Background

Preeclampsia (PE) is a special complication of pregnancy that is characterized by de novo proteinuria and hypertension after 20 gestational weeks. It affects 2 to 8% of all pregnant woman worldwide and is the leading cause of maternal and fetal morbidity and mortality [1, 2], especially in developing countries. Furthermore, women with PE are more likely to develop cardiovascular disease (CVD), hypertension, and chronic kidney disease in later life [3, 4]. As a special type of hypertension, PE is a complex multifactor disease, which is the result of the interaction between genetic factors and environmental factors. Therefore, factors that affect blood pressure may be one of the primary mechanisms underlying PE. Although many environmental risk factors are associated with the formation of hypertension, including higher sodium intake, smoking, excessive alcohol consumption, obesity, activation of the sympathetic nervous system, and endothelial dysfunction [5], genetic factors also play an important role in the maintenance of hypertension [6]. Furthermore, multiple studies have shown that many candidate hypertension genes [7,8,9], such as STOX1, IL-1, IL6, and COMT, were also shown to be linked to PE [10,11,12,13].

Recent genome-wide association studies (GWAS) identified many genetic variants or chromosomal regions that are associated with hypertension, such as ATP2B1, CYP17A1, CYP1A2, SH2B3, CACNB2, TGFB2, MTHFR, CYP11B2, and ULK4 [14,15,16,17]. Furthermore, ATP2B1, MTHFR, and CYP11B2 have been validated to have associations with the risk of PE in different ethnic groups [18,19,20]. Two large GWAS meta-analyses suggested that rs1004467 (P = 1.28 × 10−10) and rs3824755 (P = 1.21 × 10−6) in CYP17A1 were significantly associated with the risk of hypertension [14, 16]. Subsequently, Lu et al. found that rs4409766 had the strongest correlation with CYP17A1 (P = 7.33 × 10−13) and CYP21A2 (P = 3.53 × 10−11) in a large-scale hypertension GWAS, of which 80,962 subjects were Chinese Han nationality [21].

CYP17A1 encodes the cytochrome P450 protein, which has 17-alpha-hydroxylase and 17,20-lyase activities and is one of the key enzymes in the synthesis of human steroid hormones. It had been investigated to play a role in multiple complex diseases, such as cardiovascular disease [22], hypertension [14], and fetal growth restriction [23]. CYP21A2 encodes 21-hydroxylase enzyme, and mutations in this gene may lead to congenital adrenal hyperplasia, which is characterized by hypertension, hypokalemia, and sexual infantilism [24]. Because they are candidate genes for blood pressure and play crucial roles in steroid hormone biosynthesis, thus having a further effect on hypertension, we designed and conducted this study to explore the relationship between genetic variations in CYP17A1 (rs4409766, rs1004467, and rs3824755) and CYP21A2 (rs2021783) and the sensitivity of Chinese Han women to PE.

Material and methods

Subjects

A total of 5021 subjects, including 2002 PE patients (mean age 30.09 ± 5.65 years) and 3019 controls (mean age 30.31 ± 4.01 years), were recruited from the Affiliated Hospital of Qingdao University, Linyi People’s Hospital, Liaocheng People’s Hospital, Yantai Yuhuangding Hospital, Heze Municipal Hospital, Binzhou Medical University Hospital, and the Maternal and Child Health Care of Zaozhuang between January 2012 and May 2016. All women had singleton pregnancies and were of Chinese Han ethnicity. The inclusion criteria for cases were established according to ACOG (American College of Obstetricians and Gynecologists, 2013), defined as hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg on two or more occasions at least 4 h apart) with proteinuria (0.3 g/24 h, 0.3 mg/dl, or 1+ by dipstick, without renal disease or infection) after 20 gestational weeks [25]. Exclusion criteria for both groups included chronic hypertension, a history of renal and endocrinal diseases, multifetal pregnancy, and autoimmune disorder. The control groups included normal healthy pregnant women without any pregnancy disorder or fetal disorders. All PE subjects were divided into the mild PE group (n = 454) and severe PE group (n = 1548). Severe PE was diagnosed if any following symptoms appeared in the case subjects: blood pressure ≥ 160/110 mmHg or progressive renal insufficiency (proteinuria ≥ 5 g/24 h, or cr > 1.1 mg/dl), new-onset visual or cerebral disturbances, impaired liver functions, and pulmonary edema [25]. Additionally, we divided the cases into early-onset PE (n = 937, before 34 weeks of gestation) and late-onset PE (n = 1065, after 34 weeks of gestation).

The questionnaire provided clinical data, including age, history of pregnancy and childbirth, blood pressure, gestational age, clinically relevant symptoms, and some laboratory examinations. All participants were provided with an explanation of the research and then gave written informed consent. Our study was approved by the ethics committee of the Affiliated Hospital of Qingdao University.

Genotyping

Qiagen DNA extraction kit (Qiagen, Hilden, Germany) was applied to extract genomic DNA from 2 ml peripheral venous anticoagulant blood, and then stored at – 80 °C. SNP genotyping was determined by the TaqMan allele recognition real-time PCR. The probes and primers were designed and synthesized by Applied Biosystems by Life Technologies (ABI, NY, USA). The sequence of primers was provided in https://www.mysequenom.com/Tools. All polymerase chain reaction (PCR) amplification systems (thermocycler, C1000TM) were conducted in a total of 25 μl, containing DNase-free water, 11.25 μl of DNA, and 12.5 μl of 2× PCR Master Mix and 1.25 μl of 20× SNP Genotyping Assay. The cycling protocol was included in an initial denaturation at 95 °C for 3 min, and then carried out 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The fluorescent signals of VIC/FAM-labeled probes were detected in each cycle, and different genotypes were detected applying Bio-Rad CFX manager 3.0 software. We also selected 100 samples of each genetic variant to verify the genotype by Sanger sequencing.

Statistical analysis

All data were analyzed applying the software package SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Clinical data and demographic were performed using Student’s t test or the chi-square test and were described by the mean ± standard error (SE) or percentage. A P value < 0.05 was considered statistically significant. Each control group polymorphism was tested for the Hardy-Weinberg equilibrium using the goodness-of-fit χ2 test. The distributions of genotypic and allelic frequencies between the case and control groups were analyzed by Pearson’s χ2 test. Ninety-five percent confidence intervals (CIs) and odds ratios (ORs) were applied to express the risk between the groups, and P < 0.05 was considered statistically significant; however, for four SNPs, a level of P < 0.0125 or Pc < 0.05 was considered significant when using Bonferroni’s correction. The genotype-phenotype was analyzed by the one-way analysis of variance. The power analysis was calculated using the program Power and Sample Size Calculations (PS, version 3.1.2).

Results

Demographic and clinical characteristics

In Table 1, the demographic and clinical features of the two groups were shown. There were no obvious differences in the age, age of menarche, or number of abortions between the control group and the case group (P > 0.05). However, compared with normal controls, PE patients were more likely to have earlier gestational weeks at admission and gestational weeks at delivery, lower fetal birth weight, lower cholesterol, higher blood pressure, higher triglyceride, higher creatinine, elevated white blood cells, and higher blood urea nitrogen (P < 0.001).

Table 1 Demographic and clinical characteristics of the case and control groups
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Genetic analysis

The genotypic distributions of the following SNPs in the control groups were consistent with the Hardy-Weinberg equilibrium (for rs4409766, χ2 = 0.091, P = 0.763; for rs1004467, χ2 = 1.203, P = 0.272; for rs3824755, χ2 = 0.206, P = 0.886; for rs2021783, χ2 = 0.040, P = 0.841).

The genotypic and allelic frequencies of CYP17A1 (rs4409766, rs1004467, rs3824755) and CYP21A2 (rs2021783) polymorphisms are summarized in Table 2.

Table 2 Genotypic and allelic distributions in the case and control groups
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There was an obvious difference in the allelic frequencies of CYP21A2 rs2021783 between the PE patients and control group (χ2 = 7.201, P = 0.007, Pc = 0.028). The T allele was related to the occurrence of PE (OR = 1.151, 95% CI 1.039–1.275). In the genotype (CC vs. CT vs. TT, P = 0.017; however, after Bonferroni’s correction, Pc = 0.068), the difference between the groups showed no statistical significance. In contrast, there were no obvious differences in the genotypic and allelic frequencies of rs4409766, rs1004467, and rs3824755 in CYP17A1 between the groups (for rs4409766, χ2 = 2.518, P = 0.284 by genotype, and χ2 = 1.345, P = 0.246 by allele; for rs1004467, χ2 = 1.424, P = 0.491 by genotype, and χ2 = 1.414, P = 0.234 by allele; and for rs3824755, χ2 = 2.392, P = 0.302 by genotype, and χ2 = 2.386, P = 0.122 by allele).

For rs2021783 in CYP21A2, Table 3 displays that there was no difference between the patients with mild/severe PE and control groups (Pc > 0.05). However, for rs1004467, the distribution of allelic frequencies differed significantly between the mild PE and control groups (χ2 = 6.843, P = 0.009, Pc = 0.036). The allele T may thus be a risk factor for mild PE (OR = 1.219, 95% CI 1.051–1.413), and the TT genotype was associated with the risk of developing mild PE compared to those subjects who had a CT or CC genotype (χ2 = 7.002, P = 0.008, Pc = 0.032, OR = 1.306, 95% CI 1.071–1.593). Similarly, the allelic frequencies of rs3824755 differed significantly between the mild PE group and the control group (χ2 = 6.869, P = 0.009, Pc = 0.036). Further, the GG genotype appeared to be a protective effect on the occurrence of mild PE (OR = 0.766, 95% CI 0.629–0.934). For rs4409766, there was no difference in the genotypic and allelic frequencies between the mild/severe PE and control groups (P > 0.05).

Table 3 Genotype and allele distributions in the mild/severe PE and control groups
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We also found that rs2021783 was related to the risk of early-onset PE (Pc = 0.008 by genotype, Pc = 0.004 by allele). No obvious differences were found in the genotypic and allelic frequencies of CYP17A1 (rs4409766, rs1004467, and rs3824755) between the control group and early and advanced PE patients (Table 4).

Table 4 Genotype and allele distributions in the early-onset/late-onset PE and control groups
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Analysis of genotype-phenotype relationship

A laboratory-based examination of PE among several genotypes is presented in Table 5. For rs2021783, patients carrying the TT genotype had a lower menarche age (13.55 ± 1.31 vs. 14.01 ± 1.09, or vs. 14.00 ± 1.22; P = 0.027) than did those with the CT or CC genotype. Furthermore, the level of urea nitrogen was lower in patients with the CT genotype than in those carrying the CC genotype (4.49 ± 1.72 vs. 4.73 ± 2.00, P = 0.016). However, the gestational age, birth weight of children, number of abortions, systolic/diastolic blood pressure, triglyceride, total cholesterol, and creatinine showed no statistical differences among three groups (all, P > 0.05).

Table 5 Laboratory-based examination of PE among different genotypes
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Discussion

A normal pregnancy is a condition of increased extracellular fluid, plasma volume, renal blood flow, and glomerular filtration rate. In contrast, PE is characterized by hypertension, proteinuria, higher vascular resistance, and decreased intravascular volume, cardiac output, and uteroplacental flow [26]. Despite unremitting efforts that have been undertaken for several decades, the precise pathophysiology or pathogenesis of this human pregnancy disorder remains elusive. However, increasing evidence suggests that gene-environment interactions maybe play important roles in the pathogenesis of PE [6]. Because PE is a special type of hypertension, we hypothesize that the genetic factors that affect blood pressure may be the primary cause for the incidence of PE.

GWAS had been demonstrated to be an effective way for identifying genetic variants in many common traits and diseases [27]. The Framingham Heart Study enrolled 9400 participants across three generations in Framingham, and the Wellcome Trust Case Control Consortium, which examined 2000 cases and a shared set of 3000 controls of white Europeans in Great Britain, reported the first two GWAS of hypertension but did not observe genetic loci that attained genome-wide significance [28, 29]. Then, a study in 34,433 subjects of European ancestry found eight blood pressure loci, including rs11191548 in CYP17A1, rs17367504 in MTHFR, rs12946454 in PLCD3, rs1378942 in CYP1A2, rs16998073 in FGF5, rs653178 in SH2B3, rs1530440 in c10orf107, and rs16948048 in ZNF652 [15]. Since then, more genetic loci, such as ATP2B1, CYP17A1, CYP1A2, SH2B3, CACNB2, MTHFR, CYP11B2, and ULK4, were identified in a large-scale GWAS associated with blood pressure [14, 15, 30, 31].

Some genetic variants in CYP17A1 were associated with hypertension in different populations, such as rs11191548 which was found to be associated with both the She ethnic minority in China and the Chinese Han population [32, 33]. rs11191416 and rs6163 were also identified to have association with blood pressure in 4178 European ancestry participants [34]. Furthermore, rs1004467 in CYP17A1 is a common intronic variant that is associated with hypertension; it has been reported as a significant locus in the genome-wide meta-analysis and was verified in a Chinese Han cohort [15]. A study of mean arterial pressure and pulse pressure among 26,600 East Asians followed by a repeat study of 28,783 participants suggested that CYP17A1 rs3824755 was associated with blood pressure (P = 1.2 × 10−6) [16]. The other two genetic loci of CYP21A2 rs2021783 and CYP17A1 rs4409766 were only found to be associated with blood pressure in a Chinese population [21], which indicated that these SNPs were genetic marker variants of hypertension in Chinese populations.

However, in addition to genetic factors, metabolic factors such as abnormal steroid metabolism play important roles in the occurrence of PE [35]. In normal pregnancy, the aldosterone levels are elevated to contribute to sodium retention and the water retention, whereas aldosterone is reduced in PE patients [35, 36]. Additionally, PE patients had increased serum progesterone or androgen levels and decreased estrogen levels [37].

CYP17A1 and CYP21A2 play important roles in the metabolic pathways of steroid hormones. CYP17A1 encoded the cytochrome P450 proteins, which had 17-alpha-hydroxylase and 17,20-lyase activities, which was the key step in the biosynthesis of mineralocorticoids, glucocorticoids, and sex-steroid biosynthesis [38]. CYP21A2 encoded 21-hydroxylase enzyme and converted 17-hydroxyprogesterone into 11-deoxycortisol or progesterone to deoxycorticosterone [24]. Aldosterone affected blood pressure and the blood volume in the body by regulating the salt retained by the kidneys. Therefore, the mutations in CYP17A1 and CYP21A2 may lead to a loss in enzyme activity and then affect the synthesis of steroid hormones.

In our study, we investigated the associations between rs4409766, rs1004467, and rs3824755 in CYP17A1 and rs2021783 in CYP21A2 and PE; several GWAS had identified these as the genetic variants associated with hypertension in China [16, 21]. To our knowledge, this is the first study to study the relationship between patients with PE and normal pregnant women of CYP17A1 and CYP21A2 in Chinese Han women. In the present study, the C allele of rs2021783 indicated an increased risk in patients with PE compared to the control group. The CC genotype was more susceptible to experiencing severe PE or early-onset PE. The CT genotype had the effect of protecting women from severe PE or early-onset PE. We also found, for rs1004467, that the T allele was the risk allele for the occurrence of mild PE and that patients with the TT genotype had a 1.219-fold risk of developing the PE compared to patients with CT or CC genotype in Chinese Han women. For rs3824755, the frequency of the G allele was associated with a higher risk of mild PE patients, and the GG genotype was identified as a risk factor in mild PE. However, Lim et al. investigated the association between polymorphisms of -34T/C in the promoter in CYP17A1 and PE in 164 cases and 182 normal pregnancies in Korea but did not obtain significant results [39]. Additionally, Coto et al. found no relationship between CYP21A2 rs6471 polymorphism with PE in 250 cases and 250 controls from Asturias [40].

Although our study contained a sufficient number of samples, including 2002 PE patients and 3019 age-matched normal controls, and had sufficient statistical power for each SNP (rs1004467, 0.221; rs4409766, 0.214; rs3824755, 0.338) to draw conclusions, some limitations should be noted. All subjects were selected from Shandong Province of China, and a single ethnic group may yield false positives due to unmeasured collinearity in the cohort. Thus, our findings should be expanded outside the Han ethnicity and be validated in different ethnicities and regions. Although PE was generally considered a complex multifactorial disease resulting from genetic and environment risk factors, including dietary and lifestyle factors, we could not consider all of these factors. Additionally, whether other SNPs in CYP21A2 and CYP17A1 participate in the development of PE needs to be studied further.

Conclusion

CYP21A2 rs2021783 appears to be closely associated with PE susceptibility, and CYP17A1 rs1004467 and rs3824755 seem to be related to mild PE in Chinese Han women.