Intra-Monozygotic Twin Pair Discordance and Longitudinal Variation of Whole-Genome Scale DNA Methylation in Adults

<div><p>Monozygotic twins share identical genomic DNA and are indistinguishable using conventional genetic markers. Increasing evidence indicates that monozygotic twins are epigenetically distinct, suggesting that a comparison between DNA methylation patterns might be useful to approach this forensic problem. However, the extent of epigenetic discordance between healthy adult monozygotic twins and the stability of CpG loci within the same individual over a short time span at the whole-genome scale are not well understood. Here, we used Infinium HumanMethylation450 Beadchips to compare DNA methylation profiles using blood collected from 10 pairs of monozygotic twins and 8 individuals sampled at 0, 3, 6, and 9 months. Using an effective and unbiased method for calling differentially methylated (DM) CpG sites, we showed that 0.087%–1.530% of the CpG sites exhibit differential methylation in monozygotic twin pairs. We further demonstrated that, on whole-genome level, there has been no significant epigenetic drift within the same individuals for up to 9 months, including one monozygotic twin pair. However, we did identify a subset of CpG sites that vary in DNA methylation over the 9-month period. The magnitude of the intra-pair or longitudinal methylation discordance of the CpG sites inside the CpG islands is greater than those outside the CpG islands. The CpG sites located on shores appear to be more suitable for distinguishing between MZ twins.</p></div

Volunteer characteristics.

<p>^a: monozygotic.</p><p>Volunteer characteristics.</p

Relationship between position of CpG sites and differences in DNA methylation.

<p>(A) Comparison of the genomic distribution of DM CpG sites detected within at least one MZ twin pair (Group A, upper panel) or within a subject 3, 6, or 9 months apart (Group B, lower panel) under |ΔM| > 1 and FDR-adjusted <i>P</i> value < 0.05. n, the number of CpG sites satisfying the thresholds. (B-C) |ΔM| values within MZ twin pairs (B) or within the same individuals (C) plotted against their annotated locations. Black lines within each box represent the median of |ΔM|. The statistical analysis was performed using a non-parametric equivalent of one-way analysis of variance (ANOVA), the Kruskal-Wallis test, followed by a Bonferroni's/Dunn's multiple comparison test, ***<i>P</i> < 0.001.</p

DM CpG sites detected in blood samples.

<p>(A) Pink circle: The numbers of DM CpG sites detected within 10 MZ twin pairs (Group A); blue circle: the numbers of DM CpG sites from 8 individuals collected at 4 (0, 3, 6, and 9 months) or 3 (0, 6, and 9 months) time points (Group B) Intersection (purple): The number of DM CpG sites detected in both groups (B) Comparison of the genomic distribution of DM CpG sites detected within MZ twin pair only, within individual over time only, and in both groups. DM CpG sites satisfy the difference criteria (|ΔM|) of 1.0 and FDR-adjusted <i>P</i> value of 0.05.</p

Examination of the variance between the monozygotic (MZ) twin co-pairs.

<p>(A) Mean of absolute difference of the M-values (|ΔM|) within MZ twin pairs plotted against technical replicate pairs at 375,324 CpG sites (R = 0.3656). (B) Distribution of the Euclidean distance calculated based on the M-values. From left to right: technical replicate pairs (N = 10, mean Euclidean distance = 152.3, based on the dataset containing independent 5 replicates from Subject MZ 5_B), MZ twin pairs (N = 10, mean Euclidean distance = 182.3), and unrelated individual pairs (N = 11, mean Euclidean distance = 242.7). The black lines within each box represent the median of the Euclidean distance distribution. The boxes represent the inter-quartile range. The statistical analysis was performed using a non-parametric equivalent of one-way analysis of variance (ANOVA), the Kruskal-Wallis test, followed by a Bonferroni's/Dunn's multiple comparison test, *<i>P</i> < 0.05, ***<i>P</i> < 0.001.</p

The number of common differentially methylated (DM) CpG sites detected in 10 pairs of monozygotic (MZ) co-twins using the dual thresholds of absolute difference in the M-value (ΔM) and the False Discovery Rate (FDR)-adjusted <i>P</i> value.

<p>The number of common differentially methylated (DM) CpG sites detected in 10 pairs of monozygotic (MZ) co-twins using the dual thresholds of absolute difference in the M-value (ΔM) and the False Discovery Rate (FDR)-adjusted <i>P</i> value.</p

The number of CpG sites with intra-individual longitudinal changes when using the dual thresholds of absolute difference in the M-value (ΔM) and the False Discovery Rate (FDR)-adjusted <i>P</i> value.

<p>^#: E_{9 m_}1^st used</p><p>*: not applicable.</p><p>The number of CpG sites with intra-individual longitudinal changes when using the dual thresholds of absolute difference in the M-value (ΔM) and the False Discovery Rate (FDR)-adjusted <i>P</i> value.</p

Technical reproducibility of the Infinium HumanMethylation450 (HM450) BeadChip platform.

<p>MZ 5_B from Group A and E_{9 m} from Group B were independently replicated 5 and 6 times on the Infinium HM450 BeadChip platform, respectively. (A) M-values for the 3^rd versus the 4^th replicate of E_{9 m} (R = 0.9955); (B and C) The standard deviation in M-values of 5 (B) or 6 (C) replicates of all 375,324 (B) or 369,187 (C) probes. The nonlinear fit is displayed as red lines, illustrating the trends in the SD for the M-values across the entire range.</p

Distribution of euclidean distances between sample M-values.

<p>From left to right Euclidean distance between samples collected from the same individuals 0 month (N = 15 pairs, mean Euclidean distance = 163.9, based on the dataset containing independent 6 replicates from Subject E at 9 months), 3 months (0 months <i>vs</i>. 3 months, 3 months <i>vs</i>. 6 months, & 3 months <i>vs</i>. 9 months, N = 22 pairs, mean Euclidean distance = 163.9), 6 months (0 months <i>vs</i>. 6 months & 3 months <i>vs</i>. 9 months, N = 15 pairs, mean Euclidean distance = 168.8), and 9 months (0 months <i>vs</i>. 9 months, N = 8 pairs, mean Euclidean distance = 171.0) apart. Black lines within each box represent the median of the Euclidean distances. Boxes represent the inter-quartile range. The statistical analysis was performed using a non-parametric equivalent of one-way analysis of variance (ANOVA), the Kruskal-Wallis test, followed by a Bonferroni's/Dunn's multiple comparison test.</p