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Detection of Nonrandom X Chromosome Inactivation

Melissa M. Thouin¹, James M. Giron¹, Eric P. Hoffman¹

¹Children's Research Institute, Washington, D.C.

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 9.7

DOI:

10.1002/0471142905.hg0907s35

Online Posting Date:

February, 2003

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Abstract

This unit describes a PCR-based assay for distinguishing between the two X chromosomes in female cells and assessing the percentage of cells having each parental X chromosome active. Methylation of CpG residues in gene promoters is a major mechanism of transcriptional silencing. In mammalian female cells, hypermethylation is the way in which one X chromosome is inactivated. The X-inactivation assay described in the Basic Protocol relies on methylation sensitivity. In this unit, the highly polymorphic and therefore typically heterozygous (CAG)n region of the 5 end of the coding region of the human androgen receptor gene (HUMARA), at Xq11.2, is used to distinguish and compare the methylation activity of the X chromosomes.

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

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Materials

Basic Protocol: X Chromosome Inactivation Assay

Materials

NEBuffer 1 (New England Biolabs)
10 U/µl HpaII restriction endonuclease
100 ng/µl genomic DNA sample (appendix 3B) isolated, e.g., from peripheral blood or oral mucosa
100 ng/µl DNA sample that has previously shown a pattern of highly skewed X chromosome inactivation (contact authors at ehoffman@childrens-research.org)

RNase/DNase-free microcentrifuge tubes
Thermal cycler or water bath

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

Materials

Red Taq DNA polymerase and 10× PCR reaction buffer (Sigma)
1.25 mM dNTP mix (1.25 mM each dNTP; see recipe)
1 pmol/µl labeled Met-F forward primer (see recipe for primers)
5 pmol/µl unlabeled Met-R1 reverse primer (see recipe for primers)
50 ng/µl HpaII-predigested and positive and negative-control DNA (see Basic Protocol)
100-bp DNA ladder (Life Technologies)
IR2 stop solution/loading dye (LI-COR)
96-well PCR plates (e.g., Perkin-Elmer)
Thermal cycler
LI-COR 4200S DNA Analyzer
Additional reagents and equipment for PCR (unit 2.5; cpmb unit 15.1)

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Figures

Figure 9.7.1

Example of results showing extremely skewed pattern of X inactivation and random X inactivation. The upper automated sequencer traces correspond to the undigested, PCR-amplified HUMARA locus and show two alleles of the androgen receptor corresponding to the normal (paternal), and, in the case of the skewed example, mutant (maternal) X. The lower traces show the two samples after predigestion with the methylation-sensitive restriction endonuclease HpaII and PCR amplification of HUMARA. The X chromosome that is active is undermethylated, and, once digested with the restriction enzyme, cannot be PCR-amplified and thus disappears from the lower trace. In the lower right trace, because both alleles exist in the methylated and unmethylated states, both alleles appear equally decreased after enzymatic predigestion, indicating a random pattern of X inactivation.

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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 chromsome 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.
	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 dysrophyin 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. Molec. 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., Robunson, D., 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.