In a cancer-free population, we examined the association between age, gender, race/ethnicity, body composition, diet and lifestyle factors and global genomic methylation in leukocyte DNA and found significant differences in global leukocyte DNA methylation by gender and race/ethnicity.
The impact of aging on DNA methylation has been well documented.11
Aging cells show a progressive loss of 5-methylcytosine content.12,13
Recent studies have found that CpG islands tended to gain methylation whereas non-island CpGs lose methylation with increasing age.10
The overall impact of aging on methylation was to reduce the global level of genomic DNA methylation, and this age-related decline in DNA methylation appears to be similar across tissue types.10
In our study population aged 45–75 years old, the age-related trend in global DNA methylation was in the expected direction but not statistically significant. An age range wider than 45–75 years or a larger sample size may have improved power to detect a significant impact of aging on global DNA methylation.
We found women had a significantly lower level of methylation than men. Although the absolute difference in global genomic DNA methylation is small (1.8%), it is in agreement with three previous studies measuring LINE-1 methylation in peripheral leukocytes. For example, Hsiung et al. reported a 1.17% significantly lower level of LINE-1 methylation in women than in men.2
El-Maarri et al. reported the mean differences in LINE-1 methylation by gender ranging between 1.61% and 5.80%: all were significantly lower in women than in men.14
A combined analysis from five studies by Zhu et al. identified a 0.8% significantly lower level of LINE-1 methylation in women than in men.15
The reason for this gender specific difference in global DNA methylation is not clear. One of the two chromosomes in women is heavily methylated and transcriptionally inactivated. It has been proposed that X chromosome inactivation may deplete resources required for properly methylating autosomal loci.14
Lower levels of global methylation in women may also be due to different levels of dietary folate or other one-carbon nutrients in men and women. In our study population, the median level of dietary folate equivalent was lower in women than in men (397.8 vs. 423.7 µg/day); however, the difference was not statistically significant after controlling for the effect of total energy intake (p = 0.19). Women may also have a higher folate requirement than men because of regular loss of red blood cells through menstruation but it is unlikely that a higher folate requirement in this population of mostly postmenopausal women can explain the observed gender-specific difference in global methylation. Alcohol drinking and dietary intake of other one-carbon nutrients (vitamins B2
and methionine) did not differ significantly between men and women in this study.
The potential racial/ethnic difference in global DNA methylation has not been widely studied. Hsiung et al. studied 526 healthy individuals who served as controls in a case-control study of head and neck cancer and found a 1.26% significantly higher level of leukocyte LINE-1 methylation in non-Caucasians compared to Caucasians.2
In contrast to their findings, we observed a 2.2% lower level of leukocyte LINE-1 methylation in non-Hispanic blacks and 1.3% lower level of leukocyte LINE-1 methylation in Hispanics as compared to non-Hispanic whites although the association for Hispanics did not reach statistical significance. In our study, different racial/ethnic groups had comparable levels of daily intake of dietary folate equivalent and other one-carbon nutrients. Previous studies demonstrated that MTHFR C677T
polymorphism is more prevalent in Hispanics, compared to non-Hispanics and non-Hispanic blacks carry the lowest frequency of MTHFR
The MTHFR C677T
polymorphism is associated with reduced activities of methylenetetrahydrofolate reductase and disruption in DNA methylation. Hispanics may therefore have a lower level of global DNA methylation due to higher frequency of the MTHFR C677T
polymorphism. However, we observed the lowest levels of global methylation in non-Hispanic blacks despite the fact that non-Hispanics blacks carry the lowest frequency of the MTHFR
C677T polymorphism. Therefore, the different frequency in MTHFR
C677T polymorphism across racial/ethnic groups cannot account for observed differences in global DNA methylation in this study. Other genetic polymorphisms of folate-metabolizing enzymes such as MTHFR
A66G and CBS
844ins68 also vary in minor allele frequencies in different racial/ethnic groups17
but their functionality on folate metabolism is less pronounced than that of MTHFR
C677T. For example, Yang et al. reported that the MTHFR
C677T polymorphism had a significant effect on serum folate and homocysteine concentrations across all racial/ethnic groups but the impact of other genetic polymorphisms in one-carbon metabolism was heterogeneous in different racial/ethnic groups.17
The biochemistry of folate metabolism is complex and genetic polymorphisms in folate metabolism may have an interactive effect on global DNA methylation. Some unknown genetic or environmental factors may also contribute to the racial/ethnic difference in global DNA methylation. Since we did not measure genetic polymorphisms in this study, our interpretation is largely speculative. Future studies investigating genetic polymorphisms along with global DNA methylation may help elucidate the difference in global DNA methylation by race/ethnicity.
Few studies have investigated the association between obesity and global leukocyte DNA methylation. In animal models, high-fat diet-induced obesity was found to modify the methylation level of the leptin promoter in adipocytes.18
Levels of pro-inflammatory cytokines such as IL-6 and TNFα are often elevated in obese individuals. Recent studies also indicated a good correlation between levels of leukocyte DNA methylation and degrees of inflammation, suggesting DNA methylation may represent a novel pathway underlying the association between obesity, inflammation and cancer risk.19,20
Although BMI is the most commonly used measure for obesity, it does not adequately capture body fat mass and the distribution of body fat. We characterized body composition using % body fat and areas of subcutaneous fat (SAT) and visceral fat (VAT) (cm2
). Interestingly, we found central obesity, subcutaneous fat, SAT/VAT ratio, % fat mass and FM/FFM ratio were significant predictors for low levels of LINE-1 methylation but no associations remained after adjusting for age, gender and race/ethnicity. Gender and race/ethnicity were strongly associated with LINE-1 methylation and appeared to confound the association between body composition and LINE-1 methylation. And gender showed a stronger confounding effect than race/ethnicity. As shown in , the inclusion of gender in the model substantially attenuated the associations between body composition and methylation and the associations became statistically insignificant. However, the associations were only slightly attenuated and remained statistically significant or borderline significant after adjusting for race/ethnicity. Age had a minimal confounding effect of the association between body composition and global DNA methylation. Because the majority of our study population is overweight or obese, we have limited statistical power to detect the impact of obesity on global methylation.
Consistent with previous studies, our study did not support an association between smoking, alcohol drinking and leukocyte DNA methylation.2,3,5,15
However, Smith et al. reported that levels of LINE-1 methylation were significantly associated with smoking and alcohol drinking in tissues of squamous cell head and neck cancer.21
This suggested that the impact of smoking and drinking on global DNA methylation may be tissue specific. We also did not find significant associations between one-carbon nutrients (e.g., folate, vitamin B12
, vitamin B6
, riboflavin and methionine) and global DNA methylation in peripheral blood although we were not able to assess the potential effect modification by MTHFR
polymorphisms or other genetic polymorphisms involved in one-carbon metabolism. Environmental factors may modify patterns of DNA methylation during a critical window such as embryogenesis or postnatally. For example, a recent study measured global DNA methylation in umbilical cord blood and found a dose-response relationship between prenatal lead exposure and leukocyte DNA methylation.22
Although we did not find significant associations between lifestyle factors and global DNA methylation, the impact of adulthood environmental exposure over the life time cannot be ignored. Future studies with a larger sample size and a longitudinal design are needed for further examining this issue.
It has to be noted that we measured LINE-1 methylation as a surrogate for global DNA methylation by pyrosequencing. Methylation of repetitive elements has been shown to be a major contributor to total genomic DNA methylation in the human genome.23
Pyrosequencing has been used extensively to measure global DNA methylation and has been shown to be a reproducible assay with a standard deviation of 2%.23
Although the absolute difference in LINE-1 methylation identified in this study was small (2–3%), a modest and significant difference in LINE-1 methylation has previously been associated with the risk of head and neck squamous cell carcinoma (74.7% in cases vs. 75.3% in controls),2
and in individuals with different exposures to airborne benzene (65.7% in exposed groups vs. 62.3% in unexposed groups).24
These results support the notion that a small difference in global genomic DNA methylation can be etiologically important.
In conclusion, we found significant differences in global genomic DNA methylation in peripheral blood by gender and race/ethnicity in a cancer-free population. The associations are unlikely to be mediated by body composition and other behavioral risk factors such as dietary folate intake, physical activity, smoking and drinking. The biological mechanisms underlying these differences warrant further investigation. Because of the strong association between gender, race/ethnicity and global DNA methylation, confounding and effect modification by these variables needs to be taken into consideration in studies of global DNA methylation as an epigenetic marker for cancer.