RESEARCH PAPER
Application of RFLP-PCR method for molecular diagnostics of hereditary non-polyposis colorectal cancer (HNPCC)
 
More details
Hide details
1
Department of Internal Medicine, Medical University, Lublin, Poland
2
Department of Biological Bases of Animal Production, University of Life Sciences, Lublin, Poland
3
Department of General Surgery with Endoscopy Unit, Medical University, Lublin, Poland
4
Department of Anesthesiology and Intensive Care Unit, Medical University, Lublin, Poland
CORRESPONDING AUTHOR
Andrzej Prystupa   

Department of Internal Medicine, Medical University, Staszica 16, 20-081 Lublin, Poland.
 
J Pre Clin Clin Res. 2011;5(2):70–73
KEYWORDS
ABSTRACT
Colorectal cancer is one of the leading causes of cancer deaths, constituting a major public health concern. Epidemiologic studies have revealed a number of risk factors for colorectal cancer including age, family history of colon cancer or inflammatory bowel disease, smoking, alcohol consumption, obesity, and diet. Mutations in MSH2 and MLH2 genes are associated with colon cancer in many studies published to date. The aim of the presented study was to assess the associations of MSH2 and MLH1 genes mutations with colorectal cancer in a Polish population, using the PCR-RFLP method. Mutations in the exon 1 of both genes were detected using the PCR-RFLP method in colorectal patients and healthy individuals. There were no statistical differences in the presence of mutations between colorectal cancer and healthy groups. The PCR-RFLP method is not suitable for the detection of mutant alleles present in less than 5-10% of wild-type alleles. This is probably the reason why in the present study, the analysis did not allow the finding of genetic differences in the first exons of MSH2 and MLH1 genes between healthy individuals and those with the colorectal cancer. It is reasonable to continue studies based on RFLP-PCR, because the costs of this method are low compared to the sequencing method.
 
REFERENCES (27)
1.
Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Th un MJ. Cancer statistics, 2008. CA Cancer J Clin 2008;58(2):71-96.
 
2.
Colorectal Cancer Risk Factors. http://www.cdc.gov/cancer/colo... basic_info/risk_factors.htm (access:2011.08.19).
 
3.
Boutron MC, Faivre J, Quipourt V, Senesse P, Michiels C. Family history of colorectal tumours and implications for the adenomacarcinoma sequence: a case control study. Gut 1995;37:830-4.
 
4.
Marra G, Boland CR. Hereditary non-polyposis colorectal cancer: the syndrome, the genes, and historical perspectives. J Natl Cancer Inst 1995;87(15):1114-25.
 
5.
Vogelstein B. Genetic testings for cancer: the surgeon’s critical role. Familial colon cancer. J Am Coll Surg 1999;188(1):74-9.
 
6.
Kładny J, Lubiński J. Dziedziczny niepolipowaty rak jelita grubego [Hereditary non-polyposis colorectal cancer]. Pol Przegl Chir 1996;68:728-734.
 
7.
Lynch HT, de la Chapelle A. Genetic susceptibility to non-polyposis colorectal cancer. J Med Genet 1999;36(11):801-18.
 
8.
Peltomaki P, Vasen H. Mutations associated with HNPCC predisposition- Update of ICG-HNPCC/INSiGHT mutation database. Dis Markers 2004;20:269-276.
 
9.
Papp J., Kovacs M. E., Olah E. Germline MLH1 and MSH2 mutational spectrum including frequent large genomic aberrations in Hungarian hereditary non-polyposis colorectal cancer families: Implications for genetic testing. World J Gastroenterol 2007;13(19): 2727-2732.
 
10.
Taylor CF, Charlton RS, Burn J, Sheridan E, Taylor GR. Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary nonpolyposis colorectal cancer: identifi cation of novel and recurrent deletions by MLPA. Hum Mutat 2003;22(6):428-433.
 
11.
Lynch HT, de la Chapelle A. Hereditary colorectal cancer. N Engl J Med 2003; 48:919-932.
 
12.
Baudhuin LM, Burgart LJ, Leontovich O, Th ibodeau SN. Use of microsatellite instability and immunohistochemistry testing for the identifi cation of individuals at risk for Lynch syndrome. Fam Cancer 2005;4:255-265.
 
13.
Charbonnier F, Raux G, Wang Q, Drouot N, Cordier F, Limacher J-M. Saurin J-C, Puisieux A, Olschwang S, Frebourg T. Detection of exon deletions and duplications of the mismatch repair genes in hereditary nonpolyposis colorectal cancer families using multiplex polymerase chain reaction of short fl uorescent fragments. Cancer Res 2000;60(11):2760-2763.
 
14.
Choi Y-H, Cotterchio M, McKeown-Eyssen G, Neerav M, Bapat B, Boyd K, Gallinger S, McLaughlin J, Aronson M, Briollais L. Penetrance of colorectal cancer among MLH1/MSH2 carriers participating in the colorectal cancer familial registry in Ontario. Hered Cancer Clin Pract 2009;7(1):14.
 
15.
Gargiulo S, Torrini M, Ollila S, Nasti S, Pastorino L, Cusano R, Bonelli L, Battistuzzi L, Mastracci L, Bruno W, Savarino V, Sciallero S, Borgonovo G, Nystrom M, Bianchi-Scarra`G, Mareni C, Ghiorzo P. Germline MLH1 and MSH2 mutations in Italian pancreatic cancer patients with suspected Lynch syndrome. Fam Cancer 2009; 8(4):547-553.
 
16.
Hampel H, Stephens JA, Pukkala E, Sankila R, Aaltonen LA, de la Chapelle A. Cancer risk in hereditary nonpolyposis colorectal cancer syndrome. Gastroenterology 2005;129(2):415-421.
 
17.
http://frodo.wi.mit.edu/primer... (access: 2011.08.19).
 
18.
Vincze T, Posfai J, Roberts RJ. NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res 2003;31(13):3688-3691.
 
19.
Pourzand C, Cerutti P. Genotypic mutation analysis by RFLP/PCR. Mutat Res 1993;288(1):113-21.
 
20.
Castells A, Puig P, Mora J, Boadas J, Boix L, Urgell E, Sole M, Capella G, Lluis F, Fernandez-Cruz L, Navarro S, Farre A. K-ras mutations in DNA extracted from the plasma of patients with pancreatic carcinoma: diagnostic utility and prognostic signifi cance. J Clin Oncol 1999;17(2):578-84.
 
21.
Eiken H G, Odland E, Boman H, Skjelkvale L, Engebretsen L F, Apold J. Application of natural and amplifi cation created restriction sites for the diagnosis of PKU mutations. Nucleic Acids Res 1991;19(7):1427- 1430.
 
22.
Bazrafshani MR, Ollier WE, Hajeer AH. A novel PCR-RFLP assay for the detection of the single nucleotide polymorphism at position-1082 in the human IL-10 gene promoter. Eur J Immunogenet 2000;27(3):119- 120.
 
23.
Friedman KJ, Highsmith WE Jr, Prior TW, Perry TR, Silverman LM. Cystic fi brosis deletion mutation detected by PCR-mediated sitedirected mutagenesis. Clin Chem 1990;36(4):695-696.
 
24.
Parsons BL, Hefl ich RH. Genotypic selection methods for the direct analysis of point mutations. Mutat Res 1997;387(2):97-121.
 
25.
Plendl H, Siebert R, Steinemann D, Grote W. High frequency of the N34S mutation in the SPINK1 gene in chronic pancreatitis detected by a new PCR-RFLP assay. Am J Med Genet 2001;100(3):252-253.
 
26.
Medina-Arana V, Barrios Y, Fernandez-Peralta A, Herrera M, Chinea N, Lorenzo N, Jimenez A, Martin-Lopez JV, Gonzalez-Hermoso F, Salido E, Gonzalez-Aguilera JJ. New founding mutation in MSH2 associated with hereditary nonpolyposis colorectal cancer syndrome on the Island of Tenerife. Cancer Lett 2006;244(2):268-273.
 
27.
Haliassos A, Chomel JC, Grandjouan S, Kruh J, Kaplan JC, Kitzis A. Detection of minority point mutations by modifi ed PCR technique: a new approach for a sensitive diagnosis of tumor-progression markers. Nucleic Acids Res 1989;17(20):8093-8099.
 
eISSN:1898-7516
ISSN:1898-2395