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Polymorphisms in the interleukin-1 gene cluster in children and young adults with systemic meningococcemia.

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-a (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 [micro]L 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 [micro]L containing 0.75 U of AmpliTaq Gold (Applied Biosystems), 1.5 mM Mg[Cl.sub.2], 200 [micro]M 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-313 (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-113-7A (5'-CCTGCAATTGACAGAGAGCTTCC-3'), 5 pmol of the IL1B (-511)T primer IL-113-8A (5'-CTCAGA000TAATGCAATTGACAGAGAGCACCT-3'), 10 pmol of the IL1B (-511) common reverse primer IL-113-913 (5'-CGTTGTGCAGTTGATGTCCACATT-3'), 7.25 pmol of the IL1B (-31)C primer IL-113-213 (5'-CAGTTTCTCCCTCGCTGTTTTTGTG-3'), 8.5 pmol of the IL1B (-31)T primer IL-113-313 (5'-TGGTATCTGCACGTTTCTCCCTCGCTGTTTTGATA-3'), 15.5 pmol of the IL1B (-31) common reverse primer IL-113-16A (5'-AACGATTGTCAGGAAAACAATGCATATTTG-3'), 2 pmol of the IL1B (+3954)C primer IL-113-10A (5'CTCCACATTTCAGAACCTATCTTCCTC-3'), 5 pmol of the IL1B (+3954)T primer IL-113-11A (5'CAACATGTGTACCACATTTCAGAACCTATCTTGTTT-3'), 5 pmol of the IL1B (+3954) common reverse primer IL-113-1213 (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-313 (5'-CCT000AGCTTCAAACTTGAATAC-3'), and ~50 ng of DNA.

Amplifications were performed in a GeneAmp 9700 thermocycler (Applied Biosystems) under the following cycling conditions: denaturation at 94[degrees]C for 5 min, followed by 35 cycles at 94[degrees]C for 30 s, 60[degrees]C for 30 s, and 72[degrees]C for 60 s, with a final extension step at 72[degrees]C for 7 min. The allele-specific PCR products [305 by for IL1A (-889)C, 317 by for IL1A (-889)T, 170 by for IL1A (+4845)T, 183 by for IL1A (+4845)G, 279 by for IL1B (-511)C, 289 by for IL1B (-511)T, 252 by for IL1B (-31)C, 262 by for IL1B (-31)T, 230 by for IL1B (+3954)C, 240 by for IL1B (+3954)T, 204 by for IL1RA (+2018)C, and 212 by 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[degrees]C.

Categorical variables (e.g., genotypes) were compared by [chi square] 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.). [chi square] 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).

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, [chi square] 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 [greater than or equal to]_8 on admission to the hospital, 19.3% (21 of 109) of all C-allele carriers had scores [greater than or equal to] 8 (P = 0.007).

According to our data, genetic variants within IL-1RA, the physiologic antagonist of proinflammatory IL-1A and IL-113, 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-113 concentrations, whereas genetic variants within the IL1B gene were of less biological relevance for IL-113 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.

References

(1.) Riordan FA, Marzouk O, Thomson AP, Sills JA, Hart CA. The changing presentations of meningococcal disease. Eur J Pediatr 1995;154:472-4.

(2.) Toews WH, Bass JW. Skin manifestations of meningococcal infection; an immediate indicator of prognosis. Am J Dis Child 1974;127:173-6.

(3.) Bone RC. The pathogenesis of sepsis. Ann Intern Med 1991;115:457-69.

(4.) Read RC, Pullin J, Gregory S, Borrow R, Kaczmarski EB, di Giovine FS, et al. A functional polymorphism of toll-like receptor 4 is not associated with likelihood or severity of meningococcal disease. J Infect Dis 2001;184: 640-2.

(5.) Westendorp RG, Langermans JA, Huizinga TW, Elouali AH, VerweiJ CL, Boomsma DI, et al. Genetic influence on cytokine production and fatal meningococcal disease. Lancet 1997;349:170-3.

(6.) Geishofer G, Binder A, Muller M, Zohrer B, Resch B, Muller W, et al. 4G/5G promoter polymorphism in the plasminogen-activator-inhibitor-1 gene in children with systemic meningococcaemia. Eur J Pediatr 2005;164:486-90.

(7.) Dinarello CA. Interleukin-1, interleukin-1 receptors, and interleukin-1 receptor antagonist. Int Rev Immunol 1998;16:457-99.

(8.) Nicklin MJ, Weith A, Duff GW. A physical map ofthe region encompassing the human interleukin-1 a, interleukin-1 [beta], and interleukin-1 receptor antagonist genes. Genomics 1994;19:382-4.

(9.) Read RC, Cannings C, Naylor SC, Timms JM, Maheswaran R, Borrow R, et al. Variation within genes encoding interleukin-1 and the interleukin-1 receptor antagonist influence the severity of meningococcal disease. Ann Intern Med 2003;138:534-41.

(10.) Faber J MC, Gemmer C, Russo A, Finn A, Murdoch C, Zenz W, et al. Human toll-like receptor 4 mutations are associated with susceptibility to invasive meningococcal disease in infancy. Pediatr Infect Dis J 2006;25:80-1.

(11.) Campos MI, Santos MC, Trevilatto PC, Scarel-Caminaga RM, Bezerra FJ, Line SR. Evaluation of the relationship between interleukin-1 gene cluster polymorphisms and early implant failure in non-smoking patients. Clin Oral Implants Res 2005;16:194-201.

(12.) Latkovskis G, Licis N, Kalnins U. C-reactive protein levels and common polymorphisms of the interleukin-1 gene cluster and interleukin-6 gene in patients with coronary heart disease. EurJ Immunogenet 2004;31:207-13.

(13.) Reilly PL, Simpson DA, Sprod R, Thomas L. Assessing the conscious level in infants and young children: a paediatric version of the Glasgow Coma Scale. Childs Nerv Syst 1988;4:30-3.

(14.) Read RC, Camp NJ, di Giovine FS, Borrow R, Kaczmarski EB, Chaudhary AG, et al. An interleukin-1 genotype is associated with fatal outcome of meningococcal disease. J Infect Dis 2000;182:1557-60.

(15.) Santtila S, Savinainen K, Hurme M. Presence of the IL-IRA allele 2 (IL-1RN*2) is associated with enhanced IL-1[beta] production in vitro. Scand J Immunol 1998;47:195-8.

DOI: 10.1373/clinchem.2005.058537

Georg Endler, [3] ([dagger]) Rodrig Marculescu, [3] + Philipp Starkl, [3] Alexander Binder, [1] Gotho Geishofer, [1] Martin Muller, [1] Bettina Zohrer, [1] Bernhard Resch, [2] Werner Zenz, [1] ([dagger]) and Christine Mannhalter, [3] ([double dagger]) for the Central European Meningococcal Genetic Study Group (Departments of [1] General Pediatrics and [2] Neonatology, Medical University of Graz, Graz, Austria; [3] Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Vienna, Vienna, Austria; ([dagger]) G.E. and R.M. contributed equally to this work; ([double dagger]) C.M. and W.Z. contributed equally to this work; * address 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@meduniwien.ac.at)
Table 1. Genotype and allele frequencies
in patients and controls.

 Controls All patients
Genotype (n = 481) (n = 285) P (a)

IL1A (-889), n (%)
 C/C 248 (51.6) 134 (47.0)
 C/T 163 (33.9) 116 (40.7)
 T/T 70 (14.6) 35 (12.3) 0.16
Allele frequency, %
 C 69 67
 T 31 33 0.21
IL1B (-889), n (%)
 C/C 210 (43.7) 125 (43.9)
 C/T 205 (42.6) 125 (43.9)
 T/T 66 (13.7) 35 (12.3) 0.84
Allele frequency, %
 C 75 76
 T 35 34 0.67
IL1B (-31), n (%)
 C/C 68 (14.1) 36 (12.6)
 C/T 203 (42.2) 124 (43.5)
 T/T 210 (43.7) 125 (43.9) 0.83
Allele frequency, %
 C 35 34
 T 65 66 0.78
IL1B (+3954), n (%)
 C/C 272 (56.5) 161 (56.5)
 C/T 179 (37.2) 107 (37.5)
 T/T 30 (6.2) 17 (6.0) 0.99
Allele frequency, %
 C 75 76
 T 25 24 0.99
IL1RA (+2018), n (%)
 C/C 26 (5.4) 31 (10.9)
 C/T 195 (40.5) 95 (33.3)
 T/T 260 (54.1) 159 (55.8) 0.008
Allele frequency, %
 C 27 27
 T 73 73 0.45
IL1RA (+4845), n (%)
 G/G 230 (47.8) 133 (46.7)
 G/T 153 (31.8) 111 (38.9)
 T/T 98 (20.4) 41 (14.4) 0.044
Allele frequency, %
 G 63 66
 T 37 34 0.37

 Patients

 Survivors Nonsurvivors
Genotype (n = 264) (n = 21) P (a)

IL1A (-889), n (%)
 C/C 124 (47.0) 10 (47.6)
 C/T 106 (40.2) 10 (47.6)
 T/T 34 (12.9) 1 (4.8) 0.52
Allele frequency, %
 C 67 72
 T 33 28 0.66
IL1B (-889), n (%)
 C/C 117 (44.3) 8 (38.1)
 C/T 115 (43.6) 10 (47.6)
 T/T 32 (12.1) 3 (14.3) 0.85
Allele frequency, %
 C 66 62
 T 34 38 0.7
IL1B (-31), n (%)
 C/C 32 (12.1) 4 (19.0)
 C/T 115 (43.6) 9 (42.9)
 T/T 117 (44.3) 8 (37.1) 0.63
Allele frequency, %
 C 34 40
 T 66 60 0.49
IL1B (+3954), n (%)
 C/C 148 (56.1) 13 (61.9)
 C/T 100 (37.9) 7 (33.3)
 T/T 16 (6.1) 1 (4.8) 0.87
Allele frequency, %
 C 75 78
 T 25 22 0.75
IL1RA (+2018), n (%)
 C/C 30 (11.4) 1 (4.8)
 C/T 82 (31.1) 13 (61.9)
 T/T 152 (57.6) 7 (33.3) 0.015
Allele frequency, %
 C 27 36
 T 73 64 0.29
IL1RA (+4845), n (%)
 G/G 124 (47.0) 9 (42.9)
 G/T 101 (38.3) 10 (47.6)
 T/T 39 (14.8) 2 (9.5) 0.64
Allele frequency, %
 G 76 77
 T 34 33 0.99

(a) P values were calculated by [chi square] tests for
comparing patients vs controls (left-hand columns) or
survivors vs nonsurvivors of meningococcal disease
(right-hand columns).
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Title Annotation:Technical Briefs
Author:Endler, Georg; Marculescu, Rodrig; Starkl, Philipp; Binder, Alexander; Geishofer, Gotho; Muller, Mar
Publication:Clinical Chemistry
Date:Mar 1, 2006
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