Human P2X7 Pore Function Predicts Allele Linkage Disequilibrium

Abstract

Background: Innate immune response amplification is achieved by leukocyte expression of the purinergic nucleotide receptor P2X 7 , an extracellular nucleotidegated pore. Previously, low P2X 7 pore activity in whole blood was associated with loss-of-function genotypes in correlation with a decreased ratio of lipopolysaccharidestimulated tumor necrosis factor-␣ to interleukin-10, of relevance to a variety of infectious and inflammatory disorders. We hypothesized that evaluation of participants with discordance between the P2X7 genotype and pore status would disclose additional alleles, linkage disequilibrium, and novel functional correlates of genotype to phenotype. Methods: Comparison of whole-blood pore results with restriction fragment length polymorphism data for known loss-of-function genotypes from 200 healthy participants optimized the diagnostic threshold for low pore activity by ROC curve analysis. We identified novel alleles and inferred haplotypes by sequencing outlier genomic templates and by linkage analysis. Results: With a refined threshold of low activity, a normal pore result had only a 2% probability of association with known loss-of-function variants. By contrast, the positive predictive value of low pore activity was 59% for identifying known alleles. DNA samples from this discordant group contained 28 P2X7 sequence variations. Linkage analysis demonstrated that A1513C, T1729A, and G946A are inherited independently from one another, although these loss-of-function variants are

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

“…Moreover, significant synergism was observed in the chimeric variant (AE). These results indicated that A348T is a weak gain-of-function SNP, consistent with those reported by Denlinger et al [58].…”

“…Construct DR with C-terminus of the cysteine-rich domain 1 (CRD 1) of P2X 7 critical for regulation of pore formation Position 166aa is located in the C-terminal portion (around exon 5) of the cysteine-rich domain 1 (CRD1) of human P2X 7 . Both gain-of-function and loss-of-function mutations have been reported previously in this region [58,59] in addition to the A166G reported in this study. The region is located between the last two cysteine residues of CRD1.…”

“…Net dye uptake indicates the percentage dye uptake induced by BzATP with control (no BzATP treatment) subtracted. The data shown are representative of more than three independent experiments and two-tailed Student's t tests (unpaired) compared to Reference (R) yielded significance levels of <0.05(*), <0.01(**), or <0.001 (***) as shown in b-e of-function (H155Y and A166G) variations are located in the first cysteine-rich domain and only one loss-of-function SNP (R307Q) is at the C-terminus between the second cysteine-rich domain and second membrane-spanning domain (this study and references [57][58][59]). The large extracellular loop is believed to be responsible for ligandbinding and is quite conserved among the P2X receptors, although the fine structure of the ligand-binding pocket is still uncertain.…”

“…However, the majority of these have not been validated at the population genetic level and their functional effects are unclear. Five loss-of-function nonsynonymous allelic SNPs in P2X 7 have been described to date, of which three are located in the carboxy-terminal cytoplasmic tail (T357S, E496A, and I568N) and two are in the extracellular loop [54][55][56][57][58]. Cabrini et al have characterized the first gain-of-function polymorphism identified (H155Y) [59].…”

“…Cabrini et al have characterized the first gain-of-function polymorphism identified (H155Y) [59]. Another putative gain-of-function polymorphism described recently, the Q460R substitution, appears to enhance pore activity of the P2X 7 receptor [58]. Increasing evidence from genetic association studies have demonstrated that the polymorphisms of the P2X 7 gene are implicated in various diseases such as chronic lymphocytic leukemia, tuberculosis, bipolar affective disorders, and diabetes [60][61][62][63][64][65][66][67].…”