A Preliminary Study On The Role And Mechanisms Of Mitochondrial Transcription Factor A Gene Polymorphisms In High Altitude Pulmonary Edema

Background and obsjectivesWhen ascending to an altitude over3000meters, the human body starts a numberof adaptive responses to adapt to the hypoxic environment. This is termedacclimatization. Maladapation to high altitude will lead to mountain sicknesses, such ashigh altitude pulmonary edema (HAPE). Ours previous studies suggest thatmitochondria are the key points of cellular hypoxic acclimatization. Otherwise,mitochondrial structural and functional impairment will cause maladaption to hypoxicenvironment, even mountain sicknesses, for example HAPE, occur.The mechanisms of HAPE are to be unveiled. It is at present thought to beconnected with pulmonary vascular constriction; pulmonary hypertension, pulmonarycapillary permeability, and water clearance malfunction of type II epithelial cells.Recent study indicated that hypoxia could lead to mitochondrial malfunction andreactive oxygen species (ROS) over production, which consequently causes vascularconstriction and water clearance malfunction. These could contribute to themitochondrial mechanisms of HAPE.Based on these above, we presumed that HAPE could be correlated with mtDNAinstability and mitochondrial malfunction. As a key factor ot mtDNA maintenance, it isworth of further study on TFAM polymorphisms and HAPE susceptivity.Materials and methodsHan HAPE patients and the controls were recruited in Xigaze (3836m), Tibet, andperepheral blood collected. The diagnosis of HAPE was according to the diagnosticcreteria of HAPE publish by Chinese Medical Association in1995, and the controlswere healthy Han who came to Xigaze in the corresponding time period. Subjectssuffering known pulmonary diseases. Genomic DNA was extracted with the BloodDNA Kit and a supercoiling sensitive qPCR approach was used to measure mtDNAdamage. We then sequenced7TFAM exons and neighborhood noncoding regions andcompared them with the NCBI TFAM sequence (TFAM transcription factor A, mitochondrial [Homo samiens (human)], Gene ID:7019, updated on27-Mar-2014) toaquire SNPs and genotypes. Chi-square test was used to analyze distributions of allelesand genotypes in HAPE patients and the controls, and Binary logistic regression wasapplied to assess the risk of the genotypes. To explore the SNPs functions, we used anon line software Match (http://www.gene-regulation.com/pub/programs.html#match) toanalyze transcription factor binding sites of the TFAM sequence. And we further usedone way and two way ANOVA to investigate effects of the SNPs on mtDNA stability,and evaluated its role in HAPE pathophysiology.Results1. By PCRing cytochrome b (cyt b) to present the mtDNA copy number, we foundthe original mtDNA was significantly higher in HAPE patients (1.590±0.092)than in thecontrol (1.245±0.076)(p=0.005), and the heated mtDNA significantly lower in HAPEpatients (0.865±0.045) than in the control (1.079±0.057)(p=0.004). Alternatively, ByPCRing cytochrome C oxidase subunit III (COX III) to present the mtDNA copynumber, we found the original mtDNA was significantly higher in HAPEpatients(1.159±0.061) than in the control(1.044±0.039), while the heated mtDNA wasnot significantly different between the groups (1.354±0.069，1.170±0.069)(p=0.049). Acommon4977bp deletion was not significantly diferent between HAPE patients(1.291±0.154) and the control (1.167±0.094)(P=0.494).2. We found4SNPs in the7exons and neighhood noncoding regions of TFAM,which were A281T, C305T, rs1937and A3790G. Among these SNPs, rs1937has beenpreviously reported, while A281T, C305T and A3790G are newly found in this study.With the transcription binding site analysis, we found A281T was inside the bindingsequence of Staf, a factor that involves in modulation of TFAM transcription.3. In the A281T SNP, the ratio of allele T and genotype AT was significantly lowerin HAPE patients (0%，0%) than in the control (15%,30%)(p=0.000，p=0.000). Byusing allele A as the reference, allele T is a protective factor of HAPE (OR=0.850，CI：0.788-0.916). By using genotype AA as the reference, genotype AT is a protective factorof HAPE (OR=0.800，CI：0.705-0.908). The distribution of alleles and genotypes ofC305T, rs1937and A3790G was not show significant difference between HAPEpatients and the control (p>0.05).4. By PCRing cyt b to present the mtDNA copy number, we found the originalmtDNA was significantly higher in both the control281AA genotype (1.355±0.102) and the HAPE281AA genotype (1.590±0.092) than in the HAPE281AT genotype, and theheated mtDNA was significantly in the control281AA genotype (1.061±0.065) and thecontrol281AT genotype (1.118±0.117) than in the HAPE281AA genotype (p=0.015，p=0.019). By PCRing COX III to present the mtDNA copy number, we found theheated mtDNA was significantly higher in control281AA genotype (1.281±0.079) andthe HAPE281AA genotype (1.354±0.069) than in the control281AT gentype(0.912±0.058)(p=0.009，p=0.001), while the heated mtDNA was not significantlydiferent among the groups (F=1.363，p=0.260). The genotypes of C305T, rs1937andA3790G did not show significant differece among the groups (p>0.05).Conclusions1. The mtDNA instability plays an important role in HAPE development.2. It was the first time that we reported TFAM SNPs of A281T, C305T andA3790G.3. The allele T and genotype AT were protective factors of HAPE development inthe TFAM SNP A281T. This SNP could be of great importance to predict HAPE and itis worth of further study on its role in HAPE mechanisms.4. The TFAM281AT genotype may affects the binding of the transcription factorStaf and facilitate mtDNA maintenance to prevent HAPE development.