Genetic Testing for Hereditary Nonpolyposis Colorectal Cancer (HNPCC)

Babi Ramesh Reddy Nallamilli1, Madhuri Hegde1

1 Department of Human Genetics, Emory University School of Medicine, Atlanta
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 10.12
DOI:  10.1002/cphg.40
Online Posting Date:  July, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Hereditary nonpolyposis colorectal cancer (HNPCC), also called Lynch syndrome, is an autosomal dominant cancer syndrome that confers an elevated risk of early‐onset colorectal cancer (CRC) and increased lifetime risk for other cancers of the endometrium, stomach, small intestine, hepatobiliary system, kidney, ureter, and ovary. Lynch syndrome accounts for up to 3% of all CRC, making it the most common hereditary colorectal cancer syndrome. Germline mutations in methyl‐directed mismatch repair (MMR) genes give rise to microsatellite instability (MSI) in tumor DNA. Lynch syndrome is most frequently caused by pathogrenic variants in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. Germline mutations in MLH1 and MSH2 account for approximately 90% of detected mutations in families with Lynch syndrome. Pathogenic vatiants in MSH6 have been reported in approximately 7‐10% of families with Lynch syndrome. Pathogenic variants in PMS2 account for fewer than 5% of mutations in families with Lynch syndrome. This unit presents a comprehensive molecular genetic testing strategy for Lynch syndrome including MSI analysis, next generation sequencing (NGS)‐based targeted sequence analysis, PCR‐based Sanger sequencing and microarray‐based comparative genomic hybridization (array‐CGH). © 2017 by John Wiley & Sons, Inc.

Keywords: lynch syndrome; MLH1; MSH2; MSH6; PMS2; microsatellite instability; NGS‐based sequencing; Sanger sequencing; array‐CGH

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Microsatellite Instability Analysis on DNA From Colorectal Tumor Tissue
  • Support Protocol 1: Preparation of DNA for Analysis of Microsatellite Instability
  • Basic Protocol 2: Targetted Next‐Generation Sequencing of MLH1, MSH2, and MSH6 Genes Using IDT Xgen Lockdown Probes
  • Alternate Protocol 1: Sanger Sequencing Using M13‐Tailed Primers
  • Basic Protocol 3: Microarray‐Based Comparative Genomic Hybridization to Detect Deletions and Duplications in MLH1, MSH2, and MSH6 Genes
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Microsatellite Instability Analysis on DNA From Colorectal Tumor Tissue

  • Master mix containing the following (see Table 10.12.2):
    • 10× FastStart PCR buffer without MgCl 2 (Roche Applied Science)
    • 25 mM MgCl 2 (Roche Applied Science)
    • 2.5 mM dNTP mix (2.5 mM each dATP, dCTP, dGTP, dTTP)
    • 50 μM fragment‐specific forward and reverse primers (Table 10.12.1), mixed 1 part fluorescently labeled primers to 14 parts unlabeled primers
    • FastStart Taq DNA polymerase (Roche Applied Science)
    • 50 ng/μl patient normal template DNA (e.g., blood or normal tissue; see protocol 2Support Protocol)
    • 50 ng/μl patient tumor template DNA (see protocol 2Support Protocol)
  • Ice
  • Nuclease‐free water
  • GeneScan‐500 LIZ Size Standard (Applied Biosystems, cat. no. 4322682)
  • Formamide
  • 96‐well optical PCR plate (Applied Biosystems)
  • Thermal cycler with heated lid
  • ABI Prism 3100 sequence analysis system
Table 0.2.2   MaterialsPCR Reaction Mix for MSI Analysis of MLH1, MSH2, and MSH6 Genes

Reagent Stock concentration Volume/reaction (μl) Final concentration Total volume for 3 reactions (μl)
H 2O 18.0 24.0
10× FastStart buffer without Mg2+ 10× 5.0 15.0
Mg 2Cl 25 mM 5.0 5 mM 15.0
dNTP mix 2.5 mM 4.0 200 μM 12.0
FastStart Taq a 2.5 U/μl 0.5 2 U/reaction 1.5
50 μM primer mix 50 μM 1.5 200 nM 4.5
Template DNA 50 ng/μl 1.0 50 ng/reaction 3.0
Total 25.0 b 75.0 b

 aStore at –20°C in a cooler container until ready for use, then return to freezer as soon as possible; mix well before use.
 bIncludes excess for pipetting errors.

Support Protocol 1: Preparation of DNA for Analysis of Microsatellite Instability

  • Normal and tumor tissue from same patient
  • Xylene
  • 70% and 100% (v/v) ethanol
  • Cell lysis solution (Qiagen)
  • Proteinase K solution (Qiagen)
  • Protein precipitation solution (Qiagen)
  • Isopropanol
  • Gentra Glycogen Solution (Qiagen), optional
  • DNA hydration solution (Qiagen)
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge
  • Microcentrifuge tube pestle
  • Vortex mixer
  • 55°C and 65°C heating blocks
  • Additional reagents and equipment for quantifying DNA using the NanoDrop method (Nallamilli & Hegde, , unit 10.8)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Bapat, B., Berk, T., Mitri, A., Cohen, Z., Gallinger, S., & Stern, H. (1994). Characterization of two novel adenomatous polyposis coli (APC) gene mutations in patients with familial adenomatous polyposis (FAP). Human Mutation, 4, 253–256. doi: 10.1002/humu.1380040404.
  Baxevanis, A. D. (2012). Searching online mendelian inheritance in man (OMIM) for information on genetic loci involved in human disease. Current Protocols in Human Genetics. 73, 9.13.1–9.13.10.
  Calistri, D., Presciuttini, S., Buonsanti, G., Radice, P., Gazzoli, I., Pensotti, V., … Ranzani, G. N. (2000). Microsatellite instability in colorectal‐cancer patients with suspected genetic predisposition. International Journal of Cancer, 89, 87–91. doi: 10.1002/(SICI)1097‐0215(20000120)89:1%3c87::AID‐IJC14%3e3.0.CO;2‐9.
  Chin, E. L., da Silva, C., & Hegde, M. (2013). Assessment of clinical analytical sensitivity and specificity of next‐generation sequencing for detection of simple and complex mutations. BMC Genetics, 14, 6. doi: 10.1186/1471‐2156‐14‐6.
  Cottrell, S., Bicknell, D., Kaklamanis, L., & Bodmer, W. F. (1992). Molecular analysis of APC mutations in familial adenomatous polyposis and sporadic colon carcinomas. Lancet, 340, 626–630. doi: 10.1016/0140‐6736(92)92169‐G.
  de la Chapelle, A., & Peltomaki, P. (1995). Genetics of hereditary colon cancer. Annual Review of Genetics, 29, 329–348. doi: 10.1146/
  De Rosa, M., Scarano, M. I., Panariello, L., Morelli, G., Riegler, G., Rossi, G. B., … Izzo, P. (2003). The mutation spectrum of the APC gene in FAP patients from southern Italy: Detection of known and four novel mutations. Human Mutation, 21, 655–656. doi: 10.1002/humu.9151.
  De Vos, M., Hayward, B. E., Picton, S., Sheridan, E., & Bonthron, D. T. (2004). Novel PMS2 pseudogenes can conceal recessive mutations causing a distinctive childhood cancer syndrome. American Journal of Human Genetics, 74, 954–964. doi: 10.1086/420796.
  Dobbie, Z., Spycher, M., Hurliman, R., Ammann, R., Ammann, T., Roth, J., … Scott, R. J. (1994). Mutational analysis of the first 14 exons of the adenomatous polyposis coli (APC) gene. European Journal of Cancer, 30A, 1709–1713. doi: 10.1016/0959‐8049(94)00294‐F.
  Elsaleh, H., Joseph, D., Grieu, F., Zeps, N., Spry, N., & Iacopetta, B. (2000). Association of tumour site and sex with survival benefit from adjuvant chemotherapy in colorectal cancer. Lancet, 355, 1745–1750. doi: 10.1016/S0140‐6736(00)02261‐3.
  Fitzgibbons, R. J., Jr., Lynch, H. T., Stanislav, G. V., Watson, P. A., Lanspa, S. J., Marcus, J. N., … Lynch, J. F. (1987). Recognition and treatment of patients with hereditary nonpolyposis colon cancer (Lynch syndromes I and II). Annals of Surgery, 206, 289–295. doi: 10.1097/00000658‐198709000‐00007.
  Fitzsimmons, M. L. (1992). Hereditary colorectal cancers. Seminars in Oncology Nursing, 8, 252–257. doi: 10.1016/0749‐2081(92)90037‐4.
  Fornasarig, M., Campagnutta, E., Talamini, R., Franceschi, S., Boz, G., Scarabelli, C., … Valentini, M. (1998). Risk factors for endometrial cancer according to familial susceptibility. International Journal of Cancer, 77, 29–32. doi: 10.1002/(SICI)1097‐0215(19980703)77:1%3c29::AID‐IJC6%3e3.0.CO;2‐1.
  Fox, E. A. 2001. Preparation of DNA from Fixed, Paraffin‐Embedded Tissue. Current Protocols in Human Genetics. 8, A.3I.1–A.3I.5.
  Gargis, A. S., Kalman, L., Bick, D. P., da Silva, C., Dimmock, D. P., Funke, B. H., … Lubin, I. M. (2015). Good laboratory practice for clinical next‐generation sequencing informatics pipelines. Nature Biotechnology, 33, 689–693. doi: 10.1038/nbt.3237.
  Gilbert, J. R., & Vance, J. M. 2001. Isolation of Genomic DNA from Mammalian Cells. Current Protocols in Human Genetics. 19, A.3B.1–A.3B.6.
  Harrison, S.M., Riggs, E.R., Maglott, D.R., Lee, J.M., Azzariti, D.R., Niehaus, A., … Rehm, H. L. (2016). Using ClinVar as a resource to support variant interpretation. Current Protocols in Human Genetics, 89, 8.16.1‐8.16.23. doi: 10.1002/0471142905.hg0816s89
  Hegde, M., Ferber, M., Mao, R., Samowitz, W., & Ganguly, A. (2014). ACMG technical standards and guidelines for genetic testing for inherited colorectal cancer (Lynch syndrome, familial adenomatous polyposis, and MYH‐associated polyposis). Genetics in Medicine, 16, 101–116. doi: 10.1038/gim.2013.166.
  Ichikawa, Y., Lemon, S. J., Wang, S., Franklin, B., Watson, P., Knezetic, J. A., … Lynch, H. T. (1999). Microsatellite instability and expression of MLH1 and MSH2 in normal and malignant endometrial and ovarian epithelium in hereditary nonpolyposis colorectal cancer family members. Cancer Genetics and Cytogenetics, 112, 2–8. doi: 10.1016/S0165‐4608(98)00252‐0.
  Jass, J. R. (1995). Colorectal adenoma progression and genetic change: Is there a link? Annals of Medicine, 27, 301–306. doi: 10.3109/07853899509002581.
  Jass, J. R. (1998). Diagnosis of hereditary non‐polyposis colorectal cancer. Histopathology, 32, 491–497. doi: 10.1046/j.1365‐2559.1998.00442.x.
  Jass, J. R. (2000a). Familial colorectal cancer: Pathology and molecular characteristics. The Lancet Oncology, 1, 220–226. doi: 10.1016/S1470‐2045(00)00152‐2.
  Jass, J. R. (2000b). Pathology of hereditary nonpolyposis colorectal cancer. Annals of the New York Academy of Sciences, 910, 62–74. doi: 10.1111/j.1749‐6632.2000.tb06701.x.
  Jass, J. R., Stewart, S. M., Stewart, J., & Lane, M. R. (1994). Hereditary non‐polyposis colorectal cancer—morphologies, genes and mutations. Mutation Research, 310, 125–133. doi: 10.1016/0027‐5107(94)90016‐7.
  Jass, J. R., Walsh, M. D., Barker, M., Simms, L. A., Young, J., & Leggett, B. A. (2002). Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability. European Journal of Cancer, 38, 858–866. doi: 10.1016/S0959‐8049(02)00041‐2.
  Jones, P. A., & Laird, P. W. (1999). Cancer epigenetics comes of age. Nature Genetics, 21, 163–167. doi: 10.1038/5947.
  Kuismanen, S. A., Moisio, A. L., Schweizer, P., Truninger, K., Salovaara, R., Arola, J., … Peltomaki, P. (2002). Endometrial and colorectal tumors from patients with hereditary nonpolyposis colon cancer display different patterns of microsatellite instability. The American Journal of Pathology, 160, 1953–1958. doi: 10.1016/S0002‐9440(10)61144‐3.
  Liu, T., Wahlberg, S., Burek, E., Lindblom, P., Rubio, C., & Lindblom, A. (2000). Microsatellite instability as a predictor of a mutation in a DNA mismatch repair gene in familial colorectal cancer. Genes Chromosomes Cancer, 27, 17–25. doi: 10.1002/(SICI)1098‐2264(200001)27:1%3c17::AID‐GCC3%3e3.0.CO;2‐Y.
  Nallamilli, B. R. R., & Hegde, M. (2017). Detecting APC gene mutations in familial adenomatous polyposis (FAP). 92, 10.8.1‐10.8.29. doi: 10.1002/cphg.29
  Nallamilli, B. R. R., Ankala, A., & Hegde, M. 2014. Molecular Diagnosis of Duchenne Muscular Dystrophy. Current Protocols in Human Genetics. 83, 9.25.1–9.25.29.
  Nowak, N. J., Snijders, A. M., Conroy, J. M., & Albertson, D. G. (2005). The BAC resource: tools for array CGH and FISH. Current Protocols in Human Genetics, 46, 4.13.1–4.13.36.
  Papadopoulos, N. (1999). Microsatellite instability (MSI) in non‐colonic, non‐HNPCC tumors: ‘Instable’ evidence? Annals of Oncology, 10, 751–752. doi: 10.1023/A:1008389100079.
  Rodriguez‐Bigas, M. A., Boland, C. R., Hamilton, S. R., Henson, D. E., Jass, J. R., Khan, P. M., … Srivastava, S. (1997). A National Cancer Institute Workshop on hereditary nonpolyposis colorectal cancer syndrome: Meeting highlights and Bethesda guidelines. Journal of the National Cancer Institute, 89, 1758–1762. doi: 10.1093/jnci/89.23.1758.
  Rüschoff, J., Wallinger, S., Dietmaier, W., Bocker, T., Brockhoff, G., Hofstädter, F., & Fishel, R. (1998). Aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selection. Proceedings of the National Academy of Sciences of the United States of America, 95, 11301–11306. doi: 10.1073/pnas.95.19.11301.
  Sankila, R., Aaltonen, L. A., Järvinen, H. J., & Mecklin, J. P. (1996). Better survival rates in patients with MLH1‐associated hereditary colorectal cancer. Gastroenterology, 110, 943–954. doi: 10.1053/gast.1996.v110.pm8608876.
  Syngal, S., Fox, E. A., Eng, C., Kolodner, R. D., & Garber, J. E. (2000). Sensitivity and specificity of clinical criteria for hereditary non‐polyposis colorectal cancer associated mutations in MSH2 and MLH1. Journal of Medical Genetics, 37, 641–645. doi: 10.1136/jmg.37.9.641.
  Thibodeau, S. N., French, A. J., Cunningham, J. M., Tester, D., Burgart, L. J., Roche, P. C., McDonnell, S. K., Schaid, D. J., Vockley, C. W., Michels, V. V., Farr, G. H., Jr., & O'Connell, M. J. (1998). Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hMLH1. Cancer Research, 58, 1713–1718.
  van der Luijt, R. B., Khan, P. M., Vasen, H. F., Tops, C. M., van Leeuwen‐Cornelisse, I. S., Wijnen, J. T., … Fodde, R. (1997). Molecular analysis of the APC gene in 105 Dutch kindreds with familial adenomatous polyposis: 67 germline mutations identified by DGGE, PTT, and southern analysis. Human Mutation, 9, 7–16. doi: 10.1002/(SICI)1098‐1004(1997)9:1%3c7::AID‐HUMU2%3e3.0.CO;2‐8.
  Vnencak‐Jones, C. L. 2009. Bone Marrow Engraftment Studies. Current Protocols in Human Genetics. 62, 9.17.1–9.17.34.
  Wei, X., Dai, Y., Yu, P., Qu, N., Lan, Z., Hong, X., … Yi, X. (2014). Targeted next‐generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD: A multi‐population diagnostic study. European Journal of Human Genetics, 22, 110–118. doi: 10.1038/ejhg.2013.82.
  Wijnen, J., Khan, P. M., Vasen, H., van der Klift, H., Mulder, A., van Leeuwen‐Cornelisse, I., … Fodde, R. (1997). Hereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch‐repair‐gene mutations. American Journal of Human Genetics, 61, 329–335. doi: 10.1086/514847.
  Wu, Y., Berends, M. J., Mensink, R. G., Kempinga, C., Sijmons, R. H., van Der Zee, A. G., … Hofstra, R. M. (1999). Association of hereditary nonpolyposis colorectal cancer‐related tumors displaying low microsatellite instability with MSH6 germline mutations. American Journal of Human Genetics, 65, 1291–1298. doi: 10.1086/302612.
  Wu, G., Wu, W., Hegde, M., Fawkner, M., Chong, B., Love, D., … Richards, C. S. (2001). Detection of sequence variations in the adenomatous polyposis coli (APC) gene using denaturing high‐performance liquid chromatography. Genetic Testing, 5, 281–290. doi: 10.1089/109065701753617408.
PDF or HTML at Wiley Online Library