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Published ahead of print on April 26, 2007, doi:10.1164/rccm.200702-169OC
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American Journal of Respiratory and Critical Care Medicine Vol 176. pp. 181-187, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200702-169OC

Original Article

I{kappa}B Genetic Polymorphisms and Invasive Pneumococcal Disease

Stephen J. Chapman1,2, Chiea C. Khor1, Fredrik O. Vannberg1, Angela Frodsham1, Andrew Walley1, Nicholas A. Maskell2, Christopher W. H. Davies3, Shelley Segal4, Catrin E. Moore4, Stephen H. Gillespie5, Paul Denny6, Nicholas P. Day7, Derrick W. Crook8, Robert J. O. Davies2 and Adrian V. S. Hill1

1 The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom; 2 Oxford Centre for Respiratory Medicine, Churchill Hospital Site, Oxford Radcliffe Hospital, Oxford, United Kingdom; 3 Department of Respiratory Medicine, Royal Berkshire Hospital, Reading, United Kingdom; 4 Department of Paediatrics, John Radcliffe Hospital, Oxford, United Kingdom; 5 Centre for Medical Microbiology, Department of Infection, University College London, London, United Kingdom; 6 Medical Research Council UK Mouse Genome Centre and Mammalian Genetics Unit, Harwell, Oxon, United Kingdom; 7 Centre for Tropical Diseases, Cho Quan Hospital, Ho Chi Minh City, Vietnam; and 8 Department of Microbiology, John Radcliffe Hospital, Oxford, United Kingdom

Correspondence and requests for reprints should be addressed to Stephen J. Chapman, M.R.C.P., The Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN, UK. E-mail: schapman{at}well.ox.ac.uk


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Increasing evidence supports a key role for the transcription factor nuclear factor (NF)-{kappa}B in the host response to pneumococcal infection. Control of NF-{kappa}B activity is achieved through interactions with the I{kappa}B family of inhibitors, encoded by the genes NFKBIA, NFKBIB, and NFKBIE. Rare NFKBIA mutations cause immunodeficiency with severe bacterial infection, raising the possibility that common I{kappa}B gene polymorphisms confer susceptibility to common bacterial disease.

Objectives: To determine whether polymorphisms in NFKBIA, NFKBIB, and NFKBIE associate with susceptibility to invasive pneumococcal disease (IPD) and thoracic empyema.

Methods: We studied the frequencies of 62 single-nucleotide polymorphisms (SNPs) across NFKBIA, NFKBIB, and NFKBIE in individuals with IPD and control subjects (n = 1,060). Significantly associated SNPs were then studied in a group of individuals with thoracic empyema and a second control group (n = 632).

Measurements and Main Results: Two SNPs in the NFKBIA promoter region were associated with protection from IPD in both the initial study group and the pneumococcal empyema subgroup. Significant protection from IPD was observed for carriage of mutant alleles at these two loci on combining the groups (SNP rs3138053: Mantel-Haenszel 2 x 2 {chi}2 = 13.030, p = 0.0003; odds ratio [OR], 0.60; 95% confidence interval [CI], 0.45–0.79; rs2233406: Mantel-Haenszel 2 x 2 {chi}2 = 18.927, p = 0.00001; OR, 0.55; 95% CI, 0.42–0.72). An NFKBIE SNP associated with susceptibility to IPD but not pneumococcal empyema. None of the NFKBIB SNPs associated with IPD susceptibility.

Conclusions: NFKBIA polymorphisms associate with susceptibility to IPD. Genetic variation in an inhibitor of NF-{kappa}B therefore not only causes a very rare immunodeficiency state but may also influence the development of common infectious disease.

Key Words: genetic polymorphisms • pneumococcal infection • nuclear factor-{kappa}B



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
The nuclear factor (NF)-{kappa}B inflammatory pathway plays a key role in the host immune response to pneumococcal infection, and very rare genetic mutations in an NF-{kappa}B inhibitor cause immunodeficiency with severe bacterial infection.

What This Study Adds to the Field
This study describes novel associations between common genetic polymorphisms in NF-{kappa}B inhibitors and susceptibility to invasive pneumococcal disease in humans.

 
Infection with Streptococcus pneumoniae remains a significant global problem, accounting for the deaths of more than 1 million children younger than 5 years worldwide (1). Invasive pneumococcal disease (IPD) is defined by the isolation of S. pneumoniae from a normally sterile site, such as blood (septicemia), cerebrospinal fluid (meningitis), or pleural fluid (thoracic empyema). The incidence rate of IPD ranges from 10 to 100 per 100,000 persons per year, and despite advances in medical treatments, the mortality rate of IPD in adults remains at least 10 to 20% (1). Colonization of the nasopharynx by the pneumococcus is widespread, yet only a minority of individuals develop invasive disease (2). An important, and often neglected, factor that influences the development of infectious diseases such as IPD is the host's genetic profile (3).

The ubiquitous transcription factor nuclear factor (NF)-{kappa}B is central to a diverse array of cellular processes, including host innate and adaptive immune responses (4, 5). Activation of NF-{kappa}B occurs after stimulation of a variety of immune receptors, including Toll-like receptors (TLRs) and members of the interleukin (IL)-1 and tumor necrosis factor receptor superfamilies. The signaling pathway downstream of the TLR and IL-1 family of receptors is complex and incompletely understood; key mediators include the cytoplasmic adaptor molecules MyD88 and Mal/TIRAP, which activate TRAF6 via IL-1 receptor-associated kinases (IRAK1 and IRAK4) (6, 7). In unstimulated cells, NF-{kappa}B transcription factors are prevented from binding DNA due to their association with the inhibitors of NF-{kappa}B (I{kappa}B) protein family; phosphorylation of the I{kappa}B inhibitors by the I{kappa}B kinase complex leads ultimately to their degradation and the release of NF-{kappa}B, which is then capable of inducing gene transcription (5, 8). The best-studied members of the I{kappa}B family are I{kappa}B-{alpha}, I{kappa}B-beta, and I{kappa}B-{epsilon}, encoded by the genes NFKBIA (Chr14q13.2), NFKBIB (Chr19q13.2), and NFKBIE (Chr6p21.1), respectively (5). The recent identification of functional homologs of NF-{kappa}B and I{kappa}B in the horseshoe crab suggests that these components originated more than 500 million years ago and further underlines the key role of this pathway in host defense (9).

Although polymorphisms within genes encoding the activating TLRs have been associated with a number of disease states (10), the role of genetic variation within downstream components of the NF-{kappa}B pathway in disease development remains largely unexplored. Exceptions are the associations described between variants in NFKBIA with Crohn's disease, trachoma, and sarcoidosis (11–13), and the recent association of a functional polymorphism in IRAK1 with outcomes from sepsis (14).

There is increasing evidence to support a critical role for the NF-{kappa}B pathway in the host immune response to pneumococcal infection. Many of the immune receptors capable of activating NF-{kappa}B are stimulated during pneumococcal infection, and, in particular, TLR2 recognizes components of gram-positive bacteria such as S. pneumoniae (15–17) and TLR4 recognizes the pneumococcal toxin pneumolysin (18). Activation of NF-{kappa}B by pneumococci has been clearly demonstrated both in vitro and in animal models (19–22), and targeted genetic disruption of the NF-{kappa}B p50 subunit in mice has been shown to increase susceptibility to overwhelming pneumococcal infection (23). Recently, two patients have been described with mutations in the NFKBIA gene leading to impaired NF-{kappa}B activation and a primary immunodeficiency syndrome characterized by recurrent severe bacterial infections in association with the skin condition anhidrotic ectodermal dysplasia (24, 25). On the basis of these findings, we hypothesized that common polymorphisms in the I{kappa}B genes may be associated with susceptibility to the phenotype of IPD regularly encountered in clinical practice. To investigate this further, we studied the frequencies of polymorphisms in the three major I{kappa}B genes NFKBIA, NFKBIB, and NFKBIE in groups of individuals with IPD and thoracic empyema, as well as two control groups.


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Information
The IPD sample collection has been previously described in detail (26). Briefly, blood samples were collected from patients with IPD (defined by the isolation of S. pneumoniae from a normally sterile site) as part of an enhanced active surveillance program in three hospitals in Oxfordshire, United Kingdom (John Radcliffe, Horton General, and Wycombe General Hospitals). Frequencies of clinical presentation were as follows: pneumonia, 69%; isolated bacteremia, 15%; meningitis, 11%; and other presentations, 5%. This collection was designated the IPD study group (1). Individuals with thoracic empyema (suppurative infection of the pleural cavity) were recruited on entry to the U.K. MIST1 (First Multicenter Intrapleural Sepsis Trial) trial, as previously described (27–29). A bacteriological diagnosis was made in 59% of the available samples, with the breakdown of bacterial species within this group as follows: S. pneumoniae, 27%; Streptococcus intermedius-anginosus-constellatus (milleri) group, 24%; other streptococcal species, 7%; Staphylococcus aureus, 8%; anaerobes, 16%; gram-negative bacteria, 6%; and others, 12%. Individuals with pneumococcal empyema comprised the IPD study group (2).

Two control groups were available for study. The first control group (control 1) comprised a combination of 180 United Kingdom healthy adult blood donors and 590 cord blood samples, as previously described (26). The second control group (control 2) was collected independently and consisted of 370 United Kingdom healthy adult blood donors. Individuals of non-European ancestry were excluded from cases and control subjects. The study was approved by the research ethics committees of the participating hospitals.

Genotyping Techniques
Genotyping was performed using the Sequenom mass-array Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) primer extension assay (30), as described in the online supplement.

Statistical Analysis
Statistical analysis of genotype associations and logistic regression was performed using SPSS version 12.0 (SPSS, Inc., Chicago, IL). Analysis of linkage disequilibrium (LD) and haplotypes was performed using the Haploview version 3.2 program (31). Haplotype blocks were defined as regions demonstrating strong evidence of historical recombination between less than 5% of single-nucleotide polymorphism (SNP)–pair comparisons (32). All control genotype distributions were in Hardy-Weinberg equilibrium (0.05 level).


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We genotyped a total of 62 SNPs in the initial IPD study group (IPD 1) and control group (control 1). Of these SNPs, 19 were either nonpolymorphic or extremely rare (minor allele frequency < 0.01) in the population studied, leaving a total of 43 SNPs across the three I{kappa}B genes for analysis (Table E2 of the online supplement). None of the SNPs genotyped in NFKBIB were associated with IPD susceptibility. Of the remaining SNPs, six appeared to be associated with susceptibility to IPD at the 0.05 significance level (Table E2). Within NFKBIA, the associated polymorphisms were clustered particularly in the promoter region of the gene, where the adjacent SNPs rs2233406 and rs3138053 were most significantly associated with disease (Table E2). Only a single SNP in NFKBIE (rs529948) appeared to be associated with IPD susceptibility (Table E2). The genotype frequencies for the three SNPs with strongest evidence of association with IPD susceptibility are presented in Table 1 (control 1 and IPD 1 groups). In each case, conditional logistic regression confirmed that the pattern of association best fits a dominant, protective effect of the mutant allele. Logistic regression analysis demonstrated no effect of age, comorbid conditions, or sex on I{kappa}B genotype. Analysis of I{kappa}B haplotypes is described in the online supplement.


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  		TABLE 1. NFKBIA AND NFKBIE POLYMORPHISM GENOTYPE FREQUENCIES IN INDIVIDUALS WITH INVASIVE PNEUMOCOCCAL DISEASE AND CONTROL SUBJECTS

 
The three most strongly associated SNPs were then examined in the thoracic empyema and second control study groups to assess whether the associations were limited to pneumococcal disease or more generally applicable to other forms of gram-positive invasive respiratory bacterial disease. No association was observed between genotype and susceptibility to thoracic empyema overall (cases [n = 262]; for NFKBIArs3138053, {chi}2 = 1.177, p = 0.56; for NFKBIArs2233406, {chi}2 = 1.505, p = 0.471; for NFKBIErs529948, {chi}2 = 4.015, p = 0.134). Analysis of the very small (n = 42) subgroup of individuals with pneumococcal empyema, however, revealed associations between each NFKBIA genotype and disease susceptibility (Table 1, control 2 and IPD 2 groups). For each of the two SNPs in NFKBIA (rs3138053 and rs2233406), protection from pneumococcal empyema was associated with carriage of the mutant allele, the same direction of association as that seen with IPD group 1 and control 1 (Table 1). For NFKBIErs529948, on the other hand, individuals with pneumococcal empyema were overrepresented in the mutant homozygote group, a different direction of association from that previously observed for this SNP and IPD (Table 1); indeed, the association with pneumococcal empyema was not significant when analyzed using the previous dominant model (p = 0.292, Table 1). In keeping with this, significant heterogeneity of odds ratios (ORs) for NFKBIErs529948 between the two IPD-control groups was noted (Tarone's homogeneity of OR {chi}2 = 5.519, p = 0.019). Comparison of ORs for NFKBIArs3138053 and NFKBIArs2233406 did not demonstrate any evidence of heterogeneity between the two IPD-control groups for either SNP; after combining and stratifying the study groups, carriage of the mutant allele (mutant homozygotes and heterozygotes combined) was associated with protection from IPD when compared with the wild-type homozygous state (for rs3138053: Mantel-Haenszel 2 x 2 {chi}2 = 13.030, p = 0.0003; OR, 0.60; 95% confidence interval [CI] for OR, 0.45–0.79; for rs2233406: Mantel-Haenszel 2 x 2 {chi}2 = 18.927, p = 0.00001; OR, 0.55; 95% CI, 0.42–0.72).

Outcome data were available for 127 individuals with IPD and for all of the individuals with pneumococcal empyema; mortality rates were 10 and 14% in the two groups, respectively. No association was observed between genotypes and outcome (data not shown), although the number of individuals in the poor outcome groups was small and a significantly larger study would be required to examine effects of genotype on mortality.

An important question is whether the associations with IPD susceptibility are truly with polymorphisms in NFKBIA and NFKBIE, or whether these simply represent markers in LD with disease-associated polymorphisms located in other genes. To address this, we examined the pattern of LD between polymorphisms (Figures 1–3GoGo). The associated SNP in NFKBIE (rs529948) is located within a region of strong LD in this population (Figure 3); the lack of associated SNPs outside this region suggests that the observed association is localized to NFKBIE, rather than with a gene elsewhere along the chromosome. The pattern of LD across NFKBIA and its flanking regions is more complex (Figure 1). Nevertheless, the disease-associated SNPs are contained within the region studied, and the flanking markers genotyped are in only weak LD with the associated SNPs and do not themselves demonstrate any association with IPD (Table E2), again indicating that the association is likely to be with NFKBIA rather than a neighboring gene. The absence of any predicted genes in the vicinity of NFKBIA and NFKBIE (Figures 1 and 3) further suggests that the observed disease associations are with these genes, although genotyping of a significantly larger number of polymorphisms would be required to absolutely exclude long-range LD with a variant elsewhere.


Figure 1
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  		Figure 1. Genomic organization, location of single-nucleotide polymorphisms (SNPs), and linkage disequilibrium (LD) map for NFKBIA. (A) Gene structure. Exons are shown as rectangles or vertical lines and are numbered. There are no neighboring genes in the region studied. (B) LD between the polymorphisms studied. Polymorphisms are identified by their dbSNP rs numbers, and their position relative to the gene structure (A) is marked by a vertical line. Empty squares indicate a high degree of LD (LD coefficient D' = 1) between pairs of markers. Numbers indicate the D' value expressed as a percentile. Red squares indicate pairs in strong LD with logarithm of odds (LOD) scores for LD >= 2; pink squares, D' < 1 with LOD >= 2; white squares, D' < 1.0 and LOD < 2.

 

Figure 2
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  		Figure 2. Genomic organization, location of SNPs, and LD map for NFKBIB. (A) Gene structure. Exons are shown as rectangles or vertical lines and are numbered. The locations of neighboring genes in the region are shown. (B) LD between the polymorphisms studied. Polymorphisms are identified by their dbSNP rs numbers, and their position relative to the gene structure (A) is marked by a vertical line. Empty squares indicate a high degree of LD (LD coefficient D' = 1) between pairs of markers. Numbers indicate the D' value expressed as a percentile. Red squares indicate pairs in strong LD with LOD scores for LD >= 2; pink squares, D' < 1 with LOD >= 2; white squares, D' < 1.0 and LOD < 2.

 

Figure 3
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  		Figure 3. Genomic organization, location of SNPs, and LD map for NFKBIE. (A) Gene structure. Exons are shown as rectangles or vertical lines and are numbered. There are no neighboring genes in the region studied. (B) LD between the polymorphisms studied. Polymorphisms are identified by their dbSNP rs numbers, and their position relative to the gene structure (A) is marked by a vertical line. Empty squares indicate a high degree of LD (LD coefficient D' = 1) between pairs of markers. Numbers indicate the D' value expressed as a percentile. Red squares indicate pairs in strong LD with LOD scores for LD >= 2; pink squares, D' < 1 with LOD >= 2; white squares, D' < 1.0 and LOD < 2.

 
Finally, we performed logistic regression analysis to investigate possible interactions between the most statistically significant disease-associated SNPs in NFKBIA (rs2233406) and NFKBIE (rs529948), and between these SNPs and the (nonassociated) polymorphisms in NFKBIB. This analysis was performed using the IPD 1 and control 1 study groups, because the pneumococcal empyema group was too small to assess for interactions. Carriage of a protective allele (either the NFKBIArs2233406 T allele or the NFKBIErs529948 A allele) was associated with an OR for IPD of approximately 0.6 (Table 2). Carriage of protective alleles at both loci was associated with an OR for disease of 0.31 (Table 2). No evidence of epistasis was observed between NFKBIArs2233406 and NFKBIErs529948 and polymorphisms in NFKBIB (data not shown).


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  		TABLE 2. ANALYSIS OF INTERACTION BETWEEN NFKBIARS2233406 AND NFKBIERS529948 IN CONFERRING PROTECTION AGAINST INVASIVE PNEUMOCOCCAL DISEASE

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate that common polymorphisms in the I{kappa}B genes NFKBIA and NFKBIE associate with susceptibility to IPD. We consider these associations to be particularly compeling in the setting of evidence from in vitro, animal, and rare human (primary immunodeficiency) models of disease, all of which support an important role for I{kappa}B proteins in susceptibility to invasive bacterial disease. The increased risk (wild-type homozygote) genotypes for NFKBIArs2233406 and NFKBIErs529948 are present in nearly 50 and 70% of the individuals studied, respectively (Table 1). As a result, these polymorphisms might be expected to make a significant contribution to the burden of invasive pneumococcal disease in this population. The polymorphisms are located in the upstream regions of the genes and are likely to exert subtle, regulatory functional effects in comparison to the NFKBIA mutations responsible for primary immunodeficiency (24, 25). These findings raise the possibility that polymorphisms in other genes associated with rare primary immunodeficiency states may also influence susceptibility to common infectious disease.

An important cause of type I error in genetic association studies is the failure to correct significance levels when multiple independent markers have been examined. Here, we analyzed 43 polymorphisms in total, and applying a Bonferroni correction results in a threshold significance level of 0.001, rather than 0.05. Even with this corrected significance level, rs2233046 and rs529948 remain associated with IPD susceptibility. The Bonferroni correction assumes, however, that markers are independent, whereas many of the SNPs studied here are in strong or complete LD and are therefore not truly independent from each other. As a result, the Bonferroni adjustment is likely to markedly overcorrect in this case. A correction based on the total number of LD blocks and singleton (not part of an LD block) SNPs has been proposed; such a correction has been demonstrated to generate acceptable type I error rates when using the blocking algorithm described by Gabriel and colleagues across regions of moderate and high LD (33). Using this approach, 18 independent tests were performed in this study, suggesting a threshold p value of 0.0027 for statistical significance. It should be noted that even this approach is likely to be conservative because it does not take into account interblock LD.

Whichever approach is used, the observed p values of association for rs2233406 and rs529948 suggest that these are unlikely to represent artifacts of multiple testing. Population stratification is also unlikely to account for the observed association, because all cases and control subjects were of white United Kingdom descent. Further evidence for the association between NFKBIA and IPD is provided by the observation of significant associations between the linked SNPs rs3138053 and rs2233406 and susceptibility to pneumococcal empyema in an independently collected group of samples (Table 1). The direction of association in the two groups is the same, with carriage of the mutant allele in each case associated with protection from IPD. It should be noted that the number of individuals in the pneumococcal empyema subgroup is extremely small, however, and this result should therefore be interpreted with some caution. The absence of an association between the polymorphisms and susceptibility to thoracic empyema overall suggests that the effect may be specific to invasive pneumococcal disease, although the number of individuals in each of the remaining bacterial groups is also relatively small. The lack of clear replication for NFKBIErs529948 with pneumococcal empyema does not necessarily indicate that the initial association with IPD is false. This result could simply reflect sampling variation in the context of a small replication group, and attempted replication of the association with rs529948 in a larger sample set is required.

Further research is needed to identify the functional effects of the associated polymorphisms in each gene. The most statistically significant associated SNPs in NFKBIA are clustered in the upstream region of the gene, suggesting a possible effect on promoter function, although neither rs2233406 nor rs3138053 appear to interfere with predicted transcription factor binding sites (described in the online supplement). Previous studies have not investigated the functional effects of polymorphisms within NFKBIA, although disease-associated polymorphisms were located in the promoter region in the case of sarcoidosis and trachoma (12, 13), and the 3' untranslated region in the case of Crohn's disease (11). The two patients with primary immunodeficiency were each found to have a mutation in the serine residue at position 32 of I{kappa}B-{alpha}, rendering the protein resistant to phosphorylation and degradation and enhancing its inhibition of NF-{kappa}B (24, 25). This mutation appears to be extremely rare, however, and no common nonsynonymous polymorphisms have been identified on sequencing of the NFKBIA exons (11, 34). NFKBIE has not, to our knowledge, been subject to previous disease-association studies and the functional effects of polymorphisms in this gene are unknown. Although the location of rs529948 may be in keeping with an enhancer regulatory effect (see the online supplement), this SNP is within a region of strong LD extending across the gene and therefore may simply be a marker linked to a functional variant within the gene itself.

No associations were observed in our study between polymorphisms in NFKBIB and susceptibility to IPD. Furthermore, the extensive LD across NFKBIB implies that all possible haplotypic combinations are likely to have been captured, suggesting that it is unlikely that a common IPD-associated polymorphism within this gene has been missed. Why do polymorphisms in NFKBIA and NFKBIE but not NFKBIB appear to be associated with IPD susceptibility? The precise role of each of these I{kappa}B proteins in NF-{kappa}B activation remains unclear, although their degradation and resynthesis kinetics differ (35). After cellular stimulation, both I{kappa}B-{alpha} and I{kappa}B-{epsilon} are degraded and then resynthesized as a result of activation by NF-{kappa}B itself, thereby providing negative feedback control of NF-{kappa}B activity (35–38). The synthesis of I{kappa}B-beta, in contrast, is not controlled by NF-{kappa}B and consequently the degradation of this inhibitor leads to more sustained NF-{kappa}B activation (35). The temporal control of NF-{kappa}B activity appears in turn to determine the dynamics and pattern of gene expression (35, 38–40). Functional polymorphisms in the I{kappa}B genes may affect the relative levels of the different inhibitors, potentially resulting in different NF-{kappa}B activation profiles and different patterns of gene expression. Although the presence of disease-associated polymorphisms may simply reflect the historical chance occurrence of mutations in functional sites within NFKBIA and NFKBIE but not NFKBIB, it is intriguing that I{kappa}B-{alpha} and I{kappa}B-{epsilon} are both induced by NF-{kappa}B, suggesting that interference with this negative control pathway may have particularly important functional consequences.

Given the similar functions of I{kappa}B-{alpha} and I{kappa}B-{epsilon}, it would be unsurprising if polymorphisms within NFKBIA and NFKBIE interact in determining susceptibility to IPD. The risk of IPD was found to be reduced to just below 0.6 of expected in the presence of a protective allele, but further reduced to less than a third of that expected in the presence of both protective alleles (Table 2). This finding suggests that the effect of NFKBIErs529948 on susceptibility to IPD is not redundant in the setting of the NFKBIArs223306 effect. The interaction between these polymorphisms and functional genetic variants in other NF-{kappa}B pathway components is worthy of further investigation. Indeed, in a study of wild flies, naturally occurring polymorphisms in cactus (the Drosophila melanogaster homolog of I{kappa}B) were not only themselves associated with susceptibility to infection but also exhibited significant epistasis with other intracellular signaling loci in determining immune competence (41).

Our findings raise the interesting question of why the protective alleles are not more common in the population studied, given the considerable historical selective pressure exerted by IPD. One possible explanation is that these alleles confer increased susceptibility to another major cause of mortality, with the observed allele frequencies reflecting a balance between these opposing selective pressures. This theory is consistent with increasing evidence that "optimal" levels of NF-{kappa}B pathway activity may reflect a balance between the deleterious consequences of impaired or excessive pathway activation. Although, on one hand, impaired NF-{kappa}B activation is associated with immunodeficiency (24, 25), there is also considerable evidence for a central role of NF-{kappa}B activation in the pathogenesis of inflammatory disease (42, 43), and increased levels of NF-{kappa}B activation are, for example, associated with a worse outcome from sepsis (14, 44, 45). Greater understanding of the genetic control of this major inflammatory pathway is of particular relevance in the setting of growing interest in the modulation of NF-{kappa}B activity as a treatment for inflammatory disease (43, 46). Further studies investigating the role of common genetic polymorphisms in NF-{kappa}B pathway components in susceptibility to other infectious and inflammatory diseases may shed light on this issue.


   Acknowledgments
 
S.J.C. is a Wellcome Trust Clinical Research Fellow; A.V.S.H. is a Wellcome Trust Principal Fellow. CCK is a scholar of the Agency for Science, Technology, and Research (A-STAR), Singapore, and member of the MBBS-PhD programme, Faculty of Medicine, National University of Singapore.


   FOOTNOTES
 
Supported by the Wellcome Trust, UK.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200702-169OC on April 26, 2007

Conflict of Interest Statement: S.J.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.C.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.O.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.A.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.W.H.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.E.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.H.G.'s research group has been in receipt of a research grant from Wyeth Pharmaceuticals supporting the development of a pneumococcal diagnostic test. P.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.P.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.W.C. received a grant from Wyeth worth £200,000 from 2005–2007 for invasive pneumococcal surveillance. R.J.O.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.V.S.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 1, 2007; accepted in final form April 26, 2007


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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
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 DISCUSSION
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