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.


| INTRODUC TI ON
Premature aging syndromes are rare disorders with clinical features that mimic physiological aging at an early age. The known causative genes are related to the maintenance and repair of genomic DNA (Vijg & Suh, 2013). A failure of their genomic integrity is considered a primary driving force of human aging (Vijg & Suh, 2013).
Aneuploidy, the presence of an abnormal number of chromosomes, is an example of the failure of genomic integrity. The number of aneuploid cells increases with aging in various organs (Duncan et al., 2012;Jacobs et al., 1961;Rehen et al., 2005). However, it is not known whether an increased number of aneuploid cells directly drive aging in humans. To date, three causative genes, BUBR1, CEP57, and TRIP13, have been found to induce mosaic variegated aneuploidy (MVA) syndromes that show random aneuploidy in a significant percentage of somatic cells (Hanks et al., 2004;Snape et al., 2011;Yost et al., 2017). Increased risk for carcinogenesis, congenital abnormalities, including intrauterine growth retardation, and infantile death are major symptoms in those MVA syndromes. Only tissue-specific premature aging phenotypes, including cataracts, have been reported in those MVA syndromes, while Bubr1 hypomorphic mice show MVA and systemic premature aging phenotypes, including kyphosis, muscle atrophy, dermal thinning, heart failure, and loss of fat tissues (Baker et al., 2004).
Here, we present a patient exhibiting premature aging of various organs, early stem cell loss, and MVA. We identified a missense variant of CDC20 that occurred de novo and characterized the variant, which induced spindle assembly checkpoint (SAC) failure and MVA.
The findings of this case could provide the basis for a novel disease model of human aging.

| RE SULTS
The patient, a 54-year-old Japanese woman (II-1 in Figure 1a), was born healthy from healthy parents but gradually developed growth retardation without mental retardation (Figure 1b); dry and atrophic skin with hyper-and hypo-pigmented macules, atrophic uvula, and oral mucosa; and hair loss on the scalp and body before she reached 20 years of age (Figure 1c-e and Figure S1). From age 20 to 50, she developed bilateral renal atrophy, bilateral cataracts, femur head necrosis, kyphosis, anemia, cardiac insufficiency, and thyroid atrophy (Tables S1 and S2; and Supplemental Text 1). She was cancerfree until uterine endometrial carcinoma appeared at age 48, which was completely excised and without recurrence 5 years after the operation without adjuvant therapy. Bone marrow biopsy at age 50 revealed replacement of bone marrow by fat tissue and a marked decrease in colony-forming efficiency of hematopoietic cells (Figure 1f, g). The patient showed no neurodevelopmental, cognitive, memory, or other neurological defects. No immunodeficiency or autoimmunity was identified.
The patient showed complete female genitalia but had a history of surgical extrusion of streak gonads and renal failure, and thus, Frasier syndrome was first suspected (Barbaux et al., 1997).
Karyotype analyses and genomic sequencing revealed the pa-  Table S3) with no detectable chromosome breakage (0/250 cells). Premature chromatid separation (PCS) was observed in ~6% of cells (12/200 cells), while this was <2% in a normal cohort (Kajii et al., 1998). Micronuclei, the hallmark of chromosome missegregation, was observed in 2.5% of cells (Table S4, control: 0.7 ± 0.2% [mean ± SEM], n = 3). Treatment of the patient's PBMCs with nocodazole, a microtubule-depolymerizing reagent, showed the accumulation of octoploid cells (Figure 2c), indicating aberrant cell cycle exit from metaphase due to SAC failure. Thus, we diagnosed the patient with MVA syndrome and decided not to administer adjuvant chemotherapy using taxanes after surgery for uterine endometrial carcinoma at the age of 48 years.
Typical symptoms of the known MVA syndromes caused by mutations in BUBR1, CEP57, and TRIP13 are intrauterine growth retardation, mental retardation, microcephaly, and childhood cancer (Hanks et al., 2004;Snape et al., 2011;Yost et al., 2017), none of which were observed in the patient (Table S1). Exome sequencing of genomic DNA from the patient revealed no rare variants in the causative genes of known MVA syndromes and no significant accumulation of somatic mutations. Screening for genes related to premature aging syndromes revealed that the patient was a heterozygous carrier of several rare variants for Rothmund-Thomson syndrome and Fanconi anemia (Table S5), but these are autosomal-recessive diseases and their phenotypes differ from those of the patient. Exome sequencing revealed that the patient possessed nine homozygous, hemizygous, or compound heterozygous pairs of rare variants and three de novo germline monoallelic variants ( Figure S2, Table S6). Among them, we focused on the de novo c.856C>A (p.R286S) variant in CDC20 (RefSeq: NM_001255.3/ NP_001246.2) and homozygous inframe deletion of c.862+3G>C (p.N235_N287del) in CENPT (RefSeq: NM_025082.4/ NP_079358.3, see Supplemental Text 2). CDC20 is a component of the mitotic checkpoint complex (MCC) and a cofactor of the anaphase-promoting complex/cyclosome (APC/C) (Yu, 2007).
CENPT is a kinetochore-localizing protein important for kinetochore assembly and function (Nishino et al., 2013). The patient's younger sister was a heterozygous carrier of the CENPT variant and did not have the CDC20 variant ( Figure S2). The CENPT variant correctly localized to the kinetochore in the Epstein-Barr virus-transformed lymphoblastoid cell line of the patient ( Figure S3). Because the biallelic defect of CENPT causes a syndrome characterized by severe growth failure and microcephaly without chromosome number instabilities (Hung et al., 2017), which differed from the patient's phenotypes (Table S7), we further investigated the CDC20 variant in this study.
CDC20 binds to BUBR1 in the formation of the MCC and in the inhibition of the APC/C via the MCC. The R286 residue of CDC20 locates at the binding site to BUBR1 and is conserved among eukaryotes (Figure 2d-f); therefore, we examined whether the p.R286S | 3 of 13 FUJITA eT Al.
variant affects SAC via knocking in the variation to HCT116 cells ( Figure S4). Knocked-in cells showed allele dosage-dependent slippage from nocodazole-induced metaphase arrest and accumulation of octoploid cells (Figure 3a and Figure S5a). By contrast, the induction of a monoallelic frameshift mutation to CDC20 did not induce SAC failure ( Figure S6), suggesting that the p.R286S variant but not haploinsufficiency of CDC20 specifically induced SAC failure. Furthermore, a significant correlation was observed between the knocked-in allele dosage of CDC20 p.R286S and aneuploid cell ratios in HCT116 clones (F(2,6) = 5.824, p = 0.0393 by one-way ANOVA, Figure 3b and Figure S5b), although aneuploid cells were spontaneously observed in the parent clone of the HCT116 cells.
These results indicate the CDC20 p.R286S variant induces SAC failure and MVA.
To determine how the p.R286S variant induces SAC failure, we investigated the interaction between CDC20 and BUBR1. One BUBR1 molecule binds to two CDC20 molecules, namely CDC20 MCC and CDC20 APC ; the former is included in the MCC, which consists of BUBR1, CDC20 MCC , BUB3, and MAD2. The latter is included in the APC/C-CDC20 complex (APC/C CDC20 ) (Izawa & Pines, 2015). MCC binding to CDC20 APC via BUBR1 inhibits the ubiquitination activity of APC/C CDC20 and the degradation of APC/C substrates, such as cyclin B1, and therefore blocks cell cycle exit from mitosis (Izawa & Pines, 2015). 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 × 10 4 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 uniterythroid; CFU-GM, colony-forming unit-granulocytes/macrophages. . Among them, the predicted structural interaction model suggests KEN1 and KEN2 of BUBR1 are faced to the R286 residues of CDC20 MCC and CDC20 APC , respectively (Alfieri et al., 2016) (Figure 3c).
An immunoprecipitation assay using FLAG-tagged BUBR1 fragments and Venus-tagged CDC20 (Figure 3d- fragment, a middle part of BUBR1 responsible for CDC20 recruitment to the kinetochores (Lischetti et al., 2014), showed no differences  the immunoprecipitation of wild-type CDC20 (Figure 3e, f). Therefore, the KEN1-mediated binding to CDC20, but not the KEN2-mediated binding to another CDC20, was affected in the interaction between BUBR1 and the CDC20 p.R286S variant. The CDC20 p.R286A variant, which had been reported to show lower binding affinity to BUBR1 (Tian et al., 2012) and was used as a control, showed similar specific reduction in the KEN1-mediated binding between BUBR1 and CDC20 ( Figure 3e, f). These results indicate that the R286 residue of CDC20 is specifically responsible for the KEN1-mediated binding of BUBR1 to CDC20 MCC , but not KEN2-mediated binding to CDC20 APC . The p.R286S CDC20 variant was suggested to induce aberrant activation of the APC/C CDC20 , possibly via insufficient formation of MCC to inhibit APC/C CDC20 , resulting in SAC failure and MVA in the patient ( Figure 4).

| DISCUSS ION
To the best of our knowledge, this is the first reported case of systemic premature aging associated with SAC failure and MVA in humans. The de novo-occurring monoallelic p.R286S missense variant of CDC20 is considered pathogenic. The patient data suggest that aging could be accelerated by increased numbers of aneuploid cells in humans.
Several nonsense mutations of CDC20 have been registered in human exome databases (dbSNP, 2019). No SAC failure was observed in heterozygous CDC20 knockout human cells ( Figure S6), suggesting that heterozygous CDC20-knockout humans likely exist in the general population without showing SAC failure and MVA phenotype. Cdc20-null mice were embryonic lethal at the two-cell stage (Li et al., 2007;Manchado et al., 2010). Tamoxifen-induced conditional knockout of Cdc20 in mice during the developmental stage revealed severe developmental defects with widespread metaphase arrest in proliferating cells caused by anaphase onset failure (Manchado et al., 2010). In contrast, patient cells investigated in this study showed SAC failure, which accelerate cells to exit metaphase and enter anaphase.
Therefore, the cellular phenotypes induced by the monoallelic p.R286S missense mutation of CDC20 are different from the phenotypes induced by CDC20 haploinsufficiency or CDC20 deletion.
Our results strongly suggest that a specific missense mutation of the R286 residue of CDC20 induced SAC failure and MVA by affecting the KEN1-mediated binding of BUBR1 to CDC20 MCC , but not affecting the KEN2-mediated binding to CDC20 APC . This may explain the extreme rarity of the disease because only specific missense mutations on a specific residue may cause the disease.
It is likely that instability of the MCC caused by the lower binding affinity of BUBR1 to the CDC20 p.R286S mutant resulted in insufficient inhibition of the APC/C CDC20 by MCC in the patient, for example, via aberrant early dissociation of the MCC from the APC/ C CDC20 or an imbalance between the MCC and APC/C CDC20 ratio ( Figure 4). Because the patient has one normal CDC20 allele, aberrant activation of the APC/C CDC20 is supposed to be less frequent in the patient than in the MVA syndrome patients caused by biallelic BUBR1 mutations. This hypothesis likely explains why the frequency and extent of MVA and PCS were relatively low in the patient compared with BUBR1-deficient MVA syndrome patients (Table S1), and why the patient did not show severe anomalies, childhood cancer, and early death.
The phenotype of systemic premature aging and being XY female were the major clinical differences in our patient compared with known MVA syndromes (  (Baker et al., 2004(Baker et al., , 2006Wijshake et al., 2012). Various cellular stresses, including proteotoxic and metabolic stresses, are known to be induced by aneuploidy in in vitro cultured cells (Santaguida & Amon, 2015).
Early exhaustion of stem cells has been observed in tissue-specific knockout mice of Mad2, a component to form MCC with Bubr1 and Cdc20 MCC (Foijer et al., 2013;Kollu et al., 2015). where APC/C CDC20 is associated with self-renewal activity of stem cells by interacting with pluripotency-related transcription factor SOX2 (Mao et al., 2015). Further studies are required to evaluate the non-mitotic function of the APC/C CDC20 in the context of stem cell maintenance.

| Exome sequencing
Exome sequencing was performed as described previously (Kubo et al., 2013;Takenouchi et al., 2015Takenouchi et al., , 2016. Exome sequencing produced ~90,000,000 paired reads per sample, of which ~99.76% were mapped to the hs37d5 exon region of the human genome sequence assembly. The average coverage of the targeted exonic region was 97.22×, with more than 98.94% of targeted bases covered at over 10× reads. Candidate variants were screened using the auto-

| Sanger sequencing
The target DNA fragments were amplified by PCR using HotStarTaq DNA Polymerase (Qiagen) and subjected to direct Sanger sequencing. The sequences of PCR primer pairs are listed in Table S8.   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). Scientific) supplemented with 10% FBS, 100 units ml −1 penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Sigma).

F I G U R E 3
Each cell line was maintained at 95% humidified air, 5% CO 2 , and 37°C.
To construct the pCAGGS-Venus-hCDC20 plasmid encoding, the fusion protein of Venus (Nagai et al., 2002) and human CDC20, the human CDC20 open reading frame (ORF) was isolated from the pCMV7.1-3×FLAG-CDC20 plasmid (Miyamoto et al., 2011) by EcoRI digestion and subcloned into the pCAGGS vector (Niwa et al., 1991) with To construct the pCMV7.1-3×FLAG-hBUBR1 plasmid encoding 3×FLAG-tagged human BUBR1, human BUBR1 ORF was PCRamplified from the pEGFP-hBUBR1 plasmid (Miyamoto et al., 2011) using a primer set with a Kpn1 site at the 5′-end and a BamHI site at the 3'-end and then was subcloned into the pCMV7.1-3×FLAG vector (Sigma). To construct the plasmids encoding the 3×FLAGtagged truncated human BUBR1 proteins, BUBR1 fragments were PCR-amplified from pCMV7.1-3×FLAG-hBUBR1 using primer sets with a BamHI site at the 5′-end and a NotI site at the 3′-end. These fragments were subcloned into a modified pCMV7.1-3×FLAG vector, which had a HindIII-BamHI-NotI-SmaI multi-cloning site.
All constructs used in this study were verified by Sanger sequencing. The primer sequences are listed in Table S8. The molecular weight and antibody reactivity of the product proteins were verified by Western blotting ( Figure S8).

G C T T G C T T G C A T T T G G T G C T G C C A C A G A A C C T GAT TCCCT TCT T TCCTCCTCCAGTGGATCCCGT TCCGGACAC ATCC ACC ACC ATG ATG T TCG G G TAG C AG A AC ACC ATG TG G C C A C A C T G A G T G G C C A C A G C C A G G A A G T G T -3 ' ;
CDC20 p.R286S mutant ssODN for sgRNA-E2: 5'-GCTCT

G G C T T G C T T G C A T T T G G T G C T G C C A C A G A A C C T G A T T C C C T T C T T T C C T C C T C C A G T G G A T C C A G T T C G G A C A C A T C C A C C A C C A T G A T G T T C G G G T A G C A G A A C A C C A T G T G G C C A C A C T G A G T G G C C A C A
GCCAGGAAGTGT-3'; CDC20 WT ssODN for sgRNA-E3: 5'-CTGG

C T T G C T T G C A T T T G G T G C T G C C A C A G A A C C T G A T T C C C T T C T T T C C T C C T C C A G T G G A T C C C G T T C T G G T C A T A T C C A C C A C C A T G A T G T T C G G G T A G C A G A A C A C C A T G T G G C C A C A C T G A G T G
GCCACAGCCAGGAAGTGTGTG-3'; CDC20 p.R286S mutant ssODN for sgRNA-E3: 5'-CTGGCTTGCTTGCATTTGGTGCTGC

C A C A G A A C C T G A T T C C C T T C T T T C C T C C T C C A G T G G A T C C A G T T C T G G T C A T A T C C A C C A C C A T G A T G T T C G G G T A G C A G A A C A C C A T G T G G C C A C A C T G A G T G G
CCACAGCCAGGAAGTGTGTG-3.' The transfected cells were plated onto two 100-mm dishes. After 24-h incubation, the cells were treated with puromycin (0.75 μg/ml) for 48 h and then further incubated with puromycin-free media for 2 weeks, after which the colonies were picked. To screen for positive clones, genomic DNA was extracted from each clone by the phenol/ chloroform method, and the target locus was amplified by PCR using HotStarTaq and the CDC20 ex8F/R primer pair. PCR products were further digested with BamHI or BspEI restriction enzymes and electrophoresed on agarose gels. Positive clones were further confirmed by direct Sanger sequencing.

| Chromosome analyses
Karyotype analyses and premature chromatid separation analyses of the patient blood sample were performed at the LSI Medience Corporation. For chromosome count analyses of the HCT116 clones, the cell cycle was arrested with 0.12 μg/ml colcemid for 1 h and then treated with 8 ml 0.075 M KCl at room temperature for 20 min.
We added 2 ml Carnoy's solution, and the cells were incubated for 10 min and washed two or three times with Carnoy's solution. The chromosomes were spread onto glass slides using a HANABI metaphase spreader (ADSTEC).

| Cytokinesis-block micronucleus (CBMN) assay
The CBMN assay was performed as described previously (Fenech, 2007). Briefly, PBMCs were isolated from the peripheral blood of the patient and healthy controls by gradient centrifugation using

| Colony-forming unit assay of bone marrow cells
The patient bone marrow cells were obtained from the remainder of the bone marrow aspiration sample, and mononuclear cells were isolated by gradient centrifugation using Lymphoprep (Axis-Shield). An age-matched normal control bone marrow mononuclear cell sample was obtained from AllCells. Cells were cultured in MethoCult H4034 Optimum (STEMCELL Technologies) at 1 × 10 4 or 2 × 10 4 cells per 35 mm dish at 95% humidified air, 5% CO 2 , and 37°C for 14 days, after which the colony numbers were counted.

| Immunoprecipitation analyses
The 293 T cells were plated onto a 35-mm dish and transiently transfected with plasmids encoding 3×FLAG-tagged truncated BUBR1 fragments and Venus-CDC20 fusion proteins by Lipofectamine LTX with PLUS reagent (Thermo Fisher Scientific). One day after transfection, the cells were lysed with Triton X-100-based lysis buffer (0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA with a 1/100 concentration of protease inhibitor cocktail (Sigma)). The lysates were sheared with a 23 G needle, incubated on ice for 30 min, and centrifuged at 20,000 × g for 10 min at 4°C.
The supernatants were incubated with anti-FLAG M2 Affinity Gel

| Fluorescence imaging analyses of cultured cells
To analyze the localization of the Venus-CDC20 fusion protein in the HCT116 cell line, cells were seeded onto coverslips (Matsunami Glass) coated with poly-L-lysine (Sigma) and transiently transfected with plasmids encoding WT or p.R286S mutant CDC20 protein fused to Venus fluorescent protein (Nagai et al., 2002)

| Statistics
Statistical significance of the differences in the mitotic indices and the percentages of aneuploid cells between CRISPR-mediated HCT116 mutant clones shown in Figure 3 were analyzed with a oneway analysis of variance (ANOVA) and the post hoc Tukey's multiple comparison test using Prism software (ver.6; GraphPad Software).

| 3D modeling of the protein structure
All 3D images of the protein structures and their interactions were generated using the PyMOL Molecular Graphics System, Version 2.0.7 (Schrödinger LLC) (https://pymol.org). For Figure 2f, the 3D structural model of CDC20 was reconstituted from the published Protein Data Bank (PDB) file ID: 4GGD (Tian et al., 2012). For Figure 3c, the 3D structural model of the APC/C-CDC20-MCC interaction was reconstituted from the published PDB file ID: 5LCW (Alfieri et al., 2016).

ACK N OWLED G M ENTS
We are grateful to the patient and her family for their participation and kind contribution to this study. We thank for Nobuyo Nishimura, and "Program for an Integrated Database of Clinical and Genomic Information" (Grant Number JP18kk0205002).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.