Anne Fassbender

Differences in DNA methylation patterns between humans and chimpanzees

Methylation at CpG dinucleotides is important for gene regulation in mammals [1]. However, it is unknown how methylation patterns change during evolution. Here, we compare methylation patterns between humans and chimpanzees at 36 genes in the brain, the liver and in lymphocytes. We find that the extent of the change in methylation pattern is much more extensive in the brain than in the other tissues. Furthermore, of the 15 CpGs that have significantly changed methylation in the brain, 14 are more methylated in humans than in chimpanzees. This indicates that CpGs might generally be more methylated in human brains than in chimpanzee brains. Despite considerable phenotypic differences, humans and their closest living relatives, the chimpanzees, are on average 98.8% identical in their alignable genomic DNA sequences [2,3]. It is currently unknown which genotypic differences are responsible for the phenotypic differences. One possibility to tackle this question is to compare gene expression patterns between humans and chimpanzees using functional genomic approaches [4–6]. In this respect, it may also be useful to compare methylation patterns in regulatory DNA sequences, as the methylation status can be viewed as a “footprint” of the chromatin structures that are crucial for gene regulation [7,8]. In order to take a first step toward understanding the evolution of methylation patterns, we compared the methylation status of 145 CpGs in the presumed regulatory regions of 36 different genes between humans and chimpanzees in brain, liver and lymphocytes using a recently developed array technique [9–11]. Thereby, genomic DNA is treated with sodium bisulphite such that unmethylated CpGs are amplified as TpGs in the following PCR. For each CpG examined, the arrays contain two oligonucleotides: one complementary to a TpG, resembling a formerly unmethylated CpG and one complementary to a CpG, resembling a formerly methylated CpG. We identified 22 CpGs in which the ratio of the intensities of these two oligonucleotides differed significantly between human and chimpanzee in at least one tissue. By contrast, zero to three differences would be expected due to random experimental and biological variation, as is shown by permutating the species labels for each tissue (see supplemental data for all methodological details). Therefore, the differences between the two species are highly significant, whereas the differences between the individuals of the same species are within the range of the experimental error (data not shown). We also do not observe a strong correlation of methylation levels with age or time post mortem (see supplemental data), Thus, we conclude that most of the observed methylation differences between humans and chimpanzees are neither due to random measurement errors nor due to random or systematic differences in their environment. To exclude trivial genetic causes, we sequenced the region of the 22 CpG sites in the chimpanzee and excluded 4 CpGs that carried a sequence difference between the chimpanzee sequence and the human-based oligonucleotide sequence. The remaining 18 CpGs from 12 genes are shown in Figure 1. Three observations from these experiments are especially noteworthy: First, despite the limited number of CpGs studied, several significant differences in their methylation status can be found between humans and chimpanzees. Second, out of 18 differences, 15 are found between chimpanzee and human brain, whereas only six are found between the other two tissues. Third, 14 of the 15 sites differing in methylation in the brain show a higher degree of methylation in humans. The first observation indicates that — at least in humans and chimpanzees — the methylation status of many CpG sites changes during the course of evolution. The second observation indicates that more CpG sites have changed their methylation status in the brain than in liver or lymphocytes. Notably, DNA methylation seems to be especially important for the brain, as defects in methylation lead to mental retardation in humans [8] and a mouse model for one of these diseases — Rett syndrome — indicates that the symptoms can be caused solely by a defect in postmitotic neurons [12,13]. Our third observation, namely that 14 of 15 CpG sites differently methylated in the brain show a higher degree of methylation in humans, might reflect a general up-methylation of genes in the human brain, rather than several independent, gene-specific methylation changes. Although it is unclear at this point whether this up-methylation directly translates into observable changes in gene expression (supplemental data), it is tempting to speculate that such an upmethylation was important for the evolution of the human brain. However, we cannot exclude that a general tendency towards a lower degree of methylation occurred on the chimpanzee lineage. It is furthermore unclear if the change in methylation patterns is especially pronounced in the human brain or if a rapid change in methylation patterns is typical of brain evolution in many mammals. Further work has to clarify these issues.

CDO1 Promoter Methylation is a Biomarker for Outcome Prediction of Anthracycline Treated, Estrogen Receptor-Positive, Lymph Node-Positive Breast Cancer Patients

BackgroundVarious biomarkers for prediction of distant metastasis in lymph-node negative breast cancer have been described; however, predictive biomarkers for patients with lymph-node positive (LNP) disease in the context of distinct systemic therapies are still very much needed. DNA methylation is aberrant in breast cancer and is likely to play a major role in disease progression. In this study, the DNA methylation status of 202 candidate loci was screened to identify those loci that may predict outcome in LNP/estrogen receptor-positive (ER+) breast cancer patients with adjuvant anthracycline-based chemotherapy.MethodsQuantitative bisulfite sequencing was used to analyze DNA methylation biomarker candidates in a retrospective cohort of 162 LNP/ER+ breast cancer patients, who received adjuvant anthracycline-based chemotherapy. First, twelve breast cancer specimens were analyzed for all 202 candidate loci to exclude genes that showed no differential methylation. To identify genes that predict distant metastasis, the remaining loci were analyzed in 84 selected cases, including the 12 initial ones. Significant loci were analyzed in the remaining 78 independent cases. Metastasis-free survival analysis was conducted by using Cox regression, time-dependent ROC analysis, and the Kaplan-Meier method. Pairwise multivariate regression analysis was performed by linear Cox Proportional Hazard models, testing the association between methylation scores and clinical parameters with respect to metastasis-free survival.ResultsOf the 202 loci analysed, 37 showed some indication of differential DNA methylation among the initial 12 patient samples tested. Of those, 6 loci were associated with outcome in the initial cohort (n = 84, log rank test, p < 0.05).Promoter DNA methylation of cysteine dioxygenase 1 (CDO1) was confirmed in univariate and in pairwise multivariate analysis adjusting for age at surgery, pathological T stage, progesterone receptor status, grade, and endocrine therapy as a strong and independent biomarker for outcome prediction in the independent validation set (log rank test p-value = 0.0010).ConclusionsCDO1 methylation was shown to be a strong predictor for distant metastasis in retrospective cohorts of LNP/ER+ breast cancer patients, who had received adjuvant anthracycline-based chemotherapy.