Association of mitochondrial copy number variation and T16189C polymorphism with colorectal cancer in North Indian population

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1 740296TUB / Tumor BiologyKumr et l. reserch-rticle Originl Article Assocition of mitochondril copy number vrition nd T16189C polymorphism with colorectl cncer in North Indin popultion Tumor Biology November 2017: 1 7 The Author(s) 2017 Reprints nd permissions: sgepub.co.uk/journlspermissions.nv DOI: journls.sgepub.com/home/tub Bhupender Kumr 1 *, Zfr Iqbl Bht 2 *, Svit Bnsl 1, Sunil Sini 3, Afreen Nseem 2, Khushnum Whbi 2, Archn Burmn 1, Geet Trilok Kumr 1, Sundeep Singh Sluj 4 nd M Moshhid Alm Rizvi 2 Abstrct Globlly, colorectl cncer is the third most common type of cncer. Genetic instbility leding to cncer development is one of the mjor cuses for development of cncer. Altertions in mitochondril genome, tht is, muttions, singlenucleotide polymorphisms, nd copy number vritions re known to contribute in cncer development. The im of our study ws to investigte ssocition of mitochondril T16189C polymorphism nd copy number vrition with colorectl cncer in North Indin popultion. DNA isolted from peripherl blood of 126 colorectl cncer ptients nd 114 helthy North Indin subjects ws nlyzed for T16189C polymorphism nd hlf of them for mitochondril copy number vrition. Genotyping ws done using polymerse chin rection restriction frgment length polymorphism, nd copy number vrition ws estimted using rel-time polymerse chin rection, numbers of mitochondril copies nd found to be significntly higher in colorectl cncer ptients thn helthy controls (88 (58 154), p = 0.001). In the regression nlysis, incresed mitochondril copy number vrition ws ssocited with risk of colorectl cncer (odds rtio = 2.885, 95% confidence intervl = ). However, T16189C polymorphism ws found to be significntly ssocited with the risk of rectl cncer (odds rtio = 5.213, p = 0.001) nd non-significntly with colon cncer (odds rtio = 0.867, p = 0.791). Also, flse-positive report probbility nlysis ws done to vlidte the significnt findings. Our results here indicte tht mitochondril copy number vrition my be plying n importnt role in the development of colorectl cncer, nd detection of mitochondril copy number vrition cn be used s biomrker for predicting the risk of colorectl cncer in North Indin subjects. Keywords RFLP, copy number vrition, polymorphism, mitochondril DNA, colorectl cncer Dte received: 25 July 2017; ccepted: 10 October 2017 Introduction Colorectl cncer (CRC) remins the third most common cncer worldwide. In the United Sttes lone, it ws estimted to cuse 40,000 deths in After lung nd liver cncer, CRC is lso mong the leding cuse of cncerrelted deths worldwide. 2 CRC generlly progresses slowly, initilly s polyp nd lter develops into cncer. 3 As per Americn Cncer Society, diet, obesity, overweight, 1 Deprtment of Biochemistry, Institute of Home Economics, University of Delhi, New Delhi, Indi 2 Genome Biology Lbortory, Deprtment of Biosciences, Jmi Milli Islmi, New Delhi, Indi 3 School of Life Sciences, Jwhrll Nehru University, New Delhi, Indi 4 Deprtment of Gstrointestinl Surgery, Govind Bllbh Pnt Hospitl nd Muln Azd Medicl College, New Delhi, Indi *These uthors contributed eqully to this work. Corresponding uthor: M Moshhid Alm Rizvi, Genome Biology Lbortory, Deprtment of Biosciences, Jmi Milli Islmi, New Delhi , Indi. Emil: rizvijmi@gmil.com Cretive Commons Non Commercil CC BY-NC: This rticle is distributed under the terms of the Cretive Commons Attribution-NonCommercil 4.0 License ( which permits non-commercil use, reproduction nd distribution of the work without further permission provided the originl work is ttributed s specified on the SAGE nd Open Access pges (

2 2 Tumor Biology smoking, nd lcohol consumption hve been ssocited with the development of CRC. Although 70% cses of CRC ptient re reported to be spordic, 30% 35% hve been shown s fmilil nd inheritble. 4 6 Erly dignosis of different cncer types including CRC cn help in better mngement of cncers. 7 Genetic vritions ssocited with CRC would serve s n importnt dignostic tool for such mngement. Different genetic vribility including those of GSTT1, GSTP1, GSTM1, PTEN, nd IGF-1, IGFBP-3 hs been found to be ssocited with the risk of CRC GST (glutthione S-trnsferse) is wellestblished rective oxygen species (ROS) detoxifier in our cells, nd ROS detoxifiction is common nticncer strtegy in cellulr system. 13 Cncer cells exhibits high levels of ROS genertion which promotes genetic instbility, where mitochondri remins the mjor site for ROS genertion. 14 Chnges in mitochondril genome re known to promote ROS genertion in cncer cells. 14,15 Mitochondril copy number vritions (CNVs) nd mitochondril genome polymorphisms re lso further reported to contribute ROS nd cncer development. 16,17 Previous literture hve lredy shown the ssocition of mitochondril CNV in the peripherl blood leucocytes (PBLs) with risk of different cncers such s melnom, brest cncer, heptic crcinom, nd esophgel cncer The poly cytosine trct (poly-c) of D-loop region in mitochondril DNA (mtdna) is especilly importnt for repliction nd trnscription of mitochondril genome, thus vritions in this region cn directly ffect function of mitochondri. 22 T16189C vrition in this poly-c trct is one such vrition which ffects the repliction of mitochondril genome, lthough only limited informtion is vilble on the ssocition of mitochondril polymorphisms with CRC. 23,24 In Indin popultion till dte, only one study mde on south Indin popultion showed tht T16189C vrint ws ssocited with the risk of CRC. 25 In ddition, no meningful informtion is vilble on mitochondril CNV from PBLs in CRC ptients. As South Indin nd North Indin popultions re two distinct popultions, in this present cse-control study, we hve tried to figure out whether T16189C vrint nd mtdna CNVs from PBLs re somehow ssocited with the risk of CRC in North Indin popultion or not. Mteril nd methods Study subjects A totl of 126 tumor subjects who regulrly ttended Medicl Outptient Deprtment of Gstrointestinl Surgery GB Pnt Hospitl, Delhi, Indi were enrolled in this study. The ptients were divided into two groups bsed on loction of tumor either colon (n = 78) or rectl (n = 48). Dignosis of cncer ws bsed on the criteri of histologicl confirmtion. The control group ws composed of 114 subjects free from CRC with similr distribution of sex nd ge with tht of tumor ptients nd hving no fmilil history of CRC. The study ws pproved by ethicl committee of Jmi Milli Islmi. Informed written consent ws obtined from ll subjects prticipting in the study. Smple collection nd DNA extrction Tking full septic precutions, fsting blood smples were collected nd genomic DNA ws extrcted from peripherl blood leucocytes using the stndrd phenol chloroform method. All smples were gthered consecutively during the detiled time period. The qulity nd quntity of extrcted DNA were estimted first by running it on 0.8% grose nd then using NnoDrop ND-1000 spectrophotometer. After quntifiction, pproprite dilutions were mde for further experiments. Genotyping Genotyping ws performed using polymerse chin rection restriction frgment length polymorphism (PCR- RFLP) method where region of mitochondri (630 bp) ws mplified. For PCR, 25 μl rection ws setup in 200 μl PCR tubes. The rection mixture consisted s follows: 2.5 μl of 10 rection buffer (with 1.5 mm MgCl 2 ), 2 μl of deoxynucleotide triphosphte (dntp; 2.5 mm), 2 μl of primer pir ech of 12 μm, 50 ng/μl templte, 1 U/μL of Tq polymerse (Himedi), nd 14.5 μl Milli-Q wter. The primer sequences used were, FP 5 -AC CAGTCTTGTAAACCGGAG-3 nd RP 5 -GTGGGCTAT TTAGGCTTTAT-3 nd PCR cycle used for mplifiction ws s follows 95ºC for 5 min nd 32 cycles of 95ºC for 30 s, 59ºC for 30 s, 72ºC for 30 s, nd finl extension for 72ºC for 5 min. PCR product (15 μl) ws digested overnight t 37ºC in wter bth with 1 U of the Mn1I enzyme in totl rection volume of 50 µl s per mnufcturer s protocol (BioLb). The digested products were resolved on 3% grose gels t 60 V for 90 min. The T-llele gives mjor product of 287 bp, while the C-llele gives mjor product of 322 bp; 10% percent of smples were reconfirmed with direct sequencing to check the ccurcy of our RFLP dt. CRC cncer smples were compred with helthy controls, where control ref is bseline group. CNV Rel-time PCR ws performed to estimte mtdna CNV using PowerUp SYBR Green Mster Mix (#A25742; Thermo Fisher Scientific) on Rel-Time Detection System (CFX96, # ; BioRd). The reltive mtdna CNV for CRC nd norml helthy smples ws clculted s mitochondril gene/nucler genome; 5 ng of totl genomic DNA ws used s templte to crry out rel time polymerse chin rection (rel time-pcr) rection using primer for mitochondril 16s ribosoml RNA (rrna)

3 Kumr et l. 3 Tble 1. Clinicl chrcteristics nd lbortory dt of cncer ptients nd helthy subjects. Prmeters gene s mitochondril gene nd β-2 microglobulin (β- 2M) gene (single copy) s nucler reference gene. Primers used for nucler DNA (ndna) β2m re FP 5 -GCTGTCTCCATGTTTGATGTATCT-3 nd RP 5 -TC TCTGCTCCCCACCTCTAAGT-3 giving n mplicon size of 107 bp. Primers used for mtdna, 16S rrna re FP 5 -GCCTTCCCCCGTAAATGATA-3 nd RP 5 -TTATGC GATTACCGGGCTCT-3 giving n mplicon of 97 bp. The rel time-pcr cycle used consisted of initil denturtion t 95ºC for 10 min followed by 40 cycles of 15 s t 95ºC nd 1 min t 60ºC. Ech smple ws studied in duplicte on 96-well plte (BioRd). A negtive control (without templte) ws included in ech plte. Quntities of mtdna nd ndna were estimted using the eqution 2 ΔCt, where ΔCt = ΔCt mtdna ΔCt ndna. For copy number nlysis, reference is the bseline group in which mitochondril copy number is less thn or equl to 56 in cse of comprison by medin nd 36 in cse of comprison by qurtile. Sttisticl nlysis Helthy subjects Cncer ptients (CRC) p vlue Subjects (n) Age (yers) 48 ± ± b Sex (mle/femle) 78/36 72/ b Alcohol (yes/no) 54/60 70/ b Smoking (yes/no) 38/76 66/ b Pesticide infection (yes/no) 36/78 74/ b Dwelling (urbn/rurl) 64/50 64/ b Hypertension (yes/no) 32/82 63/ b CRC: colorectl cncer; SD: stndrd devition. Dt re presented by men ± SD. b Cncer ptients versus helthy subjects. Sttisticl nlysis ws crried out using stndrd sttisticl methods (SPSS softwre version 17.0). Dt were expressed s men ± stndrd devition. In cse of high inter-individul vribility nd non-norml distribution, dt were expressed s medin nd inter-qurtile rnge. Differences between mens were compred by Student s t-test or Mnn Whitney U test (where the dt were not distributed normlly). Multivrite nlysis ws done using Bonferroni djustment to negte the effects for confounding fctors like ge nd sex. Allele nd genotype frequencies of CRC ptients nd helthy controls were compred with vlues predicted by Hrdy Weinberg equilibrium using the chi-squre (χ 2 ) test. Odds rtios (ORs) of tumor subjects ssocited with genotype with 95% confidence of intervls were clculted by logistic regression nlysis ssuming dominnt mode of inheritnce. Smple size for the single-nucleotide polymorphism (SNP) t 5% Tble 2. Genotype distribution of SNPs in mitochondril T16189C polymorphism in cncer ptients nd helthy subjects. significnce level nd sttisticl power ws clculted using OSEE online clcultor (Tble 3). Flse-positive report probbility (FPRP) nlysis ws performed using R softwre for ssessing our significnt findings. FPRP threshold ws set t 0.2 long with prior probbility of 0.1 for detecting the ssocited risk. Vlues obtined below 0.2 in FPRP were considered noteworthy. 26 Logistic regression ws lso performed to find out the ssocition of copy number with the risk of cncer. For ll sttisticl tests, p < 0.05 ws considered s the level of significnce. Results Helthy control (n = 114) Cncer Colon ptients cncer (CRC; n = 126) (n = 78) In this study, 114 helthy controls with no previous history of ny type of mlignncy nd 126 CRC ptients were included. The clinicl chrcteristics of the study groups re summrized in Tble 1. There ws no significnt difference in ge nd sex distribution between the study groups. Assocition of mitochondril T16189C polymorphism with the risk of cncer development Rectl cncer (n = 48) Allele frequencies T 91.2% 82.5% 92.3% 66.6% C 8.8% 17.5% 7.7% 33.3% CRC: colorectl cncer. T represents wild form. C represents polymorphic form. Vlues re presented s n (%). Genotype distribution ws in Hrdy Weinberg equilibrium. We hve nlyzed T16189C polymorphisms in D-loop region of mitochondri to find out its ssocition with the risk of cncer development. The llelic frequency distribution of T16189C vrints in controls nd tumor subjects is shown in Tble 2. The frequency of C-llele ws found to be higher in CRC ptients (17.18%) thn control (8.77%). Also, the frequency of C-llele in rectl cncer (33%) ws lmost four-fold higher when compred to control (Tble 2). Although no significnt difference ws observed in C-llele frequency of colon cncer compred to control. The binry logistic regression nlysis reveled tht C-llele provides risk for development of CRC (OR: 2.158, p = 0.058) nd the p vlue ws found to be very close to significnt level. Interestingly when rectl nd colon smples were nlyzed individully with helthy controls, C-llele provided very high risk (OR = 5.213,

4 4 Tumor Biology Tble 3. Odds rtios nd FPRP vlues for ssocition between mitochondril T16189C polymorphism in different study groups. T16189C polymorphism OR (95% CI) p vlue # Power Prior probbility Cncer (CRC) versus control b 2.15 ( ) Rectl cncer versus control b 5.21 ( ) Colon cncer versus control b 0.86 ( ) CRC: colorectl cncer; OR: odds rtio; FPRP: flse-positive report probbility; CI: confidence intervl. Test of ssocition ws repeted in logistic regression model djusting for ge, durtion, nd gender. b Reference bseline group free from ny type cncer. # p < 0.05 considered significnt, FPRP vlues (bold vlues) below 0.2 re considered noteworthy. p = 0.001) for development of rectl cncer, while no risk ws observed for colon cncer (Tble 3). FPRP vlue ws fr below 0.2 for the rectl cncer risk suggesting tht the ssocition found ws not flse positive. However, FPRP vlues reveled tht the risk found for development of CRC ws not noteworthy. Assocition between mtdna copy number nd CRC The comprison between mtdna copy number of CRC nd helthy subjects is summrized in Tble 4. The levels of mtdna copy number were not normlly distributed in study subjects; hence, they re presented by qurtile rnge. It ws found tht the levels of mtdna copies were significntly higher (p = 0.001) in CRC thn control subjects (Tble 4). Adjustment for potentil confounding fctors, tht is, sex nd ge by the nlysis of covrince using Bonferroni djustment, reveled tht difference in mtdna copy number mong study subjects lso remined significnt (p < 0.05) s shown in Supplementry Tble 2. Seprte nlysis of mtdna copy number of colon nd rectl cncer with control subjects lso showed the significnt ssocition with colon (81 (60 143), p = 0.002) nd rectl (103 (32 178), p = 0.021) subjects, respectively (Tble 4). On dividing the tumor subjects on the bsis of their pthology into different grdes, significnt difference ws lso observed for mtdna copy number in tumor grdes I + II (83 (53 148), p = 0.005) nd tumor grdes III + IV (88 (60 190), p = 0.007) on compring with control. To find out the risk for development of CRC due to mtdna copy number, binry logistic regression ws crried out between tumor subjects nd control (reference), nd it ws found tht incresed mtdna copy number provides significnt risk for development of CRC hving odd rtios of (p = 0.009). Dt nlysis by qurtile distribution of mtdna copy number lso reveled dosedependent ssocition between copy number nd CRC risk (highest vs lowest qurtile: OR = 5, 95% confidence intervl (CI) = , p = 0.004). The presence of greter thn 73 mtdna copies per nucler genome provides highly significnt risk for development of CRC in North Indin popultion (Tble 5). Also, no significnt effect of different prmeters like smoking nd lcohol ws observed on CNV mong the ptients (Supplementry Tble 1). While ll prmeters except smoking showed significnt difference of copy number between CRC ptients nd control (Supplementry Tble 2). Assocition of mitochondril T16189C polymorphism nd mtdna copy number To confirm the role of T16189C polymorphism in controlling the mtdna copy number, ssocition of this polymorphism in different smples with their mtdna copy number ws tested. Interestingly, no ssocition ws found between the presence of T-/C-llele nd mtdna copy number in both CRC ptients nd controls (Tble 6). Discussion This study ws crried out on 230 North Indin subjects including 126 CRC ptients, dignosed between 2013 nd 2016, nd ge-, sex-, nd ethnicity-mtched helthy controls. Mitochondril muttion polymorphisms nd copy number hve been shown to ply n importnt role in development of mny diseses including cncer. Usully, these vritions in mtdna destbilize intrcellulr ROS blnce which leds to initition or progression of the disese. 27 mtdna contins hyper vrible regions (HVR I, II, nd III) which re the prime sites where such chnges occur. In the D-loop region, this T to C trnsition t position genertes poly-c trct. It hs been suggested tht this poly-c trct cn regulte mtdna copy number leding to disese phenotype. Continuous cytosine residues were ssocited with reduced mtdna copy number. 22,28 A common vrint T16189C from HVR III region hs been ssocited with different diseses such s Type II dibetes mellitus (DM) mong Indins nd other Asins, 29,30 coronry rtery disese mong middle Europens, 31 metbolic syndrome mong Cucsins, 32 insulin resistnce, 33 nd dilted crdiomyopthy. 34 T16189C provides risk nd is

5 Kumr et l. 5 Tble 4. Copy number dt of tumor ptients nd helthy subjects. Prmeters Helthy control Cncer (CRC) ptients Colon cncer Rectl cncer Tumor grde I nd II Tumor grde III nd IV Subjects (n) Copy number 56 (36 73) 88 (58 154) 81 (60 147) 103 (33 176) 83 (53 148) 88 (62 187) p vlue p = # p = # p = # p = # p = # CRC: colorectl cncer. Comprison between the groups ws performed with Mnn Whitney U Test. Dt re presented by inter-qurtile rnge. # versus helthy subjects. Tble 5. Assocition of copy number with cncer development. MtDNA Cses Control OR 95% CI # p vlue By medin Reference > By qurtile Reference > CI: confidence intervl. Reference bseline group tht do not contin the polymorphism. very significntly ssocited in South Indin CRC ptients 25 s well s brest cncer 35 nd found in higher frequency mong melnom ptients in Europens, 36 nd the T-llele correltes with shorter life expectncy mong prostte cncer ptients. 37 The frequency of minor llele (C) mong North Indin CRC ptients nd helthy subjects (17.18% in CRC ptients nd 8.77% in controls) mtched exctly with the frequency reported in South Indin popultion (18.39% in CRC ptients nd 8.82% in controls). 25 Our dt of North Indin subjects showed tht minor llele provides risk (OR = 2.158) for development of CRC which ws consistent with the dt published on South Indin popultion, lthough the significnt vlue tht we observed ws borderline non-significnt (p = 0.058) which my be due to lower smple size in this study. Since the FPRP vlues were not significnt for CRC risk, the result obtined could be flse negtive due to lower smple size. Therefore, we suggest for dditionl vlidtions with lrger smple size to further vlidte the risk of CRC development due to T16189C polymorphism. Individul nlysis of colon nd rectl smples reveled greter minor llele C frequency (33.33%) in rectl cncer cses thn in colon cncer cses, which provided highly significnt risk (p = 0.001, OR = 5.213) for development of rectl cncer. FPRP vlues for this comprison suggested tht the result obtined ws uthentic nd not flse positive. No risk or significnce ws observed for colon cncer cses. Assocition studies between mtdna copy number from cncer nd djcent norml tissues hs been described for mny cncer types such s brest cncer, cervicl cncer, leukemi, nd CRCs. 38 The stte of cncer cell depending on its needs selects n incresed or decresed copy number of mitochondril genome. Evidence from multiple studies of mtdna copy number from PBLs hs described ssocition of n incresed mtdna in melnoms, renl cell crcinom, brest cncer, nd prostte cncer 18,19,39,40 nd decresed mtdna copy number with risk of cncers such s esophgel denocrcinom nd soft tissue srcoms. 21,41 It hs been reported tht incresed mtdna from PBLs of CRC ptients shows poor survivl of ptients. 42 Counter clims where ptients with lower mtdna show low survivl hve lso been reported. 43 In this study, we tried to find out the scenrio in peripherl blood of the ptients. To the best of our knowledge, the ssocition of peripherl blood mtdna copy number s mrker nd risk of CRC hs not yet been scertined in Indin popultion. The comprison of mtdna copy number reveled tht copy number ws significntly higher mong CRC ptients in comprison to helthy controls. It ws lso significntly higher both in colon cses versus helthy controls nd in rectl cses versus helthy controls. As the C-llele is known to reduce the mtdna copy number, 22 the reduced C-llele frequency nd corresponding incresed mtdna copy number mong colon cncer smples go hnd in hnd. But the sme ws not found true for rectl cncer, thereby suggesting involvement of other vritions which might be involved with mtdna repliction. No significnt difference ws found between different prmeters such s smoking, hypertension, nd lcohol consumption mong colon nd rectl cses, thus limiting the involvement of such fctors in regulting the mtdna copy number between the two types. However, these fctors showed significnt deference in copy number between CRC ptients nd control in our smples. The fctors such s hypertension, smoking, nd lcohol consumption hve been previously reported to ffect the copy number in different studies 44,45 nd not mong others. 46,47 Incresed copy number in CRC ptients (medin >56) provided significntly incresed risk (OR: 2.885, p = 0.009) for the development of CRC. Thus, there is strong risk fctor ssocited

6 6 Tumor Biology Tble 6. Assocition of mitochondril T16189C polymorphisms nd copy number dt in cncer ptients nd helthy subjects. Prmeters Helthy control CRC ptients p vlue Copy number without 54 (33 73) 83 (58 176) polymorphism (n = 40) (n = 58) Copy number with 56 (38 81) 102 (45 138) polymorphism (n = 8) (n = 8) p vlue CRC: colorectl cncer between incresed mtdna nd development of CRC. The qurtile distributions of copy number hve further showed tht greter thn 73 copies of mtdna hve high risk. Another blood smples relted study from CRC ptients reveled high ORs for both lower nd higher qurtiles of mtdna copy number suggesting U-shped reltionship, 48 therefore supporting our dt in prt for incresed risk for CRC with incresed mtdna copy number from PBLs. Incresed mtdna my led to sitution where ROS nd other free rdicls produced by mitochondri increse which is thought to be involved in crcinogenesis. 49 Mitochondril ROS-medited retrogrde signling hs been shown to induce pro-cncerous metbolic fetures. 16 Wheres reduced mtdna hs been shown to promote resistnce to poptosis in cncer cells long with epithelil mesenchyml trnsition (EMT). 50,51 These ltertions in mtdna copy number (both incresed nd decresed) my represent diverse mechnisms by which mitochondri nd mtdna copy number ply role in different mlignncies. 17 As per the finding of this study, it would be pproprite to stte tht there is definitely some involvement of T16189C polymorphism nd mtdna copy number in providing risk for development of CRC mong North Indin popultion. Interestingly, our dt lso suggest tht C-llele t position my not be regulting mtdna copy number lone since we could not find ny sttisticlly significnt reltionship between the two, which my be due to the smller smple size employed for CNV nlysis. In conclusion, this study is the first report where the reltionship between mtdna copy number from PBLs of CRC nd helthy subjects ws estblished. Mitochondril D-loop vrition T16189C nd copy number my constitute intrinsic risk fctors for CRC development. Although future study with lrger smple size is required to fill in ny gp tht we fced. We therefore suggest tht mtdna CNV nd ltertion t could be utilized for identifiction of subjects who re t high risk for development or progression of CRC. Acknowledgements Zfr Iqbl Bht cknowledges MANF-UGC for providing fellowship. M Moshhid Alm Rizvi is thnkful to UGC for providing funds. Bhupender Kumr cknowledges Trun Kumri from Deprtment of Sttistics, University of Delhi, for vluble help in FPRP nlysis. Declrtion of conflicting interests The uthor(s) declred no potentil conflicts of interest with respect to the reserch, uthorship, nd/or publiction of this rticle. Funding The uthor(s) received no finncil support for the reserch, uthorship, nd/or publiction of this rticle. References 1. Mrley AR nd Nn H. Epidemiology of colorectl cncer. Int J Mol Epidemiol Genet 2016; 7: Kolligs FT. Dignostics nd epidemiology of colorectl cncer. Visc Med 2016; 32: Stryker SJ, Wolff BG, Culp CE, et l. Nturl history of untreted colonic polyps. Gstroenterology 1987; 93: Lichtenstein P, Holm NV, Verkslo PK, et l. Environmentl nd heritble fctors in the custion of cncer nlyses of cohorts of twins from Sweden, Denmrk, nd Finlnd. N Engl J Med 2000; 343: Jsperson KW, Tuohy TM, Neklson DW, et l. Hereditry nd fmilil colon cncer. Gstroenterology 2010; 138: Grdy WM. Genetic testing for high-risk colon cncer ptients. Gstroenterology 2003; 124: Herceg Z nd Hinut P. Genetic nd epigenetic ltertions s biomrkers for cncer detection, dignosis nd prognosis. Mol Oncol 2007; 1: Morimoto LM, Newcomb PA, White E, et l. Insulin-like growth fctor polymorphisms nd colorectl cncer risk. Cncer Epidemiol Biomrkers Prev 2005; 14: Pechlivnis S, Wgner K, Chng-Clude J, et l. Polymorphisms in the insulin like growth fctor 1 nd IGF binding protein 3 genes nd risk of colorectl cncer. Cncer Detect Prev 2007; 31: Hung K, Sndler RS, Millikn RC, et l. GSTM1 nd GSTT1 polymorphisms, cigrette smoking, nd risk of colon cncer: popultion-bsed cse-control study in North Crolin (United Sttes). Cncer Cuses Control 2006; 17: Cnby E, Khrmn OT, Bugr D, et l. Assocition between PTEN IVS4 polymorphism nd development of colorectl cncer in Turkish popultion. Expert Opin Ther Trgets 2013; 17: Sun L, Liu J, Yun Q, et l. Assocition between PTEN gene IVS4 polymorphism nd risk of cncer: met-nlysis. PLoS ONE 2014; 9: e Gorrini C, Hrris IS nd Mk TW. Modultion of oxidtive stress s n nticncer strtegy. Nt Rev Drug Discov 2013; 12: Sullivn LB nd Chndel NS. Mitochondril rective oxygen species nd cncer. Cncer Metb 2014; 2: Sbhrwl SS nd Schumcker PT. Mitochondril ROS in cncer: inititors, mplifiers or n Achilles heel? Nt Rev Cncer 2014; 14:

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