Association of APP gene polymorphisms and promoter methylation with essential hypertension in Guizhou: a case–control study

Hypertension is a multifactorial disease associated with both the environment and heredity [15]. Guizhou province is located in southwest China, with a subtropical monsoon climate, more rainfall, humid climate, and high air humidity. It is a cosmopolitan province, with Miao and Buyi being the two ethnic minorities having the largest population. Its unique geographical environment promotes the dietary preferences for sour, smoked, spicy, oil, and wine. Thus, investigating the genetic susceptibility of EH in different ethnic groups in Guizhou by a case–control method is of great importance. The MassARRAY flight mass spectrometry method is a technology that detects gene molecular weight with high accuracy, sensitivity, and throughput [16].

The APP gene is located in the 21q21.3 region of human chromosomes, with a full length of 290,579 bp, containing 20 exons, and encoding the amyloid precursor protein. APP is a transmembrane protein continuously cleaved by β and γ secretases (amyloid pathway) to produce polypeptides including Aβ40 and Aβ42 [8, 17]. Specifically, Aβ42 is prone to misfolding and forming aggregates [18]. APP gene mutations cause the occurrence of the amyloid pathway and increase Aβ production and aggregation [9]. Aβ aggregation causes abnormal cerebrovascular metabolism, increases angiotensin II and cerebrovascular resistance, and subsequently induces cerebrovascular dysfunction, resulting in decreased cerebral blood flow. To maintain cerebral perfusion in the face of these metabolic abnormalities, cerebral perfusion pressure must be increased, resulting in systemic hypertension. Meanwhile, Aβ40 causes the production of reactive oxygen species and/or downregulation of nitric oxide synthase through NADPH oxidase, which mediates an increase in sympathetic nerve activity, thereby increasing the total peripheral resistance and hypertension occurrence [10]. Thus, our case–control study for the first time explored the relationship between APP gene SNP mutation and hypertension. Consequently, we found that rs2211772 is associated with EH in the Guizhou Han population. Studies indicate that rs2211772 is associated with cholesterol and high-density lipoprotein [19], and plasma cholesterol level is a risk factor for cardiovascular disease [20], corroborating our findings. rs2211772 (chr21: 26027126, T > C) is an intronic variant of CpG-SNP, which generates a CpG site. Introns increase transcript levels by influencing the transcription rate, nuclear export, transcript stability, and mRNA translation efficiency [21]. Studies have shown that intronic SNP variants promote mRNA transcription, resulting in epigenetic gene modification [22, 23]. By predicting the transcription factors bound by the sequence where the SNP is located, we found that the sequence has several transcription factor binding sites including TBX19. It has been shown that TBX19 is a transcription factor that regulates growth and development as well as blood pressure [24]. Therefore, the binding activity of rs2211772 genotypes should be investigated using chromatin immunoprecipitation (ChIP) assay and the function of rs2211772 polymorphism needs to be explored using the luciferase reporter assay.

Lynn M Bekris reported that rs2040273 minor allele carriers have significantly lower levels of CSF Aβ42 [25]. Similarly, our study noted that the distribution of alleles and genotypes of rs2040273 in the Guizhou Miao population had a small statistical P value (P = 0.051 and P = 0.095) between the disease group and the control group. Moreover, regression analysis showed that in contrast with allele A, hypertension risk in carriers of minor allele G decreased (OR 0.533, 95%CI 0.294–0.965, P = 0.038). Thus, the relationship between rs2040273 and hypertension in a larger population is worth studying.

Elsewhere, Craig Myrum performed a functional evaluation of rs2830077 and found that the SNP is located in the active region of chromatin, which may have transcriptional enhancer activity and is a binding site for transcription factor CP2. Luciferase analysis revealed that the expression of its allele C improves APP expression [26]. However, we did not identify the relationship between rs2830077 and hypertension. rs63750921 mutation changes the encoded amino acid, and the pathological examination of the patient displayed severe cerebral amyloid angiopathy. This suggests that rs63750921 has vascular tropism [27]. This study, which for the first time reports rs63750921 mutation in a Chinese population, did not identify gene mutation of rs63750921. This indicates that the SNP is significantly conservative among the Chinese population.

Polygenic diseases including hypertension and diabetes often do not follow the common Mendelian inheritance pattern, where one gene modifies the phenotype of another gene, causing complex higher-order interactions between two or more genes [28]. Therefore, genetic interactions may induce hypertension risk. Our gene interaction analysis revealed that the interaction model made up of rs467021 and rs364051 had a cross-validation consistency of 7/10 (P = 0.006), and the interaction line was red. This indicates that rs467021 and rs364051 have a strong positive interaction effect on EH in the Guizhou populations, confirming the above standpoint.

Although human genome-wide association research has identified a large number of genetic loci associated with hypertension, these loci account for only a small fraction of its heritability [29]. Epigenetic modifications may partly explain the genetic absence of hypertension [6]. The APP promoter has multiple possible transcription factor binding sites [30, 31]. Promoter methylation has a strong correlation with transcriptional silencing of APP [32]. The APP proximal promoter region is crucial for cell-specific expression of the APP gene [33]. Herein, the methylation sequences (− 265 to − 742 bp) detected were predicted to contain 25 binding transcription factors, indicating that the target sequence promotes transcriptional regulation. Furthermore, we found that CpG_10 (− 406 bp), CpG_19 (− 613 bp), and CpG_1 (− 296 bp) of the target sequence were associated with hypertension. Regression analysis adjusted for confounding factors, and showed that for every 1% increase in CpG_10 methylation level, hypertension risk decreased by 32.4%; every 1% increase in CpG_19 methylation level was associated with a 4.1% higher risk of hypertension; every increase in CpG_1 methylation level 1%, the risk of hypertension in women reduced by 8%. This may be attributed to changes in methylation levels, which trigger changes in the sequence of transcription factor binding sites, hence affecting APP gene expression [34, 35] and abnormal metabolism of APP, ultimately resulting in hypertension [10].

Genetic variation potentially modulates DNA methylation [36]. SNP and CpG site methylation may jointly promote gene expression or alternative splicing, providing novel insights into polygenic disease research [37, 38]. This study included three SNPs in the positive regulatory region of the APP gene promoter, i.e., rs466433 (− 875 bp/T > C, generating a new CpG site), rs364048 (− 953 bp/A > C), rs364051 (− 1158/ A > C) [39]. Previous studies indicate that the transcriptional activity of haplotype TA (rs466433–rs364048) in neural cells is four times higher than that of haplotype CG, and APP mutation promoter upregulates APP gene expression and aggravates Aβ accumulation [40, 41]. Although we did not identify the relationship between promoter SNP variants and hypertension, the carriers of the minor allele of the promoter SNP in the hypertensive populations significantly reduced the CpG_19 methylation levels. Additionally, the results of MDR interaction analysis showed that CpG_11, CpG_19, and the promoter variant rs364051 interacted with EH in Guizhou populations. Thus, the variation of the APP gene promoter may influence gene expression by targeting the methylation level, hence changing the blood pressure.

Although hypertension and AD are closely related, the mechanism responsible for the association is not clear [42]. Animal experiments indicate that hypertension activates receptors for advanced glycation end products (RAGE) in the cerebrovascular system via oxidative stress, and mediates the transcytosis of Aβ across brain endothelial cells, resulting in Aβ accumulation, cognitive impairment, and memory degradation [43]. Previous research findings have also pointed out that hypertension promotes APP processing, which may be a mechanism of pathogenic interaction between hypertension and AD [44]. In this work, the polymorphism and methylation of the APP gene, closely related to AD, were associated with hypertension. This provides a genetic reference for further research on the interaction mechanism between hypertension and AD.

This work has the following limitations. First, due to sampling limitations, we did not identify cholesterol, triglyceride, and high-density lipoprotein, among other biochemical indicators contributing to hypertension for the Miao and Buyi populations, and therefore, these populations were not be included in the model for regression analysis. Secondly, environmental data, including smoking and diet, were lacking, and therefore, we could not analyze the interaction between genes and the environment. As such, the association of APP gene polymorphism and promoter methylation with EH deserves to be studied in a larger population with more comprehensive indicators.

In conclusion, we used Sequenom MassARRAY to investigate the associations of APP gene rs2040273, rs63750921, rs2211772, rs2830077, rs467021, rs368196, rs466433, rs364048, rs364051, rs438031, rs463946, and promoter methylation with EH in Guizhou populations. For the first time, we found that the APP gene rs2211772 and promoter methylation levels may be associated with EH among the Guizhou populations. Our findings provide an important reference value for a deeper understanding of genetic pathogenesis, prevention, and control strategies of EH in Guizhou.

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