Quantification of X-chromosome inactivation in fibroblast and iPSC models of UBQLN2 ALS/FTD using allele-selective qPCR

Pathogenic missense variants in the X chromosome gene UBQLN2 cause amyotrophic lateral sclerosis (ALS), often accompanied by frontotemporal dementia (FTD). As an X-linked gene, UBQLN2 is subject to X chromosome inactivation (XCI), a process wherein one X chromosome in each cell is randomly inactivated to a Barr body throughout the body in females, creating a mosaic of allelic expression in the tissues of heterozygotes. Despite heterozygous females constituting a majority of reported cases of UBQLN2-linked ALS/FTD, and the known influence of XCI on neurological disorders at large, no current disease models account for XCI. Here we report the characterisation of 12 iPSC clones carrying the ALS/FTD-causing p.T487I (c.1460C>T) UBQLN2 variant. These clones, originally derived from 3 heterozygous carrier fibroblast lines, underwent validation of homeostatic Barr body retention. Erosion of XCI in a subset of the lines was correlated with biallelic expression (of both wildtype and mutant UBQLN2), as measured through a novel allele-selective qPCR (AS-qPCR) assay and verified by amplicon-based Illumina sequencing and Sanger chromatogram quantification, enabling selection of iPSC clones best retaining XCI. Together, this UBQLN2 AS-qPCR assay and selected iPSC clones will enable studies of the role of XCI and its skew in female resilience to UBQLN2 p.T487I-linked ALS/FTD and enable development of allele-selective therapies.