Molecular Diagnosis of Myotonic Dystrophy

Sujata Chakraborty1, Matteo Vatta1, Linda L. Bachinski2, Ralf Krahe2, Stephen Dlouhy1, Shaochun Bai1

1 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, 2 Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.29
DOI:  10.1002/cphg.22
Online Posting Date:  October, 2016
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Myotonic dystrophy types 1 (DM1) and 2 (DM2) are autosomal dominant, microsatellite repeat expansion disorders that affect muscle function. Myotonic dystrophy type 1 is caused by CTG repeat expansion in the 3′ UTR region of the DMPK gene. Patients with DM2 have expansion of CCTG repeats in intron 1 of the CNBP gene. In this unit, we review and discuss the clinical phenotypes, genetic mutations causing the diseases, and the molecular diagnostic approaches and tools that are used to determine repeat sizes in DM1/2. In summary, the goal of this chapter is to provide the reader with a basic understanding of the clinical, genetic and diagnostic aspects of these disorders. © 2016 by John Wiley & Sons, Inc.

Keywords: CCTG repeat; CNBP; CTG repeat; DMPK; myotonic dystrophy type 1; Myotonic dystrophy type 2; repeat‐primed PCR (RP‐PCR); Southern Blot; triplet‐repeat primed PCR (TP‐PCR)

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Table of Contents

  • Introduction
  • Molecular Diagnosis of DM1
  • Basic Protocol 1: Screening for Expanded Allele with RP‐PCR
  • Basic Protocol 2: Southern Blot Method and Interpretation in DM1
  • Molecular Diagnosis of DM2
  • Basic Protocol 3: Screen for Expanded Allele with RP‐PCR in DM2
  • Basic Protocol 4: Detection of DM2 (CCTG)n Expansion Mutation by FIGE (Field Inversion Gel Electrophoresis) and Southern Blot Analysis
  • Support Protocol 1: Recommended Procedures for Vacuum Transferring with the Amersham Vacuum Transfer System
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Screening for Expanded Allele with RP‐PCR

  • Primers: All primers are dissolved with PCR‐grade dH 2O to make 100 μM stock solutions and stored frozen at −20°C (Minimize freeze‐thaw cycles; see Table 9.29.1)
  • DNA template
  • FailSafe PCR 2× PreMix K (Epicentre)
  • FailSafe PCR Enzyme Mix (Epicentre)
  • MapMarker ROX 1000 (BioVentures)
  • Hi‐Di formamide (Thermo Fisher Scientific)
  • Ice
  • Vortex mixer
  • Centrifuge
  • ABI 3130 XL Genetic Analyzer (Thermo Fisher Scientific) (Polymer used: FragmentAnalysis36_POP7; and Dye Set D used: to calibrate the system)
  • GeneMarker Software (SoftGenetics)
  • 96‐well septa, Reservoir septa (Thermo Fisher Scientific)

Basic Protocol 2: Southern Blot Method and Interpretation in DM1

  • 10× NE Buffer 3 (New England Biolabs)
  • Bgl1 Enzyme (New England Biolabs)
  • Genomic DNA
  • 3× BlueJuice Gel Loading Buffer (New England Biolabs)
  • Low EEO Agarose, analytical level
  • 10× TBE buffer, pH 8.0
  • 1‐kb DNA ladder (New England Biolabs)
  • 10× Stop Solution: 100 mM EDTA; 1% SDS
  • 3 M Sodium Acetate
  • 95% Ethanol
  • 40× TAE: Sodium EDTA, Tris‐Acetate, pH 8.0
  • I.D. NA Agarose, analytical level (Lonza)
  • Ethidium Bromide Solution, 10 mg/ml
  • Denaturing Solution: 0.5 M NaOH:0.5 M NaCl
  • ddH 2O
  • Neutralization Solution – 2.5 M NaCl:0.5 M Tris·Cl, pH 7.4
  • DM Probe: A targeted fragment with a size of 1.09 kb was amplified from human genomic DNA through PCR; next, the fragment was TA tailed and cloned into pCR vector (3.9 kb) (EcoRI can be used to release the fragment from the plasmid)
  • Megaprime DNA Labeling System (GE Healthcare Biosciences)
  • Ice
  • 32P‐dATP 10 uCi/µl
  • 32P‐dCTP 10 uCi/µl
  • Rapid hybridization buffer (Rapid‐Hyb Buffer)
  • Salmon Sperm, Sheared DNA (Amresco, cat. no. E213)
  • Denhardt's Solution
  • 20× SSC Solution
  • 20% SDS Solution
  • 37°C water bath
  • Centrifuge
  • Electrophoresis equipment
  • UV Transilluminator
  • Micropipets
  • 17‐well comb
  • Rotator
  • Large plastic tray
  • Sponges
  • Blotting paper
  • Paper towels
  • Whatman paper
  • Gel dryer (Bio‐Rad; model 583)
  • Thermal cycler (Thermo Fisher Scientific)
  • 37°C incubator
  • Microspin G‐50 Columns
  • 1.5‐ml microcentrifuge tubes
  • Radioactive waste container
  • Positively Charged Nylon Membrane: Biodyne B (0.45 µM; VWR, cat. no. 28150‐276)
  • 50‐ml conical tubes
  • Serological pipets
  • Hybridization oven
  • Saran wrap
  • X‐ray film

Basic Protocol 3: Screen for Expanded Allele with RP‐PCR in DM2

  • gDNA (Promega)
  • EcoRI (New England Biolabs)
  • 0.5% TBE buffer (Thermo Fisher Scientific)
  • 1% FIGE agarose gel (New England Biolabs)
  • Pulse‐field gel electrophoresis certified agarose (New England Biolabs)
  • Gel‐loading buffer (Thermo Fisher Scientific)
  • Low‐range FIGE DNA MW marker (New England Biolabs)
  • λ/HindIII marker (New England Biolabs)
  • Ethidium bromide
  • 0.25 M HCl
  • 0.5 M NaOH
  • 1.5 M NaCl
  • Hybond N+ blotting membrane (Amersham)
  • RapidHyb hybridization buffer (Amersham, cat. no. NIF 939)
  • DM2‐ZNF9 probe (Liquori, et al., )
  • [α‐32P]‐dCTP and/or [α‐32P]‐dATP (Stratagene or Amersham)
  • 2× Saline Sodium Citrate (SSC)
  • Sodium Dodecyl Sulfate (SDS)
  • 0.5 M phosphate‐buffered saline (PBS; Thermo Fisher Scientific)
  • FIGE Mapper (Bio‐Rad)
  • Vacuum transfer system (Amersham)
  • Razor blades
  • UV scanner with fluorescent ruler
  • UV cross‐linker (Agilent Stratagene StrataLinker)
  • X‐ray film

Basic Protocol 4: Detection of DM2 (CCTG)n Expansion Mutation by FIGE (Field Inversion Gel Electrophoresis) and Southern Blot Analysis

  Additional Materials (also see protocol 4)
  • 0.5 M phosphate buffer, pH 7
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Literature Cited

Literature Cited
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