A Novel NKAP Gene Frameshift Mutation in a Sardinian Family Causes Early Cell Death and Secondary Skewed X-Chromosome Inactivation and Associates with Multiple Male Miscarriages

NF-κB-activating protein (NKAP) gene maps to Xq24 and encodes a nuclear protein involved in NF-κB transcription factor activation. Missense variants in NKAP have previously been reported in males with X-linked intellectual disability. Here, we identified a recurrent, novel frameshift variant, c.172del (p.His58Ilefs74*), by targeted X-chromosome sequencing (X-TRS) in seven females from a Sardinian family showing fully skewed X-chromosome inactivation (XCI). To investigate the functional effect of the mutation, we knocked out (KO) and knocked down (KD) NKAP in healthy donor fibroblasts and mouse embryonic stem cells, respectively. We performed RNA sequencing, immunofluorescence, and Western blot analysis on fibroblasts derived from the index subject. We demonstrated that the NKAP frameshift variant leads to monoallelic wild-type (WT) transcription and does not directly affect XCI, indicating secondary rather than primary XCI skewing. NKAP-edited human fibroblasts underwent cell death, leading to the positive selection of WT cells. The NKAP correctly localized to the nucleus but showed reduced expression. We also showed that XIST expression was not affected by this NKAP mutation, further excluding a primary XCI event. Our findings demonstrate that this novel NKAP frameshift variant causes early cell death and post-mitotic negative selection of mutant cells, resulting in a fully secondary XCI. Female carriers are healthy and fertile, whereas affected males are likely nonviable. In conclusion, our results provide insights into both the role of NKAP in cells and novel clinical implications for human fertility.