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A compound heterozygous mutation in MCM10 causes NK cell deficiency

Natural killer cell deficiency (NKD) is a rare disease in which natural killer (NK) cell function is reduced, leaving affected individuals susceptible to repeated viral infections and cancer. Defects in several replication factors have been shown to cause a class of NKD that has complete loss of mature NK cells. Recently, a new NKD patient was identified with a compound heterozygous mutation in MCM10 (minichromosome maintenance 10), an essential gene required for DNA replication. In this patient, one allele contains a missense mutation of a highly conserved residue and the other has a nonsense mutation causing loss of function. To better understand how each mutation affects DNA replication, we generated MCM10 heterozygous HCT116 and hTERT‐RPE1 cell lines, mimicking the nonsense mutation in transformed and nontransformed cells. Analysis of these cell lines demonstrated that MCM10 was haploinsufficient, resulting in increased doubling time, enhanced sensitivity to genotoxic drugs, and reduced viability. Although the nonsense mutation was haploinsufficient in cellular systems, the patient inherited this allele from a healthy parent implying that the missense mutation is contributing to the clinical phenotype. Consistent with this notion, hTERT‐RPE1 cells homozygous for the missense mutation also displayed an increase in doubling time. These data suggested that the compound heterozygous MCM10 mutation led to loss of functionally mature NK cells due to reduced proliferative capacity. To better understand the molecular defects underlying the observed phenotypes, we further analyzed the heterozygous HCT116 mutants. Cell cycle analysis demonstrated delay in mitotic progression, but a relatively normal cell cycle distribution. Moreover, we observed defects in replication initiation and decreased replication fork restart following induction of fork stalling. Interestingly, these defects in DNA replication did not cause acute DNA damage suggesting that MCM10 haploinsufficiency led to minor defects in global DNA replication that remained below the threshold of checkpoint activation. Rather, the observed reduction in viability was associated with increased genomic instability over time including progressive telomere erosion. To uncover the molecular mechanisms of telomere shortening, we utilized 2D gel analyses which showed increased accumulation of telomeric “t‐complexes” in MCM10 mutants. This high molecular weight species consist of highly branched DNAs with single‐stranded regions that may result from stalled replication forks. Taken together, these data suggest that replication defects caused by MCM10 deficiency are exacerbated in hard‐to‐replicate, origin poor regions such as telomeres and cause replicative exhaustion. Currently, we are determining whether telomere erosion driven by MCM10 depletion prevents development of mature NK cells.