Colorectal adenoma and cancer detection based on altered methylation pattern of SFRP1, SFRP2, SDC2, and PRIMA1 in plasma samples

References

• Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010;127(12):2893-917. doi:10.1002/ijc.25516. PMID:21351269.
   PubMed Web of Science ®Google Scholar
• Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015;65:5-29. doi:10.3322/caac.21254.
   PubMed Web of Science ®Google Scholar
• Vermeer NC, Snijders HS, Holman FA, Liefers GJ, Bastiaannet E, van de Velde CJ, Peeters KC. Colorectal cancer screening: Systematic review of screen-related morbidity and mortality. Cancer Treat Rev. 2017;54:87-98. doi:10.1016/j.ctrv.2017.02.002.
   PubMed Web of Science ®Google Scholar
• Hassan C, Quintero E, Dumonceau JM, Regula J, Brandão C, Chaussade S, Dekker E, Dinis-Ribeiro M, Ferlitsch M, Gimeno-García A et al. Post-polypectomy colonoscopy surveillance: European Society of Gastrointestinal Endoscopy (ESGE) Guideline. Endoscopy 2013;45(10):842-51. doi:10.1055/s-0033-1344548.
   PubMed Web of Science ®Google Scholar
• Kumar M, Cash BD. Screening and Surveillance of Colorectal Cancer Using CT Colonography. Curr Treat Options Gastroenterol. 2017;15(1):168-83. doi:10.1007/s11938-017-0121-7. PMID:28130651.
   PubMedGoogle Scholar
• Winawer SJ, Stewart ET, Zauber AG, Bond JH, Ansel H, Wave JD, Hall D, Hamlin JA, Schapiro M, O'Brien MJ et al. A comparison of colonoscopy and double-contrast barium enema for surveillance after polypectomy. N Engl J Med 2000;342:1766-72. doi:10.1056/NEJM200006153422401. PMID:10852998.
   PubMed Web of Science ®Google Scholar
• Kahi CJ, Boland CR, Dominitz JA, Giardiello FM, Johnson DA, Kaltenbach T, Lieberman D, Levin TR, Robertson DJ, Rex DK. Colonoscopy Surveillance after Colorectal Cancer Resection: Recommendations of the US Multi-Society Task Force on Colorectal Cancer. Am J Gastroenterol. 2016;111(3):337-46; quiz 347. doi:10.1038/ajg.2016.22. PMID:26871541.
   PubMed Web of Science ®Google Scholar
• Ahlquist DA, Sargent DJ, Loprinzi CL, Levin TR, Rex DK, Ahnen DJ, Knigge K, Lance MP, Burgart LJ, Hamilton SR, et al. Stool DNA and occult blood testing for screen detection of colorectal neoplasia. Ann Intern Med. 2008;149:441-50. doi:10.7326/0003-4819-149-7-200810070-00004.
   PubMed Web of Science ®Google Scholar
• Robertson DJ, Lee JK, Boland CR, Dominitz JA, Giardiello FM, Johnson DA, Kaltenbach T, Lieberman D, Levin TR, Rex DK. Recommendations on Fecal Immunochemical Testing to Screen for Colorectal Neoplasia: A Consensus Statement by the US Multi-Society Task Force on Colorectal Cancer. Am J Gastroenterol. 2017;112(1):37-53. doi:10.1038/ajg.2016.492.
   PubMed Web of Science ®Google Scholar
• Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, Ahlquist DA, Berger BM. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014;370(14):1287-97. doi:10.1056/NEJMoa1311194.
   PubMed Web of Science ®Google Scholar
• Heigh RI, Yab TC, Taylor WR, Hussain FT, Smyrk TC, Mahoney DW, Domanico MJ, Berger BM, Lidgard GP, Ahlquist DA. Detection of colorectal serrated polyps by stool DNA testing: comparison with fecal immunochemical testing for occult blood (FIT). PLoS One 2014;9:e85659. doi:10.1371/journal.pone.0085659. PMID:24465639.
   PubMed Web of Science ®Google Scholar
• Kadiyska T, Nossikoff A. Stool DNA methylation assays in colorectal cancer screening. World J Gastroenterol. 2015;21(35):10057-61. doi:10.3748/wjg.v21.i35.10057.
   PubMed Web of Science ®Google Scholar
• Diaz LA Jr, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J. Clin. Oncol. 2014;32:579-86. doi:10.1200/JCO.2012.45.2011. PMID:24449238.
   PubMed Web of Science ®Google Scholar
• Mandel P, Metais P. Les acides nucleiques du plasma sanguin chez l'homme. C R Acad Sci Paris. 1948;142:241-43.
   Google Scholar
• Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 1977;37:646-50. PMID:837366.
   PubMed Web of Science ®Google Scholar
• Stroun M, Anker P, Maurice P, Lyautey J, Lederrey C, Beljanski M. Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology. 1989; 46:318-22. doi:10.1159/000226740. PMID:2779946.
   PubMed Web of Science ®Google Scholar
• Fujita S, Sugano K, Fukayama N, Moriya Y, Sugihara K, Akasu T. Detection of K-ras point mutations in mesenteric venous blood from colorectal cancer patients by enriched polymerase chain reaction and single-strand conformation polymorphism analysis. Jpn J Clin Oncol. 1996;26:417-21.; doi:10.1093/oxfordjournals.jjco.a023258. PMID: 9001346.
   PubMed Web of Science ®Google Scholar
• Kopreski MS, Benko FA, Borys DJ, Khan A, McGarrity TJ, Gocke CD. Somatic mutation screening: identification of individuals harboring K-ras mutations with the use of plasma DNA. J Natl Cancer Inst. 2000;92:918-23. doi:10.1093/jnci/92.11.918. PMID:10841827.
   PubMedGoogle Scholar
• Borchers R, Heinzlmann M, Zahn R, Witter K, Martin K, Loeschke K, Folwaczny C. K-ras mutations in sera of patients with colorectal neoplasias and long-standing inflammatory bowel disease. Scand J Gastroenterol 2002;37:715-18. doi:10.1080/00365520212505. PMID:12126252.
   PubMed Web of Science ®Google Scholar
• Ling E, Fich A, Man S, Wolfson M, Mikhailowsky R, Lamprecht SA. Detection of tumor mutant APC DNA in plasma of patients with sporadic colorectal cancer. In Vivo (Athens, Greece). 2000;14(4):543-6. PMID: 10945171.
   PubMed Web of Science ®Google Scholar
• Hammel P, Leroy-Viard K, Chaumette MT, Villaudy J, Falzone MC, Rouillard D, Hamelin R, Boissier B, Remvikos Y. Correlations between p53-protein accumulation, serum antibodies and gene mutation in colorectal cancer. Int J Cancer. 1999;81:712-18. doi:10.1002/(SICI)1097-0215(19990531)81:5<712::AID-IJC7>3.0.CO;2-0. PMID:10328221.
   PubMed Web of Science ®Google Scholar
• Patai AV, Molnár B, Kalmár A, Schöller A, Tóth K, Tulassay Z. Role of DNA methylation in colorectal carcinogenesis. Dig Dis. 2012;30(3):310-5. doi:10.1159/000337004. PMID:22722557.
   PubMed Web of Science ®Google Scholar
• deVos T, Tetzner R, Model F, Weiss G, Schuster M, Distler J, Steiger KV, Grützmann R, Pilarsky C, Habermann JK et al. Circulating methylated SEPT9 DNA in plasma is a biomarker for colorectal cancer. Clin Chem. 2009;55(7):1337-46. doi:10.1373/clinchem.2008.115808. PMID:19406918.
   PubMed Web of Science ®Google Scholar
• Tóth K, Sipos F, Kalmár A, Patai AV, Wichmann B, Stoehr R, Golcher H, Schellerer V, Tulassay Z, Molnár B. Detection of methylated SEPT9 in plasma is a reliable screening method for both left- and right-sided colon cancers. PLoS One. 2012;7(9):e46000. doi:10.1371/journal.pone.0046000. PMID:23049919.
   PubMed Web of Science ®Google Scholar
• Tóth K, Wasserkort R, Sipos F, Kalmár A, Wichmann B, Leiszter K, Valcz G, Juhász M, Miheller P, Patai ÁV et al. Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA. PLoS One. 2014;9(12):e115415. doi:10.1371/journal.pone.0115415.
   PubMed Web of Science ®Google Scholar
• Molnár B, Tóth K, Barták BK, Tulassay Z. Plasma methylated septin 9: a colorectal cancer screening marker. Expert Rev Mol Diagn. 2015;15(2):171-84. doi:10.1586/14737159.2015.975212.
   PubMed Web of Science ®Google Scholar
• Potter NT, Hurban P, White MN, Whitlock KD, Lofton-Day CE, Tetzner R, Koenig T, Quigley NB, Weiss G. Validation of a real-time PCR-based qualitative assay for the detection of methylated SEPT9 DNA in human plasma. Clin Chem. 2014;60(9):1183-91. doi:10.1373/clinchem.2013.221044.
   PubMed Web of Science ®Google Scholar
• Johnson DA, Barclay RL, Mergener K, Weiss G, König T, Beck J, Potter NT. Plasma Septin9 versus fecal immunochemical testing for colorectal cancer screening: a prospective multicenter study. PLoS One. 2014;9(6):e98238. doi:10.1371/journal.pone.0098238.
   PubMed Web of Science ®Google Scholar
• Nian J, Sun X, Ming S, Yan C, Ma Y, Feng Y, Yang L, Yu M, Zhang G, Wang X. Diagnostic Accuracy of Methylated SEPT9 for Blood-based Colorectal Cancer Detection: A Systematic Review and Meta-Analysis. Clin Transl Gastroenterol. 2017;8(1):e216. doi:10.1038/ctg.2016.66.
   PubMed Web of Science ®Google Scholar
• Patai ÁV, Valcz G, Hollósi P, Kalmár A, Péterfia B, Patai Á, Wichmann B, Spisák S, Barták BK, Leiszter K et al. Comprehensive DNA Methylation Analysis Reveals a Common Ten-Gene Methylation Signature in Colorectal Adenomas and Carcinomas. PLoS One. 2015;10(8):e0133836. doi:10.1371/journal.pone.0133836.
   PubMed Web of Science ®Google Scholar
• Valcz G, Patai AV, Kalmár A, Péterfia B, Fűri I, Wichmann B, Műzes G, Sipos F, Krenács T, Mihály E et al. Myofibroblast-derived SFRP1 as potential inhibitor of colorectal carcinoma field effect. PLoS One. 2014;9(11):e106143. doi:10.1371/journal.pone.0106143.
   PubMed Web of Science ®Google Scholar
• Kalmár A, Péterfia B, Hollósi P, Galamb O, Spisák S, Wichmann B, Bodor, Tóth K, Patai ÁV, Valcz G et al. DNA hypermethylation and decreased mRNA expression of MAL, PRIMA1, PTGDR and SFRP1 in colorectal adenoma and cancer. BMC Cancer. 2015;15:736. doi:10.1186/s12885-015-1687-x.
   PubMed Web of Science ®Google Scholar
• Oh T, Kim N, Moon Y, Kim MS, Hoehn BD, Park CH, Kim TS, Kim NK, Chung HC, An S.. Genome-wide identification and validation of a novel methylation biomarker, SDC2, for blood-based detection of colorectal cancer. J Mol Diagn. 2013;15(4):498-507. doi:10.1016/j.jmoldx.2013.03.004.
   PubMed Web of Science ®Google Scholar
• Mitchell SM, Ross JP, Drew HR, Ho T, Brown GS, Saunders NF, Duesing KR, Buckley MJ, Dunne R, Beetson I et al. A panel of genes methylated with high frequency in colorectal cancer. BMC Cancer. 2014;14:54. doi:10.1186/1471-2407-14-54.
   PubMed Web of Science ®Google Scholar
• Silva AL, Dawson SN, Arends MJ, Guttula K, Hall N, Cameron EA, Huang TH, Brenton JD, Tavaré S, Bienz M, et al. Boosting Wnt activity during colorectal cancer progression through selective hypermethylation of Wnt signaling antagonists. BMC Cancer. 2014;14:891. doi:10.1186/1471-2407-14-891.
   PubMed Web of Science ®Google Scholar
• Xie HQ, Liang D, Leung KW, Chen VP, Zhu KY, Chan WK, Choi RC, Massoulie J, Tsim KW. Targeting acetylcholinesterase to membrane rafts: a function mediated by the proline-rich membrane anchor (PRiMA) in neurons. J Biol Chem. 2010;285(15):11537-46. doi:10.1074/jbc.M109.038711.
   PubMed Web of Science ®Google Scholar
• Rapraeger AC. Syndecan-regulated receptor signaling. J Cell Biol. 2000;149(5):995-8. doi:10.1083/jcb.149.5.995.
   PubMed Web of Science ®Google Scholar
• Chen YZ, Liu D, Zhao YX, Wang HT, Gao Y, Chen Y. Aberrant promoter methylation of the SFRP1 gene may contribute to colorectal carcinogenesis: a meta-analysis. Tumour Biol. 2014;35(9):9201-10. doi:10.1007/s13277-014-2180-x.
   PubMed Web of Science ®Google Scholar
• Galamb O, Kalmár A, Péterfia B, Csabai I, Bodor A, Ribli D, Krenács T, Patai ÁV, Wichmann B, Barták BK et al. Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer. Epigenetics. 2016;11(8):588-602. doi:10.1080/15592294.2016.1190894.
   PubMed Web of Science ®Google Scholar
• Sabunciyan S, Aryee MJ, Irizarry RA, Rongione M, Webster MJ, Kaufman WE, Murakami P, Lessard A, Yolken RH, Feinberg AP et al. Genome-wide DNA methylation scan in major depressive disorder. PLoS One. 2012;7(4):e34451. doi:10.1371/journal.pone.0034451. PMID:22511943.
   PubMed Web of Science ®Google Scholar
• Vanaja DK, Ballman KV, Morlan BW, Cheville JC, Neumann RM, Lieber MM, Tindall DJ, Young CY. PDLIM4 repression by hypermethylation as a potential biomarker for prostate cancer. Clin Cancer Res. 2006;12(4):1128-36. doi:10.1158/1078-0432.CCR-05-2072.
   PubMed Web of Science ®Google Scholar
• Tong WG, Wierda WG, Lin E, Kuang SQ, Bekele BN, Estrov Z, Wei Y, Yang H, Keating MJ, Garcia-Manero G. Genome-wide DNA methylation profiling of chronic lymphocytic leukemia allows identification of epigenetically repressed molecular pathways with clinical impact. Epigenetics. 2010;5(6):499-508. doi:10.4161/epi.5.6.12179.
   PubMed Web of Science ®Google Scholar
• Kim J, Kim S. In silico Identification of SFRP1 as a Hypermethylated Gene in Colorectal Cancers. Genomics Inform. 2014;12(4):171-80. doi:10.5808/GI.2014.12.4.171.
   PubMedGoogle Scholar
• Luo Y, Wong CJ, Kaz AM, Dzieciatkowski S, Carter KT, Morris SM, Wang J, Willis JE, Makar KW, Ulrich CM et al. Differences in DNA methylation signatures reveal multiple pathways of progression from adenoma to colorectal cancer. Gastroenterology. 2014;147(2):418-29. e8. doi:10.1053/j.gastro.2014.04.039.
   PubMed Web of Science ®Google Scholar
• Razin A, Kantor B. DNA methylation in epigenetic control of gene expression. Prog Mol Subcell Biol. 2005;38:151-67. doi:10.1007/3-540-27310-7_6. PMID: 15881894.
   PubMedGoogle Scholar
• Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A. Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol. 2013;10(8):472-84. doi:10.1038/nrclinonc.2013.110.
   PubMed Web of Science ®Google Scholar
• Henriksen SD, Madsen PH, Larsen AC, Johansen MB, Drewes AM, Pedersen IS, Krarup H, Thorlacius-Ussing O. Cell-free DNA promoter hypermethylation in plasma as a diagnostic marker for pancreatic adenocarcinoma. Clin Epigenetics. 2016;8:117. doi:10.1186/s13148-016-0286-2.
   PubMed Web of Science ®Google Scholar
• Zhang Y, Wang R, Song H, Huang G, Yi J, Zheng Y, Wang J, Chen L. Methylation of multiple genes as a candidate biomarker in non-small cell lung cancer. Cancer Lett. 2011;303(1):21-8. doi:10.1016/j.canlet.2010.12.011.
   PubMed Web of Science ®Google Scholar
• Qi J, Zhu YQ, Luo J, Tao WH. Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor. World J Gastroenterol. 2006;12(44): 7113-17. doi:10.3748/wjg.v12.i44.7113. PMID:17131472.
   PubMed Web of Science ®Google Scholar
• Rawson JB, Manno M, Mrkonjic M, Daftary D, Dicks E, Buchanan DD, Younghusband HB, Parfrey PS, Young JP, Pollett A et al. Promoter methylation of Wnt antagonists DKK1 and SFRP1 is associated with opposing tumor subtypes in two large populations of colorectal cancer patients. Carcinogenesis. 2011 May;32(5):741-7. doi:10.1093/carcin/bgr020.
   PubMed Web of Science ®Google Scholar
• Lyu Z, Chen H, Jiang L, Zheng H, Hu J. Detection of RASSF2 and sFRP1 promoter region methylation in sporadic colorectal cancer patients. Zhonghua Wei Chang Wai Ke Za Zhi. 2014;17(1):41-4.
   PubMedGoogle Scholar
• Bedin C, Enzo MV, Del Bianco P, Pucciarelli S, Nitti D, Agostini M. Diagnostic and prognostic role of cell-free DNA testing for colorectal cancer patients. Int J Cancer. 2016;140(8):1888-98. doi:10.1002/ijc.30565. PMID:27943272.
   PubMed Web of Science ®Google Scholar
• Gonzalo V, Lozano JJ, Muñoz J, Balaguer F, Pellisé M, Rodríguez de Miguel C, Andreu M, Jover R, Llor X, Giráldez MD et al. Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer. PLoS One. 2010;5(1):e8777. doi:10.1371/journal.pone.0008777.
   PubMed Web of Science ®Google Scholar
• Andresen K, Boberg KM, Vedeld HM, Honne H, Hektoen M, Wadsworth CA, Clausen OP, Karlsen TH, Foss A, Mathisen O et al. Novel target genes and a valid biomarker panel identified for cholangiocarcinoma. Epigenetics. 2012;7(11):1249-57. doi:10.4161/epi.22191.
   PubMed Web of Science ®Google Scholar
• Chim CS, Pang R, Fung TK, Choi CL, Liang R. Epigenetic dysregulation of Wnt signaling pathway in multiple myeloma. Leukemia. 2007;21(12):2527-36. doi:10.1038/sj.leu.2404939.
   PubMed Web of Science ®Google Scholar
• Lu H, Huang S, Zhang X, Wang D, Zhang X, Yuan X, Zhang Q, Huang Z. DNA methylation analysis of SFRP2, GATA4/5, NDRG4 and VIM for the detection of colorectal cancer in fecal DNA. Oncol Lett. 2014;8(4):1751-1756. doi:10.3892/ol.2014.2413.
   PubMed Web of Science ®Google Scholar
• Zhang H, Zhu YQ, Wu YQ, Zhang P, Qi J. Detection of promoter hypermethylation of Wnt antagonist genes in fecal samples for diagnosis of early colorectal cancer. World J Gastroenterol. 2014;20(20):6329-35. doi:10.3748/wjg.v20.i20.6329.
   PubMed Web of Science ®Google Scholar
• Tang D, Wang D, Li H. Combination analysis of hypermethylated SFRP1 and SFRP2 gene in fecal as a novel epigenetic biomarker panel for colorectal cancer screening. J Nanjing Med Univ (Eng) 2008;22, 96-101. doi:10.1016/S1007-4376(08)60020-9.
   Google Scholar
• Huang ZH, Li LH, Yang F, Wang JF. Detection of aberrant methylation in fecal DNA as a molecular screening tool for colorectal cancer and precancerous lesions. World J Gastroenterol. 2007 Feb 14;13(6):950-4. doi:10.3748/wjg.v13.i6.950.
   PubMed Web of Science ®Google Scholar
• Zhang X, Song YF, Lu HN, Wang DP, Zhang XS, Huang SL, Sun BL, Huang ZG. Combined detection of plasma GATA5 and SFRP2 methylation is a valid noninvasive biomarker for colorectal cancer and adenomas. World J Gastroenterol. 2015 Mar 7;21(9):2629-37. doi:10.3748/wjg.v21.i9.2629.
   PubMed Web of Science ®Google Scholar
• Ryu H, Lee J, Yang S, Park H, Choi S, Jung KC, Lee ST, Seong JK, Han IO, Oh ES. Syndecan-2 functions as a docking receptor for promatrix metalloproteinase-7 in human colon cancer cells. J Biol Chem. 2009;284(51):35692-701. doi:10.1074/jbc.M109.054254.
   PubMed Web of Science ®Google Scholar
• Huang X, Xiao DW, Xu LY, Zhong HJ, Liao LD, Xie ZF, Li EM. Prognostic significance of altered expression of SDC2 and CYR61 in esophageal squamous cell carcinoma. Oncol Rep. 2009;21:1123-9. doi:10.3892/or_00000332. PMID: 19288017.
   PubMed Web of Science ®Google Scholar
• Chong Y, Mia-Jan K, Ryu H, Abdul-Ghafar J, Munkhdelger J, Lkhagvadorj S, Jung SY, Lee M, Ji SY, Choi E, Cho MY. DNA methylation status of a distinctively different subset of genes is associated with each histologic Lauren classification subtype in early gastric carcinogenesis. Oncol Rep. 2014;31(6):2535-44. doi:10.3892/or.2014.3133.
   PubMed Web of Science ®Google Scholar
• Dobbertin A, Hrabovska A, Dembele K, Camp S, Taylor P, Krejci E, Bernard V. Targeting of acetylcholinesterase in neurons in vivo: A dual processing function for the proline-rich membrane anchor subunit and the attachment domain on the catalytic subunit. J Neurosci. 2009;29(14):4519-30. doi:10.1523/JNEUROSCI.3863-08.2009.
   PubMed Web of Science ®Google Scholar
• Perrier AL, Massoulié J, Krejci E. PRiMA: The membrane anchor of acetylcholinesterase in the brain. Neuron. 33:275-285. doi:10.1016/S0896-6273(01)00584-0. PMID:11804574.
   PubMed Web of Science ®Google Scholar
• Janowsky DS, el-Yousef MK, Davis JM, Sekerke HJ. A cholinergicadrenergic hypothesis of mania and depression. Lancet. 1972;2(7778):632-5. doi:10.1016/S0140-6736(72)93021-8.
   PubMed Web of Science ®Google Scholar
• Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000;16(6):276-7. doi:10.1016/S0168-9525(00)02024-2.
   PubMed Web of Science ®Google Scholar
• Dietrich D, Lesche R, Tetzner R, Krispin M, Dietrich J, Haedicke W, Schuster M, Kristiansen G. Analysis of DNA methylation of multiple genes in microdissected cells from formalin-fixed and paraffin-embedded tissues. J Histochem Cytochem. 2009; 57(5):477-89. doi:10.1369/jhc.2009.953026. PMID:19153192.
   PubMed Web of Science ®Google Scholar
• Fackler MJ, Malone K, Zhang Z, Schilling E, Garrett-Mayer E, Swift-Scanlan T, Lange J, Nayar R, Davidson NE, Khan SA, Sukumar,  . Quantitative multiplex methylation-specific PCR analysis doubles detection of tumor cells in breast ductal fluid. S Clin Cancer Res. 2006;12(11 Pt 1):3306-10. doi:10.1158/1078-0432.CCR-05-2733.
   PubMed Web of Science ®Google Scholar
• Tusnady GE, Simon I, Varadi A, Aranyi T. BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes. Nucleic Acids Res. 2005;33(1):e9. doi:10.1093/nar/gni012.
   PubMed Web of Science ®Google Scholar