Functional polymorphisms in the MED13L and SLC4A7 genes are associated with pregnancy-induced hypertension


 Background: Pregnancy-induced hypertension (PIH), including gestational hypertension (GH) and preeclampsia (PE), might share pathophysiologic mechanisms and genetic risk factors with hypertension. This study aimed to investigate whether genetic risk factors for hypertension also been associated with PIH.
Methods: This is a case control candidate gene association study. A total of 84 cases with GH, 108 cases with PE, and 192 age-matched (±1 year) controls were recruited from the Liuyang Municipal Hospital of Maternal and Child Health, Hunan Province of China. Three candidate single nucleotide polymorphisms (SNPs) which have been found to be associated with hypertension or blood pressure (BP) were chosen. The associations between SNPs and GH and PE were analyzed by multiple logistic regression models after adjusting for fetal sex and primipara.
Results: GG genotype at mediator complex subunit 13-like (MED13L) rs11067763 was a protective factor for developing GH (GG vs. AA + AG, OR = 0.376, 95%CI = 0.158-0.892) and PE (GG vs. AA + AG, OR = 0.489, 95%CI = 0.245-0.979) in recessive model. AA genotype at solute carrier family 4 member 7 (SLC4A7) rs820430 was a risk factor for developing GH (AA vs. GG + AG, OR = 3.562, 95%CI = 1.343-9.447) and PE (AA vs. GG + AG, OR = 3.351, 95%CI = 1.352-8.303) in recessive model. But no significant association was observed between cytochrome P450 family 21 subfamily A member 2 (CYP21A2) rs2021783 and the GH/PE groups.
Conclusions: PIH and hypertension are likely to share genetic risk factors. The associations both between variants of MED13L rs11067763 and GH/PE and between variants of SLC4A7 rs820430 and GH/PE were significant.


Background Pregnancy-induced hypertension (PIH, including gestational hypertension [GH] and preeclampsia [PE]) is a
pregnancy-speci c complication affecting 5-10% of all pregnant women in the world [1]. Both GH and PE are characterized by hypertension rst appearing after 20 weeks of gestation, while PE is also characterized by new onset proteinuria [2]. PIH is a leading cause of maternal and fetal morbidity and mortality. Without knowing the exact etiology of PIH, delivery of the baby is the only effective way when the lives of the pregnant women are threatened [3,4].
Since familial association was observed for PIH, inheritance could have played a major function in the pathology of PIH. A twin study suggested the heritability of GH and PE was 24% and 54%, respectively [5]. Besides, it has been reported that maternal genetic factors contribute to PIH susceptibility [6]. Epidemiological studies also have shown that a family history of chronic hypertension is more common in PE groups compared with controls, and PIH is associated with an increased long-term risk of maternal hypertension [7]. Studies on pathophysiologic mechanisms have demonstrated endothelial dysfunction, in ammation, and vascular remodeling to be crucial to the development of chronic hypertension, as well as PIH [8,9]. In line with these ndings, it has been suggested that PIH and hypertension might share pathophysiological factors. And the genetic risks predisposing to hypertension and PIH may thus be partially overlapping [8, 10,11].
Genome-wide association studies (GWAS) have been conducted to search for hypertension-susceptibility genes.
Recently, Chinese researchers identi ed that mediator complex subunit 13-like (MED13L) rs11067763, solute carrier family 4 member 7 (SLC4A7) rs820430, and cytochrome P450 family 21 subfamily A member 2 (CYP21A2) rs2021783 were possibly genetic variants associated with blood pressure (BP) and/or hypertension [12]. Further studies found that hypertension risk SNP rs820430 functioned as an enhancer of SLC4A7 [13]. Another GWAS carried out in the USA found associations between CYP17A1/CYP1A2 and BP [14]. Considering the overlapping of genetic risks of hypertension and PIH, it is valuable to investigate the relationship between these three SNPs and PIH.
The present study aims to detect the association between three SNPs, which were previously reported having association with BP or hypertension, and PIH in a Southern Han Chinese population. week of gestation) and accompanying proteinuria (urinary protein dip sticks ≥ 2 + or ≥ 300 mg in a 24-h urine sample). GH was similarly de ned as de novo hypertension without proteinuria [15,16].
Genotyping of the three SNPs was performed using matrix-assisted laser desorption/ionization-time of ight(MALDI-TOF) mass spectrometry, according to the manufacturer's instructions [17].

Statistical analysis
Statistical analyses were conducted using IBM SPSS 19.0 software (IBM SPSS, Inc., Chicago, IL). Mean ± standard deviation (SD), or median and interquartile were used to describe the continuous variables with normal or skewed distribution, respectively. Frequency was used to describe categorical variables. To compare demographic and clinical data, the t-test or Wilcoxon rank sum test was used for continuous variables, and the Chi-square test or Fisher's exact test was used for categorical variables. The Bonferroni correction was applied for multiple comparisons (α = 0.05/3 = 0.0167). The SNP detection rate was calculated as the number of sites that were successfully genotyped for all of the samples divided by the number of genotyped sites for all samples. The Hardy-Weinberg test was conducted in control groups using the Chi-square goodness-of-t test or the Fisher's exact test (α = 0.01). The Chi-square test or Fisher's exact test was used to test the allele/genotype distribution between the case and control groups. Univariate logistic regression was used to estimate the crude odds ratio (OR) and its corresponding 95% con dence interval (95% CI) for different genetic models, including dominant model and recessive model. Through multiple logistic regression, the adjusted OR and 95% CI were also calculated after adjusting for fetal sex and pimipara [18,19].

Demographic and clinical features
In total, 84 GH cases, 108 PE cases, along with 192 controls were analyzed. The main demographic and clinical features of three groups are summarized in Table 1. No signi cant between-group differences were detected for fetal sex, primipara, and new born weight (P > 0.0167). Meanwhile, serum uric acid, in-hospital SBP, and in-hospital DBP with GH and PE were signi cantly higher than those in normotensive women. Moreover, in-hospital SBP of PE women were signi cantly higher than those in GH women, whereas there were no signi cant differences between the GH and PE groups as to other characteristics. Interestingly, creatinine of women with PE were signi cantly higher than those in GH and control groups, and gestational age of PE groups were signi cantly lower than those in GH and control groups, while no signi cant differences were found between GH and control groups on these two factors.

SNPs and PIH
The SNP detection rate was 98%. The allele frequencies of control groups showed no deviation from Hardy-Weinberg equilibrium, shown the representativeness of our control group. The distributions of genotypes and alleles among the three groups are shown in Table 2. The results of the Chi-square test showed that the distribution of MED13L rs11067763 genotype was signi cantly different between the GH and control groups (P = 0.007), and there was signi cant difference in the SLC4A7 rs820430 allele between the GH and control groups (P = 0.043), but no signi cant differences in these two SNPs genotype/allele were observed between the PE and control groups.
Furthermore, no signi cant differences in CYP21A2 rs2021783 genotype/allele were found between the case and control groups.
The univariate logistic regression results are shown in Table 3. Two SNPs showed nominal association with GH or PE. Speci cally, MED13L rs11067763 was signi cantly associated with both GH (GG vs. AA + AG, P = 0.009) and PE (GG vs. AA + AG, P = 0.034) in recessive model. Also, SLC4A7 rs820430 was signi cantly associated with both GH (AA vs. GG, P = 0.021; AA vs. GG + AG, P = 0.020) and PE (AA vs. GG + AG, P = 0.026). However, no signi cant association was observed between CYP21A2 rs2021783 and the GH/PE groups.
After adjusted for fetal sex and primipara by logistic regression, most of the results remained the same as the univariate logistic regression results (Table 3). MED13L rs11067763 was signi cantly associated with both GH and PE, being a carrier of the GG genotype was associated with a decreased susceptibility of developing GH/PE (GG vs. AA + AG, P = 0.027; and GG vs. AA + AG, P = 0.043, respectively ); SLC4A7 rs820430 was signi cantly associated with both GH and PE, being a carrier of the AA genotype was associated with an increased susceptibility of developing GH/PE (AA vs. GG, P = 0.007, AA vs. GG + AG, P = 0.011; and AA vs. GG, P = 0.026, AA vs. GG + AG, P = 0.009, respectively); being a carrier of the SLC4A7 rs820430 A allele was associated with an increased susceptibility of developing GH (A vs. G, P = 0.033). However, still no signi cant association was detected between CYP21A2 rs2021783 and the GH/PE groups in multiple logistic regression analysis.

Discussion
The typical manifestation of PIH is BP elevation. Women with a history of PIH are more susceptible to chronic hypertension and women with chronic hypertension are at increased risk for PE. Luscher demonstrated that endothelial cell injury in PIH women could profoundly affect cardiovascular function, and patients were likely to become hypertensive, hyperlipidemic, and eventually develop atherosclerosis with aging [7]. Ellen et al. reported that women with chronic hypertension had an increased rate of PE (17-25%, vs. 3-5% in the general population) [20].
Women with chronic arterial hypertension were also at increased risk for superimposed PE [21]. Except for similar metabolic bases, PIH and hypertension share some genetic risk factors. Our team had found signi cant associations between angiotensinogen (AGT) rs3789678 and GH and between angiotensin II receptor type 1 (AGTR1) rs275645 and PE after adjusting for fetal sex, maternal age, and gestational diabetes mellitus (GDM) [11]. So validate the association between hypertension candidate genes and PIH is a reasonable and effective way to nd genetic risk factors for PIH.
In this case-control study, signi cant differences were found between MED13L rs11067763 and GH/PE, and SLC4A7 rs820430 associated with the onset and development of GH/PE. In detail, carrying the GG genotype at rs11067763 was a protective factor for developing GH/PE. As for rs820430, carrying the AA genotype was a risk factor for developing GH/PE. But no signi cant association between CYP21A2 rs2021783 polymorphisms and GH/PE was found. Importantly, our study reveals for the rst time that the MED13L rs11067763 and SLC4A7 rs820430 were associated with GH/PE risk among Chinese Han women.
MED13L is a paralog of the closely related protein named Mediator complex subunit 13 (MED13), which is believed to have arisen from gene duplication events [22]. It is responsible for bridging the RNA pol II enzyme and transcription factors to initiate transcription of protein-coding genes and non-coding RNA genes [23]. Through retinoblastoma (Rb)/E2F pathways, it promotes proliferation of vascular smooth muscle cells which are important in the development of hypertension [24]. Besides, several researches have shown an indirect association between MED13L and in ammation through Wnt pathway which plays a regulatory role in immune system [25]. Moreover, MED13L regulates the expression level of activating transcription factor 4 (ATF4) which regulates the secretion of proin ammatory cytokines such as interleukin (IL)-6 and IL-8 [26,27], and these two cytokines have been considered in uencing the onset of GH and PE. As Adela et al. reported, the serum level of IL-6 decreased in GH subjects [28].
Besides, Krasnyi found that increased endothelial IL-8 expression represented endothelial dysfunctions, which are signi cant pathophysiologic features of PE [29]. In our study, we found genetic variants of MED13L rs11067763 associated with development of GH/PE in Chinese Han women. GG genotype at rs11067763 has shown to be a protective factor for GH/PE. Thus, more studies are needed to validate the association in larger population, and researches on mechanisms are necessary to uncover the molecular pathways of the association. SLC4A7 gene is on chromosome 3p24.1, it belongs to the SLC4 family [30]. SLC4A7 encodes the electroneutral Na + /HCO -3 , co-transporter NBCn1. Several researchers have demonstrated that NBCn1 has a role in controlling intracellular pH (pHi) in vascular smooth muscle cells (VSMCs), and pHi is a determinant of VSMCs contractility and endothelial function, both of which effect BP. Furthermore, through elevated steady-state pHi and accelerated recovery from intracellular acidosis, they also found genetic variants of SLC4A7 rs13082711 associated with increased SLC4A7 expression level and NBCn1 function. [31][32][33][34]. A large scale GWAS also detected association between polymorphisms of SLC4A7 rs13082711 and SBP/DBP [35]. A study included 275 participants from rural district of Shandong Province of China indicated rs820430 functioned as an enhancer of SLC4A7 [13]. Besides, NBCn1 promotes arterial remodeling which can cause luminal narrowing and obstruct blood ow [36]. It is known to us that increased vascular resistance [37], invasion of placental trophoblast, and failed remodeling of uterine spiral arterial[38] are pathological bases of PIH. Therefore variants of SLC4A7 might associate with the onset of PIH through several pathways such as BP regulation, and remodeling of arterial. We reported the polymorphisms of SLC4A7 rs820430 was associated with the onset of GH and PE in Chinese Han women for the rst time. AA genotype at rs820430 is a risk genotype for GH and PE, and A allele at rs820430 is a risk allele for GH. However, more studies are needed to identify pathophysiological path from PIH associated genetic variation, to gene expression, and subsequently to gene function of SLC4A7 that alters cellular behavior. . Also, a Spanish study included 500 Caucasians from the region of Asturias indicated that the variation in CYP3A5, CYP3A4, and CYP21A2 did not contribute to the risk for PIH [43]. Similarly, in our study no signi cant association has been observed between variants of CYP21A2 rs2021783 and PIH.
Our case-control study was limited by the small sample size. It is di cult to identify very weak associations between SNP and PIH. This issue is common in gene-association studies. Although we did not detect signi cant difference between fetal sex and PIH, the proportions of female fetus in GH and PE groups were higher than those in control group. Different from the systematic review[44], we found primipara were less likely to develop PE. It was due to the unequal proportion of primipara and multipara in our study. In order to detect accurate relations, we still set fetal sex and primipara as covariates in multiple logistic regression. Another limitation of our study is that the gestational age of PE onset and medical regimens were not regularly recorded in the medical records of our study region, we did not conduct subgroup analyses according to early-or late-onset PE and did not categorize PE cases by severity. Also, we did not adjust for family history of hypertension or PIH and pre-pregnancy body mass index (BMI) in the multiple logistic regression because the proportion of missing data for it was larger than 15%. Further researches with larger sample size and more comprehensive clinical characteristics are necessary to investigate the disease-related genes in GH and different types of PE.

Conclusions
In conclusion, PIH and hypertension may share genetic factors, which provide potential genetic evidence for patients with PIH having a higher risk for hypertension. Our study reported for the rst time that the associations between variants of MED13L rs11067763 and GH and between variants of SLC4A7 rs820430 and GH/PE were signi cant.
Further studies are needed to uncover the molecular mechanisms of these associations. The study was reviewed and approved by the Committee for Human Research Protections of Xiang-Ya School of Medicine. Informed consent was obtained from each individual included in the study, after which blood samples and medical records were collected.

Consent for publication
Not appllicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.