English to Serbian Medical Medical (general) Genetics

DISCUSSION Our present study reports several novel findings obtained through a comparative analysis of the methylation profile in buffy coat versus PMN and lymphocyte cell lines. First, the buffy coat methylation profile was representative of methylation patterns of derived cell lines. We characterized the genomewide methylation profile of the buffy coat, PMN and the lymphocytic fractions drawn from the same individual in 29 normotensive pregnancies across >450,000 CpG sites in genes across the entire genome. We performed pairwise comparisons that yielded a number of probes that are differentially methylated, but with relatively small differences in beta values. We found a very small percentage of differentially methylated CpG sites when the buffy coat was compared to the PMN fraction (2.96%) and a greater percentage of differentially methylated CpG sites between PMN and lymphocytes (34.69%), consistent with the fact that the PMN fraction is the main constituent of the buffy coat Second, differential methylation occurred in biological pathways that are specific for the derived cell lines, such as neutrophil degranulation and cytokine-mediated signaling pathways, consistent with the sensitivity of these cell-type specific pathways to perturbations during the cell separation process. Third, using a linear model we were able to identify approximately 29,000 probes for which the prediction of CpG methylation within the buffy coat is discordant from the prediction from constituent fractions. These discordant probes had a lower GC content, and the CpG sites involved also preferentially affected the open sea and shelf regions; discordant methylation seems to be an in vitro phenomenon and likely due to the separation process of the buffy coat. To date, few studies have provided comparative analyses of different epigenetic profiles across different blood fractions in pregnancy. One notable example is a study of the cord blood, which showed that methylation in whole blood is reasonably comparable to buffy coat in a small number (n 8) of paired samples (Dou et al., 2018). However, the concordance between special cell populations (such as lymphocytes or monocytes) with either whole blood or buffy coat was not studied. To address cell type composition of complex tissues, we have previously



































developed and characterized in silico epigenomic deconvolution methods (Onuchic et al., 2016; Decamps et al., 2020) that infer DNA methylation of constituent cell types by minimizing residuals. We now extend residual analysis to address the complexity of the blood samples with their multiple cell types by providing a novel approach to analyzing and interpreting methylation profiles from the tissue of origin (whole blood, buffy coat) and derived cell lines (PMN and lymphocyte cell lines). Negative residuals in the overall linear model indicate that at least one of the fractions changed its state from unmethylated to methylated in derived cell lines (i.e., PMN and lymphocytes), while the positive residuals representing the opposite process, were few. Most residuals in the interval between −0.7 and 0.7 seem balanced, in accordance with the expected normal distribution as a result of unobserved variation. On the other hand, close to 50% of discordant probes are grouped in a clear peak below −0.8 and have no substantial positive counterpart, i.e., the same probe being methylated in the buffy coat, but unmethylated in derived cell line(s), indicating strong preference towards methylation in PMN and lymphocytes. This targeted DNA inactivation, preferentially targeting isolated CpGs in the open-sea regions, could be the result of the separation process, and the mechanical/ chemical stress exerted on the cells removed from their normal medium. While our study was performed on a relatively small sample size, a pairwise comparison of samples drawn from the same individual should limit the effects of potential confounders. We studied only normotensive pregnancies within 24 h prior to delivery, thus limiting the extrapolation of our results to other conditions with vastly different white blood cell composition or with selective methylation of a certain white blood cell fraction. Our results










































have the potential to improve rigor and reproducibility of studies involving epigenomic profiling of buffy coat samples. For example, in the context of one of our previous studies, we identified a state of transient hypomethylation in normal early pregnancy compared with non-pregnancy (White et al., 2012). This hypomethylation would spontaneously revert after delivery. Our sample included patients close to delivery, suggesting a potential tendency towards global hypomethylation. DNA methylation can be affected by many factors including age (Bell et al., 2012), race, BMI, smoking, gestational diabetes (Wu et al., 2018), and preeclampsia (White et al., 2013). The patients in our study had healthy normotensive pregnancies, with a similar age range, a relatively homogeneous ethnicity, and free of major medical comorbidities that could affect DNA methylation. Some fetal DNA contamination was possible, but it likely represented a very small fraction (up to 6% of total measured DNA) (Bischoff, 2002) and, therefore, it was unlikely to have significantly changed our results. However, in our original study, we could not firmly establish the relation between the DNA methylation profiles in buffy coat samples and that of constituent cell types. Our current results help establish this relation, thus improving both the rigor and biological interpretability of our results. Moreover, our results will help integrate results obtained from buffy coat samples and those obtained from profiling of isolated constituent cell types. Finally, our results are not limited to studies of pregnancy and have implications for numerous other studies involving DNA methylation profiling of buffy coat samples and constituent cell types. A potential limitation of this study is that we did not account for the effect of DNA extraction techniques. DNA was isolated in samples using the AutoGenFlex DNA purification kit for the buffy coat and the neutrophil fraction, and manual extraction of DNA was performed for the lymphocytic fraction. The use of different methods for DNA isolation may affect methylation results, although the methylation mark is considered quite stable, but this is currently minimally discussed in the literature (Hjorthaug et al., 2018). Blood sample processing techniques, such as using Ficoll density centrifugation, can also potentially confound methylation. Finally, we used the Illumina 450K array, which covers only ∼2% of total CpGs within the human genome. The Illumina Infinium Human Methylation 450, however, controls for the confounding and takes SNPs into account when analyzing output data. Future directions include using a different sample cohort and methodology to confirm our results. We hope to reproduce our findings in a larger, independent sample, and in a different study population that would confirm the stability of the methylome in the buffy coat and its sub-fractions. Despite these limitations, our results demonstrate that the buffy coat methylation profile is representative of the methylation patterns in white blood cell types in normal pregnancy obtained using Illumina Human Methylation 450 BeadChip. Small differences in the buffy coat composition may confound the methylation analysis at a very small number of CpG sites, but this is not likely to affect most results. Current methods to adjust for cellular heterogeneity, either by excluding these differentially methylated genes, or, better yet, adjusting methylation data to account for these differences in buffy coat composition, improve the robustness of methylome analysis in buffy coat. Overall, our results support DNA methylation profiling of buffy coats as an acceptable approach for epigenomic profiling in pregnancy research and suggest that separation is likely only needed when studying lineage-specific diseases.