Diagnosis of Spinocerebellar Ataxias Caused by Trinucleotide Repeat Expansions

Joanne E. Martindale1

1 Wellington Regional Genetics Laboratory, Wellington Hospital, Wellington
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.30
DOI:  10.1002/cphg.30
Online Posting Date:  January, 2017
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Abstract

Spinocerebellar ataxias (SCAs) are a group of disorders that are both clinically and genetically heterogeneous. They usually demonstrate onset in adulthood, but some forms may have juvenile or infantile onset. There are many different types of SCA, demonstrating different modes of inheritance and types of mutation. The most common forms are due to dominantly inherited expansions in trinucleotide repeat sequences located within the coding region of the relevant genes, and these are readily identifiable by molecular genetic testing. In general, it is possible to test for these disorders using PCR‐based assays, amplifying across the trinucleotide repeat regions and sizing the PCR products to determine the number of repeats. Larger expansions are generally associated with a more severe presentation of the disorder, and alternative methods may be necessary to detect these alleles. This protocol describes methods for detecting normal and expanded triplet repeat alleles in the most common SCA genes. © 2017 by John Wiley & Sons, Inc.

Keywords: spinocerebellar ataxia; SCA; trinucleotide repeats

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of SCA Expansion Mutations by Non‐Fluorescent PCR and Silver Staining
  • Support Protocol 1: Polyacrylamide Gel Electrophoresis and Silver Staining
  • Alternate Protocol 1: Detection and Sizing of Expansion Mutations Using Fluorescent PCR
  • Alternate Protocol 2: Detection of Large Expansion Mutations Without Accurate Sizing
  • Basic Protocol 2: Sequencing of SCA Alleles to Determine Repeat Number and Presence of Interruptions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of SCA Expansion Mutations by Non‐Fluorescent PCR and Silver Staining

  Materials
  • Genomic DNA samples to be tested ‐ diluted to 100 ng/μl in injection grade distilled water
  • Distilled water, injection grade
  • RedHot Taq DNA polymerase (Thermo Fisher Scientific)
  • 10× PCR Buffer IV without added MgCl 2 (Thermo Fisher Scientific)
  • dNTP set, 100 mM (Fisher, Invitrogen product), diluted to 2.5 mM working stock
  • MgCl 2 solution (Thermo Fisher Scientific)
  • SCA‐specific primers, unlabeled (see Table 9.30.2)
  • Dimethyl sulfoxide (DMSO; Sigma‐Aldrich)
  • Betaine (MP Biomedicals)
  • 0.5‐ml PCR tubes
  • 96‐well double‐barcoded PCR plates (4titude; optional)
  • Indelible marker pen
  • Adhesive plate sealers (optional)
  • Vortex mixer for tubes or 96‐well plates
  • Thermal cycler
  • Multichannel pipetter
  • Filtered and non‐filtered pipet tips

Support Protocol 1: Polyacrylamide Gel Electrophoresis and Silver Staining

  Materials
  • 40% solution of 19:1 acrylamide/bisacrylamide (Acr/Bis)
  • 10× TBE (see recipe)
  • N,N,N′,N′‐Tetramethylethyldiamine (TEMED)
  • 20% (w/v) ammonium persulfate (APS, see recipe)
  • Orange G/xylene cyanol loading buffer (see recipe)
  • PCR products to be analyzed (see protocol 1)
  • Appropriate size standard (e.g., Φ× 174/HinfI‐digested DNA)
  • Fix solution (see recipe)
  • 0.1% (w/v) silver nitrate solution (see recipe)
  • Developer solution (see recipe)
  • Vertical gel electrophoresis apparatus (e.g., OWL P10DS Dual Gel System, Thermo Fisher Scientific) with 1.5‐mm spacers and comb
  • 50‐ml syringe
  • Parafilm M (Bemis)
  • Hamilton gel‐loading syringe (Sigma‐Aldrich) or gel‐loading pipet tips
  • Power supply
  • Plastic containing for staining solutions
  • Shaker
  • Heat‐sealable plastic bags
  • Heat sealer
  • UV transilluminator with white light source
  • Photographic equipment

Alternate Protocol 1: Detection and Sizing of Expansion Mutations Using Fluorescent PCR

  Materials
  • PCR products to be sequenced
  • ExoStar kit (GE Healthcare)
  • BigDye Terminator v1.1 Cycle Sequencing kit (ABI)
  • Better Buffer (Microzone)
  • M13 sequencing primers, HPLC purified (Eurogentec):
  • M13F1 (forward): CACGACGTTGTAAAACGAC
  • M13R1 (reverse): CAGGAAACAGCTATGACC
  • Agencourt CleanSEQ (Beckman Coulter)
  • 96% ethanol (VWR/AnalR; for use with CleanSEQ)
  • 96‐well double‐barcoded PCR plates (4titude)
  • Multichannel pipetter
  • Heating blocks at 37° and 80°C
  • 3730 DNA Analyzer, 48 capillary (Applied Biosystems)
  • Additional reagents and equipment for agarose or polyacrylamide gel electrophoresis
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Figures

Videos

Literature Cited

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Key References
  Garden, G. 1998. Spinocerebellar ataxia type 7. 1998 Aug 27 [Updated 2012 Dec 20]. In: GeneReviews [Internet] (Pagon, R.A., Adam, M.P., Ardinger, H.H., Wallace, S.E., Amemiya, A., Bean, L.J.H., Bird, T.D., Fong, C.T., Mefford, H.C., Smith, R.J.H., Stephens, K., eds.). Seattle (WA): University of Washington, Seattle; 1993‐2016. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1256/
  The following articles in GeneReviews provide descriptions of each of the SCA types included in this unit.
  Gomez, C.M. 1998. Spinocerebellar ataxia type 6. 1998 Oct 23 [Updated 2013 Jul 18]. In: GeneReviews [Internet] (Pagon, R.A., Adam, M.P., Ardinger, H.H., Wallace, S.E., Amemiya, A., Bean, L.J.H., Bird, T.D., Fong, C.T., Mefford, H.C., Smith, R.J.H., Stephens, K., eds.). Seattle (WA): University of Washington, Seattle; 1993‐2016. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1140/
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  Paulson, H. 1998. Spinocerebellar ataxia type 3. 1998 Oct 10 [Updated 2015 Sep 24]. In: GeneReviews [Internet] (Pagon, R.A., Adam, M.P., Ardinger, H.H., Wallace, S.E., Amemiya, A., Bean, L.J.H., Bird, T.D., Fong, C.T., Mefford, H.C., Smith, R.J.H., Stephens, K., eds.). Seattle (WA): University of Washington, Seattle; 1993‐2016. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1196/
  Pulst, S.M. 1998. Spinocerebellar ataxia type 2. 1998 Oct 23 [Updated 2015 Nov 12]. In: GeneReviews [Internet] (Pagon, R.A., Adam, M.P., Ardinger, H.H., Wallace, S.E., Amemiya, A., Bean, L.J.H., Bird, T.D., Fong, C.T., Mefford, H.C., Smith, R.J.H., Stephens, K., eds.). Seattle (WA): University of Washington, Seattle; 1993‐2016. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1275/
  Subramony, S.H. and Ashizawa, T. 1998. Spinocerebellar ataxia type 1. 1998 Oct 1 [Updated 2014 Jul 3]. In: GeneReviews [Internet] (Pagon, R.A., Adam, M.P., Ardinger, H.H., Wallace, S.E., Amemiya, A., Bean, L.J.H., Bird, T.D., Fong, C.T., Mefford, H.C., Smith, R.J.H., Stephens, K., eds.). Seattle (WA): University of Washington, Seattle; 1993‐2016. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1184/
  Toyoshima, Y., Onodera, O., Yamada, M., Tsuji, S., and Takahashi, H. 2005. Spinocerebellar ataxia type 17. 2005 Mar 29 [Updated 2012 May 17]. In: GeneReviews [Internet] (Pagon, R.A., Adam, M.P., Ardinger, H.H., Wallace, S.E., Amemiya, A., Bean, L.J.H., Bird, T.D., Fong, C.T., Mefford, H.C., Smith, R.J.H., Stephens, K., eds.). Seattle (WA): University of Washington, Seattle; 1993‐2016. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1438/
Internet Resources
  http://www.beckman.com/getattachment/c78b9ef3‐dd37‐40a5‐8a34‐117da9a3bcd5/Agencourt%20CleanSEQ%20Dye%20‐%20Terminator%20Removal
  Agencourt CleanSEQ Dye‐Terminator Removal Protocol 000600v032.
  http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042039.pdf
  Applied Biosystems GeneMapper Software Version 4.0 Microsatellite Analysis Getting Started Guide.
  https://catalog.coriell.org/0/Sections/BrowseCatalog/Diseases.aspx?a=S&coll=&PgId=3
  Coriell Institute for Medical Research online catalog, which can be used to order positive control samples for SCA types 1, 2, 3, and 7.
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