Deleterious NKAP Mutations Are Associated with Musculoskeletal Abnormalities in Hemizygous Males and Skewed X Chromosome Inactivation in Heterozygous Females.

Abstract

NKAP (NF-kappa-B-activating protein) is a ubiquitously expressed nuclear protein involved in multiple biological processes. Males with missensemutations have been reported to present with marfanoid features and behavioral and musculoskeletal abnormalities. We have previously reported that a disruptivemutation resulted in extremely skewed X chromosome inactivation (XCI), leading to phenotypic manifestation of hemophilia A (HA) in a HA carrier. In this study, with the aim of exploring the phenotypic manifestations of deleteriousmutations in males, as well as their involvement in the mechanism of XCI regulation in females, we generatedmutant mice using CRISPR/Cas9 technology. Gait analysis studies conducted in male mice hemizygous for mutantby the CatWalk XT system revealed significant alterations in gait parameters, consistent with hypotonia reported in human mutantpatients. By breeding mutantmice with HA mice, we generated a double heterozygous mutant NKAP/HA mouse model, i.e., female mice carrying mutantwith a WTcopy on one X chromosome, and WTwith a mutantcopy on the other X chromosome. XCI pattern analysis using methylation-sensitive restriction enzymes demonstrated that mutant NKAP/HA females exhibited significant XCI skewing of the X chromosome bearing the mutantcopy. Furthermore, these females exhibited significantly reduced F8 mRNA levels and FVIII (factor VIII) antigen levels, as demonstrated by quantitative RT-PCR and ELISA, respectively. Murine embryonic fibroblasts (MEFs) derived from a hemizygous mutantembryo exhibited markedly reduced proliferation rate and increased senescence compared to WTMEFs, suggesting that XCI skewing induced by mutantresults from secondary selection against cells with an active X chromosome bearing the mutantcopy.