Disturbed chromosome segregation and multipolar spindle formation in a patient with CHAMP1 mutation

Abstract

Background: Patients with intellectual disability (ID) typically exhibit significant defects in both intelligence and adaptive behavior. Aberration of several genes involved in proper progression of mitosis has been reported to underlie ID. Here, we report a new patient with a novel mutation of CHAMP1.

Methods: Whole exome sequencing (WES) analysis was performed. We isolated lymphoblast cells from the CHAMP1 patient and observed chromosome segregation.

Results: We identified a de novo frameshift mutation in CHAMP1. We find that these cells exhibit an increase in centrosome number and resulting multipolar spindle formation. The phenotypes observed in the patient's lymphoblastoid cells were presumably because of cytokinesis failure. We also confirm the identical phenotypes in human culture cells depleted of CHAMP1.

Conclusion: CHAMP1 encodes a protein regulating kinetochore-microtubule attachment and chromosome segregation. These data strongly support that CHAMP1 mutations cause ID, and suggest that CHAMP1 is critical for progression of cytokinesis and maintenance of centrosome number.

Keywords: CHAMP1; intellectual disability (ID); lymphoblastoid cell; multipolar spindle.

Figure 1

Clinical features and cytological abnormalities in a patient with CHAMP1 mutation. (A) Brain MRI showed cavum septum pellucidum, cavum vergae, cerebral atrophy, and decreased white matter volume. (B) Patient at 6 years old. Craniofacial dysmorphic features, including microcephaly, brachycephaly, sparse eyebrow, prominent eyes, down slanting palpebral fissures, ectropion of lower eyelids, flat nasal bridge, anteverted nostrils, short philtrum, down turned corners of mouth were noted. (C) Abnormal spindle formation and chromosome misalignment of patient cells with CHAMP1 mutation. Lymphoblast cells from a healthy person and a patient with CHAMP1 mutation were stained with antibodies against α‐tubulin (green) and Cep192 (red). Nuclei are shown in blue. Insets show approximately twofold‐magnified views of Cep192 signals. White arrow indicates misaligned chromosomes. White arrowheads indicate excess Cep192 foci representing multipolar spindle. (D) Histograms represent frequency of mitotic cells with normal bipolar and abnormal spindles in lymphoblast cells from a healthy person and a patient with CHAMP1 mutation. (E) Pie graphs represent frequency of mitotic cells with the indicated Cep192 foci in each condition. (F) Centrosome amplification of patient cells with CHAMP1 mutation. Lymphoblast cells from a healthy person and a patient with CHAMP1 mutation were stained with antibodies against GT335 (green) and Cep192 (red). Nuclei are shown in blue. (G) The graph shows the signal intensity of GT335 (green) and Cep192 (red) at the centrosomes along the dotted line in representative panels. All Cep192 signals were overlapped with GT335 signals (n = 10 cells).

Figure 2

Depletion of CHAMP1 causes chromosome misalignment and the increased number of centrosomes during mitosis in human cancer cells. Depletion of CHAMP1 induces chromosome misalignment and instability of kinetochore–microtubules. U2OS (A) and HeLa (B) cells treated with control siRNA or siRNA targeting endogenous CHAMP1 for 72 h were stained with the indicated antibodies. Nuclei are shown in blue. White arrow indicates misaligned chromosomes. (C) Histograms represent frequency of mitotic HeLa cells with the indicated category in each condition. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). ***P < 0.001 (two‐tailed t‐test). (D) Depletion of CHAMP1 promotes formation of the excess centrosome foci during mitosis. U2OS cells treated with control siRNA or siRNA targeting endogenous CHAMP1 for 48 h were stained with the indicated antibodies. Nuclei are shown in blue. Cells were treated with MG132 for the last 2 h. (E) Histograms represent frequency of mitotic cells with multipolar spindles in each condition. Values are the mean percentages ± SEM from three independent experiments (n = 50 for each condition). *P < 0.05 (two‐tailed t‐test). (F) Centrosome amplification in CHAMP1‐depleted cells. HeLa cells treated with control siRNA or siRNA targeting endogenous CHAMP1 for 48 h were stained with the indicated antibodies. Nuclei are shown in blue. Cells were treated with MG132 for the last 2 h. (G) Depletion of CHAMP1 causes cytokinesis failure. Live imaging of cycling HeLa cells expressing GFP‐centrin1 (green) and treated with control siRNA or CHAMP1 siRNA. Scale bar = 5 μm. White arrowheads indicate the increased number of centrosomes. 5.8% of control cells showed cell death (n = 17), compared with 43.3% of CHAMP1‐depleted cells; 10% of CHAMP1‐depleted cells showed cytokinesis defects (n = 30).