Study Of The Relationship Of Host Genetic Polymorphisms And HIV-1Drug Resistance

Three decades has passed since human Immunodeficiency Virus (HIV) wasconfirmed as the pathogen of the acquired immunodeficiency syndrome (AIDS), alethal infectious disease characterized of serious damage to host immune system. Thispandemichavealreadyspreadallovertheworld,infectedmillionsofpeopleandcausedenormous social and economic loss. Highly active antiretroviral therapy (HAART),once offensively suppressed HIV replication in vivo, prolonged and improves the lifequality of HIV infected persons. However, the occurrence of drug resistance weakensthe efficacy of HAARTgreatly and has become the main cause of antiretroviral therapyfailure. HIV-1drug resistance is the result of interactions among host, virus andantiretroviral drugs. HIV-1drug resistance research had been launchedinChina in2004,which mainly focused on the study of occurrence, evolution and detection methods fordrug resistant strains, yet little data was available about the relationship between hostgenetic factors and HIV-1drug resistance. In this study, we first performed a cross-sectional study on the prevalence of HIV-1drug resistance in part of HIV-1infectedpatients who received antiretroviral therapy in North China, then analyzed thedifference of drug resistance mutations among subtypes circulating in China, finallyinvestigated on the single nucleotide polymorphisms sites (SNPs) in host genes whichare associated with the absorption, distribution, metabolism and excretion ofantiretroviraldrugsandanalyzedtheirrelationshipwithdrugresistance.Thisstudymaynot only laid foundation for the option of clinical therapy and strategies to retard thedevelopment of drug resistance and individualized therapy, but contributed to long termand sustainable use of the limited antiretroviral drug resources. Furthermore, the SNPsin host BST-2gene and its influence on HIV-1Vpu polymorphisms were also analyzed.Part I. Drug resistance survey in part of HIV-1infected patients of Chinain2012To carry out the annual survey of HIV-1drug resistance, master the condition ofAIDS therapy and drug resistance in HIV-1infected patients receiving antiretroviraltherapy, HIV-1pol gene were amplified with the classical genotyping drug resistance test method as polymerase chain reaction amplification and sequencing. In this study,data about322HIV-1infected individuals from Hebei Province, Shanxi Province andBeijing in2012were collected and investigated through genotype drug resistance test,analyzed the occurrence of drug resistance and the factors associated with drugresistance. The main results are as follows:1. the drug resistance ratio of the322HIV-1infected patients was46.5%, and the drugresistance ratio of Hebei, Shanxi and Beijing were50.0%,56.5%and14.7%respectively, with significance difference being observed among areas (Fisher’sexact test, p<0.05).2. there were no significant difference among the distribution of drug resistancemutations,thetoptenmutationswereM41L,D67N,K70R,K101E,K103N,Y181C,M184V, G190A, T215F and T215Y, and the top three mutations were K103N,Y181C and M184V with frequency of17.5%、12.9%and29.0%respectively.3. the antiretroviral drugs resistance rates were NVP46.2%,3TC32.4%, EFV30.1%,d4T20.7%, AZT12.2%, TDF5.9%. Serious cross-drug resistance was alsoobserved, leading the antiretroviral drugs resistance rates for ABC, RPV and ETRwhich were never used, to reach to33.2%,24.4%and22.6%respectively.4. statistical analysis show transmission route could significantly impact drugresistance rank (Kruskal-Wallis test, p<0.05), transmission route and genderinfluence the types of drug resistance mutations significantly (Multinomial LogisticRegression, p<0.05).Part II.Analysis of the difference of drug resistance mutations among HIV-1subtypes circulating in ChinaHIV-1is characterized of high degree of variability, which including M, O, N andP groups. Group M is the main cause of global epidemic, which contains9subtypes(A～D, F～H, J and K),55circulating recombinant forms (CRF) and numerous uniquerecombinant forms (URF). For the genetic basis difference among variant strains, theoccurrence, path and kinds of drug resistance mutations may different under drug selectpressure. Almost all antiretroviral drugs and resistance data were collected based onsubtype B, which was the main virus circulating in the US and Western European.However, the HIV-1subtypes circulating in China are subtype B’, CRF01_AE,CRF07_BC and CRF08_BC. Whether those drugs and data also applicable to thosenon-B subtypes was not clear. Investigation on the difference of drug resistance mutations among subtypes is important to optimize clinical treatment and improvetherapeutic effect.5442HIV-1pol sequences were collected (according to the sourceof sequences,3624were download from the HIV sequence database,1483weregenerated in our laboratory and335were provided by AIDS care center of YunnanInfectious Disease Hospital; in accordance with subtypes, B1984, CRF01_AE1696,CRF07_BC502and CRF08_BC924), analyzed the difference of drug resistancemutations among subtypes and their mechanisms. The main results are as follows:1. the amino acids distribution of19drug resistance mutation sites in reversetranscriptase region show significant difference among subtypes(chi-square test,p<0.05), and the top five mutations for each subtype are B (103,215,181,184and190), CRF01_AE (238,184,190,181and103) and CRF08_BC (184,103,69,181and101/190). The amino acids distribution of17drug resistance mutation sites inprotease region also show significant difference and the top five mutations for eachsubtype are B (89,63,77,93and35), CRF01_AE (89,36,35,93and63),CRF07_BC (63,89,93,35and77) and CRF08_BC (93,36,63,89and82), andmost of which were polymorphism sites.2. Genetic barrier of5sites (69,138,181,215and238) in reverse transcriptase withsignificant difference (Kruskal-Wallis test, p<0.05) among subtype B, CRF01_AEand CRF08_BC, suggesting that subtypes may play certain roles in the generationof drug resistance.3. Most of the drug resistance mutation sites were also polymorphism sites, prone todrug resistance under the select pressure of antiretroviral drugs.4. All subtypes share the three highest drug resistance mutation sites (>20%) K103,Y181and M184in common, and the favor drug resistance mutation sites (>10%)weresimilaramongsubtypes,allthosedrugresistancemutationsexistedinsubtypeB also can be found in other non-B strains.5. Considering little difference of HAART therapy on population level, and almosteach drugresistant mutation in subtype B alsoexistedinotherthreesubtypes,cometo the conclusion that all drugs and therapy regimes designed based on subtype Bwere also applicable to other non-B viruses including those circulating in China.Part III. Correlation of host genetic polymorphisms with HIV-1drugresistanceHIV-1drug resistance is the result of interactions among virus, host and antiretroviral drugs. Single nucleotide polymorphism site (SNPs) in host genesassociated with the absorption, distribution, metabolism and excretion of antiretroviraldrugs could impact the pharmacokinetics and/or pharmacodynamics of certain drugsand further influence their plasma drug concentration, lead to unable to form and/ormaintain effective virus suppression in vivo and finally appear drug resistance.Analyzing and clarifying the correlations between host genetic polymorphisms andHIV-1drug resistance could provide theoretical basis and technical support for thetreatment and prevention of HIV-1drug resistance, optimize clinical therapy regimesand even individualized medicine. Most associated work were focus or limited on thechanges of pharmacokinetics and/or pharmacodynamics and for single gene or singledrug, no data about the correlation of host genetic polymorphisms and HIV-1drugresistance were reported. In this study, first adopt PCR and sequencing method to detectthe SNPs in the exons and the exon-intron junctions of CYP genes associated with themetabolism of commonly used antiretroviral drugs, then MassARRAY method for47host SNPs involved in the absorption, distribution, metabolism and excretion wasconstructed, and the detection of which in810HIV-1infected patients (600sensitivevs.210drug resistance) were completed, obtained NVP plasma drug concentration of153samples and their influence on HIV-1drug resistance in combination with clinicaldata were further analyzed. The main results are as follows:1. successfully constructed the method for detect SNPs in the exons and exon-intronjunctions of CYP (2B6,2C9and3A4), completed the detection in129HIV-1infected individuals and70SNPs were detected, of which17SNPs withfrequencies more than5%and most were enrolled in NCBI SNPdb and distributedin intron region, CYP2C9g.10417and CYP3A4g.20446were new identifiedSNPs.2. through literature reading, data analysis and antiretroviral drugs used in Chinaconfirmed47SNPs in host genes associated with drug absorption, distribution,metabolism and excretion, most of which were belong to cytochrome P450, ATP-binding cassette transporter superfamily and related toAIDS disease progression.3.141primers (47pairs primers to amplify the interested fragment containing the aimSNPs and47upstream extension primers) were designed for MassARRAY todetect the above47SNPs and completed810samples detection, enriched theirinformation in Chinese especially in HIV-1infected population; the allelesdistribution and frequencies of rs3892097(0.012vs.0.106), rs12248560(0.012vs. 0.152), rs2740574(0.002vs.0.202) and rs7439366(0.004vs.0.126)were withobvious difference between Chinese and foreigners.4. univariate analysis show the alleles distribution of11SNPs(rs8192726, rs3745274,rs2279343, rs4646437, rs776746, rs17222723, rs899494, rs2274407, rs2231142,rs2231137and rs9264942)with significant difference between ART sensitive anddrug resistance population(Fisher’s exact test，p<0.05) and multivariate analysisshow rs2779343, rs776746, rs9264942and baseline viral load were significantlycorrelated with HIV-1drug resistance(Binary Logistic analysis, p<0.05),rs2779343AG/GG, rs776746GG, rs9264942TT and high baseline viral load wererisk factors for the occurrence of drug resistance, the OR were3.536/4.283,2.141,1.316and3.038respectively and deserving further research.5. the plasma drug concentration detection of153samples were completed andfurther analyzed its relationship with HIV-1drug resistance.Part IV. Host BST-2genetic polymorphisms and its relationship with thepolymorphisms of HIV-1VpuHost bone marrow stromal cell antigen2(BST-2, also known as tetherin) is onekind of the antiviral proteins found in recent years, play important role in human innateimmune system and can effectively suppress the replication of Vpu defective HIV-1invivo. HIV-1Vpu protein is the key factor that against and eliminate host BST-2proteinas to ensure the normal replication of HIV-1. In this study, the polymorphisms of BST-2and HIV-1Vpu were both detected, their relationship and influence were alsoanalyzed. The main results are as follows:1. established a routine method to detect the polymorphisms in host BST-2gene andfound13SNPs with high frequency(>5%), of which9SNPs were included byNCBI SNPdb and/or Hapmap database and the rest4SNPs were new sites.Furthermore the distribution and Link-Disequilibrium were also analyzed.2. successfully constructed the MassARRAY system for detection of the above13SNPs and finished810samples test.3. established the PCR method for HIV-1Vpu gene amplification, acquired413sequences and amino acids distribution analysis of which show highlypolymorphisms and more than60%sites are polymorphic.4. statistical analysis show that BST-2polymorphisms could significant impact theamino acids distribution of HIV-1Vpu (Fisher’s exact test, p<0.05) and both of them show correlations with viral load at different levels (spearman’s rho test,p<0.05).