Distinct role of the CYP4F2 polymorphisms in determining the risk of HAPE among Chinese Han population

Background: CYP4F2 is potentially associated with High altitude pulmonary edema (HAPE) risk by regulating inflammatory mediator leukotriene B4 and arachidonic acid. However, the role of CYP4F2 in HAPE susceptibility remains unknown. For the first time, we conducted a case-control study to assess the potential association of CYP4F2 gene variants (rs3093193, rs12459936, rs3093144 and rs3093110) with HAPE susceptibility in Chinese Han population. Methods : The study included 238 BC patients and 230 healthy controls from northwest China. The polymorphisms selected in CYP4F2 gene were genotyped by Agena MassARRAY system. Odds ratios (ORs), 95% confidence intervals (95% CIs), and P values were used to evaluate the relationship between the two. Results : In the allele model and genotype model of the overall analysis, rs3093193 was shown to reduce the risk of HAPE ( P  0.05), while rs12459936 increased susceptibility to HAPE ( P  0.05). Age stratified analysis revealed that rs3093193 and rs12459936 were correlated with HAPE risk at age > 32 years ( P  0.05), and rs3093193 and rs3093110 were correlated with the HAPE risk at age ≤ 32 years ( P  0.05). Gender stratification analysis found that rs3093193, rs12459936 and rs3093110 were all related to HAPE risk in males ( P  0.05). Haplotype analysis illuminated that GCCG and CTC could increase HAPE risk at age ≤ 32 years and males, respectively ( P  0.05). Conclusions : Our research confirmed that CYP4F2 genes polymorphisms were implicated in HAPE susceptibility in Chinese Han population.


Introduction
High altitude pulmonary edema (HAPE) is a hypoxia-induced, non-cardiogenic pulmonary edema that may develop in otherwise healthy individuals when first quickly ascended and exposed to altitude above 2500m (1). It is characterized by excessive pulmonary arterial hypertension, which leads to pulmonary capillary stress failure and caused a high permeability type of life-threatening pulmonary edema (2,3), with common symptoms of cough, dyspnea, chest tightness, and fatigue (4). HAPE occurs and develops rapidly, and it can develop into a coma or even life-threatening in a short period of time if left untreated (5). However, the exact pathogenesis of HAPE remains is still not yet clear.
As we all know, Hypoxia induction is one of the principal element in the occurrence of HAPE (6). More often, hypoxia can also induce inflammation (7). The inflammatory mediator is a vital regulator of increased vascular permeability and vasodilatation (8). Vascular permeability increased, vasoconstriction of diastolic imbalance is a crucial mechanism of acute high altitude disease. In an earlier research to assess the cellular and biochemical components of bronchoalveolar fluid in highaltitude pulmonary edema (HAPE), they performed bronchoalveolar lavage in three climbers with HAPE in a research facility at 4400 m on Mount McKinley. They eventually found that HAPE fluid contained a large number of alveolar macrophages and detectable amounts of inflammatory factors such as leukotriene-B4 and other lipoxygenase products of arachidonic acid metabolism (9,10). family enzymes is increased during inflammation (11). Additionally, CYP4F2-encoded enzymes starts the process of inactivating and degrading leukotriene B4, a potent Mediator of inflammation (12).
What is more, CYP4F2 related pathway also includes arachidonic acid metabolism, which can be metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) (13). Arachidonic acid is also an important inflammatory cytokines, and its metabolic network is the core of the inflammatory metabolic network (13). CYP4F2-derived 20-HETE is known to have the characteristics of prehypertension and angiogenesis promotion (14). Therefore, we propose a reasonable hypothesis that the pathogenesis of HAPE is related to CYP4F2.
Single nucleotide polymorphisms (SNPs) have been proved to play a crucial role in the occurrence of HAPE. Many SNPs of inflammatory cytokines or genes associated with inflammation have been identified to be strongly correlated with susceptibility to pulmonary edema, for example, NR3C1 (15), IL-6 (8), IL1R2 (16). To our knowledge, there are no previous studies have investigated the association of risk of HAPE and CYP4F2 polymorphisms. Hence, we perform a hospital-based case-control study to explore the correlation of the 4 tag SNPs in CYP4F2 at allele, genotype, and haplotype interface with HAPE susceptibility among Chinese Han women.

SNP selection and genotyping
Peripheral blood samples of each subject was collected in tubes coated with ethylenediamine tetraacetic acid (EDTA). All samples were stored at -80 °C, after centrifugation, whole blood cells were collected for further analysis (17). Then, Genomic DNA was extracted from whole blood using the GoldMag whole blood genomic DNA purification kit (GoldMag Co. Ltd., Xi an, China) following the manufacturer's guidelines, and DNA quantity was assessed utilizing the NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA). Four tag-SNPs (rs2881766, rs9383951, rs9340799 and rs3020449) in CYP4F2 were involved in our study based on 1000 Genomes Project (http://www.1000genomes.org/) and dbSNP (https: //www. ncbi.nlm.nih.gov/projects/SNP/) database.
The minor allele frequency (MAF) value with > 0.05 in Chinese Han population was also applied to select candidate SNPs. Agena MassARRAY Assay Design 3.0 Software (San Diego, California, USA) was used to design the primers for amplification and single base extension. The corresponding primers of the selected SNPs in this study were listed in Supplementary Table S1. SNP genotyping was carried out by two laboratory personnel in a double-blinded manner using the Agena MassARRAY system (Agena, San Diego, CA, U.S.A.). Agena Bioscience TYPER version 4.0 software was used for data analysis. In order to verify the accuracy of genotyping, about 10% of the samples were randomly selected to repeat genotyping and the reproducibility was 100%.

Statistical analysis
The differences in the basic characteristics between the cases and controls were compared using Student's t-test or χ2-test. Hardy Weinberg equilibrium (HWE) for selected SNPs among controls and the differences in genotypes distribution between cases and controls were examined by exact test and χ2-test, respectively. We evaluated the relationship of the four SNPs with HAPE risk based on five different genetic models (A and a are used to represent the major and the minor alleles, respectively): the log-additive model. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by multivariate logistic regression, with adjustment for age and gender. Stratified analyses were also performed by gender and age to understand the relationship between each SNP and HAPE risk in different subgroups. SHEsis software (http://analysis2.bio-x.cn/myAnalysis.php) software was used for haplotype analysis. For each haplotype, its correlation with the risk of high-altitude pulmonary edema was also evaluated by using OR and 95% CI and P-values. The results were presented as ORs, 95% CIs and P values, and a two sided P-value < 0.05 was considered statistically significant. All the statistical analyses were performed using the software SPSS software package (version 20.0; SPSS Inc., Chicago, IL, USA) for Windows (18).

The basic characteristics of study subjects
A total of 468 subjects including 238 HAPE patients (220 males and 18 females) and 230 healthy individuals (213 males and 17 females) were enrolled in this study. The mean age and standard deviation of the cases and the controls were 32.35 ± 10.78 and 33.45 ± 9.05 years, respectively. As shown in Table 1, There was no significant difference between the case and control groups in regards to the distribution of gender and age (P = 1.000 and 0.236, respectively).

Basic information and preliminary statistics of the selected SNPs
The basic information of the four SNPs was presented in Table 2. We genotyped the four SNPs (rs3093193, rs12459936, rs3093144 and rs3093110) and the genotyping success rates of the four SNPs were > 95.0% among the HAPE cases and the controls. The genotype distribution of of all SNPs in the control groups were in accordance with Hardy-Weinberg Equilibrium (HWE) (P > 0.05). The differences in allele frequency between cases and controls were compared by Chi-squared test, and whether the P value was less than 0.05 to assess the association with the risk of developing HAPE.
The minor allele of each SNP as a risk factor was compared to the wild-type (major) allele. Statistical analysis showed that the allele frequency of rs3093193 was significantly different between the healthy control group and the HAPE patients, and rs3093193 was significantly correlated with the reduced risk of HAPE (OR = 0.70, 95% CI = 0.52 -0.93, P = 0.014). However, the other three SNPs (rs12459936, rs3093144 and rs3093110) were not significantly associated with HAPE risk in the allele model (P = 0.099, P = 0.252 and 0.050, respectively).

Associations between genotype frequencies and HAPE susceptibility
Multiple inheritance models (dominant, recessive, additive, and codominant models) were applied for analyzing the association between each SNP and HAPE risk in Table 3 were not related to HAPE susceptibility (P > 0.05).

Stratification analysis of CYP4F2 polymorphisms and HAPE risk
Stratified analysis regarding the impact of CYP4F2 polymorphisms on HAPE according to age are displayed in Table 4. The results indicated that rs3093193 was correlated with a decreased HAPE risk Furthermore, we conducted another stratified analysis of gender adjusted for age as shown in Table   5.

Associations between haplotype analyses and HAPE risk
Finally, haplotype-based association and linkage disequilibrium (LD) study were conducted to show the association between CYP4F2 haplotype and risk of HAPE. Among the subpopulation (age ≤ 32 years), three SNPs were found to exist in one LD block (rs3093193, rs12459936 and rs3093144) in CYP4F2 gene (Fig.1). The distributions of different haplotypes of CYP4F2 gene in both the patients of HAPE and controls, and its association with HAPE risk were presented in Table 6. The results showed that the haplotype CTC was found to prominently increase the risk of HAPE among age ≤ 32 years under unconditional logistic regression analysis adjusted for age and gender (OR = 1.52, 95% CI = 1.05-2.19, p = 0.026). Other haplotypes did not display the correlativity.

Discussion
Researches have been revealed that HAPE may have an important pathogenesis of inflammation (19).
A large amount of inflammatory cytokine genes and their single nucleotide polymorphisms have been identified to be associated with the risk of HAPE. However, the contribution of the SNPs in CYP4F2 gene to HAPE risk is remains unclear. Considering these, in this case-control study, allele, genotype and haplotype frequencies of four SNPs in the CYP4F2 gene between HAPE patients and healthy controls were compared and stratification analyses by age or gender were performed. In the overall, rs3093193 was shown to reduce the risk of HAPE, while rs12459936 conferred the increased susceptibility to HAPE. Age stratified analysis revealed that rs3093193 and rs12459936 were correlated with the HAPE risk at age > 32 years, and rs3093193 and rs3093110 were correlated with the HAPE risk at age ≤ 32 years. Gender stratification analysis found that rs3093193, rs12459936 and rs3093110 were all related to HAPE risk in males. Haplotype analysis illuminated that GCCG and CTC could increase HAPE risk at age ≤ 32 years and males, respectively. As far as we know, this is the first study to evaluate the correlation of CYP4F2 SNPs with HAPE risk in Chinese Han population.
HAPE was identified as a non-inflammatory hemorrhagic pulmonary edema, which may be accompanied by secondary inflammation (9) Severe hypoxia induced by HAPE is prone to immunogenic changes and involvement in the development of HAPE, leading to significant changes in the prevalence of immune function during HAPE (20). Inflammatory markers of IL-1IL-6 leukotriene B4, arachidonic acid CRP and CCR5 are upregulated in response to high altitude, and hypoxiainduced inflammation at high altitude may contribute to the development of HAPE (21,22). The CYP4F2 gene, known as a major member of the CYP450 subfamily, encodes o-hydroxylase. CYP4F2 gene is not only up-regulated in inflammatory response, but also participates in the metabolism of inflammatory factors such as leukotriene B4 and arachidonic acid. In humans, this gene is located on chromosome 19p13.12, and 225 SNPs for the human CYP4F2 gene listed in the National Center for Biotechnology Information SNP database Build 126 (http://www.ncbi.nlm.nih.gov/SNP).
Furthermore, studies show that SNPs at many loci of the CYP4F2 gene are associated with various diseases, such as hypertension (23,24), cerebral infarction (25) myocardial infarction (26)(27)(28)(29), and metabolic syndrome (30). There are reports that some SNPs in CYP4F2 gene, such as rs3093105, rs3093135, rs3093200, rs1558139, and rs2108622, can cause an increase or decrease in ohydroxylase activity, which results in altered levels of 20-HETE production (31). Studies have also shown that polymorphisms in CYP4F2, such as rs1558139 and rs2108622, can lead to an increased or decreased activity of the CYP4F2 gene (23). In the present case-control study, our results showed that two intron SNPs in the CYP4F2 gene (rs3093193 and rs12459936) were linked to the risk of HAPE, and three SNPs (rs3093193 and rs12459936) were related to the risk of HAPE in stratified analysis based on age and gender. This result demonstrated that CYP4F2 polymorphism are associated with susceptibility to LDH in Chinese Han population. Considering the established function of SNPs and their influence on gene expression, we speculated that SNPs may affect the occurrence risk of HAPE by changing the expression of CYP4F2 or its o-hydroxylase activity. However, the mechanisms still need more functional studies to testify. Moreover, we could not exclude the possibility that the deficiency of association in a certain group might be a consequence of the limited sample size.
Although in this study, we first investigated the association between CYP4F2 rs3093193, rs12459936 and rs3093110 polymorphisms and HAPE, there are still some potential limitations. Firstly, all participants were recruited from the identical hospitals, which may result in a poor representation.
Secondly, the number of cases in our study was not large and our study population was all Chinese Han people, which can't preclude false-negative results and cannot be extrapolated to other populations. Finally, we only found CYP4F2 polymorphisms may be related to the development of HAPE, but the mechanisms have not been studied.

Conclusion
To summarize, our study provides the first for the association between CYP4F2 variant and HAPE risk

Consent for publication
Not applicable.

Availability of data and meterial
All relevant data are within the manuscript.

Competing interest
Lining Si conceived and designed the experiments; Haiyuan Wang and Guifen Gan: performed the experiments; Rong Wang collected data; Qifu Long analyzed the data; Derui Zhu and Guoping Shen contributed reagents/materials/analysis tools; Yanli Zhao wrote the paper.
CYP4F2 gene is associated with cerebral infarction in Japanese men.   P a -value calculated by logistic regression analysis; P b -value calculated by logistic regression analysis with adjustments for gender and age; * P-value < 0.05 indicates statistical significance.  P-value calculated by logistic regression analysis with adjustments for gender and age; * P-value < 0.05 indicates statistical significance.  Figure 1 Haplotype block map for three SNPs in the CYP4F2 gene (D'). Linkage disequilibrium analysis of CYP4F2 in individuals at age ≤ 32 years. LD is indicated using standard color schemes with bright red signifying very strong LD (r2 < 1/D' = 1), pink red (D' < 1).

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