Nonrandom X Chromosome Inactivation Detection

Julie R. Jones1

1 Molecular Diagnostic Laboratory, Greenwood Genetic Center, Greenwood, South Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.7
DOI:  10.1002/0471142905.hg0907s80
Online Posting Date:  January, 2014
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X chromosome inactivation patterns may be clinically useful in assessing tumor clonality, determining carrier status for certain X‐linked disorders and evaluating the pathogenicity of a genetic variant identified in an X‐linked gene. The protocols in this unit utilize the highly polymorphic trinucleotide repeat within the first exon of the human androgen receptor gene (AR) and the methylation‐sensitive restriction enzyme HpaII to distinguish between the maternal and paternal alleles and simultaneously determine their methylation status. The data obtained from these protocols can be used to calculate the ratio of inactivation between the two alleles that ultimately reflects whether a female has a random or nonrandom pattern of X chromosome inactivation. Curr. Protoc. Hum. Genet. 80:9.7.1‐9.7.7. © 2014 by John Wiley & Sons, Inc.

Keywords: X chromosome inactivation; molecular; clinical; diagnostic; testing

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Table of Contents

  • Introduction
  • Basic Protocol 1: X Chromosome Inactivation Assay
  • Support Protocol 1: PCR Amplification and Labeling of Digested and Undigested DNA Templates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: X Chromosome Inactivation Assay

  • 10× NEBuffer 1 (New England Biolabs)
  • 10 U/µl HpaII restriction endonuclease (New England Biolabs)
  • 100 ng/µl genomic DNA samples for testing (isolated, e.g., from peripheral blood or oral mucosa; appendix 3B)
  • 100 ng/µl DNA sample that has previously shown a pattern of highly skewed X chromosome inactivation (contact author at )
  • Sterile microcentrifuge tubes or 96‐well plates
  • Aerosol‐barrier pipet tips
  • Thermal cycler or water baths (16° and 80°C)

Support Protocol 1: PCR Amplification and Labeling of Digested and Undigested DNA Templates

  • 10× AmpliTaq Gold Buffer (Applied Biosystems)
  • 25 mM MgCl 2 (used only with the undigested samples; Applied Biosystems)
  • dNTP mix (0.25 mM each deoxynucleotide; see recipe)
  • 10 µM forward primer (XI FOR‐FAM; see recipe for primers)
  • 10 µM reverse primer (XI REV; see recipe for primers)
  • 5 U/µl AmpliTaq Gold DNA polymerase (Applied Biosystems)
  • DMSO (Sigma)
  • Digested and undigested DNA samples and controls ( protocol 1Basic Protocol)
  • Liz 500 Size Standard (Applied Biosystems)
  • Hi‐Di Formamide (Applied Biosystems)
  • Aerosol‐barrier pipet tips
  • 0.2‐ml PCR tubes or PCR plate
  • Centrifuge (with plate carrier if plates are used)
  • Thermal cycler
  • DNA Analyzer (Applied Biosystems 3730xl or other appropriate capillary gel electrophoresis system)
  • Appropriate software: e.g., GeneMapper
  • Additional reagents and equipment for PCR (unit 2.5; Kramer and Coen, )
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Literature Cited

Literature Cited
  Allen, R., Zoghbi, H., Moseley, A., Rosenblatt, H., and Belmont, J. 1992. Methylation of HpaII and HhaI sites near the polymorphic CA repeat in the human androgen‐receptor gene correlates with X chromosome inactivation. Am. J. Hum. Genet. 51:1229‐1239.
  Busque, L., Mio, R., Mattioli, J., Brais, E., Blais, N., Lalonde, Y., Maragh, M., and Gilliland, D.G. 1996. Non‐random X‐inactivation patterns in normal females: Lyonization ratios vary with age. Blood 88:59‐65.
  Eggan, K., Akutsu, H., Hochedlinger, K., Rideout, W. 3rd, Yanagimachi, R., and Jaenisch, R. 2000. X‐chromosome inactivation in cloned mouse embryos. Science 290:1578‐1581.
  Kramer, M. F. and Coen, D. M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1–15.1.14.
  Lanasa, M., Hogge, W.A., Kubik, C.J., Ness, R.B., Harger, J., Nagel, T., Prosen, T., Markovic, N., and Hoffman, E.P. 2001. A novel X chromosome linked genetic etiology of recurrent spontaneous abortion. Am. J. Obstet. Gynecol. 185:563‐5688.
  Lupski, J., Garcia, C., Zoghbi, H., Hoffman, E., and Fenwich, R. 1991. Discordance of muscular dystrophy in monozygotic female twins: Evidence supporting asymmetrical splitting of the inner cell mass in a manifesting carrier of Duchenne dystrophy. Am. J. Med. Genet. 40:354‐364.
  Nance, W. 1990. Do twin Lyons have larger spots? Am. J. Hum. Genet. 46:646‐648.
  Parrish, J.E., Scheuerle, A.E., Lewis, R.A., Levy, M.L., and Nelson, D.L. 1996. Selection against mutant alleles in blood leukocytes is a consistent feature in incontinentia pigmenti type 2. Hum. Mol. Genet. 5:1777‐1783.
  Pegoraro, E., Schimke, R.N., Arahata, K., Hayashi, Y., Stern, H., Marks, H., Glasberg, M.R., Carroll, J.E., Taber, J.W., Wessel, H.B., Bauserman, S.C., Marks, W.A., Toriello, H.V., Higgins, J.V., Appleton, S., Schwartz, L., Garcia, C.A., and Hoffman, E.P. 1994. Detection of new paternal dystrophin gene mutations in isolated cases of dystrophinopathy in females. Am. J. Hum. Genet. 54:989‐1003.
  Pegoraro, E., Whitaker, J., Mowery‐Rushton, P., Surti, U., Lanasa, M., and Hoffman, E.P. 1997. Familial skewed X inactivation: A molecular trait associated with high spontaneous‐abortion rate maps to Xq28. Am. J. Hum. Genet. 61:160‐170.
  Puck, J.M., Stewart, C.C., and Nussbaum, R.L. 1992. Maximum‐likelihood analysis of human T‐cell X chromosome inactivation patterns: Normal women versus carriers of X‐linked severe combined immunodeficiency. Am. J. Hum. Genet. 50:742‐748.
  Sharp, A., Robinson, D., and Jacobs, P. 2000. Age‐ and tissue‐specific variation of X chromosome inactivation ratios in normal women. Hum. Genet. 107:343‐349.
  Willard, H. 2001. The sex chromosomes and x chromosome inactivation. In The Metabolic & Molecular Bases of Inherited Disease (C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.) pp. 1191‐1211. McGraw‐Hill, New York.
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