Significant differences in global genomic DNA methylation by gender and race/ethnicity in peripheral blood

Reduced levels of global DNA methylation are associated with genomic instability and are independent predictors of cancer risk. Little is known about the environmental determinants of global DNA methylation in peripheral blood. We examined the association between demographic and lifestyle factors and levels of global leukocyte DNA methylation in 161 cancer-free subjects enrolled in the North Texas Healthy Heart Study aged 45–75 years in 2008. We used in-person interviews for demographics and lifestyle factors, a self-administrated Block food frequency questionnaire for diet, and bioelectrical impedance analysis and CT-scan for body composition. We measured genomic DNA methylation using bisulfite conversion of DNA and pyrosequencing for LINE-1. Body composition measures including body mass index, waist circumference, areas of subcutaneous fat and visceral fat, percent of fat mass and fat-free mass were not associated with global genomic DNA methylation after controlling the effect of age, gender and race/ethnicity. Instead, female gender was significantly associated with a reduced level of global methylation (β = -2.77, 95% CI: -4.33, -1.22). Compared to non-Hispanic whites, non-Hispanic blacks (β = -2.02, 95% CI: -3.55, -0.50) had significantly lower levels of global methylation. No association was found with age, cigarette smoking, alcohol drinking and dietary intake of nutrients in one-carbon metabolism. Global leukocyte DNA methylation differs by gender and race/ethnicity, suggesting these variables need to be taken into consideration in studies of global DNA methylation as an epigenetic marker for cancer.     

White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.     

Physical activity and global genomic DNA methylation in a cancer-free population

Changes in DNA methylation may represent an intermediate step between the environment and human diseases. Little is known on whether behavioral risk factors may modify gene expression through DNA methylation. To assess whether DNA methylation is associated with different levels of physical activity, we measured global genomic DNA methylation using bisulfite-converted DNA and real-time PCR (MethyLight) for LINE-1 in peripheral blood of 161 participants aged 45–75 years enrolled in the North Texas Healthy Heart Study and levels of physical activity using an accelerometer (Actigraph GT1M Monitor). We found that individuals with physical activity 26–30 min/day had a significantly higher level of global genomic DNA methylation compared to those with physical activity ≤10 min/day (β = 2.52, 95% CI: 0.70, 4.35). However, the association was attenuated and became statistically insignificant after multivariate adjustment (β = 1.24, 95% CI: −0.93, 3.40). There were some suggestions of a positive association between physical activity and global genomic DNA methylation in non-Hispanics (β = 1.50, 95% CI: −0.08, 3.08) that warrants further investigation.Key words: DNA methylation, physical activity, peripheral blood     

White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.     

Global DNA methylation levels in white blood cell DNA from sisters discordant for breast cancer from the New York site of the Breast Cancer Family Registry

Lower global DNA methylation is associated with genomic instability and it is one of the epigenetic mechanisms relevant to carcinogenesis. Emerging evidence for several cancers suggests that lower overall levels of global DNA methylation in blood are associated with different cancer types, although less is known about breast cancer. We examined global DNA methylation levels using a sibling design in 273 sisters affected with breast cancer and 335 unaffected sisters from the New York site of the Breast Cancer Family Registry. We measured global DNA methylation in total white blood cell (WBC) and granulocyte DNA by two different methods, the [³H]-methyl acceptance assay and the luminometric methylation assay (LUMA). Global methylation levels were only modestly correlated between sisters discordant for breast cancer (Spearman correlation coefficients ranged from -0.08 to 0.24 depending on assay and DNA source). Using conditional logistic regression models, women in the quartile with the lowest DNA methylation levels (as measured by the [³H]-methyl acceptance assay) had a 1.8-fold (95% CI = 1.0–3.3) higher relative association with breast cancer than women in the quartile with the highest DNA methylation levels. When we examined the association on a continuous scale, we also observed a positive association (odds ratio, OR = 1.3, 95% CI = 1.0–1.7, for a one unit change in the natural logarithm of the DPM/μg of DNA). We observed no association between measures by the LUMA assay and breast cancer risk. If replicated in prospective studies, this study suggests that global DNA methylation levels measured in WBC may be a potential biomarker of breast cancer risk even within families at higher risk of cancer.     

Differences in DNA methylation by extent of breast cancer family history in unaffected women

Breast cancer clusters within families but genetic factors identified to date explain only a portion of this clustering. Lower global DNA methylation in white blood cells (WBC) has been associated with increased breast cancer risk. We examined whether WBC DNA methylation varies by extent of breast cancer family history in unaffected women from high-risk breast cancer families. We evaluated DNA methylation levels in LINE-1, Alu and Sat2 in 333 cancer-free female family members of the New York site of the Breast Cancer Family Registry, the minority of which were known BRCA1 or BRCA2 mutation carriers. We used generalized estimated equation models to test for differences in DNA methylation levels by extent of their breast cancer family history after adjusting for age. All unaffected women had at least one sister affected with breast cancer. LINE-1 and Sat2 DNA methylation levels were lower in individuals with 3 or more (3+) first-degree relatives with breast cancer relative to women with only one first-degree relative. For LINE-1, Alu, and Sat2, having 3+ affected first-degree relatives was associated with a decrease of 23.4% (95%CI = −46.8%, 0.1%), 17.9% (95%CI = −39.5%, 3.7%) and 11.4% (95% CI = −20.3%, −2.5%), respectively, relative to individuals with only one affected first-degree relative, but the results were only statistically significant for Sat2. Individuals having an affected mother had 17.9% lower LINE-1 DNA methylation levels (95% CI = −28.8%, −7.1%) when compared with those not having an affected mother. No associations were observed for Alu or Sat2 by maternal breast cancer status. If replicated, these results indicate that lower global WBC DNA methylation levels in families with extensive cancer histories may be one explanation for the clustering of cancers in these families. Family clustering of disease may reflect epigenetic as well as genetic and shared environmental factors.     

Race/Ethnicity-Specific Association of Vitamin D and Global DNA Methylation: Cross-Sectional and Interventional Findings

ObjectivesUnderstanding of the influence of vitamin D deficiency on epigenome will provide novel insights into the chronic disease risk. We tested our hypotheses that 1) vitamin D deficiency is associated with global hypomethylation and this association may be race/ethnicity dependent; and 2) vitamin D supplementation will increase global DNA methylation level.MethodsA two-stage design, cross-sectional observation followed by a 16 week randomized, double- blinded, placebo-controlled trial (RCT) of vitamin D3 supplementation, was undertaken. Global DNA methylation level (percentage of 5-methylcytosine, %5-mC) was quantified using leukocyte DNA with the MethylFlash^TM Methylated DNA Quantification kit (Epigentek). Global methylation data was obtained from 454 Caucasians and African Americans (42%) in the observation cohort and 58 African Americans with vitamin D deficiency in the dose responsive RCT.ResultsIn the cross-sectional study, African Americans had lower %5-mC than Caucasians (P = 0.04). A significant interaction was detected between plasma 25(OH)D and race on %5-mC (P = 0.05), as a positive association was observed between plasma 25(OH)D and %5-mC in African Americans (β = 0.20, p<0.01), but not in Caucasians (β = 0.03, p = 0.62). In the 16-week RCT, a dose-response benefit of vitamin D₃ supplementation was observed for %5-mC, as indicated by a significant linear upward trend (-0.01 ± 0.01%, placebo; 0.11 ± 0.01%, ~600 IU/day; 0.30 ± 0.01%, ~2,000 IU/day; and 0.65 ± 0.01%, ~4,000 IU/day group; P-trend = 0.04).ConclusionsVitamin D deficiency is associated with global hypomethylation in African Americans. Vitamin D3 supplementation increases global DNA methylation in a dose-response manner in African Americans with vitamin D deficiency.     

DNA Methylation of IGF2DMR and H19 Is Associated with Fetal and Infant Growth: The Generation R Study

Changes in epigenetic programming of embryonic growth genes during pregnancy seem to affect fetal growth. Therefore, in a population-based prospective birth cohort in the Netherlands, we examined associations between fetal and infant growth and DNA methylation of IGF2DMR, H19 and MTHFR. For this study, we selected 69 case children born small-for-gestational age (SGA, birth weight <-2SDS) and 471 control children. Fetal growth was assessed with serial ultrasound measurements. Information on birth outcomes was retrieved from medical records. Infant weight was assessed at three and six months. Methylation was assessed in DNA extracted from umbilical cord white blood cells. Analyses were performed using linear mixed models with DNA methylation as dependent variable. The DNA methylation levels of IGF2DMR and H19 in the control group were, median (90% range), 53.6% (44.5–61.6) and 30.0% (25.6–34.2) and in the SGA group 52.0% (43.9–60.9) and 30.5% (23.9–32.9), respectively. The MTHFR region was found to be hypomethylated with limited variability in the control and SGA group, 2.5% (1.4–4.0) and 2.4% (1.5–3.8), respectively. SGA was associated with lower IGF2DMR DNA methylation (β = −1.07, 95% CI −1.93; −0.21, P-value = 0.015), but not with H19 methylation. A weight gain in the first three months after birth was associated with lower IGF2DMR DNA methylation (β = −0.53, 95% CI −0.91; −0.16, P-value = 0.005). Genetic variants in the IGF2/H19 locus were associated with IGF2DMR DNA methylation (P-value<0.05), but not with H19 methylation. Furthermore, our results suggest a possibility of mediation of DNA methylation in the association between the genetic variants and SGA. To conclude, IGF2DMR and H19 DNA methylation is associated with fetal and infant growth.     

Early cartilage abnormalities at the hip are associated with obesity and body composition measures – a 3.0T MRI community-based study

IntroductionAlthough obesity is a risk factor for hip osteoarthritis (OA), the role of body composition, if any, is unclear. This study examines whether the body mass index (BMI) and body composition are associated with hip cartilage changes using magnetic resonance imaging (MRI) in community-based adults.Methods141 community-based participants with no clinical hip disease, including OA, had BMI and body composition (fat mass and fat free mass) measured at baseline (1990 to 1994), and BMI measured and 3.0 T MRI performed at follow-up (2009–2010). Femoral head cartilage volume was measured and femoral head cartilage defects were scored in the different hip regions.ResultsFor females, baseline BMI (β = −26 mm³, 95% Confidence interval (CI) -47 to −6 mm³, p = 0.01) and fat mass (β = −11 mm³, 95% CI −21 to −1 mm³, p = 0.03) were negatively associated with femoral head cartilage volume. Also, while increased baseline fat mass was associated with an increased risk of cartilage defects in the central superolateral region of the femoral head (Odds Ratio (OR) = 1.08, 95% CI 1.00–1.15, p = 0.04), increased baseline fat free mass was associated with a reduced risk of cartilage defects in this region (OR = 0.82, 95% CI 0.67–0.99; p = 0.04). For males, baseline fat free mass was associated with increased femoral head cartilage volume (β = 40 mm³, 95% CI 6 to 74 mm³, p = 0.02).ConclusionsIncreased fat mass was associated with adverse hip cartilage changes for females, while increased fat free mass was associated with beneficial cartilage changes for both genders. Further work is required to determine whether modifying body composition alters the development of hip OA.     

Correlations in global DNA methylation measures in peripheral blood mononuclear cells and granulocytes

Alterations in global DNA methylation levels have been associated with chronic diseases. Despite the increase in the number of studies measuring markers of global methylation, few have adequately examined within-individual differences by source of DNA and whether within-individual differences by source of DNA differ by age, race and other lifestyle factors. We examined correlations between peripheral mononuclear cell (PBMC) and granulocyte DNA methylation levels measured by the luminometric methylation assay (LUMA), and in LINE-1, Sat2, and Alu by MethyLight and pyrosequencing, in the same individual in 112 women participating in The New York City Multiethnic Breast Cancer Project. Levels of DNA methylation of Sat2 by MethyLight (r = 0.57; P < 0.01) and LINE-1 by pyrosequencing (r = 0.30; P < 0.01) were correlated between PBMC and granulocyte DNA of the same individuals, but LUMA and Alu levels were not. The magnitude of the correlations for Sat2 and LINE-1 varied when stratified by selected demographic and lifestyle factors, although the study sample size limited our comparisons across subgroups. These results lend further support to the importance of considering the source of DNA in epidemiologic studies of white blood cell DNA methylation. Results from studies that combine individuals with different available DNA sources need to be interpreted with caution.     

Gestational intake of methyl donors and global LINE-1 DNA methylation in maternal and cord blood: Prospective results from a folate-replete population

Maternal diet affects offspring DNA methylation in animal models, but evidence from humans is limited. We investigated the extent to which gestational intake of methyl donor nutrients affects global DNA methylation in maternal and umbilical cord blood. Among mother-infant pairs in Project Viva, a folate-replete US population, we estimated maternal intakes of vitamin B12, betaine, choline, folate, cadmium, zinc and iron periconceptionally and during the second trimester. We examined associations of these nutrients with DNA methylation, measured as %5-methyl cytosines (%5mC) in Long Interspersed Nuclear Element-1 (LINE-1), in first trimester (n = 830) and second trimester (n = 671) maternal blood and in cord blood at delivery (n = 516). Cord blood methylation was higher for male than female infants {mean [standard deviation (SD)] 84.8 [0.6] vs. 84.4 [0.7]%}. In the multivariable-adjusted model, maternal intake of methyl donor nutrients periconceptionally and during the second trimester of pregnancy was not positively associated with first trimester, second trimester or cord blood LINE-1 methylation. Periconceptional betaine intake was inversely associated with cord blood methylation [regression coefficient = −0.08% (95% confidence interval (CI): −0.14, −0.01)] but this association was attenuated after adjustment for dietary cadmium, which itself was directly associated with first trimester methylation and inversely associated with cord blood methylation. We also found an inverse association between periconceptional choline [−0.10%, 95% CI: −0.17, −0.03 for each SD (∼63 mg/day)] and cord blood methylation in males only. In this folate-replete population, we did not find positive associations between intake of methyl donor nutrients during pregnancy and DNA methylation overall, but among males, higher early pregnancy intakes of choline were associated with lower cord blood methylation.Key words: DNA methylation, pregnancy, cord blood, maternal diet, cadmium     

Social Factors and Leukocyte DNA Methylation of Repetitive Sequences: The Multi-Ethnic Study of Atherosclerosis

Epigenetic changes are a potential mechanism contributing to race/ethnic and socioeconomic disparities in health. However, there is scant evidence of the race/ethnic and socioeconomic patterning of epigenetic marks. We used data from the Multi-Ethnic Study of Atherosclerosis Stress Study (N = 988) to describe age- and gender- independent associations of race/ethnicity and socioeconomic status (SES) with methylation of Alu and LINE-1 repetitive elements in leukocyte DNA. Mean Alu and Line 1 methylation in the full sample were 24% and 81% respectively. In multivariable linear regression models, African-Americans had 0.27% (p<0.01) and Hispanics 0.20% (p<0.05) lower Alu methylation than whites. In contrast, African-Americans had 0.41% (p<0.01) and Hispanics 0.39% (p<0.01) higher LINE-1 methylation than whites. These associations remained after adjustment for SES. In addition, a one standard deviation higher wealth was associated with 0.09% (p<0.01) higher Alu and 0.15% (p<0.01) lower LINE-1 methylation in age- and gender- adjusted models. Additional adjustment for race/ethnicity did not alter this pattern. No associations were observed with income, education or childhood SES. Our findings, from a large community-based sample, suggest that DNA methylation is socially patterned. Future research, including studies of gene-specific methylation, is needed to understand better the opposing associations of Alu and LINE-1 methylation with race/ethnicity and wealth as well as the extent to which small methylation changes in these sequences may influence disparities in health.     

High Throughput Measurement of γH2AX DSB Repair Kinetics in a Healthy Human Population

The Columbia University RABiT (Rapid Automated Biodosimetry Tool) quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB) by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY) (r=0.257, P=0.02) and a negative correlation with residuals (r=-0.521, P=<0.0001). A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001). Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.     

Global Leukocyte DNA Methylation is Similar in African American and Caucasian Women Under Conditions of Controlled Folate Intake

DNA methylation is an epigenetic feature that may modify disease risk, and can be influenced by folate status as well as by methylenetetrahydrofolate reductase (MTHFR) C677T genotype. The aim of this study was to investigate the influence of ethnicity/race on global leukocyte DNA methylation under conditions of controlled folate intake. Caucasian (n=14) and African American (n-14) women (18-45y) possessing the MTHFR 677CC genotype consumed a folate restricted diet (135 μg/d as dietary folate equivalents, DFE) for 7 week followed by folate treatment with 400 or 800 μg DFE/d for 7 week. Global leukocyte DNA methylation was assessed via the cytosine extension assay at baseline (wk 0), after folate restriction (wk 7) and after folate treatment (wk 14). Ethnicity/race was not a determinant of global leukocyte DNA methylation. No differences (P>0.05) were detected in DNA methylation between African American and Caucasian women at baseline or any other study time point. In addition, folate intake did not modify global leukocyte DNA methylation. These data suggest that global leukocyte DNA methylation does not differ between Caucasian and African American women and that short-term folate restriction is not sufficient to modify methylation content in young women with the MTHFR 677CC genotype.     

Hypomethylation of Alu Elements in Post-Menopausal Women with Osteoporosis

A decrease in genomic methylation commonly occurs in aging cells; however, whether this epigenetic modification leads to age-related phenotypes has not been evaluated. Alu elements are the major interspersed repetitive DNA elements in humans that lose DNA methylation in aging individuals. Alu demethylation in blood cells starts at approximately 40 years of age, and the degree of Alu hypomethylation increases with age. Bone mass is lost with aging, particularly in menopausal women with lower body mass. Consequently, osteoporosis is commonly found in thin postmenopausal women. Here, we correlated the Alu methylation level of blood cells with bone density in 323 postmenopausal women. Alu hypomethylation was associated with advanced age and lower bone mass density, (P<0.05). The association between the Alu methylation level and bone mass was independent of age, body mass, and body fat, with an odds ratio [1]  = 0.4316 (0.2087–0.8927). Individuals of the same age with osteopenia, osteoporosis, and a high body mass index have lower Alu methylation levels (P = 0.0005, 0.003, and ≤0.0001, respectively). Finally, when comparing individuals with the same age and body mass, Alu hypomethylation was observed in individuals with lower bone mass (P<0.0001). In conclusion, there are positive correlations between Alu hypomethylation in blood cells and several age-related phenotypes in bone and body fat. Therefore, reduced global methylation may play a role in the systemic senescence process. Further evaluation of Alu hypomethylation may clarify the epigenetic regulation of osteoporosis in post-menopausal women.     

Infant peripheral blood repetitive element hypomethylation associated with antiretroviral therapy in utero

The use of combination antiretroviral therapy (cART) to prevent HIV mother-to-child transmission during pregnancy and delivery is generally considered safe. However, vigilant assessment of potential risks of these agents remains warranted. Epigenetic changes including DNA methylation are considered potential mechanisms linking the in utero environment with long-term health outcomes. Few studies have examined the epigenetic effects of prenatal exposure to pharmaceutical agents, including antiretroviral therapies, on children. In this study, we examined the methylation status of the LINE-1 and ALU-Yb8 repetitive elements as markers of global DNA methylation alteration in peripheral blood mononuclear cells obtained from newborns participating in the Pediatric HIV/AIDS Cohort Study SMARTT cohort of HIV-exposed, cART-exposed uninfected infants compared to a historical cohort of HIV-exposed, antiretroviral-unexposed infants from the Women and Infants Transmission Study Cohort. In linear regression models controlling for potential confounders, we found the adjusted mean difference of AluYb8 methylation of the cART-exposed compared to the -unexposed was −0.568 (95% CI: −1.023, −0.149) and for LINE-1 methylation was −1.359 (95% CI: −1.860, −0.857). Among those exposed to cART, subjects treated with atazanavir (ATV), compared to those on other treatments, had less AluYb8 methylation (−0.524, 95% CI: −0.025, −1.024). Overall, these results suggest a small but statistically significant reduction in the methylation of these repetitive elements in an HIV-exposed, cART-exposed cohort compared to an HIV-exposed, cART-unexposed historic cohort. The potential long-term implications of these differences are worthy of further examination.     

Prenatal Polycyclic Aromatic Hydrocarbon,Adiposity, Peroxisome Proliferator-Activated Receptor (PPAR) γ Methylation in Offspring,Grand-Offspring Mice

Rationale

Greater levels of prenatal exposure to polycyclic aromatic hydrocarbon (PAH) have been associated with childhood obesity in epidemiological studies. However, the underlying mechanisms are unclear.

Objectives

We hypothesized that prenatal PAH over-exposure during gestation would lead to weight gain and increased fat mass in offspring and grand-offspring mice. Further, we hypothesized that altered adipose gene expression and DNA methylation in genes important to adipocyte differentiation would be affected.

Materials and Methods

Pregnant dams were exposed to a nebulized PAH mixture versus negative control aerosol 5 days a week, for 3 weeks. Body weight was recorded from postnatal day (PND) 21 through PND60. Body composition, adipose cell size, gene expression of peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer-binding proteins (C/EBP) α, cyclooxygenase (Cox)-2, fatty acid synthase (FAS) and adiponectin, and DNA methylation of PPAR γ, were assayed in both the offspring and grand-offspring adipose tissue.

Findings

Offspring of dams exposed to greater PAH during gestation had increased weight, fat mass, as well as higher gene expression of PPAR γ, C/EBP α, Cox2, FAS and adiponectin and lower DNA methylation of PPAR γ. Similar differences in phenotype and DNA methylation extended through the grand-offspring mice.

Conclusions

Greater prenatal PAH exposure was associated with increased weight, fat mass, adipose gene expression and epigenetic changes in progeny.     

Race,Ethnicity, Psychosocial Factors,and Telomere Length in a Multicenter Setting

Background

Leukocyte telomere length(LTL) has been associated with age, self-reported race/ethnicity, gender, education, and psychosocial factors, including perceived stress, and depression. However, inconsistencies in associations of LTL with disease and other phenotypes exist across studies. Population characteristics, including race/ethnicity, laboratory methods, and statistical approaches in LTL have not been comprehensively studied and could explain inconsistent LTL associations.

Methods

LTL was measured using Southern Blot in 1510 participants from a multi-ethnic, multi-center study combining data from 3 centers with different population characteristics and laboratory processing methods. Main associations between LTL and psychosocial factors and LTL and race/ethnicity were evaluated and then compared across generalized estimating equations(GEE) and linear regression models. Statistical models were adjusted for factors typically associated with LTL(age, gender, cancer status) and also accounted for factors related to center differences, including laboratory methods(i.e., DNA extraction). Associations between LTL and psychosocial factors were also evaluated within race/ethnicity subgroups (Non-hispanic Whites, African Americans, and Hispanics).

Results

Beyond adjustment for age, gender, and cancer status, additional adjustments for DNA extraction and clustering by center were needed given their effects on LTL measurements. In adjusted GEE models, longer LTL was associated with African American race (Beta(β)(standard error(SE)) = 0.09(0.04), p-value = 0.04) and Hispanic ethnicity (β(SE) = 0.06(0.01), p-value = 0.02) compared to Non-Hispanic Whites. Longer LTL was also associated with less than a high school education compared to having greater than a high school education (β(SE) = 0.06(0.02), p-value = 0.04). LTL was inversely related to perceived stress (β(SE) = -0.02(0.003), p<0.001). In subgroup analyses, there was a negative association with LTL in African Americans with a high school education versus those with greater than a high school education(β(SE) = -0.11(0.03), p-value<0.001).

Conclusions

Laboratory methods and population characteristics that differ by center can influence telomere length associations in multicenter settings, but these effects could be addressed through statistical adjustments. Proper evaluation of potential sources of bias can allow for combined multicenter analyses and may resolve some inconsistencies in reporting of LTL associations. Further, biologic effects on LTL may differ under certain psychosocial and racial/ethnic circumstances and could impact future health disparity studies.     

Non-germ Line Restoration of Genomic Imprinting for a Small Subset of Imprinted Genes in Ubiquitin-like PHD and RING Finger Domain-Containing 1 (Uhrf1) Null Mouse Embryonic Stem Cells

The underlying mechanism for the establishment and maintenance of differential DNA methylation in imprinted genes is largely unknown. Previous studies using Dnmt1 knock-out embryonic stem (ES) cells demonstrated that, although re-expression of DNMT1 restored DNA methylation in the non-imprinted regions, the methylation patterns of imprinted genes could be restored only through germ line passage. Knock-out of Uhrf1, an accessory factor essential for DNMT1-mediated DNA methylation, in mouse ES cells also led to impaired global DNA methylation and loss of genomic imprinting. Here, we demonstrate that, although re-expression of UHRF1 in Uhrf1^−/− ES cells restored DNA methylation for the bulk genome but not for most of the imprinted genes, it did rescue DNA methylation for the imprinted H19, Nnat, and Dlk1 genes. Analysis of histone modifications at the differential methylated regions of the imprinted genes by ChIP assays revealed that for the imprinted genes whose DNA methylation could be restored upon re-expression of UHRF1, the active histone markers (especially H3K4me3) were maintained at considerably low levels, and low levels were maintained even in Uhrf1^−/− ES cells. In contrast, for the imprinted genes whose DNA methylation could not be restored upon UHRF1 re-expression, the active histone markers (especially H3K4me3) were relatively high and became even higher in Uhrf1^−/− ES cells. Our study thus supports a role for histone modifications in determining the establishment of imprinting-related DNA methylation and demonstrates that mouse ES cells can be a valuable model for mechanistic study of the establishment and maintenance of differential DNA methylation in imprinted genes.     

Physical inactivity is associated with narrower lumbar intervertebral discs,high fat content of paraspinal muscles and low back pain and disability

IntroductionAlthough physical inactivity has been associated with numerous chronic musculoskeletal complaints, few studies have examined its associations with spinal structures. Moreover, previously reported associations between physical activity and low back pain are conflicting. This study examined the associations between physical inactivity and intervertebral disc height, paraspinal fat content and low back pain and disability.MethodsSeventy-two community-based volunteers not selected for low back pain underwent magnetic resonance imaging (MRI) of their lumbosacral spine (L1 to S1) between 2011 and 2012. Physical activity was assessed between 2005 and 2008 by questionnaire, while low back pain and disability were assessed by the Chronic Pain Grade Scale at the time of MRI. Intervertebral disc height and cross-sectional area and fat content of multifidus and erector spinae were assessed from MRI.ResultsLower physical activity levels were associated with a more narrow average intervertebral disc height (β −0.63 mm, 95% confidence interval (CI) −1.17 mm to −0.08 mm, P = 0.026) after adjusting for age, gender and body mass index (BMI). There were no significant associations between physical activity levels and the cross-sectional area of multifidus or erector spinae. Lower levels of physical activity were associated with an increased risk of high fat content in multifidus (odds ratio (OR) 2.7, 95% CI 1.1 to 6.7, P = 0.04) and high-intensity pain/disability (OR = 5.0, 95% CI 1.5 to 16.4, P = 0.008) after adjustment for age, gender and BMI.ConclusionsPhysical inactivity is associated with narrower intervertebral discs, high fat content of the multifidus and high-intensity low back pain and disability in a dose-dependent manner among community-based adults. Longitudinal studies will help to determine the cause and effect nature of these associations.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0629-y) contains supplementary material, which is available to authorized users.