Genetic Analysis And Functional Study Of Susceptibility To Pulmonary Edema In Plateau

BackgroudsHigh-altitude pulmonary edema (HAPE) is considered to be a non-cardiogenic reversible hydrostatic edema that occurs in healthy persons exposed to high altitudes exceeding2,500-3,000m above sea level; it’s characterized by high pressure in pulmonary artery with edema in pulmonary interstitial tissue and alveolus. For a significant but unpredictable risk of recurrence, HAPE is suggested to be an independent clinical disorder with a constitutional and possibly genetic component in its etiology. Mortimer et al. have written an excellent review on the genetic underpinnings of HAPE. However, to determine how many genes and which genetic determinants are actually involved in the development of HAPE remains an interpretive challenge. So it is reasonable to expect that multiple genetic and/or physiological safeguards have developed to maintain pulmonary artery pressure and tissue and alveolus edema within a range of physiologically acceptable levels. Given gene-gene interactions are expected to be ubiquitous in the genetic architecture of complex diseases, identification and characterization of susceptibility genes for HAPE require a thorough understanding of these interactions.As yet the pathophysiology of HAPE is not fully understood, although studies have substantially contributed to the current understanding of several areas, including an exaggerated increase in pulmonary artery pressure in response to hypoxia and exercise, and the role of inflammation and alveolar-fluid clearance in HAPE. Available information about the genetic linings of HAPE is limited in the literature, with no consensus on firm conclusions, mainly because of the rareness of this disease and complicated environmental factors.Although variants in these genes have shown associations with HAPE in some studies, results are often not reproducible in other populations. These inconsistencies from previous studies of various candidate genes with HAPE might have resulted from differences in the polymorphism frequencies that construct the specific genetic architecture of different ethnic population. As a consequence, the relative contribution of any given variant varies from the different ethnic populations. One gene may be responsible for the disorder in one population, but not necessarily in another population. Therefore, reproduction of the finding in a different ethnic population is considered to be important.Genotyping of multiple diallelic sites, especially dense single nucleotide polymorphism (SNP) sites, is now available for genetic studies of human disease. It is more powerful to focus on the transmission of multilocus haplotypes, as opposed to alleles at individual loci. Therefore, haplotype analysis is mandatory in this regard and has become an increasingly popular tool for population genetic studies and disease-gene discovery.To address this issue and to extend association studies to other populations, we here conducted the largest nested case-control study to-date to explore the association between genetic polymorphisms in RAAS, αENaC, HSP70and the development of HAPE in Han Chinese.1. Population-Based Association Study ObjectsIn this context, we here conducted the largest nested case-control study to-date to explore the association between multigenic polymorphisms and the development of HAPE in Han Chinese. In this study, we analyzed18gene polymorphisms evenly interspersed in9candidate genes of RAAS, SCNN1A, HSP70. Because our genetic data were multilocus, the interactions between these polymorphisms should be evaluated and addressed. Traditionally, one approach to modeling gene-gene interactions in complex diseases is parametric-based logistic regression. The MDR method has been successfully applied to detecting gene-gene and gene-environment interactions for a variety of clinical endpoints. Accordingly, we have adopted the MDR method in the present study aiming to explore the synergistic effects of these polymorphisms on the development of HAPE in a large Han Chinese population.Methods 1) This study was a population-based nested case-control study. We recruited140HAPE patients and144controls during the construction of Qinghai-Tibet railway and genotyped18gene polymorphisms evenly interspersed in9candidate genes. Gene-gene interaction was conducted by multifactor dimensionality reduction (MDR, v.2.0.0) program.2) Using polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) method, polymerase chain reaction based single strand conformation polymorphism (PCR-SSCP) method, and polymerase chain reaction products direct sequencing method, we systematically screened polymorphisms and genotyped.ResultsSinge-locus analysis1) In the RAAS, single-locus analysis showed that CYP11B2C-344T and K173R and ACE A-240T polymorphisms were significantly associated with HAPE after the Bonferroni correction (P<0.01).2) In the SCNN1A, A663T polymorphism was associated with HAPE (P<0.05).3) In the Hsp70, HSPA1A A-110C polymorphism was associated with HAPE (P<0.05).Linkage Disequilibrium (LD) AnalysisThe LD analysis constructed a linkage block including C-344T and K173R polymorphisms, HSPA1B G2074C and HSPA1L T2437C polymorphisms in complete LD with each other, while occurred with significantly different frequencies between HAPE and controls. And, linkage blocks including T2437C and HSPA1A G190C polymorphisms, T2437C and HSPA1B A1267G polymorphisms were in complete LD with each other, occurred with significantly different frequencies in controls.Gene-Gene Interaction Analysis and Haplotype Analysis The gene-gene interaction analysis indicated the overall best model including A-240T, A2350G, C-344T, A1267G and A663T polymorphisms with strong synergistic effects. Haplotypes A-A-T-A-A (in order of A-240T, A2350G, C-344T, A1267G and A663T polymorphisms) conferred high genetic susceptibility to HAPE.2. Functional StudyObjectsThe plasma was compared with regard to the plasma proteomics between HAPE patients and controls; the differentiated proteins in patients were selected with their encoded genes as the candidate in order to explore the pathogenesis of HAPE.Proteomics StudyThe high abundant proteins of plasma proteomics were separated from each sample through the liquid chromatography. The remaining proteins with low-to-medium abundance were separated with a two-dimensional (2-D) gel electrophoresis, and analyzed with electrospay mass spectrometry approach and bioinformatics tools. We identified differentially expressed proteins in the plasma between HAPE patients and control subjects, and found that transferrin level was significantly higher in HAPE patients than controls.Genomics Study and Population-Based Genetic StudyWe analyzed the genetic structure of transferrin based on the results of proteomics, and sequenced the promoter and intron1region of transferrin gene. There are seven SNP, viz. A-740G, G-618A, G-552A, G-35T, T+463C, A+1138G, G+1649A.Genotyping was performed using PCR, RFLP and DNA sequencing techniques. Data were analyzed using single-locus, haplotype and synergistic analysis. Our results showed that, in single-locus analysis, the genotype distributions of G-35T and G+1649A polymorphisms differed statistically between HAPE patients and controls, while other genotype and allele distributions showed no differences. Using MDR method, we selected the final best model including A-740G, G-618A, G-552A and G-35T polymorphisms by evaluating the magnitude of cross-validation consistency and prediction error simultaneously. And the haplotype analysis focusing on polymorphisms selected from MDR indicated that overall haplotypes of A-740G, G-618A, G-552A and G-35T showed strong associations with HAPE. Haplotype inspection further identified the potential risk-conferring and protective haplotypes in this population, such as the risk-conferring haplotype G-G-G-T (in order of A-740G, G-618A, G-552A and G-35T polymorphisms), and the protective haplotype A-G-A-T.Transcriptional Activity AnalysisIn order to explain the results of population-based genetic study, we constructed the expression plasmid containing the luciferase reporter gene inserted with different haplotypes of TF gene promoter region. To study whether the certain polymorphisms or haplotypes in the regulation region alter TF gene transcription, the above plasmids and an intra-control were cotransfected into A549cell by eukaryote gene transfection techniques.1) Construction of expression plasmid containing the luciferase reporter geneThe insertion fragment contained different haplotypes of TF gene promoter region were obtained by PCR amplification from genome DNA and subcloned in the pMD-18T vector. The reporter expression plasmids were constructed by ligating the cohesive-end fragment produced by restriction endonuclease Kpn I and Xho I in pGL3-Basic/Enhancer vector and subcloned pMD-18T vector.2) Observation of proliferation of human lung cancer A549HAPE is considered to be a non-cardiogenic reversible hydrostatic edema that occurs in lung with healthy persons. So, we used the human lung cancer A549as the transfected host cell. Study the process of proliferation of human A549in F12(contain10%FBS). The cells grew rapidly from the3th to the8th day after plating.3) Effect of different haplotype on the transcription of TF geneTo study whether certain polymorphisms or haplotypes in the regulation region alter TF gene transcription, the above plasmids and pRL-TK as an intra-control were cotransfected into A549cell by eukaryote gene transfection techniques.The results of transfecting human A549cell in proliferation phase showed that the transcriptional activity had significant differences among haplotype H4, H6and pGL3-promoter.4) Effect of Different Haplotype on the Transcription of TF Gene under Different EnvironmentIn order to study the function of the four haplotypes of TF gene promoter region, we mimic the hypoxic environment (3%O2or100μmol/L CoCl2) and cotransfect the above plasmids and pRL-TK into A549cell by eukaryote gene transfection techniques. The results showed that the transcriptional activity had significant differences under different environment, and time-independent.In conclusion, the results here showed that we conducted the largest nested case-control study to-date to explore the association between variations in multiple genes and HAPE in Han Chinese. We recruited140HAPE patients and144controls during the construction of Qinghai-Tibet railway and genotyped18gene polymorphisms evenly interspersed in9candidate genes. The single-locus analysis showed that the single polymorphisms were not associated with HAPE significantly. The gene-gene interaction analysis found the overall best model including ACE A-240T and A2350G, CYP11B2C-344T, HSPAIB A1267G and SCNNIA A663T polymorphisms with strong synergistic effect and conferred high genetic susceptibility to HAPE, which was further strengthened by haplotype analysis. Our results add evidence for synergistic effect of multi-gene polymorphisms on HAPE susceptibility or tolerance. Meanwhile, we compared the plasma proteomics between HAPE patients and controls; the differentiated proteins in patents were selected with their encoded genes as the candidate in order to explore the pathogenesis of HAPE. Based on the results of the proteomics and the genomics, we selected the transferrin as the candidate and explored the association between variations in transferrin gene and HAPE in Chinese population. Moreover, we studied the function of relevant allele based on the results of association analysis; the results showed that the promoter region of the transferrin gene might be involved in the regulation of transcription of the gene.This study firstly adopts the population-genetics, candidate-gene study to explore the association between multigenes and HAPE, and studied the function of relevant allele based on the results of association analysis, that supplied a part of theory evidence for the pathogenesis of HAPE.