Allele-specific quantification of tumor necrosis factor alpha (TNF) transcription and the role of promoter polymorphisms in rheumatoid arthritis patients and healthy individuals.

Abstract

Interindividual variation in the expression of tumor necrosis factor alpha (TNF) suggests the existence of functionally distinct TNF alleles that could play a role in susceptibility to TNF associated diseases such as rheumatoid arthritis (RA). To determine whether differential expression of TNF alleles exists, the relative contribution of TNF alleles in total TNF RNA production in peripheral blood mononuclear cells (PBMC) of healthy individuals and synovial tissue of RA patients was analyzed. By using a Tai I restriction fragment length polymorphism (RFLP) located at position +489 in the first intron of the gene, the relative contribution of each allele in precursor transcript production in heterozygous individuals could be measured. By means of this method we studied whether differences exist between TNF alleles in TNF pre-mRNA production. The relative contribution of TNF alleles to the non-spliced RNA pool was measured in PBMC of healthy individuals which were stimulated with LPS, PMA and anti-CD3 and anti-CD28 monoclonal antibodies for different time periods. Moreover, synovial biopsy material of RA patients was analyzed. The results of this study do not reveal a difference in the contribution of distinct TNF alleles in TNF pre-mRNA production upon in vitro and physiological stimulation conditions in healthy individuals and RA patients. Since some of the individuals whose PBMC were tested were also heterozygous for either -308, -1031, -863, -857 TNF promoter/enhancer single nucleotide polymorphisms (SNPs), the data argue against functional relevance of these TNF promoter/enhancer SNPs in the regulation of transcription. In conclusion, the data do not provide evidence for the existence of transcriptionally distinct TNF alleles to explain interindividual variation in TNF expression.