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. Author manuscript; available in PMC: 2010 May 1.
Published in final edited form as: Cytokine. 2009 Apr 7;46(2):236–244. doi: 10.1016/j.cyto.2009.02.003

Cytokine SNPs: Comparison of Allele Frequencies by Race & Implications for Future Studies

Alison L Van Dyke a, Michele L Cote a,b, Angie S Wenzlaff a, Susan Land c, Ann G Schwartz a,b
PMCID: PMC2742911  NIHMSID: NIHMS98428  PMID: 19356949

Abstract

The role of inflammation is being considered in chronic diseases. Previous studies have examined SNPs in a few key inflammatory genes and have included small numbers of African American participants. Variation in the frequencies of inflammatory pathway SNPs may help to explain racial disparities in disease risk. Through a population-based study of 103 African American and 380 Caucasian unrelated, healthy women, we examined the relationships between race and allele frequencies of 70 cytokine and cytokine receptor SNPs. The associations between genotypic and haplotype frequencies and race were also analyzed. Allelic frequencies for 52 out of the 70 SNPs meeting criteria for analysis differed significantly by race. Of the 32 pro-inflammatory and 20 anti-inflammatory SNPs for which the allele frequencies varied significantly by race, variant allele frequency differences between Caucasians and African Americans ranged between 6%–37% and 7%–53% for pro-inflammatory SNPs and anti-inflammatory SNPs, respectively. Our findings suggest that while allele frequencies do vary by race, racial groups are not simplistically represented by a pro-inflammatory or anti-inflammatory genetic profile. Given the racial variability in allele frequencies in inflammatory gene SNPs, studies examining the association between these SNPs and disease should at least incorporate self-reported race in their analyses.

Keywords: Cytokines, SNPs, Racial Differences, Women

1.1. INTRODUCTION

Differences by race in incidence for diseases associated with inflammation suggest that there may be underlying racial differences in inflammatory pathway genes. For example, there is racial variation in the incidence of several autoimmune diseases such as systemic lupus erythematosus (SLE) and multiple sclerosis (MS), and infectious diseases such as tuberculosis, septicemia, and HIV/AIDs [1]. The incidence of several types of cancer associated with inflammation and/or chronic infection, including colorectal, liver and bile, lung and bronchus, prostate, and stomach, is also elevated in African Americans relative to Caucasians, especially among men [2]. Finally, several markers of inflammation, including C-reactive protein and homocysteine, have been shown to be associated with cardiovascular disease and to be elevated in African Americans [3].

One set of markers of inflammatory pathways are cytokines and cytokine receptors. Differences in protein expression of IL-6 SR between healthy, older African Americans and Caucasians have been shown in at least one study [4]. While few studies have examined the relationship between cytokine protein expression and race, studies investigating the association between cytokine single nucleotide polymorphisms (SNPs) and race have been previously published [5–18]. These studies have focused on SNPs in only a few cytokines including tumor necrosis factor (TNF), IL1A, IL1B, IL6, IL2, IFNG, IL18, and IL10.

The consensus from these published reports is that among African Americans there is an increase in the frequency of alleles associated with high production of pro-inflammatory T helper type I (TH1) cytokines and low production of anti-inflammatory T helper type II (TH2) cytokines. These results suggest the potential for systemic inflammatory upregulation in African Americans. Specifically, IL1A -889T, IL1B -3957C and -511A, IL6 -174G, IL18 -137G, TNFA -308A, and IFNG -874 alleles are associated with an increase in cytokine production and are found more frequently among African Americans [7,9,11,13]. IL10 -592A, -819T, and -1082A alleles, associated with decreased IL-10 production, are also found more frequently among African Americans [7,9,13,15]. In contrast, another study reported that the pro-inflammatory IL2 -330G allele, associated with increased IL-2 production, was found less frequently among African Americans than among Caucasians [9]. Furthermore, some studies have found no relationship between allele frequency of the TNFA -308 polymorphism and race [7,9,11].

To expand this research, we examined the association between race and 70 cytokine and cytokine receptor polymorphisms in 26 inflammation-related genes among African American and Caucasian women in a large, population-based study.

1.2. MATERIALS & METHODS

1.2.1 Study Participants

Subjects were population-based healthy controls obtained through a study comparing controls frequency matched on age and self-reported race to women with non-small cell lung cancer (NSCLC) [19]. Control participants were women without a history of NSCLC between the ages of 18 and 74 living in the Detroit metropolitan area and were identified through random-digit dialing. Of the households that completed the eligibility screening questionnaire, 69.6% (N=575) participated in the interview. 209 women refused to participate. Excluded from analyses were 11 controls whose self-reported race was not African American or Caucasian. Four-hundred, eighty-three controls provided a blood sample, were genotyped, and are included in the analyses.

1.2.2. Sample Collection & Genotyping

Blood was collected in Vacutainer Plus tubes containing EDTA. DNA was isolated from whole blood with a Qiagen AutoPure LS the Genomic DNA Purification System (Gentra Systems, Minneapolis, MN) following the manufacturer’s protocols.

Genomic DNA 250 ng was submitted to the Wayne State University Applied Genomics Technology Center for genotyping. The Illumina GoldenGate assay using the Cancer SNP Panel was utilized. This Panel consists of primers to interrogate 1421 SNPs in 408 genes, including 83 cytokine and cytokine receptor SNPs, selected from the National Cancer Institute’s Cancer Genome Anatomy Project SNP500 Cancer Database (http:/snp500cancer.nci.nih.gov/). The GoldenGate assay was run according to the manufacturer’s directions. These data were analyzed using Bead Studio software (Illumina).

1.2.3. Statistical Analysis

χ2 goodness-of-fit test was used to assess whether SNPs were in Hardy-Weinberg Equilibrium (HWE) among African American and Caucasian controls separately. To compare the distributions of allele frequencies by race, Pearson’s χ2 analysis was conducted. Cochran-Mantel-Haenszel test for trend and χ2G, or Fisher’s Exact test where appropriate, were performed to assess the association between genotype and race. FSTAT V2.9.3.2 was used to calculate the Weir & Cockerham estimations of Wright’s fixation index, Fst, which ranges from 0 to 1, with higher numbers indicating greater genetic distance between the two populations [20]. As negative unbiased estimates of Fst have no biological meaning, negative values of Fst were set to zero. PLINK V1.03 (Shaun Purcell, http://pngu.mgh.harvard.edu/purcell/plink/) was used to construct haplotypes separately for Caucasians and African Americans [21]. Haplotypes with at least a 1% frequency in either race are reported. χ2 analysis was conducted to test for differences in the distribution of haplotype frequencies between African Americans and Caucasians. p-values were corrected for multiple comparisons via the Benjamini & Hochberg False Discovery Rate (FDR) method. Except for haplotype construction, all analyses were conducted in SAS/Genetics v. 9.1 (SAS Institute, Cary, NC). Finally, to validate the allele frequencies obtained in this study, frequencies for Caucasian residents of Utah (CEU) and African American residents of the American Southwest (ASW) were downloaded from HapMap Phase II + III data released in November 2008.

2. RESULTS

2.1.1. Sample

Our sample included 103 population-based African American women (21.3%) and 380 Caucasian (78.7%) women.

2.1.2. Cytokine & Cytokine Receptor SNPs

Five (IL3-rs40401; IL12B-rs730690; TNF-rs3093661; IL10RA-rs9610; TGFβ1-rs1800471) and two (IL8RB-rs1126579; IFNGR1-rs11914) out of 83 cytokine and cytokine receptor SNPs on the cancer SNP panel were not in Hardy-Weinberg equilibrium for African Americans and Caucasians, respectively. For an additional six SNPs (IL8-rs2227549; IL8RB-rs1126579; IFNGR1-rs3799488; TNF-rs3093661; LTA-rs3093546, IL10-rs3024509; IL10-rs3021094; IL10RA-rs2229114) variant allele frequencies were less than 5%, leaving 70 cytokine and cytokine receptor SNPs for analysis.

2.1.3. Allelic Frequencies by Race

Allelic frequencies for 52 out of the 70 SNPs meeting criteria for analyses differed significantly between Caucasians and African Americans (Table 1). For 12 out of the 70 SNPs analyzed, the minor alleles differed between the two racial groups; therefore, in each table the variant allele is defined as the minor allele among Caucasians. Allelic frequencies obtained from HapMap were strikingly similar to those in our study.

Table 1.

Cytokine & Cytokine Receptor SNP Allele Frequencies by Race

Cytokine SNP Identifier Wild Type Allelea Variant Allelea Caucasians Variant Allele Frequencies Phase III HapMap CEU Allele Frequencies African Americans Variant Allele Frequencies Phase III HapMap ASW Allele Frequencies Fst Comparison of Variant Allele Freq. p-valuec
Pro-inflammatory Cytokines & Cytokine Receptors
IL1A rs17561 Ala114Ser C A 0.32 0.26 0.17 0.17 0.046 <0.0001
IL1A rs2071374 IVS4−109 A C 0.28 0.29 0.00 0.71
IL1B rs1071676 Ex7−97 G C 0.26 0.14 0.032 0.0012
IL1B rs1143634 Phe105Phe G A 0.26 0.22 0.13 0.10 0.041 0.0002
IL1B rs3136558 IVS3−123 A G 0.24 0.22 0.17 0.12 0.009 0.06
IL1B rs1143627 −31 A G 0.32 0.35 0.64 0.65 0.176 <0.0001
IL1B rs16944 −511 G A 0.32 0.35 0.59 0.57 0.137 <0.0001
IL2 rs2069763 Leu38Leu C A 0.32 0.14 0.072 <0.0001
IL2 rs2069762 −330 A C 0.32 0.10 0.111 <0.0001
IL6 rs1800797 −597 G A 0.37 0.52 0.07 0.08 0.182 <0.0001
IL6 rs1800795 −174 C G 0.38 0.53 0.07 0.10 0.190 <0.0001
IL6R rs8192284 Asp358Ala A C 0.41 0.34 0.13 0.16 0.153 <0.0001
IL7R rs1494555 Ile138Val A G 0.29 0.28 0.17 0.16 0.033 0.0009
IL7R rs7737000 His165His G A 0.12 0.15 0.06 0.13 0.016 0.02
IL8 rs4073 −251 A T 0.43 0.80 0.230 <0.0001
IL8 rs2227306 IVS1+298 G A 0.39 0.14 0.126 <0.0001
IL8RA rs2854386 1379 bp 3′ STP G C 0.06 0.32 0.283 <0.0001
IL12A rs582537 IVS2−701 C A 0.45 0.30 0.040 0.0003
IL12B rs3212227 Ex8+159 A C 0.22 0.19 0.34 0.32 0.034 0.0008
IL15 rs1493013 Ex3+163 A G 0.43 0.39 0.000 0.38
IL15 rs2254514 Ex3−92 G A 0.28 0.17 0.025 0.0034
IL15 rs2857261 IVS3+8 A G 0.45 0.48 0.000 0.52
IL15 rs1057972 Ex9−181 T A 0.45 0.65 0.068 <0.0001
IL15 rs10833 Ex9−66 G A 0.36 0.37 0.14 0.11 0.095 <0.0001
IL15RA rs2296135 Ex8−361 C A 0.49 0.53 0.21 0.28 0.141 <0.0001
IL15RA rs2296141 IVS6−242 G A 0.08 0.10 0.000 0.39
IL15RA rs2228059 Asn146Thr A C 0.48 0.44 0.76 0.68 0.136 <0.0001
IL15RA rs3136614 IVS4+32 A G 0.22 0.21 0.12 0.16 0.031 0.0012
IFNAR2 rs3153 IVS1−4640 G A 0.30 0.14 0.060 <0.0001
IFNAR2 rs7279064 Phe10Val A C 0.32 0.35 0.19 0.21 0.041 0.0002
IFNAR2 rs2236757 IVS6−50 G A 0.30 0.31 0.21 0.24 0.015 0.02
IFNG rs1861494 IVS2+284 A G 0.28 0.11 0.075 <0.0001
IFNGR2 rs1059293 Ex7−134 G A 0.45 0.48 0.80 0.78 0.207 <0.0001
TNF rs1799964 −1031 A G 0.18 0.22 0.14 0.18 0.005 0.14
TNF rs1800630 −863 C A 0.13 0.15 0.09 0.13 0.004 0.16
TNF rs1800629 −308 G A 0.17 0.18 0.14 0.07 0.000 0.38
LTA rs909253 IVS1+90 A G 0.35 0.48 0.031 0.0013
TNFRSF10A rs2235126 IVS7+218 G A 0.27 0.23 0.21 0.19 0.005 0.12
TNFRSF10A rs4871857 Arg209Thr G C 0.48 0.34 0.031 0.0011
TNFRSF1A rs1800692 IVS4−33 G A 0.40 0.10 0.177 <0.0001
TNFRSF1A rs887477 IVS1−2572 C A 0.48 0.23 0.114 <0.0001
Anti-inflammatory Cytokines & Cytokine Receptors
IL1RN rs419598 Ala57Ala A G 0.27 0.28 0.06 0.06 0.110 <0.0001
IL1RN rs454078 IVS6+59 T A 0.27 0.06 0.108 <0.0001
IL1RN rs380092 IVS6+166 T A 0.35 0.72 0.238 <0.0001
IL4 rs2243248 −1098 A C 0.07 0.09 0.14 0.18 0.029 0.0018
IL4 rs2243250 −590 G A 0.14 0.14 0.67 0.60 0.509 <0.0001
IL4 rs2070874 Ex1−168 G A 0.13 0.14 0.45 0.39 0.257 <0.0001
IL4 rs2243268 IVS2−1443 A C 0.13 0.14 0.27 0.27 0.067 <0.0001
IL4R rs2243290 IVS3−9 C A 0.13 0.14 0.36 0.34 0.158 <0.0001
IL4R rs2057768 −29429 G A 0.29 0.31 0.30 0.24 0.000 0.67
IL4R rs3024544 IVS3−85 G A 0.15 0.17 0.11 0.14 0.002 0.22
IL4R rs1805011 Glu400Ala A C 0.13 0.10 0.53 0.55 0.369 <0.0001
IL4R rs1805012 Cys431Arg A G 0.12 0.10 0.11 0.07 0.000 0.75
IL4R rs1805015 Ser503Pro A G 0.17 0.16 0.39 0.35 0.130 <0.0001
IL4R rs1801275 Gln576Arg A G 0.22 0.21 0.66 0.353 <0.0001
IL4R rs1805016 Ser752Ala A C 0.05 0.06 0.26 0.43 0.213 <0.0001
IL4R rs8832 Ex12−313 G A 0.43 0.50 0.77 0.81 0.200 <0.0001
IL10 rs3024496 Ex5+210 A G 0.47 0.51 0.28 0.36 0.066 <0.0001
IL10 rs3024491 IVS1−286 C A 0.47 0.51 0.19 0.30 0.149 <0.0001
IL10 rs1800871 −819 G A 0.23 0.44 0.106 <0.0001
IL10 rs1800896 −1082 A G 0.47 0.52 0.28 0.35 0.073 <0.0001
IL10 rs1800890 −3575 T A 0.38 0.18 0.075 <0.0001
IL13 rs1881457 −1469 A C 0.23 0.19 0.16 0.19 0.009 0.07
IL13 rs1800925 −1112 G A 0.22 0.41 0.093 <0.0001
IL13 rs1295686 IVS3−24 G A 0.22 0.22 0.68 0.57 0.371 <0.0001
IL13 rs20541 Arg144Gln G A 0.21 0.22 0.23 0.18 0.000 0.52
TGFβ1 rs1800469 308 bp 3′ STP G A 0.31 0.29 0.27 0.18 0.002 0.23
TGFβR1 rs928180 IVS3−2409 A G 0.09 0.05 0.007 0.08
TGFβR1 rs334358 IVS8+547 C A 0.22 0.19 0.000 0.40
TGFβR1 rs868 Ex9+195 A G 0.22 0.20 0.16 0.20 0.005 0.12
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a

Wild type and variant alleles for Caucasians.

b

African American variant allele frequencies in bold are for SNPs for which the variant allele differed between Caucasians and African Americans.

c

p-values adjusted by the Benjamini & Hochberg FDR method.

2.2.1. Pro-inflammatory Cytokine & Cytokine Receptor SNP Allelic Distributions by Race

Of the 41 SNPs in pro-inflammatory cytokine and cytokine receptor genes, allele frequencies differed by race for all but nine (Table 1). Three of the SNPs that did not vary by race were the three SNPs in TNF. Otherwise, the five remaining SNPs not varying by race were in genes in which other SNPs were genotyped and found to vary by race (including IL1A, IL1B, IL15, IL15RA, and TNFRSF10A). All SNPs genotyped in IL2, IL6, IL6R, IL7R, IL8, IL8RA, IL12A, IL12B, IFNAR2, IFNG, IFNGR2, LTA and TNFRSF1A showed significant variation in allele frequency by race. Of the 32 pro-inflammatory SNPs for which the allele frequencies statistically significantly varied by race, the average absolute difference in variant allele frequencies between Caucasians and African Americans was 0.20 +/− 0.08 with a range from 0.06 to 0.37. Fst estimates for the 32 pro-inflammatory SNPs that varied by race ranged from 0.016 to 0.283. Three of these estimates, for rs4073-IL8, rs2854386-IL8RA, and rs1059293-IFNGR2, exceeded 0.200.

2.2.2. Anti-inflammatory Cytokine & Cytokine Receptor SNP Allelic Distributions by Race

Of the 29 SNPs in anti-inflammatory cytokine and cytokine receptor genes, allele frequencies differed by race in all but nine. All SNPs in IL1RN, IL4 and IL10 demonstrated significant racial differences in allele frequencies. Six of the nine SNPs in IL4R and two of four SNPs in IL13 also showed variation in allele frequency by race. Of the 20 anti-inflammatory SNPs for which the allele frequencies statistically significantly varied by race, the average absolute difference in variant allele frequency between Caucasians and African Americans was 0.27 +/− 0.12 with a range from 0.07 to 0.53. Estimates of Fst for these 20 anti-inflammatory SNPs ranged from 0.029 to 0.509. Seven of these estimates, in rs380092-IL1RN, rs2243250-IL4, rs2070874-IL4, rs1805011-IL4R, rs1801275-IL4R, rs1805016-IL4R, and rs1295686-IL13, exceeded 0.200.

2.3. Genotypic Frequencies by Race

With the following exceptions, the same SNPs with statistically significant differences in allelic frequencies by race also had statistically significant differences in genotype frequencies by race (data not shown). The distribution of genotypes for IL7R-rs7737000 was no longer significantly different by race. While there was a statistically significant trend for a higher proportion of the A allele at this SNP among Caucasians (p=0.02), the χ2 test for a general association between genotype and race was not statistically significant (p=0.07). Similarly, IFNAR2-rs2236757 had an increase in allele frequency among Caucasians (p=0.02); however, the χ2 test for a general association between genotype and race was not statistically significant (p=0.08). χ2 test for a general association and Fisher’s Exact test could not be calculated for one SNP, IL15RA-rs2296141, due to sparse data.

2.4. Haplotypes by Race

The 70 SNPs meeting criteria for analysis were located in 26 genes with multiple polymorphisms in 18 of these genes. Within gene haplotypes common to both African American and Caucasian women were discerned for all 18 of these genes including: IL1A, IL1B, IL1RN, IL2, IL6, IL7R, IL8, IL15, IL15RA, IFNAR2, TNF, TNFRSF1A, TNFRSF10A, IL4, IL4R, IL10, IL13, and TGFβR1 (Table 2). Two haplotypes were identified per gene in IL4 and IL4R. Of the 20 haplotypes, the distributions of haplotype frequencies differed significantly by race for all haplotypes constructed except for the TNF haplotype rs1799964-rs1800630-rs1800629.

Table 2.

Cytokine & Cytokine Receptor Haplotype Frequencies by Race Haplotypes Common to Caucasians and African Americans

%
Cytokine Haplotype Caucasians African Americans χ2 p-valuea
Pro-inflammatory Cytokines & Cytokine Receptors
rs17561-rs2071374
IL1A C-A 40.4 53.4
A-A 31.8 17.5
C-C 27.8 29.1
Other 0 0 0.0003
rs3136558-rs1143627-rs16944
IL1B A-A-G 45.2 25.5
A-G-A 31.0 52.6
G-A-G 22.3 10.4
G-G-A 1.3 6.1
A-G-G 0.1 4.4
Other 0.1 1.0 <0.0001
rs2069763-rs2069762
IL-2 C-A 35.3 75.5
A-A 32.0 14.2
C-C 32.6 10.3
Other 0.1 0 <0.0001
rs1800797-rs1800795
IL6 G-C 61.7 92.7
A-G 37.5 7.3
Other 0.8 0 <0.0001
rs1494555-rs7737000
IL7R A-G 59.0 76.9
G-G 29.2 17.2
A-A 11.8 5.9
Other 0 0 <0.0001
rs4073-rs2227306
IL8 A-G 56.7 19.6
T-A 39.2 14.1
T-G 4.1 66.3
Other 0 0 <0.0001
rs2245414-rs2857261-rs1057972-rs10833
IL15 G-G-A-G 44.9 47.0
A-A-T-A 20.5 4.3
G-A-T-A 14.9 8.6
G-A-T-G 11.5 15.9
A-A-T-G 7.2 4.7
G-A-A-G 0.3 9.7
A-A-A-G 0.2 8.3
Other 0.5 1.5 <0.0001
rs2296135-rs2228059-rs3136614
IL15RA C-C-A 42.7 70.5
A-A-A 33.0 13.4
A-A-G 14.0 4.9
C-A-G 4.8 3.6
C-C-G 3.5 2.2
A-C-A 1.9 1.7
C-A-A 0 2.4
A-C-G 0.1 1.3
Other 0 0 <0.0001
rs3153-rs7279064
IFNAR2 G-A 67.7 81.0
A-C 29.6 13.6
G-C 2.8 5.4
Other 0 0 <0.0001
rs1799964-rs1800630-rs1800629
TNF A-C-G 64.2 71.8
A-C-A 17.3 14.6
G-A-G 13.0 9.2
G-C-G 5.4 4.4
Other 0.1 0 0.72
rs1800692-rs887477
TNFRSF1A G-C 52.2 77.2
A-A 39.6 9.7
G-A 7.9 13.1
Other 0.3 0 <0.0001
rs2235126-rs4871857
TNFRSF10A G-G 50.0 63.3
A-C 24.7 19.1
G-C 22.9 15.4
A-G 2.4 2.3
Other 0 0 0.0077
Anti-inflammatory Cytokines & Cytokine Receptors
rs419598-rs454078-rs380092
IL1RN A-T-A 33.8 72.1
A-T-T 39.2 21.1
G-A-T 25.6 5.8
G-A-A 1.1 0
Other 0.3 1.0 <0.0001
rs2243248-rs2243250
IL4 A-G 79.4 23.0
A-A 13.6 62.9
C-G 7.0 9.5
C-A 0 4.6
Other 0 0 <0.0001
rs2243268-rs2243290
IL4-IL4R A-C 86.7 63.6
C-A 13.0 27.1
A-A 0.3 9.3
Other 0 0 <0.0001
rs2057768-rs1805015-rs8832
IL4R G-A-G 43.4 12.4
A-A-A 22.5 23.9
G-A-A 12.5 22.7
G-G-A 7.7 27.6
G-G-G 7.6 7.0
A-A-G 4.8 2.2
A-G-A 0.6 3.2
Other 0.9 1.0 <0.0001
rs1805012-rs1805015-rs1801275-rs1805016
IL4R A-A-A-A 77.7 32.1
G-G-G-A 11.4 11.3
A-A-G-A 5.2 8.2
A-G-G-C 4.3 4.9
A-A-G-C 0.4 20.2
A-G-G-A 0.1 21.4
A-G-A-A 0 1.2
Other 0.9 0.7 <0.0001
rs1800871-rs1800896-rs1800890
IL10 G-G-A 37.6 14.6
G-A-T 30.0 24.5
A-A-T 22.6 44.0
G-G-T 9.8 13.1
G-A-A 0 3.8
Other 0 0 <0.0001
rs1295686-rs20541
IL13 G-G 78.3 32.0
A-A 21.0 23.3
A-G 0.6 44.7
Other 0 0 <0.0001
rs928180-rs334358-rs868
TGFβR1 A-C-A 68.9 76.6
A-A-G 21.8 16.0
G-C-A 9.3 4.4
A-A-A 0 2.1
Other 0 0.9 0.0017
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a

p-values adjusted by the Benjamini & Hochberg FDR method.

Finally, our results remained statistically significant even after adjusting for multiple comparisons.

3. DISCUSSION

We observed allelic frequency differences by race for 52 of the 70 SNPs meeting criteria for analysis. General genotypic frequency differences between African Americans and Caucasians were observed for 49 of these 52 SNPs. Significant trends in genotypic frequency by race were found for all of these 52 SNPs. While the same haplotypes were constructed for African Americans and Caucasians for all genes for which multiple SNPs were analyzed, there was marked variation in haplotype frequency by race.

3.1 Pro-inflammatory Cytokines & Cytokine Receptors

Previous studies that have examined SNPs in IL1, IL2, IL6 and TNF genes have found differences between African Americans and Caucasians in allelic frequencies for these genes except for TNF.

3.1.1. IL1

Secreted primarily by macrophages and epithelial cells, IL-1 activates macrophages and T lymphocytes. In this study, we examined two IL1A SNPs, five IL1B SNPs, and three SNPs of the IL-1 antagonist gene, IL1RN. Allele frequencies for all but one SNP were statistically significantly different by race. Three of the IL1B SNPs included in our analyses have been previously studied. For the synonymous SNP, rs1143634, the T allele has been found to be less frequent among African Americans [18], and this finding is consistent with the decreased frequency of the A allele (the complement allele genotyped) in our study. Similar findings were also obtained between the previously published increased C allele frequency of rs1143627 (−31A>G) among African Americans [18]. Complement to the G allele, the C allele is associated with decreased promoter activity and decreased gene transcription, so our observation for this one SNP is inconsistent with the general hypothesis that African Americans have increased frequencies of high producing pro-inflammatory alleles. For the previously studied third IL1B SNP, rs16944 (−511 G>A), other researchers have found that the A allele (complement to the T allele genotyped in previous studies), which has been associated with increased transcriptional activity, is increased among African Americans. In our study, the A allele was the major allele among African Americans and the minor allele among Caucasians, supporting these results [13,18].

3.1.2 IL2

IL-2 is a T cell proliferation factor that acts in an autocrine fashion on TH1 cells. The minor allele for both IL2 SNPs studied in this project was found less frequently among African Americans. At least one of these SNPs, rs2069762 (−330A>C) has been previously studied. The G allele, associated with both an increased IL-2 production in vitro and a decreased IL-2 production in vivo [22], previously was found more frequently among African Americans [6,8]. Consistent with these results, we observed that the C allele (the complement allele analyzed in our study) was less frequent among African Americans. These findings are consistent with the assertion of increase frequency of high producing pro-inflammatory alleles in African Americans.

3.1.3 IL6

Allelic distributions of one of the two IL6 SNPs examined in this study previously have been found to differ by race. The IL6 SNP rs1800795 (−174G>C) is located in the promoter 5′ flanking region. The C allele creates an endonuclease restriction site producing cleaved products with 3′ overhangs; whereas, the G allele, which has been shown to be more frequent among African Americans, is associated with higher IL-6 production [7,9,11,13,16,17]. In contrast to these findings, the G allele was found less frequently among African Americans in our study.

3.1.4.TNF & TNFRSF

Primarily produced by macrophages, tumor necrosis factor (TNF) plays a role in inflammation partly through activating NFκB, tumorigenesis, apoptosis, and immune cell regulation. The A allele of TNF-308 lies in a binding site for the transcription factor AP1 and has been shown to increase TNF-α production in vitro. Studies examining the association between TNF-308 allele distribution and race have shown either no association [7,13,14,16] or decreased frequency of the A allele among African Americans [6,9,18]. We found no differences between African Americans and Caucasians in the allele frequencies for this SNP and the other TNF SNPs. However, we did find a difference by race in three of four TNF receptor SNPs, one of which, rs4871857, lies in a coding region of the TNFRSF10A gene (also referred to as TRAIL DR4, the “death receptor”). This SNP results in an amino acid substitution from arginine to threonine in the ligand binding domain and subsequently the transition from a basic to a hydroxylic amino acid that bears a phosphorylation site. Our study suggests a decreased frequency of the C allele among African Americans in comparison with Caucasians. The functional consequence of this SNP, its association with disease, and whether allelic variation by race can explain any racial differences in disease susceptibility need to be further explored.

3.1.5. Summary of Pro-inflammatory SNPs & Race

Findings for IL1-511 and IL2-330 are consistent with the hypothesis that African Americans have an increased pro-inflammatory genetic profile associated with high cytokine production. However, results for IL1-31 and IL6-174 were contrary to this hypothesis. The relationship between these genes and race are complicated; the effect of the IL1-31 polymorphism on IL-1 production has been shown to depend on the IL1-511 SNP [23] Additionally, functional studies are not consistent; some studies of the IL6-174 polymorphism have indicated that the G allele increases cytokine production [24–26] while others found that the C allele increases IL-6 production [27–29]; no association between genotype and expression has also been reported [30]. Our findings are not uniformly illustrative of African Americans’ having a greater likelihood of a pro-inflammatory genetic profile than whites.

3.2. Anti-inflammatory Cytokines & Cytokine Receptors

Studies have previously examined anti-inflammatory cytokine SNPs in IL4 and IL10 genes. We also evaluated IL13 polymorphisms.

3.2.1. IL4

Secreted by TH2 lymphocytes and mast cells, IL-4 acts to activate B lymphocytes, stimulate IgE class switching, and suppress TH1 lymphocytes and is thought to play a role in asthma and hypersensitivity reactions. Nguyen et al (2004) found that the A allele of rs2243250 (−590 G>A), which is associated with increased IL-4 production, was more common among African Americans [14]. Our results confirmed this finding as the A allele was the major allele among African Americans but the minor allele among Caucasians. This discrepancy in allele frequencies may contribute to the increased prevalence of asthma among African Americans [31].

3.2.2. IL10

All five of the IL10 SNPs examined differed by race in terms of allelic frequency, and two of these SNPs have been examined in previous studies. The rs1800896 (−1082A>G) A allele has been associated with lower IL-10 production in vitro by stimulated T lymphocytes and in vivo [32]. Either no association between allele frequencies at this SNP [7,11,14], or an increase in the A allele among African Americans has been reported [9,13,15,18]. Consistent with these latter findings, we observed an increase in the frequency of the A allele in African Americans versus Caucasians.

Our finding of an increased frequency of the A allele for rs1800871 (−819G>A) among African Americans is consistent with previously published reports [7,9,13,15]; however, studies including a smaller number of African Americans have observed no relationship between race and allelic distribution of this SNP [11,16]. This SNP sits in an estrogen receptor response element and is part of a haplotype with IL10-1082 and IL10-592 associated with IL-10 production. The ATA haplotype, associated with lower IL-10 production, has been found to be more frequent among African Americans than Caucasians [9,13,15] or equally as frequent in a smaller study involving 42 African American women [11].

3.2.3 Summary of Anti-inflammatory SNPs & Race

Our findings of an increase in the low IL-10 producing alleles among African Americans supports the conclusion that African Americans have an increased frequency of low producing anti-inflammatory polymorphisms. Contrary to this conclusion is our finding of an increase in the IL4 A allele associated with high levels of the anti-inflammatory IL-4, which has not been examined in previous studies of the associations between cytokine allelic frequency and race. Again, our findings suggest that while allele frequencies do vary by race, racial groups are not simplistically represented by a pro-inflammatory or anti-inflammatory genetic profile.

3.3. Other Cytokine SNPs

Other cytokine and cytokine receptor gene polymorphisms examined in this study included SNPs in IL7R, IL12A and IL12B, IL8, IL15, IL15RA, IL4R, IL13, and TGFβ and TGFβR1 genes. IL-7 acts as a hemopoietic growth factor, and IL-12 plays a major role in differentiation of precursor T helper lymphocytes. The chemokine IL-8, which draws immune system cells to the site at which it is produced and is significantly increased in the lungs of chronic smokers, has been implicated in the pathogenesis of COPD. IL-15 functions similarly to IL-2, acting as a T cell and natural killer cell growth factor, and its receptor, IL-15RA has been associated with autoimmune disease. Like IL-4, IL-13 has been shown to play a role in asthma. Under normal conditions, transforming growth factor beta (TGF-β) regulates cellular proliferation and differentiation; however, it is secreted by cancer cells into the tumor environment, resulting in immunosuppression by directly acting on immune cells and converting effector T cells into suppressor T cells. While minor allele frequencies of TGFβ and TGFβR1 SNPs did not differ between African American and Caucasian women, the frequency distributions of a TGFβR1 haplotype did differ significantly between the two racial groups, supporting the assertions that haplotype analysis needs to be conducted by race and that study of individual SNPs alone may not be adequate to analyze disease-locus associations.

3.4. Strengths & Limitations

This paper has a number of strengths including the population-based design and method of recruitment, which ensure that participants are representative of the population. Our sample included a large number of African Americans. Our use of a standardized panel from the NCI’s Cancer Anatomy Genome Project allows for the future comparison of our results to those of other researchers using the same panel. Moreover, we analyzed SNPs in a wider number of cytokine and cytokine receptor genes than have previously been studied in this field.

This study is not without limitations. The relationships between a number of the cytokine and cytokine receptor SNPs included in our analysis and protein expression and function have not been well characterized. Therefore, drawing conclusions about variations in inflammatory pathway function by race cannot be made based only on these data. Another limitation is that the African American women included in this study may not be representative of all African American women in the United States [33].

3.5. Conclusions

We found that cytokine and cytokine receptor SNP allelic, genotypic and haplotype frequencies varied significantly by race. These results suggest that future analyses of the associations between cytokine and cytokine receptor SNPs and disease risk and prognosis should at least consider self-reported race, if not more complex measures such as race and socioeconomic status or genetic ancestry. Additionally, the conclusion by Ness et al (2004) that African Americans have an increased allele frequency of high producing pro-inflammatory alleles and of low producing anti-inflammatory alleles was not fully supported in this study when a larger number of SNPs were examined than in previous studies, suggesting that this general conclusion may be an oversimplification [13]. It is likely that these differences in cytokine allele frequencies may contribute to racial disparities with regard to risk and mortality of diseases such as cancer, end stage renal disease and transplantation, and preterm labor [7,9,18,34–36], but the examination of only a handful of polymorphisms and the lack of critical factors confounding such relationships, such as socioeconomic status, has limited this work. Further research should involve a wider array of cytokine genes, as included in this study, and should assess whether these differences in cytokine genes are associated with functional differences in inflammatory pathways between African Americans and Caucasians.

Acknowledgments

This research was funded by NIH grant R01-CA87895 and contracts N01-PC35145 and P30CA22453.

Footnotes

Conflicts of Interest: None

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