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Loss of δ-catenin function in severe autism

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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Figure 1: Genetic features of a sex-dependent multifactorial model.
Figure 2: Missense variants in human δ-catenin and their effect on protein function in vivo.
Figure 3: CNV in human CTNND2.
Figure 4: δ-Catenin is critical for maintaining functional neuronal networks.
Figure 5: Gene expression correlation between CTNND2 and known autism genes.

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Acknowledgements

We acknowledge the participation of all of the families in the AGRE, NIMH and SSC studies that have been a model of public participatory research. The AGRE is a program of Autism Speaks and is supported, in part, by grant 1U24MH081810 from the National Institute of Mental Health. The SSC used here was developed by the following principal investigators: A. Beaudet, R. Bernier, J. Constantino, E. Cook, E. Fombonne, D. Geschwind, D. Grice, A. Klin, D. Ledbetter, C. Lord, C. Martin, D. Martin, R. Maxim, J. Miles, O. Ousley, B. Peterson, J. Piggot, C. Saulnier, M. State, W. Stone, J. Sutcliffe, C. Walsh, E. Wijsman. We thank the Allen Brain Atlas for use of their publicly available developing human brain expression data. Finally, we thank V. Kustanovich (AGRE) for helping with access to Autism Diagnostic Observation Schedule severity score data, D. Arking for sharing DNA from the SSC for Taqman genotyping, S. Maragh for zebrafish complementary DNA (cDNA) libraries and eef1a1l1 primers, A. Kapoor for discussions, Q. Jiang for the translation of ref. 43, and J. A. Rosenfeld, L. G. Shaffer, Y. Shen and B.-L. Wu for sharing CNV data sets. Sequencing services were provided by the Johns Hopkins University Next Generation Sequencing Center, Sidney Kimmel Comprehensive Cancer Center, Illumina Sequencing Services and the Johns Hopkins University Genetic Resources Core Facility. E.C.O. is a National Alliance for Research on Schizophrenia and Depression young investigator. N.K. is a Distinguished George W. Brumley Professor. This work was funded by grants from the Simons Foundation to A.C. and to N.K., NIMH grant MH095867 to M.E.T., NIMH grants 5R25MH071584-07 and MH19961-14 to D.M.D.L. (Malison), National Institutes of Health grant RO1MH074090 to C.L.M., NIMH grant R01MH081754 to A.C. and an Autism Speaks Dennis Weatherstone pre-doctoral fellowship (number 7863) to T.T.

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Authors and Affiliations

Authors

Contributions

Designed the study and wrote the manuscript (T.T., A.C.); edited the manuscript (all authors); examined phenotype data for the female autism patients (T.T., E.C.); MECP2/CTNND2 sequencing and TaqMan genotyping (T.T., M.X.S., T.P., K.P., D.S., M.W.S.); autism exome sequencing (T.T.); Simons exome sequencing analysis (S.S., M.S.); CNV analysis (S.W.C., C.L.M., D.M.D., S.S., R.C.C., H.B., M.E.T, M.S., T.T.); CTNND2 molecular biology (T.T., M.X.S.); zebrafish gastrulation and protein-protein interaction studies (Y.P.L., E.O., N.K.); primary hippocampal neuron experiments and expression analysis (K.S., T.T., D.A.); bioinformatics analyses (T.T.,V.P.).

Corresponding author

Correspondence to Aravinda Chakravarti.

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The authors declare no competing financial interests.

Additional information

All sequence data have been deposited in the National Database for Autism Research in NDAR Study 367 and are available at http://dx.doi.org/10.15154/1171641.

Extended data figures and tables

Extended Data Figure 1 Exome sequencing workflow and quality control.

a, Workflow of exome analysis in this study. b, Variant recalibration metrics exhibiting why a 99% cutoff was used for truth sensitivity. c, Variant recalibration specificity versus sensitivity.

Extended Data Figure 2 Sanger sequencing chromatograms.

a, G34S variant in this study; b, R713C variants in this study; c, G34S in NA19020.

Extended Data Figure 3 Principal component analysis of 6,211 shared autosomal single nucleotide polymorphisms in CEU, YRI, CHB/JPT, autism and NA19020 samples.

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 Validation of deletions.

a, In AU066818; b, in AU075604; c, in AU1178301 and AU1178202; d, in AU051503.

Extended Data Figure 6 CTNND2 CNVs from patients with neurodevelopment disorders studied using methods published in ref. 11.

Extended Data Figure 7 Wnt defects in ctnnd2b zebrafish morphant embryos.

a, Relative axin2 mRNA level in the ten-somite stage in control versus morphant embryos. b, Wholemount 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: P < 0.05, P < 0.01 and P < 0.001, respectively. Sample size (n) is marked for each condition.

Extended Data Figure 8 Functional in vitro modelling of δ-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. Both P < 0.05 than GFP and significantly different from wild type, respectively.

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 (http://www.brainspan.org); shown are log2(RPKM expression) values at time-points from 8 weeks after conception to 40 years of age, with the lowest to highest expression coloured 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 Analysis of overexpression of transiently transfected neurons.

Representation of average intensity of five individual regions of interest from a selected dendritic region. Quantitative comparison does not reveal a significant difference in expression levels of different variants of CTNND2.

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Turner, T., Sharma, K., Oh, E. et al. Loss of δ-catenin function in severe autism. Nature 520, 51–56 (2015). https://doi.org/10.1038/nature14186

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