Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) as a novel strategy for identification of the skewed X inactivation pattern in balanced and unbalanced X-rearrangements

Abstract

X-chromosome inactivation occurs randomly in normal female cells. However, the inactivation can be skewed in patients with alterations in X-chromosome. In balanced X-autosome translocations, normal X is preferentially inactivated, while in unbalanced X alterations, the aberrant X is usually inactivated. Here, we present a novel strategy to verify the skewed X inactivation pattern through the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into cells, in 11 patients: five carriers of balanced X-autosome translocations and six of unbalanced X-chromosome alterations. Since EdU is a labeled nucleoside analog of thymidine, its incorporation during DNA synthesis can reveal late replication regions and the inactive X-chromosome. All EdU findings were validated by the human androgen receptor gene (HUMARA) assay. The late replication regions were easily and quickly visualized in all cells, where inactive Xs are marked with strong green fluorescence. It was observed that the normal X-chromosome was preferentially inactivated in patients with balanced X-autosome translocations; while the aberrant X-chromosome was inactivated in most cells from patients with unbalanced alterations. By performing the fluorescence-based EdU assay, the differences between the active and inactive X-chromosomes are more easily recognizable than by classic cytogenetic methods. Furthermore, EdU incorporation allows the observation of the late replication regions in autosomal segments present in X derivatives from X-autosome translocations. Therefore, EdU assay permits an accurate and efficient cytogenetic evaluation of the X inactivation pattern with a low-cost, easy to perform and highly reproducible technique.

X‐linked intellectual disability: Phenotypic expression in carrier females

“…22 However, XLID genes with female phenotypes only in X:autosome translocations is a notable exception to the above observations, and in these cases specifically, skewing of XI is a well-documented phenomenon believed to occur in order to ensure the correct gene dosage of the autosomal segment. 23 This study supports these findings with all cases of X:autosome translocations reported here having skewed XI.…”

“…While these numbers are higher than the reported prevalence of skewing in normal non‐carrier females (3.6%), it does not appear that skewed XI favoring expression of the variant allele in affected females or favoring expression of the normal allele in non‐affected carriers explains female phenotypic expression patterns in the majority of XLID syndromes . However, XLID genes with female phenotypes only in X:autosome translocations is a notable exception to the above observations, and in these cases specifically, skewing of XI is a well‐documented phenomenon believed to occur in order to ensure the correct gene dosage of the autosomal segment . This study supports these findings with all cases of X:autosome translocations reported here having skewed XI.…”

Inactivation of AMMECR1 is associated with growth, bone, and heart alterations

“…For patient 1, breakpoint localization was performed by fluorescence in situ hybridization (FISH) and array painting as described (Liehr, Heller, Starke, & Claussen, ; Moyses‐Oliveira et al., ) and validated by FISH using bacterial artificial chromosome probe CTD‐3066N24 (Thermo Fisher Scientific, Waltham, MA, USA) and whole‐genome sequencing. X‐chromosome inactivation was assessed as described (Sisdelli et al., ).…”

Unusual X‐chromosome inactivation pattern in patients with Xp11.23‐p11.22 duplication: Report and review

“…The X‐chromosome inactivation pattern was determined by human androgen receptor assay (HUMARA), as previously described by [Araujo and Ramos, ] and modified by [Sisdelli et al, ]. HUMARA was performed in the patient and her mother in order to determine the inactivation pattern in both and the parental origin of the inactive X‐chromosome in the proposita.…”