title: An X chromosome inactivation assay based on differential methylation of a CpG island coupled to a VNTR polymorphism at the 5' end of the monoamine oxidase A gene.
abstract: A CpG island has been identified just upstream of the first exon of the human monoamine oxidase A (MAOA) gene, localized to Xp11.4-Xp11.23. Southern blotting following digestion with the methylation sensitive restriction endonucleases SmaI, HpaII and HhaI, indicated that CpG dinucleotides within the CpG island were unmethylated on the active X chromosome and extensively methylated on the inactive X chromosome. These sites of differential methylation were close to a polymorphic GT-dinucleotide/VNTR region, which is located 1 kb 3' of the first exon and has a heterozygosity value of 75%. PCR primers were designed for amplification of 1.2-1.3 kb DNA fragments, encompassing both the hypervariable region and a cluster of six HpaII sites within the CpG-rich region. Cleavage of HpaII sites was found to be restricted to active X chromosomes. Therefore, following HpaII digestion, DNA fragments were exclusively amplified from inactive X chromosomes. The resulting PCR products were digested with SacI, which reduced the size of the DNA fragments containing the hypervariable region to 230-330 bp, and were subsequently analyzed on denaturating polyacrylamide gels. Because amplified fragments were exclusively derived from the inactive X chromosome, the relative densities of the two allelic fragments should reflect the proportions of cells that have either of the two X chromosome inactivated. The results of this PCR-based X chromosome inactivation assay were fully concordant with Southern blotting methylation analyses at the PGK locus. It therefore provides a rapid and informative method in tumour clonality analysis and carrier detection in X-linked diseases.
authors.affiliation: Department of Biochemistry, University of Oxford, UK.
authors.forename: R W
authors.initials: RW
authors.lastname: Hendriks
authors.forename: Z Y
authors.initials: ZY
authors.lastname: Chen
authors.forename: H
authors.initials: H
authors.lastname: Hinds
authors.forename: R K
authors.initials: RK
authors.lastname: Schuurman
authors.forename: I W
authors.initials: IW
authors.lastname: Craig
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics; D004303:Dosage Compensation, Genetic; D005260:Female; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D014960:X Chromosome
chemical_list: D009838:Oligodeoxyribonucleotides; D004247:DNA; D008995:Monoamine Oxidase