Accurate DNA Methylation Predictor for C9orf72 Repeat Expansion Alleles in the Pathogenic Range

The hexanucleotide (G ₄ C ₂ ) repeat expansion in the promoter region of C9orf72 is the most frequent genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this study, we conducted a genome-wide DNA methylation (DNAm) analysis using EPIC version 2 (EPICv2) arrays on an FTD cohort comprising 27 carriers and 250 non-carriers of the pathogenic C9orf72 repeat expansion from the Amsterdam Dementia Cohort. We identified differentially methylated CpGs probes associated with the pathogenic C9orf72 expansion and used these findings to create a DNAm Least Absolute Shrinkage and Selection Operator (LASSO) predictor to identify repeat expansion carriers. Eight CpG sites at the C9orf72 locus were significantly differentially hypermethylated in repeat expansion carriers compared to non-carriers. The LASSO model predicted repeat expansion status with an average accuracy of 98.6%. The LASSO predictor was further validated in an independent cohort of 2,548 subjects with available EPICv2 data, identifying four C9orf72 repeat expansion carriers, subsequently confirmed by repeat-primed PCR. This result not only illustrates the accuracy of the DNAm predictor of C9orf72 repeat expansion carriers but also suggests that repeat expansion carriers may be more prevalent than expected. The identification of a highly accurate DNAm biomarker for a repeat expansion locus associated with neurodegenerative disorders may provide great value for studying this locus. The approach holds significant promise for investigating this and other repeat expansion loci, particularly given the growing interest in epigenetic epidemiological studies involving large cohorts with available DNAm data.