1. bookVolume 26 (2018): Issue 2 (April 2018)
Journal Details
License
Format
Journal
eISSN
2284-5623
First Published
08 Aug 2013
Publication timeframe
4 times per year
Languages
English
access type Open Access

Sanger sequencing of MMR genes in a one-plate system

Published Online: 17 May 2018
Volume & Issue: Volume 26 (2018) - Issue 2 (April 2018)
Page range: 153 - 163
Received: 21 Aug 2017
Accepted: 10 Jan 2018
Journal Details
License
Format
Journal
eISSN
2284-5623
First Published
08 Aug 2013
Publication timeframe
4 times per year
Languages
English
Abstract

Both incidence and mortality of colorectal cancer (CRC) in Romania have shown a continuous increase during the last decades. Hereditary Non-Polyposic Colorectal Cancer (HNPCC), also known as Lynch syndrome, is mainly attributable to mismatch repair (MMR) genes MSH2, MSH6, and MLH1. Individuals carrying germ-line mutations of these genes present high lifetime risk of colorectal and other cancers, compared to non-carriers. Oncogenetics is developed worldwide nowadays, for identifying hereditary predisposition to cancer and offering appropriate clinical follow-up to patients and mutation carriers in Lynch families. Molecular oncogenetic diagnosis in Lynch syndrome is based on complete Sanger sequencing of entire MMR genes, which is time and resources consuming, therefore needing an appropriate and adapted optimization. Conventional sequencing requires a sufficient number of available samples to be processed simultaneously, which increases the waiting time for diagnostic results. Complete analysis for only one patient meets difficult technical problems due to the complex co-amplification of all gene regions of interest within the same conditions, therefore increasing the costs and reducing the cost-effectiveness of the test. Here we present an original and robust technical protocol for sequencing the entire MSH2, MSH6, and MLH1 coding sequence for one patient in a single PCR plate. Our optimized and verified system overcomes all technical problems and offers a quick, robust, and cost-effective possibility to personalize molecular oncogenetic diagnosis in Lynch syndrome.

Keywords

1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015 Mar;136(5):E359-86. DOI: 10.1002/ijc.2921010.1002/ijc.29210Open DOISearch in Google Scholar

2. Patel SG, Ahnen DJ. Familial colon cancer syndromes: an update of a rapidly evolving field. Curr Gastroenterol Rep. 2012 Oct;14(5):428-38. DOI: 10.1007/s11894-012-0280-610.1007/s11894-012-0280-6Open DOISearch in Google Scholar

3. Center MM, Jemal A, Smith RA, Ward E. Worldwide variations in colorectal cancer. CA Cancer J Clin. 2009 Nov-Dec;59(6):366-78. DOI: 10.3322/caac.2003810.3322/caac.20038Open DOISearch in Google Scholar

4. Valean S, Armean P, Resteman S, Nagy G, Muresan A, Mircea PA. Cancer mortality in Romania, 1955-2004. Digestive sites: esophagus, stomach, colon and rectum, pancreas, liver, gallbladder and biliary tree. J Gastrointestin Liver Dis. 2008 Mar;17(1):9-14.Search in Google Scholar

5. Risch N. The genetic epidemiology of cancer: interpreting family and twin studies and their implications for molecular genetic approaches. Cancer Epidemiol Biomarkers Prev. 2001 Jul;10(7):733-41.Search in Google Scholar

6. de la Chapelle A. Genetic predisposition to colorectal cancer. Nat Rev Cancer. 2004 Oct;4(10):769-80. DOI: 10.1038/nrc145310.1038/nrc1453Open DOISearch in Google Scholar

7. Lynch HT, Krush AJ. Cancer family “G” revisited: 1895-1970. Cancer. 1971 Jun;27(6):1505-11. DOI: 10.1002/1097-0142(197106)27:6<1505::AID-CNCR2820270635>3.0.CO;2-L10.1002/1097-0142(197106)27:6<1505::AID-CNCR2820270635>3.0.CO;2-Open DOISearch in Google Scholar

8. Lynch HT, Guirgis HA, Lynch PM, Lynch JF, Harris RE. Familial cancer syndromes: a survey. Cancer. 1977 Apr;39(4 Suppl):1867-81. DOI: 10.1002/1097-0142(197704)39:4+<1867::AID-CNCR2820390820>3.0.CO;2-QSearch in Google Scholar

9. Schlussel AT, Gagliano RA Jr, Seto-Donlon S, Eggerding F, Donlon T, Berenberg J, et al. The evolution of colorectal cancer genetics - Part 1: from discovery to practice. J Gastrointest Oncol. 2014 Oct;5(5):326-35.10.1155/2014/432324Search in Google Scholar

10. Schlussel AT, Gagliano RA Jr, Seto-Donlon S, Eggerding F, Donlon T, Berenberg J, et al. The evolution of colorectal cancer genetics - Part 2: clinical implications and applications. J Gastrointest Oncol. 2014Oct;5(5):336-44.Search in Google Scholar

11. Lynch HT, de la Chapelle A. Hereditary colorectal cancer. N Engl J Med. 2003 Mar;348(10):919-32. DOI: 10.1056/NEJMra01224210.1056/NEJMra012242Search in Google Scholar

12. Vasen HF, Wijnen JT, Menko FH, Kleibeuker JH, Taal BG, Griffioen G, et al. Cancer risk in families with hereditary nonpolyposis colorectal cancer diagnosed by mutation analysis. Gastroenterology. 1996 Apr;110(4):1020-7. DOI: 10.1053/gast.1996.v110.pm861298810.1053/gast.1996.v110.pm8612988Open DOISearch in Google Scholar

13. Giardiello FM, Allen JI, Axilbund JE, Boland CR, Burke CA, Burt RW, et al. Guidelines on genetic evaluation and management of Lynch syndrome: a consensus statement by the US Multi-society Task Force on colorectal cancer. Am J Gastroenterol. 2014 Aug;109(8):1159-79. DOI: 10.1038/ajg.2014.18610.1038/ajg.2014.186Search in Google Scholar

14. Peltomaki P, Vasen HF. Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Gastroenterology. 1997 Oct;113(4):1146-58. DOI: 10.1053/gast.1997.v113.pm932250910.1053/gast.1997.v113.pm9322509Search in Google Scholar

15. Ransohoff DF. Rules of evidence for cancer molecular- marker discovery and validation. Nat Rev Cancer. 2004 Apr;4(4):309-14. DOI: 10.1038/nrc132210.1038/nrc1322Open DOISearch in Google Scholar

16. Bedeir A, Krasinskas AM. Molecular diagnostics of colorectal cancer. Arch Pathol Lab Med. 2011 May;135(5):578-87.10.5858/2010-0613-RAIR.1Search in Google Scholar

17. Cunningham JM, Tester DJ, Thibodeau SN. Mutation detection in colorectal cancers: direct sequencing of DNA mismatch repair genes. Methods Mol Med.2001;50:87-98.Search in Google Scholar

18. Vasen HF, Watson P, Mecklin JP, Lynch HT. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology. 1999 Jun;116(6):1453-6. DOI: 10.1016/S0016-5085(99)70510-X10.1016/S0016-5085(99)70510-XOpen DOISearch in Google Scholar

19. Umar A, Boland CR, Terdiman JP, Syngal S, de la Chapelle A, Ruschoff J, et al. Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst. 2004 Feb;96(4):261-8. DOI: 10.1093/jnci/djh03410.1093/jnci/djh034Search in Google Scholar

20. Genuardi M, Anti M, Capozzi E, Leonardi F, Fornasarig M, Novella E, et al. MLH1 and MSH2 constitutional mutations in colorectal cancer families not meeting the standard criteria for hereditary nonpolyposis colorectal cancer. Int J Cancer. 1998 Mar;75(6):835-9. DOI: 10.1002/(SICI)1097-0215(19980316)75:6<835::AIDIJC4>3.0.CO;2-W10.1002/(SICI)1097-0215(19980316)75:6<835::AIDIJC4>3.0.CO;2-Open DOISearch in Google Scholar

21. Negură A, Matei M, Negură L, Carasevici E, Uhrhammer N, Bignon Y.-J, et al. Ethical implications in communicating molecular tests results to hereditary cancer predisposition families. Revista Romana de Bioetica. 2010 Dec;8(4):74-83.Search in Google Scholar

22. Negură L, Negura A, Coneac A, Neagoe IB, Azoicai D, Irimie A. Pathogenic intronic and deleterious benign variants: two extremes in cancer predisposition molecular diagnosis. Rev Romana Med Lab. 2012;20(4):317-26.Search in Google Scholar

23. Negură L, Uhrhammer N, Negură A, Artenie V, Carasevici E, Bignon Y-J. Complete BRCA mutation screening within breast and ovarian cancer predisposition families in North-Eastern Romanian population. Familial Cancer 2010 Dec;9(4):519-23. DOI: 10.1007/s10689-010-9361-610.1007/s10689-010-9361-6Open DOISearch in Google Scholar

24. Negură L, Carasevici E, Negură A, Uhrhammer N, Bignon Y-J. Identification of a recurrent BRCA1 mutation in two breast/ovarian cancer predisposition families with distinct phenotypes, by allele-specific multiplex- PCR. Rev Romana Med Lab. 2010;18(2):53-61.Search in Google Scholar

25. Negură L, Azoicăi D, Matei M, Popoiu G, Negură A. Screening of a novel BRCA2 mutation by rapid in-house PCR-RFLP. Rev Romana Med Lab. 2011;19(4):333-9.Search in Google Scholar

26. http://primer3.ut.ee/Search in Google Scholar

27. http://www.genomic.unimelb.edu.au/mdi/mutnomen/Search in Google Scholar

28. http://www.interactive-biosoftware.com/alamut.htmlSearch in Google Scholar

29. Jemal A, Ward E, Hao Y, Thun M. Trends in the leading causes of death in the United States, 1970-2002. JAMA. 2005 Sep;294(10):1255-9. DOI: 10.1001/jama.294.10.125510.1001/jama.294.10.1255Search in Google Scholar

30. Mardis ER. Genome sequencing and cancer. Curr Opin Genet Dev. 2012 Jun;22(3):245-50. DOI: 10.1016/j.gde.2012.03.00510.1016/j.gde.2012.03.005Open DOISearch in Google Scholar

Recommended articles from Trend MD

Plan your remote conference with Sciendo