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
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Abstract

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

     
 
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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
     
 
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Materials

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

  Materials
  • 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

  Materials
  • 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)
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Figures

Videos

Literature Cited

Literature Cited
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