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


Activating mutations in genes encoding phosphatidylinositol 3-kinase (PI3K)-AKT pathway components cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH, OMIM 603387)1,2,3. Here we report that individuals with MPPH lacking upstream PI3K-AKT pathway mutations carry de novo mutations in CCND2 (encoding cyclin D2) that are clustered around a residue that can be phosphorylated by glycogen synthase kinase 3β (GSK-3β)4. Mutant CCND2 was resistant to proteasomal degradation in vitro compared to wild-type CCND2. The PI3K-AKT pathway modulates GSK-3β activity4, and cells from individuals with PIK3CA, PIK3R2 or AKT3 mutations showed similar CCND2 accumulation. CCND2 was expressed at higher levels in brains of mouse embryos expressing activated AKT3. In utero electroporation of mutant CCND2 into embryonic mouse brains produced more proliferating transfected progenitors and a smaller fraction of progenitors exiting the cell cycle compared to cells electroporated with wild-type CCND2. These observations suggest that cyclin D2 stabilization, caused by CCND2 mutation or PI3K-AKT activation, is a unifying mechanism in PI3K-AKT–related megalencephaly syndromes.

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Figure 1: Images of children with MPPH and CCND2 mutations.
Figure 2: CCND2 mutations and cyclin D2 protein conservation.
Figure 3: CCND2 protein stabilization by MPPH-associated mutations.
Figure 4: Comparative ability of mutant and wild-type CCND2 to enhance neural progenitor proliferation in the embryonic mouse brain.
Figure 5: Phosphodeficient CCND2 more effectively promotes proliferation and prevents neural progenitor cells from exiting the cell cycle.

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We thank the study patients and their families, without whose participation this work would not be possible. We thank M. O'Driscoll (University of Sussex) for advice and help. This work was funded by the Government of Canada through Genome Canada, the Canadian Institutes of Health Research (CIHR), the Ontario Genomics Institute (OGI-049), Genome Quebec and Genome British Columbia (to K.M.B.). The work was selected for study by the FORGE Canada Steering Committee, consisting of K. Boycott (University of Ottawa), J. Friedman (University of British Columbia), J. Michaud (Université de Montreal), F. Bernier (University of Calgary), M. Brudno (University of Toronto), B. Fernandez (Memorial University), B. Knoppers (McGill University), M. Samuels (Université de Montreal) and S. Scherer (University of Toronto). Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke (NINDS) of the US National Institutes of Health under award numbers P01-NS048120 (to M.E.R.), NRSA F32 NS086173 (to K.A.G.) and R01NS058721 (to W.B.D.), by The Baily Thomas Charitable Fund (to D.T.P.) and by the Sir Jules Thorn Charitable Trust and Great Ormond Street Children's Hospital Charity (to E.G.S.).

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Authors and Affiliations




E.G.S., W.B.D., D.T.P., M.E.R., K.M.B., G.M.M., D.A.P., A.E.F. and K.A.G. designed the study and experiments. G.M.M., A.E.F., C.A., D.T.B., K.P.J.B., L.F., K.W.G., G.M.S.M., K.P., E.S., H.v.E., N.V., D.W., D.T.P., W.B.D. and E.G.S. identified, consented and recruited the study subjects and provided clinical information. J.-B.R., J.S-O. and M.S. recruited patients. G.M.M., A.E.F., D.T.P. and W.B.D. evaluated the magnetic resonance imaging. G.M.M., D.A.P., C.A.J., J.S., M.V., C.V.L. and N.R. developed the bioinformatics scripts and performed genetic data analysis and confirmation studies. R.D.H. and R.F.H. provided the AKT3 mouse mutant brain samples. D.A.P. and C.V.L. performed the protein stability experiments. K.A.G., S.S., S.S.K. and M.E.R. performed and analyzed the IUEP experiments and quantitative western blot analyses of p.Asp219Val AKT3. J.M. analyzed data. G.M.M., D.A.P., A.E.F., K.A.G., D.T.P., M.E.R., W.B.D. and E.G.S. wrote the manuscript.

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Correspondence to M Elizabeth Ross or Eamonn G Sheridan.

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The authors declare no competing financial interests.

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Membership of the Steering Committee for the Consortium is provided in the Acknowledgments section.

Integrated supplementary information

Supplementary Figure 1 Conservation of the cyclin D protein family C terminus.

Cyclin D family members were identified using Homologene and aligned using ClustalX15. The C-terminal sequence again starting from p.Ser269 of human CCND2 is shown. Proteins are identified by the corresponding human protein ortholog, species name, and NCBI protein accession identifier. Note the perfect conservation of residues corresponding to human CCND2 positions 280-284 (asterisks).

Supplementary Figure 2 Demonstration of CCND2 protein expression off the IRES-eGFP plasmid constructs.

(a-c) Cortical embryonic tissue harvested 48 hours after IUEP with either plasmid expressing only GFP (a) or plasmid expressing GFP and p.Thr280Ala CCND2 (b,c). Monoclonal anti-cyclin D2 antibody (anti-CCND2) is best localized in paraffin-embedded tissue. Therefore, tissues were processed and embedded in paraffin, sectioned at 4 μm and immunostained with anti-CCND2 and anti-GFP antibodies as described in the Methods and previously detailed2. (a) Two days after IUEP, nearly all of the GFP+ cells are postmitotic and do not co-label with anti-CCND2. (b,c) However, 48 hours post-IUEP with plasmid containing mutant CCND2, the majority of cells expressing GFP in the cytoplasm also label for CCND2 in the nucleus, including cells that have migrated into the cortical plate. Panel c shows a lower magnification that illustrates the difference between endogenous and introduced CCND2. Note that mutant CCND2 persists in cells that have migrated to the cortical plate. The Ki67 labeling in Figure 4 indicates that most of these cortical plate cells are non-dividing, implying that CDK inhibitor levels have been induced to push cells into G0. Scale bar = 50 μm. (d) Western blot of HEK293 cells transfected with the constructs demonstrating CCND2 expression off the IRES-eGFP plasmid. HEK293 cells do not express CCND2 endogenously, and therefore the GFP only transfection shows no CCND2, while all other constructs used in these experiments successfully expressed the exogenous protein.

Supplementary Figure 3 Effect of introduced mutant CCND2 on proliferation of PAX6- compared with TBR2-expressing progenitors.

(a) Expression of the phosphodeficient form of CCND2 resulted in an increase in the proportion of electroporated cells that were immunopositive for Pax6 (GFP+Pax6+/GFP+ total cells), relative to the GFP control, wild-type CCND2 and phosphomimetic groups. (b) Transfection of progenitors with the phosphodeficient form of CCND2 caused a significant increase in the proportion of electroporated cells that expressed Tbr2 compared to the three other groups (GFP, wild-type CCND2 and the phosphomimetic CCND2). Transfection with the phosphomimetic CCND2 generated results statistically similar to the wild-type CCND2 sequence. GFP/Pax6 experiment: n = 6 GFP, n = 8 wild-type CCND2, n = 5 p.Thr280Ala and n = 5 p.Thr280Asp. GFP/Tbr2 experiment: n = 6 GFP, n = 6 wild-type CCND2, n = 5 p.Thr280Ala and n = 5 p.Thr280Asp. n = embryos, each value average of 5 sections/embryo. Data are presented as mean ± standard error of measurement. Significance is tabulated below each graph.

Supplementary Figure 4 Proposed pathway for megalencephaly syndromes.

Accumulation of cyclin D2 (CCND2) may occur either through CCND2 mutations impairing phosphorylation at Thr280 or via mutations in the phosphatidylinositide 3-kinase (PI3K) pathway converging on glycogen synthase kinase 3β (GSK-3β). Under normal circumstances, GSK-3β phosphorylates CCND2 to target it for ubiquitin-mediated degradation. Constitutively activating mutations of AKT3 result in an increase in phosphorylation of its target GSK-3β, rendering GSK-3β inactive and leading to an accumulation of degradation resistant CCND2. Similarly, mutations in the regulatory (PIK3R2) or catalytic (PIK3CA) subunits of PI3K lead to over-activation of PI3K, resulting in increased phosphorylation of phosphatidylinositol-4,5-biphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), activating AKT3 and in turn inactivating GSK-3β. Somatic overgrowth in syndromes such as Cowden syndrome, resulting from loss of function mutations in the phosphatase PTEN, may also result in over-activation of AKT3 and decreased GSK-3β-mediated phosphorylation of CCND2 due to increased PIP3 levels in the absence of dephosphorylation of PIP3 to PIP2 by PTEN. Arrows highlighted in red show the 'direction' of over-activated pathways in somatic overgrowth syndromes. Phosphate groups are represented by red Ps in yellow circles. The types of mutations resulting in megalencephaly syndromes via apparent accumulation of degradation-resistant CCND2 are marked on the relevant proteins.

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Mirzaa, G., Parry, D., Fry, A. et al. De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome. Nat Genet 46, 510–515 (2014).

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