Clinical and molecular aspects of the association of skewed X chromosome inactivation and recurrent spontaneous abortion

Christy L. Beever
Recurrent spontaneous abortion (RSA), defined as three or more pregnancy losses at less than 20 weeks of gestation, is a devastating problem that affects 1-2% of couples trying to have children. Despite extensive medical testing and experimental treatments couples with RSA may go through, a clear cause for their pregnancy losses is often not found. The finding that skewed X chromosome inactivation (XCI), the non-random inactivation of one of two X chromosomes in female cells, is increased in
more » ... en with RSA has led to the exciting prospect that a cause for these pregnancy losses can be identified in a subset of women with RSA. The objective of this thesis was to explore the etiology of the association of RSA and skewed XCI and the potential use of skewed XCI in counseling of couples with RSA. The reliability of the assay for skewed XCI was also examined by comparison of different methodologies. Skewed XCI is currently used in both clinical and research settings as a biological marker for clonality and carrier status of certain X-linked diseases. DNA methylation-based XCI assays generally involve amplification by PCR of an X-linked locus which is both differentially methylated, allowing distinction of the inactive and active X via digestion with a methylation sensitive enzyme, and polymorphic, allowing discrimination of alleles of the locus. To test the reproducibility of such assays, the following aspects of the XCI assay were altered: 1) method of quantification (densitometry of silver-stained polyacrylamide gels vs. automated fluorescent analysis using an ABI 310 Genetic Analyzer); 2) the use of a secondary cutter (a restriction enzyme which is not methylation sensitive) in conjunction with the methylation sensitive enzyme; 3) locus (androgen receptor (AR) vs. FMR-1). In 60 samples tested by both quantification methods, XCI results were well correlated (r=0.839) with a mean difference in percent skewing between replicates of 6.4%±0.6% with a range of 0.0 to 21.2. By analyzing replicate meas [...]
doi:10.14288/1.0090194 fatcat:55kdb2vuurfc3osl32zkovqdem