Mitochondrial DNA Variation And The Susceptibility Of Acute Mountain Sickness In Southwestern Han Chinese

Acute mountain sickness (acute mountain sickness, AMS) is caused by hypoxia that typically affects lowlanders who ascend rapidly to high altitude or highlanders who ascend to a higher altitude. Headache is the hallmark phenotypical characteristic of AMS. AMS usually occurs within 6 to 12 hrs of arrival at high-altitude and resolves in 24 to 48 hrs. The incidence of AMS ranges from 15% to 75%, which mainly depending on the rate of ascent, the altitude attained, and individual susceptibility. AMS is the mildest form of acute high altitude disease (acute high altitude disease, AHAD), which was classified into AMS, high altitude pulmonary edema (HAPE), and high altitude cerebral edema (HACE) in the Seventh Banff hypoxia Forum in Canada in 1991. In China, AMS has also been named acute mild altitude disease (AMAD) since 1995. Prior assessment of AMS in China was based on the score systems proposed by Chinese Professor Yin and his colleagues, and the Lake Louise AMS scoring system was rarely used.As a neurological disorder caused by hypoxia, the underlying pathophysiology of AMS is still unclear. Differences in the susceptibility of individual and population, and even familial clustering are often cited as the genetic evidence that there is innate, inherited factors as an important cause of AMS. Thus far, several researches which mainly focused on nuclear DNA (nDNA) have been performed to explore the genetic susceptibility to AMS, but gained no breakthrough progress. Therefore, it is important to explore a new genetic system to reveal genetic mechanism of AMS susceptibility and to establish a sensitive and reliable predictive method of AMS.Hypoxia is major factor affecting people who ascend to altitudes. Mitochondrion is an important organelle found in most eukaryotic cells, which consumes 90% of absorbed oxygen and generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy. Since mitochondrion is in the central position of cellular metabolism and biological energy conversion, it plays important roles in the life activities. Additionally, mitochondrion also produces a small quantity of reactive oxygen species (ROS) through oxidative phosphorylation (OXPHOS) when generating ATP and heat essential for body. When oxygen is limited (hypoxia), the electron can’t be transferred to oxygen via the electron transport chain, and thus, the production of energy would reduce and ROS would dramatically increase, which may contribute to the decline in mitochondrion and cell function and eventually cause cell and tissue damage. Several studies have found that increase of ROS during hypoxia might contribute to pathophysiology of AMS. Mitochondrial DNA (mtDNA) is the genome exceptionally located outside of eukaryotic cell nuclear. It has been confirmed that mtDNA variation was associated not only with some human diseases and human adaptation to climate, but with adaptation to high altitude in human and other species.In humans, each double-stranded circular mtDNA molecular consists of 16569 base pairs, which encodes 37 genes. Of the 37 genes, 13 are for polypeptides, 22 are for tRNA and 2 are for rRNA. The polypeptides encoded by mtDNA are important component for the electron transport chain. MtDNA is maternally inherited and has a mutation rate over 10 times higher than nDNA. The mutation of mtDNA can be categorized into three groups: neutral mutations, deleterious mutations and adaptive mutations. Some deleterious mutations would be eliminated from the population by purifying selection over time. By contrast, some adaptive mutations would retained and be enriched in the population to form haplotype and haplogroup. The haplotype and haplogroup enriched by a specific circumstance may not be able to adapt to another environment, and thus, lead to the carriers vulnerable to some diseases. For example, European mtDNA haplogroups J and K are considered to be influenced by cold selection; however, both haplogroups have been linked to increased susceptibility to multiple sclerosis. Recent studies have shown that certain mtDNA haplogroups may also influence OXPHOS and change ATP and ROS production. Therefore,we speculated that mitochondrial DNA genetic variation might be involeved in AMS susceptibility.The present study enrolled 1874 male Han Chinese who were new army recruits and ascended to Lhasa city of Tibet Autonomous Region by air in November 2008. After a cross-sectional study conducted in the new army recruits from six provinces and one city of China by cluster sampling and developing the questionnaire of AMS to explore the prevalence of AMS and potential risk factor, PCR-RFLPs (Polymerase Chain Reaction–Restriction Fragment Length Polymorphisms) and mtDNA HVS I (hypervariable segment I) sequencing was applied to analyze, based on the case-control study, the association of mtDNA haplotypes in the Han lowlanders from south-western (SW) China with AMS susceptibility. Epstein-Barr virus (EBV) was used to establish lymphoblastoid cell lines which carried AMS susceptibility associated mtDNA haplotypes, which will be used to study biological functions of the mtDNA haplotypes in the future. Results:1. At 3,700 m, the incidence of AMS in Han Chinese was 37.41%. Among these regions, the subjects from Zhejiang province (ZJ) had a highest incidence of AMS (48.94%), while the subjects from Yunnan province (YN) had a lowest incidence of AMS (29.41%) (p < 0.0001, ZJ vs. YN). The subjects from Northern China represented by Shandong province (SD) and Heilongjiang province (HLJ) had a lower incidence of AMS (30.56%), and a significant difference was found in subjects from northern China and from south-eastern China represented by ZJ ( p = 0.003). The average Lake Louise AMS score in YN Han was 2.250±2.197, which was significantly lower than that in Han Chinese form ZJ, Guizhou province (GZ), Sichuan province (SC), and Chongqing city (CQ) (p = 0.0452, 0.0229, 0.0036, and 0.0446,vs. YN, respectively).2. Multivariate logistic regression analysis showed that body mass index (BMI) is a risk factor of AMS (OR = 1.378, 95%CI = 1.284-1.479, p < 0.0001) and that the subjects who resided at altitudes greater than 1,000 m had a 0.630-fold decreased risk of developing AMS (95%CI = 0.486-0.817, p < 0.0001). When compared with south-eastern Han Chinese and after adjusted by BMI and resident altitude, it was found that northern Han Chinse was associated with a 0.468-fold decreased risk of developing AMS(95%CI = 0.279-0.784, p = 0.004), while there was no significant difference between southeastern Han Chinese and south-western Han Chinese(p = 0.634).3. The subsequent analysis stratified by resident altitude confirmed that the subjects who lived at altitude between 1,000 m and1,500 m, and greater than 1,500 m in YN had a significantly lower AMS incidence than that in ZJ (p = 0.001 and < 0.0001, respectively), while there was no significant difference in Han lowlanders from ZJ and YN ( p = 0.092).4. The analysis of mtDNA haplogroup in Han lowlanders from southwestern China showed that, compared with non-AMS subjects, mtDNA haplogroups D and M9 decreased significantly (p < 0.0001 and 0.003, respectively), while haplogroup M7 increased significantly in AMS subjects (p < 0.0001).5. The multivariate logistic regression analysis showed that, after adjustment for BMI, both haplogroups D and M9 were associated with significantly decreased risks of AMS, while M7 was associated with a significantly increased risk of AMS (OR = 0.605, p < 0.0001; OR = 0.037, p = 0.001, and OR = 2.419, p = 0.001, respectively). In addition, the subsequent analysis stratified by the AMS severities indicated that haplogroup B was correlated with a 2.41-folds increased risk of developing severe AMS (95%CI = 1.288-4.514, p = 0.006).6. EBV was applied to transform lymphoblastoid cell lines carrying mtDNA haplotypes B、D、M7, and M9. The cellular karyotype has not been changed in the lymphoblastoid cell lines, and its logarithmic growth phase was on the 3rd-4th day after cell recovery. Conclusions:1. Both BMI and resident altitude lower than 1,000m are risk factors of AMS2. There are geographical differences of AMS incidence in Han Chinese populations. South-western Han Chinese and northern Han Chinese have a lower incidence of AMS, while south-eastern Han Chinese has a higher incidence of AMS.3. The higher altitude of permanent residence is one of the main reasons of lower incidence of AMS in south-western Han Chineses. Northern Han Chinese is an independent protective factor to reduce the incidence of AMS.4.There is correlation between mtDNA haplogroups and AMS susceptibility in south-western Han Chinese. MtDNA haplogroups D and M9 are related to individual tolerance to AMS, while haplogroups M7 and B are risk factors for AMS.5. Six lymphoblastoid cell lines carrying mtDNA haplotypes B、D、M7, and M9 were successfully established.