Reduced global DNA methylation in maternal peripheral blood is associated with preterm birth: an exploratory study in women with diabetes in pregnancy

Alterations in global DNA methylation have been suggested to play an important role in cancer development. We evaluated the association of global DNA methylation in peripheral blood with the risk of lung cancer in nonsmoking women from six countries in Central and Eastern Europe. This multicenter case-control study included primary, incident lung cancer cases diagnosed from 1998 to 2001 and controls frequency-matched for geographic area, sex, and age. Global methylation was assessed in peripheral blood DNA from 83 nonsmoking female cases and 181 nonsmoking female controls using the luminometric methylation assay (LUMA). Unconditional logistic regression models were used to estimate associations between DNA methylation in the blood and the risk of lung cancer. LUMA methylation level was not associated with the risk of lung cancer in nonsmoking women. Associations were not significantly different according to different strata of age, BMI, alcohol drinking, or second-hand tobacco smoke exposure status. In our study of nonsmoking women, the LUMA methylation level in peripheral blood was not associated with the risk of lung cancer. Our findings do not support an association of global blood DNA methylation with the risk of lung cancer in nonsmoking women.

Arsenic exposure from drinking water is associated with decreased gene expression and increased DNA methylation in peripheral blood

<div>Abstract<p>DNA methylation in peripheral blood is a potential biomarker of gastric cancer risk which could be used for early detection. We conducted a prospective case–control study nested within the Melbourne Collaborative Cohort Study. Genomic DNA was prepared from blood samples collected a median of 12 years before diagnosis for cases (<i>N</i> = 168). Controls (<i>N</i> = 163) were matched to cases on sex, year of birth, country of birth, and blood sample type using incidence density sampling. Genome-wide DNA methylation was measured using the Infinium HumanMethylation450K Beadchip. Global measures of DNA methylation were defined as the median methylation M value, calculated for each of 13 CpG subsets representing genomic function, mean methylation and location, and reliability of measurement. Conditional logistic regression was conducted to assess associations between these global measures of methylation and gastric cancer risk, adjusting for <i>Helicobacter pylori</i> and other potential confounders. We tested nonlinear associations using quintiles of the global measure distribution. A genome-wide association study of DNA methylation and gastric cancer risk was also conducted (<i>N</i> = 484,989 CpGs) using conditional logistic regression, adjusting for potential confounders. Differentially methylated regions (DMR) were investigated using the R package <i>DMRcate</i>. We found no evidence of associations with gastric cancer risk for individual CpGs or DMRs (<i>P</i> > 7.6 × 10<sup>−6</sup>). No evidence of association was observed with global measures of methylation (OR 1.07 per SD of overall median methylation; 95% confidence interval, 0.80–1.44; <i>P</i> = 0.65). We found no evidence that blood DNA methylation is prospectively associated with gastric cancer risk.</p><p><b>Prevention Relevance:</b> We studied DNA methylation in blood to try and predict who was at risk of gastric cancer before symptoms developed, by which stage survival is poor. We did not find any such markers, but the importance of early diagnosis in gastric cancer remains, and the search for markers continues.</p></div>

<div>Abstract<p>DNA methylation in peripheral blood is a potential biomarker of gastric cancer risk which could be used for early detection. We conducted a prospective case–control study nested within the Melbourne Collaborative Cohort Study. Genomic DNA was prepared from blood samples collected a median of 12 years before diagnosis for cases (<i>N</i> = 168). Controls (<i>N</i> = 163) were matched to cases on sex, year of birth, country of birth, and blood sample type using incidence density sampling. Genome-wide DNA methylation was measured using the Infinium HumanMethylation450K Beadchip. Global measures of DNA methylation were defined as the median methylation M value, calculated for each of 13 CpG subsets representing genomic function, mean methylation and location, and reliability of measurement. Conditional logistic regression was conducted to assess associations between these global measures of methylation and gastric cancer risk, adjusting for <i>Helicobacter pylori</i> and other potential confounders. We tested nonlinear associations using quintiles of the global measure distribution. A genome-wide association study of DNA methylation and gastric cancer risk was also conducted (<i>N</i> = 484,989 CpGs) using conditional logistic regression, adjusting for potential confounders. Differentially methylated regions (DMR) were investigated using the R package <i>DMRcate</i>. We found no evidence of associations with gastric cancer risk for individual CpGs or DMRs (<i>P</i> > 7.6 × 10<sup>−6</sup>). No evidence of association was observed with global measures of methylation (OR 1.07 per SD of overall median methylation; 95% confidence interval, 0.80–1.44; <i>P</i> = 0.65). We found no evidence that blood DNA methylation is prospectively associated with gastric cancer risk.</p><p><b>Prevention Relevance:</b> We studied DNA methylation in blood to try and predict who was at risk of gastric cancer before symptoms developed, by which stage survival is poor. We did not find any such markers, but the importance of early diagnosis in gastric cancer remains, and the search for markers continues.</p></div>

Objective: This study was undertaken to determine the clinical use of comparative genomic hybridization (CGH) for detection of fetal trisomy 21 from fetal ceIls (nucleated red blood cells; nRBCs) isolated from maternal peripheral venous blood. Methods: Maternal peripheral venous blood samples were collected in sterile tubes containing heparin. After triple density gradient centrifugation, magnetic activated cell sorting using CD45 and CD71 was used to isolate the fetal nRBCs. Fetal nRBCs were successfully isolated from maternal peripheral blood in all cases. After laser-microdissecting fetal nRBCs, degenerate oligonucleotide-primed polymerase chain reaction, and nick translation, DNA size was suitable for hybridization. Results: By CGH analysis, we diagnosed one normal male, one normal female, and one trisomy 21 male fetus. These results were confirmed by amniocentesis. Conclusions: Prenatal diagnosis from fetal cells in maternal peripheral blood by CGH shows clinical promise as an alternative or as a supplement to fluorescence in situ hybridization with chromosome-specific probes but further studies are warranted.

A-kinase-interacting protein 1 (AKIP1) has been discovered to be a pivotal signaling adaptor in the regulation of human labor and associated with preterm birth, but its effect on fetal growth was still unclear. Meanwhile, the regulation role of DNA methylation (DNAm) on placental and fetal development has been demonstrated. Therefore, we aimed to investigate the association of AKIP1 DNAm in maternal peripheral blood with placental development and full-term small for gestational age (FT-SGA) neonates, and to explore whether placenta mediate the association between AKIP1 DNAm and FT-SGA; Methods: This study was a case-control study including 84 FT-SGAs and 84 FT-AGAs derived from the Shenzhen Birth Cohort Study. The DNA methylation analysis of CpG in the target region of the AKIP1 gene was measured by the Sequenom MassARRAY EpiTYPER approach. Multiple-variable logistic and linear regression analyses were used to estimate the association between the DNAm of three validated CpG sites in the AKIP1 gene, placental thickness, and FT-SGA. Mediation analysis was used to examine the mediation effect of placental development on the association between the DNAm of AKIP1 and FT-SGA. For every increment in standard deviation in the DNAm of CpG4 (cg00061907) at AKIP1, the risk of FT-SGA elevated by 2.01-fold (aOR = 2.01, 95%CI = 1.39~3.01), and the thickness of the placenta significantly decreased by a 0.19 standard deviation (β = -0.19, 95%CI = -0.32~ -0.06). Placental thickness mediated the 22.96% of the effect of the DNAm of CpG4 at AKIP1 on the risk of FT-SGA with statistical significance. The findings in the present study suggested the mediating effect of placental thickness on the association of the DNAm of AKIP1 in maternal peripheral blood and the risk of FT-SGA, providing new evidence for the mechanism of maternal epigenetics in placental and fetal development.

Bladder cancer is a common solid tumor, with greater than 71,000 cases diagnosed in the United States last year, and greater than 14,000 deaths attributable to this disease. In addition to this mortality, the morbidity of this disease can be great, as the non-invasive form of this disease, making up >75% of the incidence, recurs frequently, and therefore requires frequent invasive monitoring through cystoscopy at a great cost to the health care system. There are critical gaps in our understanding of the predictors of its recurrence and survival, and identifying molecular alterations associated with these outcomes is essential for improving patient morbidity and the care of patients with this disease. We have shown that profiles of DNA methylation in peripheral blood are associated with human bladder cancer and have recently demonstrated that much, but not all, of the variation in DNA methylation is attributable to disease-associated changes in the underlying proportion of immune cells in the peripheral blood sample. We also have devised a novel statistical strategy that utilizes DNA methylation profiles to quantify the proportion of blood cell subtypes in archival DNA. This is able to disentangle the variability in DNA methylation profiles attributable to blood cell population differences from epigenetic variation arising within a cell population. We sought to determine if variation in DNA methylation identified in peripheral blood, reflective, in part, of altered white blood cell proportions, is associated with bladder cancer recurrence and survival in a population-based study of incident bladder cancers in New Hampshire, USA. Illumina Infinium 27K DNA methylation arrays were performed on whole peripheral blood derived DNA samples from 223 cases of bladder cancer, having a median follow-up time of approximately 13 years. Array data was subjected to quality control procedures and normalization using the ComBAT methodology to account for plate/BeadChip effects. Fitting a series of Cox proportional hazards models, stratified by histology (carcinoma in-situ, low-grade non-invasive, high-grade non-invasive, and invasive) and controlled for patient age and gender revealed 6,873 high-confidence (q < 0.05) associations with bladder cancer specific death as the endpoint. In a principle components analysis, the first principal component, accounting for a striking 46% of the variability in methylation, was significantly associated with bladder cancer specific death. Ongoing analyses are examining how changes to underlying cell proportions mediate the relationship between DNA methylation and patient survival. These data suggest that peripheral blood DNA methylation profiles proximal to the time of diagnosis are strongly prospectively predictive of bladder cancer specific survival and suggest that these biomarkers may have important use as non-invasive prognostic tools worthy of continued study. Citation Format: Devin C. Koestler, Megan A. Murphy, Eugene A. Houseman, Margaret R. Karagas, Karl T. Kelsey, Carmen J. Marsit. Profiles of DNA methylation in peripheral blood are associated with bladder cancer survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3642. doi:10.1158/1538-7445.AM2013-3642

The idea that information can be transmitted to subsequent generation(s) by epigenetic means has been studied for decades but remains controversial in humans. Epidemiological studies have established that grandparental exposures are associated with health outcomes in their grandchildren, often with sex-specific effects; however, the mechanism of transmission is still unclear. We conducted Epigenome Wide Association Studies (EWAS) to test whether grandmaternal smoking during pregnancy is associated with altered DNA methylation (DNAm) in peripheral blood from their adolescent grandchildren. We used data from a birth cohort, with discovery and replication datasets of up to 1225 and 708 individuals (respectively, for the maternal line), aged 15–17 years, and tested replication in the same individuals at birth and 7 years. We show for the first time that DNAm at a small number of loci in cord blood is associated with grandmaternal smoking in humans. In adolescents we see suggestive associations in regions of the genome which we hypothesised a priori could be involved in transgenerational transmission - we observe sex-specific associations at two sites on the X chromosome and one in an imprinting control region. All are within transcription factor binding sites (TFBSs), and we observe enrichment for TFBS among the CpG sites with the strongest associations; however, there is limited evidence that the associations we see replicate between timepoints. The implication of this work is that effects of smoking during pregnancy may induce DNAm changes in later generations and that these changes are often sex-specific, in line with epidemiological associations.

Outcomes Stratification of Head and Neck Cancer Using Pre- and Post-Treatment DNA Methylation in Peripheral Blood

Prior studies report associations of maternal serum Lamin A, encoded by the LMNA gene, with fetal congenital heart disease (CHD). It is unknown whether DNA methylation (DNAm) of cytosine-phosphate-guanine (CpG) sites in LMNA impacts the CHD susceptibility. We investigated the associations of LMNA DNAm with CHD using publicly available data of CHD cases (n = 197) and controls (n = 134) from the Gene Expression Omnibus repository. Peripheral blood DNAm was measured using Illumina 850 K BeadChip for cases and 450 K BeadChip for controls. We tested 31 LMNA CpGs to identify differences in DNAm between cases and controls using linear regression correcting for multiple testing with false discovery rate (FDR). In a case-only analysis, we tested the variations in LMNA DNAm between CHD subtypes. To identify the consistency of DNAm across tissue types we compared peripheral blood (n = 197) and heart tissue DNAm (n = 20) in CHD cases. After adjusting for age, sex, and cell types there were significant differences in 17 of the 31 LMNA CpGs between CHD cases and controls (FDR p ≤ .05). We identified lower DNAm of cg09820673 at 3' UTR for hypoplastic left heart syndrome compared to other CHD subtypes. Three CpGs exhibited uniform DNAm in blood and heart tissues in cases. Eleven CpGs showed changes in the same direction in blood and heart tissues in cases compared to controls. We identify statistically significant differences in LMNA DNAm between CHD cases and controls. Future studies should investigate the role of maternal LMNA DNAm in CHD development.

Epidemiological research suggests that paternal obesity may increase the risk of fathering small for gestational age offspring. Studies in non-human mammals indicate that such associations could be mediated by DNA methylation changes in spermatozoa that influence offspring development in utero. Human obesity is associated with differential DNA methylation in peripheral blood. It is unclear, however, whether this differential DNA methylation is reflected in spermatozoa. We profiled genome-wide DNA methylation using the Illumina MethylationEPIC array in a cross-sectional study of matched human blood and sperm from lean (discovery n = 47; replication n = 21) and obese (n = 22) males to analyse tissue covariation of DNA methylation, and identify obesity-associated methylomic signatures. We found that DNA methylation signatures of human blood and spermatozoa are highly discordant, and methylation levels are correlated at only a minority of CpG sites (~1%). At the majority of these sites, DNA methylation appears to be influenced by genetic variation. Obesity-associated DNA methylation in blood was not generally reflected in spermatozoa, and obesity was not associated with altered covariation patterns or accelerated epigenetic ageing in the two tissues. However, one cross-tissue obesity-specific hypermethylated site (cg19357369; chr4:2429884; P = 8.95 × 10−8; 2% DNA methylation difference) was identified, warranting replication and further investigation. When compared to a wide range of human somatic tissue samples (n = 5,917), spermatozoa displayed differential DNA methylation across pathways enriched in transcriptional regulation. Overall, human sperm displays a unique DNA methylation profile that is highly discordant to, and practically uncorrelated with, that of matched peripheral blood. We observed that obesity was only nominally associated with differential DNA methylation in sperm, and therefore suggest that spermatozoal DNA methylation is an unlikely mediator of intergenerational effects of metabolic traits.

Epidemiological research suggests that paternal obesity may increase the risk of fathering small for gestational age offspring. Studies in non-human mammals indicate that such associations could be mediated by DNA methylation changes in spermatozoa that influence offspring development in utero. Human obesity is associated with differential DNA methylation in peripheral blood. It is unclear, however, whether this differential DNA methylation is reflected in spermatozoa. We profiled genome-wide DNA methylation using the Illumina MethylationEPIC array in a cross-sectional study of matched human blood and sperm from lean (discovery n = 47; replication n = 21) and obese (n = 22) males to analyse tissue covariation of DNA methylation, and identify obesity-associated methylomic signatures. We found that DNA methylation signatures of human blood and spermatozoa are highly discordant, and methylation levels are correlated at only a minority of CpG sites (~1%). At the majority of these sites, DNA methylation appears to be influenced by genetic variation. Obesity-associated DNA methylation in blood was not generally reflected in spermatozoa, and obesity was not associated with altered covariation patterns or accelerated epigenetic ageing in the two tissues. However, one cross-tissue obesity-specific hypermethylated site (cg19357369; chr4:2429884; P = 8.95 × 10-8; 2% DNA methylation difference) was identified, warranting replication and further investigation. When compared to a wide range of human somatic tissue samples (n = 5,917), spermatozoa displayed differential DNA methylation across pathways enriched in transcriptional regulation. Overall, human sperm displays a unique DNA methylation profile that is highly discordant to, and practically uncorrelated with, that of matched peripheral blood. We observed that obesity was only nominally associated with differential DNA methylation in sperm, and therefore suggest that spermatozoal DNA methylation is an unlikely mediator of intergenerational effects of metabolic traits.

DNA methylation in peripheral blood is a potential biomarker of gastric cancer risk which could be used for early detection. We conducted a prospective case-control study nested within the Melbourne Collaborative Cohort Study. Genomic DNA was prepared from blood samples collected a median of 12 years before diagnosis for cases (N = 168). Controls (N = 163) were matched to cases on sex, year of birth, country of birth, and blood sample type using incidence density sampling. Genome-wide DNA methylation was measured using the Infinium HumanMethylation450K Beadchip. Global measures of DNA methylation were defined as the median methylation M value, calculated for each of 13 CpG subsets representing genomic function, mean methylation and location, and reliability of measurement. Conditional logistic regression was conducted to assess associations between these global measures of methylation and gastric cancer risk, adjusting for Helicobacter pylori and other potential confounders. We tested nonlinear associations using quintiles of the global measure distribution. A genome-wide association study of DNA methylation and gastric cancer risk was also conducted (N = 484,989 CpGs) using conditional logistic regression, adjusting for potential confounders. Differentially methylated regions (DMR) were investigated using the R package DMRcate We found no evidence of associations with gastric cancer risk for individual CpGs or DMRs (P > 7.6 × 10-6). No evidence of association was observed with global measures of methylation (OR 1.07 per SD of overall median methylation; 95% confidence interval, 0.80-1.44; P = 0.65). We found no evidence that blood DNA methylation is prospectively associated with gastric cancer risk.Prevention Relevance: We studied DNA methylation in blood to try and predict who was at risk of gastric cancer before symptoms developed, by which stage survival is poor. We did not find any such markers, but the importance of early diagnosis in gastric cancer remains, and the search for markers continues.

Several techniques for the enrichment of nucleated fetal red blood cells present in maternal blood have been reported. Here we describe the use of a quantitative fluorescence in situ hybridization (FISH) method and in vitro suspension cultures of erythroid cells from newborn cord blood and maternal peripheral blood. Together with a rapid high performance liquid chromatography (HPLC) method, that allows us to determine as few as 100 cells containing haemoglobin F (HbF), we have scrutinized the reported enrichment methods for fetal nucleated cells in peripheral maternal blood. One hundred FISH analyses on maternal peripheral blood were performed. The method comprises a cell lysis method for depletion of red cells with minimal losses of nucleated cells, uniform numbers of cells (750 000 cells each) on microscopic slides, and inclusion of internal controls to monitor the efficacy of hybridization. Twenty-six cultures of pure erythroid progenitor cells from maternal peripheral blood were analysed for the expansion of fetal cells. To generate these in vitro cultures, nucleated cells from 10-20 ml of peripheral blood from 26 pregnant women were grown in media containing growth factors and hormones to yield over 10(7) of immature erythroid cells within two weeks. Of those, 13 cultures were from pregnancies with confirmed male fetuses. A total of approximately 8x10(8) maternal cells were added into tissue culture medium for these 13 cultures, resulting in about 2x10(8) nearly pure erythroid cells after two weeks. Whereas fetal cells, alone or added into cultures of peripheral blood, grow rapidly and can be detected quantitatively, we could not find any fetal cells in cultures from maternal blood. Likewise, in 7.5x10(7) peripheral blood cells probed by FISH analysis (half of which were from pregnancies with male fetuses) no single Y chromosome was detected. In summary, suspension cultures of erythroid cells can be established routinely and easily. With the quantitative FISH technique used, 750 000 cells per slide can be screened reliably for cells with Y chromosomes. However, the stringent quality-criteria and most elaborate methods indicate that fetal cells in maternal peripheral blood can not be found using the current technology.

Objective: To characterize and compare the microbiome signature in the maternal, intrauterine, and fetal environments and the associated bacterial species in women who experienced preterm birth and term birth. Methods: A total of 140 women with singleton pregnancies were enrolled in this study. Among them, 31 experienced spontaneous preterm delivery (gestational age < 37 weeks), and 28 of them experienced vaginal delivery at term. Maternal peripheral blood, saliva, and vaginal discharge samples and fetal membrane, amniotic fluid, and cord blood samples were collected immediately after delivery under sterile conditions. DNA was isolated from the fetal membrane and umbilical cord blood samples, and the V3-V4 region of the bacterial 16S rRNA gene was sequenced. The sequence data were quality-filtered, chimera-checked, and organized into operational taxonomic units (OTUs) based on phylogeny. Principal coordinate analysis of beta diversity measures was used for visualization. The linear discriminant analysis effect size (LEfSe) algorithm and Wilcoxon test were used to differentiate the microbiomes found in the fetal membranes and cord blood in the cases of preterm birth. Results: OTU analysis based on the 16S rRNA gene showed similar microbiomes in the maternal peripheral blood, amniotic fluid, fetal membranes, and cord blood. However, the LEfSe algorithm revealed significantly different bacterial compositions in the fetal environment between the preterm and term groups, with some of the bacterial species originating from the maternal peripheral blood or saliva. Conclusions: The bacteria in the intrauterine and fetal environments may originate from other body sites through hematogenous transmission, and may cause the occurrence of preterm birth.