Genetic Susceptibility Study Of High Altitude Pulmonary Edema In Han Chinese

High-altitude pulmonary edema (HAPE) is one of idiopathic mountain sicknessthat occurs in healthy plainsman when quickly ascended and exposed to altitudeexceeding2,500m above sea level or in highlanders who reach to extreme highaltitude within1-7days. It is considered a life-threatening respiratory disease becauseit occurs in previously healthy people; it remains the major cause of death in newcomers, with0.4%~2%mortality rates in the absence of adequate emergencytreatment. For a significant but unpredictable risk of recurrence, HAPE is suggestedto be an independent clinical disorder with a constitutional and possibly geneticcomponent in its etiology.Although the mechanisms underlying the pathogenesis of HAPE are complex, thegenerally proposed paradigm is a sequential process of hypoxia-induced pulmonaryhypertension, increased permeability of pulmonary capillaries because of stressfailure, and destruction of the alveolar epithelial membrane barrier, resulting in highpermeability pulmonary edema. The exactly pathogenesis of HAPE remains unclear.Existing research results indicated that HAPE showed obvious individual susceptibletendency, because some individuals looks likely more susceptible to HAPE thanothers when exposed to the same hypoxia conditions, probably due to individualvariations in response to hypoxia. Now, it was widely demonstrated that HAPEfrequently occurs in individuals exposed to high altitudes, and several genetic studieshave demonstrated that a genetic susceptibility may play an important role in thedevelopment of HAPE. The scholars put forward that the interaction of genetic andenvironmental factors expected to be the genetic architecture of HAPE, identificationand characterization of susceptibility genes for HAPE require a thoroughunderstanding of these interactions. Growing evidence suggests geneticpredisposition involved in the pathogenesis of HAPE. But, different research groupshave reported the different association results in different enthnic groups’ entire theworld. The SNPs and genes responed to HAPE were still unclear. In addition, there is still lack of sensitive biomarkers in HAPE for early prospect and dignosis.Existing research results indicated that HAPE showed obvious raal spefity andindividual susceptible tendency, suggesting the occurrence of HAPE was associatedwith genetic factors. In recent years, the relationship between some genepolymorphisms and susceptibility to HAPE were studied at home and abroad, but theresults were inconsistent across studies for the race of partipants and researchmethods were different. In this study, association studies of susceptibility to HAPEwere carried out by qualitative analysis. Besides, molecular epidemiology,meta-analysis and bioinformatics methods were adopted to discover the relationshipbetween susceptibility genes and the onset of HAPE. The purpose of this study is toexplore the association of candidate gene polymorphisms with HAPE on the level ofgene and protein. This study is of great importance to in-depth research ofpathogenesis and prevention of HAPE.Recently, proteomic analyses have greatly falitated the comprehensive catalogingof protein expression profiles in not only cell lines but also clinical samples,including serum/plasma, urine, spinal fluid, synovial fluid and tissues. Plasma is notonly the primary clinical spemen, but also the useful targets for disease diagnosis andtherapeutic monitoring. It is the most complex human-derived proteome, containingall tissue proteomes as subsets plus very numerous distinct immunoglobulins, and ithas an extraordinary dynamic range in that more than10orders of magnitude inconcentration separate albumin and the rarest proteins now measured clinically orresearched in labs. With proteomic tools available recently, profiling of humanplasma proteome becomes more feasible in searching for disease-related markers, thepresence of a particular protein and/or its isoforms in the plasma represents thelikelihood of other biologically active molecules. Since cellular functions and theprotein expression pattern often change during different disease states individually, tothe best of our knowledge, there are no references has been reported that differentialdisplay of the plasma proteome profile of acute stage compared with that ofrecovering phase in the same patients with HAPE.Recent progress in high-throughput genotyping technology and the development ofpublic databases of human genome variations has enabled the testing of genome-wide single nucleotide polymorphisms (SNPs) for genetic association. A genome-wideassociation study (GWAS) avoids the need for a priori hypothesis for the primarycause of illness, unlike candidate gene approaches. The GWAS has been successful indetecting a number of replicated and novel marker associations with complexdiseases, and became the basic strategate for genetic study in populatin.In this study, All Han-Chinese HAPE subjects came to Yushu area as constructionworkers after the earthquake of magnitude7.1on April14th2010. We adopted2-Dgel electrophoresis differential display the plasma proteome profile of acute stagecompared with that of recovering phase in the same patients with HAPE. Thosesubjects were also scanned by Affymetrix SNP Array6.0Chips. Genome-wideassociation study (GWAS) was used to screen the susceptibility genes and geneticmarkers. Existing research results indicated that HAPE showed obviously spefity; theresults of relationship between some genes and susceptibility to HAPE wereinconsistent across studies entire the world. Those genes and single nucleotidepolymorphisms (SNPs) were also screened and analyzed.The purpose of this study is to explore the association of candidate genepolymorphisms with HAPE on the level of gene and protein. This study is of greatimportance to in-depth research of pathogenesis and prevention of HAPE.PartⅠ. Differential plasma proteome analysis in patients with high altitudepulmonary edema under different phaseAim: To investigate the differential expression plasma proteins in patients sufferingfrom HAPE under acute stage and recovery phase.METHODS: A complete proteomic profiles analysis were performed by usingtwo-dimensional (2-D) gel electrophoresis followed by mass spectrometry in3patients with HAPE in their acute stage and recovery phase.RESULTS:8protein spots were identified from the comparison (>1.5fold) betweenacute stage and recovery phase, the expression patterns changes in HAPE patientsunder different phase, which mainly were Apolipoproteins, Serum Amyloid PComponent, compliment components, and others. The Apolipoprotein A-I (Apo A-I),Serum Amyloid P Component, and Fibrinogen showed over expression in HAPEpatients in acute stage as compared to recovery phase, But the Apolipoprotein A-IV (Apo A-IV), Antithrombin-Iii showed over expression in HAPE patients in recoveryphase as compared to acute stage.CONCLUSION: The result shows that the differential plasma proteome in theHAPE patients may be related to the occurrence of HAPE, and the expressionchanged protein, Apolipoprotein A-I, would offer a meaningful biomarker forunderstanding of HAPE for precaution, diagnosis and treatment.Part Ⅱ. Genome-wide association study of HAPE by Affymetrix SNP Array6.0ChipsAim: To screen and analyze the susceptibility genes and single nucleotidepolymorphisms (SNPs) in Han Chinese HAPE.METHODS:1).The DNA samples of40patients with HAPE and33healthy controlswere scanned by Affymetrix SNP Array6.0Chips. Genome-wide association study(GWAS, by PLINK software) was used to screen the susceptibility genes and geneticmarkers.2). The Candidate SNPs from GWAS results were genotyped and analyzed.RESULTS:1). a total of57SNPs were found to be significantly different betweencase and control groups (adjust P <0.05). GO and Pathway enrichment analysis of74genes around the57SNPs indicated that these genes were significantly correlatedwith prostanoid metabolic process, arachidonic ad metabolism and nitrogenmetabolism (adjust P <0.05), which were involved in the physiopathologicmechanism of HAPE.2). The genotype TT rs4906864were significantly moreprevalent among the HAPE-p group (14.8%) than the HAPE-r group (0.08%)(P <0.05) with the odds ratio0.425(0.187-0.966). The T allele were also significantlymore prevalent among the HAPE-p group (38.5%) than the HAPE-r group (29.9%)(P<0.05) with the odds ratio0.682(0.477-0.973).CONCLUSION: These genetic polymorphisms and genes in prostanoid metabolicprocess, arachidonic acid metabolism and nitrogen metabolism were associated withHAPE. The genotypy and allelic frequencies of rs4906864were associated withHAPE pathophysiology process.Part Ⅲ Association study between Candidate genes and HAPEAim: Based on the previous reported biological properties and function of endothelialPAS domain protein1(EPAS1) in hypoxic conditions, investigations have focused the hypothesis that EPAS1gene has an important role in the pathogenesis of mountainsickness. Therefore, the association of EPAS1gene single nucleotide polymorphisms(SNP) with being susceptible to HAPE was investigated. ACE I/D polymorphism alsowere scanned.METHODS:1). The16exons of EPAS1gene were sequenced by ABI3730DNA analyzer in40HAPE patients and40Health Controls.2). Differential SNPs were verified in153HAPE-p,298HAPE-r and135HLTsubjects by polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP) and confirmed by DNA sequencing.3). The135HAPE-p,137HAPE-r and135HLT were enrolled for EPAS1SNPsgenotyping. The twenty-two SNPs of EPAS1gene were selected and studied byimproved multiple ligase detection reaction (iMLDR) allele discriminationexperiments.3) ACE-I/D polymorphism was examined by the polymerase chain reaction (PCR)with in147HAPE-p and193HAPE-r subjects.RESULTS:1). There were no difference in16exons between HAPE patients and Controls. Theallelic frequencies of EPAS1-Chr2:46441523(hg18) polymorphism were significantlydifference between the two groups (P<0.001).2).The verification experiment showed the C allele frequency of Chr2:46441523(hg18) polymorphism were significantly higher in HAPE-p group than in HAPE-rgroup (P<0.001), but the frequencies of heterozygous C/G were significantly higherin HAPE-r group than in HAPE-p group (P<0.001). Moreover, the frequencies of theEPAS1Chr2:46441523(hg18) polymorphism G allele were significantly higher inHLT group than in HAPE-p and HAPE-r groups.3). The CC genotype of rs10193827and rs4953359were significantly more prevalentamong the HAPE-p group than the HAPE-r group. The genotypes GG, TT, AA ofrs13419896, rs17035010, rs6544889and combined-genotypes GG-TT-AA differedsignificantly between HAPE-p and HAPE-r. The rs10193827were significantlyassociated with HAPE risk under the dominant model of inheritance [OR (95%): 1.653(1.023-2.669), P=0.029], while the rs6544889were significantly associatedwith HAPE risk under the recessive model of inheritance [OR (95%):4.821(1.022-22.745), P=0.029]. The genotypes CC, TT, GG, TT, TT, TT, AA, AA, GG,CC, CC, GG, AA, GG, AA, GG of rs10193827, rs11675232, rs13419896,rs17035010, rs1992846, rs4953359, rs6544889, rs7589621, rs10206434, rs13006131,rs1562453, rs1868092, rs4953354, rs4953361, rs7571218and rs7598371, prevalentin Han Chinese, and their counterpart homozygotes, prevalent in HLT group.4). The genotype frequencies of ACE-I/D for II, ID, DD in HAPE-r and HAPE-pgroups were0.430,0.446,0.124and0.435,0.469,0.095, respectively, the allelicfrequencies of I and D were0.650,0.350and0.670,0.330, respectively. The OR ofID, DD and D alleles relative to II for HAPE was0.961(0.610-1.514),1.322(0.634-2.758) and1.080(0.783-1.489). There was no significant difference of thegenotypic and the allelic frequencies in ACE-I/D polymorphism between the HAPE-pand HAPE-r groups.CONCLUSION: This genetic study provided evidence that EPAS1gene isassociated with susceptibility to HAPE in a Han Chinese population, and all of theselected SNPs are strongly associated with high altitude adaptation in the Tibetans.There is no relation between ACE-I/D polymorphism and HAPE in the Han Chinese.