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BackgroundPhthalate exposures are ubiquitous during pregnancy and may contribute to racial and ethnic disparities in preterm birth.ObjectivesWe investigated race and ethnicity in the relationship between biomarkers of phthalate exposure and preterm birth by examining: a) how hypothetical reductions in racial and ethnic disparities in phthalate metabolites might reduce the probability of preterm birth; and b) exposure-response models stratified by race and ethnicity.MethodsWe pooled individual-level data on 6,045 pregnancies from 16 U.S. cohorts. We investigated covariate-adjusted differences in nine urinary phthalate metabolite concentrations by race and ethnicity [non-Hispanic White (White, 43%), non-Hispanic Black (Black, 13%), Hispanic/Latina (38%), and Asian/Pacific Islander (3%)]. Using g-computation, we estimated changes in the probability of preterm birth under hypothetical interventions to eliminate disparities in levels of urinary phthalate metabolites by proportionally lowering average concentrations in Black and Hispanic/Latina participants to be approximately equal to the averages in White participants. We also used race and ethnicity-stratified logistic regression to characterize associations between phthalate metabolites and preterm birth.ResultsIn comparison with concentrations among White participants, adjusted mean phthalate metabolite concentrations were consistently higher among Black and Hispanic/Latina participants by 23%-148% and 4%-94%, respectively. Asian/Pacific Islander participants had metabolite levels that were similar to those of White participants. Hypothetical interventions to reduce disparities in metabolite mixtures were associated with lower probabilities of preterm birth for Black [13% relative reduction; 95% confidence interval (CI): -34%, 8.6%] and Hispanic/Latina (9% relative reduction; 95% CI: -19%, 0.8%) participants. Odds ratios for preterm birth in association with phthalate metabolites demonstrated heterogeneity by race and ethnicity for two individual metabolites (mono-n-butyl and monoisobutyl phthalate), with positive associations that were larger in magnitude observed among Black or Hispanic/Latina participants.ConclusionsPhthalate metabolite concentrations differed substantially by race and ethnicity. Our results show hypothetical interventions to reduce population-level racial and ethnic disparities in biomarkers of phthalate exposure could potentially reduce the probability of preterm birth. https://doi.org/10.1289/EHP12831.

Tumor subtype and menopausal status are strong predictors of breast cancer (BC) prognosis. We aimed to find and validate subtype- or menopausal-status-specific changes in tumor DNA methylation (DNAm) associated with all-cause mortality or BC progression. Associations between site-specific tumor DNAm and BC prognosis were estimated among The Cancer Genome Atlas participants (n = 692) with Illumina Infinium HumanMethylation450 BeadChip array data. All-cause mortality and BC progression were modeled using Cox proportional hazards models stratified by tumor subtypes, adjusting for age, race, stage, menopausal status, tumor purity, and cell type proportion. Effect measure modification by subtype and menopausal status were evaluated by incorporating a product term with DNAm. Site-specific inference was used to identify subtype- or menopausal-status-specific differentially methylated regions (DMRs) and functional pathways. The validation of the results was carried out on an independent dataset (GSE72308; n = 180). We identified a total of fifteen unique CpG probes that were significantly associated ( P ≤ 1 × 10 - 7 with survival outcomes in subtype- or menopausal-status-specific manner. Seven probes were associated with overall survival (OS) or progression-free interval (PFI) for women with luminal A subtype, and four probes were associated with PFI for women with luminal B subtype. Five probes were associated with PFI for post-menopausal women. A majority of significant probes showed a lower risk of OS or BC progression with higher DNAm. We identified subtype- or menopausal-status-specific DMRs and functional pathways of which top associated pathways differed across subtypes or menopausal status. None of significant probes from site-specific analyses met genome-wide significant level in validation analyses while directions and magnitudes of coefficients showed consistent pattern. We have identified subtype- or menopausal-status-specific DNAm biomarkers, DMRs and functional pathways associated with all-cause mortality or BC progression, albeit with limited validation. Future studies with larger independent cohort of non-post-menopausal women with non-luminal A subtypes are warranted for identifying subtype- and menopausal-status-specific DNAm biomarkers for BC prognosis.

We previously described a DNA methylation (DNAm) based biomarker of human mortality risk DNAm GrimAge. Here we describe version 2 of GrimAge (trained on individuals aged between 40 and 92) which leverages two new DNAm based estimators of (log transformed) plasma proteins: high sensitivity C-reactive protein (logCRP) and hemoglobin A1C (logA1C). We evaluate GrimAge2 in 13,399 blood samples across nine study cohorts. After adjustment for age and sex, GrimAge2 outperforms GrimAge in predicting mortality across multiple racial/ethnic groups (meta P=3.6x10-167 versus P=2.6x10-144) and in terms of associations with age related conditions such as coronary heart disease, lung function measurement FEV1 (correlation= -0.31, P=1.1x10-136), computed tomography based measurements of fatty liver disease. We present evidence that GrimAge version 2 also applies to younger individuals and to saliva samples where it tracks markers of metabolic syndrome. DNAm logCRP is positively correlated with morbidity count (P=1.3x10-54). DNAm logA1C is highly associated with type 2 diabetes (P=5.8x10-155). DNAm PAI-1 outperforms the other age-adjusted DNAm biomarkers including GrimAge2 in correlating with triglyceride (cor=0.34, P=9.6x10-267) and visceral fat (cor=0.41, P=4.7x10-41). Overall, we demonstrate that GrimAge version 2 is an attractive epigenetic biomarker of human mortality and morbidity risk.

Higher maternal pre-pregnancy body mass index (ppBMI) is associated with increased neonatal morbidity, as well as with pregnancy complications and metabolic outcomes in offspring later in life. The placenta is a key organ in fetal development and has been proposed to act as a mediator between the mother and different health outcomes in children. The overall aim of the present work is to investigate the association of ppBMI with epigenome-wide placental DNA methylation (DNAm) in 10 studies from the PACE consortium, amounting to 2631 mother-child pairs. We identify 27 CpG sites at which we observe placental DNAm variations of up to 2.0% per 10 ppBMI-unit. The CpGs that are differentially methylated in placenta do not overlap with CpGs identified in previous studies in cord blood DNAm related to ppBMI. Many of the identified CpGs are located in open sea regions, are often close to obesity-related genes such as GPX1 and LGR4 and altogether, are enriched in cancer and oxidative stress pathways. Our findings suggest that placental DNAm could be one of the mechanisms by which maternal obesity is associated with metabolic health outcomes in newborns and children, although further studies will be needed in order to corroborate these findings.

BackgroundAging and inflammation are important components of Parkinson's disease (PD) pathogenesis and both are associated with changes in hematopoiesis and blood cell composition. DNA methylation (DNAm) presents a mechanism to investigate inflammation, aging, and hematopoiesis in PD, using epigenetic mitotic aging and aging clocks. Here, we aimed to define the influence of blood cell lineage on epigenetic mitotic age and then investigate mitotic age acceleration with PD, while considering epigenetic age acceleration biomarkers.ResultsWe estimated epigenetic mitotic age using the "epiTOC" epigenetic mitotic clock in 10 different blood cell populations and in a population-based study of PD with whole-blood. Within subject analysis of the flow-sorted purified blood cell types DNAm showed a clear separation of epigenetic mitotic age by cell lineage, with the mitotic age significantly lower in myeloid versus lymphoid cells (p = 2.1e-11). PD status was strongly associated with accelerated epigenetic mitotic aging (AccelEpiTOC) after controlling for cell composition (OR = 2.11, 95 % CI = 1.56, 2.86, p = 1.6e-6). AccelEpiTOC was also positively correlated with extrinsic epigenetic age acceleration, a DNAm aging biomarker related to immune system aging (with cell composition adjustment: R = 0.27, p = 6.5e-14), and both were independently associated with PD. Among PD patients, AccelEpiTOC measured at baseline was also associated with longitudinal motor and cognitive symptom decline.ConclusionsThe current study presents a first look at epigenetic mitotic aging in PD and our findings suggest accelerated hematopoietic cell mitosis, possibly reflecting immune pathway imbalances, in early PD that may also be related to motor and cognitive progression.

BackgroundBiological 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.ResultsLeveraging 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.ConclusionThis study illuminates the genetic architecture underlying epigenetic aging and its shared genetic contributions with lifestyle factors and longevity.

BackgroundSystemic inflammation may play a role in shaping breast composition, one of the strongest risk factors for breast cancer. Pubertal development presents a critical window of breast tissue susceptibility to exogenous and endogenous factors, including pro-inflammatory markers. However, little is known about the role of systemic inflammation on adolescent breast composition and pubertal development among girls.MethodsWe investigated associations between circulating levels of inflammatory markers (e.g., interleukin-6 (IL-6), tumor necrosis factor receptor 2 (TNFR2), and C-reactive protein (CRP)) at Tanner stages 2 and 4 and breast composition at Tanner stage 4 in a cohort of 397 adolescent girls in Santiago, Chile (Growth and Obesity Cohort Study, 2006-2018). Multivariable linear models were used to examine the association between breast composition and each inflammatory marker, stratifying by Tanner stage at inflammatory marker measurement. Accelerated failure time models were used to evaluate the association between inflammatory markers concentrations at each Tanner stage and time to menarche.ResultsIn age-adjusted linear regression models, a doubling of TNFR2 at Tanner 2 was associated with a 26% (95% CI 7-48%) increase in total breast volume at Tanner 4 and a 22% (95% CI 10-32%) decrease of fibroglandular volume at Tanner 4. In multivariable models further adjusted for body fatness and other covariates, these associations were attenuated to the null. The time to menarche was 3% (95% CI 1-5%) shorter among those in the highest quartile of IL-6 at Tanner 2 relative to those in the lowest quartile in fully adjusted models. Compared to those in the lowest quartile of CRP at Tanner 4, those in the highest quartile experienced 2% (95% CI 0-3%) longer time to menarche in multivariable models.ConclusionsSystemic inflammation during puberty was not associated with breast volume or breast density at the conclusion of breast development among pubertal girls after adjusting for body fatness; however, these circulating inflammation biomarkers, specifically CRP and IL-6, may affect the timing of menarche onset.

DNA methylation (DNAm) age estimators are widely used to study aging-related conditions. It is not yet known whether DNAm age is associated with the accumulation of stochastic epigenetic mutations (SEMs), which reflect dysfunctions of the epigenetic maintenance system. Here, we defined epigenetic mutation load (EML) as the total number of SEMs per individual. We assessed associations between EML and DNAm age acceleration estimators using biweight midcorrelations in four population-based studies (total n = 6,388). EML was not only positively associated with chronological age (meta r = 0.171), but also with four measures of epigenetic age acceleration: the Horvath pan tissue clock, intrinsic epigenetic age acceleration, the Hannum clock, and the GrimAge clock (meta-analysis correlation ranging from r = 0.109 to 0.179). We further conducted pathway enrichment analyses for each participant's SEMs. The enrichment result demonstrated the stochasticity of epigenetic mutations, meanwhile implicated several pathways: signaling, neurogenesis, neurotransmitter, glucocorticoid, and circadian rhythm pathways may contribute to faster DNAm age acceleration. Finally, investigating genomic-region specific EML, we found that EMLs located within regions of transcriptional repression (TSS1500, TSS200, and 1stExon) were associated with faster age acceleration. Overall, our findings suggest a role for the accumulation of epigenetic mutations in the aging process.

Aim: Given the immunosuppressive nature of pancreatic cancer, we investigated the relationship between epigenetic modification of immune-regulatory cytokine genes and pancreatic cancer outcomes. Materials & methods: We evaluated DNA methylation of 184 pancreatic tumor samples from The Cancer Genome Atlas for 111 CpG loci in seven cytokine genes: IL10, IL6, IL8, TGFβ1, TGFβ2, TGFβ3 and TNF. We used Cox regression to evaluate the associations between methylation and overall survival, disease-specific survival and disease progression (α = 0.05). Results: Poorer survival was associated with increased methylation in fifteen CpG probes in TGFβ1, TGFβ2, TGFβ3 and TNF. We also detected improved outcomes for three loci in IL10, IL8 and IL6. Conclusion: Epigenetic regulation of cytokine-related gene expression may be associated with pancreatic cancer outcomes.

Telomere length correlates with morbidity and mortality. While telomere length appears to be influenced by hormone levels, the potential impact of exposure to endocrine-disrupting chemicals (EDCs) has not been studied. We examined the association between maternal gestational concentrations of biomarkers of EDC exposure and telomere length at birth in the Harvard Epigenetic Birth Cohort. EDC (phenols and phthalates) biomarker concentrations were measured in maternal spot urine samples during the first trimester and telomere length in maternal and cord blood collected at delivery among 181 mother-newborn singleton dyads. Maternal and newborn telomere length exhibited a positive correlation (Spearman ρ = 0.20 (p-value< 0.01). Infant telomere length was associated with maternal biomarker concentrations of specific EDCs, and most of these associations were observed to be infant sex-specific. Prenatal exposure to triclosan, a non-paraben phenol with antimicrobial properties, was one of the most strongly associated EDCs with telomere length; telomere length was 20% (95% CI 5%-33%) shorter among boys in the highest quartile of maternal biomarker concentrations compared to the lowest quartile. In contrast, we observed longer telomere length associated with increased gestational concentrations of mono-isobutyl phthalate, and among boys, with increased concentrations of mono-2-ethylhexyl phthalate. In this birth cohort, we observed associations between maternal gestational exposure to select EDC biomarkers and telomere length, most of which were sex-specific. These findings need to be confirmed in future studies.

BackgroundDuring puberty, mammary tissue undergoes rapid development, which provides a window of heightened susceptibility of breast composition to the influence of endogenous and exogenous hormones. Exposure to endocrine-disrupting chemicals (EDC) may affect breast development and composition and the risk of developing breast cancer in adulthood.MethodsWe evaluated the associations between breast density and urinary concentrations of phenols and phthalates collected at Tanner 1 (B1) and Tanner 4 (B4) in 200 Chilean girls. Total breast volume (BV), fibroglandular volume (FGV), and percent dense breast (%FGV) were evaluated at B4 using dual X-ray absorptiometry. Generalized estimating equations were used to analyze the association between concentrations of EDC biomarkers across puberty and breast density.ResultsThe geometric mean %FGV was 7% higher among girls in the highest relative to the lowest tertile of monocarboxyisooctyl phthalate [1.07; 95% confidence interval (CI), 1.01-1.14]. Monoethyl phthalate concentrations at B4 were positively associated with FGV (highest vs. lowest tertile: 1.22; 95% CI, 1.06-1.40). Bisphenol A displayed a U-shaped association with FGV; girls in the middle tertile had at least 10% lower FGV than girls in the lowest or highest tertiles. Monocarboxyisononyl phthalate showed a nonlinear association with BV. No other statistically significant associations were observed.ConclusionsOur results suggest that the developing breast tissue is susceptible to select EDCs during childhood and adolescence.ImpactThis study may spur further investigations into environmental influences on breast development during puberty and how shifts in pubertal breast density track through the life course to modify breast cancer risk.

BackgroundEstrogen receptor-α (ER-α) is a transcriptional regulator, which mediates estrogen-dependent breast development, as well as breast tumorigenesis. The influence of epigenetic regulation of ER-α on adolescent breast composition has not been previously studied and could serve as a marker of pubertal health and susceptibility to breast cancer. We investigated the association between ER-α DNA methylation in leukocytes and breast composition in adolescent Chilean girls enrolled in the Growth and Obesity Cohort Study (GOCS) in Santiago, Chile. Breast composition (total breast volume (BV; cm3), fibroglandular volume (FGV; cm3), and percent fibroglandular volume (%FGV)) was measured at breast Tanner stage 4 (B4). ER-α promoter DNA methylation was assessed by pyrosequencing in blood samples collected at breast Tanner stages 2 (B2; n = 256) and B4 (n = 338).ResultsAfter adjusting for fat percentage at breast density measurement, ER-α methylation at B2, and cellular heterogeneity, we observed an inverse association between B4 average ER-α DNA methylation and BV and FGV. Geometric mean BV was 15% lower (95% CI: - 28%, - 1%) among girls in the highest quartile of B4 ER-α methylation (6.96-23.60%) relative to the lowest (0.78-3.37%). Similarly, FGV was 19% lower (95% CI: - 33%, - 2%) among girls in the highest quartile of B4 ER-α methylation relative to the lowest. The association between ER-α methylation and breast composition was not significantly modified by body fat percentage and was not influenced by pubertal timing.ConclusionsThese findings suggest that the methylation profile of ER-α may modulate adolescent response to estrogen and breast composition, which may influence breast cancer risk in adulthood.

BACKGROUND:The age of menarche has been associated with metabolic and cardiovascular disease, as well as cancer risk. The decline in menarcheal age over the past century may be partially attributable to increased exposure to endocrine disrupting chemicals (EDCs). METHODS:We assessed the influence of 26 phenol and phthalate biomarkers on the timing of menarche in a longitudinal cohort of Chilean girls. These EDCs were quantified in urine collected prior to the onset of breast development (Tanner 1; B1), and during adolescence (Tanner 4; B4). Multivariable accelerated failure time (AFT) models were used to analyze associations between biomarker concentrations and the age of menarche adjusting for body mass index (BMI) Z-score and maternal education, accounting for within-subject correlation. RESULTS:Several biomarkers were significantly associated with the age at menarche; however, these associations were dependent on the timing of biomarker assessment. A log(ng/ml) increase in B1 concentrations of di(2-ethylhexyl) phthalate biomarkers was associated with later menarche (hazard ratio (HR): 0.77; 95% CI: 0.60, 0.98), whereas higher B1 concentrations of 2,5-dichlorophenol and benzophenone-3 were associated with earlier menarche (HR: 1.13; 95% CI: 1.01, 1.27; HR: 1.17; 95% CI: 1.06, 1.29, respectively). Elevated B4 concentrations of monomethyl phthalate were similarly associated with earlier menarche (HR: 1.30; 95% CI: 1.10, 1.53). The impact of monoethyl phthalate and triclosan concentrations on pubertal timing were significantly modified by BMI Z-score. Higher monoethyl phthalate and triclosan concentrations were associated with earlier menarche among overweight or obese girls, but not among those that were normal weight. CONCLUSIONS:This study identifies modulation of sexual maturation by specific EDC biomarkers in Latina girls.

In humans the expression of lactase changes during post-natal development, leading to phenotypes known as lactase persistence and non-persistence. Polymorphisms within the lactase gene (LCT) enhancer, in particular the -13910C > T, but also others, are linked to these phenotypes. We were interested in identifying dynamic mediators of LCT regulation, beyond the genotype at -13910C > T. To this end, we investigated two levels of lactase regulation in human intestinal samples obtained from New England children and adolescents of mixed European ancestry: differential expression of transcriptional regulators of LCT, and variations in DNA methylation, and their relation to phenotype. Variations in expression of CDX2, POU2F1, GATA4, GATA6, and HNF1α did not correlate with phenotype. However, an epigenome-wide approach using the Illumina Infinium HM450 bead chip identified a differentially methylated position in the LCT promoter where methylation levels are associated with the genotype at -13910C > T, the persistence/non-persistence phenotype and lactase enzymatic activity. DNA methylation levels at this promoter site and CpGs in the LCT enhancer are associated with genotype. Indeed, taken together they have a higher power to predict lactase phenotypes than the genotype alone.

We empirically examined the strengths and weaknesses of two human genome-wide DNA methylation platforms: rapid multiplexed reduced representation bisulfite sequencing and Illumina's Infinium BeadChip. Rapid multiplexed reduced representation bisulfite sequencing required less input DNA, offered more flexibility in coverage, and interrogated more CpG loci at a higher regional density. The Infinium covered slightly more protein coding, cancer-associated and mitochondrial-related genes, both platforms covered all known imprinting clusters, and rapid multiplexed reduced representation bisulfite sequencing covered more microRNA genes than the HumanMethylation450, but fewer than the MethylationEPIC. Rapid multiplexed reduced representation bisulfite sequencing did not always interrogate exactly the same CpG loci, but genomic tiling improved overlap between different libraries. Reproducibility of rapid multiplexed reduced representation bisulfite sequencing and concordance between the platforms increased with CpG density. Only rapid multiplexed reduced representation bisulfite sequencing could genotype samples and measure allele-specific methylation, and we confirmed that Infinium measurements are influenced by nearby single-nucleotide polymorphisms. The respective strengths and weaknesses of these two genome-wide DNA methylation platforms need to be considered when conducting human epigenetic studies.

IntroductionThe prevalence of type 2 diabetes (T2D) and the metabolic syndrome (MetS) is increasing and several studies suggested an involvement of DNA methylation in the development of these metabolic diseases. This study was designed to investigate if differential DNA methylation in blood can function as a biomarker for T2D and/or MetS.MethodsPyrosequencing analyses were performed for the candidate genes KCNJ11, PPARγ, PDK4, KCNQ1, SCD1, PDX1, FTO and PEG3 in peripheral blood leukocytes (PBLs) from 25 patients diagnosed with only T2D, 9 patients diagnosed with T2D and MetS and 11 control subjects without any metabolic disorders.ResultsNo significant differences in gene-specific methylation between patients and controls were observed, although a trend towards significance was observed for PEG3. Differential methylation was observed between the groups in 4 out of the 42 single CpG loci located in the promoters regions of the genes FTO, KCNJ11, PPARγ and PDK4. A trend towards a positive correlation was observed for PEG3 methylation with HDL cholesterol levels.DiscussionAltered levels of DNA methylation in PBLs of specific loci might serve as a biomarker for T2D or MetS, although further investigation is required.

BackgroundThere is increasing concern that early-life exposure to endocrine-disrupting chemicals (EDCs) can influence the risk of disease development. Phthalates and phenols are two classes of suspected EDCs that are used in a variety of everyday consumer products, including plastics, epoxy resins, and cosmetics. In utero exposure to EDCs may affect disease propensity through epigenetic mechanisms.ObjectiveThe objective of this study was to determine whether prenatal exposure to multiple EDCs is associated with changes in miRNA expression of human placenta, and whether miRNA alterations are associated with birth outcomes.MethodsOur study was restricted to a total of 179 women co-enrolled in the Harvard Epigenetic Birth Cohort and the Predictors of Preeclampsia Study. We analyzed associations between first-trimester urine concentrations of 8 phenols and 11 phthalate metabolites and expression of 29 candidate miRNAs in placenta by qRT-PCR.ResultsFor three miRNAs--miR-142-3p, miR15a-5p, and miR-185--we detected associations between Σphthalates or Σphenols on expression levels (p < 0.05). By assessing gene ontology enrichment, we determined the potential mRNA targets of these microRNAs predicted in silico were associated with several biological pathways, including the regulation of protein serine/threonine kinase activity. Four gene ontology biological processes were enriched among genes significantly correlated with the expression of miRNAs associated with EDC burden.ConclusionsOverall, these results suggest that prenatal phenol and phthalate exposure is associated with altered miRNA expression in placenta, suggesting a potential mechanism of EDC toxicity in humans.

BackgroundGestational diabetes mellitus (GDM) affects approximately 10 % of pregnancies in the United States and increases the risk of adverse health outcomes in the offspring. These adult disease propensities may be set by anatomical and molecular alterations in the placenta associated with GDM.ResultsTo assess the mechanistic aspects of fetal programming, we measured genome-wide methylation (Infinium HumanMethylation450 BeadChips) and expression (Affymetrix transcriptome microarrays) in placental tissue of 41 GDM cases and 41 matched pregnancies without maternal complications from the Harvard Epigenetic Birth Cohort. Specific transcriptional and epigenetic perturbations associated with GDM status included alterations in the major histocompatibility complex (MHC) region, which were validated in an independent cohort, the Rhode Island Child Health Study. Gene ontology enrichment among gene regulation influenced by GDM revealed an over-representation of immune response pathways among differential expression, reflecting these coordinated changes in the MHC region. This differential methylation and expression may be capturing shifts in cellular composition, reflecting physiological changes in the placenta associated with GDM.ConclusionsOur study represents the largest investigation of transcriptomic and methylomic differences associated with GDM, providing comprehensive insight into how GDM shapes the intrauterine environment, which may have implications for fetal (re)programming.

IntroductionBisulfite treatment of DNA introduces methylation-dependent sequence changes through selective chemical conversion of nonmethylated cytosine to uracil and serves as pretreatment step for the majority of DNA methylation analysis methods.MethodsWe have evaluated the conversion performance of five of the most commonly used bisulfite treatment kits [MethylDetector (Active Motif), Epitect+ (Qiagen), Zymo Methylation, Zymo Gold and Zymo Lightning (all from Zymo Research)] by pyrosequencing four different regions with variable methylation levels, including: a repetitive element (ALUSX), a gene with low levels of methylation (IL6ST), an imprinted gene expected to be approximately 50% methylated (IGF2), and a fully methylated gene (ST3GAL2). In addition, we have studied the influence of duration (3 vs. 16 h) and type (fixed temperature vs. cycling program) of incubation protocol on the conversion efficiency of each evaluated kit.ResultsAll kits produced similar conversion rates of ALUSX, IGF2 and ST3GAL2, while the conversion of the low methylated IL6ST gene was variable between kits. The Zymo kits were highly consistent in their performance even when different protocols of incubation were applied, generating full conversion at the low methylated gene IL6; this was not true for the MethylDetector and Epitect+ kits. However, long-cycling incubation could produce similar conversion rates for the same locus in combination with Active Motif and Qiagen kits.ConclusionsThe selection of a long-cycling protocol during conversion permits standardization of protocols, improving the reproducibility of methylation estimates across laboratories for gene-specific, genome-wide and bisulfite-based sequencing analyses.

Background: Many patients with colon cancer cannot fully adhere to postoperative chemotherapy due to dose-limiting toxicities, resulting in lower relative dose intensity (RDI) and potentially compromising overall survival. This study examined whether home-based resistance training (RT) during adjuvant chemotherapy improves RDI and patient-reported toxicities versus usual care (UC) in colon cancer patients. Methods: Multicenter, randomized control trial (RCT) conducted at community and academic practices. Enrollment of patients receiving postoperative chemotherapy for colon cancer occurred between February 23, 2018, and September 29, 2021; final follow-up was March 21, 2022. Participants were randomized to RT (n = 90) or UC (n = 91) for the duration of chemotherapy. Participants in the RT group engaged in twice weekly home-based progressive RT. At the end of the study, UC was given an online exercise program. Results: Among 181 randomized patients (mean age, 55.2 [SD, 12.8] years, 95 [52.5%] were men), there were no differences in the mean RDI among those in RT (79% [SD, 19%]) and those in UC (82% [SD, 19%]); (mean difference -0.04 [95% confidence interval (CI), -0.09 to 0.02]). Assignment to RT did not significantly reduce the number of moderate/severe symptoms per week across follow-up (relative rate: 0.94 [95% CI, 0.72-1.22]). Additionally, time since randomization did not significantly modify the effect of RT on the overall number of symptoms (p = .06). Conclusions: Among patients with colon cancer, these results do not support homebased RT as an adjunct to chemotherapy specifically to improve planned treatment intensity. [ABSTRACT FROM AUTHOR]

BackgroundInvestigation of the biological mechanism by which folate acts to affect fetal development can inform appraisal of expected benefits and risk management. This research is ethically imperative given the ubiquity of folic acid fortified products in the US. Considering that folate is an essential component in the one-carbon metabolism pathway that provides methyl groups for DNA methylation, epigenetic modifications provide a putative molecular mechanism mediating the effect of folic acid supplementation on neonatal and pediatric outcomes.ResultsIn this study we use a Mendelian Randomization Unnecessary approach to assess the effect of red blood cell (RBC) folate on genome-wide DNA methylation in cord blood. Site-specific CpG methylation within the proximal promoter regions of approximately 14,500 genes was analyzed using the Illumina Infinium Human Methylation27 Bead Chip for 50 infants from the Epigenetic Birth Cohort at Brigham and Women's Hospital in Boston. Using methylenetetrahydrofolate reductase genotype as the instrument, the Mendelian Randomization approach identified 7 CpG loci with a significant (mostly positive) association between RBC folate and methylation level. Among the genes in closest proximity to this significant subset of CpG loci, several enriched biologic processes were involved in nucleic acid transport and metabolic processing. Compared to the standard ordinary least squares regression method, our estimates were demonstrated to be more robust to unmeasured confounding.ConclusionsTo the authors' knowledge, this is the largest genome-wide analysis of the effects of folate on methylation pattern, and the first to employ Mendelian Randomization to assess the effects of an exposure on epigenetic modifications. These results can help guide future analyses of the causal effects of periconceptional folate levels on candidate pathways.

Background: Despite evidence that low muscle increases the risk of chemotoxicity, most chemotherapies are dosed on body surface area without considering body composition. Among 178 patients with colon cancer, we assessed muscle and adipose tissue with multiple techniques and examined their associations with relative dose intensity (RDI) and adverse events. Methods: We estimated (i) cross-sectional skeletal muscle area (SMA) and total adipose tissue (TAT) area at L3 from computed tomography (CT); (ii) appendicular lean mass (ALM) and total body fat (TBF) mass from dual-energy X-ray absorptiometry (DXA); and (iii) total body skeletal muscle mass using D3-creatine (D3Cr) dilution. We standardized each measurement by its sex-specific standard deviation (SD). The primary outcome was reduced RDI (RDI <85%). The secondary outcome was the number of moderate and severe adverse events during each cycle of chemotherapy. We estimated the associations of muscle and adipose tissue measurements (per SD increase) with reduced RDI using logistic regression and adverse events using generalized estimating equations for repeated measures. Results: Higher CT SMA and DXA ALM were significantly associated with a lower risk of reduced RDI [odds ratios: 0.56 (0.38-0.81) for CT SMA; 0.56 (0.37-0.84) for DXA ALM]. No measurements of muscle or adipose tissue were associated with adverse events. Conclusions: More muscle was associated with improved chemotherapy completion among patients with colon cancer, whereas muscle and adipose tissue were not associated with adverse events. Impact: Considering body composition may help personalize dosing for colon cancer chemotherapy by identifying patients at risk for poor chemotherapy outcomes. [ABSTRACT FROM AUTHOR]