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
8 affiliations
- 1Medical Genetics Unit, Department of Medical Sciences, University of Ferrara, 44121 Ferrara, Italy
- 2Department of Life Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy
- 3Center for Regenerative Medicine, “Stefano Ferrari” University of Modena and Reggio Emilia, 41125 Modena, Italy
- 4Epigenetics and Neurobiology Unit, EMBL Rome, 00015 Monterotondo, Italy
- 5Department of Biology, University of Pisa, 56123 Pisa, Italy
- 6Department of Biomedical Sciences and Biotechnology, University of Cagliari, 09124 Cagliari, Italy
- 7BGI-Shenzhen, Shenzhen 518083, China
- 8Institute of Genetics and Biophysics (IGB) “Adriano Buzzati-Traverso”, Consiglio Nazionale delle Ricerche (CNR), 80131 Naples, Italy
Abstract
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.
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BibTeX
@article{mietto2026novel,
title = {{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}},
author = {Martina Mietto and Maria Sofia Falzarano and Annarita Armaroli and Fernanda Fortunato and Stefania Bigoni and Roberta Schininà and Beatrice Vianello and Federico Grasso and Daniela Benati and Andrea Cerase and Milena Cau and Maria Antonietta Melis and Xiao Dang and Mingyan Fang and Alessandra Recchia and Salvatore Fioriniello and Floriana Della Ragione and Alessandra Ferlini},
journal = {Cells},
year = {2026},
month = oct,
doi = {10.3390/cells15211960},
url = {https://www.mdpi.com/2073-4409/15/21/1960},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - 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
AU - Martina Mietto
AU - Maria Sofia Falzarano
AU - Annarita Armaroli
AU - Fernanda Fortunato
AU - Stefania Bigoni
AU - Roberta Schininà
AU - Beatrice Vianello
AU - Federico Grasso
AU - Daniela Benati
AU - Andrea Cerase
AU - Milena Cau
AU - Maria Antonietta Melis
AU - Xiao Dang
AU - Mingyan Fang
AU - Alessandra Recchia
AU - Salvatore Fioriniello
AU - Floriana Della Ragione
AU - Alessandra Ferlini
PY - 2026
DA - 2026/10/28
JO - Cells
DO - 10.3390/cells15211960
UR - https://www.mdpi.com/2073-4409/15/21/1960
AB - 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.
SN - 2073-4409
ER -