Targeted Next-Generation Bisulfite Sequencing Identifies Differential Methylation in a South African ASD Cohort

Background: DNA methylation is a well-recognised epigenetic modifier of gene expression that mediate transcription via conformational chromatin changes. DNA methylation is implicated in various biological processes, and has been investigated in the aetiology of disease, including Autistic Spectrum Disorders (ASD). A recent study done by our group, using a whole-genome bead-chip approach, identified over 800 genes to be differentially methylated between ASD and controls in a South African cohort, with many of these genes in canonical pathways involved in mitochondrial function. Mitochondrial homeostasis is fundamental and has been associated with numerous neurological disorders. A 50-fold increase in mitochondrial disease in children with ASD compared to the general population has been reported, implicating mitochondrial dysfunction in the pathophysiology of ASD.

Objectives: Firstly, to set up targeted Next-Generation Bisulfite Sequencing (tNGBS) for 30 to 40 loci using buccal cells, to identify assays that can accurately measure methylation at high quantitative resolution. Secondly, to confirm DNA methylation observed in our earlier whole-genome methylation screen in a locus-specific manner using pyrosequencing for the loci optimised using tNGBS. Lastly, this study aims to examine methylation of genes implicated in mitochondrial dysfunction in ASD.

Methods: The 898 genes from our methylation screen were compared with genes from the SFARI database and we identified 59 common genes. These included seven high confidence genes based on SFARI rankings, as well as genes involved in mitochondrial function and ubiquitination pathways, and these were selected for tNGBS. Only loci that passed assay validation QC criteria (e.g. r² >0.9) and minimal PCR bias were carried through for subsequent locus-specific pyrosequencing analysis. DNA samples were collected from the buccal cells of a homogenous, mixed race cohort of South African boys, aged 6 to 12, with 46 ASD cases, and 46 neurotypical controls.

Results: From the 59 common genes in the methylation screen -SFARI overlay, 40 met criteria to be selected for tNGBS and four loci were pyrosequenced. We confirmed differential methylation between ASD and controls, highlighting the importance of epigenetic changes. These results accentuate the role of mitochondrial and ubiquitination pathways in ASD.

Conclusions: This study showed that pyrosequencing is a cost- and labour-effective way to identify loci that show differential methylation of the promotor regions of genes. It demonstrates the importance of epigenetics in ASD and the dysfunction in pathways maintaining mitochondrial homeostasis.