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Association between allelic variants in cytokine genes and preeclampsia
American Journal of Obstetrics and Gynecology (2005) 193, 209–15
www.ajog.org
Association between allelic variants in cytokine genes and preeclampsia Catherine L. Haggerty, PhD, MPH,a,b,* Robert E. Ferrell, PhD,a Carl A. Hubel, PhD,b Nina Markovic, PhD,a,b Gail Harger, MS,a Roberta B. Ness, MD, MPHa,b University of Pittsburgh,a Magee-Womens Research Institute,b Pittsburgh, PA Received for publication August 13, 2004; revised October 1, 2004; accepted November 3, 2004
KEY WORDS Preeclampsia Cytokine Genetic Inflammation Pregnancy
Objective: The purpose of this study was to examine the relationship between cytokine genotypes and preeclampsia. Study design: We conducted a case-control study that examined cytokine genotypes among 150 primiparous preeclamptic women and 661 primiparous, normotensive women. Analyses were adjusted for age, prepregnancy cigarette smoking, and education. Results: Preeclamptic white women were more likely than normotensive white women to carry the up-regulating tumor necrosis factor–a–308 A/A (odds ratio, 4.1; 95% CI, 1.1-15.3) genotype. Both black and white women with preeclampsia were more likely than normotensive control subjects to carry the interleukin-1a–producing–4845 G/G genotype (black odds ratio, 11.6; 95% CI, 1.5-89.3; white odds ratio, 1.7; 95% CI, 0.7-3.9), –889 C/C genotype (black odds ratio, 5.1; 95% CI, 0.6-41.6; white odds ratio, 1.9; 95% CI, 0.8-4.7), and the interleukin-1a–4845/ interleukin-1a–889/interleukin-1b–3957 GCC/GCC haplotype (black odds ratio, 3.4; 95% CI, 1.3-8.7; white odds ratio, 2.1; 95% CI, 1.4-3.2). Conclusion: Cytokine genotypes were associated with preeclampsia and may identify women who are at high risk for preeclampsia. Ó 2005 Elsevier Inc. All rights reserved.
Preeclampsia is a systemic maternal disease that is characterized by hypertension and proteinuria1,2 and is a leading cause of maternal and neonatal morbidity.3,4 Although the cause of preeclampsia is not known Supported by grants AI44151 and AI48909 from the National Institute of Allergy and Infectious Diseases, HD30367 from the National Institute of Child Health and Human Development, HS10592 from the Agency for Healthcare Research and Quality, 5M01 RR00056 from the Magee-Womens Clinical Research Center, and HL64144 from the National Heart, Lung, and Blood Institute. * Reprint requests: Catherine L. Haggerty, PhD, MPH, Assistant Professor, University of Pittsburgh, Department of Epidemiology, 130 DeSoto St, 516B Parran Hall, Pittsburgh, PA 15261. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.11.008
completely, a central pathophysiologic feature of preeclampsia is systemic inflammation, which secondarily involves widespread endothelial dysfunction.5 Redman et al5 have proposed that endothelial dysfunction is part of a generalized inflammatory response to pregnancy, which is exaggerated in cases of preeclampsia. Levels of plasma and placental proinflammatory cytokines (such as tumor necrosis factor–a (TNF-a) and interleukin (IL)-1b are higher in preeclamptic women than in normotensive pregnant women.6-12 Further, the level of IL-10, an immunosuppressor cytokine, is lower in the plasma and placenta of preeclamptic women, compared with normotensive, pregnant control subjects.13-15
210 An exaggerated inflammatory response to pregnancy may occur among genetically susceptible women, such as those women who carry cytokine gene polymorphisms that are known to up-regulate cytokine production.16-24 Two previous studies have examined the relationship between functionally relevant IL-1 genotypes and preeclampsia, but with small sample sizes; no significant differences were found.25,26 Although a small study reported no relationship between TNF-a genotype and subsequent preeclampsia risk,27 Chen et al28 reported that women with preeclampsia were more likely to carry the TNF-1 allele (87 base pair) than pregnant women without preeclampsia. No published studies have examined IL-10 polymorphisms and preeclampsia. We examined whether primiparous women with preeclampsia are more likely than normotensive primiparous pregnant women to carry allelic variants that are linked to increased production of the proinflammatory cytokines IL-1a (-889T and -4845G), IL-1b (-3957C), and TNF-a (-308A) and known to down-regulate the inflammatory-modulating cytokine IL-10 (-819T and -1082A).16-24
Material and methods Subjects Maternal peripheral venous blood samples were obtained from the Pregnancy Exposures and Preeclampsia Prevention (PEPP) study. The study was approved by the Magee-Womens Hospital Institutional Review Board, and all the women signed informed consent. The PEPP study is comprised of 2 cohorts: 1 cohort was recruited at labor and delivery and the other cohort was recruited at !20 weeks of gestation and followed through delivery. All women with rule-out preeclampsia who were transferred to the perinatal referral service or who were delivered at Magee-Womens Hospital from February 1994 through May 1998 and were R14 years of age were invited to participate in the cross-sectional study. Of these 750 women, 744 women were juried by chart abstraction. Four women (1%) were delivered at another hospital, and 2 women (0.5%) rescinded consent. Of the 744 women, 243 women (33%) were juried as preeclamptic, and 105 women (14%) were juried as normotensive control subjects. From 1997 through 2001, healthy primiparous women ages 14 to 44 years were enrolled when they were seeking prenatal care at !20 weeks of gestation at Magee-Womens Hospital in Pittsburgh, Pa. Of the 6444 women who were screened for possible enrollment at !20 weeks of gestation, 2399 women (37.2%) were ineligible because of maternal age, gestational age, planned delivery at another hospital, or not being pregnant; 4045 women were eligible for participation, and 2891 women (71.5%) were enrolled.
Haggerty et al An additional 680 women were excluded from analysis on the basis that they were delivered at another hospital (n = 173), had a spontaneous abortion (n = 194), had a pregnancy termination or 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 2211 patients in the longitudinal cohort, 38 women were defined as preeclamptic, and 1560 women were classified as normal control subjects. In both cohorts, women were not classified as preeclamptic cases or normotensive control subjects if they had any of the following conditions: gestational hypertension; chronic hypertension; chronic hypertension with superimposed preeclampsia; intrauterine growth restriction; systemic lupus erythematosus; diabetes mellitus; multiple gestation; a positive toxicology screen; HIV; or a molar pregnancy. Women also were not categorized as cases or control subjects if they met some, but not all, of the preeclamptic criteria. Because allelic distributions vary widely by race, all analyses were stratified with the racial categorizations of white and black. Because only 15 women were categorized as Asian or other race, they were excluded from the analyses. Blood for analysis was available and sufficient from 150 preeclamptic and 661 normotensive black and white primiparous women from the 2 PEPP study cohorts and is the subject of this analysis.
Data collection Trained professional interviewers assessed maternal age, race, education, smoking, recalled prepregnancy weight, and height. Preeclampsia was defined as an elevation of blood pressure plus proteinuria and hyperuricemia. Hypertension was defined as an absolute blood pressure of 140 systolic or 90 diastolic on 2 occasions after 20 weeks of gestation and an increase of blood pressure of O15 mm Hg diastolic or 30 mm Hg systolic after 20 weeks of gestation over the average of all blood pressure at !20 weeks of gestation. Proteinuria was defined as O300 mg/24 hours or O0.3 protein/creatinine ratio or a 1C on a catheterized specimen or 2C on a random collection. Hyperuricemia was defined as serum uric acid concentration O1 standard deviation from normal for gestational age. Pregnancy control subjects were normotensive and without proteinuria or hyperuricemia throughout gestation. Whole blood samples that were collected in 5% EDTA were obtained at or around the time of delivery, processed within 2 hours, and stored at –80(C. High molecular weight DNA was isolated with the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, Minn). The cytokine polymorphisms of interest were selected on the basis of previous evidence of functionality or of linkage disequilibrium with a functional cytokine variant. All single nucleotide polymorphism
Haggerty et al assays were genotyped with the 50 -nuclease assay and the TaqMan (Applied Biosystems Inc, Forest City, Calif) protocol. Each assay was designed with the ABI Assays-on-Demand service (Applied Biosystems Inc), and the fidelity of the assays was confirmed by resequencing of samples of representative genotypes. A 5% random sample from each plate was genotyped independently for quality control.
Statistical analyses Allele frequencies were tested for Hardy-Weinberg equilibrium with the chi-squared goodness-of-fit statistic. Sociodemographic characteristics were compared between preeclamptic cases and normotensive control subjects with analysis of variance. Chi-squared tests of proportion or Fisher’s exact tests were used to compare each genotype frequency between preeclamptic cases and normotensive control subjects. Logistic regression models were fit after an adjustment for any risk factor that was associated with preeclampsia at a level of probability of .10. Models included age (continuous), prepregnancy cigarette smoking (yes/no), and education (!high school, high school graduate, Ohigh school) as covariates. Finally, because the IL-1 polymorphisms that were examined occur within a small genomic region, multilocus haplotypes were tested for linkage disequilibrium as measured by their pairwise D0 values with the Estimated Haplotype program (Rockefeller University, New York, NY). Loci in strong linkage disequilibrium were combined into haplotypes. All analyses were stratified by race.
Results Within the cases of preeclampsia, 35% were defined with early preeclamptic, delivering before 35 weeks of gestation. Women with preeclampsia were older, less likely to smoke cigarettes, and more educated (Table I). There was no difference in prepregnancy body mass index between women with and without preeclampsia. A larger proportion of preeclamptic cases were recruited as rule-out preeclamptic cases from labor and delivery. These women were also more likely to be private practice patients and represent a higher socioeconomic status group than the women who were recruited prospectively through outpatient clinics. There were no differences in the IL-10 allele distribution between preeclamptic and normotensive black women (Table II). White women who were homozygous for the down-regulating -1082A IL-10 allele were more likely to be preeclamptic (odds ratio [OR], 1.7), although results were of borderline significance (Table III). Preeclamptic white women were significantly more likely than normotensive white women to carry the upregulating TNF-a –308 A/G (OR, 1.7) and A/A (OR,
211 2.8) genotypes. The TNF-a –308A variant was not associated with preeclampsia in black women, but the A/A genotype was almost absent. Both black and white women with preeclampsia were more likely to carry the IL-1a–producing –4845 G/G (black women OR, 11.7; white women OR, 1.9) and –889 C/C (black woman OR, 5.9); white woman OR, 2.3) genotypes than their normotensive control subjects. Adjustment for maternal age, prepregnancy smoking status, and education did not significantly change the results. Adjusted probability values for trend were significant across the allelic variants of IL-1a –889 among black women, and TNF-a –308, IL-1a –4845, and IL-1a –889 in white women. At each of these loci, women who were homozygous for the up-regulating allele had the greatest risk for preeclampsia. All 3 IL-1 loci were in significant to near complete linkage disequilibrium with one another: IL-1a –4845 and IL-1a –889, D0 = 0.996; IL-1a –889 and IL-1b –3957, D0 = 0.7504; and IL-1a –4845 and IL-1b –3957, D0 = 0.7526. Black women with preeclampsia were O3 times as likely and white women with preeclampsia were twice as likely to carry the IL-1a –4845, IL-1a –889, IL-1b –3957 GCC/GCC haplotype than women without preeclampsia (Table IV).
Comment Preeclamptic white women were more likely than their normotensive control subjects to carry allelic variants that are known to increase the expression of the proinflammatory cytokines IL-1a and TNF-a. Among black women, preeclamptic cases were more likely than normotensive control subjects to carry allelic variants that were associated with the up-regulated expression of IL-1a. Both black and white women with preeclampsia more commonly carried an up-regulating IL-1a/IL-1b haplotype. An overly aggressive inflammatory response may result in numerous adverse reproductive outcomes, including preeclampsia.5,29-31 Abnormal placentation and genetic predisposition for exaggerated inflammation may interact to trigger the syndrome of preeclampsia. Defective placentation leads to ischemic tissue, which may be damaged by hypoxia and release uric acid, thereby activating the production of cytokines.32 Exaggerated cytokine expression in genetically predisposed women may promote an inflammatory response, which leads to oxidative stress, endothelial dysfunction, and preeclampsia. Other maternal inflammatory factors may exaggerate the normal inflammatory response to pregnancy, predisposing women to preeclampsia. Bacterial and viral infections, known to elicit an overall up-regulation of immune mediators, also lead to oxidative stress and endothelial dysfunction.5,31,33,34 Several infections such
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Table I
Demographic characteristics among black and white primiparous normotensive women
Characteristic Age (y)* Prepregnancy body mass index (kg/m2)* Smoke cigarettes (%) Yes No Education (%) OHigh school High school OHigh school
Preeclamptic women (n = 150)
Normotensive women (n = 661)
P value
26.8 G 6.1 24.4 G 7.9
23.6 G 5.6 24.8 G 6.4
!.001 .578
23.3 76.7
55.1 44.9
!.001
6.6 30.9 62.5
15.4 38.8 45.8
!.001
* Data are given as mean G SD.
Table II
Effect of cytokine gene polymorphisms on preeclampsia among primiparous black women
Cytokine genotype IL-10–819 C/C (n = 84) C/T (n = 88) T/T (n = 26) IL-10–1082 G/G (n = 23) G/A (n = 86) A/A (n = 88) TNFa–308 G/G (n = 152) A/G (n = 43) A/A (n = 2) IL-1a–4845 T/T (n = 5) T/G (n = 67) G/G (n = 126) IL-1a–889 T/T (n = 29) T/C (n = 93) C/C (n = 75) IL-1b–3957 T/T (n = 4) C/T (n = 43) C/C (n = 149)
Normotension (%)*
Preeclampsia (%)y
42.1 43.8 14.0
45.0 50.0 5.0
11.9 42.9 45.2
10.0 50.0 40.0
76.8 22.0 1.1
80.0 20.0 0.0
2.8 37.1 60.1
0.0 5.0 95.0
15.8 49.2 35.0
5.0 30.0 65.0
1.7 24.4 73.9
5.0 0.0 95.0
Adjusted P value trendz
Pairwise odds ratio (95% CI)
Adjusted pairwise odds ratio (95% CI)z
.380
Referent group 1.0 (0.4, 2.7) 0.3 (0.04, 2.6)
Referent group 1.0 (0.4, 2.8) 0.3 (0.04, 3.0)
.913
Referent group 1.4 (0.3, 6.8) 1.1 (0.2, 5.3)
Referent group 1.2 (0.2, 6.1) 0.9 (0.2, 4.7)
N/A
Referent group 0.9 (0.3, 2.8) N/A
Referent group 0.8 (0.3, 2.6) N/A
N/A
N/A Referent group 11.7 (1.5, 89.6)
N/A Referent group 11.6 (1.5, 89.3)
.035
Referent group 1.9 (0.2, 16.7) 5.9 (0.7, 47.1)
Referent group 1.9 (0.2, 17.5) 5.1 (0.6, 41.6)
N/A
Referent group N/A 0.4 (0.04, 4.4)
Referent group N/A 0.5 (0.1, 5.2)
N/A, Not applicable. * N = 199. y N = 20. z Adjusted for maternal age, smoking and education.
as Chlamydia pneumoniae,35 herpes simplex virus-2,36,37 Epstein-Barr virus,36 malaria,38 urinary tract infection,39-41 bacterial vaginosis,42 C trachomatis,42 Trichomonas vaginalis,42 Gardnerella vaginalis,42 group B streptococcus,42and cytomegalovirus36,37,43 have been associated with preeclampsia. The interaction of bacterial and viral infections with cytokine polymorphisms may further modify the risk of preeclampsia and should be examined in future studies.
Although we found no association between body mass index and preeclampsia, elevated prepregnancy body mass index, and waist circumference have been associated strongly with preeclampsia in previous studies.40,44-51 In our study, women with preeclampsia were of higher socioeconomic status, and this likely biased the relationship between body mass index and preeclampsia toward the null. Obesity is associated with chronic inflammation and oxidative stress.52 Thus, elevated
Haggerty et al Table III
213
Effect of cytokine gene polymorphisms on preeclampsia among primiparous white women
Cytokine genotype IL-10–819 C/C (n = 291) C/T (n = 210) T/T (n = 27) IL-10–1082 G/G (n = 101) G/A (n = 277) A/A (n = 142) TNFa–308 G/G (n = 372) A/G (n = 143) A/A (n = 13) IL-1a–4845 T/T (n = 49) T/G (n = 208) G/G (n = 259) IL-1a -889 T/T (n = 50) T/C (n = 214) C/C (n = 260) IL-1b–3957 T/T (n = 25) C/T (n = 171) C/C (n = 329)
Normotension (%)*
Preeclampsia (%)y
54.8 39.9 5.3
56.3 39.3 4.5
20.5 53.7 25.9
15.5 51.8 32.7
73.1 25.0 1.9
60.7 34.8 4.5
10.1 43.2 46.7
7.2 29.7 63.1
10.4 43.6 46.0
6.3 30.6 63.1
4.8 36.0 59.2
4.5 19.8 75.7
Adjusted P value trendz
Pairwise odds ratio (95% CI)
Adjusted pairwise odds ratio (95% CI)z
.900
Referent group 1.0 (0.6, 1.5) 1.0 (0.4, 2.7)
Referent group 0.9 (0.5, 1.4) 1.1 (0.4, 2.9)
.101
Referent group 1.3 (0.7, 2.3) 1.7 (0.9, 3.2)
Referent group 1.4 (0.7, 2.7) 1.9 (1.0, 3.9)
.011
Referent group 1.7 (1.1, 2.6) 2.8 (0.9, 8.8)
Referent group 1.7 (1.0, 2.7) 4.1 (1.1, 15.3)
.042
Referent group 1.0 (0.4, 2.2) 1.9 (0.9, 4.3)
Referent group 0.9 (0.4, 2.2) 1.7 (0.7, 3.9)
.025
Referent group 1.2 (0.5, 2.8) 2.3 (1.0, 5.3)
Referent group 1.1 (0.4, 2.7) 1.9 (0.8, 4.7)
.905
Referent group 0.6 (0.2, 1.7) 1.4 (0.5, 3.8)
Referent group 0.6 (0.2, 1.9) 1.4 (0.5, 4.1)
* N = 462. y N = 130. z Adjusted for maternal age, smoking and education.
Table IV Frequency of the IL-1a–4845, IL-1a–889, IL-1b–3957 GCC/GCC haplotype among primiparous black and white women with and without preeclampsia Variable Black women* GCC/GCC All other haplotypes White womeny GCC/GCC All other haplotypes
Normotension (%)
Preeclampsia (%)
Odds ratio (95% CI)
30.9 69.1
60.0 40.0
3.4 (1.3, 8.7) d
42.1 57.9
60.4 39.6
2.1 (1.4, 3.2) d
* Normotension, n = 175; preeclampsia, n = 20. y Normotension, n = 401; preeclampsia, n = 111.
body fat may trigger excessive cytokine production among genetically susceptible pregnant women. The primary strength of our study is the large number of preeclamptic women who were studied, which allowed us to examine relatively uncommon alleles. Our study also exhibited strong internal consistency, in that all of the cytokine alleles that are more common among women with preeclampsia are those that up-regulate inflammation. A weakness of our study is the smaller number of black women, which limited interpretation in this subgroup. However, strengths of association were generally similar between white and black women,
which adds consistency to our findings. Further studies that use larger cohorts, including adequate numbers of black women, and examine a broader range of inflammatory cytokine and growth factor genes are needed. The main limitation of the study is the lack of circulating cytokine measures that are needed to prove a functional relationship between polymorphisms, elevated cytokines, and preeclampsia. Because serum samples were obtained at or around the time of delivery, we did not measure circulating cytokines, because they would be highly elevated and unrepresentative of
214 normal pregnancy levels. Rather, we selected cytokine alleles that were based on previously published functional relevance. Specifically, the IL-10 –1082G and – 819C allelic variants have been shown to increase IL-10 production in vitro.16-18 Similarly, the –308A TNFa variant has been associated with higher protein levels.19-22 Although the functional significances of the IL-1a –889 and –4845 polymorphisms are unknown, the IL-1a G –4845 polymorphism leads to an alanine to serine amino acid substitution at codon 114 of the IL-1a protein53 and has been associated with an increased risk of atopy.23 Finally, the –3957 C/C genotype is associated with increased IL-1b secretion in activated macrophages in vitro.24 Additional studies of cytokine polymorphisms and circulating cytokines with early pregnancy serum samples would confirm the relationship between genetic polymorphisms, excessive inflammation, and preeclampsia. We conclude that genetic polymorphisms that lead to increases in proinflammatory cytokine production were associated with preeclampsia in the Pittsburgh-based PEPP cohort. Polymorphisms that are associated with cytokine up-regulation may increase the normal inflammatory response to pregnancy or cause excessive immune system stimulation in response to defective placentation, infection, or elevated body fat. In either case, this genetic susceptibility to preeclampsia suggests an inheritability for preeclampsia and underscores the importance played by a predisposition to disease that precedes pregnancy. Cytokine genotyping may prove to be an effective early pregnancy preeclampsia risk-screening tool. Future research is needed to confirm our findings in other populations and to identify additional susceptibility genes. Further, the concept that inflammation may cause the maternal syndrome preeclampsia, as evidenced in this and previous reports,5,31 supports the need for interventions directed at modulating inflammation. Aspirin and antioxidants, which are known to reduce inflammation and oxidative stress, may be important preemptive therapies for high-risk women. Disrupting the pathway that promotes inflammation, oxidative stress, and endothelial dysfunction may reduce the incidence of preeclampsia and its consequential maternal and neonatal morbidity and death.
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215 37. Rustveld L, Ness R, Costantino J, Roberts J. Serological association between primary infections with herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), cytomegalovirus (CMV) and Epstein Barr virus (EBV) and the risk of preeclampsia [Abstract]. Am J Epidemiol 2003;157:S74. 38. Sartelet H, Rogier C, Milko-Sartelet I, Angel G, Michel G. Malaria associated pre-eclampsia in Senegal. Lancet 1996; 347:1121. 39. Hsu CD, Witter FR. Urogenital infection in preeclampsia. Int J Gynaecol Obstet 1995;49:271-5. 40. Mittendorf R, Lain KY, Williams MA, Walker CK. Preeclampsia: a nested, case-control study of risk factors and their interactions. J Reprod Med 1996;41:491-6. 41. Hill JA, Devoe LD, Bryans CI Jr. Frequency of asymptomatic bacteriuria in preeclampsia. Obstet Gynecol 1986;67:529-32. 42. Herrera JA, Chaudhuri G, Lopez-Jaramillo P. Is infection a major risk factor for preeclampsia? Med Hypotheses 2001;57:393-7. 43. Carreiras M, Montagnani S, Layrisse Z. Preeclampsia: a multifactorial disease resulting from the interaction of the feto-maternal HLA genotype and HCMV infection. Am J Reprod Immunol 2002;48:176-83. 44. Knuist M, Bonsel GJ, Zondervan HA, Treffers PE. Risk factors for preeclampsia in nulliparous women in distinct ethnic groups: a prospective cohort study. Obstet Gynecol 1998;92:178. 45. Sibai BM, Ewell M, Levine RJ, Klebanoff MA, Esterlitz J, Catalano PM, et al. Risk factors associated with preeclampsia in healthy nulliparous women: the Calcium for Preeclampsia Prevention (CPEP) Study group. Am J Obstet Gynecol 1997;177:1010. 46. Odegard RA, Vatten LJ, Nilsen ST, Salvesen KA, Austgulen R. Risk factors and clinical manifestations of pre-eclampsia. BJOG 2000;107:1410-6. 47. Parazzini F, Bortolus R, Chatenoud L, Restelli S, Ricci E, Marozio L, et al. Risk factors for pregnancy-induced hypertension in women at high risk for the condition. Epidemiology 1996;7:306-8. 48. Stone JL, Lockwood CJ, Berkowitz GS, Alvarez M, Lapinski R, Berkowitz RL. Risk factors for severe preeclampsia. Obstet Gynecol 1994;83:357-61. 49. Mostello D, Catlin TK, Roman L, Holcomb WL Jr, Leet T. Preeclampsia in the parous woman: Who is at risk? Am J Obstet Gynecol 2002;187:425-9. 50. Eskenazi B, Fenster L, Sidney S. A multivariate analysis of risk factors for preeclampsia. JAMA 1991;266:237-41. 51. Baeten JM, Bukusi EA, Lambe M. Pregnancy complications and outcomes among overweight and obese nulliparous women. Am J Publ Health 2001;91:436-40. 52. Reilly MP, Rader DJ. The metabolic syndrome: more than the sum of its parts? Circulation 2003;108:1546-51. 53. van den Velden PA, Reitsma PH. Amino acid dimorphism in IL1A is detectable by PCR amplification. Hum Mol Genet 1993;2:1753.
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Association between allelic variants in cytokine genes and preeclampsia Catherine L. Haggerty, PhD, MPH,a,b,* Robert E. Ferrell, PhD,a Carl A. Hubel, PhD,b Nina Markovic, PhD,a,b Gail Harger, MS,a Roberta B. Ness, MD, MPHa,b University of Pittsburgh,a Magee-Womens Research Institute,b Pittsburgh, PA Received for publication August 13, 2004; revised October 1, 2004; accepted November 3, 2004
KEY WORDS Preeclampsia Cytokine Genetic Inflammation Pregnancy
Objective: The purpose of this study was to examine the relationship between cytokine genotypes and preeclampsia. Study design: We conducted a case-control study that examined cytokine genotypes among 150 primiparous preeclamptic women and 661 primiparous, normotensive women. Analyses were adjusted for age, prepregnancy cigarette smoking, and education. Results: Preeclamptic white women were more likely than normotensive white women to carry the up-regulating tumor necrosis factor–a–308 A/A (odds ratio, 4.1; 95% CI, 1.1-15.3) genotype. Both black and white women with preeclampsia were more likely than normotensive control subjects to carry the interleukin-1a–producing–4845 G/G genotype (black odds ratio, 11.6; 95% CI, 1.5-89.3; white odds ratio, 1.7; 95% CI, 0.7-3.9), –889 C/C genotype (black odds ratio, 5.1; 95% CI, 0.6-41.6; white odds ratio, 1.9; 95% CI, 0.8-4.7), and the interleukin-1a–4845/ interleukin-1a–889/interleukin-1b–3957 GCC/GCC haplotype (black odds ratio, 3.4; 95% CI, 1.3-8.7; white odds ratio, 2.1; 95% CI, 1.4-3.2). Conclusion: Cytokine genotypes were associated with preeclampsia and may identify women who are at high risk for preeclampsia. Ó 2005 Elsevier Inc. All rights reserved.
Preeclampsia is a systemic maternal disease that is characterized by hypertension and proteinuria1,2 and is a leading cause of maternal and neonatal morbidity.3,4 Although the cause of preeclampsia is not known Supported by grants AI44151 and AI48909 from the National Institute of Allergy and Infectious Diseases, HD30367 from the National Institute of Child Health and Human Development, HS10592 from the Agency for Healthcare Research and Quality, 5M01 RR00056 from the Magee-Womens Clinical Research Center, and HL64144 from the National Heart, Lung, and Blood Institute. * Reprint requests: Catherine L. Haggerty, PhD, MPH, Assistant Professor, University of Pittsburgh, Department of Epidemiology, 130 DeSoto St, 516B Parran Hall, Pittsburgh, PA 15261. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.11.008
completely, a central pathophysiologic feature of preeclampsia is systemic inflammation, which secondarily involves widespread endothelial dysfunction.5 Redman et al5 have proposed that endothelial dysfunction is part of a generalized inflammatory response to pregnancy, which is exaggerated in cases of preeclampsia. Levels of plasma and placental proinflammatory cytokines (such as tumor necrosis factor–a (TNF-a) and interleukin (IL)-1b are higher in preeclamptic women than in normotensive pregnant women.6-12 Further, the level of IL-10, an immunosuppressor cytokine, is lower in the plasma and placenta of preeclamptic women, compared with normotensive, pregnant control subjects.13-15
210 An exaggerated inflammatory response to pregnancy may occur among genetically susceptible women, such as those women who carry cytokine gene polymorphisms that are known to up-regulate cytokine production.16-24 Two previous studies have examined the relationship between functionally relevant IL-1 genotypes and preeclampsia, but with small sample sizes; no significant differences were found.25,26 Although a small study reported no relationship between TNF-a genotype and subsequent preeclampsia risk,27 Chen et al28 reported that women with preeclampsia were more likely to carry the TNF-1 allele (87 base pair) than pregnant women without preeclampsia. No published studies have examined IL-10 polymorphisms and preeclampsia. We examined whether primiparous women with preeclampsia are more likely than normotensive primiparous pregnant women to carry allelic variants that are linked to increased production of the proinflammatory cytokines IL-1a (-889T and -4845G), IL-1b (-3957C), and TNF-a (-308A) and known to down-regulate the inflammatory-modulating cytokine IL-10 (-819T and -1082A).16-24
Material and methods Subjects Maternal peripheral venous blood samples were obtained from the Pregnancy Exposures and Preeclampsia Prevention (PEPP) study. The study was approved by the Magee-Womens Hospital Institutional Review Board, and all the women signed informed consent. The PEPP study is comprised of 2 cohorts: 1 cohort was recruited at labor and delivery and the other cohort was recruited at !20 weeks of gestation and followed through delivery. All women with rule-out preeclampsia who were transferred to the perinatal referral service or who were delivered at Magee-Womens Hospital from February 1994 through May 1998 and were R14 years of age were invited to participate in the cross-sectional study. Of these 750 women, 744 women were juried by chart abstraction. Four women (1%) were delivered at another hospital, and 2 women (0.5%) rescinded consent. Of the 744 women, 243 women (33%) were juried as preeclamptic, and 105 women (14%) were juried as normotensive control subjects. From 1997 through 2001, healthy primiparous women ages 14 to 44 years were enrolled when they were seeking prenatal care at !20 weeks of gestation at Magee-Womens Hospital in Pittsburgh, Pa. Of the 6444 women who were screened for possible enrollment at !20 weeks of gestation, 2399 women (37.2%) were ineligible because of maternal age, gestational age, planned delivery at another hospital, or not being pregnant; 4045 women were eligible for participation, and 2891 women (71.5%) were enrolled.
Haggerty et al An additional 680 women were excluded from analysis on the basis that they were delivered at another hospital (n = 173), had a spontaneous abortion (n = 194), had a pregnancy termination or 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 2211 patients in the longitudinal cohort, 38 women were defined as preeclamptic, and 1560 women were classified as normal control subjects. In both cohorts, women were not classified as preeclamptic cases or normotensive control subjects if they had any of the following conditions: gestational hypertension; chronic hypertension; chronic hypertension with superimposed preeclampsia; intrauterine growth restriction; systemic lupus erythematosus; diabetes mellitus; multiple gestation; a positive toxicology screen; HIV; or a molar pregnancy. Women also were not categorized as cases or control subjects if they met some, but not all, of the preeclamptic criteria. Because allelic distributions vary widely by race, all analyses were stratified with the racial categorizations of white and black. Because only 15 women were categorized as Asian or other race, they were excluded from the analyses. Blood for analysis was available and sufficient from 150 preeclamptic and 661 normotensive black and white primiparous women from the 2 PEPP study cohorts and is the subject of this analysis.
Data collection Trained professional interviewers assessed maternal age, race, education, smoking, recalled prepregnancy weight, and height. Preeclampsia was defined as an elevation of blood pressure plus proteinuria and hyperuricemia. Hypertension was defined as an absolute blood pressure of 140 systolic or 90 diastolic on 2 occasions after 20 weeks of gestation and an increase of blood pressure of O15 mm Hg diastolic or 30 mm Hg systolic after 20 weeks of gestation over the average of all blood pressure at !20 weeks of gestation. Proteinuria was defined as O300 mg/24 hours or O0.3 protein/creatinine ratio or a 1C on a catheterized specimen or 2C on a random collection. Hyperuricemia was defined as serum uric acid concentration O1 standard deviation from normal for gestational age. Pregnancy control subjects were normotensive and without proteinuria or hyperuricemia throughout gestation. Whole blood samples that were collected in 5% EDTA were obtained at or around the time of delivery, processed within 2 hours, and stored at –80(C. High molecular weight DNA was isolated with the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, Minn). The cytokine polymorphisms of interest were selected on the basis of previous evidence of functionality or of linkage disequilibrium with a functional cytokine variant. All single nucleotide polymorphism
Haggerty et al assays were genotyped with the 50 -nuclease assay and the TaqMan (Applied Biosystems Inc, Forest City, Calif) protocol. Each assay was designed with the ABI Assays-on-Demand service (Applied Biosystems Inc), and the fidelity of the assays was confirmed by resequencing of samples of representative genotypes. A 5% random sample from each plate was genotyped independently for quality control.
Statistical analyses Allele frequencies were tested for Hardy-Weinberg equilibrium with the chi-squared goodness-of-fit statistic. Sociodemographic characteristics were compared between preeclamptic cases and normotensive control subjects with analysis of variance. Chi-squared tests of proportion or Fisher’s exact tests were used to compare each genotype frequency between preeclamptic cases and normotensive control subjects. Logistic regression models were fit after an adjustment for any risk factor that was associated with preeclampsia at a level of probability of .10. Models included age (continuous), prepregnancy cigarette smoking (yes/no), and education (!high school, high school graduate, Ohigh school) as covariates. Finally, because the IL-1 polymorphisms that were examined occur within a small genomic region, multilocus haplotypes were tested for linkage disequilibrium as measured by their pairwise D0 values with the Estimated Haplotype program (Rockefeller University, New York, NY). Loci in strong linkage disequilibrium were combined into haplotypes. All analyses were stratified by race.
Results Within the cases of preeclampsia, 35% were defined with early preeclamptic, delivering before 35 weeks of gestation. Women with preeclampsia were older, less likely to smoke cigarettes, and more educated (Table I). There was no difference in prepregnancy body mass index between women with and without preeclampsia. A larger proportion of preeclamptic cases were recruited as rule-out preeclamptic cases from labor and delivery. These women were also more likely to be private practice patients and represent a higher socioeconomic status group than the women who were recruited prospectively through outpatient clinics. There were no differences in the IL-10 allele distribution between preeclamptic and normotensive black women (Table II). White women who were homozygous for the down-regulating -1082A IL-10 allele were more likely to be preeclamptic (odds ratio [OR], 1.7), although results were of borderline significance (Table III). Preeclamptic white women were significantly more likely than normotensive white women to carry the upregulating TNF-a –308 A/G (OR, 1.7) and A/A (OR,
211 2.8) genotypes. The TNF-a –308A variant was not associated with preeclampsia in black women, but the A/A genotype was almost absent. Both black and white women with preeclampsia were more likely to carry the IL-1a–producing –4845 G/G (black women OR, 11.7; white women OR, 1.9) and –889 C/C (black woman OR, 5.9); white woman OR, 2.3) genotypes than their normotensive control subjects. Adjustment for maternal age, prepregnancy smoking status, and education did not significantly change the results. Adjusted probability values for trend were significant across the allelic variants of IL-1a –889 among black women, and TNF-a –308, IL-1a –4845, and IL-1a –889 in white women. At each of these loci, women who were homozygous for the up-regulating allele had the greatest risk for preeclampsia. All 3 IL-1 loci were in significant to near complete linkage disequilibrium with one another: IL-1a –4845 and IL-1a –889, D0 = 0.996; IL-1a –889 and IL-1b –3957, D0 = 0.7504; and IL-1a –4845 and IL-1b –3957, D0 = 0.7526. Black women with preeclampsia were O3 times as likely and white women with preeclampsia were twice as likely to carry the IL-1a –4845, IL-1a –889, IL-1b –3957 GCC/GCC haplotype than women without preeclampsia (Table IV).
Comment Preeclamptic white women were more likely than their normotensive control subjects to carry allelic variants that are known to increase the expression of the proinflammatory cytokines IL-1a and TNF-a. Among black women, preeclamptic cases were more likely than normotensive control subjects to carry allelic variants that were associated with the up-regulated expression of IL-1a. Both black and white women with preeclampsia more commonly carried an up-regulating IL-1a/IL-1b haplotype. An overly aggressive inflammatory response may result in numerous adverse reproductive outcomes, including preeclampsia.5,29-31 Abnormal placentation and genetic predisposition for exaggerated inflammation may interact to trigger the syndrome of preeclampsia. Defective placentation leads to ischemic tissue, which may be damaged by hypoxia and release uric acid, thereby activating the production of cytokines.32 Exaggerated cytokine expression in genetically predisposed women may promote an inflammatory response, which leads to oxidative stress, endothelial dysfunction, and preeclampsia. Other maternal inflammatory factors may exaggerate the normal inflammatory response to pregnancy, predisposing women to preeclampsia. Bacterial and viral infections, known to elicit an overall up-regulation of immune mediators, also lead to oxidative stress and endothelial dysfunction.5,31,33,34 Several infections such
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Table I
Demographic characteristics among black and white primiparous normotensive women
Characteristic Age (y)* Prepregnancy body mass index (kg/m2)* Smoke cigarettes (%) Yes No Education (%) OHigh school High school OHigh school
Preeclamptic women (n = 150)
Normotensive women (n = 661)
P value
26.8 G 6.1 24.4 G 7.9
23.6 G 5.6 24.8 G 6.4
!.001 .578
23.3 76.7
55.1 44.9
!.001
6.6 30.9 62.5
15.4 38.8 45.8
!.001
* Data are given as mean G SD.
Table II
Effect of cytokine gene polymorphisms on preeclampsia among primiparous black women
Cytokine genotype IL-10–819 C/C (n = 84) C/T (n = 88) T/T (n = 26) IL-10–1082 G/G (n = 23) G/A (n = 86) A/A (n = 88) TNFa–308 G/G (n = 152) A/G (n = 43) A/A (n = 2) IL-1a–4845 T/T (n = 5) T/G (n = 67) G/G (n = 126) IL-1a–889 T/T (n = 29) T/C (n = 93) C/C (n = 75) IL-1b–3957 T/T (n = 4) C/T (n = 43) C/C (n = 149)
Normotension (%)*
Preeclampsia (%)y
42.1 43.8 14.0
45.0 50.0 5.0
11.9 42.9 45.2
10.0 50.0 40.0
76.8 22.0 1.1
80.0 20.0 0.0
2.8 37.1 60.1
0.0 5.0 95.0
15.8 49.2 35.0
5.0 30.0 65.0
1.7 24.4 73.9
5.0 0.0 95.0
Adjusted P value trendz
Pairwise odds ratio (95% CI)
Adjusted pairwise odds ratio (95% CI)z
.380
Referent group 1.0 (0.4, 2.7) 0.3 (0.04, 2.6)
Referent group 1.0 (0.4, 2.8) 0.3 (0.04, 3.0)
.913
Referent group 1.4 (0.3, 6.8) 1.1 (0.2, 5.3)
Referent group 1.2 (0.2, 6.1) 0.9 (0.2, 4.7)
N/A
Referent group 0.9 (0.3, 2.8) N/A
Referent group 0.8 (0.3, 2.6) N/A
N/A
N/A Referent group 11.7 (1.5, 89.6)
N/A Referent group 11.6 (1.5, 89.3)
.035
Referent group 1.9 (0.2, 16.7) 5.9 (0.7, 47.1)
Referent group 1.9 (0.2, 17.5) 5.1 (0.6, 41.6)
N/A
Referent group N/A 0.4 (0.04, 4.4)
Referent group N/A 0.5 (0.1, 5.2)
N/A, Not applicable. * N = 199. y N = 20. z Adjusted for maternal age, smoking and education.
as Chlamydia pneumoniae,35 herpes simplex virus-2,36,37 Epstein-Barr virus,36 malaria,38 urinary tract infection,39-41 bacterial vaginosis,42 C trachomatis,42 Trichomonas vaginalis,42 Gardnerella vaginalis,42 group B streptococcus,42and cytomegalovirus36,37,43 have been associated with preeclampsia. The interaction of bacterial and viral infections with cytokine polymorphisms may further modify the risk of preeclampsia and should be examined in future studies.
Although we found no association between body mass index and preeclampsia, elevated prepregnancy body mass index, and waist circumference have been associated strongly with preeclampsia in previous studies.40,44-51 In our study, women with preeclampsia were of higher socioeconomic status, and this likely biased the relationship between body mass index and preeclampsia toward the null. Obesity is associated with chronic inflammation and oxidative stress.52 Thus, elevated
Haggerty et al Table III
213
Effect of cytokine gene polymorphisms on preeclampsia among primiparous white women
Cytokine genotype IL-10–819 C/C (n = 291) C/T (n = 210) T/T (n = 27) IL-10–1082 G/G (n = 101) G/A (n = 277) A/A (n = 142) TNFa–308 G/G (n = 372) A/G (n = 143) A/A (n = 13) IL-1a–4845 T/T (n = 49) T/G (n = 208) G/G (n = 259) IL-1a -889 T/T (n = 50) T/C (n = 214) C/C (n = 260) IL-1b–3957 T/T (n = 25) C/T (n = 171) C/C (n = 329)
Normotension (%)*
Preeclampsia (%)y
54.8 39.9 5.3
56.3 39.3 4.5
20.5 53.7 25.9
15.5 51.8 32.7
73.1 25.0 1.9
60.7 34.8 4.5
10.1 43.2 46.7
7.2 29.7 63.1
10.4 43.6 46.0
6.3 30.6 63.1
4.8 36.0 59.2
4.5 19.8 75.7
Adjusted P value trendz
Pairwise odds ratio (95% CI)
Adjusted pairwise odds ratio (95% CI)z
.900
Referent group 1.0 (0.6, 1.5) 1.0 (0.4, 2.7)
Referent group 0.9 (0.5, 1.4) 1.1 (0.4, 2.9)
.101
Referent group 1.3 (0.7, 2.3) 1.7 (0.9, 3.2)
Referent group 1.4 (0.7, 2.7) 1.9 (1.0, 3.9)
.011
Referent group 1.7 (1.1, 2.6) 2.8 (0.9, 8.8)
Referent group 1.7 (1.0, 2.7) 4.1 (1.1, 15.3)
.042
Referent group 1.0 (0.4, 2.2) 1.9 (0.9, 4.3)
Referent group 0.9 (0.4, 2.2) 1.7 (0.7, 3.9)
.025
Referent group 1.2 (0.5, 2.8) 2.3 (1.0, 5.3)
Referent group 1.1 (0.4, 2.7) 1.9 (0.8, 4.7)
.905
Referent group 0.6 (0.2, 1.7) 1.4 (0.5, 3.8)
Referent group 0.6 (0.2, 1.9) 1.4 (0.5, 4.1)
* N = 462. y N = 130. z Adjusted for maternal age, smoking and education.
Table IV Frequency of the IL-1a–4845, IL-1a–889, IL-1b–3957 GCC/GCC haplotype among primiparous black and white women with and without preeclampsia Variable Black women* GCC/GCC All other haplotypes White womeny GCC/GCC All other haplotypes
Normotension (%)
Preeclampsia (%)
Odds ratio (95% CI)
30.9 69.1
60.0 40.0
3.4 (1.3, 8.7) d
42.1 57.9
60.4 39.6
2.1 (1.4, 3.2) d
* Normotension, n = 175; preeclampsia, n = 20. y Normotension, n = 401; preeclampsia, n = 111.
body fat may trigger excessive cytokine production among genetically susceptible pregnant women. The primary strength of our study is the large number of preeclamptic women who were studied, which allowed us to examine relatively uncommon alleles. Our study also exhibited strong internal consistency, in that all of the cytokine alleles that are more common among women with preeclampsia are those that up-regulate inflammation. A weakness of our study is the smaller number of black women, which limited interpretation in this subgroup. However, strengths of association were generally similar between white and black women,
which adds consistency to our findings. Further studies that use larger cohorts, including adequate numbers of black women, and examine a broader range of inflammatory cytokine and growth factor genes are needed. The main limitation of the study is the lack of circulating cytokine measures that are needed to prove a functional relationship between polymorphisms, elevated cytokines, and preeclampsia. Because serum samples were obtained at or around the time of delivery, we did not measure circulating cytokines, because they would be highly elevated and unrepresentative of
214 normal pregnancy levels. Rather, we selected cytokine alleles that were based on previously published functional relevance. Specifically, the IL-10 –1082G and – 819C allelic variants have been shown to increase IL-10 production in vitro.16-18 Similarly, the –308A TNFa variant has been associated with higher protein levels.19-22 Although the functional significances of the IL-1a –889 and –4845 polymorphisms are unknown, the IL-1a G –4845 polymorphism leads to an alanine to serine amino acid substitution at codon 114 of the IL-1a protein53 and has been associated with an increased risk of atopy.23 Finally, the –3957 C/C genotype is associated with increased IL-1b secretion in activated macrophages in vitro.24 Additional studies of cytokine polymorphisms and circulating cytokines with early pregnancy serum samples would confirm the relationship between genetic polymorphisms, excessive inflammation, and preeclampsia. We conclude that genetic polymorphisms that lead to increases in proinflammatory cytokine production were associated with preeclampsia in the Pittsburgh-based PEPP cohort. Polymorphisms that are associated with cytokine up-regulation may increase the normal inflammatory response to pregnancy or cause excessive immune system stimulation in response to defective placentation, infection, or elevated body fat. In either case, this genetic susceptibility to preeclampsia suggests an inheritability for preeclampsia and underscores the importance played by a predisposition to disease that precedes pregnancy. Cytokine genotyping may prove to be an effective early pregnancy preeclampsia risk-screening tool. Future research is needed to confirm our findings in other populations and to identify additional susceptibility genes. Further, the concept that inflammation may cause the maternal syndrome preeclampsia, as evidenced in this and previous reports,5,31 supports the need for interventions directed at modulating inflammation. Aspirin and antioxidants, which are known to reduce inflammation and oxidative stress, may be important preemptive therapies for high-risk women. Disrupting the pathway that promotes inflammation, oxidative stress, and endothelial dysfunction may reduce the incidence of preeclampsia and its consequential maternal and neonatal morbidity and death.
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