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

NKAP (NF-kappa-B-activating protein) is a ubiquitously expressed nuclear protein involved in multiple biological processes. Males with missense <i>NKAP</i> mutations have been reported to present with marfanoid features and behavioral and musculoskeletal abnormalities. We have previously reported that a disruptive <i>NKAP</i> mutation 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 deleterious <i>NKAP</i> mutations in males, as well as their involvement in the mechanism of XCI regulation in females, we generated <i>NKAP</i> mutant mice using CRISPR/Cas9 technology. Gait analysis studies conducted in male mice hemizygous for mutant <i>NKAP</i> by the CatWalk XT system revealed significant alterations in gait parameters, consistent with hypotonia reported in human mutant <i>NKAP</i> patients. By breeding mutant <i>NKAP</i> mice with HA mice, we generated a double heterozygous mutant NKAP/HA mouse model, i.e., female mice carrying mutant <i>NKAP</i> with a WT <i>F8</i> copy on one X chromosome, and WT <i>NKAP</i> with a mutant <i>F8</i> copy 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 mutant <i>NKAP</i> copy. 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 mutant <i>NKAP</i> embryo exhibited markedly reduced proliferation rate and increased senescence compared to WT <i>NKAP</i> MEFs, suggesting that XCI skewing induced by mutant <i>NKAP</i> results from secondary selection against cells with an active X chromosome bearing the mutant <i>NKAP</i> copy.