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Genomic Polymorphic Profiles in an Irish Population with Meningococcaemia: Is it Possible to Predict Severity and Outcome of Disease?

April 14, 2004

Abstraction Template
     
Key variables & Description Article

Reference
Complete the bibliographic reference for the article according to AJE format.

 

Balding J, Healy CM, Livingstone WJ, et al. Genomic polymorphic profiles in an Irish population with meningococcaemia: is it possible to predict severity and outcome of disease? Genes Immun 2003;4:533-40.

Category of HuGE information
Specify the types of information (from the list below) available in the article:

  1. Prevalence of gene variant
  2. Gene-disease association
  3. Gene-environment interaction
  4. Gene-gene interaction
  5. Genetic test evaluation/monitoring

 

The article includes the prevalence of seven inflammatory cytokine polymorphisms in a Northern Ireland population; gene-disease susceptibility, severity, and outcome associations; and a multipolymorphism analysis.

Study hypotheses or purpose
The authors study hypotheses or main purpose for conducting the study.

 

Cytokines are key regulators of the inflammation response to Neisseria meningitidis endotoxin, and the overall pathophysiology of invasive meningococcal disease (IMD). The study examines the associations between seven cytokine polymorphisms and susceptibility, severity, and outcome of IMD.

Gene(s)
Identification of the following:

  1. Gene name
  2. Chromosome location
  3. Gene product/function
  4. Alleles
  5. OMIM #

 

  1. Gene name: Interleukin 6 (IL-6)
  2. Chromosome location: 7p21
  3. Gene product/function: interleukin 6, a multifunctional cytokine, with proinflammation and procoagulation effects, and roles in regulation of the immune response, and hematopoiesis.
  4. Alleles: IL-6 - 174 G/G, IL-6 - 174 G/C, and IL-6 - 174 C/C
  5. OMIM #: 147620
  1. Gene name: Interleukin 10 (IL-10)
  2. Chromosome location: 1q31-q32
  3. Gene product/function: interleukin 10, an anti-inflammatory cytokine that downregulates proinflammatory cytokines and upregulates IL-1Ra (a receptor antagonist of
    IL-1 ß).
  4. Alleles: IL-10- 1082 G/G, IL-10 - 1082 G/A, and IL-10 - 1082 A/A
  5. OMIM #: 124092
  1. Gene name: Interleukin 1 receptor antagonist (IL-1RN)
  2. Chromosome location: 2q14.2
  3. Gene product/function: interleukin 1 Ra, which binds to IL1 receptors to inhibit IL1- alpha and IL1-ß.
  4. Alleles: IL-1RN 1/1, IL-1RN 1/2, IL-1RN 2/2, IL-1RN 1/3, and IL-1RN 2/3
  5. OMIM #: 147679
  1. Gene name: Interleukin 1ß (IL-1B)
  2. Chromosome location: 2q14
  3. Gene product/function: associated with variations in interleukin 1B levels, which act synergistically with and similar to TNF, causing severe disease via polymorphonulear activity, upregulation of endothelial adhesion molecules, procoagulation, prostaglandin production, cytokine activity, and other proinflammation properties.
  4. Alleles: IL-1B +3953 C/C, IL-1B +3953 C/T, and IL-1B +3953 T/T
  5. OMIM #: 147720
  1. Gene name: Interleukin 10 (IL-10)
  2. Chromosome location 1q31-q32
  3. associated with interleukin 10, an anti-inflammatory cytokine that downregulates proinflammatory cytokines and upregulates IL-1Ra (a receptor antagonist of IL-1ß).
  4. Alleles: IL-10 -592 C/C, IL-10 -592 C/A, and IL-10 -592 A/A
  5. OMIM #: 124092
  1. Gene name: Tumor necrosis factor (TNF)
  2. Chromosome location: 6p21.3
  3. Gene product/function: associated with variations in TNFalpha levels, which act synergistically with and similar to IL-1B, causing severe disease via polymorphonulear activity, upregulation of endothelial adhesion molecules, procoagulation, prostaglandin production, cytokine activity, and other proinflammation properties.
  4. Alleles: TNF -308 G/G, TNF -308 G/A, and TNF -308 A/A
  5. OMIM #: 191160
  1. Gene name: lymphotoxinalpha (LTA)
  2. Chromosome location: 6p21.3
  3. Gene product/function: associated with lymphotoxin alpha (LTA), a proinflammatory cytokine.
  4. Alleles: LTA +252 G/G, LTA +252 G/A, and LTA +252 A/A
  5. OMIM #: 153440

For more information on cytokine gene polymorphisms in human disease, see the University of Bristol database at http://bris.ac.uk/pathandmicro/services/GAI/cytokine4.htm

 

Environmental factor(s)
Identification of the major environmental factors studied (infectious, chemical, physical, nutritional, and behavioral)

 

The major environmental factors in the study are Neisseria meningitidis (a Gram-negative, bacterial pathogen) and its virulence factor, a lipooligosaccharide (LOS) endotoxin. In this study, all cases were exposed because a PCR test for the bacterial citrA gene was a component of the case definition.

Health outcome(s)
Identification of the major health outcome(s) studied

 

The study explores three health outcomes:

  1. Susceptibility to invasive meningococcal disease (IMD) in cases versus controls;
  2. Severity of IMD in mild versus severe groups (as measured by a scoring system of prognostic predictors, additional therapies, clinical indicators, sequelae, and mortality); and
  3. Outcome of IMD, survivors versus nonsurvivors.

 

Study design
Specification of the type of study design(s)
  1. Case-control
  2. Cohort 
  3. Cross-sectional
  4. Descriptive or case series
  5. Clinical trial
  6. Population screening

 

The study has two design components. By comparing genotype and allele frequencies in cases and controls, gene-disease susceptibility association is investigated for the seven cytokine polymorphisms. In addition, a case-series design split cases into groups to analyze the seven cytokine polymorphisms in:

  • Patients with mild invasive meningococcal disease (IMD) and no sequelae vs. severe disease (poor prognostic indicators, sequelae, and death), and
  • IMD survivors vs. nonsurvivors.
Case definition
For study designs 1, 4, and 5, define the following if available:
  1. Disease case definition
  2. Exclusion criteria
  3. Gender
  4. Race/ethnicity
  5. Age
  6. Time period
  7. Geographic location
  8. Number of participants

 

  1. Disease case definition: The study included cases of invasive meningococcal disease (IMD) that were positive for the meningococcal citrA gene by polymerase chain reaction (PCR) analysis of blood, cerebrospinal fluid, or other sterile sites; present in the Irish Meningococcal and Meningitis Reference Laboratory (IMMRL) database; subsequently genotyped; and either sought care at one of two tertiary referral pediatric hospitals or were patients known to have died at another hospital.
  2. Exclusion criteria: All Irish cases of IMD that did not meet the criteria in the above case definition. This could include patients for whom sterile specimens were not obtained, cases of invasive meningococcal disease who were PCR negative, children who sought care for IMD at other hospitals, and adults who survived IMD.
  3. Gender: Males and females. Though gender information is not available, as samples were irreversibly unlinked to these data as a condition of ethical approval of the research, this information could have been tabulated prior to unlinking (as was done for age and other clinical characteristics).
  4. Race/ethnicity: Presumably consistent with the racial and ethnic composition Ireland , predominantly Caucasian (data not available)
  5. Age: 89% are children between 2 months and 15 years of age, with a weighted average age of approximately 22 months (age data on the remainder are not available).
  6. Time period: 89% of cases were obtained from the IMMRL database between January 1997 and February 2000 (remainder data not available).
  7. Geographic location: 89% of cases sought care in one of two Dublin hospitals (remainder data not available).
  8. Number of participants: N= 183 (surveillance data for meningococcal disease* suggest that this is a high percentage of the total eligible, but the actual number of IMD cases is not known).

    * Communicable Disease Surveillance Centre of Northern Ireland

 

Control definition
For study design 1, define the following if available:
  1. Control selection criteria
  2. Matching variables
  3. Exclusion criteria
  4. Gender
  5. Race/ethnicity
  6. Age
  7. Time period
  8. Geographic location
  9. Number of participants
  1. Control selection criteria: Control samples were blood donors, and obtained from the Northern Ireland Blood Transfusion Service (15%) and the Irish Blood Transfusion Service (85%)
  2. Matching variables: none
  3. Exclusion criteria: not applicable
  4. Gender: 58% male, 42% female
  5. Race/ethnicity: Caucasian
  6. Age: Mean age is 37.1 years (age range is 18-65 years).
  7. Time period: not specified
  8. Geographic location: Ireland
  9. Number of participants: 389 (percent of total eligible not known)

 

 

Assessment of environment factors
For studies that include gene-environment interactions, define the following, if available:
  1. Environmental factor
  2. Exposure assessment
  3. Exposure definition
  4. Number of participants with exposure data (%
    of total eligible)

 

  1. Environmental factor: Not applicable
  2. Exposure assessment:
  3. Exposure definition:
  4. Number of participants with exposure data: N (% of total eligible)

 

Genotyping
Specify the following:
  1. Gene
  2. DNA source
  3. Methodology
  4. Number of participants genotyped (% of total eligible) 
  1. Gene: Interleukin 6 (IL-6) G-174C
  2. DNA source: DNA was isolated from case blood samples via the Puregene extraction procedure, and isolated from EDTA anti-coagulated control blood samples via a large-volume extraction protocol.
  3. Methodology: Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays were used to genotype all seven polymorphisms. The PCR reaction mix included Taq DNA polymerase buffer with MgCL 2 , 0.4 U of DNA Taq polymerase, 2 µ l of genomic DNA, 4% DMSO, 30 µM each of dNTPs, and 0.2 µM each of sense and antisense primer. For genotyping the IL-6 G-174C polymorphism, a IL-6 - 174 F and IL-6 - 174 R primers, Hsp92II restriction enzyme, 3% agarose gel with ethidium bromide, and specific cycling parameters (94° C 60 s, 58° C 60 s, 72 ° C 90 s) were used.
  4. Number of participants genotyped: 183 cases (95% of collected samples), 389 controls

 

  1. Gene: Interleukin 10 (IL-10) - 1082
  2. DNA source: as above.
  3. Methodology: As above, except with IL-10 - 1082 F and IL-10 - 1082 R primers, MnlI restriction enzyme, 4% agarose gel with ethidium bromide, specific cycling parameters (94° C 60 s, 58° C 60 s, 72° C 60 s), and no DMSO
  4. Number of participants genotyped: as above.

 

  1. Gene: Interleukin 1 receptor antagonist (IL-1RN) VNTR
  2. DNA source: as above.
  3. Methodology: As above, except with IL-1RN F and IL-1RN R primers and 1 µM each primer , no restriction enzyme, 2% agarose gel with ethidium bromide, specific cycling parameters (94° C 60 s, 58° C 60 s, 72° C 90 s), and 6% DMSO
  4. Number of participants genotyped: as above.

 

  1. Gene: Interleukin 1 ß (IL-1B) +3953
  2. DNA source: as above.
  3. Methodology: As above, except with IL-1B +3953 F and IL-1B +3953 R primers, TaqI restriction enzyme, 3% agarose gel with ethidium bromide, and specific cycling parameters (94° C 60 s, 58° C 60 s, 72° C 60 s).
  4. Number of participants genotyped: as above.

 

  1. Gene: Interleukin 10 (IL-10) -592
  2. DNA source: as above.
  3. Methodology: As above, except with IL-10 -592 F and IL-10 -592 R primers and 1 µM each primer , RsaI restriction enzyme, 3% agarose gel with ethidium bromide, and specific cycling parameters (94° C 60 s, 64° C 60 s, 72 ° C 60 s).
  4. Number of participants genotyped: as above.

 

  1. Gene: Tumor necrosis factor (TNF) -308
  2. DNA source: as above.
  3. Methodology: As above, except with TNF -308 F and TNF -308 R primers, NcoI restriction enzyme, 2% agarose gel with ethidium bromide, and specific cycling parameters (94° C 60 s, 65° C 60 s, 72° C 60 s).
  4. Number of participants genotyped: as above.

 

  1. Gene : lymphotoxin alpha (LTA) +252
  2. DNA source: as above.
  3. Methodology: As above, except with LTA +252 F and LTA +252 R and 1 µM each primer , HinfI restriction enzyme, 2% agarose gel with ethidium bromide, and specific cycling parameters (95° C 30 s, 68° C 30 s, 74° C 42s).
  4. Number of participants genotyped: as above.

 

Results
Describe the major results under each of the following HuGE categories. Include tables when data are provided:
  1. Prevalence of gene variant
  2. Gene-disease association
  3. Gene-environment interaction
  4. Gene-gene interaction
  5. Genetic test evaluation/monitoring


Prevalence of seven inflammatory cytokine polymorphisms: Based on genotyping data from 389 control samples obtained from Irish blood banks, the prevalence of seven cytokine polymorphisms are shown in Table 1. Among alleles for the IL-6 G-174C polymorphism, the heterozygous allele (G/C) was most common (51%). Similarly, the heterozygous allele (G/A) was most common for the IL-10- 1082 polymorphism (46%). Genotypes 1/1 (47%) and 1/2 (41%) were common among the IL-1RN polymorphisms, while the other three genotypes were relatively uncommon. The homozygous C/C variant of the IL-1B +3953 polymorphism was most common (62%); as was the homozygous C/C form of the IL-10 -592 gene (60%) and the homozygous G/G form of the TNF -308 gene (60%). The G/A was the most common variant of the LTA +252 gene (53%).

Susceptibility to invasive meningococcal disease (IMD): In comparing cases to controls, a statistically significant difference was found in the prevalence of the IL-1RN 2/2 genotype, which was identified in 14% of cases and 8% of controls (p=0.033) (Table 2).

Severity of IMD: Cases with severe IMD more often carried the IL-6 - 174 G/G allele (41%) compared to cases with mild disease (26%), a difference with statistical significance (p=0.037). In addition, the IL-10 - 1082 A/A allele was more common in severe IMD cases (29%) than mild cases (13%) (p=0.0078); severe cases were 2.7 times more likely to carry this variant than other variants of the same gene (odds ratio (OR) = 2.7, 95% confidence interval (CI) =2.3 - 3.6) (Table 2).

Outcome of IMD (mortality): The IL-6 - 174 G/G variant, which was associated with severe disease, was also associated with mortality (p=0.023). IMD patients who did not survive were 2.6 times more to carry this genotype (OR=2.64, CI=1.12-6.22). Similarly, in comparing mild IMD to cases of IMD where patients did not survive, there was a strong association with the homozygous G/G allele (p=0.012). The IL-1RN 1/1 and IL-1RN 1/2 genotypes also had significant associations with mortality. Fatal cases of IMD more often carried the 1/1 variant (60% vs. 43% in survivors) and, accordingly, survivors more often had the 1/2 variant (41% versus 20% in nonsurvivors) (p=0.043).

:: Click here to view Table 1 ::

Multipolymorphism analysis: Among persons carrying all three deleterious genotypes ( IL-6 - 174 G/G, IL-10 - 1082 A/A, and IL-1RN 1/1), differences with respect to severity of disease or survival were not found using logistic regression, though this combination was relatively uncommon (data not shown). The two-way combinations of the same genotypes, however, were significant. The combination of IL-6 - 174 G/G and IL-10 - 1082 A/A, while also uncommon, was associated with severity of disease (p=0.016) and the combination of IL-6 - 174 G/G and IL-1RN 1/1 was strongly associated with fatality (p=0.0008)

:: Click here to view Table 2 ::

 

Conclusion
State the author's overall conclusions from the study

The authors conclude that key findings in the study were, in general, statistically significant but not highly statistically significant - thereby necessitating further investigation. They note that severe meningococcemia is certainly multifactorial, and therefore their study should be viewed as a preliminary, exploratory study.

Nevertheless, the data suggest roles for the IL-6 - 174 G/G and IL-10 - 1082 A/A genotypes with disease severity. In addition, the IL-1RN 1/2 and 2/2 variants may be important to disease susceptibility as well as mortality. When in combination, the IL-6 - 174 G/G and IL-10 - 1082 A/A were also related to disease severity, and the IL-6 - 174 G/G and IL-1RN 1/2 were related to mortality. Other polymorphisms related to cytokines, however, did not appear to be related to susceptibility, severity, or outcome of IMD. These include the interleukin 1 ß (IL-1B) +3953, interleukin 10 (IL-10) -592, tumor necrosis factor (TNF) -308, and lymphotoxin alpha (LTA) +252 polymorphisms.

 

Comments
Provide additional insight, including methodologic issues and/or concerns about the study

The authors note that patients who died of invasive meningococcal disease (IMD), but were not captured by the Irish Meningococcal and Meningitis Reference Laboratory (IMMRL) database, were excluded from the study, and therefore could have biased the study results. In fact, the study is focused toward pediatric IMD, as 89% of cases are children between 2 months and 15 years of age. Meningococcal disease, however, not only disproportionately affects children, but also the elderly and persons with complement deficiencies and chronic medical conditions. Therefore, cases in the study may not be representative of all IMD cases in Ireland . Similarly, controls were not population-based; whether persons who donate blood are representative of the general Irish population is not known. Due to selection strategies for cases and controls, these two groups differed markedly with respect to age, their ability to produce antimeningococcal antibodies, and possibly with respect to other factors that relate to the study outcomes.

This exploratory study tests multiple hypotheses. Seven cytokines are compared for three outcomes, such that at least 21 tests of significance are performed. Yet, at the alpha =.0.05 significance level, at least one of these tests might be expected to be significant due to chance alone. The authors reason that potential associations necessarily require interpretation, and note that they provide sufficient information for reader's to calculate adjusted p-values (e.g., Bonferroni's correction). While this is true, the onus of statistical analysis still lies with the authors.

There may be biological plausibility for the study findings, but the cytokine network and its regulatory function in response to meningococcal endotoxin is complex. Untangling the influence of these and other genetically variant cytokine mediators, many of which were not evaluated in this study, will likely be a challenge for years to come. While understanding the role of cytokines may be prove valuable in predicting susceptibility, severity, and outcome of meningococcemia, it is unlikely to be the only useful information. Research related to host characteristics beyond the cytokine network, meningococcal virulence factors, and key genetically-defined invasive clonal groups (e.g., ET-37 and ET-5 complexes, and the A4 cluster) offer equal promise for advancing our understanding of clinical variability in meningococcemia.

 

Last Updated August 24, 2004