Allele-specific quantification of tumor necrosis factor α (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.

Allelic effects on gene regulation at the autoimmunity-predisposing CTLA4 locus: a re-evaluation of the 3′ +6230G>A polymorphism

“…Ueda et al, 5 used allele-specific restriction digestion with densitometric quantification of bands, a technique less sophisticated than fluorescent primer extension, which was not validated by assaying known allele mixtures as we did in Figure 4b. It is worth noting that the results of the study given as the methodological reference for this method used by Ueda et al, evaluating a TNF-alpha promoter polymorphism for which no allelic differences were found, 15 have been contradicted in another report. 16 In addition, Ueda et al examined only three þ 6230G4A heterozygotes and their result may be a statistical artifact of small numbers.…”

Is there a future for TNF promoter polymorphisms?

“…We applied this method, known as allele-specific transcript quantification (ASTQ), to determine the direct transcriptional activity of each allele. While we initially used the rare þ 70 SNP, 196 which allows the ratios of mRNA transcripts to be estimated, we later switched to the use of the common intron SNP at þ 489, 197 because this can be found in conjunction with various promoter SNPs, allowing the study of possible differential transcription mediated by these SNPs. As prespliced mRNA is measured, this estimation is independent of the possible influence of a higher turnover of a given allele.…”

Disease association of two distinct interleukin-18 promoter polymorphisms in Caucasian rheumatoid arthritis patients

“…Despite numerous data at the protein and cellular level implicating these cytokines in RA pathogenesis, genetic polymorphism analyses and their functional consequences have proven contradictory. [9][10][11][12][13] We have speculated that cytokine genes that regulate a Th1 dominant pathway may associate with RA. In particular, we have focused on the innate response cytokine interleukin (IL)-18.…”