The present systematic study examined the polymorphism of TNF-α gene promoter, circulating TNF-α levels, and traditional cardiovascular risk factor for ischemic stroke in the Chinese Han population. Our findings show that the polymorphisms of TNF-α promoter may function as an important risk factor for ischemic stroke and our meta-analysis data confirmed that patients with acute ischemic stroke compared to controls without stroke have significantly higher median serum levels of TNF-α. TNF-α level was also positively associated with some established traditional cardiovascular risk factors, age, BMI, total cholesterol, blood pressure, and inversely with HDL in our study. Additional functional analyses showed that variants in TNF-α gene promoter region may interact together to determine the overall activity of the TNF-α gene promoter. The flow chart illustrating our study process was shown in Additional file 1: Figure S5.
SNPs at the promoter region of the TNF-α gene have been commonly studied. Resequencing of 96 unrelated Chinese Han individuals revealed high linkage disequilibrium between the −863 C/A and the -1031T/C and no other SNPs have any significant LD with each other. But complete allelic associations were observed between the -238G/A and the -1031T/C, and between the −863 C/A and the -1031T/C in Europeans . The partial LD was also observed between the TNF-α -1031T/C and TNF-α -863C/A polymorphisms (D’ =0.75) in an Indian population and the similar results were also found between -308G/A and -1031T/C and -308G/A and -863C/A in this study (D’ = 0.74 and D’ =0.73, respectively) . In addition, five polymorphisms (−308G/A, -238G/A, -857C/T, -863C/A, -1031T/C) located in the human TNF-α gene promoter have been identified and the allele frequencies of these polymorphisms observed in this study in a Chinese Han population are quite distinct from those reported by Skoog et al. in a European population . The different allelic frequency and partial LD may be due to the differences in the genetic backgrounds and these differences also indicated that these polymorphisms of TNF-α promoter may act on different function in different ethnic group.
In our next case–control study and meta-analysis, we confirmed the significant association between TNF-α promoter variant -308G/A and stroke in the Asian population. Recently, several GWASs for stroke have been reported [29–34], but most of these study populations were of European origin and they did not detect the association of rs1800629 in TNF-α with stroke. Indeed, the similar negative results were obtained from the European populations reported by Freilinger et al. . Recently, due to the small sample size in stage 1 of GWAS screening (131 cases and 135 controls), the GWASs performed in the Japanese population  did not have sufficient power to observe a positive association. These data imply that the variant rs1800629 might have different effects on stroke (or other biological and pathological processes) among different populations; however, up to now, no GWASs results for stroke were reported in Chinese Han populations.
Inflammatory processes have increasingly been shown to be involved in the pathogenesis of cerebrovascular diseases, including ischemic stroke and cerebral hemorrhage. We demonstrate elevated serum TNF-α level was associated with higher risk of ischemic stroke in the Chinese Han population. By contrast, the TNF-α was not associated with risk of stroke in a British prospective study . Another meta-analysis also confirmed that TNF-α was linked to a 1.6-fold increase in ischemic stroke risk in adult Asian subjects but had no effect on European ancestry . Likewise, we found a weakly association between TNF-α level and different genotypes of -308G/A. Indeed, we cannot formally exclude the possibility that other functional SNPs, linked to the -308G/A, may influence the expression of TNF-α or other functional SNPs may interact together with -308G/A polymorphism to play a different role in different pathological processes. On the other hand, there was no clear signal for an effect of TNF-α gene variant on cardiovascular risk factors but TNF-α level itself was correlated with a range of baseline characters in our study subjects, and the findings are consistent with those of the previous reports in other ethnic groups [14, 21, 36]. However, we can see an inverse correlation between Q3 of TNF-α level and blood pressure, and this may be due to the smaller cohort size in our study.
To date, there is no consensus regarding the functional significance of TNF gene polymorphisms. A number of explanations for this discrepancy have been offered including cell types, stimulants, and reporter gene constructs. Therefore, these polymorphisms may serve as markers for additional polymorphisms in the TNF-α locus or neighboring genes that may influence disease severity or functional activity. In this study, we provided additional evidence that limited data reported so far do not provide strong evidence in favor of a physiological role of -308G/A and -238G/A in the regulation of TNF-α promoter activity. We also detected a -1031C allele had a significantly reduced luciferase activity compared to -1031 T allele which had a strong partial LD with -863C/A polymorphism and the similar results were reported by Skoog et al. . Specifically, functional study indicated a novel mutation -1376T/C identified in our study may have a large effect on the TNF-α promoter activity. However, this rare mutation need to be verified in a larger population and assayed in other ethic subjects. In particular, our results show, for the first time, the polymorphisms of TNF-α promoter may act in a cooperative manner of interact together to determine the overall activity of the TNF-α gene promoter. Therefore, the new potential transcription factor and regulation mechanism needed to be elucidated in the future.
Our conclusions must be interpreted in the context of important potential limitations of our study. Not all variants at the TNF-α gene were assessed in this study. Complete sequencing will be necessary for systematic identification of potentially causative mutations in TNF-α whole gene function region. Another limitation of the present study is the relatively small sample size used for TNF-α level analysis, which may give rise to false associations by chance (type one error), or may fail to detect true differences. Serum TNF-α levels are affected by multiple environmental, genetic factors and their interactions. Although we have observed the effects of several metabolism characters and SNP in TNF-α on serum levels in this study, there are still many other correlative environmental and genetic factors need to be determined. Our meta-analysis results must be viewed with careful because of potential biases. Perhaps because positive results have a better chance of being accepted for publication than small studies with non-significant or negative findings. Therefore, conclusions based on these published work might be misleading . Finally, it is important to confirm these findings in prospective cohort studies both in Chinese Han populations and other ethnic groups.
To conclude, this may be the first systematic and comprehensive analysis of TNF-α and stroke especially in an Asian population. Our results strongly support the involvement of TNF-α in the pathogenesis of stroke, and may have potentially important scientific, clinical, and public health implications.