Molecular Analysis of Fragile X Syndrome

Monica J. Basehore1, Michael J. Friez1

1 Greenwood Genetic Center, Greenwood, South Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.5
DOI:  10.1002/0471142905.hg0905s80
Online Posting Date:  January, 2014
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The gene responsible for Fragile X syndrome, fragile X mental retardation‐1 (FMR1), contains an unstable sequence of CGG trinucleotide repeats in its promoter region. Expansions of >200 trinucleotide repeats are considered full mutations and typically lead to abnormal methylation of the region, resulting in loss of FMR1 expression. Males with loss of FMR1 protein are expected to be affected by Fragile X syndrome, while females may or may not clinically manifest features of the condition. The protocols in this unit outline the complementary use of polymerase chain reaction (PCR) and methylation‐sensitive Southern blot hybridization to accurately measure trinucleotide repeat size and methylation status. These protocols are also used to evaluate CGG repeat size in two adult‐onset conditions known for their association with FMR1 premutation alleles, Fragile X Tremor/Ataxia (FXTAS) syndrome and Premature Ovarian Failure (POF). Curr. Protoc. Hum. Genet. 80:9.5.1‐9.5.19. © 2014 by John Wiley & Sons, Inc.

Keywords: Fragile X; molecular; diagnostic; clinical; testing

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Table of Contents

  • Introduction
  • Basic Protocol 1: FMR1 Trinucleotide Fluorescent PCR Amplification Using Capillary Gel Electrophoresis
  • Basic Protocol 2: FMR1 Trinucleotide PCR Amplification Using Polyacrylamide Gel Electrophoresis
  • Basic Protocol 3: Detection of FMR1 CGG Repeat Amplification and Methylation Status by Southern Blot Hybridization
  • Basic Protocol 4: Screening for the Presence or Absence of FMR1 CGG Repeat Amplification by Triplet Repeat‐Primed (TRP) PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: FMR1 Trinucleotide Fluorescent PCR Amplification Using Capillary Gel Electrophoresis

  • No‐template control (NTC)
  • Positive control with a known CGG repeat length
  • 6 M betaine (Sigma)
  • 10× PCR Gold Buffer (Applied Biosystems)
  • 25 mM MgCl 2 (Applied Biosystems)
  • Deoxynucleotides (dNTPs; 3 mM each dATP and dTTP, 0.75 mM each dCTP and dGTP)
  • 7‐deaza‐2′‐deoxyguanosine 5′‐triphosphate (5 mM)
  • Forward primer: (100 ng/µl) 5′GACGGAGGCGCCGCTGCCAGG
  • Reverse primer: (100 ng/µl) 5′GTGGGCTGCGGGCGCTCGAGG (FAM‐labeled)
  • Dimethyl sulfoxide (DMSO; Sigma)
  • 5 U/µl AmpliTaq Gold DNA polymerase (Applied Biosystems)
  • Purified DNA sample ( appendix 3B) in 10 mM Tris·Cl, pH 7.5 ( appendix 3D)/1 mM EDTA
  • Hi‐Di Formamide (Applied Biosystems)
  • LIZ fluorescent size standard (Applied Biosystems)
  • 1.5‐ml sterile microcentrifuge tube
  • Sterile PCR tubes/plate
  • Thermal cycler
  • Applied Biosystems 3100/3130 or 3730 Genetic Analyzer or other appropriate capillary gel electrophoresis system

Basic Protocol 2: FMR1 Trinucleotide PCR Amplification Using Polyacrylamide Gel Electrophoresis

  • 10× PCR amplification buffer: 100 mM Tris·Cl (pH 8.3)/500 mM KCl
  • 25 mM MgCl 2
  • 10 mM dNTP mix of dATP, dCTP, dTTP and 7‐deaza‐2′‐deoxyguanosine‐ 5′‐triphosphate (7‐deaza‐2′‐dGTP; Roche Diagnostics; also see appendix 2D)
  • 10 µM oligonucleotide primers 1 and 3 in 10 mM Tris·Cl, pH 7.5 ( appendix 2D)/1 mM EDTA
  • DMSO (Sigma)
  • 5 U/µl Taq DNA polymerase (Applied Biosystems)
  • 0.3 to 1 µg/ml purified sample DNA ( appendix 3B) in 10 mM Tris·Cl, pH 7.5 ( appendix 2D)/1 mM EDTA
  • 0.67× TBE buffer ( appendix 2D)
  • Loading buffer (see recipe)
  • 32P‐labeled DNA size markers: pBR322 digested with MspI
  • 0.4 N NaOH
  • Quick Light Hybridization kit (Orchid Biosciences) containing:
    • Quick Light buffer concentrate
    • Wash I and II solutions
    • Hybridization solution
  • Quick Light Genome Mapping Probe kit (Orchid Biosciences) containing alkaline phosphatase–labeled (CGG) n oligonucleotide probe
  • Lumi‐Phos spray
  • 0.2‐ and 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Thermal cycler
  • 100°C water baths
  • Vertical gel apparatus (e.g., BRL V15.17, Life Technologies)
  • Sequa Gel‐6 (optional; National Diagnostics)
  • Plastic wrap
  • Spatula
  • Positively charged nylon membrane (Biotrans+, ICN Biomedicals, precut to 12 × 16–cm)
  • Whatman 3 MM filter paper
  • Blotting paper (Owl Separation Systems), precut to 12 × 16–cm
  • Electroblot apparatus (Integrated Separation Systems)
  • Additional reagents and equipment for PCR (Kramer and Coen, ), denaturing PAGE ( appendix 3F), electroblotting from a polyacrylamide gel to a nylon membrane (Brown, ), and chemiluminescent detection of nonisotopic probes (see unit 9.6 and Perry‐O'Keefe and Kissinger, )

Basic Protocol 3: Detection of FMR1 CGG Repeat Amplification and Methylation Status by Southern Blot Hybridization

  • 0.5 to 1 µg/ml genomic DNA ( appendix 3B)
  • 5× restriction buffer (see recipe)
  • 0.1 M DTT (Sigma)
  • 3 U/ml RNase A (Sigma)
  • 100 U/µl EcoRI (New England Biolabs)
  • 50 U/µl EagI (New England Biolabs)
  • 10× Ficoll loading buffer (see recipe)
  • 30 to 50 µCi/ml [α‐32P] StB12.3 probe (3000 mCi/mmol, Amersham; or unlabeled from Dr. J.L. Mandel, Institut de Chimie Biologique; )
  • 1× TAE buffer, pH 8.5 ( appendix 2D)
  • Denaturing buffer (see recipe)
  • Transfer buffer (see recipe)
  • Neutralizing buffer (see recipe)
  • Hybridization buffer (see recipe)
  • 2× SSPE/0.1% (w/v) SDS ( appendix 2D; prepare fresh)
  • 1× SSPE/0.1% (w/v) SDS ( appendix 2D; prepare fresh)
  • 0.1× SSPE/1.0% (w/v) SDS, 60°C ( appendix 2D; prepare fresh)
  • 20× SSPE ( appendix 2D)
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • Vortex mixer
  • Whatman 3 MM filter paper
  • Nytran SuPercharge Turboblotter Rapid Downward Transfer System (Schleicher & Schuell)
  • 60°C hybridization oven
  • 80°C oven
  • UV‐transparent plastic wrap
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), Southern blotting with alkaline buffer (see unit 2.7 and Brown, ), and radiolabeling of DNA ( appendix 3E)
CAUTION: 32P is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Basic Protocol 4: Screening for the Presence or Absence of FMR1 CGG Repeat Amplification by Triplet Repeat‐Primed (TRP) PCR

  • High GC PCR buffer (Abbott Molecular)
  • FMR1 Primers‐2 (Abbott Molecular)
  • TR PCR Enzyme Mix (Abbott Molecular)
  • DNase/RNase‐free water
  • Hi‐Di Formamide
  • ROX 1000 size standard (Abbott Molecular)
  • 1.5‐ml sterile microcentrifuge tubes
  • Aerosol‐resistant pipet tips
  • Micropipettors
  • 0.2‐ml sterile PCR tubes
  • PCR tray/base
  • Thermal cycler
  • 96‐well plates
  • Applied Biosystems 3730 Genetic Analyzer or other appropriate capillary gel electrophoresis system
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  •   FigureFigure 9.5.1 The fragile X region with normal, premutation, and full mutation CGG repeats. The CGG repeat region is represented by a jagged line. Restriction sites are indicated by solid arrows for EcoRI and a dashed arrow for methylation‐sensitive EagI. Full mutation alleles are typically methylated and are resistant to digestion at the EagI site.
  •   FigureFigure 9.5.2 Restriction map of the fragile X region. Probes used in Southern analysis of the region are shown.
  •   FigureFigure 9.5.3 Top: Fluorescent PCR () electropherogram traces for (A) a normal male with 31 repeats, (B) a normal female with 25 and 31 repeats, (C) a normal female with 30 and 31 repeats, and (D) a male with a premutation‐sized allele of 75 repeats. (E) PCR analysis () of the FMR1 CGG repeat in the general population and in families with Fragile X. Lane 1: premutation male with 115 repeats. Lane 2: normal male with 29 repeats. Lane 3: normal male with 30 repeats. Lanes 4 to 6: family with Fragile X including premutation mother with 30 and 95 repeats (lane 4); full mutation female fetus with 31 and >200 repeats (lane 5); and normal father with 31 repeats (lane 6). Lanes 7 to 9: family with Fragile X: normal father with 30 repeats (lane 7); prenatal sample of normal female fetus with 27 and 30 repeats (lane 8); and premutation mother with 27 and 145 repeats (lane 9). Lane 10: normal male with 33 repeats. Lane 11: full mutation male with >200 repeats. Lane 12: negative control with no DNA. Lane 13: size standard (pBR322 digested with MspI) with corresponding CGG repeat numbers shown.
  •   FigureFigure 9.5.4 Fragile X Southern analysis of genomic DNA digested with EcoRI and EagI, and hybridized with probe StB12.3 (). Lane 1: full mutation male with methylation mosaicism. Lane 2: premutation male. Lane 3: premutation female. Lane 4: full mutation male. Lane 5: full mutation female. Lane 6: Full mutation male. Lanes 7 to 9: normal females. Lane 10: full mutation male. DNA size markers are indicated on the right.
  •   FigureFigure 9.5.5 Triplet repeat‐primed FMR1 PCR screening test for FMR1 CGG alleles (). The forward PCR primer is located upstream of the FMR1 CGG region while the fluorescently labeled reverse primer randomly binds inside the FMR1 CGG repeat region. These PCR primers generate different sized amplicons depending on the size of the CGG repeat region present. The presence or absence of a trinucleotide “ladder” that extends beyond a threshold of 55 CGG repeats can be easily recognized and is typically used to identify expanded FMR1 alleles. Example 1 shows the trace for a female with two normal alleles, neither of which results in the characteristic ladder motif present for expanded alleles. Example 2 illustrates the typical pattern for a female with one normal allele and one expanded allele while Example 3 illustrates the typical pattern for a male with an expansion at the FMR1 locus.


Literature Cited

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