Background: The P2X7 receptor is ubiquitiously expressed and following ATP stimulation, this ion channel leads to a large calcium influx and immediate secretion of inflammatory cytokines. Functional single nucleotide polymorphisms (SNP) cause loss-of-function of the P2X7R such as E496A (1513A>C), I568N (1729T>A) and R307Q (946G>A). Homozygous mutations at these sites appear to be compensated by a gain-of-function SNP H155Y (489C>T) which stimulates P2X7 activity. We investigated the haplotypes of these non synonymous polymorphisms located either in the extracellular part of the receptor, such as the H155Y and R307Q (ATP-binding domain) variants, or in the long cytoplasmic tail of the receptor E496A and I568N (ion channel domain) variants in healthy caucasoids. Methods: DNA was isolated using EZ1 automated DNA extraction (Qiagen) and PCR was performed with one biotinylated primer for sequencing of the P2X7 specific single nucleotide polymorphisms (SNPs): H115Y, R307Q, E496A, and I568N. In addition, we investigated the Q460R (1405A>G) SNP which has not been described to alter P2X7 function, all by pyrosequencing using a PSQ-96MA (Biotage). Results: Individual frequencies of SNPs were as follows: H115Y: 34.4% (wt) and 65.6% (mut); R307Q: 97.4%(wt) and 2.6% (mut); E496A 65.2% (wt) and 34.8% (mut); I560N 93.5% (wt) and 6.5% (mut), and of the neutral SNP Q460R 70.9% (wt) and 29.1% (mut). Interestingly, no individual who was homozygously mutated at E496A had a mutation in Q460R and vice versa. In addition, all homozygous mutations of either E496A or Q460R were also mutated in the gain-of-function SNP H115Y. Conclusions: These results strongly imply, that the H115Y gain-of-function SNP compensates not only for the homozygous loss of function E496A SNP, but also for an as yet non-defined deficiency given by the Q460R mutation. Since ATP is released by tissue trauma, we think that P2X7 polymorphisms play a major role in SIRS associated local cytokine release.
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