Differential Distribution of Allelic Variants in Cytokine Genes among African Americans and White Americans

Roberta B. Ness , Catherine L. Haggerty, Gail Harger and Robert Ferrell

From the Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA.

Received for publication March 19, 2004; accepted for publication June 3, 2004.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Racial disparities in health are largely unexplained. Because many diseases causing premature mortality among African Americans are mediated by the immune system, the authors explored the race-specific distribution of allelic variants in cytokine genes known to stimulate inflammation. The authors studied women seeking prenatal care and delivering singletons in uncomplicated first births at a US hospital in 1997–2001. A total of 179 African-American women and 396 White women were evaluated for functionally relevant allelic variants in cytokine genes. African-American women were significantly more likely to carry allelic variants known to up-regulate proinflammatory cytokines; odds ratios increased with allele dose. Odds ratios for African Americans versus Whites in genotypes up-regulating proinflammatory interleukin (IL) 1 (IL1A-4845G/G, IL1A-889T/T, IL1B-3957C/C, and IL1B-511A/A) ranged from 2.1 to 4.9. The proinflammatory cytokine interleukin-6 IL6-174 G/G genotype was 36.5 times (95% confidence interval (CI): 8.8, 151.9) more common among African Americans. Genotypes known to down-regulate the antiinflammatory interleukin-10 (IL10-819 T/T and IL10-1082 A/A) were elevated 3.5-fold (95% CI: 1.8, 6.6) and 2.8-fold (95% CI: 1.6, 4.9) in African Americans. Cytokine genotypes found to be more common in African-American women were consistently those that up-regulate inflammation.

continental population groups; cytokines; polymorphism (genetics)

Abbreviations: Abbreviations: CI, confidence interval; IL, interleukin; IL1A, interleukin-1{alpha}; IL1B, interleukin-1ß; IL6, interleukin-6; IL10, interleukin-10; IL18, interleukin-18; OR, odds ratio; Th-1, T-helper type 1; TNFA, tumor necrosis factor-{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Racial disparities in health are ubiquitous (13) yet poorly understood. Health behaviors, socioeconomic status, access to health care, and community-based factors explain some but not all of the racial differences (46). Genetic explanations have been largely rejected, because genetic polymorphisms tend to be more variable within-race than between-race (7) and because, to date, few studies have demonstrated racial differences in allelic frequencies for multiple genes affecting a single biologic mechanism such that these multiple small differences might accumulate to produce a biologically plausible effect (8).

Inflammation is a pathophysiology common to several disorders, such as preterm birth and cardiovascular disease, each of which is known to contribute disproportionately to the burden of mortality borne by African Americans (911). Inflammation also mediates autoimmunity and graft rejection, which are more common in African Americans (12, 13). Cytokines are key mediators of the inflammatory response, and collectively they are likely to influence its robustness (14).

We hypothesized that African Americans would be more likely than White Americans to carry allelic variants demonstrated to increase production by stimulated lymphocytes/macrophages of the proinflammatory cytokines interleukin (IL)-1{alpha} (IL1A) (-889*T), interleukin-1ß (IL1B) (-3957*C and -511*A), interleukin-6 (IL6) (-174*G), interleukin-18 (IL18) (-137*G), and tumor necrosis factor-{alpha} (TNFA) (-308*A) and known to down-regulate the inflammation-modulating cytokine interleukin-10 (IL10) (-819*T and -1082*A) (1522).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Healthy primiparous women aged 14–44 years who were seeking prenatal care prior to 20 weeks’ gestation at Magee-Womens Hospital in Pittsburgh, Pennsylvania, in 1997–2001 were approached for participation in a cohort study that followed them through delivery. The study was approved by the hospital’s institutional review board, and all women signed informed consent forms. Of the 6,444 women screened for possible enrollment, 2,399 (37.2 percent) were ineligible because of maternal age, gestational age, planned delivery at another hospital, or not being pregnant. Of the 4,045 women eligible for participation, 2,891 (71.5 percent) were enrolled. An additional 680 women were excluded from analysis because they delivered at another hospital (n = 173), had a spontaneous abortion (n = 194), terminated the pregnancy or had an ectopic pregnancy (n = 71), were determined after enrollment to be ineligible (n = 26), rescinded consent (n = 94), or were lost to follow-up (n = 122). Of the 2,211 women included in the study, 867 African-American or non-Hispanic White women had a full-term singleton during an uncomplicated first delivery. Plasma was available and of sufficient quantity for 575 of these women, and they were the subjects of this analysis. In comparison with all primiparous women enrolled, those who had plasma analyzed were not significantly different (at p < 0.10) with respect to age, prepregnancy body mass index (weight (kg)/height (m)2), current tobacco smoking, education, or race. Compared with all women enrolled (primiparous or multiparous), those with plasma analyzed differed significantly only by age.

Data collection
All participants completed a baseline interview that included collection of information on sociodemographic characteristics and medical, gynecologic, and reproductive history. All but 1.8 percent of women enrolled in this study identified themselves as Black or non-Hispanic White, reflecting the demographic profile of Pittsburgh. Adjudication of whether a woman had had an uncomplicated, full-term, singleton delivery was determined by a jury of at least three clinicians on the basis of a thorough review of the antepartum and intrapartum medical records.

Whole blood samples collected in 5 percent ethylenediaminetetraacetic acid were obtained at or around the time of delivery, processed within 2 hours, and stored at –70°C. High-molecular-weight DNA was isolated using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, Minnesota). The cytokine polymorphisms of interest were selected on the basis of prior evidence of functionality or linkage disequilibrium with a functional cytokine variant. All single nucleotide polymorphisms were assayed for genotype using the 5'-nuclease assay and the TaqMan protocol (Applied Biosystems, Inc., Forest City, California). Each assay was designed using the Applied Biosystems Assays-on-Demand service (Applied Biosystems, Inc.), and the fidelity of the assays was confirmed through resequencing of samples of known genotypes. A 5 percent random sample from each plate was independently genotyped for quality control.

Statistical analyses
Allele frequencies were generated and tested for fit to the expectations of Hardy-Weinberg equilibrium using the chi-squared goodness-of-fit statistic. Descriptive analyses were conducted comparing White and African-American women with respect to sociodemographic and behavioral characteristics. Contingency tables were constructed to compare frequencies of the candidate genotypes between African Americans and Whites. Chi-squared statistics or Fisher’s exact tests were used to test the null hypothesis of independence in each contingency table. Because the IL1A/IL1B and IL10 polymorphisms of interest each occur within relatively small genomic regions, multilocus haplotypes were tested for linkage disequilibrium as measured by their pairwise D' values using the estimated haplotype program. Loci in strong linkage disequilibrium were combined into haplotypes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We compared 179 African-American women with 396 White women. Black women were younger, heavier, less likely to smoke, and of lower socioeconomic status (table 1).


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TABLE 1. Demographic characteristics of pregnant Black women and White women, Pittsburgh, Pennsylvania, 1997–2001
 
Allelic distributions were characterized in genes regulating the proinflammatory cytokines IL1A, IL1B, IL6, IL18, and TNFA and the inflammatory-modulating cytokine IL10 (table 2). The down-regulating IL10-819 T/T genotype was 3.5 times (95 percent confidence interval (CI): 1.8, 6.6) more common and the down-regulating IL10-1082 A/A genotype was 2.8 times (95 percent CI: 1.6, 4.9) more common in African-American women than in White women. Moreover, the probability of carrying the -819T and -1082A alleles was dose-dependent, with carriage of one allele 1.5 and 1.4 times more common and carriage of two alleles 3.5 and 2.8 times more common in African-American women than in White women, respectively. That is, African-American women had a statistically significant genetic susceptibility to lower production of the immune-system-suppressant cytokine IL10.


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TABLE 2. Relation between race and cytokine gene polymorphisms among pregnant women, Pittsburgh, Pennsylvania, 1997–2001
 
In the proinflammatory IL1 genes, the up-regulating IL1A-4845 G/G and -889 T/T genotypes were significantly more common among African-American women than among White women. The odds of African-American women’s carrying a single -4845*G allele (odds ratio (OR) = 3.0, 95 percent CI: 1.1, 8.0) or -889*T allele (OR = 1.4, 95 percent CI: 1.0, 2.1) were marginally elevated, and the odds of homozygous carriage of the -4845 G/G genotype (OR = 4.3, 95 percent CI: 1.6, 11.3) or the -889 T/T genotype (OR = 2.1, 95 percent CI: 1.2, 3.6) were further elevated. Associations were also found for African-American versus White women with regard to the distribution of the IL1B-3957 C/C (OR = 3.1, 95 percent CI: 0.9, 10.9) and IL1B-511 A/A genotypes (OR = 4.9, 95 percent CI: 2.9, 8.5); again, the greater the dose of *C or *A, the greater the association with race.

The proinflammatory IL6 gene -174 C/C low-producer variant was markedly uncommon (1.2 percent) in African-American women. Carriage of a single high-producer *G allele was significantly (OR = 5.9, 95 percent CI: 1.4, 25.5) more common in African-American women than in White women, and carriage of the G/G genotype was 36.5 times (95 percent CI: 8.8, 151.9) more common in African-American women than in White women.

The allelic distributions for the remaining two proinflammatory cytokines, IL18 and TNFA, did not differ significantly between African-American and White women. Nonetheless, the high-producing G/G genotype at IL18 position -137 was somewhat elevated among African-American women (OR = 2.0, 95 percent CI: 0.9, 4.5).

Two loci, IL1A-4845 and IL1B-3957, were in significant linkage disequilibrium with one another (D' = 0.7361), whereas the other IL1A and IL1B alleles were not. The IL1A-4845*G/IL1B-3957*C haplotype was 40 percent more common in African-American women than in White women (95 percent CI: 1.03, 2.1) (table 3). The IL10-1082 and IL10-819 loci were in complete linkage disequilibrium (D' = 1.0000). Black women were at threefold increased risk of carrying the *A/*T haplotype (95 percent CI: 1.6, 5.3).


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TABLE 3. Relations between interleukin-1 and interleukin-10 haplotypes and race among pregnant women, Pittsburgh, Pennsylvania, 1997–2001
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
African-American women in our study were more likely than White women to carry allelic variants known to increase the expression of the proinflammatory cytokines IL1A, IL1B, IL6, and IL18 and less likely to carry allelic variants known to increase expression of the immune-system-suppressant cytokine IL10.

Cytokines generated by T-helper type 1 (Th-1) cells, such as IL1A, IL1B, IL6, IL18, and TNFA, acting as cytokine receptor ligands, up-regulate immune cell division, growth and differentiation, and migration—all proinflammatory effects (14). Expressed in the presence of many infections and inflammants, Th-1 cytokines also activate prostaglandins, which in turn act on surrounding tissues to decrease cell differentiation, inhibit apoptosis, and induce angiogenesis through growth factors and matrix metalloproteases (23). These actions contribute locally to the pathogenesis of conditions as disparate as atherosclerosis and premature delivery. Cytokines act in a complex and coordinated network in which they induce or repress their own synthesis (24). In particular, antiinflammatory cytokines generated by T-helper type 2 cells, including IL10, inhibit the production of Th-1 cytokines, and Th-1 cytokines decrease the production of IL10.

Immune-system-modulating genes are the most highly variant characteristic within the population, and genes encoding cytokines are no exception (24). The cytokine alleles studied here were chosen for their functional relevance. In particular, the -819*C and -1082*G alleles have been shown to increase IL10 protein production in stimulated peripheral blood lymphocytes in vitro (15, 16). Although the functional significance of the IL1A-889 polymorphism is unknown, the IL1A-4845 G/G polymorphism produces an alanine-to-serine amino acid substitution at codon 114 of the IL1A cytokine protein and has been associated with increased risk of atopy (17). The IL1B-3957*C allele increases IL1B secretion in activated macrophages in vitro (18, 19). Similarly, the IL1B-511*A allele has been found to occur in almost total linkage disequilibrium with the IL1B-31*T allele, a polymorphism that involves a TATA sequence in the IL1B promoter and induces a multifold increase in DNA binding in lipopolysaccharide-activated monocytes (20). The IL6-174 C/C variant is associated with a low-producer phenotype (21). Finally, the IL18-137*G and TNFA-308*A alleles have been shown to produce higher levels of mRNA or protein for those cytokines (22). All of this suggests that the statistically significant and dose-dependent distribution of alleles found in African-American women would all translate into functionally greater proinflammatory cytokine expression. Immune system hyperresponsiveness and inflammation have been associated with a number of disorders that more commonly plague African Americans. Examples include preterm birth (6, 25, 26), atherosclerosis (1, 5, 27, 28), autoimmune diseases (24, 29, 30), and transplant rejection (13).

Hoffman et al. (13) recently sought to examine systematically ethnic differences in cytokine gene polymorphisms among patients enrolled in National Institutes of Health transplant studies and volunteers (among controls, 78 were White and 19 were African-American). Consistently with our findings, Hoffman et al. reported that African Americans differed significantly from Whites in the distribution of genotypes for IL6 (C->G transversion at -174 and IL10 (-1082 [G/A], -819 [C/T], -592 [C/A]) but not TNFA (G->A transversion at -308). They also found African Americans to be more likely to carry IL2*G at position -330. The functional consequences of these polymorphisms would be a predisposition to higher levels of proinflammatory cytokines and lower levels of immunosuppressant cytokines. Two other recent studies, each conducted among non-population-based convenience samples of approximately 120 and 160 women, respectively (31, 32), showed racial variation in allelic distributions in cytokine genes, including TNFA, IL1A, IL1B, IL2, IL6, IL12, and IFNG (interferon-{gamma}). Our study evaluated a substantially larger and unselected population, allowing us to conclude that the distribution of polymorphisms, congruent with earlier findings, would predispose people to a systematic inflammatory up-regulation. Indeed, the antigen-presenting cells from African Americans, as compared with Whites, express more CD80 and CD86 costimulatory molecules (33). Moreover, T-cell costimulation during IL1B activation is elevated in African Americans.

Strengths of our study included a sample size large enough to examine relatively uncommon alleles and a population that was homogeneous in terms of health and health-care-seeking behavior. Participants were women receiving routine prenatal care at a single hospital in Pittsburgh. Women with pregnancy complications were excluded, which eliminated confounding by diseases that may be associated with inflammation. Weaknesses of our study include the genotyping of a limited number of cytokine alleles and, therefore, the limited modeling of complex inflammation-mediating pathways. Furthermore, we relied on self-reports of race, despite the known 7–20 percent White admixture within African Americans (34), which would tend to reduce observed associations. Nonetheless, the internal consistency of our findings is compelling: All of the cytokine alleles that were more common in African-American women were those that tend to up-regulate the inflammatory response, which suggests that a genetic contribution to this biologically important pathway may be race-specific.


    ACKNOWLEDGMENTS
 
This study was funded by grants AI44151 and AI48909 from the National Institute of Allergy and Infectious Diseases; grant HD30367 from the National Institute of Child Health and Human Development; and grant HS10592 from the Agency for Health Care Research and Quality.


    NOTES
 
Correspondence to Dr. Roberta B. Ness, Room A527, Crabtree Hall, University of Pittsburgh Graduate School of Public Health, 130 DeSoto Street, Pittsburgh, PA 15261 (e-mail: repro{at}edc.pitt.edu). Back


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 DISCUSSION
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