Haploinsufficiency of MeCP2-interacting transcriptional co-repressor SIN3A causes mild intellectual disability by affecting the development of cortical integrity

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Abstract

Numerous genes are associated with neurodevelopmental disorders such as intellectual disability and autism spectrum disorder (ASD), but their dysfunction is often poorly characterized. Here we identified dominant mutations in the gene encoding the transcriptional repressor and MeCP2 interactor switch-insensitive 3 family member A (SIN3A; chromosome 15q24.2) in individuals who, in addition to mild intellectual disability and ASD, share striking features, including facial dysmorphisms, microcephaly and short stature. This phenotype is highly related to that of individuals with atypical 15q24 microdeletions, linking SIN3A to this microdeletion syndrome. Brain magnetic resonance imaging showed subtle abnormalities, including corpus callosum hypoplasia and ventriculomegaly. Intriguingly, in vivo functional knockdown of Sin3a led to reduced cortical neurogenesis, altered neuronal identity and aberrant corticocortical projections in the developing mouse brain. Together, our data establish that haploinsufficiency of SIN3A is associated with mild syndromic intellectual disability and that SIN3A can be considered to be a key transcriptional regulator of cortical brain development.

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Figure 1: Haploinsufficiency of SIN3A causes a distinct syndrome.
Figure 2: Sin3a is expressed by cortical progenitors.
Figure 3: Sin3a downregulation decreases the number of cortical progenitors.
Figure 4: The proliferation phenotype can be rescued by co-electroporation with shRNA-insensitive Sin3a.
Figure 5: Diminished Sin3a levels result in altered layer-specific identity for cortical progenitors.
Figure 6: Knockdown of Sin3a leads to a neurite outgrowth defect of corticocortical projections.
Figure 7: Knockdown of Sin3a mRNA in N2a cells by shRNAs affects Nanog and E2f1 expression.

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Acknowledgements

We thank laboratory members and colleagues for critically reading this manuscript and members of the various laboratories for helpful editing and discussions. We express thanks to W. Hendriks (Radboud University) for sharing plasmids and N. Nadif Kasri (Radboud University Medical Center) for kindly providing antibody to mouse Ki-67. We are grateful for the mouse Sin3a cDNA clone from R. Floyd and B.D. Hendrich (Wellcome Trust Centre for Stem Cell Research and MRC Centre for Stem Cell Biology and Regenerative Medicine). We thank the Radboud Institute for Molecular Life Sciences microscopy platform (see URLs) for excellent support and maintenance of the equipment. We are grateful to the families and subjects participating in this study for their involvement. This work was supported by funding from Science without Borders, CAPES-Brasil (BEX 12044/13-0) to T.C.D.D., complemented by extra support from the Educational Institute of Biosciences at Radboud University, by grants from the Netherlands Organization for Health Research and Development, ZonMw (grant 907-00-365) to T.K., by the Dutch Brain Foundation (HsN F2014(1)-16) to J.E.V. and by the German Ministry of Research and Education (grants 01GS08164, 01GS08167 and 01GS08163 German Mental Retardation Network) to H.E. and T.S., as part of the National Genome Research Network.

Author information

The study was designed and directed by T.K. and S.M.K. Patient ascertainment and recruitment were carried out by T.K., M.H.W., H.E.V.-K., C.M.A.v.R.-A., D.V., J.S.K.W.-R., M.V., A.D., J.S., P.R., N.F., K.C., S.A.d.M., C.L.C. and H.G.B. Microarray analyses, DNA sequencing, validation and genotyping were carried out and interpreted by W.M.N., T.S., A.M.Z., H.E. and J.S.W. C.G. was responsible for the bioinformatics of human genetic data analyses. R.P., T.C.D.D., N.H.M.v.B. and E.J.R.J. performed the in vitro functional assays, cloning and mouse experiments. J.E.V. interpreted the in vitro functional assays, cloning and mouse experiments. J.A.v.H. was invaluable in mouse care. The manuscript was written by J.S.W., M.H.W., T.C.D.D., G.J.M.M., T.K. and S.M.K., with all authors refining and approving the final version of it.

Correspondence to Tjitske Kleefstra or Sharon M Kolk.

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Integrated supplementary information

Supplementary Figure 1 Schematic of the deleted regions on chromosome 15q24 in individuals 1– 4 and two previously reported individuals with a 15q24 microdeletion.

The chromosomal 15q24 region contains several segmental duplication blocks, including breakpoints A, B, C, D and E (breakpoints C and D are indicated), which are thought to predispose to the occurrence of deletions and duplications in this region by non-allelic homologous recombination (NAHR) during meiosis. Different deletions mediated by different combinations of segmental duplication blocks were previously reported. In addition, some individuals have an atypical deletion in which one or both of the breakpoints are not located in a segmental duplication block. Consequently, the clinical features of the 15q24 microdeletion syndrome are heterogeneous. Genotype–phenotype studies of individuals with typical overlapping deletions have suggested the 1.1-Mb region between segmental duplication blocks B and C (72.2–73.3 Mb, hg18; 74.4–75.5 Mb, hg19) as the critical region for the core phenotype. However, Mefford et al. reported two small de novo deletions only involving the region between breakpoints C and D. These two individuals shared only five genes in the deleted region: PTPN9, SIN3A, MAN2C, NEIL1 and COMMD4. The phenotype of these individuals was reported to be milder with less pronounced speech delay than in individuals with the larger deletions between breakpoints B and C.

Supplementary Figure 2 Developmental transcriptome and LMD microarray analysis show high expression levels of SIN3A in proliferative regions of human developing cortex.

(a) Overview of the online database of the Allen Institute for Brain Science showing RNA-seq data across developmental stages (from 4–7 weeks post-conception (wpc) into adulthood) of human brain development showing low to moderate expression of SIN3A across development in various brain structures. (b) Detail of the expression levels of SIN3A in cortical regions 9 wpc, with a higher expression level in temporal neocortex (TGx). (c) Detail of the expression levels of SIN3A in cortical regions 9 wpc, with a higher expression level in the primary motor-sensory cortex (M1C-S1C). (d) Overview of the online database of the Allen Institute for Brain Science showing LMD microarray data across developmental stages (from 4–7 wpc into adulthood) of human brain development showing low to moderate expression of SIN3A 21 wpc in the ventricular zone (VZ) of the posterior frontal cortex (motor cortex). (e) Comparison of the expression levels of SIN3A in deeper cortical regions (e.g., VZ) and more superficial cortical regions (e.g., marginal zone, MZ) 21 wpc. Details on the complete SIN3A transcriptome profile can be found at http://www.brainspan.org/.

Supplementary Figure 3 Protein expression of Sin3a over time and Nissl validation.

(a) Immunostaining for Sin3a (green) at E14.5, E16.5, E18.5, P7, P14 and P21 and counterstaining with fluorescent Nissl (blue). (b) Coronal sections showing the somatosensory cortical area (S1) of E14.5 (left) and E16.5 (right) mouse brains with immunostaining for Sin3a (green) and Ki-67 (red); sections were counterstained with fluorescent Nissl (blue). Arrows and insets show colocalization (yellow). (c) Representative images of Nissl staining of a control (Ctrl)-electroporated cortical area (left) and an shSin3a-electroporated cortical area (right) immunostained for GFP (green) and counterstained with fluorescent Nissl (blue) flanked by a black-and-white image of Nissl staining (asterisks in the cell sparser area).

Supplementary Figure 4 Validation of Sin3a knockdown at the mRNA level.

(a) Relative expression levels (percentage) of Sin3a (tested with two primer pairs (PP1 and PP2; two shades of grey) in comparison to β-actin (black) in mouse brain at P35 and N2a cells. (b) Normalized expression levels of Sin3a mRNA in N2a cells transfected with two siRNAs targeting Sin3a mRNA (siSin3a-ex13 and siSin3a-ex16), two scrambled siRNAs (sc-siSin3a-ex13 and sc-siSin3a-ex16) in comparison to mock (no construct) as a control determined by qPCR using two primer pairs (PP1, black; PP2, grey). (cg) Schematic of the constructs containing the shRNA for Sin3a exon 13 and exon 16 with accompanying scrambled constructs and the pCAB expression vector with the shRNA-insensitive mSin3a*. (hj) Normalized mRNA expression levels of Cdkn1a, Mecp2 and Ccnd1 (n = 3 biological replicates) did not change 48 h after knockdown of Sin3a. Data are presented as normalized mean transcript levels ± s.e.m. Student’s t test.

Supplementary Figure 5 Validation of in vivo Sin3a knockdown at the protein level.

(a) Representative images are shown of the electroporated (shSin3a-ex13, green) area double labeled with Sin3a (red) and counterstained with fluorescent Nissl (blue; left). At the site of electroporation with shRNA, Sin3a protein levels are downregulated (white arrows; right). (b) Representative image of an electroporated (shSin3a-ex13, green) area double labeled with cleaved caspase 3 (CC3) showing an apoptotic cell double labeled with GFP (arrowhead) and in the vicinity of GFP-labeled cells (arrow). (c) Positive control showing an area (septal area E17.5) positive for CC3. (d) Quantification of the number of CC3-positive cells in the electroporated area; n = 4 for Ctrl, n = 5 for shSin3a and n = 2 for shSin3a + mSin3a*. Data are presented as the number of cells per mm2 ± s.e.m. One-way ANOVA (α = 0.05). (e) Quantification of the number of GFP+CC3+ cells in the electroporated area as an indication for cell-autonomous effects; n = 4 for Ctrl, n = 5 for shSin3a and n = 2 for shSin3a + mSin3a*. Data are presented as the number of cells per mm2 ± s.e.m. One-way ANOVA (α = 0.05).

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Witteveen, J., Willemsen, M., Dombroski, T. et al. Haploinsufficiency of MeCP2-interacting transcriptional co-repressor SIN3A causes mild intellectual disability by affecting the development of cortical integrity. Nat Genet 48, 877–887 (2016) doi:10.1038/ng.3619

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