DNA methylation patterns contribute to changes of cellular differentiation pathways in leukocytes with LOY from patients with Alzheimer´s disease

Abstract

Alzheimer's disease (AD) is a common and increasing societal problem due to the extending human lifespan. In males, loss of chromosome Y (LOY) in leukocytes is strongly associated with AD. We studied here DNA methylation and RNA expression in sorted monocytes and granulocytes with and without LOY from male AD patients. Through multi-omic analysis, we identified new candidate genes along with those previously associated with AD. Global analyses of DNA methylation in samples with LOY vs. normal state showed that hypomethylation dominated both in granulocytes and monocytes. Our findings highlight LOY-related differences in DNA methylation that occur in gene regulatory regions. Specifically, we observed alterations in key genes involved in leukocyte differentiation: FLI1, involved in early hematopoiesis; RUNX1, essential for blood cell development; RARA, regulating gene expression in response to retinoic acid; CANX, crucial for protein folding; CEBPB, a transcription factor important for immune responses; and MYADM, implicated in cell adhesion and migration. Moreover, protein-protein interaction analysis in granulocytes identified that products of two of these genes, CANX and CEBPB, are key hub proteins. This research underscores the potential of multi-omic approach in pure hematopoietic cell populations to uncover the molecular underpinnings of AD. Finally, our results link previous analysis showing impact of LOY on leukocyte differentiation, LOY-associated transcriptional dysregulation and GWAS studies of LOY.

Keywords: Alzheimer’s disease; CpG dinucleotide methylation; DNA methylation; Gene expression regulation; Loss of chromosome Y.

Fig. 1

Graphical abstract of the study. We selected 43 AD patients and collected their blood. Leukocytes were then sorted on FACS for two main groups of cells, i.e. granulocytes and monocytes. Estimation of %LOY in each sample (% of cells without Y chromosome) was done using SNP array. ROY stands for Retention Of chromosome Y group of samples. In parallel, samples were subjected to genome-wide DNA methylation profiling using the Illumina EPIC Beadchip. We further incorporated bulk RNA-seq data from our previous project for the correlation of variation in DNA methylation with the gene expression levels in samples from the same patients

Fig. 2

Distribution of LOY levels among the studied samples. A General distribution of %LOY cells among the two sampled cell types. P-value from Mann–Whitney (MW) pairwise test is present at the top, which is not significant, with no major differences in %LOY between granulocytes and monocytes. B Distribution of %LOY in granulocytes (X-axis) and monocytes (Y-axis). Every point in the main panel represents a measurement of %LOY for matched samples collected from the same patients. Narrow panels along each of the axes show results for LOY levels for unmatched samples in each cell type, due to insufficient DNA availability. The red dotted line denotes a 30% threshold used to divide the samples into LOY and ROY groups

Fig. 3

Results from analyses of differential methylation in LOY vs. ROY among AD patients. A Volcano plots of the genome-wide differential methylation analyses in granulocytes and monocytes. Black points denote all tested CpG sites. Colored dots represent the significant DMPs (FDR < 0.05, avDiff > 0.1). The direction of methylation change (X-axis) is shown in reference to the LOY samples (hypo—less methylated in cells with LOY, hyper—more methylated in cells with LOY). B Venn diagram comparing the number of hyper- and hypomethylated DMPs in granulocytes and monocytes identified by comparing LOY to ROY samples among AD patients

Fig. 4

Results from differential expression analyses and the correspondence of promoter-related methylation with the observed expression patterns. A Volcano plots of the genome-wide differential expression analyses using bulk RNA-seq data from corresponding patients’ samples. Colored dots represent genes with significant change in expression (DEGs, FDR < 0.05). Direction of expression change (X-axis) refers to the LOY samples (up—higher in LOY, down—lower in LOY). B Venn diagrams comparing gene sets derived from the differential expression and differential methylation analyses. Red numbers in the circles below the diagram represent the genes with LOY-associated methylation change in promoter region. C Results of KEGG pathway enrichment analyses for DEGs upregulated in AD-LOY granulocytes. The size of the circle corresponds to the number of genes representing the given pathway. The color of the circle denotes the adjusted p-value from the enrichment analysis

Fig. 5

Genomic neighborhood of selected genes from granulocytes with changes in both DNA methylation and gene expression. A Genomic neighborhood of the promoter region of selected differentially expressed genes from granulocytes. All genes are upregulated in the LOY group in AD patients and have a hypomethylated site within their promoters. Top panel (DNA methylation) shows neighboring CpG probes located in the vicinity of the promoter region and their DNA methylation values in LOY (orange) and ROY (green) samples in AD. Black arrows point to differentially methylated probes. Second panel from top shows the identified significant differentially methylated sites. Panel named “UCSC genes” shows annotations of the gene’s structure. Blue rectangles show exons and introns are shown as thin lines. Arrows on introns show the direction of the gene from its 5’ side. TSS denotes the site of the start of transcription. The bottom-most panel shows CpG islands located in the vicinity of TSS. Dark green denotes CpG islands, and their shores are in light green. Chromosome name and the genomic coordinates are shown above each plot. B Promoter-associated CpGs with significant change in methylation in granulocytes juxtaposed with a relevant change in expression of the associated gene. Boxplots show methylation and expression values in one of the two compared groups—LOY or ROY in AD patients. Hypomethylation in LOY is associated with upregulation of the corresponding gene’s expression in LOY. BH-adjusted p-values are shown

Fig. 6

String protein–protein interaction network based on genes that display both variations in DNA methylation (within promoter region) and gene expression. Lines represent different types of evidence used in predicting the associations: the presence of fusion evidence (red), neighborhood (green), cooccurrence (blue), experimental (purple), text mining (yellow), database (light blue), coexpression (black). A 153 genes with changes in granulocytes were used as input for the interaction analyses (see Material and Methods). Edges represent known or predicted protein–protein interactions, e.g. from curated databases and experiments. B Selected hub protein encoded by the CANX gene (calnexin, calcium-binding protein), which has 12 known interactions with other products of DMG/DEG genes from granulocytes. C Selected hub protein encoded by the CEBPB gene (CCAAT Enhancer Binding Protein Beta), which has 11 known interactions with other products of DMG/DEG genes from granulocytes