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Association between Single-Nucleotide Polymorphisms in Mal/TIRAP and Interleukin-10 Genes and Susceptibility to Invasive Haemophilus influenzae Serotype b Infection in Immunized Children

  1. Shamez N. Ladhani1,2,
  2. Sonia Davila5,
  3. Martin L. Hibberd5,
  4. Paul T. Heath3,
  5. Mary E. Ramsay2,
  6. Mary P. E. Slack2,
  7. Andrew J. Pollard4, and
  8. Robert Booy1,6
  1. 1Academic Unit of Paediatrics, Barts and The London School of Medicine and Dentistry, London
  2. 2Centre for Infections, Health Protection Agency, London
  3. 3Vaccine Institute and Division of Child Health, St George's, University of London, London
  4. 4Oxford Vaccine Group, Department of Paediatrics, Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Oxford United Kingdom
  5. 5Infectious Diseases Unit, Genome Institute of Singapore, Republic of Singapore
  6. 6National Centre for Immunisation Research & Surveillance, Children's Hospital at Westmead and The University of Sydney, Australia
  1. Reprints or correspondence: Dr Shamez Ladhani, Immunisation Dept, Centre for Infections, Health Protection Agency, 61 Colindale Ave, London NW9 5EQ (shamez.ladhani{at}hpa.org.uk).

The introduction of the Haemophilus influenzae serotype b (Hib) conjugate vaccine into national immunization programmes has resulted in a dramatic reduction in the incidence of invasive Hib disease in many countries, including the United Kingdom [1,2]. Prior to the vaccine's use, Hib was a major childhood cause of meningitis, epiglottitis, septicemia, and other invasive infections in young children [2]. Because the vaccine is so effective, the development of invasive Hib disease after a course of immunization (ie, Hib vaccine failure) is rare [1, 3]. In the UK, for example, the vaccine failure rate during the period 1992–1999 among 4.4 million infants who received 3 doses of vaccine in infancy was only 2.2 cases per 100,000 vaccinees [1]. Although there have been concerns regarding the lack of a booster dose in the second year of life and the use of a less immunogenic Hib combination vaccine in 2000–2001 [4], the UK immunization campaign has resulted in a remarkable reduction in the burden of invasive Hib disease [5]. The annual number of cases, for example, decreased from almost 1000 cases in the prevaccine era to 37 cases in 1998, whereas the subsequent increase after 1999 peaked at 264 cases only in 2004 and decreased significantly thereafter [5].

Children with Hib vaccine failure, therefore, are a unique and important cohort. Although a proportion of these children have comorbidities, such as malignancy or immunosuppression, the vast majority appear to have no underlying reasons for developing Hib disease [3]. We, therefore, hypothesized that such a rare event may be influenced by rare recessive genotypes in a small selection of immune response genes. The UK Health Protection Agency currently holds the largest database of data on Hib vaccine failure cases in the world [6, 7]. The objective of this study was to identify any genetic susceptibility to invasive Hib disease or its clinical presentations in previously immunized children.

 
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METHODS

Cases. Hib vaccine failure was defined as invasive Hib disease occurring any time after receipt of 3 doses of Hib conjugate vaccine given in the first year of life, or >1 week after 2 doses in the first year, or >2 weeks after a single dose given after 12 months of age [1, 6]. The recruitment of children with Hib vaccine failure identified through national surveillance has been described elsewhere [8, 9]. In brief, general practitioners caring for children with Hib vaccine failure who survived their infection were approached for permission to approach the family. If we received a positive response, after obtaining written consent, the families were invited to participate in the study, which involved completing a questionnaire and obtaining a blood sample from the child.

Single-nucleotide polymorphism (SNP) selection. A literature search was performed to identify studies published before June 2007 on functional SNPs associated with sepsis, systemic inflammatory response syndrome, invasive bacterial infection, or immune-mediated conditions [10]. Because we chose to use the Wellcome Trust Case Control Consortium (WTCCC) cohort as controls, we selected SNPs that were in either the Illumina-300k-v1 (Illumina) or Affymetrix-500k (Affymetrix) genotyping chips. If a corresponding functional SNP was not identified on either chip, the HapMap database (http://www.hapmap.org; dbSNP126) was used to identify another SNP in strong linkage disequilibrium (r2>0.80) with the SNP of interest in white populations that was also present in one of the chips [11]. Functional SNPs for which we were unable to find an appropriate corresponding SNP in either genotyping platform were not included.

Controls. Two independent control groups were studied: healthy adult white persons from the British 1958 Birth Cohort, and blood donors recruited as part of the WTCCC project [12]. After obtaining written permission, WTCCC phase 1 and phase 2 control data were used in our analysis. These groups were chosen as our control subjects because (1) they were from the same geographic location and ethnic background as our case patients, (2) they are known to be healthy, (3) genotypes for the WTCCC cohort have already been determined on 2 different genotyping platforms for other studies [12], and (4) very few would have experienced invasive Hib disease: the cumulative risk of developing invasive Hib disease before 5 years of age in the prevaccine era was 1 case per 560 persons and much lower after this age [13].

DNA extraction. Peripheral blood samples were collected into ethylenediamine tetra-acetic acid-anticoagulated (EDTA) bottles and stored at −70°C within 24 h. DNA was extracted from whole blood using a standardized salting-out protocol [14]. DNA quality was assessed by visual inspection after running 1.2% agarose gels and by calculating absorbance ratio optical density, OD260nm/280nm. DNA quantification was performed using Picogreen dsDNA reagent. None of the DNA samples were degraded.

Genotyping and statistical analysis. Genotyping was initially performed on the Sequenom MassARRAY iPLEX Platform. Nineteen SNPs were included in the final multiplex reaction (Table 1). All 19 SNPs had >95% call rates for the case patients and were, therefore, included in the analysis. Three case patients had overall SNP call rates <95% and, thus, were excluded from the study. Confirmation of linkage disequilibrium between the rs1893352 and Ser180Leu (rs8177374) SNP in TIRAP was obtained by using the Taqman SNP Genotyping Assay (Applied Biosystems). The corresponding control genotypes for 9 of the functional SNPs were obtained from WTCCC Phase 1 (Affymetrix-500k) and included 1504 individuals from the 1958 British Birth cohort and 1500 from anonymous British blood donors. This data set had already undergone quality-control, whereby those samples that did not meet predefined criteria had been excluded in the data set made available by the WTCCC [12]. Therefore, 2938 samples were included for comparison. Control genotypes for the 10 remaining SNPs were obtained from WTCCC Phase 2 (Illumina-1.2M), which included 2482 from the 1958 British Birth cohort and 2587 from anonymous British blood donors. Quality control for this control data set was performed in house. After performing principal component analysis to detect population stratification, 268 samples from nonwhite individuals were excluded. Differences in the number of controls stated for individual SNPs were due to failed genotyping for that particular SNP only.

Table 1
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Table 1

Functional Single-Nucleotide Polymorphisms (SNPs) Identified from the Literature That Were Also in the Illumina-300k-v1 (Illumina) or Affymetrix-500k (Affymetrix) Genotyping Chips

Hardy-Weinberg equilibrium was tested separately for cases and controls using the χ2 test in HelixTree software, version 4.4.1 (Golden Helix), and was found to be in agreement (P>.05) for all SNPs after Bonferroni correction. Each SNP was tested using the allelic, genotypic, dominant, additive, and recessive models in HelixTree software. In all, 19 SNPs were analyzed using 5 genetic models for 3 clinical outcomes. Allelic and genotypic P values were calculated by means of 2×2 and 3×2 Pearson χ2 tables, respectively, or the Fisher exact test if counts in any cell were <5. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to measure any associations between case and control subjects. Uncorrected P values are given in the text and tables.

Ethics agreement. Ethics agreement was obtained from Thames Valley Multi-Centre Research Ethics Committee, UK (MREC Reference Number 05/MRE12/50). The UK Health Protection Agency has approval under Section 60 of the Health and Social Care Act to process confidential patient information for monitoring efficacy and safety of vaccination programmes (http://www.legislation.hmso.gov.uk/si/si2002/20021438.htm).

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RESULTS

Of the 323 families approached, 175 (54.2%) provided a bloom sample and returned a completed questionnaire, but 3 case patients were excluded because their overall SNP call rates were <95%. The final study cohort comprised 169 children whose parents self-reported as ethnically white (Table 2). There was no difference in age at onset of Hib disease, sex, geographic location, underlying medical conditions, or clinical presentation between children who provided a blood sample and those who declined (data not shown).

Table 2
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Table 2

Demographic Characteristics, Underlying Medical Conditions, and Clinical Presentations of 169 Children with Haemophilus influenzae Serotype b (Hib) Vaccine Failure Who Were Included in the Study

The 2 control cohorts (1958 Birth Cohort and anonymous blood donors) for each of the genotyping platforms were initially analyzed separately against the case patients, but because there was no statistical difference, they were merged for all subsequent analyses. Exclusion of children with comorbidities and/or immunoglobulin deficiency did not affect any of the observed genetic associations and were, therefore, included in all the analyses.

TIRAP gene. It was not possible to directly compare the Ser180Leu SNP (rs8177374) in TIRAP between case and control subjects because this polymorphism was not present in either Affymetrix or Illumina SNP chips. We, therefore, selected rs1893352 that was in strong linkage disequilibrium (r2=0.93) with Ser180Leu with allelic (0.86/0.14 vs 0.86/0.14) and genotypic (0.74/0.24/0.017 vs 0.73/0.25/0.017) frequencies that were comparable with the European Caucasian population (http://www.hapmap.org). Our results indicate that the genotype distribution for this SNP was significantly associated with Hib vaccine failure and that there was an excess of the recessive homozygous genotype among case patients, compared with control subjects (Table 3). This association was only seen among nonmeningitis cases (Table 3) and was primarily attributable to invasive infections other than epiglottitis, such as bacteremia and bacteremic pneumonia (genotype P value was 5.6×10−6 for genotype; OR for recessive homozygous genotype, 7.6 [95% CI, 2.9–19.9; P=1.3×10−6). The frequency of the recessive homozygous genotype among the 33 children (19.5%) with Hib vaccine failure who had ⩾1 other serious infection requiring hospitalization [8] was similar to that among children who developed invasive Hib disease only (3 [9.1%] of 33 vs. 9 [6.6%] of 136; P=.71). The heterozygous genotype was associated with protection against invasive Hib disease overall (OR, 0.68; 95% CI, 0.45–0.99; P=.045) but not with any of its clinical presentations and did not remain significant after Bonferroni correction.

Table 3
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Table 3

Genotype Distribution and the Recessive Homozygote Model for Single-Nucleotide Polymorphisms (SNPs) in TIRAP and IL-10

Because of the strong association between this SNP and Hib vaccine failure, we subsequently determined the genotype for the Ser180Leu (rs8177374) SNP using the Taqman SNP Genotyping Assay, which as expected, showed very high concordance, with only 3 exceptions. One dominant homozygous genotype in rs1893352 was genotyped as heterozygous in the Ser180Leu SNP, and 2 heterozygous genotypes were genotyped as homozygous dominant. More importantly, there was 100% concordance among the recessive homozygous genotypes between the 2 SNPs.

Interleukin-10 (IL-10) gene. The rs1554286 SNP was selected to investigate functional polymorphisms within the promoter region of the IL-10 gene because it was present in the Affymetrix-500k chip and was in strong linkage disequilibrium with both the C-891T (rs1800871; r2=0.87) and the C-592A (rs1800872; r2=0.75) IL-10 functional promoter polymorphisms. The selected SNP also has similar allelic (0.83/0.17 vs 0.83/0.17 vs 0.80/0.20, respectively) and genotypic (0.68/0.31/ 0.017 vs 0.67/0.31/0.02 vs 0.63/0.33/0.04, respectively) frequencies as the 2 IL-10 promoter polymorphisms among European Caucasian population (http://www.hapmap.org). When compared with controls, the recessive homozygous genotype was significantly associated with nonmeningitis cases only (Table 3). In particular, this association was only seen among patients with epiglottitis (6 [12.8%] of 47 cases; OR, 5.8; 95% CI, 2.4–14.2; P=1.1×10−5) and not among those presenting with meningitis (P=.90) or other invasive Hib infections (P=.88). Among Hib vaccine failure cases, 6 (66.7%) of 9 children with the IL-10 recessive homozygous genotype developed epiglottitis, compared with 41 (25.6%) of 160 with the heterozygous or dominant homozygous genotypes (relative risk, 5.2; 95% CI, 1.3–19.9; P=.01). Only 1 child among all case patients had both the IL-10 and the TIRAP homozygous recessive genotypes; this child presented with epiglottitis.

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DISCUSSION

This study provides evidence of a genetic susceptibility for invasive Hib disease in some vaccinated children. Two recessive SNPs in TIRAP and IL10 show evidence of association with invasive Hib disease in previously vaccinated children and, in particular, point towards a genetic basis for specific clinical presentations. TIRAP is an essential adaptor molecule that binds to the intracellular domain of 2 toll-like receptors, TLR2 and TLR4, and initiates a cascade of reactions that ultimately leads to the production of pro-inflammatory cytokines including, tumor necrosis factor-α and IL-12 [22, 23]. TLR2 recognizes different microbial products including lipoteichoic acid of Gram positive bacteria, whereas TLR4 is a critical sensor for lipopolysaccharide (LPS) of gram-negative pathogens [24]. TLR2 has also been shown to have a role in development of protective antibodies after vaccination against Borrelia burgdorferi, the pathogen causing Lyme disease [25].

In one of the largest studies of its kind, the heterozygous Ser180/Leu180 variant on TIRAP was associated with protection against pneumococcal disease, bacteremia, malaria, and tuberculosis in >6000 cases (P⩽9.6×10−8) [26]. Other studies have demonstrated a protective role for the heterozygous TIRAP variant in immune-mediated rheumatological conditions [27–29]. A recent study involving intravenous injection of LPS into healthy volunteers reported that individuals with the heterozygous Ser180/Leu180 variant produced significantly more pro-inflammatory cytokines in vivo, compared with those with the homozygous Ser180 variant [30]. The authors also demonstrated that cells isolated from individuals with the recessive homozygous Leu180/Leu180 variant produced the highest amount of pro-inflammatory cytokines after stimulation of TLR2 and TLR4 with appropriate ligands [30]. In our study, the protection provided by the heterozygous genotype was barely statistically significant. However, the recessive homozygous genotype was associated with an increased risk of invasive Hib disease (OR, 5.6; 95% CI, 2.7–11.5; P = 1.2×10−7), primarily among children with Hib septicemia and bacteremic pneumonia. Thus, it is conceivable that, in children with the recessive homozygous genotype, the presence of Hib in the bloodstream triggers unusually high pro-inflammatory cytokine release, which may lead to systemic inflammatory response syndrome, septic shock, and even death [31]. This association appears to be specific to Hib, because the frequency of the recessive homozygous genotype was similar among children with multiple serious infections, compared with those who developed a single episode of Hib disease.

Despite the small number of cases, the highly significant P value approaching genome-wide significance suggests that the association between the TIRAP polymorphism and systemic Hib infection is unlikely to be attributable to chance. We have shown a 100% concordance in the recessive homozygous genotype for the Ser180Leu polymorphism and the SNP we selected. In addition, the frequency of the recessive homozygous genotype for our selected SNP in the WTCCC control cohort (2.1%) was similar to the frequency of the recessive homozygous Leu180/Leu180 variant among healthy white UK controls—2.0% in one cohort comprising 567 UK cord blood samples from healthy neonates, 76 from healthy adult blood donors, and 96 from healthy adults from a Human Random Control Panel (the genotype frequencies between the adult controls and cord blood controls were reported to be nearly identical) [26] and 2.2% in another cohort of 965 healthy adults [28].

Khor et al [26] were only able to demonstrate a trend towards increased risk of invasive pneumococcal disease (OR, 2.39; 95% CI, 0.95–5.92) and pneumococcal empyema (OR, 4.01; 95% CI, 0.65–17.7) among 191 and 36 UK cases, respectively, for the recessive Leu180/Leu180 variant. There are several possible explanations why our study demonstrated a stronger genetic association for this SNP. First, children with Hib vaccine failure may be more likely to have an underlying genetic susceptibility to infection than unvaccinated children. Second, the lipoteichoic acid of the gram-positive Streptococcus pneumoniae primarily activates TLR2, whereas LPS from the gram-negative H. influenzae activates TLR4, and although TIRAP forms a common intracellular pathway for TLR2 and TLR4, interactions between different ligands and TLRs or between TLRs and TIRAP may be different [32]. Third, susceptibility to pneumococcal disease may lie further downstream in the TLR signalling pathway, as has recently been reported in children with IRAK-4 and NEMO mutations [33].

Interestingly, the recessive homozygous Leu180/Leu180 variant is extremely rare in developing countries [26, 27, 30] and our study supports recent speculations that the homozygous recessive TIRAP variant may increase susceptibility to specific infectious diseases to such an extent that it may have selected itself out of populations with a high burden of infections [26, 30].

There was also a strong genetic association between a SNP in the IL-10 promoter region and clinical presentation with epiglottitis. The IL-10 SNP (rs1554286) selected for in our study is in strong linkage disequilibrium with both the C-891T and the C-592A functional promoter polymorphisms, whose recessive homozygous genotypes are associated with lower IL-10 production [34, 35]. IL-10 is a potent, obligatory anti-inflammatory cytokine [36, 37] that is primarily induced by proinflammatory cytokines [38]. IL-10 has multiple biological effects, including inhibition of pro-inflammatory cytokines by T helper 1 lymphocytes, phagocytes, and natural killer cells [36, 37]. In mice, pretreatment with a single dose of IL-10 prevented death due to LPS-induced toxic shock [39]. Epiglottitis involves an acute and rapidly progressive inflammation of the supraglottic structures characteristically caused by Hib, that leads to acute airway obstruction and cardiorespiratory arrest if not treated promptly [40]. In epiglottitis, therefore, local invasion of Hib in the upper respiratory tract may lead to a rapid and intense inflammatory process. In individuals with the low IL-10-producing phenotype this process may remain unchecked, resulting in local swelling and inflammation that may progress to acute airway obstruction. Interestingly, both unvaccinated and vaccinated children with epiglottitis have the highest Hib antibody concentrations after infection compared with other Hib clinical presentations, supporting the argument for a strong proinflammatory response in children with epiglottitis [2, 9, 13]. Although this recessive IL-10 genotype may increase the risk of epiglottitis, it may also serve to localize the infection to the upper airways and, thus, prevent the pathogen from entering the bloodstream or the central nervous system.

We report the largest study (to our knowledge) of genetic association in children with conjugate vaccine failure. The uniqueness of this cohort, however, also means that our sample size is relatively small for a genetic association study and we have been unable to find a second cohort to replicate our findings. Also, the use of preselected control genotypes from the Wellcome Trust Cohort limited the polymorphisms we could study. Moreover, children who died—the group most likely to have an underlying genetic susceptibility—were not included, because this was a long-term follow-up study.

In spite of these drawbacks, the finding of such highly significant associations merits further attention. The inclusion of children with underlying medical conditions in our study can be justified, because although many were at increased risk of infection, the fact that they developed invasive Hib disease suggests that they may be particularly susceptible to this organism. Because we did not include unvaccinated children with invasive Hib disease in our study, it is difficult to distinguish whether the identified genetic associations are risk factors for Hib in general or to vaccine failure in particular. The former is more likely but, by selecting vaccine failure cases only, we increased the probability of identifying significant genetic associations. It is interesting to note that none of the 19 polymorphisms were associated with meningitis in this cohort, suggesting that genetic susceptibility to Hib meningitis is different to other clinical presentations, an important finding in its own right. Although this study may be difficult to replicate in a similar cohort, our findings may be applicable to infections or vaccine failures caused by other (particularly gram-negative)

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Acknowledgments

We thank the general practitioners, pediatricians, and families of all children with Hib vaccine failure who participated in this study. We are grateful to our colleagues at the Genome Institute of Singapore: Chui Chin Lim and Chang Hua Wong for sample handling; Carine Bonnard and Jason Ong for genotyping; and Erwin Tantoso and Jieming Chen for helping on the management of the genotyping data. This study makes use of data generated by the Wellcome Trust Case-Control Consortium 1 and 2. A full list of the investigators who contributed to the generation of the data is available at the WTCCC Web site (http://www.wtccc.org.uk).

Financial support. S.N.L. was awarded a competitive 2-year European Society for Paediatric Infectious Diseases (ESPID) Fellowship to complete this study. A.J.P. is a Jenner Institute Investigator and is funded by the NIHR Oxford Biomedical Research Centre. This study did not receive any external funding.

Potential conflicts of interest. S.N.L. and M.P.S. have received assistance from vaccine manufacturers to attend scientific meetings, as has R.B., who has also conducted clinical trials sponsored by pharmaceutical companies (Sanofi-Pasteur, GSK, Wyeth, CSL, and Roche). Payments from industrial sources are not personally accepted by R.B. but placed in a University account for research and education. P.T.H. is an investigator for clinical trials conducted on behalf of St. George's, University of London, sponsored by vaccine manufacturers, including manufacturers of Hib vaccines. He has also received assistance from vaccine manufacturers to attend scientific meetings. A.J.P. acts as chief investigator for clinical trials conducted on behalf of Oxford University and runs educational activities sponsored by vaccine manufacturers but does not accept any personal remuneration from pharmaceutical companies. Industry-sourced honoraria for consultancy or travel expenses are paid to an educational/administrative fund held by the Department of Pediatrics, University of Oxford. All other authors: no conflicts.

  • Received February 6, 2010.
  • Accepted June 24, 2010.
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  1. Clin Infect Dis. (2010) 51 (7): 761-767. doi: 10.1086/656236
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