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Mitochondrial DNA Haplogroups and Susceptibility to Prostate Cancer in a Colombian Population

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Prostate cancer (PC) is one of the most common cancers and the second leading cause of mortality from cancer in Colombian men. Mitochondrial DNA (mtDNA) haplogroups have been associated with the risk of PC. Several studies have demonstrated dramatic
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  Research Article Mitochondrial DNA Haplogroups and Susceptibility toProstate Cancer in a Colombian Population D.Cano, 1 C.F.Gomez, 2 N.Ospina, 1 J.A.Cajigas, 3 H.Groot, 1 R.E.Andrade, 4 andM.M.Torres 1 󰀱 Human Genetics Laboratory, Science Faculty, Universidad de los Andes, Bogot ´a, Colombia 󰀲 Prostate Clinic, Fundaci´on Santa Fe de Bogot ´a University Hospital, Bogot ´a, Colombia 󰀳 Urology Department, Hospital Militar Central, Bogot ´a, Colombia 󰀴 Pathology and Laboratory Department, Fundaci´on Santa Fe de Bogot ´a University Hospital, Bogot ´a, Colombia Correspondence should be addressed to M. M. orres; maritorr@uniandes.edu.coReceived 󰀲 September 󰀲󰀰󰀱󰀳; Accepted 󰀲󰀸 October 󰀲󰀰󰀱󰀳; Published 󰀲󰀸 January 󰀲󰀰󰀱Academic Editors: P. Parrella and L. SaragoniCopyright © 󰀲󰀰󰀱 D. Cano et al.TisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,whichpermits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.Prostatecancer(PC)isoneothemostcommoncancersandthesecondleadingcauseomortalityromcancerinColombianmen.Mitochondrial DNA (mtDNA) haplogroups have been associated with the risk o PC. Several studies have demonstrated dramaticdifferences regarding the risk o PC among men rom different ethnic backgrounds. Te present study was aimed at assessing therelationship between mtDNA haplogroups and PC. Te mitochondrial DNA hypervariable segment I (HSV-󰀱) was sequenced in apopulation-basedstudycovering󰀱󰀶󰀸cases(CA)and󰀱󰀰unrelatedhealthyindividualsasacontrolgroup(CG).Atotalo󰀹󰀲differentmtDNAsequenceswereoundinCAand󰀵󰀹wereoundintheCG.AccordingtothegeographicalsrcinattributedtoeachmtDNAhaplogroup,󰀸󰀲%othemtDNAsequencesoundinbothgroupswereNativeAmericans(A,B,C,andD).TemostrequentwasA(󰀱.󰀱%CA–󰀲.󰀱%CG), ollowed by B (󰀲󰀲.󰀰%CA–󰀲󰀱.%CG), C (󰀱󰀲.󰀰%CA–󰀱󰀱.%CG), and D (󰀶%CA–󰀱󰀰.󰀰%CG). A lower percentageo European haplogroups (U, H, K, J, M, , and HV) were also ound (󰀱󰀳.󰀱%CA–󰀱󰀲.󰀹%CG), likewise Arican haplogroups (L󰀰, L󰀱,L󰀲, and L󰀳) (󰀶.󰀵%CA–󰀲.󰀱%CG). Tere were no statistically significant differences between the distribution o mtDNA haplogroupsin CA and the CG in this study. 1. Introduction Teprecisemoleculareventsleadingtoprostatecarcinogene-sisarecurrentlynotwellknown.Tegeneticcharacterizationo this neoplasm has mainly been ocused on the nucleargenome, showing complex chromosomalinstability as one o themainchanges;however,thecauseothediversityochro-mosomal alterations detected in patients is still unclear. Tepresence o mutations in oncogenes and tumor suppressorgenes has been associated with late events in the progressiono prostate cancer (PC) [󰀱].Mitochondrial DNA (mtDNA) is the main target or cel-lular reactive oxygen species (ROS) and it has been observedthat the level o oxidative damage is more extensive and per-sistent in this than in the nuclear genome, thereby leading totheaccumulationogreaternumbersomutations[󰀲].Recentstudies have shown that the presence o multiple homoplas-mic point mutations in the mitochondrial genome is com-mon in many human tumors, such as those ound in colonand prostate cancers [󰀳, ]. Tese mutations could also lead to mitochondrial dys-unction due to alteration o the intermediary metabolism,which could be interpreted as a signal or inducing tumorpathogenesis [󰀵].Te location o these mutations within the genome hasbeen correlated with different types o cancer. Te presenceo mutations in the cytochrome oxidase I (COI) gene occursin around 󰀱󰀱% o PC patients [󰀶]. On the other hand, thenoncoding displacement loop (D-loop) region proved to bea critical site or the presence o mutations (mutational hot-spot) in neoplasm o the bladder, lung, head, and neck.Tese mutations are associated with the D-loop unction as Hindawi Publishing CorporationISRN Oncology Volume 2014, Article ID 530675, 11 pageshttp://dx.doi.org/10.1155/2014/530675  󰀲 ISRN Oncology a regulatory site or this genome’s replication and expression[󰀷].mtDNA is characterized by a strictly maternal mode o inheritance, the absence o recombination, a rapid mutationrate, and high level o population-specific polymorphisms.Mutation accumulation in mtDNA is tenold greater thanin nuclear DNA. Tis eature has created and characterizedgroups defined by having a maternal lineage legacy, makingmtDNA a useul tool or studying srcin and migration inhumanpopulations;itiswidelyapplicableorstudyingevolu-tionary relationships among human ethnic groups [󰀸]. Tecontrol region (D-loop) is the most variable region in themitochondrialgenomeandthemostpolymorphicnucleotidesites are concentrated in two hypervariable segments (HVS-I and HSV-II). Individuals’ geographical srcin has beenidentified by high-resolution RFLP analysis and HSV-Isequencing [󰀹–󰀲󰀰]. Studying mtDNA haplogroups has been o great interestas this presents a potential disease susceptibility biomarkerin different population groups. Te growing number o pub-licationsdescribingtheriskocancerassociatedwithvariousmtDNA haplogroups in the human population has chal-lenged the reported mutations’ validity and their use assusceptibility biomarkers [󰀲󰀱–󰀲󰀳]. Systematic errors are re- quently reported in anthropological and orensic sciencestudies because o the multiple steps involved in analyzingmtDNA sequences. More than hal o the sequences containobviouserrors[󰀲].IdentiyinglegitimatemtDNAmutationsofenbecomesconusedbyaheteroplasmyevent,aconditionin which both wild and mutant genomes coexist within thesame cell [󰀲󰀵]. “Phantom” mutations (systematic errors orartiacts produced during sequencing) can also create adifferent mutation pattern rom that produced in the cell innatural conditions [󰀲󰀶]. Tese mutations have been called“innovative” by some authors and erroneous conclusionshave led to a alse interpretation o results regarding theirassociation with different diseases [󰀲󰀷, 󰀲󰀸]. Some authors have suggested that this type o study should be analyzed inthelightohaplogroupphylogeny,takingtheirdiversificationin younger clades and those having limited geographical andethnicdistributioninto account,as well as identiyingsharedrequent mutations, to avoid such errors [󰀹, 󰀲󰀹]. Several studies have linked PC susceptibility to individu-als’ ethnic srcin which could suggest a relationship betweenpopulationvariabilityandthegeneticsocancer[󰀳󰀰–󰀳󰀲].Pre-  vious research has shown that the incidence is two to tentimes greater in North Americans o Arican descent whencompared to Caucasians and Asians, respectively [󰀳󰀳]. Inthis regard, studies by Booker et al. [󰀳] have shown thathaplogroup U (European srcin) is associated with abouttwice the risk o PC and 󰀲.󰀵 times the risk o renal carcinomainAmericanindividualshavingEuropeanancestry.Contrary to this finding, Kim et al. [󰀳󰀵] did not reveal any associationbetween Asian and PC lineages or the Korean population.Similarly, research by Muller et al. [󰀳󰀶] ound no associationbetweenthesehaplogroupsandPCinaEuropeanpopulation.AncestralstudiesintheColombianpopulationhavebeenconducted in the past, ocusing on determining the popula-tion’sorigin;however,veryewworkshavebeencarriedouttodate which have ocused on complex diseases such as PC. Nostudy which has been carried out on admixed Latin-Amer-ican populations has sought to associate maternal lineage(mtDNAhaplogroup)withsusceptibilitytoPC;thiswouldbeogreatinterest,astheseadmixedpopulationsarehistorically shaped by three major human geographical groups: NativeAmericans, Europeans, and Aricans. Bearing this in mind,the present study was aimed at establishing ethnic srcinbased on 󰀱󰀶󰀸 sequences rom the mtDNA hypervariablesegment I (HSV-󰀱) in a group o PC patients compared to 󰀹󰀰sequences rom healthy patients, thus correlating requency and possible disease susceptibility to one o the recognizedmtDNA haplogroups. 2. Methods 󰀲.󰀱. Study Population.  Tis work orms part o a case-controlstudy seeking to identiy PC susceptibility biomarkers. Testudy population thereore started rom a group o 󰀳󰀱󰀰patients having a confirmed diagnosis o PC and who hadundergone radical prostatectomy and 󰀱󰀵󰀲 individuals whowere seeking medical attention at the same medical centers(Fundaci´on Santa Fe de Bogot´a and Hospital Militar Central, Bogot´a, Colombia) who did not have clinical and/or para-clinical PC (controls) and who were randomly selected (notrelatedtothecases)romthesameplaceobirth.󰀱󰀶󰀸patientsand 󰀱󰀰 controls were selected rom this group or analyzingmtDNA HSV-󰀱 sequences. Clinical inormation was alsoavailable which took into account clinical-pathological vari-ablessuchasprostate-specificantigen(PSA)level[󰀳󰀷],tumoraggressiveness parameters and grade according to Gleasonscore [󰀳󰀸], and tumor status (MN), according to WorldHealth Organization (WHO) recommendations. Likewise, asurveywascarriedoutoridentiyingtheoriginothestudiedspecimensbyamily.TeUniversidaddelosAndes’ResearchEthics Committee had already approved a research proposalentitled“Asearchorgeneticmarkersabletoidentiyprostatecancer susceptible individuals.” All participants signed aninormed consent orm and the study was carried out in-linewith the Declaration o Helsinki principles (󰀲󰀰󰀰󰀰). 󰀲.󰀲. DNA Isolation and Sequence Analysis.  DNA was extract-ed rom whole blood collected rom each individual by the salting-out method. mtDNA sequences were analyzedrom position 󰀱󰀶󰀰󰀶 to 󰀱󰀶󰀳󰀷󰀳 (a 󰀳󰀲󰀸bp ragment). PCR wasperormed with primers L󰀱󰀵󰀹󰀹󰀶 (CCCACCAAGCAC-CCAAAG) and H󰀱󰀶󰀰󰀱 (GACACGGAGGG) orampliying the mtDNA ragment. Each reaction was carriedout in 󰀲󰀵  L containing 󰀱󰀲.󰀵  L Master Mix, 󰀱.󰀲󰀵  L o eachprimer, 󰀹  L distilled water, and 󰀱  L DNA sample. Termalcycle conditions were 󰀹󰀵 ∘ C or 󰀳min, 󰀳󰀵 cycles o 󰀹󰀵 ∘ C or󰀱min, 󰀵 ∘ C or 󰀱min, 󰀷󰀲 ∘ C or 󰀱min, and a final extensionstep at 󰀷󰀲 ∘ C or 󰀵min. Te mutations in the sequences werethoroughly reviewed to veriy their existence using chro-matograms and Geneious sofware [󰀳󰀹]. MUSCLE multiplealignments (deault parameters) were separately made withthe patients and controls’ sequences, using the consensus  ISRN Oncology 󰀳 󰁡󰁢󰁬󰁥 󰀱: Clinical-pathological characteristics o Colombian prostate cancer patients and the control group.Characteristic Category PC patients ( 󽠵 = 168 ) Controls ( 󽠵 = 140 )Mean (SD 󰀱 ) age 󰀶󰀸.󰀰󰀲 (󰀹.󰀱󰀸) 󰀶󰀲.󰀶󰀲 (󰀱󰀱.󰀹󰀷) 󽠵 = 162 󽠵 = 133 Mean (SD 󰀱 ) PSA 󰀲 󰀲.󰀱󰀶 (󰀰.󰀸󰀷) 󰀱.󰀱󰀲 (󰀰.󰀳󰀹) 󽠵 = 160 󽠵 = 140≤  󰀳󰀵 (󰀲󰀱.󰀹%) 󰀱󰀲󰀵 (󰀸󰀹.󰀳%).󰀱–󰀱󰀰.󰀰 󰀷󰀹 (󰀹.%) 󰀱󰀲 (󰀸.󰀶%)󰀱󰀰.󰀱–󰀲󰀰.󰀰 󰀳󰀱 (󰀱󰀹.%) 󰀳 (󰀲.󰀱%) > 󰀲󰀰 󰀱󰀵 (󰀹.󰀳%) 󰀰 (󰀰.󰀰%)Gleason score 󽠵 = 155  —Gleason score  <  󰀷 󰀱󰀰󰀰 (󰀶.󰀵%) —Gleason score  ≥  󰀷 󰀵󰀵 (󰀳󰀵.󰀵%) —MN 󽠵 = 146  —󰀱 󰀵 (󰀳󰀷.󰀰%) —󰀲 󰀵󰀸 (󰀳󰀹.󰀷%) —󰀳 󰀳󰀰 (󰀲󰀰.󰀵%) —  (󰀲.󰀸%) — 1 SD: standard deviation. 2 PSA: prostate-specific antigen. revised Cambridge reerence sequence (CRS) [󰀰]. Phyloge-netic analysis was perormed or determining relationshipsbetween the HSV-󰀱 region sequences using a phylogenetictreebuiltwiththeNeighbor-JoiningmethodusingMEGA.󰀰sofware [󰀱] with the Kimura-󰀲P evolutionary model whichledtoorganizingthehaplogroupsintodistinctcladesaccord-ingtotheirmutations.AtablewascreatedinExcel(MicrosofOffice 󰀲󰀰󰀰󰀷) showing the segregating sites or each haplotypeand their location in the genome or better visualization o thespecificmutationsdeterminingeachhaplogroup.Currentreerences were used or assigning the haplogroups to eachsequence or achieving a much more specific subhaplogroupclassification. A haplotype network was constructed withNetwork .󰀵 (Fluxus echnology) or visualizing phylogeny which showed haplogroup ramifications depending on caseand control requencies. 󰀲.󰀳.DataAnalysis.  mtDNAhaplogrouprequencieswerecal-culatedbydirectlycountingtheobservedphenotypes.AChi-square test was made in a two-by-two table, between pairso patient and control samples using PASW Statistics 󰀱󰀸.󰀰sofware (SPSS GmbH Sofware, 󰀸󰀰 󰀳󰀳󰀹 Munich, Germany)totestwhetherthepopulationhadsignificantdifferentiation.Tetest’ssignificancelevelwasappliedwith < 󰀰.󰀰󰀵probability as cutoff. A proportions and odds ratios (OR) test was thenmade with 󰀹󰀵% confidence interval. 3. Results 󰀳.󰀱. Population Characteristics.  able 󰀱 gives a description o the characteristics o the population being studied. Mean ageatonsetwas󰀶󰀸.󰀰󰀲( ± 󰀹.󰀱󰀸)yearsorPCpatientsand󰀶󰀲( ± 󰀱󰀲.󰀰)or the control group. Regarding PSA range, only 󰀲󰀲% o thepatients had PSA levels below  (remaining normal), whilethevastmajorityopatientshadlevelsabovenormal,rangingrom .󰀱 to 󰀱󰀰.󰀰ng/mL (󰀹.%), rom 󰀱󰀰.󰀱 to 󰀲󰀰.󰀰ng/mL(󰀱󰀹.%), and above 󰀲󰀰.󰀰ng/mL (󰀹.%). Most o the controlgroup had normal PSA levels (󰀸󰀹.󰀳%). Te clinical andhistological parameters regarding histological grade showedthat 󰀶.󰀵% o the specimens had a Gleason score below 󰀷and 󰀳󰀵.󰀵% had a value greater than or equal to 󰀷. Regardingtumor status (MN), the vast majority o patients were instages 󰀱 and 󰀲. Te case-control population surveyed herewas characterized by having a high requency o individuals(󰀹󰀰%) rom Andean region departments, most rom theCundinamarca-Boyaca plateau, and less requently rom theCaribbean area and abroad. 󰀳.󰀲. Analyzing mtDNA Genetic Diversity in the Study Pop-ulation.  Ninety-two different haplotypes were identified inthe group o patients (󰀱󰀶󰀸) which showed 󰀹󰀰 polymorphicsites.Fify-ninedifferenthaplotypeswereoundinthecontrolgroup (󰀱󰀰) in which 󰀶󰀷 polymorphic sites were observed;󰀱󰀷 haplotypes were shared in the cases and 󰀱󰀰 in the controlgroup (able 󰀲). A haplotype network was constructed usingthe Median-Joining method (Figure 󰀱) with all the haplo-types rom both the patients and control group to establishrelationships between HSV-󰀱 region sequences and identiy distinctive haplogroups according to their mutations sharedby clades. Te network arrangement showed our groupseaturing Amerindian haplogroups, A󰀲, B󰀲, C󰀱, and D󰀱.European-srcin sequences (U, H, HV, M, and ) were alsooundinagroupmorecloselyrelatedtotherevisedreerencesequence(CRS-Anderson).Somesequenceshadmanymuta-tions which generated outstanding long branches that weresubsequently identified as belonging to Arican haplogroups(L). Te different literature reerences [󰀲] were used orassigning haplogroups to find their characteristic mutations;   ISRN Oncology  󰁡󰁢󰁬󰁥 󰀲: Variable nucleotide positions or the HVS󰀱 mtDNA sequences obtained and their requency in cases and control groups.Control CasesHap Haplogroup  󽠵  HSV-󰀱 (󰀱󰀶󰀰󰀰󰀰+) Hap Haplogroup  󽠵  HSV-󰀱 (󰀱󰀶󰀰󰀰󰀰+)󰀱 H 󰀳 CRS 󰀱 H 󰀳 CRS󰀲 H 󰀱 󰀲󰀷󰀱, 󰀳󰀰 󰀲 H 󰀱 󰀱󰀸󰀸G󰀳 H 󰀱 󰀲󰀳, 󰀳󰀱󰀱 󰀳 H 󰀱 󰀲󰀳󰀰, 󰀲󰀵󰀶, 󰀳󰀵󰀲 H 󰀱 󰀱󰀸󰀹  H 󰀱 󰀱󰀲󰀹, 󰀳󰀱󰀶󰀵 H 󰀱 󰀱󰀸󰀹, 󰀲󰀲󰀳 󰀵 H 󰀱 󰀱󰀲󰀹, 󰀲󰀵󰀷, 󰀳󰀱󰀶󰀶 H 󰀱 󰀲, 󰀲󰀹󰀵󰀷 J 󰀱 󰀰󰀶󰀹, 󰀱󰀲󰀶󰀸 J 󰀱 󰀰󰀶󰀹, 󰀱󰀲󰀶, 󰀲󰀷󰀸󰀶 HV 󰀱 󰀲󰀷󰀸, 󰀳󰀱󰀱 󰀹 HV 󰀱 󰀱󰀳, 󰀳󰀶󰀲󰀷 K 󰀱 󰀲󰀲, 󰀳󰀱󰀱 󰀱󰀰 HV 󰀲 󰀳󰀶󰀲󰀸 U󰀱b 󰀱 󰀱󰀰, 󰀱󰀱󰀱, 󰀲󰀹, 󰀳󰀲󰀷 󰀱󰀱 M󰀱 󰀱 󰀱󰀲󰀹, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀳󰀱󰀱󰀹 U󰀲e 󰀱 󰀰󰀵󰀱, 󰀱󰀲󰀶C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀳󰀶󰀲 󰀱󰀲  󰀱 󰀱󰀲󰀶, 󰀱󰀶󰀳, 󰀱󰀸󰀶, 󰀱󰀸󰀹N, 󰀲󰀹󰀱󰀳  󰀱 󰀱󰀲󰀶, 󰀲󰀹󰀲, 󰀲󰀹󰀱  󰀱 󰀱󰀱󰀱, 󰀱󰀲󰀶, 󰀲󰀹, 󰀳󰀰󰀱󰀰 U󰀵a 󰀱 󰀱󰀹󰀲, 󰀲󰀷󰀰 󰀱󰀵 U󰀵a 󰀱 󰀱󰀹󰀲, 󰀲󰀷󰀰󰀱󰀶 U󰀵a 󰀱 󰀱󰀹󰀲, 󰀲󰀵󰀶, 󰀲󰀷󰀰󰀱󰀷 U󰀵b 󰀱 󰀱󰀸󰀹, 󰀱󰀹󰀲, 󰀲󰀳, 󰀲󰀷󰀰, 󰀳󰀱󰀱, 󰀳󰀳󰀶󰀱󰀸 U󰀶a 󰀱 󰀱󰀷󰀲, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀹, 󰀲󰀷󰀸, 󰀲󰀹󰀵󰀱󰀹 L󰀰a󰀱 󰀱󰀱󰀲󰀹, 󰀱󰀸, 󰀱󰀶󰀸, 󰀱󰀷󰀲, 󰀱󰀸󰀷, 󰀱󰀸󰀸G,󰀱󰀸󰀹, 󰀲󰀱, 󰀲󰀲󰀳, 󰀲󰀳󰀰, 󰀲󰀷󰀸, 󰀲󰀹󰀳,󰀳󰀱󰀱, 󰀳󰀲󰀰󰀲󰀰 L󰀱c󰀱a 󰀱 󰀱󰀲󰀹, 󰀱󰀸󰀷, 󰀱󰀸󰀹, 󰀲󰀲󰀳, 󰀲󰀷, 󰀲󰀷󰀸,󰀲󰀹󰀳, 󰀲󰀹, 󰀳󰀱󰀱, 󰀳󰀶󰀰󰀱󰀱 L󰀲d󰀲 󰀱 󰀰󰀹󰀳, 󰀱󰀱󰀱A, 󰀱󰀵, 󰀱󰀸, 󰀲󰀲󰀳,󰀲󰀳󰀹, 󰀲󰀷󰀸, 󰀲󰀹󰀲, 󰀳󰀵󰀵, 󰀳󰀶󰀲 󰀲󰀱 L󰀲a 󰀱 󰀱󰀳󰀱, 󰀱󰀸󰀹, 󰀲󰀲󰀳, 󰀲󰀲󰀵, 󰀲󰀳, 󰀲󰀷󰀸,󰀲󰀹, 󰀳󰀰󰀹󰀲󰀲 L󰀲a 󰀱 󰀰󰀹󰀳, 󰀱󰀸󰀹, 󰀱󰀹󰀲, 󰀲󰀲󰀳, 󰀲󰀷󰀸, 󰀲󰀹,󰀳󰀰󰀹󰀲󰀳 L󰀲a 󰀱 󰀱󰀸󰀹, 󰀲󰀲󰀳, 󰀲󰀳󰀰, 󰀲󰀷󰀸, 󰀲󰀹󰀲 L󰀲b󰀱 󰀱 󰀱󰀱A, 󰀱󰀲󰀹, 󰀲󰀱󰀳, 󰀲󰀲󰀳, 󰀲󰀷󰀸, 󰀳󰀵󰀲󰀵 L󰀲c 󰀱 󰀲󰀲󰀳, 󰀲󰀷󰀸󰀱󰀲 L󰀳b󰀱 󰀱 󰀱󰀲, 󰀲󰀲󰀳, 󰀲󰀳, 󰀲󰀷󰀸, 󰀳󰀶󰀲 󰀲󰀶 L󰀳e󰀱 󰀱 󰀱󰀸󰀹, 󰀲󰀰󰀷, 󰀲󰀲󰀳, 󰀳󰀲󰀷󰀲󰀷 L󰀳e󰀱a 󰀱 󰀱󰀸󰀵, 󰀲󰀰󰀹, 󰀲󰀲󰀳, 󰀳󰀲󰀷󰀲󰀸 L󰀳d 󰀱 󰀱󰀱󰀱, 󰀱󰀲, 󰀲󰀲󰀳󰀱󰀳 L󰀳 󰀱 󰀱󰀸󰀹, 󰀲󰀰󰀹, 󰀲󰀲󰀳, 󰀲󰀹󰀲, 󰀳󰀱󰀱 󰀲󰀹 L󰀳 󰀱 󰀲󰀰󰀹, 󰀲󰀲󰀳, 󰀲󰀹󰀲, 󰀲󰀹󰀵, 󰀳󰀱󰀱󰀱 B󰀲 󰀱 󰀱󰀸󰀲C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷 󰀳󰀰 B󰀲 󰀱 󰀰󰀸󰀶, 󰀱󰀸󰀲C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷󰀱󰀵 B󰀲 󰀱 󰀰󰀸󰀶, 󰀱󰀸󰀲C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷 󰀳󰀱 B󰀲 󰀱 󰀱󰀸󰀲C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀳󰀰󰀱,󰀳󰀰󰀱󰀶 B󰀲 󰀱 󰀱󰀲󰀹, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀸󰀳C 󰀳󰀲 B󰀲 󰀲 󰀱󰀸󰀲C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀳,󰀳󰀶󰀲󰀱󰀷 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀶󰀱, 󰀲󰀸 󰀳󰀳 B󰀲 󰀶 󰀱󰀸󰀲C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷󰀱󰀸 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀶󰀶 󰀳 B󰀲 󰀱 󰀰󰀹󰀷, 󰀰󰀹󰀸, 󰀱󰀸󰀲C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹,󰀲󰀱󰀷󰀱󰀹 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀷 󰀳󰀵 B󰀲 󰀱 󰀰󰀹󰀷, 󰀰󰀹󰀸, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷󰀲󰀰 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀳󰀲 󰀳󰀶 B󰀲 󰀱 󰀱󰀸󰀲C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀳󰀲󰀲󰀱 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀷󰀸, 󰀳󰀵󰀵 󰀳󰀷 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀳󰀲, 󰀳󰀶󰀶󰀲󰀲 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀷󰀰, 󰀳󰀰 󰀳󰀸 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀳󰀲󰀲󰀳 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀹󰀹 󰀳󰀹 B󰀲 󰀱 󰀱󰀵, 󰀱󰀵󰀷, 󰀱󰀸󰀲C, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷,󰀲󰀹, 󰀳󰀱󰀱  ISRN Oncology 󰀵 󰁡󰁢󰁬󰁥 󰀲: Continued.Control CasesHap Haplogroup  󽠵  HSV-󰀱 (󰀱󰀶󰀰󰀰󰀰+) Hap Haplogroup  󽠵  HSV-󰀱 (󰀱󰀶󰀰󰀰󰀰+)󰀲 B󰀲 󰀹 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷 󰀰 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀳󰀱󰀱󰀲󰀵 B󰀲 󰀳 󰀰󰀹󰀸, 󰀱󰀰󰀶, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷,󰀳󰀶󰀲 󰀱 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀵󰀶󰀲󰀶 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀳, 󰀳󰀶󰀲 󰀲 B󰀲 󰀱 󰀰󰀹󰀳, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷󰀳 B󰀲 󰀱 󰀱󰀷󰀹, 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀶, 󰀱󰀸󰀹, 󰀲󰀱󰀶, 󰀲󰀱󰀷󰀵 B󰀲 󰀱󰀵 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷󰀶 B󰀲 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀱󰀷, 󰀲󰀷󰀸󰀲󰀷 C󰀱 󰀱 󰀰󰀵󰀱, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀰, 󰀳󰀱󰀱,󰀳󰀲󰀵, 󰀳󰀲󰀷 󰀷 C󰀱 󰀱 󰀲󰀰󰀵G, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀲󰀸 C󰀱 󰀱 󰀰󰀵󰀱, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷 󰀸 C󰀱 󰀱 󰀲󰀲󰀳, 󰀲󰀶󰀵, 󰀲󰀹󰀸, 󰀳󰀱󰀹, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀲󰀹 C󰀱 󰀱 󰀰󰀵󰀱, 󰀲󰀰󰀹, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵,󰀳󰀲󰀷 󰀹 C󰀱 󰀲 󰀱󰀵󰀵, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀳󰀰 C󰀱 󰀱 󰀱󰀶󰀹, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷 󰀵󰀰 C󰀱 󰀱 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀳󰀱 C󰀱 󰀱 󰀱󰀹󰀲, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷 󰀵󰀱 C󰀱 󰀱 󰀰󰀵󰀱, 󰀱󰀷󰀲, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀵󰀲 C󰀱 󰀳 󰀰󰀵󰀱, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀵󰀳 C󰀱 󰀱 󰀰󰀵󰀱, 󰀲󰀰󰀹, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀰󰀰, 󰀳󰀲󰀵,󰀳󰀲󰀷󰀵 C󰀱 󰀱 󰀰󰀵󰀱, 󰀲󰀰󰀹, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀵󰀵 C󰀱  󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀵󰀶 C󰀱 󰀱 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀵󰀷 C󰀱 󰀱 󰀰󰀸󰀶, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀵󰀸 C󰀱 󰀱 󰀰󰀹󰀳, 󰀲󰀲󰀳, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀵󰀹 C󰀱 󰀱 󰀲󰀲󰀳, 󰀲󰀷󰀸, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀶󰀰 C󰀱 󰀱 󰀲󰀲󰀳, 󰀲󰀵󰀹, 󰀲󰀹󰀸, 󰀳󰀲󰀵, 󰀳󰀲󰀷󰀳󰀲 D󰀱 󰀱 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀲󰀳, 󰀳󰀲󰀵, 󰀳󰀶󰀲 󰀶󰀱 D󰀱 󰀳 󰀱󰀸󰀳C, 󰀱󰀸󰀹, 󰀲󰀲󰀳, 󰀳󰀲󰀵, 󰀳󰀶󰀲󰀳󰀳 D󰀱 󰀱 󰀱󰀸󰀹, 󰀲󰀲󰀳, 󰀲󰀷󰀲, 󰀳󰀲󰀵, 󰀳󰀶󰀲 󰀶󰀲 D󰀱 󰀱 󰀱󰀲󰀹, 󰀲󰀲󰀳, 󰀲󰀷, 󰀳󰀲󰀵, 󰀳󰀶󰀲󰀳 D󰀱 󰀱 󰀱󰀲, 󰀲󰀰󰀷, 󰀲󰀲󰀳, 󰀳󰀲󰀵, 󰀳󰀶󰀲 󰀶󰀳 D󰀱 󰀱 󰀱󰀲󰀹, 󰀱󰀸󰀹, 󰀲󰀲󰀳, 󰀳󰀲󰀵, 󰀳󰀶󰀲󰀳󰀵 D󰀱 󰀱 󰀰󰀹󰀳, 󰀱󰀲, 󰀲󰀲󰀳, 󰀳󰀲󰀵, 󰀳󰀶󰀲 󰀶 D󰀱 󰀱 󰀱󰀲󰀶, 󰀲󰀲󰀳, 󰀲󰀵, 󰀳󰀲󰀵, 󰀳󰀶󰀲󰀳󰀶 D󰀱 󰀲 󰀲󰀲󰀳, 󰀳󰀲󰀵, 󰀳󰀶󰀲 󰀶󰀵 D󰀱 󰀱 󰀱󰀲, 󰀲󰀲󰀳, 󰀳󰀲󰀵, 󰀳󰀶󰀲󰀳󰀷 D󰀱 󰀱 󰀱󰀲󰀹, 󰀲󰀲󰀳, 󰀲󰀷, 󰀳󰀲󰀵, 󰀳󰀶󰀲 󰀶󰀶 D󰀱 󰀳 󰀲󰀲󰀳, 󰀳󰀲󰀵, 󰀳󰀶󰀲󰀳󰀸 A󰀲 󰀲 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀲󰀹󰀳, 󰀳󰀱󰀹, 󰀳󰀶󰀲 󰀶󰀷 A󰀲 󰀱󰀱 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀶󰀲󰀳󰀹 A󰀲 󰀱 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀶󰀲 󰀶󰀸 A󰀲 󰀱 󰀰󰀹󰀲, 󰀱󰀱󰀱, 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀵󰀶,󰀳󰀶󰀲󰀰 A󰀲 󰀱 󰀲󰀲󰀳, 󰀲󰀵󰀸N, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀶󰀲 󰀶󰀹 A󰀲 󰀱 󰀱󰀱󰀱, 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀵󰀶, 󰀳󰀶󰀲󰀱 A󰀲 󰀱 󰀲󰀲󰀳, 󰀲󰀶󰀶, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀶󰀲 󰀷󰀰 A󰀲 󰀱 󰀱󰀱󰀱, 󰀲󰀲󰀳, 󰀲󰀶󰀶, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀵󰀶,󰀳󰀶󰀲󰀲 A󰀲 󰀱 󰀱󰀱󰀱,󰀲󰀲󰀳, 󰀲󰀳󰀹, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀶󰀲 󰀷󰀱 A󰀲 󰀱 󰀰󰀹󰀲, 󰀱󰀱󰀱, 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀶󰀰,󰀳󰀶󰀲󰀳 A󰀲 󰀲 󰀱󰀱󰀱, 󰀲󰀲󰀳, 󰀲󰀳󰀹, 󰀲󰀹󰀰, 󰀳󰀱󰀱, 󰀳󰀱󰀹,󰀳󰀶󰀲 󰀷󰀲 A󰀲 󰀱 󰀰󰀷󰀵, 󰀱󰀱󰀱, 󰀱󰀷󰀵, 󰀲󰀲󰀳, 󰀲󰀵󰀹, 󰀲󰀹󰀰,󰀳󰀰󰀰, 󰀳󰀱󰀹, 󰀳󰀶󰀲 A󰀲 󰀱 󰀱󰀱󰀱, 󰀱󰀸󰀹, 󰀲󰀲󰀳, 󰀲󰀳󰀹, 󰀲󰀹󰀰,󰀳󰀱󰀹, 󰀳󰀶󰀲 󰀷󰀳 A󰀲  󰀱󰀱󰀱, 󰀱󰀷󰀵, 󰀲󰀲󰀳, 󰀲󰀵󰀹, 󰀲󰀹󰀰, 󰀳󰀰󰀰,󰀳󰀱󰀹, 󰀳󰀶󰀲󰀵 A󰀲 󰀲 󰀱󰀱󰀱, 󰀱󰀲󰀹, 󰀲󰀲󰀳, 󰀲󰀵󰀶, 󰀲󰀹󰀰,󰀳󰀱󰀹, 󰀳󰀶󰀲 󰀷 A󰀲 󰀱 󰀱󰀱󰀱, 󰀱󰀷󰀵, 󰀲󰀲󰀳, 󰀲󰀵󰀹, 󰀲󰀹󰀰, 󰀳󰀱󰀹,󰀳󰀶󰀲󰀶 A󰀲 󰀱 󰀱󰀱󰀱, 󰀲󰀲󰀳, 󰀲󰀸󰀷, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀶󰀲 󰀷󰀵 A󰀲 󰀱 󰀱󰀱󰀱, 󰀱󰀲󰀹, 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀳󰀱󰀹, 󰀳󰀶󰀲󰀷 A󰀲 󰀱 󰀱󰀱󰀱, 󰀲󰀲󰀳, 󰀳󰀱󰀹, 󰀳󰀶󰀰, 󰀳󰀶󰀲 󰀷󰀶 A󰀲 󰀱 󰀱󰀱󰀱, 󰀱󰀲󰀹, 󰀱󰀷󰀲, 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀳󰀱󰀹,󰀳󰀶󰀲
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