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Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women
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EURO 8965 1–5 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
Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women
2 3 4
Wang a, G. Wang a, C. Guo a, X. Cao a, L. An a, M. Du a, Y. Qiu a, Y. Yang a, Y. Wang b, S. Wang b, X. Wang a,*, X. Ma a,c,d
Q1 Q.
5 6 7 8 9
a
National Research Institute for Family Planning, Beijing, China Department of Obstetrics and Gynaecology, Shengjing Hospital of China Medical University, Shenyang, China Graduate School of Peking Union Medical College, Beijing, China d World Health Organization Collaborating Centre for Research in Human Reproduction, Beijing, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 December 2014 Received in revised form 11 February 2015 Accepted 2 April 2015
Context and objective: Pre-eclampsia (PE) is the most common medical complication of pregnancy encountered worldwide. A previous genome-wide association study showed that three single nucleotide polymorphisms (SNPs) located near the inhibin beta B gene (INHBB) – rs12711941 (T>G), rs7576192 (A>G) and rs7579169 (T>C) – were associated with PE in Australian women. The present study was undertaken to assess the genetic association between these three SNPs and the risk of PE in Han Chinese women. Patients: One hundred and eighty-one pregnant women with PE and 203 healthy pregnant women (controls) were recruited from the Department of Obstetrics and Gynaecology at the Shengjing Hospital of China Medical University from October 2012 to June 2013. Study design: PE patients and controls underwent clinical and biochemical examination. The three SNPs were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PE patients were divided into various subgroups for comparison with controls. Results: The rs7579169 CC genotype was found to have a significantly higher frequency in PE patients than in controls [TT vs TC + CC: odds ratio (OR) = 2.278, p = 0.040], indicating that the C allele of rs7579169 confers a risk for PE [OR = 1.4440, 95% confidence interval (CI) 1.0517–1.9827, p = 0.0229]. The rs7576192 genotype showed significant differences between controls and multiparous PE patients (x2 = 4.6088, df = 1, P = 0.0319, OR = 0.6379, 95% CI 0.4223–0.9634). However, neither allele nor genotype frequencies of rs12711941 differed significantly between patients and controls in any genetic models or subgroup analyses. Haplotype analysis revealed that the H1 haplotype (G-G-C) was associated with PE (OR = 1.437, 95% CI 1.034–1.997, p < 0.05), whereas the H2 haplotype (G-G-T) offered protection against PE (OR = 0.482, 95% CI 0.232–1.001, p < 0.05). Conclusion: Pregnant Han Chinese women carrying the rs7579169 CC genotype and G-G-C haplotype are significantly more likely to develop PE, especially late-onset and multiparous cases. ß 2015 Published by Elsevier Ireland Ltd.
Keywords: SNP Pre-eclampsia Genome-wide association study INHBB
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Introduction Q2
Pre-eclampsia (PE) is a disorder of pregnancy characterized by high blood pressure and high levels of protein in the urine. It occurs in approximately 3–7% of pregnancies, especially first pregnancies or those of twins [1,2]. It is one of the most important causes of
* Corresponding author at: Centre for Genetics, National Research Institute for Family Planning, 12 Dahuisi Road, Haidian, Beijing 100081, China. Tel.: +86 010 62117712; fax: +86 010 62179151. E-mail address: [email protected] (X. Wang).
maternal and neonatal mortality, leading to 50,000 maternal deaths annually [3]. PE is thought to be related to several factors, including abnormalities of trophoblast cell infiltration, impaired placental perfusion, vascular endothelial injury and oxidative stress [4–8]. Genetic factors have been suggested to be responsible for more than 50% of PE liability [9–11], but the inheritance pattern remains unclear. The tendency for PE to be passed on from mother to daughter was the first pattern to be observed [12–14], although fathers also appear to contribute to PE susceptibility, possibly through paternal transmission of genes to the fetus [15,16]. To date, many maternal susceptibility loci have been identified by genome-wide association studies (GWAS) [3,17–20]. A previous
http://dx.doi.org/10.1016/j.ejogrb.2015.04.001 0301-2115/ß 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Wang Q, et al. Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.04.001
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GWAS in unrelated Australian women of Caucasian ancestry genotyped 648,175 single nucleotide polymorphisms (SNPs) in 538 PE patients and 540 controls, and identified three that were in linkage disequilibrium: rs12711941, rs7576192 and rs7579169 [3]. These SNPs reside in an intergenic region less than 15 kb downstream from the 30 terminus of the inhibin beta B gene (INHBB) on 2q14.2. The INHBB protein is the b subunit of inhibin, a pituitary follicle-stimulating hormone secretion inhibitor that has been suggested to play a role in PE development together with other maternal susceptibility genes [21]. The present study was undertaken to research the relationship between these three SNPs and PE in Han Chinese women.
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Materials and methods
42
Patients and controls
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
This study was approved by the Ethics Committee of the National Research Institute for Family Planning. Written informed consent was obtained from each pregnant woman and her family. The research agreement was in accordance with the Declaration of Helsinki [22]. With no diversity in genetic background, the participants were all from northeastern China (Heilongjiang, Liaoning and Jilin provinces). Due to the harsh cold weather in these areas (minimum temperatures range from 34 to 28 8C), the incidence of PE is higher than in other areas of China [1,22]. All control participants were selected at random from the same region. They did not have proteinuria, and delivered a healthy neonate at term (37 weeks of gestation) with no obstetric complications, such as diabetes, renal insufficiency and chronic hypertension. PE patients were defined as women with gestational hypertension with proteinuria (blood pressure >140/90 mmHg on two measurements at least 6 h apart, 24 h urinary protein >300 mg) after 20 weeks of gestation. PE patients were divided into various subgroups: mild PE (n = 97, two separate blood pressure measurements 140/ 90 mmHg and proteinuria 300 mg protein/24 h) and severe PE (n = 84, at least three separate blood pressure measurements 160/100 mmHg combined with proteinuria 2 g protein/24 h); early-onset PE (n = 68, gestational age 34 weeks) and lateonset PE (n = 113, gestational age >34 weeks); and primiparas (n = 78, women who had only conceived once) and multiparas (n = 103, women who had conceived more than once). The clinical characteristics of PE patients and controls are shown in Table 1.
DNA extraction and genotyping
72
Blood samples from patients and healthy donors were collected and stored at 20 8C. Genomic DNA was extracted from peripheral blood leukocytes according to conventional proteinase K digestion and phenol/chloroform extraction methods [23]. Primers were designed using https://www.mysequenom.com/Tools, and the genotypes of the three SNPs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [24].
73 74 75 76 77 78 79
Statistical analysis
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Chi-squared tests were used to compare the clinical data. Allele frequency and genotype distribution of the three SNPs were analyzed for deviation from Hardy–Weinberg equilibrium (HWE; p > 0.05) using Chi-squared test. Comparison of allele and genotype frequencies between PE patients and controls was evaluated using Pearson’s Chi-squared test in Statistical Package for the Social Sciences Version 13.0 (SPSS Inc., Chicago, IL). Odds ratios (ORs) and 95% confidence intervals (CIs) were computed to evaluate the strength of the relationship with allele distribution. Genetic models were divided into additive (+/+ vs +/ vs /), dominant (+/+ plus +/ vs /) and recessive (+/+ vs +/plus /) groups. Finally, linkage disequilibrium, haplotype construction and genetic association of the polymorphic loci were analyzed using SHEsis software [25,26]. Significant differences between PE patients and controls were defined by p < 0.05, and all statistical tests were two sided.
81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Results
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One hundred and eighty-one PE patients and 203 controls were analyzed in this study, and their demographic and clinical characteristics are displayed in Table 1. Significant differences in several characteristics were observed between the groups. PE patients had significantly higher maternal age, blood pressure and number of pregnancies compared with the controls (p < 0.05). Similar to previous epidemiological studies, PE patients had significantly higher body mass index (p < 0.05) [27]. Moreover, in accordance with PE leading to intra-uterine growth restriction and preterm birth, offspring born to PE patients had significantly lower fetal weight and gestational age at delivery (p < 0.05) [27]. The number of primiparous women did not differ significantly between the two groups. Allele frequencies did not deviate from HWE in either PE patients or controls. As shown in Table 2, a significant difference was observed in the allele frequency of rs7579169 between PE
98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113
Table 1 Clinical characteristics (mean standard deviation) of the study population. Characteristics
Controls (n = 203)
PE (n = 181)
Maternal age Primiparous SBP (mmHg) DBP (mmHg) Gestational age at delivery (weeks) Number of pregnancies Body mass index Newborn weight (g)
29.4 4.3 124 (61.1%) 117.8 10.9 77.5 6.9 39.1 1.0 1.5 0.6 23.71 0.06 3359 28.81
31 6a 130 (71.8%)a,c 164.5 23a 104.7 16.2 34.3 3.4a 1.9 1.2a 26.48 0.12a 2201 64.93a
PE Mild (n = 97)
Severe (n = 84)
29.42 5.2 67 (69.1%)c 149.5 12.4a 94.9 8.5a 35.4 2.6a 1.8 1.2a 25.76 0.13a 1974 83.00a
31.85 6.1a,b 63 (75%)a,c 181.5 18.9a,b 115.8 14.5a,b 33.1 3.9a,b 2.1 1.2a,b 27.39 0.17a,b 2493 121.3a
SBP, systolic blood pressure; DBP, diastolic blood pressure; PE, pre-eclampsia. a p < 0.05 vs control. b p < 0.05 vs mild PE. c Differences between discrete variables evaluated with Chi-squared test.
Please cite this article in press as: Wang Q, et al. Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.04.001
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EURO 8965 1–5 Q. Wang et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2015) xxx–xxx Table 2 Allele frequencies of rs7579169 in women with and without pre-eclampsia (PE). Group
No.
Allele (C/T)
MAF
OR (95% CI)
p-value
Controls PE
203 181
249/131 258/94
0.345 0.267
1.44 (1.05–1.98)
0.02
97 84
139/51 119/43
0.268 0.265
1.43 (0.98–2.11) 1.46 (0.97–2.19)
0.07 0.07
Early-onset Late-onset
68 113
92/42 166/52
0.313 0.239
1.15 (0.76–1.76) 1.68 (1.15–2.45)
0.51 0.007
Primiparas Multiparas
78 103
105/47 153/47
0.309 0.235
1.18 (0.78–1.76) 1.71 (1.16–2.53)
0.43 0.006
PE Mild Severe
MAF, minor allele frequency; OR, odds ratio between case and control groups; CI, confidence interval.
114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144
patients and controls (x2 = 5.1808, df = 1, p = 0.0229, OR = 1.4440, 95% CI 1.0517–1.9827). In the subgroup analysis, rs7579169 C/T allele frequency was significantly different in multiparous (x2 = 7.4182, df = 1, p = 0.0065, OR = 1.7126, 95% CI 1.1603– 2.5279) and late-onset PE patients (x2 = 7.3575, df = 1, p = 0.0067, OR = 1.6795, 95% CI 1.1525–2.4473), but not in primiparous (x2 = 0.6155, df = 1, p = 0.4328, OR = 1.1753, 95% CI 0.7848–1.7601) and early-onset PE patients (x2 = 0.4348, df = 1, p = 0.5097, OR = 1.1524, 95% CI 0.7558–1.7572), compared with controls, suggesting that the C allele of rs7579169 is a risk factor for PE, especially in late-onset and multiparous cases. Genotype frequencies were also analyzed in this study, and a significant difference was observed between PE patients and controls in the dominant genetic model analysis of rs7579169 (p = 0.040, Table 3). Subgroup analysis showed that the genotype frequency of rs7579169 differed significantly in late-onset PE patients in both the dominant and recessive genetic models (p = 0.035 and p = 0.049, respectively). Similar to the gestational subgroup, the genotype frequency of rs7579169 was significantly different in multiparous PE patients compared with controls in both the dominant and recessive genetic models (p = 0.041 and p = 0.043, respectively). Rs7576192 showed no significant differences between controls and PE patients, apart from allele analysis in multiparous PE patients (x2 = 4.6088, df = 1, p = 0.0319, OR = 0.6379, 95% CI 0.4223–0.9634, Table 4). No significant difference in allele or genotype frequency was observed between PE patients and controls for rs12711941 (Table 5). Haplotype analysis was adopted based on the measured Q3 genotypes (Table 6). The order of SNPs was as follows: rs12711941, rs7576192 and rs7579169. All frequencies <0.03
3
Table 3 Genotype frequencies of rs7579169 in women with and without pre-eclampsia (PE). Group
No.
Frequency (CC/CT/TT)
padd
pdom
prec
Controls PE
190 176
98/53/39 104/50/22
0.107
0.040
0.149
95 81
57/25/13 47/25/9
0.285 0.178
0.159 0.063
0.178 0.330
Early-onset Late-onset
67 109
35/22/10 69/28/12
0.540 0.064
0.316 0.035
0.926 0.049
Primiparas Multiparas
76 100
40/25/11 64/25/11
0.465 0.065
0.254 0.041
0.877 0.043
PE Mild Severe
Padd, p-value of additive model (three genotypes). Pdom, p-value of dominant model [(homozygotes of risk allele + heterozygotes) vs homozygotes of non-risk allele]. prec, p-value of recessive model [homozygotes of risk allele vs (heterozygotes + homozygotes of non-risk allele)]. The T allele is the risk allele.
were excluded from analysis. The frequency of the H1 haplotype (G-G-C) was significantly different in PE patients compared with controls (p = 0.0304, OR = 1.437, 95% CI 1.034–1.997). Moreover, the H2 haplotype (G-G-T) had a significantly lower frequency in PE patients than in controls (p = 0.0460, OR = 0.482, 95% CI 0.232– 1.001).
145 146 147 148 149 150
Comments
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PE is currently thought to be a syndrome rather than a definite disease. Although family studies suggest that it may have a heritable component, its underlying cause and pathogenesis remain to be uncovered [12–30]. In the present study, three SNPs (rs12711941, rs7576192 and rs7579169) previously associated with PE in an Australian population demonstrated different associations with PE in Chinese women. Weak relationships were found between rs12711941 and rs7576192 and PE, while a significant association was observed between rs7579169 and PE. Following subgroup analysis, rs12711941 maintained its negative association with PE, and rs7576192 was only found to be significantly associated with PE in multiparous PE patients. However, a strong association was observed in the late-onset and multiparous subgroups compared with controls in both the dominant and recessive models. These findings suggest that the C allele of rs7579169 is a risk allele for PE (p = 0.0229, OR = 1.4440, 95% CI 1.0517–1.9827).
152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168
Table 4 Allele and genotype frequencies of rs7576192 in women with and without pre-eclampsia (PE). Group
No.
Allele (A/G)
MAF
OR (95% CI)
p-value
Controls PE
203 181
81/269 107/273
0.282 0.231
0.77 (0.55–1.07)
0.12
96 85
44/144 37/125
0.234 0.228
0.78 (0.52–1.17) 0.76 (0.49–1.16)
Early-onset Late-onset
68 113
35/99 46/170
0.261 0.213
Primiparas Multiparas
78 103
41/109 40/160
0.273 0.200
PE Mild Severe
Frequency (AA/AG/GG)
padd
pdom
prec
15/77/98 10/61/104
0.298
0.132
0.410
0.23 0.20
6/32/56 4/29/48
0.631 0.118
0.203 0.246
0.647 0.383
0.90 (0.58–1.41) 0.69 (0.47–1.03)
0.65 0.07
3/29/35 7/32/69
0.444 0.437
0.926 0.040
0.346 0.654
0.96 (0.63–1.47) 0.64 (0.42–0.96)
0.85 0.03
6/29/40 4/32/64
0.961 0.101
0.797 0.043
0.977 0.203
MAF, minor allele frequency; OR, odds ratio between case and control group; CI, confidence interval; pdom, p-value of dominant model [(homozygotes of risk allele + heterozygotes) vs homozygotes of non-risk allele]; prec, p-value of recessive model [homozygotes of risk allele vs (heterozygotes + homozygotes of non-risk allele)]. The A allele is the risk allele.
Please cite this article in press as: Wang Q, et al. Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.04.001
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Table 5 Allele and genotype frequencies of rs1271941 in women with and without pre-eclampsia (PE). Group
No.
Allele (G/T)
MAF
OR (95% CI)
p-value
Controls PE
203 181
284/96 270/78
0.253 0.224
1.17 (0.83–1.65)
0.37
97 84
147/43 123/35
0.226 0.222
1.16 (0.77–1.74) 1.19 (0.76–1.85)
Early-onset Late-onset
68 113
97/33 173/45
0.254 0.206
Primiparas Multiparas
78 103
108/40 162/38
0.270 0.190
PE Mild Severe
Frequency (GG/GT/TT)
padd
pdom
prec
106/72/12 106/58/10
0.610
0.820
0.322
0.49 0.44
57/33/5 49/25/5
0.785 0.612
0.724 0.997
0.498 0.346
1.00 (0.63–1.57) 1.30 (0.87–1.94)
0.98 0.20
36/25/4 70/33/6
0.996 0.358
0.933 0.777
0.955 0.154
0.91 (0.59–1.40) 1.44 (0.95–2.20)
0.68 0.09
40/28/6 66/30/4
0.869 0.229
0.604 0.412
0.926 0.092
MAF, minor allele frequency; OR, odds ratio between case and control group; CI, confidence interval; pdom, p-value of dominant model [(homozygotes of risk allele + heterozygotes) vs homozygotes of non-risk allele]; prec, p-value of recessive model [homozygotes of risk allele vs (heterozygotes + homozygotes of non-risk allele)]. The T allele is the risk allele.
Table 6 Haplotype frequency estimates and their association with pre-eclampsia. Allotypea
H1 H2 H3
SNP SNP1
SNP2
SNP3
G G T
G G A
C T T
Frequency
Odds ratio (95% CI)
p-value
0.725 0.032 0.214
1.437 (1.034–1.997) 0.482 (0.232–1.001) 0.796 (0.563–1.127)
0.030 0.046 0.199
CI, confidence interval. Frequency <0.03 in both controls and patients with pre-eclampsia was ignored in the analysis. a The single nucleotide polymorphism (SNP) order was SNP1 = rs19711941, SNP2 = rs7576192 and SNP3 = rs7579169.
169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197
Haplotype analysis indicated that the H1 haplotype (G-G-C) is a risk factor (p = 0.0304), and the H2 haplotype (G-G-T) is a protective factor against PE (p = 0.0460). It is noteworthy that these two haplotypes differ only at the third SNP site (rs7579169), which is consistent with the findings of allele analysis. The experimental design of this study is a particular strength, because it involved the unbiased detection of cases and controls, and used a well-defined phenotype of PE. Moreover, all participants were Han Chinese women from northeastern China with no variation in their genetic background. To the best of the authors’ knowledge, this is the first report to show that SNPs located near INHBB are associated with PE in the Han Chinese population. However, several limitations of this study should be taken into consideration. First, genetic factors that influence the pathogenesis of PE differ by ethnic group, so the association between rs12711941, rs7576192, rs7579169 and PE should be tested in a larger sample size composed of different racial groups. Second, the functional significance of rs7579169 in PE remains unknown. Third, no significant differences in allele and genotype frequencies of rs12711941 and rs7576192 were found between PE patients and controls, despite using the same statistical methods as studies that reported significant differences. Therefore, future studies should evaluate the associations between PE and these two genetic variants, particularly rs12711941. In conclusion, a significant association was found between rs7579169 and PE in Han Chinese women. Women who carry the CC genotype are therefore at higher risk of PE, especially multiparous and late-onset cases. However, additional studies are required to confirm and extend these findings.
198
Acknowledgments
199 200 201
The authors wish to thank all subjects for participating in this study, and are grateful to Professor Hui Li from the Department of Obstetrics and Gynecology at the Shengjing Hospital of China
Medical University for sharing the samples. This work was supported by the National International Scientific and Technologi- Q4 cal Cooperation Program 2012DFB30130.
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EURO 8965 1–5 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
Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women
2 3 4
Wang a, G. Wang a, C. Guo a, X. Cao a, L. An a, M. Du a, Y. Qiu a, Y. Yang a, Y. Wang b, S. Wang b, X. Wang a,*, X. Ma a,c,d
Q1 Q.
5 6 7 8 9
a
National Research Institute for Family Planning, Beijing, China Department of Obstetrics and Gynaecology, Shengjing Hospital of China Medical University, Shenyang, China Graduate School of Peking Union Medical College, Beijing, China d World Health Organization Collaborating Centre for Research in Human Reproduction, Beijing, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 December 2014 Received in revised form 11 February 2015 Accepted 2 April 2015
Context and objective: Pre-eclampsia (PE) is the most common medical complication of pregnancy encountered worldwide. A previous genome-wide association study showed that three single nucleotide polymorphisms (SNPs) located near the inhibin beta B gene (INHBB) – rs12711941 (T>G), rs7576192 (A>G) and rs7579169 (T>C) – were associated with PE in Australian women. The present study was undertaken to assess the genetic association between these three SNPs and the risk of PE in Han Chinese women. Patients: One hundred and eighty-one pregnant women with PE and 203 healthy pregnant women (controls) were recruited from the Department of Obstetrics and Gynaecology at the Shengjing Hospital of China Medical University from October 2012 to June 2013. Study design: PE patients and controls underwent clinical and biochemical examination. The three SNPs were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PE patients were divided into various subgroups for comparison with controls. Results: The rs7579169 CC genotype was found to have a significantly higher frequency in PE patients than in controls [TT vs TC + CC: odds ratio (OR) = 2.278, p = 0.040], indicating that the C allele of rs7579169 confers a risk for PE [OR = 1.4440, 95% confidence interval (CI) 1.0517–1.9827, p = 0.0229]. The rs7576192 genotype showed significant differences between controls and multiparous PE patients (x2 = 4.6088, df = 1, P = 0.0319, OR = 0.6379, 95% CI 0.4223–0.9634). However, neither allele nor genotype frequencies of rs12711941 differed significantly between patients and controls in any genetic models or subgroup analyses. Haplotype analysis revealed that the H1 haplotype (G-G-C) was associated with PE (OR = 1.437, 95% CI 1.034–1.997, p < 0.05), whereas the H2 haplotype (G-G-T) offered protection against PE (OR = 0.482, 95% CI 0.232–1.001, p < 0.05). Conclusion: Pregnant Han Chinese women carrying the rs7579169 CC genotype and G-G-C haplotype are significantly more likely to develop PE, especially late-onset and multiparous cases. ß 2015 Published by Elsevier Ireland Ltd.
Keywords: SNP Pre-eclampsia Genome-wide association study INHBB
10 11 12 13 14 15
Introduction Q2
Pre-eclampsia (PE) is a disorder of pregnancy characterized by high blood pressure and high levels of protein in the urine. It occurs in approximately 3–7% of pregnancies, especially first pregnancies or those of twins [1,2]. It is one of the most important causes of
* Corresponding author at: Centre for Genetics, National Research Institute for Family Planning, 12 Dahuisi Road, Haidian, Beijing 100081, China. Tel.: +86 010 62117712; fax: +86 010 62179151. E-mail address: [email protected] (X. Wang).
maternal and neonatal mortality, leading to 50,000 maternal deaths annually [3]. PE is thought to be related to several factors, including abnormalities of trophoblast cell infiltration, impaired placental perfusion, vascular endothelial injury and oxidative stress [4–8]. Genetic factors have been suggested to be responsible for more than 50% of PE liability [9–11], but the inheritance pattern remains unclear. The tendency for PE to be passed on from mother to daughter was the first pattern to be observed [12–14], although fathers also appear to contribute to PE susceptibility, possibly through paternal transmission of genes to the fetus [15,16]. To date, many maternal susceptibility loci have been identified by genome-wide association studies (GWAS) [3,17–20]. A previous
http://dx.doi.org/10.1016/j.ejogrb.2015.04.001 0301-2115/ß 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Wang Q, et al. Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.04.001
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GWAS in unrelated Australian women of Caucasian ancestry genotyped 648,175 single nucleotide polymorphisms (SNPs) in 538 PE patients and 540 controls, and identified three that were in linkage disequilibrium: rs12711941, rs7576192 and rs7579169 [3]. These SNPs reside in an intergenic region less than 15 kb downstream from the 30 terminus of the inhibin beta B gene (INHBB) on 2q14.2. The INHBB protein is the b subunit of inhibin, a pituitary follicle-stimulating hormone secretion inhibitor that has been suggested to play a role in PE development together with other maternal susceptibility genes [21]. The present study was undertaken to research the relationship between these three SNPs and PE in Han Chinese women.
41
Materials and methods
42
Patients and controls
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
This study was approved by the Ethics Committee of the National Research Institute for Family Planning. Written informed consent was obtained from each pregnant woman and her family. The research agreement was in accordance with the Declaration of Helsinki [22]. With no diversity in genetic background, the participants were all from northeastern China (Heilongjiang, Liaoning and Jilin provinces). Due to the harsh cold weather in these areas (minimum temperatures range from 34 to 28 8C), the incidence of PE is higher than in other areas of China [1,22]. All control participants were selected at random from the same region. They did not have proteinuria, and delivered a healthy neonate at term (37 weeks of gestation) with no obstetric complications, such as diabetes, renal insufficiency and chronic hypertension. PE patients were defined as women with gestational hypertension with proteinuria (blood pressure >140/90 mmHg on two measurements at least 6 h apart, 24 h urinary protein >300 mg) after 20 weeks of gestation. PE patients were divided into various subgroups: mild PE (n = 97, two separate blood pressure measurements 140/ 90 mmHg and proteinuria 300 mg protein/24 h) and severe PE (n = 84, at least three separate blood pressure measurements 160/100 mmHg combined with proteinuria 2 g protein/24 h); early-onset PE (n = 68, gestational age 34 weeks) and lateonset PE (n = 113, gestational age >34 weeks); and primiparas (n = 78, women who had only conceived once) and multiparas (n = 103, women who had conceived more than once). The clinical characteristics of PE patients and controls are shown in Table 1.
DNA extraction and genotyping
72
Blood samples from patients and healthy donors were collected and stored at 20 8C. Genomic DNA was extracted from peripheral blood leukocytes according to conventional proteinase K digestion and phenol/chloroform extraction methods [23]. Primers were designed using https://www.mysequenom.com/Tools, and the genotypes of the three SNPs were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [24].
73 74 75 76 77 78 79
Statistical analysis
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Chi-squared tests were used to compare the clinical data. Allele frequency and genotype distribution of the three SNPs were analyzed for deviation from Hardy–Weinberg equilibrium (HWE; p > 0.05) using Chi-squared test. Comparison of allele and genotype frequencies between PE patients and controls was evaluated using Pearson’s Chi-squared test in Statistical Package for the Social Sciences Version 13.0 (SPSS Inc., Chicago, IL). Odds ratios (ORs) and 95% confidence intervals (CIs) were computed to evaluate the strength of the relationship with allele distribution. Genetic models were divided into additive (+/+ vs +/ vs /), dominant (+/+ plus +/ vs /) and recessive (+/+ vs +/plus /) groups. Finally, linkage disequilibrium, haplotype construction and genetic association of the polymorphic loci were analyzed using SHEsis software [25,26]. Significant differences between PE patients and controls were defined by p < 0.05, and all statistical tests were two sided.
81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Results
97
One hundred and eighty-one PE patients and 203 controls were analyzed in this study, and their demographic and clinical characteristics are displayed in Table 1. Significant differences in several characteristics were observed between the groups. PE patients had significantly higher maternal age, blood pressure and number of pregnancies compared with the controls (p < 0.05). Similar to previous epidemiological studies, PE patients had significantly higher body mass index (p < 0.05) [27]. Moreover, in accordance with PE leading to intra-uterine growth restriction and preterm birth, offspring born to PE patients had significantly lower fetal weight and gestational age at delivery (p < 0.05) [27]. The number of primiparous women did not differ significantly between the two groups. Allele frequencies did not deviate from HWE in either PE patients or controls. As shown in Table 2, a significant difference was observed in the allele frequency of rs7579169 between PE
98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113
Table 1 Clinical characteristics (mean standard deviation) of the study population. Characteristics
Controls (n = 203)
PE (n = 181)
Maternal age Primiparous SBP (mmHg) DBP (mmHg) Gestational age at delivery (weeks) Number of pregnancies Body mass index Newborn weight (g)
29.4 4.3 124 (61.1%) 117.8 10.9 77.5 6.9 39.1 1.0 1.5 0.6 23.71 0.06 3359 28.81
31 6a 130 (71.8%)a,c 164.5 23a 104.7 16.2 34.3 3.4a 1.9 1.2a 26.48 0.12a 2201 64.93a
PE Mild (n = 97)
Severe (n = 84)
29.42 5.2 67 (69.1%)c 149.5 12.4a 94.9 8.5a 35.4 2.6a 1.8 1.2a 25.76 0.13a 1974 83.00a
31.85 6.1a,b 63 (75%)a,c 181.5 18.9a,b 115.8 14.5a,b 33.1 3.9a,b 2.1 1.2a,b 27.39 0.17a,b 2493 121.3a
SBP, systolic blood pressure; DBP, diastolic blood pressure; PE, pre-eclampsia. a p < 0.05 vs control. b p < 0.05 vs mild PE. c Differences between discrete variables evaluated with Chi-squared test.
Please cite this article in press as: Wang Q, et al. Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.04.001
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EURO 8965 1–5 Q. Wang et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology xxx (2015) xxx–xxx Table 2 Allele frequencies of rs7579169 in women with and without pre-eclampsia (PE). Group
No.
Allele (C/T)
MAF
OR (95% CI)
p-value
Controls PE
203 181
249/131 258/94
0.345 0.267
1.44 (1.05–1.98)
0.02
97 84
139/51 119/43
0.268 0.265
1.43 (0.98–2.11) 1.46 (0.97–2.19)
0.07 0.07
Early-onset Late-onset
68 113
92/42 166/52
0.313 0.239
1.15 (0.76–1.76) 1.68 (1.15–2.45)
0.51 0.007
Primiparas Multiparas
78 103
105/47 153/47
0.309 0.235
1.18 (0.78–1.76) 1.71 (1.16–2.53)
0.43 0.006
PE Mild Severe
MAF, minor allele frequency; OR, odds ratio between case and control groups; CI, confidence interval.
114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144
patients and controls (x2 = 5.1808, df = 1, p = 0.0229, OR = 1.4440, 95% CI 1.0517–1.9827). In the subgroup analysis, rs7579169 C/T allele frequency was significantly different in multiparous (x2 = 7.4182, df = 1, p = 0.0065, OR = 1.7126, 95% CI 1.1603– 2.5279) and late-onset PE patients (x2 = 7.3575, df = 1, p = 0.0067, OR = 1.6795, 95% CI 1.1525–2.4473), but not in primiparous (x2 = 0.6155, df = 1, p = 0.4328, OR = 1.1753, 95% CI 0.7848–1.7601) and early-onset PE patients (x2 = 0.4348, df = 1, p = 0.5097, OR = 1.1524, 95% CI 0.7558–1.7572), compared with controls, suggesting that the C allele of rs7579169 is a risk factor for PE, especially in late-onset and multiparous cases. Genotype frequencies were also analyzed in this study, and a significant difference was observed between PE patients and controls in the dominant genetic model analysis of rs7579169 (p = 0.040, Table 3). Subgroup analysis showed that the genotype frequency of rs7579169 differed significantly in late-onset PE patients in both the dominant and recessive genetic models (p = 0.035 and p = 0.049, respectively). Similar to the gestational subgroup, the genotype frequency of rs7579169 was significantly different in multiparous PE patients compared with controls in both the dominant and recessive genetic models (p = 0.041 and p = 0.043, respectively). Rs7576192 showed no significant differences between controls and PE patients, apart from allele analysis in multiparous PE patients (x2 = 4.6088, df = 1, p = 0.0319, OR = 0.6379, 95% CI 0.4223–0.9634, Table 4). No significant difference in allele or genotype frequency was observed between PE patients and controls for rs12711941 (Table 5). Haplotype analysis was adopted based on the measured Q3 genotypes (Table 6). The order of SNPs was as follows: rs12711941, rs7576192 and rs7579169. All frequencies <0.03
3
Table 3 Genotype frequencies of rs7579169 in women with and without pre-eclampsia (PE). Group
No.
Frequency (CC/CT/TT)
padd
pdom
prec
Controls PE
190 176
98/53/39 104/50/22
0.107
0.040
0.149
95 81
57/25/13 47/25/9
0.285 0.178
0.159 0.063
0.178 0.330
Early-onset Late-onset
67 109
35/22/10 69/28/12
0.540 0.064
0.316 0.035
0.926 0.049
Primiparas Multiparas
76 100
40/25/11 64/25/11
0.465 0.065
0.254 0.041
0.877 0.043
PE Mild Severe
Padd, p-value of additive model (three genotypes). Pdom, p-value of dominant model [(homozygotes of risk allele + heterozygotes) vs homozygotes of non-risk allele]. prec, p-value of recessive model [homozygotes of risk allele vs (heterozygotes + homozygotes of non-risk allele)]. The T allele is the risk allele.
were excluded from analysis. The frequency of the H1 haplotype (G-G-C) was significantly different in PE patients compared with controls (p = 0.0304, OR = 1.437, 95% CI 1.034–1.997). Moreover, the H2 haplotype (G-G-T) had a significantly lower frequency in PE patients than in controls (p = 0.0460, OR = 0.482, 95% CI 0.232– 1.001).
145 146 147 148 149 150
Comments
151
PE is currently thought to be a syndrome rather than a definite disease. Although family studies suggest that it may have a heritable component, its underlying cause and pathogenesis remain to be uncovered [12–30]. In the present study, three SNPs (rs12711941, rs7576192 and rs7579169) previously associated with PE in an Australian population demonstrated different associations with PE in Chinese women. Weak relationships were found between rs12711941 and rs7576192 and PE, while a significant association was observed between rs7579169 and PE. Following subgroup analysis, rs12711941 maintained its negative association with PE, and rs7576192 was only found to be significantly associated with PE in multiparous PE patients. However, a strong association was observed in the late-onset and multiparous subgroups compared with controls in both the dominant and recessive models. These findings suggest that the C allele of rs7579169 is a risk allele for PE (p = 0.0229, OR = 1.4440, 95% CI 1.0517–1.9827).
152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168
Table 4 Allele and genotype frequencies of rs7576192 in women with and without pre-eclampsia (PE). Group
No.
Allele (A/G)
MAF
OR (95% CI)
p-value
Controls PE
203 181
81/269 107/273
0.282 0.231
0.77 (0.55–1.07)
0.12
96 85
44/144 37/125
0.234 0.228
0.78 (0.52–1.17) 0.76 (0.49–1.16)
Early-onset Late-onset
68 113
35/99 46/170
0.261 0.213
Primiparas Multiparas
78 103
41/109 40/160
0.273 0.200
PE Mild Severe
Frequency (AA/AG/GG)
padd
pdom
prec
15/77/98 10/61/104
0.298
0.132
0.410
0.23 0.20
6/32/56 4/29/48
0.631 0.118
0.203 0.246
0.647 0.383
0.90 (0.58–1.41) 0.69 (0.47–1.03)
0.65 0.07
3/29/35 7/32/69
0.444 0.437
0.926 0.040
0.346 0.654
0.96 (0.63–1.47) 0.64 (0.42–0.96)
0.85 0.03
6/29/40 4/32/64
0.961 0.101
0.797 0.043
0.977 0.203
MAF, minor allele frequency; OR, odds ratio between case and control group; CI, confidence interval; pdom, p-value of dominant model [(homozygotes of risk allele + heterozygotes) vs homozygotes of non-risk allele]; prec, p-value of recessive model [homozygotes of risk allele vs (heterozygotes + homozygotes of non-risk allele)]. The A allele is the risk allele.
Please cite this article in press as: Wang Q, et al. Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.04.001
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Table 5 Allele and genotype frequencies of rs1271941 in women with and without pre-eclampsia (PE). Group
No.
Allele (G/T)
MAF
OR (95% CI)
p-value
Controls PE
203 181
284/96 270/78
0.253 0.224
1.17 (0.83–1.65)
0.37
97 84
147/43 123/35
0.226 0.222
1.16 (0.77–1.74) 1.19 (0.76–1.85)
Early-onset Late-onset
68 113
97/33 173/45
0.254 0.206
Primiparas Multiparas
78 103
108/40 162/38
0.270 0.190
PE Mild Severe
Frequency (GG/GT/TT)
padd
pdom
prec
106/72/12 106/58/10
0.610
0.820
0.322
0.49 0.44
57/33/5 49/25/5
0.785 0.612
0.724 0.997
0.498 0.346
1.00 (0.63–1.57) 1.30 (0.87–1.94)
0.98 0.20
36/25/4 70/33/6
0.996 0.358
0.933 0.777
0.955 0.154
0.91 (0.59–1.40) 1.44 (0.95–2.20)
0.68 0.09
40/28/6 66/30/4
0.869 0.229
0.604 0.412
0.926 0.092
MAF, minor allele frequency; OR, odds ratio between case and control group; CI, confidence interval; pdom, p-value of dominant model [(homozygotes of risk allele + heterozygotes) vs homozygotes of non-risk allele]; prec, p-value of recessive model [homozygotes of risk allele vs (heterozygotes + homozygotes of non-risk allele)]. The T allele is the risk allele.
Table 6 Haplotype frequency estimates and their association with pre-eclampsia. Allotypea
H1 H2 H3
SNP SNP1
SNP2
SNP3
G G T
G G A
C T T
Frequency
Odds ratio (95% CI)
p-value
0.725 0.032 0.214
1.437 (1.034–1.997) 0.482 (0.232–1.001) 0.796 (0.563–1.127)
0.030 0.046 0.199
CI, confidence interval. Frequency <0.03 in both controls and patients with pre-eclampsia was ignored in the analysis. a The single nucleotide polymorphism (SNP) order was SNP1 = rs19711941, SNP2 = rs7576192 and SNP3 = rs7579169.
169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197
Haplotype analysis indicated that the H1 haplotype (G-G-C) is a risk factor (p = 0.0304), and the H2 haplotype (G-G-T) is a protective factor against PE (p = 0.0460). It is noteworthy that these two haplotypes differ only at the third SNP site (rs7579169), which is consistent with the findings of allele analysis. The experimental design of this study is a particular strength, because it involved the unbiased detection of cases and controls, and used a well-defined phenotype of PE. Moreover, all participants were Han Chinese women from northeastern China with no variation in their genetic background. To the best of the authors’ knowledge, this is the first report to show that SNPs located near INHBB are associated with PE in the Han Chinese population. However, several limitations of this study should be taken into consideration. First, genetic factors that influence the pathogenesis of PE differ by ethnic group, so the association between rs12711941, rs7576192, rs7579169 and PE should be tested in a larger sample size composed of different racial groups. Second, the functional significance of rs7579169 in PE remains unknown. Third, no significant differences in allele and genotype frequencies of rs12711941 and rs7576192 were found between PE patients and controls, despite using the same statistical methods as studies that reported significant differences. Therefore, future studies should evaluate the associations between PE and these two genetic variants, particularly rs12711941. In conclusion, a significant association was found between rs7579169 and PE in Han Chinese women. Women who carry the CC genotype are therefore at higher risk of PE, especially multiparous and late-onset cases. However, additional studies are required to confirm and extend these findings.
198
Acknowledgments
199 200 201
The authors wish to thank all subjects for participating in this study, and are grateful to Professor Hui Li from the Department of Obstetrics and Gynecology at the Shengjing Hospital of China
Medical University for sharing the samples. This work was supported by the National International Scientific and Technologi- Q4 cal Cooperation Program 2012DFB30130.
202 203 204
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Please cite this article in press as: Wang Q, et al. Single nucleotide polymorphisms near the inhibin beta B gene on 2q14 are associated with pre-eclampsia in Han Chinese women. Eur J Obstet Gynecol (2015), http://dx.doi.org/10.1016/j.ejogrb.2015.04.001
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