Detection of DNA methylation from buccal swabs using nanopore sequencing to study stunting

Stunting is the result of chronic malnutrition due to the lack of micronutrient-based methyl donors required for epigenetic programming during the first 1000 days of life. Methylation studies using bisulfite conversion from blood DNA are invasive and may not be practical for large-scale epidemiological investigation or nutrition intervention programs. Buccal epithelial methylation may reflect early germline methylation. Therefore, buccal cells can serve as convenient sample sources to collect biomarkers associated with the risk of stunting. This study aims to describe the feasibility of nanopore adaptive sampling in detecting DNA methylation from children's buccal DNA. We used adaptive sampling of Oxford Nanopore Technology on barcoded samples to describe differential methylation associated with malnutrition. Overall, the level of 5-methylcytosine (5mC) was lower in stunted children than in normal children. We also found differentially methylated regions at the MIR6724 and RNA45SN1 gene loci on chromosome 21, which was higher in stunted children than in normal children. We described and detected differential DNA methylation in the locus previously not known to be associated with stunting. Interestingly, this locus on chromosome 21 has been implicated in the stunted phenotype of Down syndrome.