The relationship between Polymorphisms of PTEN Gene and High Altitude Polycythemia in Tibetans at the Qinghai-Tibetan Plateau #

XU Jin，YANG Ying-zhong，Li Yong-zhen，TANG Feng，WANG Zhan

(1.Department of the Basic Medical Sciences，Qinghai University Medical College，Qinghai 810001，China；2.Research Center for High Altitude Medical Sciences，Qinghai University Medical College，Qinghai 810001，China； 3.Department of Biomedical Engineering，Tsinghua University，Beijing 100000，China)

TherelationshipbetweenPolymorphismsofPTENGeneandHighAltitudePolycythemiainTibetansattheQinghai-TibetanPlateau#

XU Jin1，YANG Ying-zhong2，Li Yong-zhen1，TANG Feng2，WANG Zhan3※

(1.Department of the Basic Medical Sciences，Qinghai University Medical College，Qinghai 810001，China；2.Research Center for High Altitude Medical Sciences，Qinghai University Medical College，Qinghai 810001，China； 3.Department of Biomedical Engineering，Tsinghua University，Beijing 100000，China)

ObjectivePTENgenewere identified that exhibit evidence of local positive selection of Tibetan adaptation by previous works.Although Tibetans adapt to high altitude environments through a lower blood hemoglobin concentration compared to other populations，some Tibetans still develop high altitude polycythemia(HAPC).To explore whether the polymorphisms of Phosphatase and tensin homolog(PTEN)gene are associated with the susceptibility to high altitude polycythemia(HAPC)in Tibetans at the Qinghai-Tibetan Plateau.MethodsWe enrolled 63 HAPC-p(HAPC patient)and 131 HAPC-r(HAPC resistant)from the Yushu area in Qinghai province where the altitude is over 3500 m above sea level.Blood samples were collected from each of the HAPC-p and HAPC-r groups.Information about physiological phenotypes was obtained via fieldwork investigation.Seven SNPs of thePTENgene(rs1234212，rs1234213，rs1234219，rs2299939，rs2299941，rs532678 and rs555895)were genotyped in HAPC-p and HAPC-r groups by the Sequenom MassARRAY SNP assays.ResultsThe incidence of HAPC was higher in men than women，and the SaO2was significantly lower，while Hb and Hct were much higher in HAPC-p group compared to the HAPC-r group.There was no significant difference of the genotypic and the allelic frequencies in the selected polymorphisms ofPTENgenebetween the HAPC-p and HAPC-r groups.ConclusionsThere is no relation betweenPTENgenepolymorphisms and HAPC in the Tibetans.

HAPCPTENPolymorphism

1 INTRODUCTION

Qinghai-Tibetan plateau，the highest plateau in the world，has an average elevation of 4 000 meters above sea level.It was estimated that 53% Tibetans live at an altitude over 3 500 meters[1].The Tibetans，who have been live permanently in Qinghai-Tibetan plateau，possess heritable adaptations to the hypoxic environment，as indicated by lower hemoglobin(Hb)levels，lower haematocrit(Hct)[2]，higher oxygen saturation of blood in infants[3]and high work performance[4].Although most of the Tibetans do not exhibit the high hemoglobin level to cope up with oxygen deficiency as observed in other populations who have moved temporarily or permanently at high altitudes，some Tibetans living at high altitude do show elevated hemoglobin concentration[5]and even develop high altitude polycythemia(HAPC)[6-8].males，Hb≥21 g/dL；females，Hb≥19 g/dL.When high-altitude dwellers compensate for high altitude associated hypoxia，hypoxia enhances erythropoiesis，consequently，increases circulating red blood cells to deliver more oxygen to their tissues.This compensatory response in excess is termed high altitude polycythemia(HAPC)and it is encountered in 5% to 18% of the population residing at the Qinghai-Tibetan Plateau[9，10].HAPC leads to a significant increase in blood viscosity，microcirculation disturbance，extensive organ damage[11，12].Although hypobaric hypoxia is recognized as the primary initiator of HAPC in high altitude dwellers，the precise mechanisms underlying the pathogenesis and adverse outcomes in subjects with HAPC are not well understood.

Growing evidence suggests that the positive directional selection genes ofPTEN，EPAS1，EGLN1，PPARAgenes et al.play an important role in the adaptation of Tibetans to long-term residence at high-altitudes[13-18].PTEN(Phosphatase and tensin homolog)gene was identified by XP-EHH and iHS test in a set of regions that exhibit evidence of local positive selection of Tibetan adaptation[16].It is an important regulator of cell cycle progression and cellular survival via the AKT signaling pathway[19].It negatively regulates the AKT pathway and is essential to homeostatis[20].

Nevertheless，whether all Tibetans carry the same genotype ofPTENgene，and whether polymorphisms influence HAPC susceptibility remains unknown.We hypothesized that polymorphisms in thePTENgenewould be associated with susceptibility to HAPC patients in Tibetans.We also supposed that an analysis of these associations would inform new biology regarding the roles ofPTENin the pathogenesis of HAPC in susceptible Tibetans.To test this hypothesis，we examined seven SNPs of thePTENgene(rs1234212，rs1234213，rs1234219，rs2299939，rs2299941，rs532678 and rs555895)in 63 HAPC-p(HAPC patient)and 131 HAPC-r(HAPC resistant)Tibetan from the Yushu area in Qinghai province.

2 MATERIALS AND METHODS

2.1 Subjects

We studied HAPC in high altitude native Tibetans and matched control subjects.All HAPC patients had been diagnosed at Yushu Peoples Hospital between March 2011 and June 2013.The inclusion criteria were：Tibetan ethnicity with polycythemia [a hemoglobin(Hb)concentration ≥21g/dL in males and ≥19g/dL in females].The exclusion criteria were：none of the participants had a history of respiratory or cardiovascular disease，such as chronic obstructive pulmonary disease，asthma，shunt，or congenital heart disease.Healthy control Tibetan subjects，who matched HAPC patients in age，gender，and working conditions from the Yushu area were randomly selected from outpatient clinics.All participants in this provided informed consent.The research protocol was approved by the human subject protection committee at the Qinghai University School of Medicine(Xining，China).Overall，63 patients with HAPC(mean age，45.51±10.07yr)and 131 control subjects(mean age，45.14±11.78yr)participated in this study.SaO2levels were tested by the Pulse Oximeter(Ohmeda 3700 Pulse Oximeter，Datex-Ohmeda，Boulder，Colorado，USA).Hemoglobin(Hb)concentration，hematocrit(Hct)were determined from venous blood samples using the Mindray Hematology Analyzer(BC-2300，Shenzhen，China).

2.2 DNA extraction and genotyping assays

Genomic DNA was extracted from venous blood by Gentra Puregene Blood Kit(Qiagen，158389，Germany)according to standard procedures.Then it was quantified using a spectrophotometer and adjusted to 50 ng/μL.All selected SNPs were genotyped by the Sequenom MassARRAY SNP assays(Capital Bio Corporation，Beijing，China).The multiple PCR reaction consisted of 4.0 μL of PCR Master Mixture(Takara Bio Inc.，Japan)，1 μL of 8 paired PCR primers mixture(supplementary)，1.0 μL of gDNA，1.9 μL of ddH2O，and 0.1 μL of dNTPs.The reaction was incubated under the following thermal cycling conditions：94 ℃ for 4 min，94 ℃ for 20 s，56 ℃ for 30 s，72 ℃ for 1 min，45 cycles，then followed by 72 ℃ for 4 min.The PCR product was treated with 2.0 μL of SAP(shrimp alkaline phosphatase)mix to remove free dNTPs(1.53L of ddH2O，0.17μL of SAP buffer，0.3μL of SAP enzyme)，then incubated under 37 ℃ for 40 min，followed by 85 ℃ for 5 min.The single base extension reaction was performed using the following thermal cycling conditions：94 ℃ for 30 s，94 ℃ for 5 s，52 ℃ for 5 s，80 ℃ for 5 s，40 cycles，then followed by 72 ℃ for 3 min.We use MassARRAY Nanodispenser RS1000 to dispense the purified extension products onto a 384-element Spectro CHIP bioarray，then for acquiring spectra using the MALDI-TOF(matrix-assisted laser desorption/ionization-time of flight)(Sequenom，USA).

2.3 Statistical analysis

We use SPSS software(version 17.0，SPSS，Inc，Chicago，USA)to analyze the clinical data and their associations withPTENSNPgenotypes.The allele，genotype frequencies，and the P values among the seven polymorphisms in both study groups are reported.The genetic distances between the HAPC-p and HAPC-r were computed.Allele frequencies were calculated based on genotype frequencies in HAPC patients and control subjects and inter-group differences were estimated using the chi-square test.The criterion for significance wasP<0.05 for all comparisons.Deviations from Hardy-Weinberg equilibrium(HWE)were assessed by processing the chi-square test for genotype frequency.We also computed the odds ratio(OR)，confidence intervals(CI)and P values between the HAPC-p and HAPC-r subjects for alleles of polymorphic loci in pre-specified secondary analyses.

3 RESULTS

3.1 Phenotype characteristics

The gender，average age，arterial oxygen saturation(SaO2)，hemoglobin(Hb)and hematocrit(Hct)for the HAPC-p and HAPC-r subjects are listed (Table 1).The incidence of HAPC was higher in men than women，consistent with the known male predisposition for HAPC(Wu 2005).The SaO2was significantly lower，while Hb and Hct were much higher in HAPC groups compared to their respective controls(P<0.05).

3.2 Genotype and allele distribution

We examined the genotypic distributions，allelic frequencies and associations of seven SNPs of thePTENgene(rs1234212，rs1234213，rs1234219，rs2299939，rs2299941，rs532678，rs555895) .All polymorphisms were found to be in Hardy-Weinberg equilibrium(HWE)for both HAPC-p and HAPC-r groups.We identified no SNPs that differed in prevalence between the two groups and were associated with HAPC.There is no significant difference in the genotype，allele and dominant model of inheritance in all the seven SNPs when comparing the HAPC group and control group(P>0.05)(Table 2).

Table 1 Gender， average ages and physiological parameters for the study groups

The data represent mean and standard error of HAPC-p and HAPC-r subjects. The physiological variables measured were，arterial oxygen saturation(SaO2)，hemoglobin(Hb)and hematocrit(Hct).N is the number of subjects sampled. *P<0.05 vs control.

Table 2 Comparison of genotype distributions，alleles frequencies，associated with HAPC

续表：

SNPgenotype&allelesHAPC-pn(%)HAPC-rn(%)OR(95%CI)χ2PDominantmodelAA2(3．3%)6(4．7%)AC+CC59(96．7%)123(95．3%)0．695(0．136-3．547)0．1930．66GenotypeGG9(14．5%)19(14．7%)AG25(40．3%)56(43．4%)1．061(0．422-2．670)0．0160．9AA28(45．2%)54(41．9%)0．914(0．366-2．281)0．0380．846AlleleG43(34．7%)94(36．4%)A81(65．3%)164(63．6%)0．926(0．592-1．450)0．1120．737DominantmodelGG9(14．5%)19(14．7%)AG+AA53(85．5%)110(85．3%)0．983(0．417-2．319)0．0020．969rs532678GenotypeCC13(20．6%)28(21．7%)CT32(50．8%)59(45．7%)0．856(0．390-1．879)0．150．698TT18(28．6%)42(32．6%)1．083(0．459-2．557)0．0330．855AlleleC58(46．0%)115(44．6%)T68(54．0%)143(55．4%)1．061(0．691-1．627)0．0730．787DominantmodelCC13(20．6%)28(21．7%)CT+TT50(79．4%)101(88．3%)0．938(0．447-1．966)0．0290．865rs555895GenotypeGG18(29．0%)42(32．6%)GT31(50．0%)60(46．5%)0．829(0．411-1．674)0．2730．602TT13(21．0%)27(20．9%)0．890(0．376-2．107)0．070．791AlleleG67(54．0%)144(55．8%)T57(46．0%)114(44．2%)0．931(0．605-1．431)0．1080．743DominantmodelGG18(29．0%)42(32．6%)GT+TT44(71．0%)87(67．4%)0．945(0．490-1．823)0．0290．866

The data are shown odds ratio(OR)，95% confidence interval(CI)and P values for seven SNPs comparing HAPC-p and HAPC-r group.

4 DISCUSSION

We genotyped seven SNPs of thePTENgene(rs1234212，rs1234213，rs1234219，rs2299939，rs2299941，rs532678，rs555895)in Tibetans with and without HAPC.Of these seven SNPs，we identified no significant differences in genotypic distributions and allelic frequencies between the two groups.Moreover，all the polymorphisms were not associated with HAPC risk under the dominant model.

PTENwas originally identified as a tumor suppressor gene that has been found to be mutated in a large percentage of human cancers.It is involved in the regulation of multiple cellular functions，such as cell growth，differentiation and proliferation，apoptosis，migration，invasion，as well as angiogenesis[21-23].PTENwas selected as gene likely involved in high altitude adaptation in Tibetans[16]，however，when analyzed for association with HAPC，there was no correlation found.Thus，this gene likely have adaptive benefits for high altitude environment but are not protective against HAPC in Tibetans.

Based on previous researches around the world，we need to adjust our strategies working on association between HAPC genetic mechanism.Combine different research groups around the world；enlarge the participant pool to re-genotype more SNPs reported in this gene.Our future studies should focus on epigenetics，micro RNA，aiming to detect systematic mechanism of HAPC，thus shed light on prevention and medication of mountain disease for clinical application.

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PTEN基因多态性与青藏高原藏族人群高原红细胞增多症易感性#

胥 瑾1，杨应忠2，李永臻1，汤 锋2，王 展3※

目的青藏高原藏族通过血液中较低的血红蛋白浓度来很好地适应高海拔环境，但仍有部分藏族罹患高原红细胞增多症(HAPC)。本研究探讨PTEN基因(gene of phosphate and tension homology deleted on chromsome ten，PTEN)多态性是否与藏族高原红细胞增多症(HAPC)相关。方法将来自青海海拔超过3 500米的玉树地区63位HAPC患者(HAPC-p)和131位健康者(HAPC-r)作对照。用Sequenom MassARRAY法对PTEN基因的七个SNP位点(rs1234212，rs1234213，rs1234219，rs2299939，rs2299941，rs532678和rs555895)进行基因分型检测。结果HAPC-p组和HAPC-r组相比，血氧饱和度明显降低，而Hb和HCT明显升高。PTEN基因被测的所有SNP位点的基因型和等位基因分布频率无显著差异。结论PTEN基因多态性与青藏高原藏族高原红细胞增多症易感性无关。

高原红细胞增多症PTEN多态性

R392

A

2017-2-3

10.13452/j.cnki.jqmc.2017.03.002

#：This work was supported by grants from Natural Science Foundation of China(No. 81641078)，Qinghai Science & Technology Department Funds for Young Scientists(2016-ZJ-924Q).

※：Corresponding author：physician-in-charge，Tel：15202513261，E-mail：ufofu01@163.com Department of Biomedical Engineering， Tsinghua University， Beijing 100000， China XU Jin(1987～)，female，Tu，Ph.D

(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； 3.清华大学生物医学工程系，北京 100000)