Premature aging syndrome showing random chromosome number instabilities with CDC20 mutation

Abstract

Damage to the genome can accelerate aging. The percentage of aneuploid cells, that is, cells with an abnormal number of chromosomes, increases during aging; however, it is not clear whether increased aneuploidy accelerates aging. Here, we report an individual showing premature aging phenotypes of various organs including early hair loss, atrophic skin, and loss of hematopoietic stem cells; instability of chromosome numbers known as mosaic variegated aneuploidy (MVA); and spindle assembly checkpoint (SAC) failure. Exome sequencing identified a de novo heterozygous germline missense mutation of c.856C>A (p.R286S) in the mitotic activator CDC20. The mutant CDC20 showed lower binding affinity to BUBR1 during the formation of the mitotic checkpoint complex (MCC), but not during the interaction between MCC and the anaphase-promoting complex/cyclosome (APC/C)-CDC20 complex. While heterozygous knockout of CDC20 did not induce SAC failure, knock-in of the mutant CDC20 induced SAC failure and random aneuploidy in cultured cells, indicating that the particular missense mutation is pathogenic probably via the resultant imbalance between MCC and APC/C-CDC20 complex. We postulate that accelerated chromosome number instability induces premature aging in humans, which may be associated with early loss of stem cells. These findings could form the basis of a novel disease model of the aging of the body and organs.

Keywords: Cdc20 proteins; M phase cell cycle checkpoints; aging; chromosomal instability; chromosome segregation; genomic instability; premature.

Figure 1

Clinical and cellular phenotypes of the patient. (a) The patient's family tree. (b) The patient's growth curve of height and weight plotted on a standard growth chart of a Japanese girl (Ministry of Health, 2010a, 2010b). Colored lines correspond to the 97th, 90th, 75th, 50th, 25th, 10th, and 3rd percentiles from top to bottom. (c–e) Clinical photographs of the patient showing the loss of scalp hair, eyelashes and eyebrows (c), dry, atrophic skin (d), and atrophic tonsils and uvula (e) at age 51. (f) Hematoxylin and eosin staining of a bone marrow biopsy section. Scale = 100 µm. (g) The numbers of colonies per 1 × 10⁴ bone marrow mononuclear cells in the patient and an age‐matched control (means ± SEM from six technical replicates from each subject). BFU‐E, burst‐forming unit–erythroid; CFU‐E, colony‐forming unit–erythroid; CFU‐GM, colony‐forming unit–granulocytes/macrophages.

Figure 2

Chromosome number instability and CDC20 mutation in the patient's peripheral blood mononuclear cells. (a) Chromosome number instability observed in the patient's peripheral blood mononuclear cells (PBMCs). (b) A representative karyotype of the patient's PBMCs. Red squares indicate chromosome gain or loss. (c) Flow‐cytometric analyses of DNA contents in the PBMCs of the patient and a healthy control with or without mitotic stimulation and nocodazole treatment. X‐axes indicate nuclear DNA contents (2C, diploid; 4C, tetraploid; 8C, octoploid) stained with 7‐amino‐actinomycin D (7‐AAD) on a linear scale. (d) A schematic representation of the human CDC20 protein. The R286 amino acid residue is indicated with a black arrowhead. MIM, MAD2 interacting motif. (e) A cross‐species amino acid sequence comparison of CDC20 around the R286 residue (red). Identical and conserved amino acids are marked with black and gray boxes, respectively. (f) Side and top view of the WD40 repeat propeller domains (WD1–7) of the human CDC20 protein. The R286 residue (orange stick) is located at the top of WD3.

Figure 3

The impact of the CDC20 variant on spindle assembly checkpoint and interaction with BUBR1. (a) The mitotic indices after 12‐h nocodazole treatment of an HCT116 parent clone and its CRISPR‐mediated mutant clones harboring either the wild‐type (WT) silence mutation or the p.R286S mutation in CDC20 (means ± SEM from three independent experiments). The numbers are the code numbers of the mutant clones. (b) The percentage of aneuploid cells in the parent and mutant clones (50 metaphase spreads were counted per clone). (c) The 3D structural model of the mitotic checkpoint complex (MCC) interacting with anaphase‐promoting complex/cyclosome (APC/C) (left panel) through the binding of BUBR1 with CDC20 s consisting of the APC/C‐CDC20 complex and MCC (CDC20_APC and CDC20_MCC, respectively, right upper panel). KEN boxes (KEN1 and KEN2), destruction boxes (D1 and D2), and ABBA motifs (A1, A2, and A3) on BUBR1 are shown with red, blue, and yellow, respectively. The detailed spatial relationships of KEN boxes with the R286 residue of CDC20_MCC and CDC20_APC are shown in right lower panels. (d) Schematics of the BUBR1 deletion constructs used in immunoprecipitation. Colored lines indicate KEN1, KEN2, destruction boxes (D box; R224STL and R555RPL), and ABBA motifs (I272TVFDE, F340TPYVE, and F528SIFDE). (e) Immunoprecipitation of Venus‐tagged wild type (WT), p.R286S, and p.R286A variants of CDC20 precipitated by 3×FLAG‐tagged BUBR1 deletion constructs shown in d using anti‐FLAG antibody. Upper three rows show Western blotting of the total cell lysates, and lower two rows show Western blotting of the immunoprecipitants. Tags used for the detection and molecular weight indicators (kilodaltons) are shown on the left and right sides of the blot, respectively. The uncropped images are shown in Figure S9. (f) The relative ratios of the band densities for the p.R286S and p.R286A variants of CDC20 compared with WT CDC20 in e (means ± SEM from three independent experiments). p‐values, ANOVA/Tukey's test.

Figure 4

Hypothetical models of the aberrant activation of the APC/C‐CDC20 complex. Hypothetical models of APC/C inhibition by MCC focusing on the interaction of BUBR1 with wild type (blue) and p.R286S variant (red) of CDC20. BUB3 and MAD2 in MCC are not shown for simplification. (a) The increased frequency of stochastic dissociation of the p.R286S variant of CDC20_MCC from BUBR1 induces aberrant dissociation of MCC from the APC/C‐CDC20 complex (APC/C^CDC20), resulting in aberrant entry into anaphase and unequal chromosome segregation (WT/R286S). Haploinsufficiency of CDC20 (WT/KO) does not induce this aberrant dissociation of MCC. (b) Balanced inhibition of the APC/C^CDC20 by MCC prohibits aberrant entry into anaphase in wild type (WT/WT) and in haploinsufficiency of CDC20 (WT/KO). Reduction in the binding affinity of the CDC20 p.R286S variant to BUBR1 specifically in the formation of MCC but not of the APC/C^CDC20 induces a molecular imbalance between MCC and the APC/C^CDC20, resulting in aberrant entry into anaphase (WT/R286S).