Epigenome-wide analysis in newborn blood spots from monozygotic twins discordant for cerebral palsy reveals consistent regional differences in DNA methylation

Namitha Mohandas, Sebastian Bass-Stringer, Jovana Maksimovic, Kylie Crompton, Yuk J. Loke, Janet Walstab, Susan M. Reid, David J. Amor, Dinah Reddihough, Jeffrey M. Craig

Cerebral palsy (CP) is a clinical description for a group of motor disorders that are heterogeneous with respect to causes, symptoms and severity. A diagnosis of CP cannot usually be made at birth and in some cases may be delayed until 2-3 years of age. This limits opportunities for early intervention that could otherwise improve long-term outcomes. CP has been recorded in monozygotic twins discordant for the disorder, indicating a potential role of non-genetic factors such as intrauterine infection, hypoxia-ischaemia, haemorrhage and thrombosis. The aim of this exploratory study was to utilise the discordant monozygotic twin model to understand and measure epigenetic changes associated with the development of CP. We performed a genome-wide analysis of DNA methylation using the Illumina Infinium Human Methylation 450 BeadChip array with DNA from newborn blood spots of 15 monozygotic twin pairs who later became discordant for CP. Quality control and data preprocessing were undertaken using theminfiR package. Differential methylation analysis was performed using the remove unwanted variation (RUVm) method, taking twin pairing into account in order to identify CP-specific differentially methylated probes (DMPs), andbumphunterwas performed to identify differentially methylated regions (DMRs). We identified 33 top-ranked DMPs based on a nominalpvalue cut-off ofp < 1 × 10-4and two DMRs (p < 1 × 10-3) associated with CP. The top-ranked probes related to 25 genes includingHNRNPL,RASSF5,CD3DandKALRNinvolved in immune signalling pathways, in addition toTBC1D24,FBXO9andVIPR2previously linked to epileptic encephalopathy. Gene ontology and pathway analysis of top-ranked DMP-associated genes revealed enrichment of inflammatory signalling pathways, regulation of cytokine secretion and regulation of leukocyte-mediated immunity. We also identified two top-ranked DMRs including one on chromosome 6 within the promoter region ofLTAgene encoding tumour necrosis factor-beta (TNF-β), an important regulator of inflammation and brain development. The second was within the transcription start site of theLIME1gene, which plays a key role in inflammatory pathways such as MAPK signalling. CP-specific differential DNA methylation within one of our two top DMRs was validated using an independent platform, MassArray EpiTyper. Ours is the first epigenome-wide association study of CP in disease-discordant monozygotic twin pairs and suggests a potential role for immune dysfunction in this condition.