DNA methylation signature in peripheral blood reveals distinct characteristics of human X chromosome numerical aberrations

Abstract

Background: Abnormal sex chromosome numbers in humans are observed in Turner (45,X) and Klinefelter (47,XXY) syndromes. Both syndromes are associated with several clinical phenotypes, whose molecular mechanisms are obscure, and show a range of inter-individual penetrance. In order to understand the effect of abnormal numbers of X chromosome on the methylome and its correlation to the variable clinical phenotype, we performed a genome-wide methylation analysis using MeDIP and Illumina's Infinium assay on individuals with four karyotypes: 45,X, 46,XY, 46,XX, and 47,XXY.

Results: DNA methylation changes were widespread on all autosomal chromosomes in 45,X and in 47,XXY individuals, with Turner individuals presenting five times more affected loci. Differentially methylated CpGs, in most cases, have intermediate methylation levels and tend to occur outside CpG islands, especially in individuals with Turner syndrome. The X inactivation process appears to be less effective in Klinefelter syndrome as methylation on the X was decreased compared to normal female samples. In a large number of individuals, we verified several loci by pyrosequencing and observed only weak inter-loci correlations between the verified regions. This suggests a certain stochastic/random contribution to the methylation changes at each locus. Interestingly, methylation patterns on some PAR2 loci differ between male and Turner syndrome individuals and between female and Klinefelter syndrome individuals, which possibly contributed to this distinguished and unique autosomal methylation patterns in Turner and Klinefelter syndrome individuals.

Conclusions: The presented data clearly show that gain or loss of an X chromosome results in different epigenetic effects, which are not necessary opposite.

Keywords: DNA methylation; Epigenetics; Klinefelter; PAR region; Turner; X chromosome inactivation.

Fig. 1

Hierarchical clustering and principal component analysis (PCA) of the Illumina 27K data. The figure is based only on autosomal loci. a Supervised analysis testing for differences between groups using ANOVA and applying a cutoff of <5 % false discovery rate (FDR), corresponding to a p value of 3.11E-4 and 148 CpG sites; b Unsupervised hierarchical clustering on the normalized data without applying a statistical hypothesis. The heat maps in the upper part of the figure correspond to one sample for each column and one locus for each horizontal line; red to green corresponds to relatively hypermethylated (red) to hypomethylated (green). In the lower part of the figure, each of the spheres corresponds to one sample and the connecting lines correspond to the two nearest neighbors (more similar) of a particular sample

Fig. 2

Volcano plots of the Illumina 27K data showing pairwise comparisons of Turner and Klinefelter samples to male and female samples. The differences in beta values between the respective comparisons is on the X-axis, while the –log10 (p values) is on the Y-axis. Vertical dashed red/green and horizontal blue lines represent the 10 % methylation differences and the 5 % false discovery rates cutoff, respectively. The differentially methylated autosomal CpGs (>10 % differences and FDR <5 %) are represented by red and green filled circles for hypo and hyper methylated loci respectively; their corresponding number is also shown. The verified CpGs by pyrosequencing are represented as blue triangles (1 = cg2487174, 2 = cg16848873, 3 = cg24169822, 4 = cg11418559, 5 = cg20191453, 6 = cg09697795, 7 = cg18059933, 8 = cg26306976, 9 = cg04451770, 10 = cg06812844, 11 = cg04452095; loci labels are as in Fig. 5). Cross-reactive loci, as defined by Chen Y. et al, [54] are labeled with a black “x”. The numbers in each figure correspond to the number of individual CpG sites on autosomes that are hypomethylated (left) and hypermethylated (right); below each number (in parenthesis) is the corresponding number of differentially methylated CpG cites on the X chromosome

Fig. 3

Characteristics of the differentially methylated CpGs. a The level of methylation of differentially methylated regions (red and green curves represent the normal methylation frequency of the hypomethylated and the hypermethylated CpG sites, respectively) in comparison to their methylation levels in male and female samples (blue curves represent the frequency of methylation distribution of all analyzed CpG sites). The p values of the Chi-square test for trend between the methylation frequency distribution of either the hypo- or hypermethylated loci in comparison to all CpG sites on the array are also shown (when significant), together with the average methylation. The autosomal (left column) and X-linked loci (right column) were analyzed separately. b CpG island status: the differentially methylated regions were investigated for their CpG island status and were divided into three groups, blue if the CpG occurs in a CpG island based on both the Illumina array annotation and the UCSC, red if the CpG occurs in a CpG island based on only Illumina array annotation, and green if the CpG occurs in no CpG island. Next, this data of each comparison is compared to the distribution of all CpGs in the array, and Chi-square p values are shown at the right. Also, p values of the comparison between the hypo- and hyper-locations are shown

Fig. 4

Overlaps in affected loci between Turner and Klinefelter samples. a Venn diagram showing the intersections between four comparisons. The comparisons are abbreviated as T-M, T-F, K-M, and K-F, where M male, F female, T Turner, K Klinefelter. Different intersection groups are labeled from G1 to G15. Below each group name the total number of genes is given as well as the number of genes on the X chromosome (in parenthesis). b Relative direction of methylation changes between the four different comparisons. The X-linked (upper right) and autosomal (lower left) loci are shown separately. The numbers in the Table correspond to the common genes between the X- and the Y-axis categories. The red two-headed arrows point to the gene numbers that switched orientation in their methylation changes. Blue dashed boxes highlight the X-linked genes that are hypomethylated in Klinefelter samples in comparison to female samples, suggesting weaker X inactivation in Klinefelter than in female samples

Fig. 5

Detailed methylation data at selected loci in a large number of samples. The data at each locus, corresponding to 22 Turner (pink filled circles), 28 male (blue triangles), 28 female (red inverted triangles), and 40 Klinefelter samples (sky blue diamonds), are represented as vertical scatter plots with means (red horizontal bars) and standard deviation (grey horizontal bars). The black filled stars represent the samples used for the pool of DNA in the methylation array experiments. a Hypomethylated loci in Klinefelter samples, b hypermethylated loci in Turner samples, c hypomethylated in Turner and hypermethylated in Klinefelter samples, and d hypomethylated in Turner as well as Klinefelter samples. The Illumina ID number is shown at the top of each plot followed by the genetic name of the loci that appear in parenthesis and then by the p values of Kruskal-Wallis test for multiple group comparison; the p values of individual comparisons when significant are shown in grey

Fig. 6

Trend in methylation differences at X chromosome between 46,XX and 47,XXY. a MeDIP data of the X chromosome in all four groups; all data are aligned to the transcription start site (TSS): 7 kb upstream and 3 kb downstream. b Illumina 27K methylation values; the data are divided into 10 pins depending on the degree of X inactivation of the corresponding locus (according to Carrel and Willard [2]); average methylation at each pin is represented in the plot and the significant p value between female and Klinefelter samples at 0/9 is shown; all other comparisons between either female and Klinefelter or between male and Turners were not significant. c Detailed pyrosequencing data on two X-linked loci (TEF3 and SLC35A2) showing the differences in average methylation between female and Klinefelter samples

Fig. 7

Methylation at PAR regions. a MeDIP-based data. Lower panel shows the scatter plot with smooth curve fitted by Loess (without gaps). Three different parts separately represent the PAR1, PAR2 and the non-PAR region. The upper panel highlights the raw MeDIP data as shown by the SignalMap software for the loci that were also studied by pyrosequencing (shown in part B of the Figure). b Pyrosequencing at two loci in PAR2: SYBL1 and CpG29. The p values of Kruskal-Wallis test for multiple group comparison are shown below the name of the locus; the Mann-Whitney p values of individual comparisons when significant are shown in grey. In the middle of the figure, a diagram of the X chromosome is displayed together with the transcripts and their level of expression (on a scale of 0 to 9 to the left of the figure). T.S Turner syndrome