Human NK cell deficiency as a result of biallelic mutations in MCM10
Emily M. Mace, … , Anja K. Bielinsky, Jordan S. Orange
Emily M. Mace, … , Anja K. Bielinsky, Jordan S. Orange
Published August 31, 2020
Citation Information: J Clin Invest. 2020;130(10):5272-5286. https://doi.org/10.1172/JCI134966.
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Research Article Immunology

Human NK cell deficiency as a result of biallelic mutations in MCM10

Abstract

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function, or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here, we report a cause of NKD resulting from compound heterozygous mutations in minichromosomal maintenance complex member 10 (MCM10) that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. Through evaluation of patient primary fibroblasts, modeling patient mutations in fibroblast cell lines, and MCM10 knockdown in human NK cell lines, we have shown that loss of MCM10 function leads to impaired cell cycle progression and induction of DNA damage–response pathways. By modeling MCM10 deficiency in primary NK cell precursors, including patient-derived induced pluripotent stem cells, we further demonstrated that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function. Together, these data define MCM10 as an NKD gene and provide biological insight into the requirement for the DNA replisome in human NK cell maturation and function.

Authors

Emily M. Mace, Silke Paust, Matilde I. Conte, Ryan M. Baxley, Megan M. Schmit, Sagar L. Patil, Nicole C. Guilz, Malini Mukherjee, Ashley E. Pezzi, Jolanta Chmielowiec, Swetha Tatineni, Ivan K. Chinn, Zeynep Coban Akdemir, Shalini N. Jhangiani, Donna M. Muzny, Asbjørg Stray-Pedersen, Rachel E. Bradley, Mo Moody, Philip P. Connor, Adrian G. Heaps, Colin Steward, Pinaki P. Banerjee, Richard A. Gibbs, Malgorzata Borowiak, James R. Lupski, Stephen Jolles, Anja K. Bielinsky, Jordan S. Orange

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Figure 6

Cell cycle arrest in a cell line model of MCM10 KD.

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Cell cycle arrest in a cell line model of MCM10 KD. MCM10 expression in ...
MCM10 expression in NK92 cells was reduced by CRISPR/Cas9 gene editing, as described in Methods. Single clones were expanded and validated, and 1 was selected for further analysis. (A) RNA was isolated from WT NK92 or MCM10-KD cells and qPCR measurement of MCM10 mRNA was performed. Pooled data from 4 independent experiments done in quadruplicate. (B) WT and MCM10-KD NK92 cells were lysed and probed for MCM10 protein and actin as a loading control. Quantification of MCM10 protein normalized to loading control is shown below each lane. Data are representative of 3 technical replicates performed on different days. (C) WT and MCM10-KD NK92 cells were labeled with BRDU and 7-AAD, and cell cycle was analyzed by FACS. (D) Quantification of the frequency of cells in early S phase relative to the WT control is shown from 3 technical replicates performed on different days. *P < 0.05, 1-sample t test and Wilcoxon’s test. (E) Cell-doubling time was calculated by enumerating cells in culture. Data show 6 technical replicates performed on different days. **P < 0.01, Mann-Whitney U test. Data are represented as mean ± 95% CI.