Human P2X7 pore function predicts allele linkage disequilibrium.
BACKGROUND
Innate immune response amplification is achieved 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-alpha 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 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.
Denlinger
Loren C
LC
Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA. ldenling@wisc.edu
Coursin
Douglas B
DB
Schell
Kathleen
K
Angelini
Giuditta
G
Green
Dawn N
DN
Guadarrama
Arturo G
AG
Halsey
Jennifer
J
Prabhu
Usha
U
Hogan
Kirk J
KJ
Bertics
Paul J
PJ
D000483:Alleles; D015415:Biomarkers / Q000097:blood; D005838:Genotype; D006239:Haplotypes; D006801:Humans; D015810:Linkage Disequilibrium; D009000:Monocytes / Q000378:metabolism / Q000648:ultrastructure; D022022:Nuclear Pore / Q000502:physiology*; D010641:Phenotype; D012150:Polymorphism, Restriction Fragment Length; D011237:Predictive Value of Tests; D011336:Probability; D012372:ROC Curve; D018048:Receptors, Purinergic P2 / Q000097:blood / Q000235:genetics / Q000502:physiology*; D058486:Receptors, Purinergic P2X7
D015415:Biomarkers; C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7