杜 慧，赵 婧，嘎 琴，刘玉红，汤 锋，杨应忠，刘永年*

(1.青海大学医学院基础医学部，青海 西宁810001;2.青海大学高原医学研究中心，青海西宁810001)

1 INTRODUCTION

High－altitude pulmonary edema(HAPE)is noncardiogenic pulmonary edema that usually occurs at altitudes above 2 500 m in rapidly ascending nonacclimatized individuals within the first week after arrival［1，2］.It is generally known that HAPE tends to be associated with exaggerated pulmonary hypertension，with edema in pulmonary interstitial tissue and alveoli.It was defined as a noninflammatory hemorrhagic pulmonaryedema，which may evolve with time infeatures of secondary inflammation［3］.Investigations showed circulating inflammatory markers are upregulated at high altitude［4］.HAPE patients showed the immune cells，and inflammatory markers markedly elevated in the bronchoalveolar lavage fluid，and speculated that hypoxiainduced inflammation at high altitude may contribute to the development of HAPE［5］.

NR3C1(Nuclear Receptor Subfamily 3，Group C，Member 1)gene，which is located on chromosome 5q31－32，codes for the human glucocorticoid receptor(GR)，plays an important role in inflammation.Glucocorticoids(GCs)has various effects，which are exclusively mediated by the intracellular GR.The binding of GCs to cytoplasmic GR molecules is translocated to the nucleus where it interacts with glucocorticoid－responsive elements of different genes，initiates signaling pathways strongly linked to changes in the cellular transcriptional status.GCinhibit production of proinflammatory cytokines and stimulate production of anti－inflammatory cytokines［6］.Variants in the NR3C1 gene may contribute to the spectrum of GC responses in diseases observed at clinic，and also have been found to modulate individual GC sensitivity in invitro investigations and in studies with healthy individuals［7］.A biallelic polymorphism(Bcl1，C＞G)was identified at downstream of the exon 2－intron 2 junction of NR3C1 gene.The polymorphism was named as rs41423247，might affect processing of GCR primary transcripts and also cause increased GC sensitivity，and has also been linked to a cluster of cardiovascular risk factors，such as hypertension，visceral obesity and steroid sensitivity［8］.Therefore，We performed a genetic genotyping in HAPE to investigate if this genetic variant of NR3C1 gene is associated with HAPE.

2 MATERIAL ANDMEDTHODS

2.1 Subjects

HAPE patients(HAPE)had been hospitalized during 2010 to 2017 from the Yushu city(3760m)of Qinghai province.The patients were diagnosed with HAPE depend on the diagnostic criteria［9］.In this study，there were 133 cases of HAPE patient(HAPE－p)，all of which were han nationality and male，with an average age of 40.20±9.91 years old.In the Healthy controls(HAPE －r)were enrolled from the same area，matched HAPE with age，sex，workplace.135 cases were selected，and the average age was 40.92±5.15 years old.The routine physical examination and related physiological indexes were detected.This study was approved by Ethics Committee of Medical College of Qinghai University，and every patient signed written consent before blood specimens were collected.All subjects were of Chinese Han ethnicity and had no blood relationship with any other enrolled subject.

2.2 Sample collection and DNA extraction

We collected 5 mL of venous blood samples from each participant.Blood samples were anticoagulated by EDTA.Genomic DNA was extracted from blood by Gentra Puregene Blood Kit(Qiagen，158389，Germany)according to standard procedures.DNA samples were stored at－20 ℃ until use.

2.3 Genotyping

The rs41423247 polymorphism of NR3C1 gene were genotyped by the Single base extension detecting technology(iPLEX)(Capital Bio Corporation，Beijing，China).SNP loci－ tested polymerase chain reaction(PCR)primers and single base extension primers were designed by using the Sequenom MassARRAY Assay Design Genotyping Software and Tools(Sequenom，San Diego，CA，USA).PCR reaction consisted of 4.0 μL of PCR Master Mixture(Takara Bio Inc.，Japan)，1 μL of primers mixture，1.0 μL of gDNA，1.9 μL of ddH2O，and 0.1 μL of dNTPs.PCR was performed under the following thermal cycling conditions:94℃for 4 minutes，then 94 ℃ for 20 seconds，56 ℃ for 30 seconds，and 72℃ for 1 minute for 45 cycles，and 72 ℃ for 4 minutes.PCR products were treated with shrimp alka-line phosphatase to remove free deoxyribonucleoside triphosphates，and single base extension reaction was performed in 2.0 μL of EXTEND MIX，0.619 μL of ddH2O，0.94 μL of Extend primer mix，0.2 μL of iPLEX buffer plus，0.2 μL of iPLEX terminator，and 0.041 μL of iPLEX enzyme(Sequenom，San Diego，CA，USA).The thermal cycling conditions were as follows:94 ℃ for 30 seconds，then 94 ℃ for 5 seconds，52 ℃ for 5 seconds，and 80℃ for 5 seconds for 40 cycles，and 72 ℃ for 3 minutes.The MassARRAY Nanodispenser RS1000(Capital Bio Corporation，Beijing，China)was used for dispensing the purified extension products onto a 384－element SpectroCHIP bioarray(Sequenom，San Diego，CA，USA)，and mass spectrometric analysis was performed using the MALDI－TOF(matrix－assisted laser desorption/ionization－time of flight)(Sequenom，San Diego，CA，USA).The results were analyzed using TYPER 4.0 software(Sequenom，San Diego，CA，USA).

2.4 Statistical analysis

SPSSsoftware(version 17.0;SP SS，Inc，Chicago，IL，USA)was used for statistical analysis.Hardy－Weinberg equilibrium(HWE)was used to assess the representativeness of the participants.Allele frequencies were calculated based on genotype frequencies in HAPE－p and HAPE－r groups，and the intergroup difference was estimated with chi－square test.Odds ratios(ORs)and 95%confidence intervals(95%CIs)were used to evaluate the strength of association between the variants and HAPE.P＜0.05 was considered as statistically significant.

3 RESULTS

As shown in table 1，the Rs41423247 genotype of the two groups was in the Hardy－Weinberg equilibrium(HAPE－p:χ2=0051，P=0.821;HAPE－r:χ2=0.287，P=0.592).

Table 1 SNP Hardy Weinberg equilibrium test

The genotype frequencies of rs41423247 for GG，GC，CC in HAPE－p and HAPE－r groups were 0.699，0.271，0.030 and 0.563，0.363，0.074，respectively were shown in Table 2.The allelic frequencies of G and C in HAPE－p and HAPE－r groups were 0.835，0.165 and 0.744，0.256，respectively.The GG genotype were significantly more prevalent among the HAPE－p group than the HAPE－r group(P＜0.05).The Gallele was also significantly more prevalent among the HAPE－p group than the HAPE－r group(P＜0.05)with an odds ratio of 1.732(95%CI=1.134－2.645).

Table 2 Distribution of the Genotypic and Allelic Frequencies of the SNPs between HAPE and Health Groups

4 DISCUSSION

Investigations showed circulating inflammatory markers are upregulated at high altitude［10］，the serum levels of IL－6，IL－6 receptor，and C－reactive protein were increased in healthy volunteers who spent 3 nights at an elevation higher than 3 400 m［4］.The climbers at 8 400 m had severe hypoxemia，and were found with subclinical HAPE［11］.The inflammatory cells and markers elevated in the bronchoalveolar lavage fluid in HAPE patients［5］.Moreover，accumulations of inflammatory cells in multiple organs，and elevated serum levels of cytokines also were found in mice after shortterm exposure to low oxygen concentrations［12，13－17］.Those studies indicated that high－land environment induces the inflammation，and the inflammation happened in pulmonary contribute to the development of HAPE.The balance of the inflammation in pulmonary play an important role in the pathogenesis of HAPE.Anti－inflammatory medication are useful for prevention and treatment of AMS［18］.

Glucocorticoids(GCs)，endogenous“dexamethasone”，regulate a broad spectrum of physiologic functions essential for life and play an important role in the maintenance of basal and stress－related homeostasis，including inflammatory reactions.The effects of GCs is mediated by the GR.The human GR belongs to the steroid/thyroid/retinoic acid nuclear receptor superfamily，it regulates the expression of glucocorticoid－responsive genes positively or negatively［6，19］.

Variations in NR3C1 gene may contribute to GC responses.This polymorphism is coupled with two other SNPs in intron B，rs33389 and rs33388［7，8］.All three variants are common substitutions and may potentially alter the processing of GC primary transcripts and increases sensitivity to GCs.SNP enhances the GC－dependent genomic mechanisms which in complexes with GR influence glucocorticoid response element(GRE)sequences in promoter and regulatory parts of gene coding protein synthesized in response of cells to glucocorticosteroid action［7］.Yan et al indicated the NR3C1 gene is associated with cardiovascular risk factors in a healthy Chinese Han population［20］.The NR3C1 gene became a hot－spot to explain the pathogenesis of diseases at the clinic.Therefore，we tried to do this hypothesis to find out some clues to be used as a baseline study for future experiments in this field.In current study，We had done the correlation between the rs41423247 of NR3C1 gene with HAPE in Han Chinese.This SNP is correlated with HAPE susceptibility in current study.GG genotype and G allele of rs41423247 polymorphism might increase the risk of HAPE in Han Chinese.

5 ACKNOWLEDGMENTS

We thanks those physicians for sample collection，Dr Jiangxi from the Yushu People's Hospital，Dr Wei Guan from the Affiliated Hospital of Qinghai University.We also extend our appreciation to Professor Ge Rili，director of the Research Center for High Altitude Medicine，Qinhai University，given us vigorously supporting anddisinterested assistance.