Whole genome bisulfite sequencing of Down syndrome brain reveals regional DNA hypermethylation and novel disorder insights

Abstract

Down Syndrome (DS) is the most common genetic cause of intellectual disability, in which an extra copy of human chromosome 21 (HSA21) affects regional DNA methylation profiles across the genome. Although DNA methylation has been previously examined at select regulatory regions across the genome in a variety of DS tissues and cells, differentially methylated regions (DMRs) have yet to be examined in an unbiased sequencing-based approach. Here, we present the first analysis of DMRs from whole genome bisulfite sequencing (WGBS) data of human DS and matched control brain, specifically frontal cortex. While no global differences in DNA methylation were observed, we identified 3,152 DS-DMRs across the entire genome, the majority of which were hypermethylated in DS. DS-DMRs were significantly enriched at CpG islands and de-enriched at specific gene body and regulatory regions. Functionally, the hypermethylated DS-DMRs were enriched for one-carbon metabolism, membrane transport, and glutamatergic synaptic signaling, while the hypomethylated DMRs were enriched for proline isomerization, glial immune response, and apoptosis. Furthermore, in a cross-tissue comparison to previous studies of DNA methylation from diverse DS tissues and reference epigenomes, hypermethylated DS-DMRs showed a strong cross-tissue concordance, while a more tissuespecific pattern was observed for the hypomethylated DS-DMRs. Overall, this approach highlights that low-coverage WGBS of clinical samples can identify epigenetic alterations to known biological pathways, which are potentially relevant to therapeutic treatments and include metabolic pathways. These results also provide new insights into the genome-wide effects of genetic alterations on DNA methylation profiles indicative of altered neurodevelopment and brain function.

StableDNMT3LOverexpression in SH-SY5Y Neurons Recreates a Facet of the Genome-Wide Down Syndrome DNA Methylation Signature

“…To determine the similarities between the DNMT3L-specific DMRs and the DNA methylation changes previously observed in DS, a pan-tissue comparison with differentially methylated DS sites across diverse tissues was performed (30,39–47). These results revealed significant overlap with DS differentially methylated sites, predominantly within the hypermethylated DNMT3L DMRs ( Figure 6C ).…”

Low-Pass Whole Genome Bisulfite Sequencing of Neonatal Dried Blood Spots Identifies a Role for RUNX1 in Down Syndrome DNA Methylation Profiles

“…Previously, in order to assay a larger proportion of the DS methylome, we performed the first whole genome bisulfite sequencing (WGBS) analysis of human DS samples, specifically postmortem brain. 7 Our low-pass sequencing analysis of postmortem brains identified 3,152 DMRs, where 75% were hypermethylated and involved in neurodevelopment and metabolism. The hypermethylated DMRs showed a cross-tissue signal, where we replicated many DS pan-and multi-tissue genes and expanded them to larger DMRs, while the hypomethylated DMRs showed a more tissue-specific profile.…”

“…In order to determine if the DS DMRs we identified in newborn blood are similar to those found in other DS tissues and studies, we performed enrichment analyses separately for hyper-and hypo-methylated DS DMRs for each comparison group ( Figure 4A and Supplementary Table 3). DS hyper-or hypo-methylated CpGs or DMRs were identified from 13 datasets from 8 different studies: 1) Neonatal whole-blood CpGs assayed by Illumina's Infinium Human Methylation 450K BeadChip array (450K) from Henneman et al 10 , 2) whole-blood CpGs within DMRs assayed on the 450K from Bacalini et al 31 , 3) sorted adult peripheral Tlymphocytes (CD3 + ), adult frontal cortex, sorted adult frontal cortex neurons (NeuN + ), sorted adult frontal cortex glia (NeuN -), adult cerebellar folial cortex, and mid-gestation fetal cerebrum assayed by 450K from Mendioroz et al 9 , 4) buccal epithelium CpGs assayed by 450K from Jones et al 32 , 5) placenta DMRs assayed by Reduced Representation Bisulfite Sequencing (RRBS) from Jin et al 5 6) neural induced pluripotent stem cell (iPSC) derivative CpGs assayed by 450K from Laan et al 33 , 7) fetal frontal cortex CpGs assayed by 450K from El Hajj et al 6 , and 8) adult frontal cortex DMRs assayed by WGBS from Laufer et al 7 As expected, the strongest overall significant (q < 0.05) enrichments for the DS NDBS DMRs were within differentially methylated sites from previous DS blood studies, where sorted adult peripheral T-lymphocytes (CD3 + ), whole blood, and neonatal blood were the top ranked. However, in tissues other than blood, the hyper-and hypo-methylated DMRs displayed divergent enrichment profiles with sites from previous DS studies.…”

“…4 Global or region-specific DNA CpG hypermethylation has been observed in a number of DS studies and is most pronounced in the brain and placenta. [5][6][7][8] A meta-analysis of DS DNA methylation array studies uncovered a pan-and multi-tissue CpG methylation signature of DS, where 24 out of the 25 differential genes were hypermethylated in at least 3 of the following tissues: adult brain, fetal brain, placenta, buccal epithelial, and adult blood. 4 While global hypermethylation (~1% difference) has been observed in DS adult and fetal brain tissue and sorted cells using arrays, the same study demonstrated that sorted T-lymphocytes (CD3 + ) from adult DS peripheral blood showed the opposite; a trend for global hypomethylation (~0.3% difference, p = 0.186) in DS compared to control.…”

Stable DNMT3L overexpression in SH-SY5Y neurons recreates a facet of the genome-wide Down syndrome DNA methylation signature

“…To test the hypothesis that the DNMT3L DMRs represent a facet of the differentially methylated genes observed in DS, cross-tissue and pan-tissue comparisons with differentially methylated DS sites across diverse tissues were performed [26][27][28][29][30][31][32][33][34][35]. These results revealed a significant (q < 0.05) enrichment of the DNMT3L DMRs within DS differentially methylated sites identified from multiple tissues (Fig.…”