- Email: [email protected]
Interleukin-1 alpha variation is associated with the risk of developing preeclampsia
G Model
EURO 9029 1–4 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb 1
Interleukin-1 alpha variation is associated with the risk of developing preeclampsia
2 3 4
Ghasemi a,1, Elham Kashani b,1, Azadeh Fayyaz c, Marzieh Attar c, Majid Shahbazi c,*
Q1 Masoumeh
5 6 7 8 9 10
a
Department of Human Genetics and Medical Cellular & Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran Department of Obstetrics & Gynecology, Sayyad Shirazi Hospital and Medical Cellular & Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran c Medical Cellular & Molecular Research Center, Talghani Children Hospital, Golestan University of Medical Sciences, Bolv Janbazan, Zip code: 4916668197 Gorgan, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 April 2015 Received in revised form 3 June 2015 Accepted 16 June 2015
Objective: Preeclampsia is a syndrome that affects 5% of all pregnancies, producing substantial maternal and prenatal morbidity and mortality. Several studies have reported that cytokine genes are associated with the persistence of preeclampsia or the severity of the disease. The aim of this study is to investigate the relationships between the polymorphisms of interleukin-1 alpha-889 (IL-1A) gene and preeclampsia. Method: Genomic DNA was extracted from the peripheral blood of 305 patients with preeclampsia and 325 normal controls from Sayyad Shirazi Hospital of Golestan University. Then subjected to SSP-PCR amplification. STATA software and the chi square test were used for statistic calculations. Results: The frequencies of IL-1A -889 genotypes C/C, T/T and C/T in preeclampsia cases were 34.8%, 8.2%, 57% and in controls were 20.9%, 7.6% and 71.3% respectively. There was a significant 1.5 fold excess frequency in genotype C/C in cases (CI = 1.44–3.07, OR = 2.1, P = 0.0001). There was a significant difference in the frequencies of alleles or genotypes in IL-1A promoter regions between patients with preeclampsia and the control group. Turkomans showed the highest frequency of the C allele and Sistanies had the lowest frequency of the C allele in preeclampsia compared to control groups (CI = 1.5– 3.9, OR = 2.48, P = 0.0001). Conclusion: Our findings suggest that the IL-1A-899C/C genotype and C allele are associated with susceptibility to preeclampsia. ß 2015 Published by Elsevier Ireland Ltd.
Keywords: Interleukin-1 alpha Polymorphism Preeclampsia Predisposing factor Susceptibility
11 12 13 14 15 16 17 18 19 20
Introduction Q2
Preeclampsia (PE) is a placenta-dependent disease that causes mother and fetus systemic disorders [1,2]. It is characterized by endothelial dysfunction throughout the maternal circulation, resulting in hypertension and proteinuria which typically occurs after 20 week gestation to after delivery during the postpartum period [3–6]. PE2 is a major cause of maternal and fetal mortality and morbidity worldwide [4]. The prevalence of this disease is 5– 7.5% in the USA and 4–4.5% in Iran [4,7]. Several studies on PE
* Corresponding author. Tel.: +98 171 235 17 35; fax: +98 171 235 17 35. E-mail address: [email protected] (M. Shahbazi). 1 These two authors have got equal right as a first author. 2 Preeclampsia.
indicated that ethnic and genetic factors may contribute to the increased risk of PE [1,8,9]. Also, the role of immune system variations in the pathophysiology of preeclampsia has been reported in many studies [10]. Cytokines are a major group of extracellular signal proteins that regulate, proliferate and make cell-to-cell connections in the immune system. Cytokine signals have an important role in the different stages of health and diseases and increases in inflammatory cytokines such as IL-1, IL-10, IL-6 and INF-gamma that are produced in response to local hypoxia and ischemia of the placenta which might trigger endothelial and result in preeclampsia [11–13]. Therefore, the gene encoding pre-inflammatory cytokines participating in the regulation of the immune response are good candidates for further studying maternal genetic susceptibility to preeclampsia. IL-1 is one of cytokines that plays a central role in the inflammation and endothelial function. All family of these genes
http://dx.doi.org/10.1016/j.ejogrb.2015.06.020 0301-2115/ß 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Ghasemi M, et al. Interleukin-1 alpha variation is associated with the risk of developing preeclampsia. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.06.020
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
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EURO 9029 1–4 M. Ghasemi et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2015) xxx–xxx
2
37 38 39 40 41 42 43 44 45 46 47
(IL-1A, IL-1B, IL-1RN) on chromosome (2q13:14) are polymorphic. These polymorphisms are in regulatory regions; they have an important role in regulating the production proteins of IL-1, and are associated with several diseases [14,15]. IL-1A is a subtype of pre-inflammatory cytokines which induce the expression of 40 several inflammatory factors [16]. Haggeri et al. (2005) found an association between this position and preeclampsia; but there are not more studies in the position of IL-1A apparently [17]. In this study, the polymorphism in IL-1A gene was investigated in preeclamptic women to elucidate the association between IL-1A gene polymorphism (-889) and susceptibility to preeclampsia.
48
Materials and methods
49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85
In this study, the blood samples were collected from 305 preeclampsia women and 325 control women with no history of preeclampsia who referred to Sayyad Shirazi Hospital of Golestan University between March 2010 and May 2013. Women with inflammatory, autoimmune and chronic diseases or infections such as HBV were excluded. Controls were selected from among healthy pregnant women who were admitted to the labor ward. Preeclampsia was defined as the development of hypertension of above 140 and 90 mm Hg of systolic and diastolic pressure with proteinuria of +1 or >300 mg in 24 h without renal diseases and infection (according to the report of National High Blood Pressure Education Program Working Group, 2000). Women with chronic hypertension or HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count) without preeclampsia were also excluded from the study. Ethical approval for data collection and the project was approved by the ethics committee of Golestan University of Medical Sciences (Registration Number: 351599012254 and date of issue: 2/ 16/2012). DNA was extracted from the whole blood using a standard phenol-chloroform extraction method and diluted to 50 ng/ml. IL1A gene polymorphism (-889C/T) was genotyped by specific sequence primer polymerase chain reaction (SSP-PCR). Details of primer sequences and fragment size for determining of IL-1A gene polymorphism and human growth hormone (HGH) gene as an internal control are provided in Table 1. The PCR (polymerase chain reaction) amplification was followed by a 15 ml reaction mixture containing 1 ml of genomic DNA, 0/9 ml of 25 mm mgcl2 (Qiagen, USA), 1/5 ml of each 10x buffer (Qiagen, USA), 1/5 ml of 10 mm dNTP, 2/2 ml of sucrose 60%, 0/5 VL of each 10pm specific primer, 0/ 5 ml of each 10pm HGH primers and 0/2 of taq polymerase (Qiagen, USA). The cycling protocol was included in an initial denaturation at 968C for 1 min, 10 cycles for 15 s at 96 8C, 50 s at 63 8C, 40 s at 72 8C (loop 1) and was followed by 20 cycles for 1 min at 96 8C, 50 s at 58 8C, 40 s at 72 8C (loop 2). A total of 30 cycles was carried out, with a final extension at 72 8C for 5 min. The PCR products were subjected to electrophoresis on 1/5% agarose gels and photographed under a UV trans-illuminator (2006 bio-red gel doc) after
Table 1 PCR primer sequences for IL-1A (-889). Gene
Annealing temperature
Fragment size (bp)
Primers
IL-1A
63
221
HGH
64
429
FW: 50 -CTTTAATAATAGTAACCAGGCAACAT-30 FM: 50 -CTTTAATAATAGTAACCAGGCAACAC-30 R: 50 -AAGTAGCCCTCTACCAAGGA-30 HGH1: 50 -GCC TTC CCA ACC ATT CCC TTA-30 HGH2: 50 -TCA CGG ATT TCT GTT GTG TTT C-30
staining with ethidium bromide. Allele frequencies were tested for hardy-Weinberg equilibrium using Chi-squared test and/or Fisher exact test. These tests were used to compare the genotype frequency between preeclamptic and control groups. SPSS 16 software (IBM, Armonk, NY, USA) was also applied, the level of statistical significance was defined as P < 0.05, odds ratios (ORs) were calculated on a 95% confidence interval (CI).
86 87 88 89 90 91 92
Results
93
Using SSP-PCR, the fragments containing the positions where the IL-1/_889 SNP occurred in 305 patients with preeclampsia and 325 control subjects were amplified. Statistical analysis showed a significant difference between preeclampsia and control groups in terms of allelic and genotype frequency (Table 2) so that the C/C genotype frequency was significantly higher in the patients (CI = 1.44–3.07, OR = 2.1, P = 0.0001). Also, the C allele frequency was significantly higher in the patients than control group (CI = 1.04–1.66, OR = 1.32, P = 0.016), while T allele did not show any significant difference between the two groups. However the present study demonstrated a significant relationship between of the IL-1A/-889 gene polymorphism and preeclampsia. Case and control groups of this work were separated into three groups based on reproductive age that included: <18, between 18 and 40 and >40 years old. In terms of ethnicity, they were divided into four subgroups of Persian, Turkoman, Sistani and others3 based on the participant’s biography. Statistical observations are summarized in Table 3. Investigation in the mentioned age groups showed no statistically significant differences between the case and control groups. But, studying the ethnicity provided the following results: Although genotyping of the following ethnic groups was slightly different, these changes were not statistically significant and needed to be studied in a larger population. However, studies demonstrated significant allelic differences so that the C allele significantly increased in the sub-groups of Turkomans, Persians, and Sistanies respectively. Turkomans showed the highest frequency of the C allele and Sistanies had the lowest frequency of the C allele in preeclampsia compared to control groups. However, T allele was significantly increased in the sub-groups of Sistanies, Persians, and Turkomans respectively. The obtained results are separately shown in Table 4.
94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125
Comments
126
In this study, the region of the IL1a gene position -889 was analyzed using SSP-PCR and a T to C polymorphism was identified in this position relative to preeclampsia. Highly significant correlation was found between homozygosity for the IL1a 889*C allele and development of PE. Also, the investigation of subethnic groups in this study showed that Turkomans had the highest percentage of the C allele and Sistani ethnicity had the lowest percentage in PE compared with control groups. These data suggested that the C allele might be as a susceptibility factor for preeclampsia in Turkoman ethnicity and T allele can have a protective role against PE in Sistani ethnicity. Both genetics and inflammatory factors play a vital role in the pathogenesis of preeclampsia [8,18]. Numerous studies have demonstrated the role of inheritance in preeclampsia [19,20]; but, the way genetics and molecular mechanisms work in preeclampsia is still unknown. An aggressive inflammatory response may result in numerous adverse reproductive outcomes including preeclampsia [21–24]. An abnormal genetic predisposition, for example, exaggerates
127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145
3
Other groups included Baluchistan and Afghanistan ethnicities.
Please cite this article in press as: Ghasemi M, et al. Interleukin-1 alpha variation is associated with the risk of developing preeclampsia. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.06.020
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EURO 9029 1–4 M. Ghasemi et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2015) xxx–xxx Table 2 Allele and genotype frequencies of IL-1A/-889 in preeclampsia and healthy pregnant controls. IL1a polymorphism
Controls N = 325
PE N = 305
OR
Allele C T
368 (56.6%) 282 (43.4%)
386 (63.2%) 224 (36.7%)
1.32 1
Genotype C/C C/T T/T
68 (20.9%) 232 (71.3%) 25 (7.6%)
106 (34.8%) 174 (57%) 25 (8.2%)
2.1 1 NS
Comparison
PE
Control
OR
95%CI
P value
1.04–1.66 –
0.016 –
1.44–3.07 – NS
0.0001 – NS
C/Turkoman vs. C/Persian C/Persian vs. C/Sistani C/Turkoman vs. C/Sistani T/Sistani vs. T/Persian T/Persian vs. T/Turkoman T/Sistani vs. T/Turkoman
82/188 188/97 82/97 57/112 112/42 57/42
46/167 167/135 46/135 107/133 133/28 107/28
1.58 1.56 2.48 0.63 0.56 0.35
1.02–2.46 1.10–2.21 1.55–3.97 0.41–0.96 0.31–0.99 0.19–0.65
0.0376 0.0089 0.0001 0.031 0.0491 0.0003
cytokine and may promote an inflammatory response which leads to oxidative stress, endothelial dysfunction, and preeclampsia [25]. Instead, cytokine allele that was of functional relevance was selected here, because it was assumed that genetic polymorphisms that lead to increased pre-inflammatory cytokine production may be associated with preeclampsia. IL-1alpha is an important pre-inflammatory factor for beginning of inflammatory diseases [16,26]. Based on the promoter role of (-889) position of the IL-1a gene, the expected molecular variation of this region leads to promoter activation, increases gene transcription, and thus promotes the inflammatory response [27]. Therefore, the present study revealed that polymorphism of this gene may influence susceptibility factor for preeclampsia. The only study which has been done on the IL-1alpha (-889) gene polymorphism was by Haggeri et al. (2005) who investigated several polymorphisms of cytokines and provided evidence for the relationship between IL-1A genotype in -889 position in both white and black women with PE compared to the control subjects so that the frequency of CC genotype was associated with the increased risk of PE [17]. Also, Goodard et al. investigated IL-1A polymorphism in three positions that were different from those of the present study and demonstrated a relationship between polymorphism in 4 and 6 intron positions and PE along with a lack of relationship between polymorphism in 5 exone position and PE [4]. Although the functional significances of the IL-1A -889 and 4845 polymorphisms lead to the variation of amino acids in IL-1A protein and are associated with the increased risk of atopy [28], the
Table 3 Demographics in preeclamptic and healthy women. Variables
Healthy women n = 325 (%)
Preeclamptic women n = 305 (%)
P value
Classification of preeclampsia Mild preeclamptic – Sever preeclamptica –
218 (71.47) 87 (25.57)
Maternal age (year) <18 18–40 >40
10 (3.07) 314 (96.61) 1 (0.30)
7 (2.29) 294 (96.39) 4 (1.31)
>0.05
Ethnic Persian Turkoman Sistani Other groups
150 37 121 17
150 62 77 16
<0.05b
(46.1) (11.3) (37.2) (5.2)
Table 4 Frequencies of risk alleles (IL-1 -889C and T) in PEa patients and control ethnics subgroups.
P valuea
95% CI
OR = odds ratio; 95% CI = 95% confidence interval. a Fisher’s exact test.
146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173
3
(49.1) (20.3) (25.2) (5.2)
–
a The presence of any one of these findings: Blood pressure 160/110 mmHg, proteinuria > 5 g on a 24 h collection, liver and renal dysfunction, pulmonary edema, fatal growth restriction. b Based on chi square test; p < 0.05 was considered significant.
a
PE, preeclampsia; OR, odds ratio; 95% CI, 95% confidence interval.
relationship between IL-1A gene polymorphism and preeclampsia may indicate the functional role of C allele in sustaining the immune response. Although this gene may be assumed as a strong candidate which is associated with PE, it may vary in different populations and is associated with multi-factorial diseases. In conclusion, an association was founded between this polymorphism and susceptibility to preeclampsia in the Iranian population. However, future studies on different ethnic populations and on other polymorphic regions in cytokine genes would provide a more accurate definition for the involvement of cytokine genetic polymorphisms in the development of preeclampsia and cytokine genotyping may prove to be an effective early pregnancy preeclampsia risk-screening tool.
174 175 176 177 178 179 180 181 182 183 184 185 186
Conflicts of interest
187
All contributing authors declare no conflicts of interest Acknowledgments
188 189
We thank all the women that participated in this study. We 190 thank the Medicine Cellular and Molecular Research Centre 191 (MCMRC) for recruiting study subjects. We wish to thank all 192 gynecologists from Sayyad Shirazi Hospital for enabling us to 193 collect samples and maintaining the study database. Our study was 194 financially supported by Golestan University of Medical Science Q3195 (Grant Number: 35/262). 196 References
197
[1] Landi B, Tranquilli AL. HELLP syndrome and placental inflammatory pathology. Minerva Ginecol 2008;60(October (5)):389–98. [2] Gibbs Rif, Karlan BY, Haney AF, Nygaard IE, editors. Danforth’s obstetrics and gynecology. tenth edition, Lippincott Williams & Wilkins; 2008. [3] Roberts JM, Taylor RN, Goldfien A. Clinical and biochemical evidence of endothelial cell dysfunction in the pregnancy syndrome preeclampsia. Am J Hypertens 1991;4(August (8)):700–8. [4] Goddard KA, Tromp G, Romero R, et al. Candidate-gene association study of mothers with pre-eclampsia, and their infants, analyzing 775 SNPs in 190 genes. Hum Hered 2007;63(1):1–16. [5] Szarka A, Rigo Jr J, Lazar L, Beko G, Molvarec A. Circulating cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia determined by multiplex suspension array. BMC Immunol 2010;11:59. [6] Dekker GA, Sibai BM. Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstet Gynecol 1998;179(November (5)):1359–75. [7] Mohammadi B, Moghadam Banaem L, Asghari M. The relationship between CRP levels during the first half of pregnancy with preeclampsia. J Reprod Infertil 2010 [Article in Persian]. [8] Cnattingius S, Reilly M, Pawitan Y, Lichtenstein P. Maternal and fetal genetic factors account for most of familial aggregation of preeclampsia: a populationbased Swedish cohort study. Am J Med Genet Part A 2004;130(4):365–71. [9] Salonen Ros H, Lichtenstein P, Lipworth L, Cnattingius S. Genetic effects on the liability of developing pre-eclampsia and gestational hypertension. Am J Med Genet 2000;91(April (4)):256–60. [10] James DSP, Weiner C, Gonik B. High risk pregnancy. third edition, Saunders; 2005. [11] Benyo DF, Miles TM, Conrad KP. Hypoxia stimulates cytokine production by villous explants from the human placenta. J Clin Endocrinol Metab 1997;82(May (5)):1582–8.
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[12] Malek A, Sager R, Schneider H. Effect of hypoxia, oxidative stress and lipopolysaccharides on the release of prostaglandins and cytokines from human term placental explants. Placenta 2001;22(Suppl. A):S45–50. [13] Lachmeijer AM, Dekker GA, Pals G, Aarnoudse JG, ten Kate LP, Arngrimsson R. Searching for preeclampsia genes: the current position. Eur J Obstet Gynecol Reprod Biol 2002;105(November (2)):94–113. [14] Ben-Ari Z, Mor E, Papo O, et al. Cytokine gene polymorphisms in patients infected with hepatitis B virus. Am J Gastroenterol 2003;98(January (1)): 144–50. [15] Hamajima N, Yuasa H. Genetic polymorphisms related to interleukin-1 beta production and disease risk. Nihon Koshu Eisei Zasshi 2003;50(March (3)): 194–207. [16] Lok CA, Boing AN, Reitsma PH, et al. Expression of inflammation-related genes in endothelial cells is not directly affected by microparticles from preeclamptic patients. J Lab Clin Med 2006;147(June (6)):310–20. [17] Haggerty CL, Ferrell RE, Hubel CA, Markovic N, Harger G, Ness RB. Association between allelic variants in cytokine genes and preeclampsia. Am J Obstet Gynecol 2005;193(July (1)):209–15. [18] Fenstad MH, Johnson MP, Roten LT, et al. Genetic and molecular functional characterization of variants within TNFSF13B, a positional candidate preeclampsia susceptibility gene on 13q. PLoS ONE 2010;5(9). [19] Arngrimsson R, Siguroardottir S, Frigge ML, et al. A genome-wide scan reveals a maternal susceptibility locus for pre-eclampsia on chromosome 2p13. Hum Mol Genet 1999;8(September (9)):1799–805.
[20] Skjaerven R, Vatten LJ, Wilcox AJ, Ronning T, Irgens LM, Lie RT. Recurrence of pre-eclampsia across generations: exploring fetal and maternal genetic components in a population based cohort. Br Med J 2005;331(October (7521)):877. [21] Redman CW, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 1999;180(2 Pt 1):499–506. [22] Ness RB. The consequences for human reproduction of a robust inflammatory response. Q Rev Biol 2004;79(December (4)):383–93. [23] Marzi M, Vigano A, Trabattoni D, et al. Characterization of type 1 and type 2 cytokine production profile in physiologic and pathologic human pregnancy. Clin Exp Immunol 1996;106(October (1)):127–33. [24] Sacks GP, Studena K, Sargent K, Redman CW. Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis. Am J Obstet Gynecol 1998;179(July (1)):80–6. [25] Redman CW, Sargent IL. Preeclampsia and the systemic inflammatory response. Semin Nephrol 2004;24(November (6)):565–70. [26] Dunn E, Sims JE, Nicklin MJ, O’Neill LA. Annotating genes with potential roles in the immune system: six new members of the IL-1 family. Trends Immunol 2001;22(October (10)):533–6. [27] Li Y, Xu P, Zhang PA, Li XY, Huang CX. Study on the polymorphism of interleukin-1 gene and the susceptibility to essential hypertension. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2004;21(October (5)):491–3. [28] Pessi T, Karjalainen J, Hulkkonen J, Nieminen MM, Hurme M. A common IL-1 complex haplotype is associated with an increased risk of atopy. J Med Genet 2003;40(May (5)):e66.
Please cite this article in press as: Ghasemi M, et al. Interleukin-1 alpha variation is associated with the risk of developing preeclampsia. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.06.020
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EURO 9029 1–4 European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb 1
Interleukin-1 alpha variation is associated with the risk of developing preeclampsia
2 3 4
Ghasemi a,1, Elham Kashani b,1, Azadeh Fayyaz c, Marzieh Attar c, Majid Shahbazi c,*
Q1 Masoumeh
5 6 7 8 9 10
a
Department of Human Genetics and Medical Cellular & Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran Department of Obstetrics & Gynecology, Sayyad Shirazi Hospital and Medical Cellular & Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran c Medical Cellular & Molecular Research Center, Talghani Children Hospital, Golestan University of Medical Sciences, Bolv Janbazan, Zip code: 4916668197 Gorgan, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 April 2015 Received in revised form 3 June 2015 Accepted 16 June 2015
Objective: Preeclampsia is a syndrome that affects 5% of all pregnancies, producing substantial maternal and prenatal morbidity and mortality. Several studies have reported that cytokine genes are associated with the persistence of preeclampsia or the severity of the disease. The aim of this study is to investigate the relationships between the polymorphisms of interleukin-1 alpha-889 (IL-1A) gene and preeclampsia. Method: Genomic DNA was extracted from the peripheral blood of 305 patients with preeclampsia and 325 normal controls from Sayyad Shirazi Hospital of Golestan University. Then subjected to SSP-PCR amplification. STATA software and the chi square test were used for statistic calculations. Results: The frequencies of IL-1A -889 genotypes C/C, T/T and C/T in preeclampsia cases were 34.8%, 8.2%, 57% and in controls were 20.9%, 7.6% and 71.3% respectively. There was a significant 1.5 fold excess frequency in genotype C/C in cases (CI = 1.44–3.07, OR = 2.1, P = 0.0001). There was a significant difference in the frequencies of alleles or genotypes in IL-1A promoter regions between patients with preeclampsia and the control group. Turkomans showed the highest frequency of the C allele and Sistanies had the lowest frequency of the C allele in preeclampsia compared to control groups (CI = 1.5– 3.9, OR = 2.48, P = 0.0001). Conclusion: Our findings suggest that the IL-1A-899C/C genotype and C allele are associated with susceptibility to preeclampsia. ß 2015 Published by Elsevier Ireland Ltd.
Keywords: Interleukin-1 alpha Polymorphism Preeclampsia Predisposing factor Susceptibility
11 12 13 14 15 16 17 18 19 20
Introduction Q2
Preeclampsia (PE) is a placenta-dependent disease that causes mother and fetus systemic disorders [1,2]. It is characterized by endothelial dysfunction throughout the maternal circulation, resulting in hypertension and proteinuria which typically occurs after 20 week gestation to after delivery during the postpartum period [3–6]. PE2 is a major cause of maternal and fetal mortality and morbidity worldwide [4]. The prevalence of this disease is 5– 7.5% in the USA and 4–4.5% in Iran [4,7]. Several studies on PE
* Corresponding author. Tel.: +98 171 235 17 35; fax: +98 171 235 17 35. E-mail address: [email protected] (M. Shahbazi). 1 These two authors have got equal right as a first author. 2 Preeclampsia.
indicated that ethnic and genetic factors may contribute to the increased risk of PE [1,8,9]. Also, the role of immune system variations in the pathophysiology of preeclampsia has been reported in many studies [10]. Cytokines are a major group of extracellular signal proteins that regulate, proliferate and make cell-to-cell connections in the immune system. Cytokine signals have an important role in the different stages of health and diseases and increases in inflammatory cytokines such as IL-1, IL-10, IL-6 and INF-gamma that are produced in response to local hypoxia and ischemia of the placenta which might trigger endothelial and result in preeclampsia [11–13]. Therefore, the gene encoding pre-inflammatory cytokines participating in the regulation of the immune response are good candidates for further studying maternal genetic susceptibility to preeclampsia. IL-1 is one of cytokines that plays a central role in the inflammation and endothelial function. All family of these genes
http://dx.doi.org/10.1016/j.ejogrb.2015.06.020 0301-2115/ß 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Ghasemi M, et al. Interleukin-1 alpha variation is associated with the risk of developing preeclampsia. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.06.020
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
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EURO 9029 1–4 M. Ghasemi et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2015) xxx–xxx
2
37 38 39 40 41 42 43 44 45 46 47
(IL-1A, IL-1B, IL-1RN) on chromosome (2q13:14) are polymorphic. These polymorphisms are in regulatory regions; they have an important role in regulating the production proteins of IL-1, and are associated with several diseases [14,15]. IL-1A is a subtype of pre-inflammatory cytokines which induce the expression of 40 several inflammatory factors [16]. Haggeri et al. (2005) found an association between this position and preeclampsia; but there are not more studies in the position of IL-1A apparently [17]. In this study, the polymorphism in IL-1A gene was investigated in preeclamptic women to elucidate the association between IL-1A gene polymorphism (-889) and susceptibility to preeclampsia.
48
Materials and methods
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In this study, the blood samples were collected from 305 preeclampsia women and 325 control women with no history of preeclampsia who referred to Sayyad Shirazi Hospital of Golestan University between March 2010 and May 2013. Women with inflammatory, autoimmune and chronic diseases or infections such as HBV were excluded. Controls were selected from among healthy pregnant women who were admitted to the labor ward. Preeclampsia was defined as the development of hypertension of above 140 and 90 mm Hg of systolic and diastolic pressure with proteinuria of +1 or >300 mg in 24 h without renal diseases and infection (according to the report of National High Blood Pressure Education Program Working Group, 2000). Women with chronic hypertension or HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count) without preeclampsia were also excluded from the study. Ethical approval for data collection and the project was approved by the ethics committee of Golestan University of Medical Sciences (Registration Number: 351599012254 and date of issue: 2/ 16/2012). DNA was extracted from the whole blood using a standard phenol-chloroform extraction method and diluted to 50 ng/ml. IL1A gene polymorphism (-889C/T) was genotyped by specific sequence primer polymerase chain reaction (SSP-PCR). Details of primer sequences and fragment size for determining of IL-1A gene polymorphism and human growth hormone (HGH) gene as an internal control are provided in Table 1. The PCR (polymerase chain reaction) amplification was followed by a 15 ml reaction mixture containing 1 ml of genomic DNA, 0/9 ml of 25 mm mgcl2 (Qiagen, USA), 1/5 ml of each 10x buffer (Qiagen, USA), 1/5 ml of 10 mm dNTP, 2/2 ml of sucrose 60%, 0/5 VL of each 10pm specific primer, 0/ 5 ml of each 10pm HGH primers and 0/2 of taq polymerase (Qiagen, USA). The cycling protocol was included in an initial denaturation at 968C for 1 min, 10 cycles for 15 s at 96 8C, 50 s at 63 8C, 40 s at 72 8C (loop 1) and was followed by 20 cycles for 1 min at 96 8C, 50 s at 58 8C, 40 s at 72 8C (loop 2). A total of 30 cycles was carried out, with a final extension at 72 8C for 5 min. The PCR products were subjected to electrophoresis on 1/5% agarose gels and photographed under a UV trans-illuminator (2006 bio-red gel doc) after
Table 1 PCR primer sequences for IL-1A (-889). Gene
Annealing temperature
Fragment size (bp)
Primers
IL-1A
63
221
HGH
64
429
FW: 50 -CTTTAATAATAGTAACCAGGCAACAT-30 FM: 50 -CTTTAATAATAGTAACCAGGCAACAC-30 R: 50 -AAGTAGCCCTCTACCAAGGA-30 HGH1: 50 -GCC TTC CCA ACC ATT CCC TTA-30 HGH2: 50 -TCA CGG ATT TCT GTT GTG TTT C-30
staining with ethidium bromide. Allele frequencies were tested for hardy-Weinberg equilibrium using Chi-squared test and/or Fisher exact test. These tests were used to compare the genotype frequency between preeclamptic and control groups. SPSS 16 software (IBM, Armonk, NY, USA) was also applied, the level of statistical significance was defined as P < 0.05, odds ratios (ORs) were calculated on a 95% confidence interval (CI).
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Results
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Using SSP-PCR, the fragments containing the positions where the IL-1/_889 SNP occurred in 305 patients with preeclampsia and 325 control subjects were amplified. Statistical analysis showed a significant difference between preeclampsia and control groups in terms of allelic and genotype frequency (Table 2) so that the C/C genotype frequency was significantly higher in the patients (CI = 1.44–3.07, OR = 2.1, P = 0.0001). Also, the C allele frequency was significantly higher in the patients than control group (CI = 1.04–1.66, OR = 1.32, P = 0.016), while T allele did not show any significant difference between the two groups. However the present study demonstrated a significant relationship between of the IL-1A/-889 gene polymorphism and preeclampsia. Case and control groups of this work were separated into three groups based on reproductive age that included: <18, between 18 and 40 and >40 years old. In terms of ethnicity, they were divided into four subgroups of Persian, Turkoman, Sistani and others3 based on the participant’s biography. Statistical observations are summarized in Table 3. Investigation in the mentioned age groups showed no statistically significant differences between the case and control groups. But, studying the ethnicity provided the following results: Although genotyping of the following ethnic groups was slightly different, these changes were not statistically significant and needed to be studied in a larger population. However, studies demonstrated significant allelic differences so that the C allele significantly increased in the sub-groups of Turkomans, Persians, and Sistanies respectively. Turkomans showed the highest frequency of the C allele and Sistanies had the lowest frequency of the C allele in preeclampsia compared to control groups. However, T allele was significantly increased in the sub-groups of Sistanies, Persians, and Turkomans respectively. The obtained results are separately shown in Table 4.
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Comments
126
In this study, the region of the IL1a gene position -889 was analyzed using SSP-PCR and a T to C polymorphism was identified in this position relative to preeclampsia. Highly significant correlation was found between homozygosity for the IL1a 889*C allele and development of PE. Also, the investigation of subethnic groups in this study showed that Turkomans had the highest percentage of the C allele and Sistani ethnicity had the lowest percentage in PE compared with control groups. These data suggested that the C allele might be as a susceptibility factor for preeclampsia in Turkoman ethnicity and T allele can have a protective role against PE in Sistani ethnicity. Both genetics and inflammatory factors play a vital role in the pathogenesis of preeclampsia [8,18]. Numerous studies have demonstrated the role of inheritance in preeclampsia [19,20]; but, the way genetics and molecular mechanisms work in preeclampsia is still unknown. An aggressive inflammatory response may result in numerous adverse reproductive outcomes including preeclampsia [21–24]. An abnormal genetic predisposition, for example, exaggerates
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3
Other groups included Baluchistan and Afghanistan ethnicities.
Please cite this article in press as: Ghasemi M, et al. Interleukin-1 alpha variation is associated with the risk of developing preeclampsia. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.06.020
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EURO 9029 1–4 M. Ghasemi et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2015) xxx–xxx Table 2 Allele and genotype frequencies of IL-1A/-889 in preeclampsia and healthy pregnant controls. IL1a polymorphism
Controls N = 325
PE N = 305
OR
Allele C T
368 (56.6%) 282 (43.4%)
386 (63.2%) 224 (36.7%)
1.32 1
Genotype C/C C/T T/T
68 (20.9%) 232 (71.3%) 25 (7.6%)
106 (34.8%) 174 (57%) 25 (8.2%)
2.1 1 NS
Comparison
PE
Control
OR
95%CI
P value
1.04–1.66 –
0.016 –
1.44–3.07 – NS
0.0001 – NS
C/Turkoman vs. C/Persian C/Persian vs. C/Sistani C/Turkoman vs. C/Sistani T/Sistani vs. T/Persian T/Persian vs. T/Turkoman T/Sistani vs. T/Turkoman
82/188 188/97 82/97 57/112 112/42 57/42
46/167 167/135 46/135 107/133 133/28 107/28
1.58 1.56 2.48 0.63 0.56 0.35
1.02–2.46 1.10–2.21 1.55–3.97 0.41–0.96 0.31–0.99 0.19–0.65
0.0376 0.0089 0.0001 0.031 0.0491 0.0003
cytokine and may promote an inflammatory response which leads to oxidative stress, endothelial dysfunction, and preeclampsia [25]. Instead, cytokine allele that was of functional relevance was selected here, because it was assumed that genetic polymorphisms that lead to increased pre-inflammatory cytokine production may be associated with preeclampsia. IL-1alpha is an important pre-inflammatory factor for beginning of inflammatory diseases [16,26]. Based on the promoter role of (-889) position of the IL-1a gene, the expected molecular variation of this region leads to promoter activation, increases gene transcription, and thus promotes the inflammatory response [27]. Therefore, the present study revealed that polymorphism of this gene may influence susceptibility factor for preeclampsia. The only study which has been done on the IL-1alpha (-889) gene polymorphism was by Haggeri et al. (2005) who investigated several polymorphisms of cytokines and provided evidence for the relationship between IL-1A genotype in -889 position in both white and black women with PE compared to the control subjects so that the frequency of CC genotype was associated with the increased risk of PE [17]. Also, Goodard et al. investigated IL-1A polymorphism in three positions that were different from those of the present study and demonstrated a relationship between polymorphism in 4 and 6 intron positions and PE along with a lack of relationship between polymorphism in 5 exone position and PE [4]. Although the functional significances of the IL-1A -889 and 4845 polymorphisms lead to the variation of amino acids in IL-1A protein and are associated with the increased risk of atopy [28], the
Table 3 Demographics in preeclamptic and healthy women. Variables
Healthy women n = 325 (%)
Preeclamptic women n = 305 (%)
P value
Classification of preeclampsia Mild preeclamptic – Sever preeclamptica –
218 (71.47) 87 (25.57)
Maternal age (year) <18 18–40 >40
10 (3.07) 314 (96.61) 1 (0.30)
7 (2.29) 294 (96.39) 4 (1.31)
>0.05
Ethnic Persian Turkoman Sistani Other groups
150 37 121 17
150 62 77 16
<0.05b
(46.1) (11.3) (37.2) (5.2)
Table 4 Frequencies of risk alleles (IL-1 -889C and T) in PEa patients and control ethnics subgroups.
P valuea
95% CI
OR = odds ratio; 95% CI = 95% confidence interval. a Fisher’s exact test.
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3
(49.1) (20.3) (25.2) (5.2)
–
a The presence of any one of these findings: Blood pressure 160/110 mmHg, proteinuria > 5 g on a 24 h collection, liver and renal dysfunction, pulmonary edema, fatal growth restriction. b Based on chi square test; p < 0.05 was considered significant.
a
PE, preeclampsia; OR, odds ratio; 95% CI, 95% confidence interval.
relationship between IL-1A gene polymorphism and preeclampsia may indicate the functional role of C allele in sustaining the immune response. Although this gene may be assumed as a strong candidate which is associated with PE, it may vary in different populations and is associated with multi-factorial diseases. In conclusion, an association was founded between this polymorphism and susceptibility to preeclampsia in the Iranian population. However, future studies on different ethnic populations and on other polymorphic regions in cytokine genes would provide a more accurate definition for the involvement of cytokine genetic polymorphisms in the development of preeclampsia and cytokine genotyping may prove to be an effective early pregnancy preeclampsia risk-screening tool.
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Conflicts of interest
187
All contributing authors declare no conflicts of interest Acknowledgments
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We thank all the women that participated in this study. We 190 thank the Medicine Cellular and Molecular Research Centre 191 (MCMRC) for recruiting study subjects. We wish to thank all 192 gynecologists from Sayyad Shirazi Hospital for enabling us to 193 collect samples and maintaining the study database. Our study was 194 financially supported by Golestan University of Medical Science Q3195 (Grant Number: 35/262). 196 References
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