An assay for X inactivation based on differential methylation at the fragile X locus, FMR1

Laura Carrel, Huntington F. Willard

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

We describe an assay analyzing methylation at the fragile X mental retardation gene, FMR1, to examine patterns of random or non-random X chromosome inactivation. Digestion of genomic DNA with the methylation- sensitive enzyme HpaII cleaves two restriction sites near the CGG repeat of the FMR1 gene if they are unmethylated on the active X chromosome, but fails to digest these sites on the inactive chromosome. Subsequent PCR using primers that flank the sites and the variable CGG repeat within the FMR1 gene amplifies alleles only on undigested, methylated inactive X chromosomes. Amplification of the hypervariable CGG repeat distinguishes alleles in heterozygous samples, while the relative ratio of alleles within a HpaII- digested sample reflects the randomness or non-randomness of inactivation. To demonstrate that methylation of the HpaII sites within the amplified FMR1 fragment correlates strictly with the activity state of the X chromosome, we have tested the validity of this assay by comparing DNA from normal males and females, as well as DNA from mouse/human somatic cell hybrids carrying either active or inactive human X chromosomes. The data demonstrate that this assay provides a reliable means of assessing the inactivation status of X chromosomes in individuals with X-linked disorders or X chromosome abnormalities.

Original languageEnglish (US)
Pages (from-to)27-30
Number of pages4
JournalAmerican Journal of Medical Genetics
Volume64
Issue number1
DOIs
StatePublished - Jul 12 1996

Fingerprint

X Chromosome Inactivation
X Chromosome
Methylation
Alleles
Chromosomes, Human, X
Genes
Hybrid Cells
DNA
DNA Methylation
Chromosome Aberrations
Intellectual Disability
Digestion
Chromosomes
Polymerase Chain Reaction
Enzymes

All Science Journal Classification (ASJC) codes

  • Genetics(clinical)

Cite this

@article{3203f557abc6472da1e9855418cb163a,
title = "An assay for X inactivation based on differential methylation at the fragile X locus, FMR1",
abstract = "We describe an assay analyzing methylation at the fragile X mental retardation gene, FMR1, to examine patterns of random or non-random X chromosome inactivation. Digestion of genomic DNA with the methylation- sensitive enzyme HpaII cleaves two restriction sites near the CGG repeat of the FMR1 gene if they are unmethylated on the active X chromosome, but fails to digest these sites on the inactive chromosome. Subsequent PCR using primers that flank the sites and the variable CGG repeat within the FMR1 gene amplifies alleles only on undigested, methylated inactive X chromosomes. Amplification of the hypervariable CGG repeat distinguishes alleles in heterozygous samples, while the relative ratio of alleles within a HpaII- digested sample reflects the randomness or non-randomness of inactivation. To demonstrate that methylation of the HpaII sites within the amplified FMR1 fragment correlates strictly with the activity state of the X chromosome, we have tested the validity of this assay by comparing DNA from normal males and females, as well as DNA from mouse/human somatic cell hybrids carrying either active or inactive human X chromosomes. The data demonstrate that this assay provides a reliable means of assessing the inactivation status of X chromosomes in individuals with X-linked disorders or X chromosome abnormalities.",
author = "Laura Carrel and Willard, {Huntington F.}",
year = "1996",
month = "7",
day = "12",
doi = "10.1002/(SICI)1096-8628(19960712)64:1<27::AID-AJMG3>3.0.CO;2-O",
language = "English (US)",
volume = "64",
pages = "27--30",
journal = "American Journal of Medical Genetics, Part A",
issn = "1552-4825",
publisher = "Wiley-Liss Inc.",
number = "1",

}

An assay for X inactivation based on differential methylation at the fragile X locus, FMR1. / Carrel, Laura; Willard, Huntington F.

In: American Journal of Medical Genetics, Vol. 64, No. 1, 12.07.1996, p. 27-30.

Research output: Contribution to journalArticle

TY - JOUR

T1 - An assay for X inactivation based on differential methylation at the fragile X locus, FMR1

AU - Carrel, Laura

AU - Willard, Huntington F.

PY - 1996/7/12

Y1 - 1996/7/12

N2 - We describe an assay analyzing methylation at the fragile X mental retardation gene, FMR1, to examine patterns of random or non-random X chromosome inactivation. Digestion of genomic DNA with the methylation- sensitive enzyme HpaII cleaves two restriction sites near the CGG repeat of the FMR1 gene if they are unmethylated on the active X chromosome, but fails to digest these sites on the inactive chromosome. Subsequent PCR using primers that flank the sites and the variable CGG repeat within the FMR1 gene amplifies alleles only on undigested, methylated inactive X chromosomes. Amplification of the hypervariable CGG repeat distinguishes alleles in heterozygous samples, while the relative ratio of alleles within a HpaII- digested sample reflects the randomness or non-randomness of inactivation. To demonstrate that methylation of the HpaII sites within the amplified FMR1 fragment correlates strictly with the activity state of the X chromosome, we have tested the validity of this assay by comparing DNA from normal males and females, as well as DNA from mouse/human somatic cell hybrids carrying either active or inactive human X chromosomes. The data demonstrate that this assay provides a reliable means of assessing the inactivation status of X chromosomes in individuals with X-linked disorders or X chromosome abnormalities.

AB - We describe an assay analyzing methylation at the fragile X mental retardation gene, FMR1, to examine patterns of random or non-random X chromosome inactivation. Digestion of genomic DNA with the methylation- sensitive enzyme HpaII cleaves two restriction sites near the CGG repeat of the FMR1 gene if they are unmethylated on the active X chromosome, but fails to digest these sites on the inactive chromosome. Subsequent PCR using primers that flank the sites and the variable CGG repeat within the FMR1 gene amplifies alleles only on undigested, methylated inactive X chromosomes. Amplification of the hypervariable CGG repeat distinguishes alleles in heterozygous samples, while the relative ratio of alleles within a HpaII- digested sample reflects the randomness or non-randomness of inactivation. To demonstrate that methylation of the HpaII sites within the amplified FMR1 fragment correlates strictly with the activity state of the X chromosome, we have tested the validity of this assay by comparing DNA from normal males and females, as well as DNA from mouse/human somatic cell hybrids carrying either active or inactive human X chromosomes. The data demonstrate that this assay provides a reliable means of assessing the inactivation status of X chromosomes in individuals with X-linked disorders or X chromosome abnormalities.

UR - http://www.scopus.com/inward/record.url?scp=0030007789&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030007789&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1096-8628(19960712)64:1<27::AID-AJMG3>3.0.CO;2-O

DO - 10.1002/(SICI)1096-8628(19960712)64:1<27::AID-AJMG3>3.0.CO;2-O

M3 - Article

VL - 64

SP - 27

EP - 30

JO - American Journal of Medical Genetics, Part A

JF - American Journal of Medical Genetics, Part A

SN - 1552-4825

IS - 1

ER -