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. 2015 Apr 2;520(7545):51-6.
doi: 10.1038/nature14186. Epub 2015 Mar 25.

Loss of δ-catenin function in severe autism

Tychele N Turner  1 , Kamal Sharma  2 , Edwin C Oh  3 , Yangfan P Liu  3 , Ryan L Collins  4 , Maria X Sosa  5 , Dallas R Auer  5 , Harrison Brand  6 , Stephan J Sanders  7 , Daniel Moreno-De-Luca  8 , Vasyl Pihur  5 , Teri Plona  9 , Kristen Pike  9 , Daniel R Soppet  9 , Michael W Smith  10 , Sau Wai Cheung  11 , Christa Lese Martin  12 , Matthew W State  7 , Michael E Talkowski  6 , Edwin Cook  13 , Richard Huganir  2 , Nicholas Katsanis  3 , Aravinda Chakravarti  5
Affiliations

Loss of δ-catenin function in severe autism

Tychele N Turner et al. Nature. .

Abstract

Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from female-enriched multiplex families with severe disease, enhancing the detection of key autism genes in modest numbers of cases. Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 null mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as female-enriched multiplex families, are of innate value in multifactorial disorders.

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Figures

Extended Data Figure 1
Extended Data Figure 1
(a) Workflow of exome analysis in this study. (b) Variant recalibration metrics exhibiting why a 99% cutoff was utilized for truth sensitivity. (c) Variant recalibration specificity vs. sensitivity.
Extended Data Figure 2
Extended Data Figure 2
Sanger sequencing chromatograms for (a) G34S variant in this study; (b) R713C variants in this study; (c) R713C in NA19020.
Extended Data Figure 3
Extended Data Figure 3
Principal components analysis of 6,211 shared autosomal SNPs in CEU, YRI, CHB/JPT, autism and NA19020 samples.
Extended Data Figure 4
Extended Data Figure 4
Read and Amplicon Metrics in CTNND2 Sequencing. (a) Histogram of Reads per Sample; (b) Average quality scores across the read across all samples with each sample represented by a separate line; (c) Boxplot of Coverage per Amplicon.
Extended Data Figure 5
Extended Data Figure 5
Validation of deletions in (a) AU066818, (b) AU075604, (c) AU1178301 and AU1178202, (d) AU051503.
Extended Data Figure 6
Extended Data Figure 6
CTNND2 copy number variants from patients with neurodevelopment disorders (NDD) studied using methods published in Talkowski et al. (2012).
Extended Data Figure 7
Extended Data Figure 7
Wnt defects in ctnnd2b zebrafish morphant embryos. (a) Relative axin2 mRNA level in 10-somite stage in control vs. morphant embryos. (b) Whole mount RNA in situ hybridization of chordin. Dorsal view in upper panels with the anterior aspect at the apex. The dorsal axis is marked with a red dashed line and regions with high expression are marked (arrows) in control embryos. Lateral view in lower panels, length (L) and width (W) of chordin expression domains were measured. (c) Quantification of chordin expression domains (length/width ratio) in injected embryos. (d) Immunoblot showing a macromolecular interaction between Flag-tagged CTNNB1 and GFP-tagged CTNND2 with the corresponding variants. Two-sided t-tests were conducted with *, ** and *** indicating P < 0.05, P < 0.01 and P < 0.001, respectively. Sample size (n) is marked for each condition.
Extended Data Figure 8
Extended Data Figure 8
Functional in vitro modeling of delta catenin missense variants in embryonic rat hippocampal neurons. (a) Representation of spines along the dendrite in control and overexpression GFP vectors (empty or fused with wild type or variant allele containing CTNND2 (G34S, R713C, A482T (control)). Cell counts for each construct were as follows: GFP (N=32), GFP-WT (N=27), GFP-G34S (N=29), GFP-R713C (N=26), and GFP-A482T (N=29). (b) Quantification of dendritic spine numbers and statistical comparisons by Tukey’s Honestly Significant test following ANOVA. * and ** both indicate P<0.05 than GFP and significantly different than wild type, respectively.
Extended Data Figure 9
Extended Data Figure 9
Gene expression of CTNND2 and co-expression with known autism genes. (a) Expression of CTNND2 in various human fetal and adult tissues, shown as fold difference relative to adult brain. (b) RNA-Seq-based CTNND2 gene expression in the developing human brain (www.brainspan.org); shown are log2RPKM expression values at time-points from 8 post-conception weeks to 40 years of age, with the lowest to highest expression colored from navy blue to red. Controls for high expression, low to no expression and known autism genes are GAPDH, CFTR, and MECP2, respectively.
Extended Data Figure 10
Extended Data Figure 10
Analysis of overexpression of transiently transfected neurons: Representation of average intensity of 5 individual ROIs from a selected dendritic region. Quantitative comparison does not reveal a significant difference in expression levels of different variants of CTNND2.
Figure 1
Figure 1. Genetic features of a sex-dependent multifactorial model
(a) Hypothetical sex-dependent liability distributions for autism under a multifactorial model of inheritance with a fixed biological threshold for affection. (b) Percent of Hirschsprung disease patients with damaging coding mutations within different risk classes characterized by gender, segment length, and familiality. The risk class is labeled 3,2,1,0 and is an additive score based on the number of factors with higher risk (female, long segment, multiplex) and comprise 13, 46, 60 and 55 patients, respectively (proportion trend test, P=3.1x10−6).
Figure 2
Figure 2. Missense variants in human delta catenin and their effect on protein function in vivo
(a) CTNND2 annotated with validated missense mutations in autism patients; G34S, G275C, Q507P, R713C, T862M variants are conserved to zebrafish. (b) Expression of two CTNND2 zebrafish orthologs (ctnnd2a, ctnnd2b) in development. Aberrant phenotypes are observed with ctnnd2b (the only ortholog expressed at these gastrulation time points) morpholino (MO) knockdown only at key stages of gastrulation (50% epiboly, 75% epiboly, bud). Elongation factor alpha (eef1a1l1) is shown as a control for ubiquitous expression. (c) Representative lateral and dorsal images of Class I and Class II ctnnd2b morphants (2 ng MO) at the 8–10 somite stage reveal defective gastrulation movements. (d) Quantification of gastrulation phenotype in control, MO and rescue constructs: wild type, autism variants (G34S, P189L, P224L, G275C, R454H, Q507P, R713C, T862M), and control variants (R330H, D465N, A482T, G810R) are indicated. (e) Quantification of gastrulation phenotype in overexpression constructs: wild type, autism variants (G34S, P189L, P224L, G275C, R454H, Q507P, R713C, T862), and control variants (R330H, D465N, A482T, G810R) are indicated. Chi-square tests were conducted with *, ** and *** indicating P < 0.05, P < 0.01 and P < 0.001, respectively, and ## indicating no rescue and worse than MO alone (P < 0.01). Sample size (n) is marked for each condition.
Figure 3
Figure 3. Copy number variants (CNV) in human CTNND2
CNVs at the 1.1 Mb CTNND2 locus (chr5:10905332-12034584, hg19), the chromosomal location, extent of each deletion and duplication, patient gender, parental origin and citation, are shown for each variant identified in autism patients and individuals with other neurodevelopmental disorders. Extensive genomic sequence conservation across the entire region in selected vertebrates is shown.
Figure 4
Figure 4. Delta catenin is critical for maintaining functional neuronal networks
(a) Gain of function: (i) Over expression of CTNND2 leads to an increase in the number of excitatory synapses. Primary dendrites from neurons transfected with GFP alone, GFP fusion with wild type CTNND2, or mutant isoforms, and immunolabeled with vGluT1 and PSD95. (ii) Quantification of number of PSD95+vGluT1 positive puncta per 100 μm of dendritic length (N=12 each). (b) Loss of function: (i) Neurons from Ctnnd2 null mutants have a significant reduction in synapse density. Synapses are identified as puncta with PSD95 and vGluT1 overlap. (ii) Quantification of the number of PSD95+vGluT1 positive puncta per 100 μm of dendritic length (N=13 each). (iii) Alternatively, neurons were immunolabeled with GluA and vGluT1 to identify active functional excitatory synapses. (iv) Quantification of the number of GluA+vGluT1 positive puncta per 100 μm of dendritic length (N=15 each). (c) Rescue of loss of function: (i) WT CTNND2 but not its mutant isoforms can rescue the loss of phenotype in neurons from CTNND2 null mutants. Primary dendrites from neurons transfected with GFP alone, GFP fusion with wild type CTNND2, or mutant isoforms, and immunolabeled with vGluT1 and PSD95. (ii) Quantification of the number of PSD95+vGluT1 positive puncta per 100 μm of dendritic length (N=14 each). Color used for merged panels are GFP (green) PSD95 (red), GluA (Red) and vGluT1 (blue). Student’s t-test were conducted with * and ** represents P<.05 and P<.001, respectively. Error bars represent standard error of mean.
Figure 5
Figure 5. Gene expression correlation between CTNND2 and known autism genes
(a) Plot of all autism genes significantly (positive and negative) correlated with CTNND2 in the developing human brain (microarray data from www.brainspan.org). (b) Pathway analysis of the autism genes positively correlated with delta catenin reveals significant enrichment of genes involved in chromatin modification and dendrite morphogenesis.
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