Analysis of Repetitive Regions in Myotonic Dystrophy Type 1 and 2

Nancy L. Carson1

1 Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.6
DOI:  10.1002/0471142905.hg0906s61
Online Posting Date:  April, 2009
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Abstract

Myotonic dystrophy is an autosomal dominant disorder characterized by myotonia, progressive muscle wasting, and cataracts. There are two forms identified: myotonic dystrophy type 1 (DM1), caused by an expansion of a CTG repeat in the 3′ untranslated region of the myotonin‐protein kinase (DMPK) gene on chromosome 19, and myotonic dystrophy type 2 (DM2), caused by an expansion of a CCTG repeat in intron 1 of the cellular nucleic acid–binding protein (CNBP) gene on chromosome 3. There is no single method that can identify all ranges of repeats in both disorders. Protocols in this unit describe the analysis of PCR‐amplified CTG repeats from the DMPK gene and CCTG repeats from the CNBP gene, respectively, using a fluorescent‐labeled primer followed by capillary electrophoresis. An additional protocol describes the analysis of genomic DNA by Southern blot and hybridization for DM1, while yet another describes a similar technique to analyze the repeat in DM2 using field‐inversion gel electrophoresis. Both techniques identify 100% of cases of these two disorders. Curr. Protoc. Hum. Genet. 61:9.6.1‐9.6.19. © 2009 by John Wiley & Sons, Inc.

Keywords: myotonic dystrophy type 1; myotonic dystrophy type 2; DMPK; CNBP

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: PCR Amplification of DMPK CTG Repeats
  • Basic Protocol 2: PCR Amplification of CNBP Repeat
  • Basic Protocol 3: Hybridization Analysis of Myotonic Dystrophy Type 1 (DM1) CTG Repeats in Genomic DNA
  • Support Protocol 1: PCR Analysis of the Alu Insertion Polymorphism
  • Basic Protocol 4: Hybridization Analysis of Myotonic Dystrophy Type 2 (DM2) CCTG Repeats in Genomic DNA
  • Support Protocol 2: ZNF9 Probe Synthesis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: PCR Amplification of DMPK CTG Repeats

  Materials
  • 1.25 mM dNTP mix ( appendix 2D)
  • 10× PCR amplification buffer ( appendix 2D)
  • 50 mM MgCl 2
  • 20 ng/µl primer 1 (5′‐CAG AGC AGG GCG TCA TGC ACA‐3′)
  • 20 ng/µl primer 2 (FAM 5′‐GAA GGG TCC TTG TAG CCG GGA A‐3′) labeled with the fluorescent tag FAM (ABI)
  • DMSO (molecular biology grade; Sigma)
  • 1.5 µg/µl T4 gene protein 32 (Amersham)
  • 5 U/µl Taq DNA polymerase
  • 200 ng/ml DNA (minimum concentration) sample and positive control (known 70 to 80 repeats): prepared using a salting out or column procedure (e.g., a Gentra kit; Qiagen)
  • Deionized formamide (unit 4.3)
  • Liz500 internal lane marker (ABI)
  • POP‐7 polymer (ABI)
  • 0.2‐ml PCR tubes
  • Thermal cycler (e.g., ABI 9700)
  • Capillary electrophoresis system (e.g., ABI 3130)

Basic Protocol 2: PCR Amplification of CNBP Repeat

  Materials
  • 1.25 mM dNTP mix ( appendix 2D)
  • 10× PCR amplification buffer ( appendix 2D)
  • 50 mM MgCl 2
  • 20 ng/µl primer 1 (5′‐GCC TAG GGG ACA AAG TGA GA‐3′)
  • 20 ng/µl primer 2 (FAM‐5′‐GGC CTT ATA ACC ATG CAA ATG‐3′) labeled with the fluorescent tag FAM
  • 5 U/µl Taq DNA polymerase
  • 10% (w/v) Triton X‐100 (Roche)
  • 2 ng/ml DNA (minimum concentration) sample and positive controls (with two different repeat sizes): prepared using a salting out or column procedure (e.g., a Gentra kit; Qiagen)
  • Deionized formamide (unit 4.3)
  • Liz500 internal lane marker (ABI)
  • POP‐7 polymer (ABI)
  • 0.2‐ml PCR tubes
  • Thermal cycler (e.g., ABI 9700)
  • Capillary electrophoresis system (e.g., ABI 3130)

Basic Protocol 3: Hybridization Analysis of Myotonic Dystrophy Type 1 (DM1) CTG Repeats in Genomic DNA

  Materials
  • 50 U/µl EcoRI restriction enzyme and 10× restriction endonuclease buffer
  • Spermidine
  • 3 to 4 µg per lane purified genomic DNA samples and controls (no CTG expansion and heterozygous for Alu, 80 repeats and a normal 10‐kb allele, and an expanded allele with >1000 repeats): prepared using a salting out or column procedure (e.g., a Gentra kit; Qiagen)
  • 0.6% agarose gel (unit 2.7)
  • 1× TBE buffer ( appendix 2D)
  • 10× gel loading buffer ( appendix 2D)
  • Molecular‐size markers (e.g., λ‐HindIII digested DNA, Invitrogen)
  • 1 µg/ml ethidium bromide in 1× TBE buffer
  • 0.4 M NaOH
  • Neutralization solution: 0.2 M Tris·Cl, pH 7.5/2× SSC (see appendix 2D)
  • Prehybridization/hybridization buffer (see recipe)
  • pGB2.2 probe (available from the author)
  • [α‐32P]dCTP (Perkin‐Elmer)
  • Salmon sperm DNA (see recipe)
  • Wash buffer: 0.1× SSC/1.0% SDS
  • 0.5‐ml microcentrifuge tubes
  • 20 ×20–cm casting tray with 1.5‐mm‐thick comb
  • Nylon membrane, positively charged (e.g., Biotrans+, ICN Biomedicals)
  • 55°C to 65°C heating block
  • Filter paper (Whatman 3 MM)
  • Plastic wrap
  • X‐AR autoradiographic film with intensifying screen
  • Additional reagents and equipment for preparing of genomic DNA ( appendix 3B& UNIT ), performing agarose gel electrophoresis and Southern blots (unit 2.7), and labeling DNA by random‐hexamer priming and column‐purifying ( appendix 3E)
CAUTION: Radiolabeled probes are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 1: PCR Analysis of the Alu Insertion Polymorphism

  Materials
  • 1.25 mM dNTP mix ( appendix 2D)
  • 10× PCR amplification buffer ( appendix 2D)
  • 50 mM MgCl 2
  • 20 ng/µl primer 1 (5′‐AAA TAG GCT GCA CCG CGG‐3′)
  • 20 ng/µl primer 2 (5′‐CTG TAT ACT CAG CTA CTA GGG T‐3′)
  • 20 ng/µl primer 3 (5′‐CTC AGG GGT TAT CTA AAG TGG C‐3′)
  • 5 U/µl Taq DNA polymerase
  • 200 ng/ml DNA (minimum concentration) sample and positive controls (known Alu‐plus and Alu‐minus alleles): prepared using a salting out or column procedure (e.g., a Gentra kit; Qiagen)
  • 1% (w/v) agarose gel
  • 10 mg/ml ethidium bromide
  • 1× TBE
  • 123‐bp molecular size marker (e.g., Invitrogen)
  • 0.2‐ml PCR tubes
  • Thermal cycler (e.g., ABI 9700)
  • Additional reagents and equipment for performing agarose gel electrophoresis (unit 2.7)

Basic Protocol 4: Hybridization Analysis of Myotonic Dystrophy Type 2 (DM2) CCTG Repeats in Genomic DNA

  Materials
  • 50 U/µl EcoRI restriction enzyme and 10× restriction endonuclease buffer
  • 3 to 4 µg purified genomic DNA per lane (≥50 kb fragments), sample and controls (no CCTG expansion and an expanded repeat): prepared using a salting out or column procedure (e.g., a Gentra kit; Invitrogen)
  • Agarose
  • 0.5× TBE buffer (see appendix 2D)
  • 10× gel loading buffer
  • Molecular‐size markers (e.g., λ‐HindIII digested DNA; Invitrogen)
  • 10 µg/ml ethidium bromide in 0.5× TBE buffer
  • 0.25 M HCl
  • 0.4 M NaOH/1.5 M NaCl
  • Neutralization solution: 0.2 M Tris·Cl (see appendix 2D), pH 7.5/2× SSC (see appendix 2D)
  • Prehybridization/hybridization buffer (see recipe)
  • ZNF9 probe ( protocol 6)
  • [α‐32P]dCTP (Perkin‐Elmer)
  • Human placental DNA (see recipe)
  • Salmon sperm DNA (see recipe)
  • Wash buffer: 0.1× SSC/0.1% SDS
  • 0.5‐ml microcentrifuge tubes
  • Field‐inversion gel‐electrophoresis (FIGE) unit (e.g., Hoefer) or pulse‐field gel‐electrophoresis unit, and gel casting tray
  • Nylon membrane, positively charged (e.g., Biotrans+, ICN Biomedicals)
  • Filter paper (Whatman 3MM)
  • 50°C and 65°C heating block
  • X‐AR autoradiographic film with intensifying screen
  • Additional reagents and equipment for preparing genomic DNA ( appendix 3B& UNIT ), performing agarose gel electrophoresis and Southern blots (unit 2.7), and labeling DNA by random‐hexamer priming and column‐purifying ( appendix 3E)

Support Protocol 2: ZNF9 Probe Synthesis

  Materials
  • 1.25 mM dNTP mix ( appendix 2D)
  • 10× PCR amplification buffer ( appendix 2D)
  • 50 mM MgCl 2
  • 20 ng/µl primer 1 (5′‐GAG AAC CTT GCC ATT TTT CG‐3′)
  • 20 ng/µl primer 2 (5′‐CAC CTA CAG CAC TGG CAA CA‐3′)
  • 5 U/µl Taq DNA polymerase
  • 10% (w/v) Triton X‐100 (Roche)
  • Cloned ZNF9 DNA (available by request from the author)
  • 1% (w/v) agarose gel with ethidium bromide
  • 0.2‐ml PCR tubes
  • Thermal cycler
  • Transilluminator 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for performing agarose gel electrophoresis (unit 2.7)
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Figures

Videos

Literature Cited

Literature Cited
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   Day, J. W., Ricker, K., Jacobsen, J.F., Rasmussen, L.J., Dick, B.A., Kress, B.A., Schneider, C., Koch, M.C., Beilman, G.J., Harrison, A.R., Dalton, J.C., and Ranum, L.P.W. 2003. Myotonic Dystrophy Type 2: Molecular, diagnostic and clinical spectrum. Neurol. 60:657‐664.
   Michael Finney, M. 2000. Pulsed‐field gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5B.1‐2.5B.9.
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   Harper, P.S. 1989. Myotonic Dystrophy, 2nd ed. W.B. Saunders, Philadelphia.
   Hunter, A., Tsilfidis, C., Mettler, G., Jacob, P., Mahadevan, M., Surh, L., and Korneluk, R. 1992. The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy. J. Med. Genet. 29:774‐779.
   Jakubiczka, S., Vielhaber, S., Kress, W., Kupferling, P., Reuner, U., Kunath, B., and Wieacker, P. 2004. Improvement of the diagnostic procedure in proximal myotonic myopathy/myotonic dystrophy type 2. Neurogenet. 5:55‐59.
   Krahe, R., Eckhart, M., Ogunniyi, A.O., Osuntokun, B.O., Siciliano, M.J., and Ashizawa, T. 1995. De novo myotonic dystrophy mutation in a Nigerian kindred. Am. J. Hum. Genet. 56:1067‐1074.
   Liquori, C.L., Ricker, K., Moseley, M.L., Jacobsen, J.F., Kress, W., Naylor, S.L., Day, J.W., and Ranum, L.P.W. 2001. Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293:864‐867.
   Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., Barcelo, J., O'Hoy, K., Leblond, S., Earle‐MacDonald, J., De Jong, P.J., Wieringa, B., and Korneluk, R. 1992. Myotonic dystrophy mutation: An unstable CTG repeat in the 3′ untranslated region of the gene. Science 255:1253‐1255.
   Ranum, L.P.W., Rasmussen, P.F., Benzow, K.A., Koob, M.D., and Day, J.W. 1998. Genetic mapping of a second myotonic dystrophy locus. Nat. Genet. 19:196‐198.
   Second International Myotonic Dystrophy Consortium (IDMC). 2000. New nomenclature and DNA testing guidelines for myotonic dystrophy type 1 (DM1). Neurol. 54:1218‐1221.
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