Journal: Nature genetics

Article Title: De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome

doi: 10.1038/ng.2948

Figure Lengend Snippet: Comparative ability of mutant and WT cyclin D2 (CCND2) to enhance neural progenitor proliferation in embryonic mouse brain. Two patient mutations (p.Thr280Ala and p.Pro281Arg) are compared to WT and phosphomimetic (p.Thr280Asp) CCND2. WT and mutant cDNAs were cloned into an IRES eGFP vector affording independent translation of both CCND2 and, from an internal ribosome entry site, eGFP. Electroporated (GFP+) cells expressed either GFP alone or together with human WT or mutant CCND2. Constructs (1 μg each) were delivered to cortex by IUEP at E13.5 and embryos were harvested at E15.5. ( a-e ) Cortical sections were immunostained with an anti-GFP antibody. ( f-j ) The same sections were double immunolabeled (yellow) with anti-GFP (green) and proliferation marker, anti-Ki67 (red), antibodies. ( a,f ) Cells receiving vector without CCND2 are mostly no longer dividing and many have migrated up to the cortical plate. ( b,g ) More cells continue to divide (yellow cells, mostly in the SVZ/IZ) when overexpressing WT CCND2. ( c,h,e,j ) Progenitors expressing the phosphodeficient p.Thr280Ala or putative phosphodeficient p.Pro281Arg CCND2 found in MPPH patients show strikingly more proliferation and fewer GFP+ cells residing in the cortical plate. ( d,i ) A phosphomimetic (p.Thr280Asp) form of CCND2 is significantly less effective in promoting proliferation than the patient mutations. ( k ) Quantification of the proportions of transfected (GFP+) cells that are proliferating (GFP+Ki67+/GFP+ total cells) * p <0.05, ** p <0.001 with respect to GFP-only (control); § p <0.0001 with respect to WT-CCND2 or p.Thr280Asp as indicated. Full statistical summary is shown in . n =7 embryos (a), n =5 embryos each (b-e), n =embryos, each value average of 5 sections/embryo. MZ=marginal zone, CP=cortical plate, IZ=intermediate zone, SVZ=subventricular zone, VZ=ventricular zone. Scale bar = 20 μm.

Article Snippet: A C-terminal myc-tagged cDNA clone of CCND2 transcript NM_001759 was purchased from Origene (Origene, Rockville, MD, USA, cat. RC210316).

Techniques: Mutagenesis, Clone Assay, Plasmid Preparation, Construct, Immunolabeling, Marker, Expressing, Transfection

Journal: Nature genetics

Article Title: De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome

doi: 10.1038/ng.2948

Figure Lengend Snippet: Summary of the phenotypic and molecular data of 12 MPPH individuals with CCND2 mutations

Article Snippet: A C-terminal myc-tagged cDNA clone of CCND2 transcript NM_001759 was purchased from Origene (Origene, Rockville, MD, USA, cat. RC210316).

Techniques: