The TNF-α gene occurs in the class III region of the major histocompatibility locus on human chromosome 6 (see (31
) for review). Here, we have described three new TNF-α single nt promoter polymorphisms, two of which are in linkage disequilibrium with extended HLA haplotypes. The finding that these polymorphisms occur in genetically separated groups of Caucasian and Asian descent and in independently derived homozygous cell lines indicates that these mutations are ancestral and did not arise recently. Given our sample size of 61 promoters, there is greater than a 95% chance that we would have detected any single nt polymorphism occurring in the human TNF-α promoter between −977 and +89 if the hetero-zygosity of that polymorphism were 0.048 or greater.
Previous studies have shown that the major regulatory elements necessary for the inducible regulation of the TNF-α gene via multiple stimuli in a variety of cell types are contained within a relatively compact promoter region contained within −200 base pairs relative to the TNF-α mRNA cap site (12
). Consistent with this observation, the −862, −856, and −574 polymorphisms did not have an effect upon the transcriptional regulation of the gene in T and B lymphocytes.
Our results also demonstrate conclusively that the −307 variant does not influence TNF-α gene expression in activated lymphocytes, in agreement with two studies (32
) and at variance with two other studies (34
). Others have speculated that the −237 and −243 TNF-α variants would have an effect upon TNF-α gene regulation because they occur within a stretch of DNA that bears a resemblance to a ‘Y-box’ (CCAAT) enhancer motif (7
). Here, we have formally shown that the −237 variant also has no effect upon TNF-α gene expression in activated T or B cells using promoter-reporter fusion genes. This data is consistent with functional studies, using the mouse TNF-α promoter, which have demonstrated that mutations in the murine equivalent of these sequences had no effect upon the transcriptional activity of the TNF-α promoter in lipopolysaccharide-stimulated monocytes (36
Using homozygous cell lines (21
), we have shown that the −856 and −862 TNF-α promoter variants are associated with extended HLA haplotypes. Previous studies have shown an association between the −307, −237, and −243 variants and extended HLA haplotypes (7
). Thus, the human TNF-α promoter polymorphisms located at positions −237, −243, −307, −862, and −856 relative to the TNF-α transciption start site are all non-randomly associated with other neighboring genes on chromosome 6 and all serve as markers for extended HLA haplotypes.
Interestingly, different patterns of TNF-α secretion have previously been associated with particular extended HLA haplotypes (37
). Our data argue that differential patterns of TNF-α secretion are not due to upregulation of TNF-α gene transcription secondary to the TNF-α promoter polymorphisms tested. Thus, the increased TNF-α bioactivity observed in association with extended HLA haplotypes must involve a different aspect of regulation of TNF-α.
As would be anticipated from the critical role that TNF-α plays in inflammation and lymphocyte biology, the regions of the promoter involved in the regulation of the gene are highly conserved. In fact, our analysis revealed no nt variability near any of the TNF-α upstream promoter elements previously shown to be involved in the regulation of the gene. It is interesting to speculate that TNF-α promoter variants that are in linkage disequilibrium with certain HLA genes are tolerated only in regions of the TNF-α promoter that are functionally silent.