Received on December 22, 2005
Accepted on March 16, 2006

Molecular Diagnostics and Genetics

Human P2X₇ Pore Function Predicts Allele Linkage Disequilibrium

¹ Departments of Medicine, Biomolecular Chemistry, and Anesthesiology, University of Wisconsin School of Medicine and Public Health, Madison, WI
² Departments of Medicine and Anesthesiology, University of Wisconsin School of Medicine and Public Health, Madison, WI
³ Comprehensive Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI
⁴ Department of Anesthesiology, University of Wisconsin School of Medicine and Public Health, Madison, WI
⁵ Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI
⁶ Department of Biomolecular Chemistry, and Comprehensive Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI

^* To whom correspondence should be addressed. E-mail: ldenling{at}wisc.edu.

Background: Innate immune response amplification is performed by leukocyte expression of the purinergic nucleotide receptor P2X₇, an extracellular nucleotide-gated pore. Previously, low P2X₇ pore activity in whole blood was associated with loss-of-function genotypes in correlation with a decreased ratio of lipopolysaccharide-stimulated 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 of 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 demonstrates that A1513C, T1729A, and G946A are inherited independently from one another, although these loss-of-function variants are in disequilibrium with other alleles. When we segregated pore activity data according to genotypes, nonsynonymous sequence variations (G474A and A1405G) appeared to exhibit modulatory effects on P2X₇ pore activity.

Conclusions: Direct analysis of pore activity demonstrates functional interactions between P2X7 alleles. The performance characteristics of the whole-blood pore assay enables correlation of genomic variation with concomitant investigation of functional performance in clinical studies.