Full text of DOI:10.1086/315889

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557
CONCISE COMMUNICATION
An Interleukin-1 Genotype Is Associated with Fatal Outcome
of Meningococcal Disease
1
1,a
1
Robert C. Read, Nicola J. Camp,
Division of Genomic Medicine, University of Sheffield
2
1
2
Medical School, Sheffield, and Meningococcal Reference Unit,
Manchester Public Health Laboratory, Withington Hospital,
Manchester, United Kingdom
Franco S. di Giovine, Raymond Borrow,
Edward B. Kaczmarski, Adeel G. A. Chaudhary,
Andrew J. Fox, and Gordon W. Duff
2
1
2
1
To determine whether known variants of the interleukin-1 (IL-1) and tumor necrosis factor
TNF) gene families are associated with severe manifestations of meningococcal disease, 276
(
white patients 4–70 years of age (median, 17 years) were genotyped. All patients had micro-
biologically proven Neisseria meningitidis infection; 39 died and 237 survived. A significant
association (P ! .001) was found between fatal outcome and genotype at IL1B (nucleotide
position Ϫ511). Homozygous individuals, both for the common (1/1) and the rare (2/2) alleles,
had increased odds ratios (ORs) for death, compared with heterozygous individuals (1/2):
ORs (95% confidence intervals [CIs]) were 3.39 (1.39–8.29) and 7.35 (2.51–21.45), respectively.
The mortality rates according to genotype at IL1B (Ϫ511) were 18.0% (1/1), 6.1% (1/2), and
32.3% (2/2), compared with 14.2% overall. The composite genotype, consisting of heterozy-
gosity of IL1B (Ϫ511) together with homozygosity of the common allele of the IL-1 receptor
antagonist gene (IL1RN) at +2018, was significantly associated with survival (P p .018; OR,
7.78 [95% CI, 1.05–59.05]). There was no association between TNF genotype and fatal out-
come. These data suggest that IL-1 genotype influences the severity of meningococcal disease.
The death rate from meningococcal disease is 5%–20%, de-
example, TNFA has a biallelic polymorphism at position Ϫ308
(i.e., 308 nt upstream of the transcriptional start), which is
associated with a higher rate of transcription of TNF [3]. Like-
wise, there are SNPs within IL1A at position ϩ4845 [4] (i.e.,
4845 nt downstream of the transcriptional start) and within
IL1B at positions ϩ3954 [5] and Ϫ511 [6]. The polymorphism
at position Ϫ511 is in 99.5% linkage disequilibrium with an-
other at position Ϫ31 that has been shown to affect the tran-
scription-initiation complex of IL1B and is associated with in-
creased risk of hypochlorhydria and gastric cancer in patients
infected with Helicobacter pylori [7]. In addition, there is a
variable-number tandem repeat in IL1RN, of which the less
common allele 2 (IL1RN∗2) is also associated with an adverse
outcome of Helicobacter infection [7]. This allele is in linkage
disequilibrium with several polymorphisms in IL1RN, includ-
ing one at position ϩ2018 within exon 2, which was previously
denoted “(ϩ8006)” [8].
spite antimicrobial therapy and intensive care treatment. The
host’s cytokine response, which is genetically determined, re-
flects the severity of the disease [1]. The cytokines tumor ne-
crosis factor (TNF)–a and interleukin (IL)–1 are key mediators
of the inflammatory response. Two genes, IL1A and IL1B, lo-
cated on human chromosome 2q13 encode the proinflamma-
tory cytokines IL-1a and IL-1b, respectively, and a third gene,
IL1RN, encodes the IL-1 receptor antagonist (IL-1ra) [2].
Single-nucleotide polymorphisms (SNPs) are substitutions of
a single base at a particular site. These are stably carried by a
proportion of the population and have the potential to affect
the protein product of the gene to which they are related. For
Received 5 June 2000; revised 19 July 2000; electronically published 9
October 2000.
Presented in part: 39th Interscience Conference on Antimicrobial Agents
and Chemotherapy, San Francisco, 26–29 September 1999 (abstract 496).
This study was approved by the Research Ethics Committee of the Public
Health Laboratory Service for England and Wales.
To test whether known polymorphisms within the IL-1 and
TNF genes are related to the outcome of meningococcal dis-
ease, we compared their distributions among DNA samples
derived from survivors and fatal cases drawn from the popu-
lations of England and Wales.
Financial support: Special Trustees for the Former United Sheffield Hos-
pitals (grant 87753); Ralph Sutcliffe Fund; National Meningitis Trust.
a
Present affiliation: Genetic Research, Intermountain Health Care, Salt
Lake City, Utah.
Reprints or correspondence: Dr. Robert C. Read, Infection andImmunity,
Division of Molecular and Genetic Medicine, F Fl., University of Sheffield
Medical School, Beech Hill Rd., Sheffield S10 2RX, United Kingdom
Methods
(r.c.read@sheffield.ac.uk).
Patients. Since October 1996, the Meningococcal Reference
Unit (MRU) for England and Wales has offered a nationwide
service for the detection of Neisseria meningitidis by polymerase
The Journal of Infectious Diseases 2000;182:1557–60
2000 by the Infectious Diseases Society of America. All rights reserved.
022-1899/2000/18205-0040$02.00
᭧
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Read et al.
JID 2000;182 (November)
chain reaction (PCR) in blood or cerebrospinal fluid (CSF) samples,
in addition to strain characterization in culture-proven cases and
meningococcal serology. From January 1997 to June 1998, we ar-
chived consecutively received whole-blood samples from white pa-
tients 4–70 years of age who had had meningococcal disease. Sam-
ples were included in the study if meningococcal disease was verified
by culture or PCR detection of N. meningitidis in blood or CSF.
Once clinical information had been collated, samples were coded
so that individuals could no longer be identified.
survived were B (91 patients [39%]), C (104 patients [44%]),
W135 (2 patients [1%]), and ungrouped (40 patients [17%]).
Genotype distributions. The distribution of gene polymor-
phisms among subjects with meningococcal disease are shown
in table 1. Allelic distributions for IL1A (ϩ4845), IL1B (ϩ3954),
and TNFA (Ϫ308) were similar. However, IL1B (Ϫ511) geno-
types were significantly different between the groups (P ! .001).
Individuals homozygous (1/1 or 2/2) at IL1B (Ϫ511) were more
likely to die from meningococcal disease, and heterozygotes were
more likely to survive. The percentage of deaths in genotype
groups 1/1, 1/2, and 2/2 were 18.0%, 6.1%, and 32.3%, respec-
tively, and the ORs for death of individuals homozygous for
allele 1 or 2 (1/1 and 2/2), compared with the ORs for hetero-
zygous individuals, were 3.39 (95% confidence interval [CI],
Laboratory methods. DNA was extracted by standard methods.
We probed for 5 SNPs: IL1A (ϩ4845), IL1B (ϩ3954), IL1B (Ϫ511),
0
IL1RN (ϩ2018), and TNFA (Ϫ308) using a validated 5 nuclease
assay (TaqMan allelic discrimination test; Perkin Elmer Biosystems,
Foster City, CA) [7]. Probes, primer sequences, and cyclingconditions
are available in an Appendix published only in the electronic edition
of the Journal (http://www.journals.uchicago.edu/JID/). We indicate
the frequent allele of each gene variant as “1” and the rarer allele
as “2,” as is the standard nomenclature. Homozygosity is indicated
as “1/1” or “2/2,” and heterozygosity is indicated as “1/2.” Carriage
of one particular allele is indicated, for example, as “2ϩ” for the
rarer allele, meaning individuals who are either 2/2 or 1/2.
1.39–8.29) and 7.35 (95% CI, 2.51–21.48), respectively.
We also observed an increased, but not significant, risk of
death for individuals carrying the rare allele (2ϩ) of IL1RN
ϩ2018): Mortality rates were 11.3% and 17.3% for 1/1 and
ϩ, respectively. Composite genotype analysis was therefore
(
2
done over IL1B (Ϫ511) and IL1RN (ϩ2018). Table 2 illustrates
Statistical analysis. Heterozygous and homozygous odds ratios
2
(ORs) for survival and death were calculated for each locus as
the 2 ϫ 6 contingency table that was generated. A x analysis
estimates of the genotypic relative risks [9]. Subsequently, data were
either analyzed by x analysis in a 2 ϫ 3 genotype table or a
(5 df) illustrated significant evidence for an association between
the composite genotype over these loci and risk of death or
survival (P ! .001). For each genotype combination across these
two loci, the mortality rate was higher when the individual was
carrying the rare allele at IL1RN (ϩ2018) (table 2). The lowest
mortality rate (2.4%) was for the composite genotype 1/2 at
IL1B (Ϫ511) and 1/1 at IL1RN (ϩ2018), and the highest mor-
tality rate (42.1%) was for the composite 2/2 and 2ϩ. The OR
for mortality, when compared between these two genotype com-
binations only, was extremely strong (OR, 29.09 [95% CI,
3.28–258.21]; P ! .001).
2
2
ϫ 2 table for carriage of a specific allele. Composite genotype
analyses over multiple loci were also done where appropriate.
Monte Carlo techniques were used to calculate empirical P values
[
10] (Monte Carlo composite genotype method [MCCG]) when the
2
standard x test on the relevant contingency table was invalid be-
cause of small expected values. A study-wide significance level of
.01 was considered a reasonable threshold and is probably con-
servative, since we have previously shown a high degree of linkage
disequilibrium in this region [11] and, therefore, individual tests
are not strictly independent.
This increased risk of mortality was also observed when the
“
protective” composite (1/2 and 1/1) was compared against all
other groups of genotype combinations together (OR, 7.78
95% CI, 1.05–59.05]; MCCG P p .0183); only 1 (2.4%) of 41
patients with this composite genotype had died versus 38
16.8%) of 226 who did not have this genotype. We found no
Results
[
Patients and microbiologic data. During the study period,
(
1
120 microbiologically proven cases of meningococcal disease
in England and Wales were verified, among patients 4–70 years
of age, by the MRU. Over the same period, whole-blood sam-
ples for PCR detection of N. meningitidis were received from
Table 1. Genotype distributions for the 5 markers tested in the
tumor necrosis factor (TNF) gene TNFA and in the interleukin (IL)–1
genes IL1A, IL1B, and IL1RN.
5
075 patients in this age group with suspected meningococcal
No. (%) of patients genotyped, by marker
infection. Of these, we identified 276 with culture- or PCR-
proven meningococcal disease. Of the DNA samples, 39 were
from patients who had died, and 237 were from patients who
had survived. The mean age of patients who had died
Patient group,
genotype
TNFA
(Ϫ308)
IL1A
(ϩ4845)
IL1B
(ϩ3954)
IL1B
(Ϫ511)
IL1RN
(ϩ2018)
Deceased (n p 39)
1
1
2/2
/1
/2
27 (71)
11 (29)
0
17 (45)
18 (47)
3 (9)
21 (55)
15 (39)
2 (5)
22 (56)
7 (18)
10 (26)
16 (41)
18 (46)
5 (13)
(
26.4 ע 19.7 years) was not significantly greater than that for
those who had survived (20.6 ע 14.9 years; P p .2, Mann-
Whitney U test). The median age in both cases was 17 years.
The serogroups of N. meningitidis detected by culture or PCR
of samples from patients who had died were B (11 patients
Survivors (n p 237)
1
1
2/2
/1
/2
135 (65)
67 (32)
5 (3)
112 (57)
87 (39)
22 (10)
.458
123 (57)
78 (36)
14 (7)
.050
100 (44)
108 (47)
21 (9)
15.632
.0005
125 (53)
86 (37)
24 (10)
1.982
2
x
.488
.485
[
28%]), C (20 patients [51%]), W135 (1 patient [3%]), and un-
P
.499
.823
.159
grouped (7 patients [18%]). The serogroups from those who had

JID 2000;182 (November)
IL-1 Genotype and Meningococcal Disease
1559
Table 2. Composite genotype analysis of the interleukin (IL)–1 gene polymorphisms IL1B
(Ϫ511) and IL1RN (ϩ2018).
No. (%) of patients, by composite genotype
a
IL1B (Ϫ511) and IL1RN (ϩ2018)
1/1
1/2
2/2
Total no.
Patient group
1/1
2ϩ
1/1
2ϩ
1/1
2ϩ
of patients
Deceased
Survivors
Total
13 (33)
72 (31.6)
85
9 (23)
27 (12)
36
1 (2.6)
40 (17.5)
41
6 (15.4)
68 (29.8)
74
2 (5)
10 (44)
12
8 (20.4)
11 (48)
19
39
228
267
14.6
Mortality rate, %
15.3
25.0
2.4
8.1
16.7
42.1
2
NOTE. P p .0005, x , 5 df.
a
Upper line (1/1, 1/2, and 2/2) is IL1B (Ϫ511), and lower line (1/1 and 2ϩ) is IL1RN (ϩ2018). 1, Carriage
of frequent allele; 2, carriage of rare allele; 2ϩ, carriage of rare allele of IL1RN (ϩ2018) (either homozygous
or heterozygous).
effect of age, sex, or year of presentation on genotype distri-
bution, at any of the loci tested, among those who had survived
and those who had died.
For technical reasons, not all samples were successfully geno-
typed: Of the 276 samples analyzed, 31 could not be genotyped
for TNFA (Ϫ308), 17 for IL1A (ϩ4845), 23 for IL1B (ϩ3954),
important determinant of IL-1 activity. Although this work dem-
onstrates a significant association of IL1B (Ϫ511) with death
from meningococcal disease, it is intriguing that persons ho-
mozygous for both the common and the rare alleles appear to
be at increased risk. This is a surprising observation and should
be confirmed in a larger study. It is possible that the advantage
for persons heterozygous at IL1B (Ϫ511) is based on a gene dose
effect on the early IL-1 response to N. meningitidis infection. The
IL-1b:IL-1ra molar ratio in heterozygotes may be neither too
great nor too small to overcome meningococcal infection without
contributing to dangerous metabolic disruption. Theinvolvement
of homozygosity at IL1RN (ϩ2018) in a “protective” composite
genotype may reflect the fact that variation within IL1RN has
been shown to enhance IL-1b release in vitro [13].
8
for IL1B (Ϫ511), and 2 for IL1RN (ϩ2018). Of the 276 blood
samples analyzed, 28 (26 survivors, 2 deaths) were collected
from a single infectious disease facility. To investigate the pos-
sibility of false-positive results due to population stratification,
we reanalyzed the data after exclusion of this cohort. All sig-
nificant associations remained.
Discussion
Other cytokine gene polymorphisms may also contribute to
the severity or outcome of meningococcal disease. Nadel et al.
In patients with meningococcal disease, we observed that
those heterozygous at IL1B (Ϫ511) were more likely than ho-
mozygotes to survive (OR, 4.08 [95% CI, 1.73–9.62]; P ! .001).
The mortality rate for individuals with genotype 1/2 was 6.1%,
compared with 20.9% for those who were homozygous. The
polymorphism at position Ϫ511 is a substitution of cytosine
by thymidine; 43.5% of individuals in a northern England pop-
ulation are heterozygous at this position. We also observed an
association between fatal outcome and composite genotype
over IL1B (Ϫ511) and IL1RN (ϩ2018) (P ! .001). The com-
posite genotype consisting of heterozygosity at IL1B (Ϫ511)
plus homozygosity for the common allele at IL1RN (ϩ2018)
appeared to be protective against death in patients with me-
ningococcal disease (OR, 7.78 [95% CI, 1.05–59.95]; MCCG
P p .0183). This composite was present in 16.8% of the sur-
vivors but in only 2.4% of the patients who had died.
[
1
14] studied 98 infants and children with meningococcal disease,
8 of whom died. Among the children who carried the rarer
allele of TNFA (Ϫ308), there were more deaths (OR, 2.5,
P p .03) and a higher incidence of severe disease (OR, 1.6,
P p .02). Our study of a different and older population did
not confirm this association and included sufficient numbers of
patients to detect a 1.2 increase in allelic frequencies (of this
polymorphism), with a power of 80%. Severity of meningo-
coccal disease is likely to be under polygenic influence. Vari-
ations in the gene encoding plasminogen-activator–inhibitor–1
are also associated with severity of meningococcal sepsis [15].
Some caution should be applied to the interpretation of data
derived from specimens referred to a reference laboratory rather
than from a population-based sample. We were careful to in-
clude only DNA from patients with microbiologically proven
disease, but this may have introduced bias caused by an effect
of cytokine genotype on diagnostic yield (unlikely) or some
other unidentified effect related to referral of the sample. The
only outcome variables we sought to identify were death and
survival, as these are easily defined and verifiable, but we ac-
knowledge that sociomedical factors, such as access to medical
care, inevitably influence individual clinical outcomes. We geno-
typed patients between the ages of 4 and 70 years, to exclude
The potential importance of IL1B and IL1RN polymorphisms
in other infectious diseases, notably periodontitis [12] and infec-
tion by H. pylori [7], has been demonstrated. The same (or linked)
proinflammatory polymorphisms at IL1B (Ϫ511) and IL1RN
have been shown to be involved in the interaction between hy-
pochlorhydria, gastric cancer, and infection by H. pylori [7]. IL-
1b and IL-1ra are proinflammatory and anti-inflammatory, re-
spectively, in vivo, and the IL-1b:IL-1ra molar ratio is the most

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Read et al.
JID 2000;182 (November)
4
. van den Velden PA, Reitsma PH. Amino acid dimorphism in IL-1A is de-
tectable by PCR amplification. Hum Mol Genet 1993;2:1753.
. Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup J. A Taq I polymorphism
in the human interleukin IL-1b gene correlates with secretion in vitro. Eur
J Clin Invest 1992;22:396–402.
patients at the extremes of age who may have had features that
distort the influence of genetic factors on outcome. In the
United Kingdom during 1995–1998, 3.2% of identified cases
were 170 years of age, but this group represented 10% of deaths
from meningococcal disease, a discrepancy that increases with
age. Patients !4 years of age represented 43% of cases but only
5
6
. di Giovine FS, Takhsh E, Blakemore A, Duff GW. Single-base polymorphism
at Ϫ511 in the human interleukin-1b gene (IL-b). Hum Mol Genet 1992;1:
450.
∼
32% of deaths (E.B.K., unpublished observations derived
7
. El-Omar EM, Carrington M, Chow WH, et al. Interleukin-1 polymorphisms
associated with increased risk of gastric cancer. Nature 2000;404:398–402.
. Clay FE, Tarlow JK, Cork MJ, Cox A, Nicklin MJH, Duff GW. Novel
interleukin-1 receptor antagonist exon polymorphisms and their use in
allele-specific mRNA assessment. Hum Genet 1996;97:723–6.
from data from the Public Health Laboratory Service).
In summary, our data suggest that IL-1 allelic variants may
be important disease modifiers of meningococcal disease, and
polymorphisms of the IL-1 genes may be among a number of
genetic markers of poor prognosis in N. meningitidis infection.
8
9. Schaid DJ, Sommer SS. Genotype relative risks: methods for design and analysis
of candidate-gene association studies. Am J Hum Genet 1993;53:1114–26.
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We thank Michael McKendrick, Stephen Green, John Anson, Si-
mone Gregory, and Pauline Whitaker for their clinical, laboratory, or
administrative expertise.
11. Cox A, Camp NJ, Nicklin MJH, di Giovine FS, Duff GW. An analysis of
linkage disequilibrium in the interleukin-1 gene cluster, using a novel group-
ing method for multiallelic markers. Am J Hum Genet 1998;62:1180–8.
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2. Kornman KS, Crane A, Wang HY, et al. The interleukin-1 gene type as a
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