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Technical Briefs |
(Departments of1 General Pediatrics and2 Neonatology, Medical University of Graz, Graz, Austria;3 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Vienna, Vienna, Austria;
aaddress correspondence to this author at: Clinical Institute of Medical and Chemical Laboratory Diagnostic, Medical University Vienna, Vienna, Austria, Waehringer Guertel 18-20, 1090 Wien, Austria; fax 43-1-40400-2097, e-mail christine.mannhalter{at}meduniwien.ac.at)
Abstract
Background: An association has been described between mortality in children with meningococcal disease and functional polymorphisms in the interleukin-1 (IL1) cluster. We undertook a multicenter study to evaluate associations of these polymorphisms in a Central European population.
Patients and Methods: The study involved 95 Middle European pediatric hospitals. We collected blood samples from, and clinical information about, 285 previously healthy children with meningococcal infection. We used a newly developed multiplexed mutagenic separated PCR assay to analyze 6 polymorphisms within the IL1 cluster: IL1A (–889)C/T, IL1A (+4845)G/T, IL1B (–511)C/T, IL1B (–31)C/T, IL1B (+3954), and IL1RA (+2018)C/T. We studied the same polymorphisms in a comparison group of 481 healthy newborns.
Results: Genotype frequencies between patients and the comparison group differed significantly only for the IL1RA (+2018)C/T variant: The CC genotype was more frequent in patients (11%) than in healthy controls (5%; P = 0.008). In the patient group, the C allele was significantly more prevalent (67%) in nonsurvivors than in survivors (42%; P = 0.02).
Conclusion: The IL1RA (+2018)C/T polymorphism is associated with the risk of meningococcal disease and with its outcome.
Fulminant meningococcemia is a life-threatening disease with acute onset, septic shock, and progressive hemorrhagic necrosis of the skin. The mortality rate is variable, ranging from 5% to 20% (1), and is highest for patients with severe septic shock and the clinical syndrome of purpura fulminans (2). One of the early characteristics of patients with severe sepsis syndrome is the overactivation of proinflammatory cytokines, leading to unusually high plasma concentrations and to symptoms of septic shock and multiorgan failure (3).
There is a strong genetic component for sensitivity to infectious disease other than exposure to infectious agents. As of this writing, several polymorphisms in candidate genes have been investigated, including the genes for toll-like receptor 4 (4), tumor necrosis factor-
(5), and plasminogen-activator inhibitor-1 (6). Our knowledge of the genetic factors contributing to the genetic susceptibility, however, is limited.
Interleukin-1 (IL-1) is the key player in the early innate response to bacterial challenge (7). The IL-1 family(8) comprises 3 members: IL-1A and IL-1B, which act as proinflammatory agonists, and their competitive antagonist, the IL-1 receptor antagonist (IL-1RA). It is reasonable to speculate that genetic variations affecting expression activities of members of the IL1 gene cluster represent genetic risk factors for susceptibility and outcome in meningococcal disease.
Several polymorphisms within the IL1 gene cluster have been reported to influence the transcription of IL-1, including polymorphisms within the IL1A gene [(–889)C/T and (+4845)G/T], the IL1B gene [(–511)C/T, (–31)C/T, and (+3954)C/T], and the IL1RA gene [(+2018)C/T]. IL1B (–511)C/T and IL1RA (+2018)C/T have been associated with either susceptibility or outcome in meningococcal disease in the British population (9). In Great Britain, meningococcal disease is relatively frequent, whereas the incidence in continental Europe is relatively low. Although the reasons for this difference are still poorly understood, we may speculate that environmental factors and genetic background contribute to the different incidences.
Patients.
To evaluate the role of polymorphisms within the IL1 gene cluster on the pathophysiology of meningococcal disease in central Europe, we studied patients in a multicenter study in Germany, Switzerland, Italy, and Austria.
Between March 2000 and October 2002, blood samples and clinical data were prospectively collected for patients with meningococcal disease at 95 pediatric hospitals and assembled in the Central European Meningococcal Research (CEMR) Cohort (6). In addition, archived blood samples and clinical data from 24 patients from 8 centers, collected during the planning stage of the study before March 2000 (6), were included in the study. In total, 285 patients could be tested. The 24 patients whose blood samples had been archived did not differ significantly from the rest of the group by age or any other variables.
The age at disease onset varied from 1 month to 27 years (median age, 35 months). The patients tested included 147 men and 138 women. Clinical information was collected from each patient according to a defined protocol (demographic data, diagnosis, and outcome) (10).
Caucasian controls.
Cord blood of healthy, unrelated newborns (n = 481) was collected between March 2002 and August 2002 at the Department of Neonatology of the Medical University of Graz and was used for genotyping and determination of prevalences. For all participants, blood samples were collected after parental consent. The study was approved by the ethics committees of all participating hospitals.
Genomic DNA was isolated from 200 µL of whole blood by use of a Magna Pure LC automated nucleic acid isolation system (Roche). The IL1A (–889)C/T, IL1A (+4845)G/T, IL1B (–511)C/T, IL1B (–31)C/T, IL1B (+3954)C/T, and IL1RA (+2018)C/T single-nucleotide polymorphisms were genotyped by a multiplexed mutagenic separated-PCR assay. Briefly, PCR mixtures were prepared in 25 µL containing 0.75 U of AmpliTaq Gold (Applied Biosystems), 1.5 mM MgCl2, 200 µM each of the deoxynucleoside triphosphates (Amersham Pharmacia Biotech), 14 pmol of the IL1A (–889)C primer IL-1A-8B (5'-CATGGATTTTTACATATGAGCCTTCGATGG-3'), 9 pmol of the IL1A (–889)T primer IL-1A-9B (5'-GAGAAAGGAAGGTGTGGATTTTTACATATGAGCCTTCAGTGA-3'), 22 pmol of the IL1A (–889) common reverse primer IL-1A-4A (5'-TTTTAGAGATGGGGGCTTCACTATG-3'), 9 pmol of the IL1A (+4845)T primer IL-1A-2B (5'-CACATTGCTCAGGAAGCTAAAAGATGA-3'), 9 pmol of the IL1A (+4845)G primer IL-1A-3B (5'-TAAAGTTGTATTTGTCATTGCTCAGGAATCTAAAAGGAGC-3'), 18 pmol of the IL1A (+4845) common reverse primer IL-1A-7A (5'-CTCAGCTGGATTGGAATATTCCTAATA-3'), 5 pmol of the IL1B (–511)C primer IL-1B-7A (5'-CCTGCAATTGACAGAGAGCTTCC-3'), 5 pmol of the IL1B (–511)T primer IL-1B-8A (5'-CTCAGAGGCTAATGCAATTGACAGAGAGCACCT-3'), 10 pmol of the IL1B (–511) common reverse primer IL-1B-9B (5'-CGTTGTGCAGTTGATGTCCACATT-3'), 7.25 pmol of the IL1B (–31)C primer IL-1B-2B (5'-CAGTTTCTCCCTCGCTGTTTTTGTG-3'), 8.5 pmol of the IL1B (–31)T primer IL-1B-3B (5'-TGGTATCTGCACGTTTCTCCCTCGCTGTTTTGATA-3'), 15.5 pmol of the IL1B (–31) common reverse primer IL-1B-16A (5'-AACGATTGTCAGGAAAACAATGCATATTTG-3'), 2 pmol of the IL1B (+3954)C primer IL-1B-10A (5'-CTCCACATTTCAGAACCTATCTTCCTC-3'), 5 pmol of the IL1B (+3954)T primer IL-1B-11A (5'CAACATGTGTACCACATTTCAGAACCTATCTTGTTT-3'), 5 pmol of the IL1B (+3954) common reverse primer IL-1B-12B (5'-GAATTAGCAAGCTGCCAGGAG-3'), 6 pmol of the IL1RA (+2018)C primer IL-1RA-1A (5'-CTGAGGAACAACCAACTAGTTTCC-3'), 6 pmol of the IL1RA (+2018)T primer IL-1RA-2A (5'-CCTTCTATGAGAGGAACAACCAACTAGTAGCT-3'), 12 pmol of the IL1RA common reverse primer IL-1RA-3B (5'-CCTCCCAGCTTCAAACTTGAATAC-3'), and 
50 ng of DNA.
Amplifications were performed in a GeneAmp 9700 thermocycler (Applied Biosystems) under the following cycling conditions: denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s, with a final extension step at 72 °C for 7 min. The allele-specific PCR products [305 bp for IL1A (–889)C, 317 bp for IL1A (–889)T, 170 bp for IL1A (+4845)T, 183 bp for IL1A (+4845)G, 279 bp for IL1B (–511)C, 289 bp for IL1B (–511)T, 252 bp for IL1B (–31)C, 262 bp for IL1B (–31)T, 230 bp for IL1B (+3954)C, 240 bp for IL1B (+3954)T, 204 bp for IL1RA (+2018)C, and 212 bp for IL1RA (+2018)T] were separated on PolyNAT 12% S-50 Wide Mini Gels (Elchrom Scientific) by electrophoresis for 2.5 h at 144 V and 55 °C.
Categorical variables (e.g., genotypes) were compared by 
2 test, and Yates correction was applied in case of 2 x 2 tables. Calculations were performed with the SPSS statistical software package, Ver. 12.0 (SPSS Inc.). 2 statistics are given as two-tailed Pearson likelihood ratios for cross-tables.
We genotyped 285 patients and 481 healthy newborns. The frequencies of the IL1A variants [(–889)C/T and (+4845)G/T] as well as the polymorphisms within the IL1B gene [(–511)C/T, (–31)C/T, and (+3954)C/T] did not differ significantly between the patient group and the control population (Table 1
) or between survivors and nonsurvivors. Allele frequencies of all polymorphisms within the control group were similar to previously published results (9)(11)(12).
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Only the distribution of the IL1RA gene polymorphism at position (+2018) differed significantly between patients and controls (Table 1
). Among patients, the rare C/C genotype was more frequent (10.9%) than among the control population (5.4%), yielding a 2-fold increase in the odds [95% confidence interval (CI), 1.1–3.4] for C/C carriers to get meningococcal disease. Heterozygous C/T carriers showed no significant increase in risk (odds ratio = 0.8; 95% CI, 0.6–1.1) compared with T/T carriers. In total, 21 patients suffered fatal outcomes: 14 of them carried the C/T or the C/C genotype, and 7 were homozygous T/T. Among nonsurvivors, carriers of the IL1RA (+2018)C allele (either C/T or C/C genotype) were overrepresented (66.7%) compared with survivors (42.5%), giving a 1.5-fold increased risk for fatal outcome in C-allele carriers (95% CI, 1.1–2.2; P = 0.054, 2 test after Yates correction). Probably because of the small number of nonsurvivors, we found only 1 individual homozygous for IL1RA (+2018)C/C. Of all C-allele carriers, 4.8% (6 of 125) required amputation or major skin transplantation, which was comparable to T/T homozygotes [3.8% (6 of 158); P = 0.54]. Interestingly, the Glasgow Meningococcal Septicaemia Prognostic Score (GMSPS) (13) was higher in C-allele carriers. Whereas only 6.8% (9 of 132) of T/T homozygotes had a score 
8 on admission to the hospital, 19.3% (21 of 109) of all C-allele carriers had scores 8 (P = 0.007).
According to our data, genetic variants within IL-1RA, the physiologic antagonist of proinflammatory IL-1A and IL-1B, might be associated with an increased susceptibility for meningococcal disease. Individuals homozygous for the IL1RA (+2018)C/C genotype had a 2-fold increased risk for suffering meningococcal disease. In contrast to disease susceptibility, the disease outcome seemed to be worse in heterozygous and homozygous IL1RA (+2018)C allele carriers. We did not find any associations between polymorphisms within the IL1B gene and susceptibility or outcome in meningococcal disease (9)(14). Our data are supported by an observation of Santilla et al. (15), who reported that genetic variants within the IL1RA gene are associated with markedly increased IL-1B concentrations, whereas genetic variants within the IL1B gene were of less biological relevance for IL-1B up-regulation.
In summary, we propose that genetic variants within the IL1 cluster modulate the manifestation and outcome of meningococcal infection in a patient population with low prevalence of this disease.
Footnotes
1 G.E. and R.M. contributed equally to this work; 
2 C.M. and W.Z. contributed equally to this work;
References
, interleukin-1 ß, and interleukin-1 receptor antagonist genes. Genomics 1994;19:382-384.[CrossRef][ISI][Medline]
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