Revealing Causal Genes in Nasopharyngeal Carcinoma: An Integrated Approach of Mendelian Randomization and Single-Cell Sequencing

Abstract

Objective: This study employed Mendelian randomization (MR) to identify genes associated with nasopharyngeal carcinoma (NPC) and utilized single-cell sequencing to analyze their expression patterns within the tumor microenvironment.

Methods: Transcriptomic datasets (GSE53819 and GSE12452) were obtained from the Gene Expression Omnibus (GEO) database. Differential expression analysis was performed using R software to identify common differentially expressed genes. Summary-level data for expression quantitative trait loci (eQTLs) of these genes and for NPC genome-wide association studies (GWAS) were sourced from the OpenGWAS and FinnGen databases, respectively. Using gene-associated single nucleotide polymorphisms (SNPs) as instrumental variables (IVs), MR analyses—including the Wald ratio, inverse variance weighted (IVW), MR-Egger regression, and weighted median methods—were conducted to evaluate the causal effects of these genes on NPC, adhering to the three core assumptions of MR. Additionally, single-cell RNA sequencing datasets (GSE150825 and GSE120926) from GEO were processed for quality control, cell clustering, and annotation. The expression of MR-identified genes across cell subtypes was visualized using DotPlot.

Results: Differential expression analysis identified 494 common differentially expressed genes, of which 313 were available for MR analysis. The MR results indicated that MFSD4 (Wald ratio) as well as PSPH and VPREB3 (IVW) were associated with an increased risk of NPC, whereas TMEM200A and THBS2 (IVW) were associated with a reduced risk. Single-cell expression analysis revealed that PSPH was predominantly expressed in epithelial cells, TMEM200A was highly expressed in fibroblasts and T cells, THBS2 was mainly expressed in fibroblasts, MFSD4 showed higher expression in endothelial cells, and VPREB3 was broadly highly expressed in tumor cells, plasma B cells, and B cells—with the highest expression observed in B cells.

Conclusion: By integrating Mendelian randomization with single-cell transcriptomic analysis, this study identifies MFSD4, PSPH, and VPREB3 as potential risk genes for NPC, and TMEM200A and THBS2 as potential protective genes. These findings clarify their cellular expression characteristics within the NPC microenvironment and provide novel insights into the molecular mechanisms of NPC, suggesting potential targets for future gene-targeted therapeutic strategies.