[Identification and validation of plasma biomarkers for mild cognitive impairment based on mRNA N⁶-methyladenosine...

Objective: To identify and validate differential mRNA N⁶-methyladenosine (m⁶A) methylation sites associated with mild cognitive impairment (MCI), providing potential molecular evidence for its early prevention and intervention. Methods: A case-control study was conducted among older adults recruited from multiple community health centers in Shenzhen, including a pilot cohort (5 pairs) and a validation cohort (25 pairs). Single-based m⁶A quantitative polymerase chain reaction was used to quantify plasma mRNA m⁶A methylation levels. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed, followed by the construction of a protein-protein interaction network to identify hub genes. The differential methylation levels of key genes were validated in an expanded cohort. For normally distributed data, t-tests were used; for non-normal data, Mann-Whitney U or χ² tests were applied. Partial correlation analysis and Firth logistic regression were conducted to verify the associations further. Results: A total of 126 differential mRNA m⁶A methylation sites were identified, including 73 upregulated and 53 downregulated sites. Five hub genes (ZFHX3, PARD3, ARHGEF2, GLB1, and LRIG3) were selected, based on protein-protein interaction network topology. Validation experiments showed that the mRNA m⁶A methylation levels of these five genes were significantly higher in the MCI group than in healthy controls (all P<0.001). After adjustment for potential confounders, partial correlation analysis revealed that the methylation levels of all five genes were negatively correlated with MMSE scores (r=-0.719- -0.426, all P<0.05). Firth logistic regression confirmed that these genes were independently associated with MCI (OR=1.57-2.88, all P<0.001). Conclusion: This study screened and verified a range of MCI-related differential m⁶A methylation sites, suggesting that abnormal m⁶A methylation of specific gene mRNAs may be associated with MCI, providing research clues for further exploration of the molecular mechanism of MCI and potential plasma molecular markers.