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 5

R426C/R582X mutations lead to increased nuclear area and γH2AX staining in immortalized fibroblasts.

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R426C/R582X mutations lead to increased nuclear area and γH2AX staining ...
(A) Immortalized fibroblasts were fixed, permeabilized, and incubated with primary anti-MCM10 antibody followed by goat anti-mouse Alexa Fluor 488 secondary antibody and directly conjugated anti-γH2AX Alexa Fluor 647. Slides were mounted with ProLong Gold antifade media with DAPI and imaged by confocal microscopy. Scale bars: 10 μm. (B) MFI of γH2AX staining (left) and area of positive γH2AX signal (right) were measured in 28 to 35 cells per condition. ****P < 0.0001, unpaired t test (left) or Mann-Whitney U test (right). Data are represented as mean ± 95% CI. (C) Nuclear area was measured by positive DAPI staining in 31 (patient) and 46 (healthy donor) cells per condition. ****P < 0.0001, Mann-Whitney U test. Data are represented as mean ± 95% CI. (D) R426C or R582X variants were transiently overexpressed in 293T cells and prepared for microscopy, as described above. Nuclear area determined by DAPI staining was measured. *P < 0.05, Kruskal-Wallis with Dunn’s multiple comparison test. Data are represented as mean ± 95% CI. n = 69 (R426C); n = 95 (R582X, WT). (E) Healthy donor–derived (left) or patient-derived (right) immortalized fibroblast cells were labeled with BRDU and 7-AAD, and cell cycle was analyzed by FACS. All data shown are of 1 representative experiment of 3 technical replicates performed on different days.