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

BACKGROUND

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.

METHODS

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 the R 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), and was performed to identify differentially methylated regions (DMRs).

RESULTS

We identified 33 top-ranked DMPs based on a nominal value cut-off of  < 1 × 10 and two DMRs ( < 1 × 10) associated with CP. The top-ranked probes related to 25 genes including , , and involved in immune signalling pathways, in addition to , and previously 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 of gene encoding tumour necrosis factor-beta (TNF-β), an important regulator of inflammation and brain development. The second was within the transcription start site of the gene, 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.

CONCLUSIONS

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.