Abstract
Preeclampsia, characterized by hypertension and proteinuria, remains a leading cause of maternal morbidity and mortality. Recently, a genome-wide association study (GWAS) identified the single-nucleotide polymorphism, rs2681472, as a new hypertension susceptibility genetic variant. The purpose of this study was to evaluate the association between preeclampsia and rs268172 in a Northern Han Chinese population. We genotyped 1218 unrelated Northern Han Chinese women, including 515 patients with preeclampsia and 703 healthy controls. No significant differences were detected in the allele frequencies between patients and controls (P = .23). When patients were divided into early-onset and late-onset preeclampsia according to gestational age of disease onset, the allele frequencies significantly differed between controls and patients with early-onset preeclampsia (P = .02). Genotype frequencies also were significantly different between controls and patients early-onset preeclampsia when data were analyzed under additive (P = .03) and dominant (P = .009) models. We replicated this association in an independent Northern Han Chinese population and observed a significant difference in the allele frequencies between patients with early-onset preeclampsia and controls (P = .011). We report that rs2681472 is associated with early-onset preeclampsia in Northern Han Chinese women.
Keywords: preeclampsia, genetics, ATP2B1, polymorphisms
Introduction
Preeclampsia, occurring in 3% to 5% of all pregnancies, is a major cause of preterm birth, intrauterine growth restriction, and stillbirth and increases the maternal risk of eclampsia; hemolysis, elevated liver enzymes, low platelet count (HELLP) syndrome; renal failure; pulmonary edema; stroke; and death. The exact etiology of preeclampsia is unknown, but a genetic predisposition is thought to play a crucial role in its development. The estimated heritability of preeclampsia is 54%.1
Recently, a genome-wide association study (GWAS) that included a large cohort of Europeans identified the single-nuclear polymorphism (SNP) rs2681472, near ATP2B1, as the only genetic variant associated with hypertension (odds ratio [OR] = 1.17, P = 1.7×10−8).2 Numerous subsequent studies have evaluated the role of the ATP2B1 gene in the pathogenesis of hypertension among various ethnic populations.3–10 A large-scale meta-analysis of East Asian populations confirmed a significant association between rs2681472 and hypertension.11 In the present study, we considered rs2681472 as a genetic risk marker for preeclampsia and evaluated its distribution in 2 independent Northern Han Chinese case–control cohorts.
Materials and Methods
Patients
This study was approved by the Institutional Review Board of Provincial Hospital Affiliated to Shandong University. Each participant provided written informed consent. We conducted a 2-stage study to identify the association between rs2681472 and preeclampsia in a Northern Han Chinese population. The first stage consisted of 1218 unrelated Northern Han Chinese pregnant women who had been recruited from Provincial Hospital Affiliated to Shandong University from December 2009 to November 2011. The second stage of the replication study included 166 patients with early-onset preeclampsia and 178 controls recruited from January 2012 to March 2013. The following information was collected: blood pressure, maternal age, gestational age, fetal weight, prepregnancy body mass index (pre-BMI), and parity. Preeclampsia was defined as the presence of hypertension (blood pressure values ≥140/90 mm Hg at 2 measurements occurring ≥6 hours apart) in combination with proteinuria (24-hour urinary protein ≥300 mg or urine dipstick protein ≥++) after the 20th week of pregnancy in a previously normotensive woman.12 Women with preeclampsia were further divided into early-onset and late-onset subgroups according to gestational age of disease onset. Early-onset preeclampsia was defined as occurring before 34 gestational weeks and late-onset preeclampsia developed at or beyond 34 weeks. Controls had normal blood pressure, lacked antenatal medical or obstetric complications, and presented normal fetal growth at delivery (defined as birth weight between the 10th and 90th percentiles at ≥37 weeks of gestation). Participants were excluded for the following reasons: chronic hypertension; multiple pregnancies; previous renal, autoimmune, metabolic, or cardiovascular disease; and loss to follow-up. Women with gestational hypertension, defined as elevated blood pressure without proteinuria, were also excluded from this study.
DNA Extraction and Genotyping
Maternal venous whole blood (5 mL) was drawn into EDTA-containing tubes and stored at −80°C. Genomic DNA was extracted using a QIAamp DNA mini kit (Qiagen, Hilden, Germany). Genotyping for the ATP2B1 rs2681472 SNP was performed using a TaqMan-MGB probe assay (Invitrogen Trading, Shanghai, China; Table 1). Reactions were carried out on a Roche LightCycler 480 with the following program: preincubation at 95°C for 4 minutes followed by 42 cycles of denaturation at 95°C for 15 seconds, annealing, extension, and detection for 40 seconds at 60°C. Randomly selected samples (5%) were further genotyped by direct sequencing to validate the genotyping assays.
Table 1.
Probes and Primers of the SNP rs2681472.
| SNP | Probes | Primers |
|---|---|---|
| rs2681472 | FAM-TCTGCCATGTAAATAG-MGB | F-GAGTTGCAATCTGATGGTTCATAGTG |
| VIC-TCTGCCATGTAAACAG-MGB | R-GCATTTCTCGTTTTGCTTGGA |
Statistical Analysis
The basic characteristics of the patients were expressed as mean ± standard deviation. Concordance with Hardy-Weinberg proportions was tested using Haploview software (Broad Institute, Cambridge, Massachusetts). The chisquare test was performed to compare allele frequencies. Genetic models were divided into additive (+/+ vs +/− vs −/−), dominant (+/+ plus +/− vs −/−), and recessive (+/+ vs +/− plus −/−) groups and then were analyzed by 1-way analysis of variance. Unconditional logistic regression analysis was used to adjust for parity, pre-BMI, and maternal age using SPSS version 17.0 software (SPSS Inc, Chicago, Illinois). P < .05 was regarded as statistically significant.
Results
The clinical characteristics of the participants are summarized in Table 2. No significant differences were detected regarding parity between patients and controls (P > .05). The mean maternal age and pre-BMI were higher among patients than controls (P < .05). The gestational age at birth and fetal weight were significantly lower among patients than controls (P < .05).
Table 2.
Characteristics of Women With and Without Preeclampsia.
| Characteristic | First Stage Study | Replication | ||
|---|---|---|---|---|
| Controls (N = 703) | PE (N = 515) | Controls (N = 178) | Early-onset (N = 166) | |
| Maternal age | 28.5 ± 4.9 | 29.8 ± 6.0 | 27.8 ± 3.9 | 30.7 ± 3.5 |
| Pre-BMI | 21.9 ± 3.2 | 24.3 ± 3.7 | 22.6 ± 3.2 | 24.4 ± 3.3 |
| Primiparas | 365 (51.9%) | 276 (53.6%) | 77 (43.3%) | 81 (48.8%) |
| SBP, mm Hg | 119.4 ± 10.1 | 160.2 ± 19.3 | 117.7 ± 9.4 | 158.4 ± 19.9 |
| DBP, mm Hg | 77.1 ± 7.4 | 108.2 ± 14.2 | 74.9 ± 6.9 | 105.9 ± 14.5 |
| Delivery weeks | 39.2 ± 1.4 | 35.7 ± 3.5 | 39.2 ± 1.2 | 29.0 ± 6.1 |
| Fetal weight, g | 3413.3 ± 473.2 | 2622.9 ± 928.5 | 3335.4 ± 368.0 | 1654.7 ± 814.7 |
Abbreviations: PE, preeclampsia; pre-BMI, prepregnancy body mass index; SBP, systolic blood pressure; DBP, Diastolic blood pressure.
The allele frequencies of the participants showed no deviation from Hardy-Weinberg equilibrium. As shown in Table 3, we observed no difference in the allele frequencies between preeclamptic women and controls (P = .23; OR = 1.11; 95% confidence interval [CI] = 0.94-1.31, Table 3). When early- and late-onset preeclampsia subgroups were considered, the allele frequencies were significantly different between early-onset preeclampsia and controls (P = .02; OR = 1.29; 95% CI = 1.04-1.59, Table 4). In contrast, no significant difference was observed between patients with late-onset preeclampsia and controls (data not shown).
Table 3.
Genotype and Allele Frequencies of rs2681472 in Women With and Without Preeclampsia in First Stage Study.
| Model | Control (N = 703) | Preeclampsia (N = 515) | P | OR (95% CI) |
|---|---|---|---|---|
| Additive | .193 | NA | ||
| CC | 89 | 66 | ||
| CT | 334 | 219 | ||
| TT | 280 | 230 | ||
| Dominant | .09 | 1.22 (0.97-1.54) | ||
| CC + CT | 423 | 285 | ||
| TT | 280 | 230 | ||
| Recessive | .94 | 0.99 (0.70-1.39) | ||
| CC | 89 | 66 | ||
| CT + TT | 614 | 449 | ||
| Allele | .23 | 1.11 (0.94-1.31) | ||
| C | 512 | 351 | .30a | |
| T | 894 | 679 |
Abbreviations: OR, odds radio between case and control group; CI, confidence interval; NA, not applicable.
a P value was adjusted for maternal age, prepregnancy body mass index, and primiparity.
Table 4.
Genotype and Allele frequencies of rs2681472 in Women With Early-Onset Preeclampsia and Controls in First Stage Study.
| Model | Control (N = 703) | Early-onset PE (N = 260) | P | OR (95% CI) |
|---|---|---|---|---|
| Additive | .032 | NA | ||
| CC | 89 | 28 | ||
| CT | 334 | 104 | ||
| TT | 280 | 128 | ||
| Dominant | .009 | 1.47 (1.10-1.95) | ||
| CC + CT | 423 | 132 | ||
| TT | 280 | 128 | ||
| Recessive | .425 | 1.20 (0.77-1.89) | ||
| CC | 89 | 28 | ||
| CT + TT | 614 | 232 | ||
| Allele | .02 | 1.29 (1.04-1.59) | ||
| C | 512 | 160 | .035a | |
| T | 894 | 360 |
Abbreviations: PE, preeclampsia; OR, odds radio between case and control group; CI, confidence interval; NA, not applicable.
a P value was adjusted for maternal age, prepregnancy body mass index, and primiparity.
Genotype frequencies were further analyzed under additive, dominant, and recessive models. No significant differences were detected between patients with preeclampsia and controls under additive (P = .193, Table 3), dominant (P = .09, Table 3), or recessive models (P = .94, Table 3). However, we detected a significant difference between patients with early-onset preeclampsia and controls under additive (P = .03, Table 4) and dominant models (P = .009, Table 4).
A total of 344 unrelated Northern Han Chinese women were included in the replication study. We observed a significant difference in the allele frequencies between early-onset preeclampsia and controls (P = .011, Table 5). There was also a significant difference in genotype frequencies when analyzed under additive (P = .014, Table 5) and recessive models (P = .002, Table 5).
Table 5.
Genotype and Allele frequencies of rs2681472 in Women With Early-Onset Preeclampsia and Controls in Replication Study.
| Model | Control (N = 178) | Early-onset PE (N = 166) | P | OR (95% CI) |
|---|---|---|---|---|
| Additive | .014 | NA | ||
| CC | 31 | 12 | ||
| CT | 78 | 76 | ||
| TT | 69 | 78 | ||
| Dominant | .123 | 1.40 (1.91-2.15) | ||
| CC +CT | 109 | 88 | ||
| TT | 69 | 78 | ||
| Recessive | .002 | 2.90 (1.44-5.85) | ||
| CC | 31 | 12 | ||
| CT +TT | 147 | 165 | ||
| Allele | .011 | 1.50 (1.10-2.06) | ||
| C | 140 | 100 | .034a | |
| T | 216 | 232 |
Abbreviations: PE, preeclampsia; OR, odds radio between case and control group； CI, confidence interval; NA, not applicable.
a P value was adjusted for maternal age, pre-pregnancy body mass index, and primiparity.
Discussion
An association between rs2681472 and hypertension was originally reported by a GWAS of hypertension in European populations. This association was subsequently confirmed by a large-scale meta-analysis of East Asian populations. In the present study, no significant difference in rs2681472 was detected between patients with preeclampsia and controls, suggesting that genes associated with hypertension are not necessarily the same genes that cause preeclampsia, even in a single ethnic group. When patients were divided into early-onset and late-onset subgroups, a significant difference in the allele frequency was detected between patients with early-onset preeclampsia and controls. To further validate this finding, we genotyped patients from an independent replication set and again observed a significant difference between patients with early-onset preeclampsia and controls.
The ATP2B1 gene, located at 12q21.3, encodes a plasma membrane Ca2+ pump (PMCA1) belonging to the family of P-type primary ion transport adenosine triphosphatases. This pump plays a crucial role in the fine regulation of intracellular free calcium concentration. Its role in blood pressure regulation has been explored extensively.13 A recent report by Kobayashi et al demonstrated a functional role of ATP2B1 in blood pressure control in vivo.14 Specifically, mice expressing a vascular smooth muscle cell-specific knockout (KO) of ATP2B1 expressed significantly lower levels of ATP2B1 messenger RNA and protein in the aorta compared to control mice, and KO mice exhibited higher systolic blood pressures.14
Preeclampsia is characterized by a disturbance in calcium metabolism that is evidenced by a decreased calcium concentration in plasma and increased calcium concentrations in the cell membrane and cytosol.15–18 In patients with preeclampsia, the activity of PMCA was reduced by approximately 50% in maternal and neonatal red blood cell ghosts, myometria, and syncytiotrophoblast basal (fetal-facing) plasma membranes compared to normal controls.19–22 The decreased activity of PMCA may be responsible, at least in part, for observed increases in peripheral resistance and blood pressure among patients with preeclampsia.
Numerous studies have indicated that preeclampsia is a syndrome rather than a definitive disease. In this study, rs2681472 was significantly associated with early-onset preeclampsia but not with late-onset preeclampsia. Early-onset preeclampsia is associated with more recurrences than late-onset preeclampsia and is thought to be affected by environmental or genetic factors.23 Conversely, late-onset preeclampsia is closely associated with a failed adaption of the maternal cardiovascular system to changes in late pregnancy and is regarded as a maternal phenotype.23
This study evaluated only Han Chinese women so the genetic background of the study population was consistent. However, this study had several limitations. Genetic factors that influence the development of preeclampsia differ by ethnic group, so our observed association between rs2681472 and early-onset preeclampsia needs to be confirmed in a larger sample size composed of various ethnic groups. Second, preeclampsia is thought to be a maternal–fetal disease. We only considered maternal genotype in this study and future studies should focus on the role of fetal or paternal genes in the development of preeclampsia.
We identified a significant association between rs268172 and early-onset preeclampsia in Northern Han Chinese women. Additional studies are warranted to confirm and extend our findings.
Acknowledgments
We are grateful to Tao Li, Lei Cheng, Peng Wang, Yue-Hong Bian, and Guang-Yu Li of Provincial Hospital Affiliated to Shandong University for technical support. We especially thank all the patients for participating in this study.
Authors’ Note: Ji-Peng Wan, Hong Wang, and Chang-Zhong Li contributed equally to this article.
Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This study was supported by National Basic Research Program of China (973 Program; 2011CB944502); Natural Science Foundations of Shandong Province (ZR2011HQ046); and National Natural Science Foundation of China (81170590, 81100417, 81200426).
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