Associations between use of chemical hair products and epigenetic age: Findings from the Sister Study.

Background: Hair products may be a source of harmful chemicals and have been linked to age-related health outcomes. We investigated whether the use of hair products is related to epigenetic age in a sample of Black (both Hispanic and non-Hispanic) and non-Hispanic White women. Methods: In a subset of 4358 participants aged 35-74 years from the Sister Study, we estimated cross-sectional associations between self-reported use of four chemical hair products (permanent dye, semipermanent dye, straighteners/relaxers, and hair permanents/body waves) in the year before enrollment (2003-2009) and three DNA methylation-based measures of epigenetic age (DunedinPACE, GrimAge age acceleration [GrimAgeAccel], and PhenoAge age acceleration [PhenoAgeAccel]) using survey-weighted multivariable linear regressions. Associations were estimated both overall and by self-identified race and ethnicity, adjusting for chronological age, socioeconomic and lifestyle factors, body mass index, menopausal status, and DNA methylation platform. Results: Associations between the use of hair products and the three epigenetic age measures were largely null. Use of hair permanents/body waves was modestly associated with higher DunedinPACE among all participants (ßever-never = 0.010; 95% confidence interval [CI] = 0.001, 0.019) and with lower PhenoAgeAccel among Black women (ßever-never = -1.53; 95% CI = -2.84, -0.21). Conclusion: In this US-based study, we found little evidence of associations between chemical hair product use and epigenetic age in Black and non-Hispanic White women. Observed associations were modest and largely not supported by dose-response relationships or were inconsistent across epigenetic age measures. Previously observed associations between chemical hair product use and aging-related health outcomes may not be explained by the biological aging pathways captured by DunedinPACE, GrimAgeAccel, or PhenoAgeAccel. Alternative biological pathways are worth investigating in racially diverse samples.

methscore: a comprehensive R function for DNA methylation-based health predictors.

MOTIVATION: DNA methylation-based predictors of various biological metrics have been widely published and are becoming valuable tools in epidemiologic studies of epigenetics and personalized medicine. However, generating these predictors from original source software and web servers is complex and time consuming. Furthermore, different predictors were often derived based on data from different types of arrays, where array differences and batch effects can make predictors difficult to compare across studies. RESULTS: We integrate these published methods into a single R function to produce 158 previously published predictors for chronological age, biological age, exposures, lifestyle traits and serum protein levels using both classical and principal component-based methods. To mitigate batch and array differences, we also provide a modified RCP method (ref-RCP) that normalize input DNA methylation data to reference data prior to estimation. Evaluations in real datasets show that this approach improves estimate precision and comparability across studies. AVAILABILITY AND IMPLEMENTATION: The function was included in software package ENmix, and is freely available from Bioconductor website (https://www.bioconductor.org/packages/release/bioc/html/ENmix.html).

Circulating Leukocyte Subsets Before and After a Breast Cancer Diagnosis and Therapy.

Importance: Changes in leukocyte composition often precede chronic disease onset. Patients with a history of breast cancer (hereinafter referred to as breast cancer survivors) are at increased risk for subsequent chronic diseases, but the long-term changes in peripheral leukocyte composition following a breast cancer diagnosis and treatment remain unknown. Objective: To examine longitudinal changes in peripheral leukocyte composition in women who did and did not develop breast cancer and identify whether differences in breast cancer survivors were associated with specific treatments. Design, Setting, and Participants: In this prospective cohort study, paired blood samples were collected from 2315 women enrolled in The Sister Study, a US-nationwide prospective cohort study of 50 884 women, at baseline (July 2003 to March 2009) and follow-up (October 2013 to March 2015) home visits, with a mean (SD) follow-up interval of 7.6 (1.4) years. By design, approximately half of the included women had been diagnosed and treated for breast cancer after enrollment and before the second blood draw. A total of 410 women were included in the present study, including 185 breast cancer survivors and 225 who remained free of breast cancer over a comparable follow-up period. Data were analyzed from April 21 to September 9, 2022. Exposures: Breast cancer status and, among breast cancer survivors, cancer treatment type (chemotherapy, radiotherapy, endocrine therapy, or surgery). Main Outcomes and Measures: Blood DNA methylation data were generated in 2019 using a genome-wide methylation screening tool and deconvolved to estimate percentages of 12 circulating leukocyte subsets. Results: Of the 410 women included in the analysis, the mean (SD) age at enrollment was 56 (9) years. Compared with breast cancer-free women, breast cancer survivors had decreased percentages of circulating eosinophils (-0.45% [95% CI, -0.87% to -0.03%]; P = .03), total CD4+ helper T cells (-1.50% [95% CI, -2.56% to -0.44%]; P = .01), and memory B cells (-0.22% [95% CI, -0.34% to -0.09%]; P = .001) and increased percentages of circulating naive B cells (0.46% [95% CI, 0.17%-0.75%]; P = .002). In breast cancer survivor-only analyses, radiotherapy was associated with decreases in total CD4+ T cell levels, whereas chemotherapy was associated with increases in naive B cell levels. Surgery and endocrine therapy were not meaningfully associated with leukocyte changes. Conclusions and Relevance: In this cohort study of 410 women, breast cancer survivors experienced lasting changes in peripheral leukocyte composition compared with women who remained free of breast cancer. These changes may be related to treatment with chemotherapy or radiotherapy and could influence future chronic disease risk.

Trans-ancestry epigenome-wide association meta-analysis of DNA methylation with lifetime cannabis use.

Cannabis is widely used worldwide, yet its links to health outcomes are not fully understood. DNA methylation can serve as a mediator to link environmental exposures to health outcomes. We conducted an epigenome-wide association study (EWAS) of peripheral blood-based DNA methylation and lifetime cannabis use (ever vs. never) in a meta-analysis including 9436 participants (7795 European and 1641 African ancestry) from seven cohorts. Accounting for effects of cigarette smoking, our trans-ancestry EWAS meta-analysis revealed four CpG sites significantly associated with lifetime cannabis use at a false discovery rate of 0.05 [Formula: see text]: cg22572071 near gene ADGRF1, cg15280358 in ADAM12, cg00813162 in ACTN1, and cg01101459 near LINC01132. Additionally, our EWAS analysis in participants who never smoked cigarettes identified another epigenome-wide significant CpG site, cg14237301 annotated to APOBR. We used a leave-one-out approach to evaluate methylation scores constructed as a weighted sum of the significant CpGs. The best model can explain 3.79% of the variance in lifetime cannabis use. These findings unravel the DNA methylation changes associated with lifetime cannabis use that are independent of cigarette smoking and may serve as a starting point for further research on the mechanisms through which cannabis exposure impacts health outcomes.

Air pollution and epigenetic aging among Black and White women in the US.

BACKGROUND: DNA methylation-based measures of biological aging have been associated with air pollution and may link pollutant exposures to aging-related health outcomes. However, evidence is inconsistent and there is little information for Black women. OBJECTIVE: We examined associations of ambient particulate matter <2.5 µm and <10 µm in diameter (PM2.5 and PM10) and nitrogen dioxide (NO2) with DNA methylation, including epigenetic aging and individual CpG sites, and evaluated whether associations differ between Black and non-Hispanic White (NHW) women. METHODS: Validated models were used to estimate annual average outdoor residential exposure to PM2.5, PM10, and NO2 in a sample of self-identified Black (n=633) and NHW (n=3493) women residing in the contiguous US. We used sampling-weighted generalized linear regression to examine the effects of pollutants on six epigenetic aging measures (primary: DunedinPACE, GrimAgeAccel, and PhenoAgeAccel; secondary: Horvath intrinsic epigenetic age acceleration [EAA], Hannum extrinsic EAA, and skin & blood EAA) and epigenome-wide associations for individual CpG sites. Wald tests of nested models with and without interaction terms were used to examine effect measure modification by race/ethnicity. RESULTS: Black participants had higher median air pollution exposure than NHW participants. GrimAgeAccel was associated with both PM10 and NO2 among Black participants, (Q4 versus Q1, PM10: ß=1.09, 95% CI: 0.16-2.03; NO2: ß=1.01, 95% CI 0.08-1.94) but not NHW participants (p-for-heterogeneity: PM10=0.10, NO2=0.20). In Black participants, we also observed a monotonic exposure-response relationship between NO2 and DunedinPACE (Q4 versus Q1, NO2: ß=0.029, 95% CI: 0.004-0.055; p-for-trend=0.03), which was not observed in NHW participants (p-for-heterogeneity=0.09). In the EWAS, pollutants were significantly associated with differential methylation at 19 CpG sites in Black women and one in NHW women. CONCLUSIONS: In a US-wide cohort study, our findings suggest that air pollution is associated with DNA methylation alterations consistent with higher epigenetic aging among Black, but not NHW, women.

Long-term exposure to ambient fine particulate components and leukocyte epigenome-wide DNA Methylation in older men: the Normative Aging Study.

BACKGROUND: Epigenome-wide association studies of ambient fine particulate matter (PM2.5) have been reported. However, few have examined PM2.5 components (PMCs) and sources or included repeated measures. The lack of high-resolution exposure measurements is the key limitation. We hypothesized that significant changes in DNA methylation might vary by PMCs and the sources. METHODS: We predicted the annual average of 14 PMCs using novel high-resolution exposure models across the contiguous U.S., between 2000-2018. The resolution was 50 m × 50 m in the Greater Boston Area. We also identified PM2.5 sources using positive matrix factorization. We repeatedly collected blood samples and measured leukocyte DNAm with the Illumina HumanMethylation450K BeadChip in the Normative Aging Study. We then used median regression with subject-specific intercepts to estimate the associations between long-term (one-year) exposure to PMCs / PM2.5 sources and DNA methylation at individual cytosine-phosphate-guanine CpG sites. Significant probes were identified by the number of independent degrees of freedom approach, using the number of principal components explaining > 95% of the variation of the DNA methylation data. We also performed regional and pathway analyses to identify significant regions and pathways. RESULTS: We included 669 men with 1,178 visits between 2000-2013. The subjects had a mean age of 75 years. The identified probes, regions, and pathways varied by PMCs and their sources. For example, iron was associated with 6 probes and 6 regions, whereas nitrate was associated with 15 probes and 3 regions. The identified pathways from biomass burning, coal burning, and heavy fuel oil combustion sources were associated with cancer, inflammation, and cardiovascular diseases, whereas there were no pathways associated with all traffic. CONCLUSIONS: Our findings showed that the effects of PM2.5 on DNAm varied by its PMCs and sources.

Changes in methylation-based aging in women who do and do not develop breast cancer.

BACKGROUND: Breast cancer survivors have increased incidence of age-related diseases, suggesting that some survivors may experience faster biological aging. METHODS: Among 417 women enrolled in the prospective Sister Study cohort, DNA methylation data were generated on paired blood samples collected an average of 7.7 years apart and used to calculate 3 epigenetic metrics of biological aging (PhenoAgeAccel, GrimAgeAccel, and Dunedin Pace of Aging Calculated from the Epigenome [DunedinPACE]). Approximately half (n = 190) the women sampled were diagnosed and treated for breast cancer between blood draws, whereas the other half (n = 227) remained breast cancer-free. Breast tumor characteristics and treatment information were abstracted from medical records. RESULTS: Among women who developed breast cancer, diagnoses occurred an average of 3.5 years after the initial blood draw and 4 years before the second draw. After accounting for covariates and biological aging metrics measured at baseline, women diagnosed and treated for breast cancer had higher biological aging at the second blood draw than women who remained cancer-free as measured by PhenoAgeAccel (standardized mean difference [ß] = 0.13, 95% confidence interval [CI) = 0.00 to 0.26), GrimAgeAccel (ß = 0.14, 95% CI = 0.03 to 0.25), and DunedinPACE (ß = 0.37, 95% CI = 0.24 to 0.50). In case-only analyses assessing associations with different breast cancer therapies, radiation had strong positive associations with biological aging (PhenoAgeAccel: ß = 0.39, 95% CI = 0.19 to 0.59; GrimAgeAccel: ß = 0.29, 95% CI = 0.10 to 0.47; DunedinPACE: ß = 0.25, 95% CI = 0.02 to 0.48). CONCLUSIONS: Biological aging is accelerated following a breast cancer diagnosis and treatment. Breast cancer treatment modalities appear to differentially contribute to biological aging.

In utero exposure to diethylstilbestrol and blood DNA methylation in adult women: Results from a meta-analysis of two cohort studies.

BACKGROUND: Prenatal exposure to diethylstilbestrol (DES) is associated with several adverse health outcomes. Animal studies have shown associations between prenatal DES exposure and DNA methylation. OBJECTIVE: The aim of this study was to explore blood DNA methylation in women exposed and unexposed to DES in utero. METHODS: Sixty women (40 exposed and 20 unexposed) in the National Cancer Institute's Combined DES Cohort Study and 199 women (99 exposed and 100 unexposed women) in the Sister Study Cohort were included in this analysis. Within each study, robust linear regression models were used to assess associations between DES exposure and blood DNA methylation. Study-specific associations were combined using fixed-effect meta-analysis with inverse variance weights. Our analysis focused on CpG sites located within nine candidate genes identified in animal models. We further explored whether in utero DES exposure was associated with age acceleration. RESULTS: Blood DNA methylation levels at 10 CpG sites in six of the nine candidate genes were statistically significantly associated with prenatal DES exposure (P < 0.05) in this meta-analysis. Genes included EGF, EMB, EGFR, WNT11, FOS, and TGFB1, which are related to cell proliferation and differentiation. The most statistically significant CpG site was cg19830739 in gene EGF, and it was associated with lower methylation levels in women prenatally exposed to DES compared with those not exposed (P < 0.0001; false discovery rate<0.05). The association between prenatal DES exposure in utero and age acceleration was not statistically significant (P = 0.07 for meta-analyzed results). CONCLUSIONS: There are few opportunities to investigate the effects of prenatal DES exposure. These findings suggest that in utero DES exposure may be associated with differential blood DNA methylation levels, which could mediate the increased risk of several adverse health outcomes observed in exposed women. Our findings need further evaluation using larger data sets.

Accelerated epigenetic aging and myopenia in young adult cancer survivors.

BACKGROUND: Young adult cancer survivors experience early aging-related morbidities and mortality. Biological aging biomarkers may identify at-risk survivors and increase our understanding of mechanisms underlying this accelerated aging. METHODS: Using an observational study design, we cross-sectionally measured DNA methylation-based epigenetic age in young adult cancer survivors at a tertiary, academic state cancer hospital. Participants were a convenience sample of consecutively enrolled survivors of childhood, adolescent, and young adult cancers treated with either an anthracycline or alkylating agent, and who were at least 3 months post-treatment. Similarly aged healthy comparators were consecutively enrolled. Cancer treatment and treatment intensity were compared to DNA methylation-based epigenetic age and pace of aging. RESULTS: Sixty survivors (58 completing assessments, mean age 20.5 years, range 18-29) and 27 comparators (mean age 20 years, range 17-29) underwent DNA methylation measurement. Survivors were predominantly female (62%) and white (60%) and averaged nearly 6 years post-treatment (range 0.2-25 years). Both epigenetic age (AgeAccelGrim: 1.5 vs. -2.4, p < 0.0001; AgeAccelPheno 2.3 vs. -3.8, p = 0.0013) and pace of aging (DunedinPACE 0.99 vs. 0.83, p < 0.0001) were greater in survivors versus comparators. In case-case adjusted analysis, compared to survivors with normal muscle mass, myopenic survivors had higher AgeAccelGrim (2.2 years, 95% CI 0.02-4.33, p = 0.02), AgeAccelPheno (6.2 years, 2.36-10.09, p < 0.001), and DunedinPACE (0.11, 0.05-0.17, p < 0.001). CONCLUSIONS: Epigenetic age is older and pace of aging is faster in young adult cancer survivors compared to noncancer peers, which is evident in the early post-therapy period. Survivors with physiological impairment demonstrate greater epigenetic age advancement. Measures of epigenetic age may identify young adult survivors at higher risk for poor functional and health outcomes.

Beadchip technology to detect DNA methylation in mouse faithfully recapitulates whole-genome bisulfite sequencing.

Aim: To facilitate wide-scale implementation of Illumina Mouse Methylation BeadChip (MMB) technology, array-based measurement of cytosine methylation was compared with the gold-standard assessment of DNA methylation by whole-genome bisulfite sequencing (WGBS). Methods: DNA methylation across two mouse strains (C57B6 and C3H) and both sexes was assessed using the MMB and compared with previously existing deep-coverage WGBS of mice of the same strain and sex. Results & conclusion: The findings demonstrated that 93.3-99.2% of sites had similar measurements of methylation across technologies and that differentially methylated cytosines and regions identified by each technology overlap and enrich for similar biological functions, suggesting that the MMB faithfully recapitulates the findings of WGBS.

Intermediate and long-term exposure to air pollution and temperature and the extracellular microRNA profile of participants in the normative aging study (NAS).

BACKGROUND: The molecular effects of intermediate and long-term exposure to air pollution and temperature, such as those on extracellular microRNA (ex-miRNA) are not well understood but may have clinical consequences. OBJECTIVES: To assess the association between exposure to ambient air pollution and temperature and ex-miRNA profiles. METHODS: Our study population consisted of 734 participants in the Normative Aging Study (NAS) between 1999 and 2015. We used high-resolution models to estimate four-week, eight-week, twelve-week, six-month, and one-year moving averages of PM2.5, O3, NO2, and ambient temperature based on geo-coded residential addresses. The outcome of interest was the extracellular microRNA (ex-miRNA) profile of each participant over time. We used a longitudinal quantile regression approach to estimate the association between the exposures and each ex-miRNA. Results were corrected for multiple comparisons and ex-miRNAs that were still significantly associated with the exposures were further analyzed using KEGG pathway analysis and Ingenuity Pathway Analysis. RESULTS: We found 151 significant associations between levels of PM2.5, O3, NO2, and ambient temperature and 82 unique ex-miRNAs across multiple quantiles. Most of the significant results were associations with intermediate-term exposure to O3, long-term exposure to PM2.5, and both intermediate and long-term exposure to ambient temperature. The exposures were most often associated with the 75th and 90th percentile of the outcomes. Pathway analyses of significant ex-miRNAs revealed their involvement in biological pathways involving cell function and communication as well as clinical diseases such as cardiovascular disease, respiratory disease, and neurological disease. CONCLUSION: Our results show that intermediate and long-term exposure to all our exposures of interest were associated with changes in the ex-miRNA profile of study participants. Further studies on environmental risk factors and ex-miRNAs are warranted.

Short-term air pollution and temperature exposure and changes in the extracellular microRNA profile of Normative Aging Study (NAS) participants.

BACKGROUND: While the health effects of air pollution and temperature are widely studied, the molecular effects are poorly understood. Extracellular microRNAs (ex-miRNAs) have the potential to serve as diagnostic or prognostic biomarkers and/or to act as intercellular signaling molecules that mediate the effects of environmental exposures on health outcomes. METHODS: We examined the relationship between short-term exposure to air pollution and ambient temperature and the ex-miRNA profiles of participants in the Normative Aging Study (NAS) from 1999 to 2015. Our exposures were defined as same-day, two-day, three-day, one-week, two-week, and three-week moving averages of PM2.5, NO2, O3, and temperature which were derived from high-resolution spatio-temporal models. The ex-miRNA profiles of the subjects were obtained during follow-up visits. We analyzed the data using a longitudinal quantile regression model adjusted for individual covariates, batch effects, and time trends. We adjusted for multiple comparisons using a false discovery rate (FDR) correction. Ex-miRNAs that were significantly associated with exposures were further investigated using pathway analyses. RESULTS: We found that all the examined exposures were associated with changes in ex-miRNA profiles in our study, particularly PM2.5 which was responsible for most of the statistically significant results. We found 110 statistically significant exposure-outcome relationships that revealed associations with the levels of 52 unique ex-miRNAs. Pathway analyses showed these ex-miRNAs have been linked to target mRNAs, genes, and biological mechanisms that could affect virtually every organ system, and as such may be linked to multiple clinical disease presentations such as cardiovascular disease, respiratory disease, and neurological disease. CONCLUSIONS: Air pollution and temperature exposures were significantly associated with alterations in the ex-miRNA profiles of NAS subjects with possible biological consequences.

Peripheral Immune Cell Composition is Altered in Women Before and After a Hypertension Diagnosis.

BACKGROUND: The development and consequences of hypertension involve multiple biological systems that may include changes in immune profiles. Whether hypertension is related to peripheral immune cell composition has not been examined in large human cohorts. METHODS: We estimated circulating proportions of 12 leukocyte subsets from the lymphoid and myeloid lineages by deconvolving cell-type-specific DNA methylation data from 4124 women. Hypertension status at baseline was defined by current use of antihypertensive medication and blood pressure measurements while new incident cases were identified during follow-up via annual health questionnaires. RESULTS: Among hypertension-free women at baseline, higher B cell and lower naïve CD4+ helper T cell proportions were associated with subsequent increased hazard of hypertension incidence (B cells; adjusted HR: 1.17 [95% CI: 1.02-1.35]; P=0.03; naïve CD4+ T cell, adjusted HR: 0.88 [95% CI: 0.78-0.99]; P=0.04). Blood pressure measurements at baseline were similarly positively associated with B cells and inversely associated with naïve CD4+ helper T cells. Compared to normotensive women, women with hypertension had higher circulating proportions of neutrophils (adjusted odds ratio: 1.18 [95% CI: 1.07-1.31]; P=0.001) and lower proportions of CD4+ helper T cells (adjusted odds ratio: 0.90 [95% CI: 0.81-1.00] P=0.05), natural killers (adjusted odds ratio: 0.82 [95% CI: 0.74-0.91]; P<0.001), and B cells (adjusted odds ratio: 0.84 [95% CI: 0.74-0.96]; P=0.01). CONCLUSIONS: These observations suggest that shifts in lymphocyte subsets occur before hypertension development, followed by later changes to neutrophils and additional lymphocytes.

Epigenome-wide DNA methylation in leukocytes and toenail metals: The normative aging study.

BACKGROUND: Environmental metal exposures have been associated with multiple deleterious health endpoints. DNA methylation (DNAm) may provide insight into the mechanisms underlying these relationships. Toenail metals are non-invasive biomarkers, reflecting a medium-term time exposure window. OBJECTIVES: This study examined variation in leukocyte DNAm and toenail arsenic (As), cadmium (Cd), lead (Pb), manganese (Mn), and mercury (Hg) among elderly men in the Normative Aging Study, a longitudinal cohort. METHODS: We repeatedly collected samples of blood and toenail clippings. We measured DNAm in leukocytes with the Illumina HumanMethylation450 K BeadChip. We first performed median regression to evaluate the effects of each individual toenail metal on DNAm at three levels: individual cytosine-phosphate-guanine (CpG) sites, regions, and pathways. Then, we applied a Bayesian kernel machine regression (BKMR) to assess the joint and individual effects of metal mixtures on DNAm. Significant CpGs were identified using a multiple testing correction based on the independent degrees of freedom approach for correlated outcomes. The approach considers the effective degrees of freedom in the DNAm data using the principal components that explain >95% variation of the data. RESULTS: We included 564 subjects (754 visits) between 1999 and 2013. The numbers of significantly differentially methylated CpG sites, regions, and pathways varied by metals. For example, we found six significant pathways for As, three for Cd, and one for Mn. The As-associated pathways were associated with cancer (e.g., skin cancer) and cardiovascular disease, whereas the Cd-associated pathways were related to lung cancer. Metal mixtures were also associated with 47 significant CpG sites, as well as pathways, mainly related to cancer and cardiovascular disease. CONCLUSIONS: This study provides an approach to understanding the potential epigenetic mechanisms underlying observed relations between toenail metals and adverse health endpoints.

Blood DNA methylation profiles improve breast cancer prediction.

Although blood DNA methylation (DNAm) profiles are reported to be associated with breast cancer incidence, they have not been widely used in breast cancer risk assessment. Among a breast cancer case-cohort of 2774 women (1551 cases) in the Sister Study, we used candidate CpGs and DNAm estimators of physiologic characteristics to derive a methylation-based breast cancer risk score, mBCRS. Overall, 19 CpGs and five DNAm estimators were selected using elastic net regularization to comprise mBCRS. In a test set, higher mBCRS was positively associated with breast cancer incidence, showing similar strength to the polygenic risk score (PRS) based on 313 single nucleotide polymorphisms (313 SNPs). Area under the curve for breast cancer prediction was 0.60 for self-reported risk factors (RFs), 0.63 for PRS, and 0.63 for mBCRS. Adding mBCRS to PRS and RFs improved breast cancer prediction from 0.66 to 0.71. mBCRS findings were replicated in a nested case-control study within the EPIC-Italy cohort. These results suggest that mBCRS, a risk score derived using blood DNAm, can be used to enhance breast cancer prediction.

The ENmix DNA methylation analysis pipeline for Illumina BeadChip and comparisons with seven other preprocessing pipelines.

BACKGROUND: Illumina DNA methylation arrays are high-throughput platforms for cost-effective genome-wide profiling of individual CpGs. Experimental and technical factors introduce appreciable measurement variation, some of which can be mitigated by careful "preprocessing" of raw data. METHODS: Here we describe the ENmix preprocessing pipeline and compare it to a set of seven published alternative pipelines (ChAMP, Illumina, SWAN, Funnorm, Noob, wateRmelon, and RnBeads). We use two large sets of duplicate sample measurements with 450 K and EPIC arrays, along with mixtures of isogenic methylated and unmethylated cell line DNA to compare raw data and that preprocessed via different pipelines. RESULTS: Our evaluations show that the ENmix pipeline performs the best with significantly higher correlation and lower absolute difference between duplicate pairs, higher intraclass correlation coefficients (ICC) and smaller deviations from expected methylation level in mixture experiments. In addition to the pipeline function, ENmix software provides an integrated set of functions for reading in raw data files from mouse and human arrays, quality control, data preprocessing, visualization, detection of differentially methylated regions (DMRs), estimation of cell type proportions, and calculation of methylation age clocks. ENmix is computationally efficient, flexible and allows parallel computing. To facilitate further evaluations, we make all datasets and evaluation code publicly available. CONCLUSION: Careful selection of robust data preprocessing methods is critical for DNA methylation array studies. ENmix outperformed other pipelines in our evaluations to minimize experimental variation and to improve data quality and study power.

Clinical value of contrast-enhanced computed tomography (CECT) combined with contrast-enhanced ultrasound (CEUS) for characterization and diagnosis of small nodular lesions in liver.

OBJECTIVES: To explore the clinical value of contrast-enhanced computed tomography (CECT) combined with contrast-enhanced ultrasound (CEUS) for characterization and diagnosis of small nodular lesions in the liver and investigate the association between such small nodular lesions and the degree of tumor differentiation. METHODS: Combined imaging modalities were performed on 120 patients who were admitted by Linyi Maternal and Child Health hospital from December 2018 to December 2020 and diagnosed with hepatic nodular lesions. The CT scans were interpreted by two senior imageologists while the ultrasound scans were analyzed by two senior sonographers. A comparative analysis was carried out on different scan modes and the postoperative or post-puncture pathological results using the t-test, the χ2 test, and the Pearson's correlation analysis. RESULTS: Compared to the pathological results, definite diagnoses of 55 malignant cases were made using CECT alone, with the coincidence rate of 78.6%; CECT combined with CEUS formed correct diagnoses in 64 cases, and the coincidence rate was up to 91.4%. The difference between the two scan modes was statistically significant (p= 0.03). Based on pathological diagnosis, seventy out of the 120 cases of small nodular lesions were identified as malignant, while the other 50 cases were benign. The single imaging modality diagnosed 63 malignant and 57 benign nodules, whereas the combined modalities identified 68 malignancies and 52 benign conditions. Compared to CECT as a single imaging modality, the combined modalities showed a higher degree of sensitivity and accuracy, and the difference was statistically significant (sensitivity: p= 0.03; accuracy: p= 0.02); in the malignant cases, the magnitudes of contrast enhancement of CT and ultrasound imaging decreased with an increase in the degree of differentiation, indicating a negative correlation between these factors. CONCLUSIONS: CECT combined with CEUS has a higher coincidence rate, greater sensitivity, and better diagnostic accuracy when being used for characterization and diagnosis of small nodular lesions in the liver. A higher degree of tumor differentiation means a decreased magnitude of contrast enhancement and a blurrier boundary, which indicates that CECT and CEUS are complementary to each other in classifying malignant liver nodules. The use of the combined imaging modalities shows clinical value for characterizing small liver nodules and predicting the degree of malignancy.

Differential Gene Expression in Bladder Tumors from Workers Occupationally Exposed to Arylamines.

Occupational exposure to the arylamines benzidine and ß-naphthylamine increase bladder cancer risk up to 100-fold, making them some of the most powerful human carcinogens. We hypothesize that tumors arising in people with occupational exposures have different patterns of gene expression than histologically similar tumors from people without such exposures. In a case-case study, we compare gene expression in 22 formalin-fixed paraffin-embedded (FFPE) bladder tumors from men with high-level occupational exposure to arylamines to that in 26 FFPE bladder tumors from men without such exposure. Gene expression analysis was performed on the NanoString nCounter system using a PanCancer Progression Panel comprised of 740 cancer progression-related genes and a custom panel of 69 arylamine- and bladder cancer-related genes which were chosen from in vitro studies. Although fold differences were small, there was evidence of differential expression by exposure status for 17 genes from the Progression Panel and 4 genes from the custom panel. In total, 10 genes showed dose-response association at a p < 0.01, of which 4 genes (CD46, NR4A1, BAX, and YWHAZ) passed a false discovery rate (FDR) q value cutoff of 0.05 but were not significant after Bonferroni correction. Overall, we find limited evidence for differentially expressed genes in pathways related to DNA damage signaling and epithelial-to-mesenchymal transition (EMT).

Genome-wide association studies identify 137 genetic loci for DNA methylation biomarkers of aging.

BACKGROUND: Biological aging estimators derived from DNA methylation data are heritable and correlate with morbidity and mortality. Consequently, identification of genetic and environmental contributors to the variation in these measures in populations has become a major goal in the field. RESULTS: Leveraging DNA methylation and SNP data from more than 40,000 individuals, we identify 137 genome-wide significant loci, of which 113 are novel, from genome-wide association study (GWAS) meta-analyses of four epigenetic clocks and epigenetic surrogate markers for granulocyte proportions and plasminogen activator inhibitor 1 levels, respectively. We find evidence for shared genetic loci associated with the Horvath clock and expression of transcripts encoding genes linked to lipid metabolism and immune function. Notably, these loci are independent of those reported to regulate DNA methylation levels at constituent clock CpGs. A polygenic score for GrimAge acceleration showed strong associations with adiposity-related traits, educational attainment, parental longevity, and C-reactive protein levels. CONCLUSION: This study illuminates the genetic architecture underlying epigenetic aging and its shared genetic contributions with lifestyle factors and longevity.

Alcohol Consumption and Methylation-Based Measures of Biological Age.

Epigenetic age acceleration is considered a measure of biological aging based on genome-wide patterns of DNA methylation. Although age acceleration has been associated with the incidence of diseases and death, less is known about how it is related to lifestyle behaviors. Among 2316 women, we evaluate associations between self-reported alcohol consumption and various metrics of epigenetic age acceleration. Recent average alcohol consumption was defined as the mean number of drinks consumed per week within the past year; lifetime average consumption was estimated as the mean number of drinks per year drinking. Whole-blood genome-wide DNA methylation was measured with HumanMethylation450 BeadChips and used to assess 4 epigenetic clocks (Hannum, Horvath, PhenoAge, and GrimAge) and their corresponding metrics of epigenetic age acceleration (Hannum AgeAccel, Horvath AgeAccel, PhenoAgeAccel, and GrimAgeAccel). Although alcohol consumption showed little association with most age acceleration metrics, both lifetime and recent average consumption measures were positively associated with GrimAgeAccel (lifetime, per additional 135 drinks/year: ß = 0.30 years, 95% confidence interval [CI]: 0.11, 0.48, p = .002; recent, per additional 5 drinks/week: ß = 0.19 years, 95% CI: 0.01, 0.37, p = .04). In a mutually adjusted model, only average lifetime alcohol consumption remained associated with GrimAgeAccel (lifetime, per additional 135 drinks/year: ß = 0.27 years, 95% CI: 0.04, 0.50, p = .02; recent, per 5 additional drinks/week: ß = 0.05 years, 95% CI: -0.16, 0.26, p = .64). Although alcohol use does not appear to be strongly associated with biological age measured by most epigenetic clocks, lifetime average consumption is associated with higher biological age assessed by the GrimAge epigenetic clock.