Biallelic TRIP13 mutations predispose to Wilms tumor and chromosome missegregation

Abstract

Through exome sequencing, we identified six individuals with biallelic loss-of-function mutations in TRIP13. All six developed Wilms tumor. Constitutional mosaic aneuploidies, microcephaly, developmental delay and seizures, which are features of mosaic variegated aneuploidy (MVA) syndrome, were more variably present. Through functional studies, we show that TRIP13-mutant patient cells have no detectable TRIP13 and have substantial impairment of the spindle assembly checkpoint (SAC), leading to a high rate of chromosome missegregation. Accurate segregation, as well as SAC proficiency, is rescued by restoring TRIP13 function. Individuals with biallelic TRIP13 or BUB1B mutations have a high risk of embryonal tumors, and here we show that their cells display severe SAC impairment. MVA due to biallelic CEP57 mutations, or of unknown cause, is not associated with embryonal tumors and cells from these individuals show minimal SAC deficiency. These data provide insights into the complex relationships between aneuploidy and carcinogenesis.

Figure 1. TRIP13 loss-of-function mutations cause chromosome segregation errors and SAC deficiency

(a) Representative anaphases of immortalized control (upper) and TRIP13-mutant patient (lower) lymphoblasts expressing H2B-mNeon, showing a lagging chromosome in the lower panels indicated with a white arrow. (b) Quantification of chromosome segregation errors of lymphoblasts as visualized in (a). Each bar depicts the mean of 3-4 experiments ±SEM, >60 cells in total. P-values ≤ 0.05, from unpaired Student’s t-tests, are shown. Patient 1 cells show increased levels of chromosome segregation errors. (c) Representative images of H2B-mNeon expressing control (upper) and patient (lower) lymphoblasts going through mitosis (time in hours with mitotic entry at t=0.0) in the presence of nocodazole. Unlike control cell lines both patient cell lines have exited from mitosis after 1.5 hours (third column of second and third row panels). (d) Analysis of mitotic delay of cells as visualized in (c), indicating the cumulative percentage of cells that exited from mitosis as a function of time (mean of three experiments ±SEM, 10-30 cells per experiment). Both patient cell lines escaped mitotic arrest within one hour. Key: patient 1, ID_0644; patient 2, ID_7054; SEM, standard error of the mean

Figure 2. TRIP13 loss-of-function mutations cause reduced levels of MAD2 on unattached kinetochores

(a-c) Immunofluorescence labelling (a) and quantification (b-c) of indicated proteins in nocodazole-arrested control or patient lymphoblasts. Each dot represents one cell, with dots from separate experiments in different shades of grey. The red bar depicts the mean of four experiments ±SEM, >70 cells in total. P-values ≤ 0.05, from unpaired Student’s t-tests, are shown. Patient 1 cells have reduced kinetochore levels of MAD2, but not MAD1, compared to the controls. Key: patient 1, ID_0644; SEM, standard error of the mean

Figure 3. SAC deficiency and CIN caused by TRIP13 loss-of-function is rescued with wild type but not mutant TRIP13

(a) Quantification of chromosome segregation errors of TRIP13-mutated patient lymphoblasts expressing H2B-mNeon or co-expressing GFP-TRIP13 wt. Each bar depicts the mean of 3-4 experiments ±SEM, >85 cells in total. P-values ≤ 0.05 from an unpaired Student’s t-test are shown. Addition of GFP-TRIP13 wt to patient 1 cells reduced the rate of chromosome missegregation. (b) Analysis of mitotic delay as in (Fig 1d) of nocodazole-treated patient lymphoblasts expressing H2B-mNeon or co-expressing GFP-TRIP13 wt. Mean of three experiments ±SEM, >35 cells in total. Patient 1 cells expressing GFP-TRIP13 now maintain mitotic arrest. (c) Analysis of mitotic delay as in (Fig 1d) and (b) of nocodazole-treated HCT116 wt or HCT116 TRIP13 KO cells expressing H2B-mNeon, co-expressing GFP-TRIP13 wt, or co-expressing TRIP13 p.Arg354X. Mean of three experiments ± SEM, >45 cells in total. HCT116 wt and TRIP13 KO + TRIP13 wt cells both maintain mitotic arrest unlike TRIP13 KO and TRIP13 KO + TRIP13 p.Arg354X cells. Key: patient 1, ID_0644; wt, wild-type; KO, knockout; CIN, chromosomal instability; SAC, spindle assembly checkpoint; SEM, standard error of the mean

Figure 4. Patient cells with TRIP13 or BUB1B mutations have severely compromised SAC

Analysis of mitotic delay as in Fig 1d and Fig 3b of nocodazole-treated MVA patient lymphoblasts treated with far-red DNA dye to visualize the DNA, showing the cumulative percentage of cells that exited from mitosis as a function of time (mean of three experiments ±SEM, 20 cells per experiment). Only TRIP13-mutant and BUB1B-mutant patient cells rapidly escape from mitotic arrest. Key: SAC, spindle assembly checkpoint; SEM, standard error of the mean