
resulting from lung inflammatory responses can contribute to
lung damage and decreased lung, while also resulting in oxida-
tive stress that may activate OGG1 as suggested by our results.
In contrast, Liu et al. found no association between OGG1 poly-
morphisms and COPD susceptibility.41 CRAT is an enzyme play-
ing an important role in metabolic processes. Carnitine decrease
has been associated with insulin resistant states and aging.42
We found that decreased methylation of ICAM, IFNγ and
IL6 was associated with a better lung function. As these three
genes are related to pro-inflammatory processes, we would have
expected lower DNA methylation to be associated with lower
lung function. ICAM encodes a cell surface glycoprotein that is
more expressed during inflammatory responses.43 IFNγ is a cyto-
kine, whose hypomethylation has been shown to suppress gene
expression.44 IFNγ is a mediator in innate and adaptive immu-
nity that regulates a variety of pro-inflammatory parameters.
However, it also has anti-inflammatory properties, which con-
fer an ambivalent role,45 and reduced IFNγ production has been
shown in asthmatic patients.46 In the same cohort we investigated
here, serum levels of IFNγ and IL6 were not associated with pul-
monary function of healthy subjects47 although another study
found serum level of IL6 associated with impaired FEV1.48 IL6
encodes a protein that acts as both a pro-inflammatory and anti-
inflammatory cytokine.49 The consistent results between IFNγ
and IL6 might reflect their concomitant variation in pro- and
anti-inflammatory processes. Inflammatory processes involve the
production of a myriad of signaling cells, which makes difficult
disentangling the specific role of each gene.
Results for iNOS, OGG1, IL6 and ICAM sometimes differed
among the individual positions. We searched for Transcription
Factor Binding Sites (TFBS) in these genes using the University
of California Santa Cruz (UCSC) genome browser (genome.
ucsc.edu). There was no TFBS and no SNP near our target
iNOS sequence. For OGG1, the CpG positions analyzed were
in proximity of a variety of TFBS including: HA-E2F1, NRSF,
Pol2, Pol2-48H, AP-2Alpha, AP-2Gamma, TAF7, TAF1, TBP,
P300, CTCF, HMGN3, NFKB, SMC3, CCNT2 (positions
2–4), Sin3Ak-20 (positions 2–4) and HEY1 (position 4). For
IL6, TFBS (BAF155, Inil, c-Myc, BAF170, Max, NRSF and
Nrf1) were present for position 1 and one SNP was observed
(C/T), whereas position 2 was free of TFBS and SNP, which
may explain the different results we observed for these two
positions. For ICAM, all positions were SNPs free and TFBS
located near positions 1 and 2 were similar (NFκB, Pol2-4H8,
PAX5-C20, EBF, Pol2, IRF4, BCL11A, PAX5-N19, TCF12,
E2F6, ELF1, MEF2a, p300, EBF1 (position 2 only)). However,
TFBS Pol2-4H8, PAX5-C20, EBF, Pol2, PAX5-N19, E2F6,
ELF1, MEF2a and EBF1 were not observed near position 3 and
different TFBS were observed (POU2F2, BCL3, STAT1, PU.1,

www.landesbioscience.com Epigenetics 267
or known to be associated with aging and age-related diseases;
and (2) expressed at variable degree in leukocytes,58 the DNA
source used in our study. The procedure for DNA methylation
analysis is detailed in the supplementary material online. For
each gene, we measured between 1–5 CpG sites (positions) and
used the mean of DNA methylation at all positions for our base-
line analysis. Individual positions were examined in a secondary
analysis.
Statistical analysis. Lung function measurements FVC, FEV1
and MMEF were log-transformed to increase normality and sta-
bilize variance. A mixed linear model was used to account for the
correlation among measurements within the same subject:
Yit = β0 + ui + β1 DNA methylationit + β2X2it + … + βpXpit + εit
(1)
where Yit was the lung function measurement for subject i at visit
t, β0 was the overall intercept, ui was the separate random inter-
cept for subject i, X2 it -Xpit were the p - 1 covariates for subject i at
visit t. Adjustment factors included in the model were age, height
(log) and standardized weight (linear and quadratic term), race
(white, black), education level (<12, 12, 13–15 and >15 y), ciga-
rette smoking (current, former, never) and pack-years, chronic
lung conditions (asthma, emphysema, chronic bronchitis),
methacholine responsiveness, medication use in yes/no (cortico-
steroids, sympathomimetics α and β, anticholinergics), percent
lymphocytes and neutrophils, season (indicator variables) and
day of the week. A p value of < 0.05 was considered statistically
significant. To exclude associations between DNA methylation
and lung function merely due to differences in the proportions of
white blood cell types, we adjusted all models below for percent
neutrophils and lymphocytes. Separate models were fit for each
combination of lung function and DNA methylation measures.
An association between the dependent variable and a covariate
was considered to be significant if the covariate had a p value <
0.05 in the model.
To determine if the association between DNA methylation
and lung function was modified by age, models with an interac-
tion term between DNA methylation and age, along with the
main effects, were run. If there was a significant interaction
(p < 0.05), effects estimates of methylation on lung function were
then calculated as the effect of DNA methylation at the 25th and
75th percentile of the distribution of age. We present the estimated
effect of methylation on lung function as the percent change in
FVC, FEV1 and MMEF and as the unit change in FEV1/FVC for
an interquartile range decrease in methylation.
We excluded from the main analysis participants with asthma,
emphysema, chronic bronchitis or positive methacholine tests or
with missing values for these chronic conditions. A sensitivity
analysis further adjusted for these medical conditions was per-
formed on the whole sample. In addition, we further adjusted
for cardiovascular diseases, diabetes and hypertension. For each
gene, we performed another sensitivity analysis adjusting the
models for the cell type proportions of white blood cells that were
associated with gene’s methylation.
To adjust for the fact that healthier men are more likely to
come back to subsequent visits, we used inverse probability
weighting to correct for a potential survival bias.59 We calculated
Blood is an easily accessible biological sample and because the
neutrophilic inflammation is an early component of lung func-
tion decline and because the genes under study are known to be
expressed in neutrophils, measuring DNA methylation in white
blood cells may be a relevant marker of inflammatory processes
in the lungs.
Since we performed multiple tests with 4 related outcomes
and 34 DNA methylation exposures (9 related genes with sev-
eral positions for each), we would expect 7 false positives, which
means that among the 48 significant associations we observed,
7 of them might be wrong. Moreover, since our cohort consists
of mainly white elderly men, our findings may not be general-
izable to other populations. How DNA methylation would be
associated with lung function in younger population, women or
in other ethnicities remain to be determined.
Epigenetic mechanisms such as DNA methylation are increas-
ingly recognized to play a role in chronic diseases. For the first
time, methylation differences in CR AT, F3, IFNγ, IL6, INOS
and TLR 2 genes have been associated with lung function char-
acteristics, particularly with large airways flow, in a cohort of
elderly men. These associations were somewhat modified by
age. Our results provide new insights regarding the biological
processes related to changes in lung function which strongly
determine mortality by complex mechanisms that are not fully
understood.
Materials and Methods
Study population. This study included 756 men examined
between March 1999–December 2006, in the NAS, a lon-
gitudinal closed-cohort of aging established by the Veterans
Administration in 1963.55 Participants were free of known
chronic medical conditions at enrolment, returned for examina-
tions every 3 –5 y, and were asked to give a DNA sample from
7-ml blood at each visit between 1999 and 2006. Height, weight
and medication use were assessed and pulmonary disorders
(asthma, chronic bronchitis, emphysema) and smoking history
were collected through American Thoracic Society question-
naire.56 Participants provided written informed consent and the
study protocol was approved by the Institutional Review Boards
of all participating institutions.
Lung function. Spirometric tests were performed as previously
reported in reference 57, following a strict protocol in accordance
with American Thoracic Society guidelines.
We used data from the most recent (1999–2000) methacho-
line challenge tests available for each subject at that visit.
DNA methylation analysis. Given the strong relation between
aging and both DNA methylation and lung function and given
the lack of studies reporting the links between DNA methyla-
tion and lung function, we leveraged an ongoing study on aging
in which methylation was measured in nine genes: CR AT, F3,
GCR, ICAM, IFNγ, IL6, iNOS, OGG1, TLR2. The aim of the
cohort was to study respiratory and cardiovascular outcomes. In
order to cover the broad range of these outcomes, we selected
nine genes among those: (1) related to respiratory and cardiovas-
cular outcomes; or related to inflammation and oxidative stress;

268 Epigenetics Volume 7 Issue 3
Institute of Environmental Health Sciences grants ES015172-01,
2RO1 ES015172-6 and ES00002.
The VA Normative Aging Study is supported by the
Cooperative Studies Program/Epidemiology Research and
Information Center of the US. Department of Veterans Affairs
and is a component of the Massachusetts Veterans Epidemiology
Research and Information Center, Boston, MA.
Note
Supplemental material can be found at:
www.landesbioscience.com/journals /epigenetics/article/19216/
the probability of having a second visit or a third visit using logis-
tic regressions given all relevant factors at the previous visit: age,
education level, body mass index, smoking status and pack-years,
hypertension, cholesterol, diabetes, FEV1 (further adjusted for
asthma, emphysema, chronic bronchitis and result of methacho-
line test for the sample including participants with these chronic
conditions). The probability at first visit was 1. We then used the
inverse of the predicted probabilities as the weights. We used SAS
version 9.2 (SAS Institute, Cary, NC).
Acknowledgments
We thank Tania Kotlov, data programmer at the Harvard
School of Public Health. We also thank the participants for their
collaboration. Financial Support
This work was supported by the US Environmental Protection
Agency grants R832416 and RD83479801 and by National
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