Molecular Diagnosis of Duchenne Muscular Dystrophy

Babi Ramesh Reddy Nallamilli1, Arunkanth Ankala1, Madhuri Hegde1

1 Department of Human Genetics, Emory University School of Medicine, Atlanta
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.25
DOI:  10.1002/0471142905.hg0925s83
Online Posting Date:  October, 2014
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Abstract

Duchenne Muscular Dystrophy (DMD) is an X‐linked inherited neuromuscular disorder caused by mutations in the dystrophin gene (DMD; locus Xp21.2). The mutation spectrum of DMD is unique in that 65% of causative mutations are intragenic deletions, with intragenic duplications and point mutations (along with other sequence variants) accounting for 6% to 10% and 30% to 35%, respectively. The strategy for molecular diagnostic testing for DMD involves initial screening for deletions/duplications using microarray‐based comparative genomic hybridization (array‐CGH) followed by full‐sequence analysis of DMD for sequence variants. Recently, next‐generation sequencing (NGS)–based targeted gene analysis has become clinically available for detection of point mutations and other sequence variants (small insertions, deletions, and indels). This unit initially discusses the strategic algorithm for establishing a molecular diagnosis of DMD and later provides detailed protocols of current molecular diagnostic methods for DMD, including array‐CGH, PCR‐based Sanger sequencing, and NGS‐based sequencing assay. Curr. Protoc. Hum. Genet. 83:9.25.1‐9.25.29 © 2014 by John Wiley & Sons, Inc.

Keywords: Duchenne Muscular Dystrophy; molecular diagnosis; comparative genomic hybridization; DMD

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Microarray‐Based Comparative Genomic Hybridization
  • Basic Protocol 2: Sanger Sequencing of Entire DMD Coding Region to Detect Point Mutations
  • Basic Protocol 3: Targeted Next‐Generation Sequencing of DMD Using IDT xGen Lockdown Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microarray‐Based Comparative Genomic Hybridization

  Materials
  • DNA sample from affected individual suspected to have DMD
  • Control DNA sample from healthy individual (patient and controls should be same gender)
  • Spike‐ins (BlueGnome)
  • Cytosure Labeling Kit (OGT) containing:
    • Random primers
    • dCTP labeling mix
    • Cy5‐dCTP
    • Cy3‐dCTP
    • Reaction buffer
    • Klenow polymerase
  • 1× Tris‐EDTA (Fisher)
  • Human Cot‐1 DNA (Invitrogen)
  • Oligo array‐CGH/ChIP‐on‐chip Hybridization Kit (Agilent Technologies/OGT) containing:
    • 10× blocking buffer (lyophilized Blocking agent, see recipe))
    • Hi‐RPM hybridization buffer
  • Array‐CGH wash buffer 2 (see recipe)
  • Array‐CGH wash buffer 1 (see recipe)
  • Acetonitrile
  • Stabilization and drying solution (Agilent Technologies)
  • NanoDrop spectrophotometer
  • Branson sonifier 450
  • PCR strip tubes
  • Thermal cycler
  • 1‐ml microcentrifuge tubes
  • MultiScreen HTS PCR 96‐well plates (Millipore)
  • MultiScreen vacuum manifold (Millipore)
  • Plate mixer
  • 65°C hybridization oven and oven rotator
  • Custom HD‐CGH Microarray 8×60K slides (OGT or Agilent technologies)
  • Hybridization gasket slide kit (100)‐8‐plex (Agilent Technologies/OGT)
  • Hybridization chamber (Agilent SureHyb)
  • 37°C water bath
  • Slide‐staining jars
  • Glass‐slide holder
  • Magnetic stir bars and stir plate
  • High‐resolution microarray scanner (Agilent Technologies)
  • CytoSure Interpret software (Oxford Genetic Technology)

Basic Protocol 2: Sanger Sequencing of Entire DMD Coding Region to Detect Point Mutations

  Materials
  • DMD exon–specific forward primer (5 μM) (required for all 79 exons)
  • DMD exon–specific reverse primer (5 μM) (required for all 79 exons)
  • DNA from DMD patient (25 ng/μl)
  • 10× PCR buffer with MgCl 2 (Roche)
  • 10 mM dNTPs (Roche)
  • Faststart Taq DNA polymerase (Roche)
  • 2% agarose gel
  • BigDye Terminator v3.1 cycle sequencing kit (Life Technologies) containing:
    • 5× sequencing buffer
    • BigDye Terminator v3.1 ready reaction mix
    • M13 forward or M13 reverse primer
  • Melatonin solution (Sigma)
  • Nanodrop spectrophotometer
  • 96‐well PCR plate
  • Thermal cycler
  • Centrifuge with plate rotor
  • MultiScreen HTS PCR plate (Millipore)
  • Multichannel pipettor
  • MultiScreen vacuum manifold (Millipore)
  • Plate mixer
  • Gel electrophoresis apparatus
  • Edge PCR 96‐well Performa Version 3 purification plates (Egde Biosystems)
  • ABI 3730 sequencer
  • Mutation Surveyor software (SoftGenetics LLC)

Basic Protocol 3: Targeted Next‐Generation Sequencing of DMD Using IDT xGen Lockdown Probes

  Materials
  • DNA samples
  • Distilled water, HPLC‐grade
  • 1× low‐TE buffer (Life Technologies)
  • Agilent High Sensitivity Kit (Agilent) containing:
    • DNA ladder
  • BIOO Scientific Adapters and Primer Mix kit containing:
    • BIOO Scientific Indexed Adapters 1 to 48
    • BIOO Scientific Primer Mix
  • KAPA HiFi HotStart ReadyMix (KAPA)
  • Agencourt Ampure Beads (Beckman Coulter)
  • 70% ethanol (Sigma)
  • Human Cot‐1 DNA (Invitrogen)
  • xGen Universal Blocking Oligo TS‐p5 (IDT)
  • xGen Universal Blocking Oligo TS‐p7 (IDT)
  • SeqCap EZ Hybridization and Wash Kits (Nimblegen) containing:
    • 10× Wash Buffer I (Nimblegen)
    • 10× Wash Buffer II (Nimblegen)
    • 10× Wash Buffer III (Nimblegen)
    • Stringent Wash Buffer (Nimblegen)
    • 2× Hybridization Buffer (Nimblegen)
    • Hybridization Component A (Nimblegen)
    • 2.5× Bead Wash Buffer (Nimblegen)
  • xGen lockdown probe mix (IDT)
  • Dynabeads M‐270 Streptavidin (Life Technologies)
  • 10 M sodium hydroxide (NaOH, Sigma)
  • 1 M Tris·Cl, pH 8.8 (Sigma)
  • 10 μM Illumina P5 primer (IDT)
  • 10 μM Illumina P7 primer (IDT)
  • HT1 buffer (Illumina), cold
  • Illumina PhiX control (Illumina)
  • Spectrophotometer (NanoDrop)
  • Covaris E series sonicator (Covaris) connected to a computer with SonoLab software
  • Covaris micro tubes and tube holder (Covaris)
  • 1.5‐ml LoBind tubes (Eppendorf)
  • Microcentrifuge
  • High‐sensitivity bioanalyzer chips (Agilent)
  • 2100 Bioanalyzer (Agilent)
  • SPRI‐TE Nucleic Acid Extractor (Beckman Coulter)
  • SPRIworks Illumina Method Card (Beckman Coulter)
  • SPRIworks 2.0‐ml tubes (Beckman Coulter)
  • SPRIworks Reagent Cartridge (Beckman Coulter)
  • SPRIworks adapter tubes (Beckman Coulter)
  • SPRIworks Piercing Tips (Beckman Coulter)
  • SPRIworks 1‐ml tips (Beckman Coulter)
  • DynaMag magnetic stand (Life Technologies)
  • HardShell PCR plates (Biorad)
  • Snap caps (Biorad)
  • Centrifuge with plate rotor
  • Thermal cyclers (Biorad T‐100)
  • Qubit fluorometer (Life Technologies)
  • Vacufuge
  • Aluminum foil
  • Vortexer
  • 47°C incubator (SciGene, Fisher)
  • HiSeq2500 (Illumina)
  • NextGENe software v2.3.4 (SoftGenetics LLC)
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Figures

Videos

Literature Cited

Literature Cited
  Ankala, A., Kohn, J.N., Hegde, A., Meka, A., Ephrem, C.L., Askree, S.H., Bhide, S., and Hegde, M.R. 2012. Aberrant firing of replication origins potentially explains intragenic nonrecurrent rearrangements within genes, including the human DMD gene. Genome Res. 22:25‐34.
  Armour, J.A., Sismani, C., Patsalis, P.C., and Cross, G. 2000. Measurement of locus copy number by hybridisation with amplifiable probes. Nucleic Acids Res. 28:605‐609.
  Askree, S.H., Chin, E.L., Bean, L.H., Coffee, B., Tanner, A., and Hegde, M. 2013. Detection limit of intragenic deletions with targeted array comparative genomic hybridization. BMC Genet. 14:116.
  Beggs, A.H., Koenig, M., Boyce, F.M., and Kunkel, L.M. 1990. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum. Genet. 86:45‐48.
  Chin, E.L., da Silva, C., and Hegde, M. 2013. Assessment of clinical analytical sensitivity and specificity of next‐generation sequencing for detection of simple and complex mutations. BMC Genet. 14:6.
  del Gaudio, D., Yang, Y., Boggs, B.A., Schmitt, E.S., Lee, J.A., Sahoo, T., Pham, H.T., Wiszniewska, J., Chinault, A.C., Beaudet, A.L., and Eng, C.M. 2008. Molecular diagnosis of Duchenne/Becker muscular dystrophy: Enhanced detection of dystrophin gene rearrangements by oligonucleotide array‐comparative genomic hybridization. Hum. Mutat. 29:1100‐1107.
  Flanigan, K.M., Dunn, D.M., von Niederhausern, A., Soltanzadeh, P., Gappmaier, E., Howard, M.T., Sampson, J.B., Mendell, J.R., Wall, C., King, W.M., Pestronk, A., Florence, J.M., Connolly, A.M., Mathews, K.D., Stephan, C.M., Laubenthal, K.S., Wong, B.L., Morehart, P.J., Meyer, A., Finkel, R.S., Bonnemann, C.G., Medne, L., Day, J.W., Dalton, J.C., Margolis, M.K., Hinton, V.J., and Weiss, R.B. 2009. Mutational spectrum of DMD mutations in dystrophinopathy patients: Application of modern diagnostic techniques to a large cohort. Hum. Mutat. 30:1657‐1666.
  Hamed, S.A. and Hoffman, E.P. 2006. Automated sequence screening of the entire dystrophin cDNA in Duchenne dystrophy: Point mutation detection. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 141B:44‐50.
  Hegde, M.R., Chin, E.L., Mulle, J.G., Okou, D.T., Warren, S.T., and Zwick, M.E. 2008. Microarray‐based mutation detection in the dystrophin gene. Hum. Mutat. 29:1091‐1099.
  Janssen, B., Hartmann, C., Scholz, V., Jauch, A., and Zschocke, J. 2005. MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: Potential and pitfalls. Neurogenetics 6:29‐35.
  Mehler, M.F. 2000. Brain dystrophin, neurogenetics and mental retardation. Brain Res. Brain Res. Rev. 32:277‐307.
  Mendell, J.R., Buzin, C.H., Feng, J., Yan, J., Serrano, C., Sangani, D.S., Wall, C., Prior, T.W., and Sommer, S.S. 2001. Diagnosis of Duchenne dystrophy by enhanced detection of small mutations. Neurology 57:645‐650.
  Stockley, T.L., Akber, S., Bulgin, N., and Ray, P.N. 2006. Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies. Genet. Test. 10:229‐243.
  Valencia, C.A., Ankala, A., Rhodenizer, D., Bhide, S., Littlejohn, M.R., Keong, L.M., Rutkowski, A., Sparks, S., Bonnemann, C., and Hegde, M. 2013. Comprehensive mutation analysis for congenital muscular dystrophy: A clinical PCR‐based enrichment and next‐generation sequencing panel. PLoS One 8:e53083.
  Wei, X., Dai, Y., Yu, P., Qu, N., Lan, Z., Hong, X., Sun, Y., Yang, G., Xie, S., Shi, Q., Zhou, H., Zhu, Q., Chu, Y., Yao, F., Wang, J., He, J., Yang, Y., Liang, Y., Qi, M., Yang, L., Wang, W., Wu, H., Duan, J., Shen, C., Cui, L., and 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. Eur. J. Hum. Genet. 22:110‐118.
  White, S.J., Aartsma‐Rus, A., Flanigan, K.M., Weiss, R.B., Kneppers, A.L., Lalic, T., Janson, A.A., Ginjaar, H.B., Breuning, M.H., and den Dunnen, J.T. 2006. Duplications in the DMD gene. Hum. Mutat. 27:938‐945.
  Worton, R.G. and Thompson, M.W. 1988. Genetics of Duchenne muscular dystrophy. Annu. Rev. Genet. 22:601‐629.
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