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

  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󰀲 󰀱 󰀱󰀱󰀱, 󰀱󰀲󰀹, 󰀱󰀷󰀲, 󰀲󰀲󰀳, 󰀲󰀹󰀰, 󰀳󰀱󰀹,󰀳󰀶󰀲