Integrated Identification of NAD⁺ Metabolism-Associated Candidate Genes

Migraine is one of the most disabling neurological diseases globally; however, a significant proportion of patients do not respond to current treatments, or treatment efficacy diminishes over time, highlighting the need to identify additional molecular candidates for further investigation. Recent studies have increasingly focused on the role of neuro-immune interactions in migraine. This study aimed to identify peripheral immune-related candidate genes associated with NAD⁺ metabolism-related transcriptional features in migraine through integrated bulk and single-cell transcriptomic analyses. Using the PRJEB40032 dataset, samples were stratified according to ssGSEA-derived NAD⁺ metabolism scores, followed by differential expression analysis, weighted gene co-expression network analysis and protein-protein interaction network construction. LASSO regression, SVM-RFE, and XGBoost were subsequently applied to prioritize candidate genes. CXCL8, CXCR4, CXCL1, and TNFAIP3 showed cross-algorithm consistency and were subsequently incorporated into an exploratory nomogram, which exhibited apparent discriminatory performance within the PRJEB40032 discovery cohort. Functional enrichment analysis revealed that these genes were associated with immune and inflammatory pathways, including IL-17, NF-κB, TNF, and Toll-like receptor signaling. Immune cell-composition analysis indicated significantly increased estimated abundances or activity scores for NK cells, mast cells, eosinophils, and neutrophils in migraine samples, with positive associations between the prioritized candidate genes and pro-inflammatory immune features. Single-cell RNA sequencing further showed that CXCL1 and CXCL8 were predominantly expressed in monocytes, while TNFAIP3 and CXCR4 were widely expressed across various immune cell types. qPCR assessment in a migraine mouse model showed group-dependent expression alterations in the functionally related murine CXCL1/CXCR2 axis. Overall, this study prioritized peripheral immune-related candidate genes associated with migraine and NAD⁺ metabolism-related transcriptional features, providing exploratory evidence for a potential link among systemic immune alterations, NAD⁺ metabolism-related processes, and migraine.