From genes to clinical application: a circulating four-gene signature for early diagnosis model of refractory

Objective: Refractory Methods: A total of 349 children (117 Normal, 123 GMPP, and 109 RMPP) were chronologically divided into a prospective training cohort (n=295) for model development and a prospective validation cohort (n=54) for external validation. Single-cell RNA sequencing (scRNA-seq) was performed on PBMCs from a discovery cohort (n=8) randomly selected from the training cohort. Differentially expressed genes that were specifically and significantly upregulated in RMPP groups were screened as candidate early diagnostic biomarkers. After primer validation, expressions of these candidate genes were subsequently measured using RT-qPCR in the entire study population. A multinomial logistic regression model with backward selection was developed on the training set, externally validated in the validation set, and its internal validation was further assessed via 1000 bootstrap resamples of the full dataset. Result: scRNA-seq identified eight specifically upregulated genes in the RMPP group. Subsequent RT-qPCR validation in the training cohort confirmed four genes- Conclusion: We established a robust PBMC-based four-gene signature diagnostic model that accurately discriminates among Normal, GMPP, and RMPP statuses at an early disease stage. This model provides a clinically accessible and precise tool to facilitate early intervention and improve patient management.