Identification of a highly polymorphic tetranucleotide repeat locus (DXpS) at Xp and development of a DXpS/HUMARA biplex methylation-based PCR assay that enhances detection of X-chromosome inactivation

Abstract

Here, we report on the identification of a highly polymorphic tetranucleotide repeat (named DXpS) mapping to within a CpG island on the Xp. This island is 191 bp upstream from the start of the repeat element, and contains sites for the HhaI, HpaII and BstUI methyl-sensitive restriction enzymes ( Figure 1). We developed the DXpS and the HUMARA markers into a biplex methylation-based quantitative fluorescent PCR assay. For DXpS we observed twelve alleles with negligible stuttering (Figure 2). DXpS exhibited a heterozygosity rate of 87% (n = 60), matching that of HUMARA. The combined informativeness of the biplex assay was 98%. Random and nonrandom X-inactivation patterns inferred with DXpS in phenotypically normal females and haemophiliac females carrying a defective F8 gene were highly concordant (r 2 = 0.96) with HUMARA patterns (Figures 3, 4 and 5).