Fig 1.
Phenotypic presentation of CENPT defect.
(A) The left panel shows height measurements on female primordial dwarfism growth curve and MRI of IA. The right panel shows height measurements on male primordial dwarfism growth curve and MRI of IC. (B) Pedigree and (C) Sanger sequence trace. (D) RT-PCR data on a BioAnalyzer chip of index patients (red and blue peaks) and control (green) are shown. The wild-type isoform is only seen in the normal control. Among the alternatively spliced isoforms (red circle) the in-frame isoform (left peak) shows highest expression. This endogenously present isoform is only minimally expressed in the normal control. (E) Schematic of human CENPT. Red marking annotates the splice-mutation in the index cases. Pink markings represent the variants found in 9 individuals of 8 families identified through database searches and clinical contacts. The green box encircles the C-terminal conserved histone-fold domain (HFD). On the bottom, the three alternative isoforms are schematically represented. In all isoforms part of exon 12 is spliced out. The out-of-frame isoforms result in premature termination codons, which obliterate most of the HFD.
Fig 2.
Cellular aberrations in patient fibroblast lines.
(A) Immunofluorescence staining of immortalized fibroblasts of index IA; blue—DAPI; magenta—CENPT; green—CENPA or phosphorylated CENPA. Scale bars represent 10μm. Multinucleated cells are depicted in columns 1–3. Micronuclei (solid arrowheads) and chromosome lagging (arrow). (B) Normal mitosis on the left. Examples of multipolar mitosis on the right in immortalized fibroblast of index IA. Blue—DAPI; magenta—CENPT; green–γ-tubulin. Scale bars represent 10μm.
Fig 3.
(A) Nuclear size distribution and CENPT signal intensity in immortalized fibroblasts measured by immunofluorescence. CENPT signal intensities are age-corrected. (B) Right-shifted histograms of patient (red) and parent (blue) EBV-transformed lymphoblast cell lines in relation to three different controls (green) by flow cytometry staining. Propidium iodide was used as DNA stain.
Fig 4.
(A) Comparison of morpholino target effects at 24hpf and localization of morpholino targets in relation to the zebrafish cenpt gene and the conserved histone-fold domain (HFD, green box). (B) Effect of Mo8 (Mo8/8) on transcription of zebrafish cenpt in comparison to control morpholino (CoMo). (C) Dose response curve of Mo12 (Mo12/12) at 75hpf. On the bottom RT-PCR of Mo12 dose response curve. The upper band represents the wild-type band. (D) Head and eye measurements in Mo12 injected zebrafish at 48hpf. (E) Left panel shows a normal mitosis. The three right panels are examples of mitotic aberrations seen in Mo12 injected Tg(h2afv:GFP) zebrafish live imaging. (F) Time lapse of aberrant mitosis in Mo12 injected Tg(h2afv:GFP) zebrafish.