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 <it>Sma</it>I, <it>Hpa</it>II and <it>Hha</it>I, 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 <it>Hpa</it>II sites within the CpG-rich region. Cleavage of <it>Hpa</it>II sites was found to be restricted to active X chromosomes. Therefore, following <it>Hpa</it>II digestion, DNA fragments were exclusively amplified from inactive X chromosomes. The resulting PCR products were digested with <it>Sac</it>I, 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.