Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency
Laure Gineau, … , Jean-Laurent Casanova, Emmanuelle Jouanguy
Laure Gineau, … , Jean-Laurent Casanova, Emmanuelle Jouanguy
Published February 22, 2012
Citation Information: J Clin Invest. 2012;122(3):821-832. https://doi.org/10.1172/JCI61014.
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Research Article

Partial MCM4 deficiency in patients with growth retardation, adrenal insufficiency, and natural killer cell deficiency

Abstract

Natural killer (NK) cells are circulating cytotoxic lymphocytes that exert potent and nonredundant antiviral activity and antitumoral activity in the mouse; however, their function in host defense in humans remains unclear. Here, we investigated 6 related patients with autosomal recessive growth retardation, adrenal insufficiency, and a selective NK cell deficiency characterized by a lack of the CD56dim NK subset. Using linkage analysis and fine mapping, we identified the disease-causing gene, MCM4, which encodes a component of the MCM2-7 helicase complex required for DNA replication. A splice-site mutation in the patients produced a frameshift, but the mutation was hypomorphic due to the creation of two new translation initiation methionine codons downstream of the premature termination codon. The patients’ fibroblasts exhibited genomic instability, which was rescued by expression of WT MCM4. These data indicate that the patients’ growth retardation and adrenal insufficiency likely reflect the ubiquitous but heterogeneous impact of the MCM4 mutation in various tissues. In addition, the specific loss of the NK CD56dim subset in patients was associated with a lower rate of NK CD56bright cell proliferation, and the maturation of NK CD56bright cells toward an NK CD56dim phenotype was tightly dependent on MCM4-dependent cell division. Thus, partial MCM4 deficiency results in a genetic syndrome of growth retardation with adrenal insufficiency and selective NK deficiency.

Authors

Laure Gineau, Céline Cognet, Nihan Kara, Francis Peter Lach, Jean Dunne, Uma Veturi, Capucine Picard, Céline Trouillet, Céline Eidenschenk, Said Aoufouchi, Alexandre Alcaïs, Owen Smith, Frédéric Geissmann, Conleth Feighery, Laurent Abel, Agata Smogorzewska, Bruce Stillman, Eric Vivier, Jean-Laurent Casanova, Emmanuelle Jouanguy

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Figure 4

Functionality of the MCM4 isoforms detected in the cells of the patient.

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Functionality of the MCM4 isoforms detected in the cells of the patient....
(A) The Triton X–extractable fraction from patient and control SV40 fibroblasts was subjected to immunoprecipitation with a monoclonal antibody against MCM2. Protein extracts and immunoprecipitates were analyzed by immunoblotting with antibodies against MCM4, MCM3, MCM5, and MCM6. MCM2 was used as a loading control. (B) The chromatin-bound fraction was subjected to immunoprecipitation with a monoclonal antibody against MCM2. This procedure was carried out on the DNase I–extracted fractions of both control cells and cells from the patient. Protein extracts and immunoprecipitates were analyzed by immunoblotting with antibodies against MCM4, MCM3, MCM5, and MCM6. MCM2 was used as a loading control. Immunoprecipitation with IgG was used as a negative control. (C) Representative flow cytometry plots of the cell cycle of SV40 fibroblasts from controls and patients. Control (left), P1.3 (middle), and P2.1 (right) cell cycles in the absence of treatment. Transformation of cells with SV40 T antigen causes increased ploidy of all cells (59). P1 corresponds to normal G1 phase, P2+P3+P4 correspond to normal S phase, P5 corresponds to normal G2 phase plus abnormal G1 or failed mitosis, P6+P7 correspond to re-replication S phase, and P8 corresponds to 8C (P8) DNA content. Patients’ SV40 fibroblasts with or without 0.3 μM aphidicolin (Aph) treatment. (D) Representative chromosome spreads of chromosome breaks induced with or without aphidicolin. A WT metaphase chromosome without aberrations (left); a P1.3 metaphase with some aberrations indicated by blue arrowhead (middle); and a P1.3 metaphase after aphidicolin treatment, with chromosomal aberrations indicated by arrowheads (right). Blue arrowheads indicate chromatid breaks, and red arrowheads indicate chromosome exchanges. Bottom: Chromosome breaks (mean) per metaphase in P1.3 and P2.1 SV40 fibroblasts and in control SV40 fibroblasts. Complementation by lentiviral transduction with the WT MCM4 allele, the empty vector, or the c.70_71insG allele in P1.3 SV40 fibroblasts. Error bars indicate SEM. ***P < 0.0005, Student’s t test.