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Clinical Chemistry 52: 995-1004, 2006. First published April 13, 2006; 10.1373/clinchem.2005.065425
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(Clinical Chemistry. 2006;52:995-1004.)
© 2006 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Human P2X7 Pore Function Predicts Allele Linkage Disequilibrium

Loren C. Denlinger1,2,3,a, Douglas B. Coursin1,3, Kathleen Schell4, Giuditta Angelini3, Dawn N. Green3, Arturo G. Guadarrama2, Jennifer Halsey3, Usha Prabhu2, Kirk J. Hogan3 and Paul J. Bertics2,4

Departments of1 Medicine,2 Biomolecular Chemistry, and3 Anesthesiology, and4 Comprehensive Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI.

aAddress correspondence to this author at: Section of Allergy, Pulmonary & Critical Care Medicine, University of Wisconsin School of Medicine and Public Health, 1300 University Ave., Rm 4285 MSC, Madison, WI 53705. Fax 608-263-4969; e-mail ldenling{at}wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The purinergic nucleotide receptor P2X7, the seventh member of the P2X receptor family, is a homotrimeric, ligand-gated cation channel (1) expressed on most classes of leukocytes that acts as an amplification loop for innate immune responses (2)(3). Activation of P2X7 by platelet granule– and/or cytolytic parenchymal cell–derived adenine nucleotides leads to nonselective cation permeability or dilatation to a size restriction of ~900 Da, a phenomenon referred to as pore activity (4)(5)(6). P2X7-dependent events trigger diverse signaling effectors and transcription factors, including the p38 member of the mitogen-activatable protein kinase family and nuclear factor-{kappa}B (7). Ultimately, P2X7-sensitive messengers synergize with lipopolysaccharide (LPS), 1 giving rise to monocyte and macrophage production of inflammatory mediators, including tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin-1ß (IL-1ß), IL-18, and nitric oxide (8)(9)(10). Conversely, P2X7 2 genetic variants are associated with an early reduction in the LPS-primed, ATP-stimulated monocyte processing of IL-1ß and IL-18 (11)(12)(13), and reduced macrophage capacity to kill the BCG strain of Mycobacteria or 2 species of Chlamydia (14)(15)(16)(17). In this way, P2X7 genetic variability may contribute to subject-specific differences in the strength of innate immune responses.

The human P2X7 gene localizes to a 55-kilobase region of chromosome 12q24, has 13 exons, and encodes a 595–amino acid protein (18). Recent sequencing efforts (e.g., HapMap) have identified hundreds of sequence variations for this gene, but the majority of these have not been validated at the population genetic level and their functional effects are unclear (19). Three loss-of-function alleles (G946A, A1513C, and T1729A) have been characterized in recombinant expression systems, and similar methods have recently been used to characterize a gain-of-function allele (C489T) (20)(21)(22)(23). In addition, in the absence of a direct index of receptor function, genetic association studies have linked 2 P2X7 sequence variations (A1513C and T–762C) to human diseases (chronic lymphocytic leukemia and tuberculosis, respectively) (24)(25)(26)(27). However, as is typical of designs lacking correlative functional assays to bridge DNA sequence variations and clinical phenotypes, there are reports in different populations refuting the association (28), and in vitro correlation for the T–762C allele failed to show a functional effect (29), suggesting the existence of 1 or more unidentified gain-of-function allele(s) in linkage disequilibrium with the promoter locus.

We previously described the design of a P2X7 pore assay in whole blood with several features permitting high-throughput functional screening in clinical studies (30). This assay preserves the allele dose dependency expected for the A1513C variant and correlates with the ratio of LPS-stimulated TNF-{alpha} to IL-10 in whole blood after 6 and 24 h of treatment (31). In the present study, we hypothesized that evaluation of persons with discordance between P2X7 genotype and pore status would disclose additional alleles, linkage disequilibrium, and novel functional correlates of genotype to phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
participants
All investigations were approved by the Human Subjects Committee of the University of Wisconsin’s Institutional Review Board, with written, informed consent and in accordance with the guidelines established by the revised Declaration of Helsinki. Recruitment of 200 healthy persons, P2X7 A1513C allele frequencies, and the effects of low pore activity on LPS-induced cytokine generation in whole blood have been reported previously (31).

Selection of 40 participants for the P2X7 genomic characterization and cytokine phase was performed according to the following protocol (also see Fig. A in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/issue6/). Concordance between predicted and observed pore activity was correlated with P2X7 A1513C genotype from the screening phase for all 200 individuals. From the 177 concordant individuals, 21 were enrolled in the P2X7 genomic characterization and cytokine phase, including all 7 persons with the variant 1513 CC genotype, plus 7 participants selected at random from each of the groups with 1513 AA and AC genotypes. Of the 23 discordant individuals (200 – 177 = 23) having low pore activity despite wild-type homozygous 1513 AA or heterozygous AC genotypes, 19 were available for participation, and 4 were lost to recontact.

performance characteristics for the whole-blood pore assay
The whole-blood P2X7 pore assay evaluating 2'-3'-O-(4-benzoylbenzoyl (Bz)-ATP–stimulated YO-PRO-1 uptake by CD14+ monocytes has been described previously (31). Genotypes for the validated loss-of-function P2X7 alleles G946A, A1513C, and T1729A were determined for all 200 participants in the screening phase, as described below. Standard methods were used for calculating the sensitivity, specificity, positive predictive value, and negative predictive value, repeated with serial low pore definition thresholds (32). With this method, the optimal threshold is selected as the point of the ROC curve demonstrating a 45-degree tangent slope (32).

P2X7 genotyping and allele detection
DNA was harvested from whole blood as described previously (31). In addition to the evaluation of the pore assay performance characteristics mentioned above, the following methods for data acquisition and analysis are specific to this report. Exon 13 of P2X7 and its boundaries were sequenced by use of genomic DNA samples from all 40 persons participating in the P2X7 genomic characterization and cytokine phase (see Table A in the online Data Supplement). Bidirectional sequencing was performed with the BigDye Kit (Applied Biosystems) and Cleanseq magnetic beads (Agencourt Bioscience). Additionally, the remaining 12 exons and their respective boundaries were sequenced in full by use of genomic DNA templates from the 19 discordant individuals. If a nonsynonymous variant was identified, the associated exon was also sequenced by use of 8 sequentially enrolled samples from the concordant group participating in the P2X7 genomic characterization and cytokine phase, to provide an estimate of allele distributions in the 1513 concordant and discordant groups.

All newly identified nonsynonymous alleles were then genotyped by restriction fragment length polymorphism analysis to complete the data set for all 200 persons (see Tables B and C in the online Data Supplement). Additionally, genotypes for the promoter allele T–762C were performed as described previously (29). The GenBank accession numbers for the P2X7 reference sequences used for comparison are as follows: Y12851, exon 1; Y12852, exons 2 and 3; Y12853, exons 4–8; Y12854, exons 9–11; Y12855, exons 12 and 13. The newly derived genetic data from this report are being submitted to GenBank.

statistical analysis
Hardy–Weinberg equilibrium and linkage analysis were calculated by use of the LDA program, Ver. 1.0 [ (33); available from the Chinese National Genome Center (http://www.chgb.org.cn/lda/lda.htm)]. The GCG Wisconsin Package software (available at http://www.biocomp.doit.wisc.edu/) was used to align the 12q24 portion of the human chromosome 12 contig (NT_009775) with the clones listed previously (exon 1 starts at contig bp 12 138 574, and exon 13 ends at 12 192 983). For the purposes of linkage analysis, an arbitrary numbering scheme was adapted that included information about intron sizes, with the following allele numbers: 1 = T–762C, 30227 = G474A, 30242 = C489T, 35344 = G835A, 43218 = G946A, 45092 = A1068G, 45120 = C1096G, 52184 = A1405G, 52293 = A1513C, and 52509 = T1729A. The LDA program was used to determine linkage disequilibrium with the likelihood ratio test and 10 000 iterations. Haplotypes were computationally inferred using PHASE (Ver. 2.1) software [ (34)(35); available at http://www.stat.washington.edu/stephens/software.html], a Bayesian method with 100 iterations and 3 independent runs. Finally, comparisons of pore activity among genotypes were performed by use of 1-factor ANOVA and unpaired Student t-tests with unequal variance. A significance of P <0.05 was adopted for all comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
distribution of monocyte pore activity and performance characteristics of the screening assay for identifying individuals with loss-of-function P2X7 alleles
Because the clinical validity of genetic association studies is often contradictory among different populations in the absence of an independent phenotypic index (36) and because P2X7 is a candidate gene for a variety of infectious and inflammatory disorders (17)(26)(27)(37)(38), we sought to develop a functional and practical assay based on P2X7 pore activity to resolve disparate genetic and phenotypic data. The design and validation of this assay for the P2X7 A1513C allele have been reported previously (30)(31). As shown in Fig. 1A , the distribution of pore activity data among the 200 individuals was normalized with outliers observed at the high end of the range. The mean (SD) pore activity was a 57.6 (39.5)-fold increase of agonist-induced uptake of YO-PRO-1 (interquartile boundaries, 27-, 51-, and 83-fold increases in uptake). Low pore activity was defined previously solely on the basis of predictions from the A1513C genotype, in which a confidence interval of pore activity was generated from the data associated with the 7 persons possessing the variant 1513 CC genotype (31). The thresholds constructed by this method identified 23 discordant individuals with low pore activity despite the presence of at least one wild-type (normal) 1513 dominant A allele (Fig. 1A ).


Figure 1
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  		Figure 1. Distribution of P2X7 pore activity in healthy persons (A), and performance characteristics of the screening assay at different thresholds of detection for loss-of-function alleles (B).

(A), P2X7 pore activity was measured by the whole-blood screening assay described in the Materials and Methods section. Results are displayed as a histogram for the number of persons with each respective range of pore activities. The hatched area in the first column indicates that 23 persons had low pore activity despite the presence of at least one 1513 A allele, labeled as "discordants". (B), using genotype data from 200 healthy persons for the 3 biochemically validated loss-of-function alleles G946A, A1513C, and T1729A, we varied the threshold for declaring low pore activity, then calculated the sensitivity and specificity for detecting persons with these variant genotypes. The results of a failed test traverse the line of identity (diagonal line) such that every increase in sensitivity is accompanied by a similar decrease in specificity. For the P2X7 pore assay, a threshold of 22-fold agonist-induced dye uptake is the point on the curve closest to the ideal test with both 100% sensitivity and specificity. PPV, positive predictive value; NPV, negative predictive value.

During our enrollment and data analysis periods, 2 additional P2X7 alleles, G946A and T1729A, were validated by others to confer a decrement of receptor function, (21)(22). These observations rendered an A1513C-based definition (31) of low pore activity incomplete, requiring additional analysis of the optimal threshold to identify potential loss-of-function variants. To meet this need, we constructed an ROC curve by varying the threshold below which the pore assay would detect persons carrying validated (i.e., known) loss-of-function alleles (Fig. 1BUp ). The optimal diagnostic threshold was found to be ≤22-fold increased Bz-ATP-induced dye uptake. The performance characteristics at this threshold were as follows: sensitivity, 85; specificity, 91%; negative predictive value, 98%; positive predictive value, 59%. This threshold identified 39 (19.5%) of 200 healthy persons with low P2X7 pore activity. Moreover, with this threshold, 16 of 39 persons in the low pore-activity group were found to have G946A, A1513C, and T1729A genotypes predicting normal pore activity, suggesting the existence of additional loss-of-function alleles or loci. Lastly, monocytes from 2 persons with the variant 946 GA and 2 with the 1729 TA genotype had normal pore activity, suggesting the influence of other mitigating alleles.

P2X7 allele detection
Sequence analysis from the samples of the A1513C-discordant persons and controls revealed 26 single-base variations, 1 inversion, and 1 duplication/insertion by this approach (Table 1 ). Twelve of these alterations were within introns, 5 were synonymous exonic variants, and 11 were found to confer predicted amino acid substitutions. Allele frequency differences were observed between the A1513C concordant and discordant groups, as expected for the G946A and T1729A alleles, but also for the A1068G, C1096G, and A1405G alleles, raising the likelihood for their candidacy as functional variants. Conversely, of the 27, 19, and 40 samples sequenced for exons 9, 12, and 13, respectively, all contained the homozygous mutant allele at the IVS8 GA-2AG, T1287C, and A1469C loci, suggesting that the published nucleotides in these positions of the reference sequence are uncommon or incorrect. Finally, after genotyping of the 200 persons from the screening phase for 9 of the nonsynonymous variations and 1 promoter single-base variation, 32 P2X7 haplotypes were inferred, none of which was more predictive of low pore activity than genotype information at the individual G946A, A1513C, and T1729A loci (see Tables C and D in the online Data Supplement).


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  		Table 1. Sequence identification of P2X7 alleles.1

nonsynonymous P2X7 allele characterization
Of the nonsynonymous variants with frequencies greater than 1%, only G474A was not in Hardy–Weinberg equilibrium (P = 0.004), suggesting a lack of bias in our sampling methods. Linkage analysis for 10 P2X7 alleles distributed in the promoter through the distal portion of the last exon is shown in Fig. 2 . In general, D' is a standardized metric of linkage disequilibrium ranging from 0 to 1, where low values are associated with evidence of independent inheritance patterns for pairwise comparisons of two alleles (39). For example, A1513C and T1729A have a D' <0.3, suggesting independence consistent with previous estimates (31). The other characterized loss-of-function allele, G946A, is likely independent from these 2. In this case, D' is >0.7, possibly because of a mathematical effect observed with rare alleles (39); however, the differences between the observed and expected frequencies were small (P = 0.162 and 1.000, respectively). On the basis of similar logic, G474A and C1096G are likely inherited independently from G946A, A1513C, and T1729A (Fig. 2 ). Conversely, a recently described gain-of-function allele, C489T (23), was in linkage disequilibrium in our population with T–762C, A1405G, and A1513C (D' >0.7; P <0.001), whereas A1068G was linked to C1096 and A1513C (D' >0.7; P <0.001; Fig. 2 ).


Figure 2
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  		Figure 2. Linkage analysis for 10 P2X7 alleles.

The LDA program (see Materials and Methods) was used to assess linkage disequilibrium for pairwise comparisons of the listed alleles with the likelihood ratio test. Low values of D' (A) are associated with independent inheritance, whereas high values may indicate linkage if the P value is also <0.001 (B), allowing adjustment for multiple comparisons (39).

We used the pore assay to rapidly identify putative functional variants (Fig. 3 ). Similar to preservation of allele dose dependency for A1513C by the whole-blood pore assay (31), the known loss-of-function variants G946A and T1729A (21)(22) had very low pore activity associated with heterozygous genotypes (Fig. 3 ). Despite amino acid substitutions sufficiently different to alter immunoreactivity, the mutant C489T and G835A alleles did not affect monocyte pore activity in the context of whole-blood samples from all 200 individuals (Fig. 3 ). Subset analysis showed that the 489 TT genotype was associated with 13% and 19% increases in pore activity for the 1513 AA and 1513 AC individuals, respectively, relative to similar groups with the 489 CC genotype; however, these results were not statistically significant. To the contrary, the uncommon G474A variant strongly attenuated pore function. Additionally, the A1068G- and C1096G-induced substitutions may have weakly negative effects, although as noted above, the former allele is in linkage disequilibrium with A1513C. Interestingly, the A1405G change was associated with significantly increased P2X7 pore activity, suggesting that this is, or is linked to, a putative gain-of-function allele.


Figure 3
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  		Figure 3. Distribution of pore activity according to P2X7 genotype.

Restriction fragment length polymorphism analysis of all 200 individuals revealed genotype data for the 9 nonsynonymous variants, displayed with the number of persons in each group listed at the top of each panel. The results of the pore assay are categorized according to genotype to begin to determine the effects of a given allele on receptor function. Data represent the mean (SE; error bars). For reference, the mean pore activity for the entire group was 57.6-fold increased in agonist-induced dye uptake, and previous validation for the A1513C allele revealed group statistics of 69 (4)-fold (n = 124), 42 (4)-fold (n = 69), and 6 (1)-fold (n = 7) stimulation of YO-PRO-1 uptake (Bz-ATP–induced) for wild-type, heterozygous, and homozygous recessive individuals, respectively (31).

We could make similar predictions by comparing the variant P2X7 allele frequencies in the low and normal pore-activity groups, using the threshold of ≤22-fold increase in agonist-induced dye uptake to identify persons with variant alleles (Table 2 ). The validated loss-of-function alleles G946A, A1513C, and T1729A showed 6.2-, 2.5-, and 26.8-fold enrichments, respectively, in the low pore-activity group. The variant G474A allele was disproportionately represented by 8.3-fold in the low pore-activity group, consistent with its apparent effects on pore activity. The enrichments in the low pore-activity group for the C489T, A1068G, and C1096G alleles were of smaller magnitudes: 1.2-, 1.4-, and 1.7-fold, respectively. Although the T985C substitution appeared to be disproportionate in the low pore-activity group by this method, only 1 of 200 persons expressed this variant, and the common allele is not conserved among species in this case (40), suggesting that this change may be linked to another unidentified loss-of-function allele.


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  		Table 2. P2X7 allele frequencies separated by low and normal pore activity.1

Consistent with its apparent increase in pore activity, the A1405G transition was relatively excluded from the low pore-activity group (Table 2Up and Fig. 3Up ). To evaluate this variant’s putative functional effects, we examined whether co-inheritance of the 1405 G allele offsets the phenotype associated with the 946 A, 1513 C, or 1729 A alleles. Accordingly, all persons with these validated loss-of-function genotypes were compared in groups with segregation according to the presence or absence of the 1405 G allele (Fig. 4 ). In these data blocks, the 1405 heterozygotes had higher pore activity than their wild-type counterparts who also carried the 1513 C or 1729 A alleles, whereas there was not a significant effect on the pore activity associated with the 946 GA genotype (Fig. 4 ). By contrast, 3 persons with low pore activity were 1405 heterozygotes (see Table C in the online Data Supplement), suggesting that the 1405 G allele is a gain-of-function variant capable of suppressing the loss-of-function effects of the variant 1513 C or 1729 A alleles. This supposition will require further in vitro analysis in a recombinant expression system for validation.


Figure 4
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  		Figure 4. Effects of heterozygosity of A1405G on pore activity associated with co-inheritance of various loss-of-function P2X7 alleles.

{square}, 1405 AA; Figure 4, 1405 AG. Of the 27 persons with 946 GA, 1513 CC, or 1729 TA genotypes, 4 also inherited an uncommon P2X7 1405 G allele. Similarly, of the 69 persons heterozygous at the 1513 locus, 20 also carried the 1405 variant. Pore activity is displayed as the mean, with SE (error bars) where appropriate, according to 1405 genotype. *, P <0.001 and <0.005 for the 1513 AC and 1729 TA groups, respectively (unpaired Student t-test comparisons). ** indicates that the single 1405 heterozygous individual had pore activity that was outside of the 95% confidence interval constructed from the data generated by the 1405 wild-type group.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A rate-limiting step in biomarker validation is the characterization of molecular and cellular phenotypes capable of bridging the gap between genomic data and clinical descriptors (36), a process requiring phenotypic tools that are precise, reliable, robust, affordable, safe, and suitable for a clinical laboratory (36). The P2X7 pore assay fulfilled these criteria, allowing efficient screening for loss-of-function alleles with <1 mL of whole blood, with a processing time of <3 h. The reproducibility and performance characteristics of this assay demonstrated high analytical validity [see Fig. 1BUp and Ref. (31)]. Because the 946 A, 1513 C, and 1729 A alleles all have strong negative effects of P2X7 pore activity (20)(21)(22), the negative predictive value of 98% provides solid assurance that a pore result within the reference interval is not associated with any these variants. By contrast, the relatively low positive predictive value of 59% pointed to targeted sequencing efforts to identify new loss-of-function variants. As new alleles are validated (i.e., clinically, cellularly, or transgenically), the diagnostic threshold distinguishing pore activity below the reference limit from activity within the reference interval may be successively refined by this method.

Cabrini et al. (23) recently evaluated the pore function of 5 P2X7 variants in purified lymphocytes from 62 patients with chronic lymphocytic leukemia and 100 healthy controls. Of note, the loss of pore function by the 1513 C allele and the weakly negative effects of variant A1068G and C1096G genotypes in their study were similar to the results shown in Fig. 3Up in this report, although additional persons would have been required to detect significant differences for the latter 2 sequence variations. Cabrini et al. (23) also reported on a gain-of-function allele, C489T, giving the appearance of disagreement with the data shown in Fig. 3Up of this report. However, the gain-of-function effect in primary lymphocytes described by Cabrini et al. (23) was seen only in a subset of participants (36 of 162 total) and could not be demonstrated in the presence of a 1513 C allele or in the study population analyzed collectively. In this regard, our observations are consistent with those of Cabrini et al. (23), and point out the importance of evaluating linkage disequilibrium when performing functional studies in primary cells. Similarly, because A1405G is also linked to A1513C (Fig. 2Up ), analysis of functional data exclusively from persons with the 1513 AA genotype (23) increases the risk of missing a functional interaction between the 2 alleles. Although validation of the effects of the 1405 G allele on pore activity will require additional in vitro investigations to exclude the possibility of linkage to another unidentified gain-of-function allele, this does not diminish functional observations made in analyzing the entire data set (Fig. 3Up ).

Although the existence of a rare P2X7 splice variant has been demonstrated recently (41), we did not detect it in our sample; by contrast, several of the common nonsynonymous mutants presented here encode amino acid substitutions in domains of the P2X7 protein with purported functional significance [Fig. 5 ; Ref. (42)]. Of the 3 previously known loss-of-function variants, those encoding the R307Q and E496A substitutions have normal surface expression but defective pore activity, possibly because of attenuated ligand binding and multimerization, respectively (20)(22). The I568N substitution affects receptor trafficking by disrupting a lipid-interaction/pore-enabling motif that may also be important for membrane recycling (21)(42)(43)(44)(45). The G150R variant has a strong negative influence on pore activity (Fig. 3Up ), potentially by disrupting a cysteine-rich region important for protein folding. Finally, the Q460R substitution appears to enhance pore activity (Fig. 3Up ) and may suppress the negative effects of having amino acids Ala496 or Asn568 (Fig. 4Up ). Similarly, the Q460R substitution falls at the boundary of an SH3 binding domain (42), which may be important for recruiting the scaffolding protein MAGuK and phosphatidylinositol-4 kinase involved in subunit assembly of proteins involved in exocytosis and membrane trafficking (45)(46). One model potentially explaining the suppression of other dominant-negative alleles (e.g., I568N) is that P2X7 proteins containing Arg460 may tightly associate soon after translation with activation of phosphatidylinositol-4 kinase and/or phospholipase D to override defects in receptor trafficking.


Figure 5
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  		Figure 5. Schematic representation of the P2X7 protein with putative functional domains and locations of nonsynonymous sequence variations.

The P2X7 protein contains 595 amino acids with 2 predicted transmembrane domains ({blacksquare}), 2 extracellular cysteine-rich regions possibly involved in folding (Figure 5), and an intracellular trafficking and pore-enabling domain overlapping a lipid-interaction motif (Figure 5). Ligand binding is thought to occur with participation from Lys64, Lys66, Arg294, and Lys311 (not shown) by homology to other P2X receptors. The C-terminal portion of the protein is enhanced for greater detail and contains a variety of previously described putative signaling motifs (42). The nonsynonymous variations are also included with approximate locations relative to these functional domains.

The present study facilitates testing of clinically relevant hypotheses involving combined modeling of genomic, functional, and clinical data, such as whether the A1405G variant is genetically linked in a larger population to the silent T–762C P2X7 promoter sequence variation associated with protection from smear-positive tubercular disease (14)(27)(47), as suggested by pairwise comparisons in the present data set (Fig. 2Up ). In this model, high P2X7 pore activity would predict a high ratio TNF-{alpha} to IL-10 (31), with such a balance known to confer an advantage in the killing of mycobacteria (48) and chlamydia (17)(49). Future characterization of larger population samples could improve estimates of haplotype incidence and possible effects of compound heterozygosity, penetrance, and expressivity for each allele as well as potential medication-induced epigenetic effects on receptor expression and pore activity.

In conclusion, we demonstrated the use of a genomically validated cellular assay for identifying and bridging apparently disparate genetic and phenotypic results and facilitating disease predictions. Our results suggest that variation in P2X7 pore activity is a biomarker of infectious, inflammatory, and autoimmune disorders.


   Acknowledgments
 
Dr. Denlinger is supported as a research fellow by the Will Rogers Institute, by mentored awards from NIH (1 K12 RR01761401) and the Departments of Medicine and Anesthesiology and the University of Wisconsin General Clinical Research Center (NIH M01 RR03186), and by the American College of Chest Physicians’ Ortho-Biotech Research Award in Critical Care. Drs. Coursin and Hogan receive funding from a Department of Anesthesiology career award, and Dr. Hogan is a recipient of a Doris Duke Innovation in Clinical Research Award. Dr. Bertics is supported by NIH Grants HL56396 and MO1 RR03186. We thank Lisa Pharo and Nicole Page for technical support. We also thank Dr. Richard A. Proctor for invaluable discussions regarding study design.


   Footnotes
 
1 Nonstandard abbreviations: LPS, lipopolysaccharide; TNF-{alpha}, tumor necrosis factor-{alpha}; IL, interleukin; and Bz-ATP, 2'-3'-O-(4-benzoylbenzoyl)-ATP.

2 Human genes: P2X7, purinergic receptor P2X, ligand-gated ion channel, 7.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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L. C. Denlinger, L. Shi, A. Guadarrama, K. Schell, D. Green, A. Morrin, K. Hogan, R. L. Sorkness, W. W. Busse, and J. E. Gern
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