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.


Cell culture
Preparation of Lymphoblast cells. Lymphoblast cells from the blood cells of a healthy person and a patient with CHAMP1 mutation were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS) at 37 ºC in a 5% CO 2 atmosphere. HeLa and U2OS cells were obtained from the ECACC (European collection of cell cultures). HeLa cells stably expressing GFP-centrin1 were generated as previously described [Piel, M et al. 2000]. HeLa and U2OS cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS at 37 ºC in 5% CO 2 incubator.

Antibodies
The following primary antibodies were used in this study: Guinea pig polyclonal

Microscopy
For immunofluorescence analysis, HeLa and U2OS cells cultured on coverslips (Matsunami: No 1 for confocal microscope) were fixed using -20ºC methanol for 10 minutes and washed with PBS. The cells were permeabilized after fixation with PBS/0.05% TritonX-100 (PBSX) for 5 minutes three times, and incubated for blocking in 1% BSA in PBSX for 30 minutes at room temperature (RT). The cells were then incubated with primary antibodies for 24 hours at 4 °C, washed with PBSX three times, and incubated with secondary antibodies for 1 hour at RT. The cells were thereafter washed with PBSX twice, stained with 0.2 µg ml -1 Hoechst 33258 (DOJINDO) in PBS for 5 minutes at RT, washed again with PBSX and mounted onto glass slides.
For specimen slide preparation of Lymphoblast cells, cell suspension was mixed 6 with Smear Gell (GenoStaff) and spread on the surface of slide. The same steps as above were repeated to perform immunofluorescence analysis of specimen slide preparation.
Counting the number of immunofluorescence signals was done using an Axioplan2 fluorescence microscope (Carl Zeiss) with a 100x/1.4 NA plan-APOCHROMAT objective. We assessed cells from several fields for each experiment. The investigators were normally blinded to the sample ID during experiments and outcome assessment.
Once a field was determined, we counted all cells which match the criteria within the field.
Confocal microscopy images were taken by the Leica TCS SP8 HSR system equipped with a Leica HCX PL APO ×63 / 1.4 oil CS2 objectives and excitation wavelength 405, 488, 561 and 647 nm. Scan speed was set to 200 Hz in combination with 5 fold line average in 1024 x 500 format. The images were collected at 300 nm z steps. For deconvolution, Huygens essential software (SVI; Scientific Volume Imaging) was used.

Live cell imaging.
A Confocal Scanner Box, Cell Voyager CV1000 (Yokogawa Electric Corp) equipped with a 63× oil immersion objective lens and the stage incubator for 35mm dish was used for live cell imaging. HeLa cells stably expressing GFP-centrin1 were treated with control siRNA or CHAMP1 siRNA for 24 hours and cultured on 35 mm glass-bottom dishes (MatTek Co.) at 37 ºC in a 5% CO 2 atmosphere. Images were taken 7 by Back-illuminated EMCCD camera. After 24 hours from transfection, the cells were visualized every 10 min over 24-36 hours. The images were collected at 1 m z steps (from 25 to 30 Z-planes and generated using ImageJ (National Institutes of Health)).

SUPPORTING FIGURE
Supp Figure S1. The increased number of centrosomes in a patient with CHAMP1 mutation.
Three color staining of centrioles in lymphoblast cells from a healthy person and a patient with CHAMP1 mutation. The cells were stained with the indicated antibodies.
The number indicates centrosomes containing mother and daughter centriole and having MTOC activity. Scale bar, 5 m.