Exploring the Bio-Functional Effect of Single Nucleotide Polymorphisms in the Promoter Region of the TNFSF4, CD28, and PDCD1 Genes

In a prior study, we discovered that hematopoietic stem cell transplantation (HSCT) and/or autoimmune diseases, such as systemic lupus erythematosus, were associated with the rs1234314 C/G and rs45454293 C/T polymorphisms of TNFSF4, the rs5839828 C > del and rs36084323 C > T polymorphisms of PDCD1, and the rs28541784C/T, rs200353921A/T, rs3181096C/T, and rs3181098 G/A polymorphisms of CD28. However, the association does not imply causation. These single nucleotide polymorphisms (SNPs) are all located in the promoter region of these genes, so we used the dual-luminescence reporter assay to explore the effect of single nucleotide polymorphisms (SNPs) on transcriptional activity. For each promoter–reporter with a single SNP mutation, more than 10 independent experiments were carried out, and the difference in transcription activity was compared using one-way ANOVA and Tukey’s honestly significant difference test. The results showed that the G-allele of rs1234314 had 0.32 ± 0.09 times the average amount of relative light units (RLU) compared to the C-allele (p = 0.003), the T-allele of rs45454293 had 4.63 ± 0.92 times the average amount of RLU compared to the C-allele (p < 0.001), the del-allele of rs5839828 had 1.37 ± 0.24 times the average amount of RLU compared to the G-allele (p < 0.001), and the T-allele of rs36084323 had 0.68 ± 0.07 times the average amount of RLU compared to the C-allele (p < 0.001). The CD28 SNPs studied here did not affect transcriptional activity. In conclusion, the findings of this study could only confirm that the SNP had a bio-functional effect on gene expression levels. According to the findings, several SNPs in the same gene have bio-functions that affect transcriptional activity. However, some increase transcriptional activity while others decrease it. Consequently, we inferred that the final protein level should be the integration result of the co-regulation of all the SNPs with the effect on transcriptional activity.