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Loss of δ-catenin function in severe autism.

Turner TN, Sharma K, Oh EC, Liu YP, Collins RL, Sosa MX, Auer DR, Brand H, Sanders SJ, Moreno-De-Luca D, Pihur V, Plona T, Pike K, Soppet DR, Smith MW, Cheung SW, Martin CL, State MW, Talkowski ME, Cook E, Huganir R, Katsanis N, Chakravarti A - Nature (2015)

Bottom Line: 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.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 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Complex Disease Genomics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA [2] Predoctoral Training Program in Human Genetics and Molecular Biology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA [3] National Institute of Mental Health (NIMH) Autism Centers of Excellence (ACE) Genetics Consortium at the University of California, Los Angeles, Los Angeles, California 90095, USA.

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 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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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.
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Figure 12: 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.

Mentions: To assess the in vivo functional consequences of autism CTNND2 variants, we used a complementation assay in zebrafish embryos. Zebrafish has two genes for delta catenin that are as divergent from each other (18.3%) as they are from humans (19.9%, 20.7%), at the protein level. We examined expression of both genes by RT-PCR at six developmental time points (Figure 2b) and focused on ctnnd2b because it was expressed at all stages. Using a splice-blocking morpholino (MO) targeting ctnnd2b, we injected 1–8 cell embryos and analyzed at the 8–10 somite stage. Morphant embryos had gastrulation phenotypes consistent with abnormal Wnt signaling (shortened body axes, longer somites, and broad and kinked notochords) (Figure 2c). RT-PCR of axin2 mRNA, a direct target of canonical Wnt signaling12, from 10-somite ctnnd2b morphants, showed significant decrease (P<0.01) reinforcing the hypothesis of defective Wnt signaling (Extended Data Figure 7a). Specificity of the MO was tested by co-injection of wild-type mRNA to observe significant (P<0.001) rescue (Figure 2d). To investigate the effect of each variant on protein function, injection cocktails containing MO and mutant variants were injected and compared to rescue with wild-type mRNA: five variants (G34S,P189L,P224L,R454H,Q507P) were better than morpholino alone (P<0.001) but worse than wild-type rescue (P<0.001), implicating these as hypomorphic (Figure 2d). One variant (R713C) was functionally while G275C and T862M were benign, and all four controls were benign, demonstrating specificity. To preclude the possibility of mRNA toxicity, we injected mutant mRNA corresponding to all alleles and observed no significant differences in the gastrulation phenotypes (Figure 2e).


Loss of δ-catenin function in severe autism.

Turner TN, Sharma K, Oh EC, Liu YP, Collins RL, Sosa MX, Auer DR, Brand H, Sanders SJ, Moreno-De-Luca D, Pihur V, Plona T, Pike K, Soppet DR, Smith MW, Cheung SW, Martin CL, State MW, Talkowski ME, Cook E, Huganir R, Katsanis N, Chakravarti A - Nature (2015)

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.
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Related In: Results  -  Collection

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Figure 12: 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.
Mentions: To assess the in vivo functional consequences of autism CTNND2 variants, we used a complementation assay in zebrafish embryos. Zebrafish has two genes for delta catenin that are as divergent from each other (18.3%) as they are from humans (19.9%, 20.7%), at the protein level. We examined expression of both genes by RT-PCR at six developmental time points (Figure 2b) and focused on ctnnd2b because it was expressed at all stages. Using a splice-blocking morpholino (MO) targeting ctnnd2b, we injected 1–8 cell embryos and analyzed at the 8–10 somite stage. Morphant embryos had gastrulation phenotypes consistent with abnormal Wnt signaling (shortened body axes, longer somites, and broad and kinked notochords) (Figure 2c). RT-PCR of axin2 mRNA, a direct target of canonical Wnt signaling12, from 10-somite ctnnd2b morphants, showed significant decrease (P<0.01) reinforcing the hypothesis of defective Wnt signaling (Extended Data Figure 7a). Specificity of the MO was tested by co-injection of wild-type mRNA to observe significant (P<0.001) rescue (Figure 2d). To investigate the effect of each variant on protein function, injection cocktails containing MO and mutant variants were injected and compared to rescue with wild-type mRNA: five variants (G34S,P189L,P224L,R454H,Q507P) were better than morpholino alone (P<0.001) but worse than wild-type rescue (P<0.001), implicating these as hypomorphic (Figure 2d). One variant (R713C) was functionally while G275C and T862M were benign, and all four controls were benign, demonstrating specificity. To preclude the possibility of mRNA toxicity, we injected mutant mRNA corresponding to all alleles and observed no significant differences in the gastrulation phenotypes (Figure 2e).

Bottom Line: 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.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 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Complex Disease Genomics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA [2] Predoctoral Training Program in Human Genetics and Molecular Biology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA [3] National Institute of Mental Health (NIMH) Autism Centers of Excellence (ACE) Genetics Consortium at the University of California, Los Angeles, Los Angeles, California 90095, USA.

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 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.

Show MeSH
Related in: MedlinePlus