RESUMEN
Natural killer (NK) cell deficiency (NKD) is a rare disease in which NK cell function is reduced, leaving affected individuals susceptible to repeated viral infections and cancer. Recently, a patient with NKD was identified carrying compound heterozygous variants of MCM10 (minichromosome maintenance protein 10), an essential gene required for DNA replication, that caused a significant decrease in the amount of functional MCM10. NKD in this patient presented as loss of functionally mature late-stage NK cells. To understand how MCM10 deficiency affects NK cell development, we generated MCM10 heterozygous (MCM10+/-) induced pluripotent stem cell (iPSC) lines. Analyses of these cell lines demonstrated that MCM10 was haploinsufficient, similar to results in other human cell lines. Reduced levels of MCM10 in mutant iPSCs was associated with impaired clonogenic survival and increased genomic instability, including micronuclei formation and telomere erosion. The severity of these phenotypes correlated with the extent of MCM10 depletion. Significantly, MCM10+/- iPSCs displayed defects in NK cell differentiation, exhibiting reduced yields of hematopoietic stem cells (HSCs). Although MCM10+/- HSCs were able to give rise to lymphoid progenitors, these did not generate mature NK cells. The lack of mature NK cells coincided with telomere erosion, suggesting that NKD caused by these MCM10 variants arose from the accumulation of genomic instability including degradation of chromosome ends.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Diferenciación Celular , Genes Esenciales , Inestabilidad Genómica , Células Asesinas Naturales , Proteínas de Mantenimiento de MinicromosomaRESUMEN
Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients.
Asunto(s)
Alelos , Cardiomiopatías/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/inmunología , Acortamiento del Telómero , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Humanos , Células Asesinas NaturalesRESUMEN
Deoxyribonucleic acid (DNA) replication can be divided into three major steps: initiation, elongation and termination. Each time a human cell divides, these steps must be reiteratively carried out. Disruption of DNA replication can lead to genomic instability, with the accumulation of point mutations or larger chromosomal anomalies such as rearrangements. While cancer is the most common class of disease associated with genomic instability, several congenital diseases with dysfunctional DNA replication give rise to similar DNA alterations. In this review, we discuss all congenital diseases that arise from pathogenic variants in essential replication genes across the spectrum of aberrant replisome assembly, origin activation and DNA synthesis. For each of these conditions, we describe their clinical phenotypes as well as molecular studies aimed at determining the functional mechanisms of disease, including the assessment of genomic stability. By comparing and contrasting these diseases, we hope to illuminate how the disruption of DNA replication at distinct steps affects human health in a surprisingly cell-type-specific manner.
Asunto(s)
Replicación del ADN/genética , Inestabilidad Genómica/genética , Neoplasias/genética , RecQ Helicasas/genética , Craneosinostosis/genética , Craneosinostosis/inmunología , Replicación del ADN/inmunología , Inestabilidad Genómica/inmunología , Humanos , Mutación/genética , Neoplasias/epidemiología , Fenotipo , RecQ Helicasas/inmunologíaRESUMEN
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.
Asunto(s)
Células Asesinas Naturales/inmunología , Proteínas de Mantenimiento de Minicromosoma/genética , Mutación , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/inmunología , Alelos , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Codón sin Sentido , Daño del ADN/genética , Daño del ADN/inmunología , Resultado Fatal , Femenino , Técnicas de Silenciamiento del Gen , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Lactante , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Masculino , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Modelos Inmunológicos , Mutación Missense , Linaje , Enfermedades de Inmunodeficiencia Primaria/patologíaRESUMEN
Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga de MutS/genética , Proteína 3 Homóloga de MutS/metabolismo , Mutación , Mapeo de Interacción de Proteínas , Proteína de Replicación A/genética , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/genética , Rayos UltravioletaRESUMEN
Cytomegalovirus (CMV) subunit vaccine candidates include glycoprotein B (gB), and phosphoprotein ppUL83 (pp65). Using a guinea pig cytomegalovirus (GPCMV) model, this study compared immunogenicity, pregnancy outcome, and congenital viral infection following pre-pregnancy immunization with a three-dose series of modified vaccinia virus Ankara (MVA)-vectored vaccines consisting either of gB administered alone, or simultaneously with a pp65 homolog (GP83)-expressing vaccine. Vaccinated and control dams were challenged at midgestation with salivary gland-adapted GPCMV. Comparisons included ELISA and neutralizing antibody responses, maternal viral load, pup mortality, and congenital infection rates. Strikingly, ELISA and neutralization titers were significantly lower in the gB/GP83 combined vaccine group than in the gB group. However, both vaccines protected against pup mortality (63.2% in controls vs. 11.4% and 13.9% in gB and gB/GP83 combination groups, respectively; p<0.0001). Reductions in pup viral load were noted for both vaccine groups compared to control, but preconception vaccination resulted in a significant reduction in GPCMV transmission only in the monovalent gB group (26/44, 59% v. 27/34, 79% in controls; p<0.05). We conclude that, using the MVA platform, the addition of GP83 to a gB subunit vaccine interferes with antibody responses and diminishes protection against congenital GPCMV infection, but does not decrease protection against pup mortality.