Three new loci for determining x chromosome inactivation patterns

Abstract

The analysis of X chromosome inactivation (XCI) patterns is a widely used diagnostic tool in clinical practice when investigating X-linked diseases. The most commonly used assay to determine XCI patterns takes advantage of a locus within the androgen receptor (AR) gene. This PCR-based assay relies on two differentially methylated restriction enzyme sites (HpaII) and a polymorphic repeat located within this locus. Although highly informative, this locus is not always sufficient to evaluate the X-inactivation status in X-linked disorders. We have identified three new loci that can be used to determine XCI patterns in a methylation-sensitive PCR-based assay. All three loci contain polymorphic repeats and a methylation-sensitive restriction enzyme (HpaII) site, methylation of which was shown to correlate with XCI. DNA from 60 females was used to estimate the heterozygosity of these new loci. The reliability of the loci was validated by showing a high correlation between the results obtained by employing the new loci and the AR locus using DNA from 15 females who were informative for all four loci. Altogether, we show that these loci can be applied easily in molecular diagnostic laboratories, either as a supplement or as an alternative to the existing AR assay.

Figure 1

Fragment analysis results for the ZDHHC15, SLITRK4, and PCSK1N loci. Undigested (−HpaII) or digested (+HpaII) DNA was amplified with PCR, using the respective primer sets for the three loci. The fragment analysis was performed with GeneMapper software (Applied Biosystems). A: After HpaII digestion, DNA from the male individual (M1) cannot be amplified with PCR. B: Three phenotypically normal females (F1–F3) with extremely skewed X chromosome inactivation. F1 and F3 have Xq28 duplication and F2 has partial deletion of the NEMO gene. As seen on the lower panel, one of the alleles (as represented by PCR fragments, blue peaks) cannot be amplified by PCR after HpaII digestion. The red peaks represent the size standard 500 ROX.