Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission

Hui Guo^{1

2}, Ying Li¹, Lu Shen¹, Tianyun Wang^{1

2}, Xiangbin Jia¹, Lijuan Liu³, Tao Xu⁴, Mengzhu Ou³, Kendra Hoekzema², Huidan Wu¹, Madelyn A Gillentine², Cenying Liu¹, Hailun Ni¹, Pengwei Peng¹, Rongjuan Zhao¹, Yu Zhang⁵, Chanika Phornphutkul⁶, Alexander P A Stegmann⁷, Carlos E Prada⁸, Robert J Hopkin⁸, Joseph T Shieh⁹, Kirsty McWalter¹⁰, Kristin G Monaghan¹⁰, Peter M van Hasselt¹¹, Koen van Gassen¹¹, Ting Bai¹, Min Long¹, Lin Han¹, Yingting Quan¹, Meilin Chen¹, Yaowen Zhang¹, Kuokuo Li¹, Qiumeng Zhang¹, Jieqiong Tan¹, Tengfei Zhu¹, Yaning Liu¹, Nan Pang¹², Jing Peng¹², Daryl A Scott^{13

14}, Seema R Lalani¹³, Mahshid Azamian¹³, Grazia M S Mancini¹⁵, Darius J Adams¹⁶, Malin Kvarnung^{17

18}, Anna Lindstrand^{17

18}, Ann Nordgren^{17

18}, Jonathan Pevsner^{19

20}, Ikeoluwa A Osei-Owusu^{19

20}, Corrado Romano²¹, Giuseppe Calabrese²¹, Ornella Galesi²¹, Jozef Gecz²², Eric Haan²³, Judith Ranells²⁴, Melissa Racobaldo²⁴, Magnus Nordenskjold^{17

18}, Suneeta Madan-Khetarpal²⁵, Jessica Sebastian²⁵, Susie Ball²⁶, Xiaobing Zou²⁷, Jingping Zhao²⁸, Zhengmao Hu¹, Fan Xia^{13

29}, Pengfei Liu^{13

29}, Jill A Rosenfeld¹³, Bert B A de Vries³⁰, Raphael A Bernier³¹, Zhi-Qing David Xu⁴, Honghui Li⁵, Wei Xie³, Robert B Hufnagel³², Evan E Eichler^{2

33}, Kun Xia^{1

34

35

36}

Affiliations

¹ Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China.
² Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA.
³ Institute of Life Sciences, Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing, Jiangsu, China.
⁴ Department of Neurobiology, Beijing Key Laboratory of Neural Regeneration and Repair, Beijing Laboratory of Brain Disorders (Ministry of Science and Technology), Beijing Institute of Brain Disorders, Capital Medical University, Beijing, China.
⁵ Key Laboratory of Developmental Disorders in Children, Liuzhou Maternity and Child Healthcare Hospital, Liuzhou, Guangxi, China.
⁶ Division of Human Genetics, Warren Alpert Medical School of Brown University, Hasbro Children's Hospital/Rhode Island Hospital, Providence, RI, USA.
⁷ Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, Netherlands.
⁸ Department of Pediatrics, University of Cincinnati College of Medicine, Division of Human Genetics, Cincinnati Children's Hospital, Cincinnati, OH, USA.
⁹ Institute for Human Genetics and Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA.
¹⁰ GeneDx, Gaithersburg, MD, USA.
¹¹ University Medical Center Utrecht, Utrecht, Netherlands.
¹² Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, China.
¹³ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
¹⁴ Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA.
¹⁵ Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam, Netherlands.
¹⁶ Goryeb Children's Hospital, Atlantic Health System, Morristown, NJ, USA.
¹⁷ Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden.
¹⁸ Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden.
¹⁹ Department of Neurology, Kennedy Krieger Institute, Baltimore, MD, USA.
²⁰ Program in Human Genetics, Johns Hopkins School of Medicine, Baltimore, MD, USA.
²¹ Oasi Research Institute-IRCCS, Troina, Italy.
²² School of Medicine and the Robinson Research Institute, University of Adelaide at the Women's and Children's Hospital, Adelaide, South Australia, Australia.
²³ Adult Genetics Unit, Royal Adelaide Hospital, and School of Medicine, University of Adelaide, Adelaide, South Australia, Australia.
²⁴ Department of Pediatrics, University of South Florida, Tampa, FL, USA.
²⁵ Division of Medical Genetics, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA.
²⁶ Central Washington Genetics Program, Virginia Mason Memorial, Yakima, WA, USA.
²⁷ Children Development Behavior Center of the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China.
²⁸ Mental Health Institute of the Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
²⁹ Baylor Genetics, Houston, TX, USA.
³⁰ Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands.
³¹ Department of Psychiatry, University of Washington, Seattle, WA, USA.
³² Ophthalmic Genetics and Visual Function Branch, National Eye Institute, NIH, Bethesda, MD, USA.
³³ Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA.
³⁴ Key Laboratory of Medical Information Research, Central South University, Changsha, Hunan, China.
³⁵ CAS Center for Excellence in Brain Science and Intelligences Technology (CEBSIT), Chinese Academy of Sciences, Shanghai 200030, China.
³⁶ Hunan Key Laboratory of Animal Models for Human Diseases, Central South University, Changsha, Hunan 410078, China.

PMID: 31579823
PMCID: PMC6760934
DOI: 10.1126/sciadv.aax2166

Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission

Hui Guo et al. Sci Adv. 2019.

. 2019 Sep 25;5(9):eaax2166.

doi: 10.1126/sciadv.aax2166. eCollection 2019 Sep.

Authors

Affiliations

¹ Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China.
² Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA.
³ Institute of Life Sciences, Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing, Jiangsu, China.
⁴ Department of Neurobiology, Beijing Key Laboratory of Neural Regeneration and Repair, Beijing Laboratory of Brain Disorders (Ministry of Science and Technology), Beijing Institute of Brain Disorders, Capital Medical University, Beijing, China.
⁵ Key Laboratory of Developmental Disorders in Children, Liuzhou Maternity and Child Healthcare Hospital, Liuzhou, Guangxi, China.
⁶ Division of Human Genetics, Warren Alpert Medical School of Brown University, Hasbro Children's Hospital/Rhode Island Hospital, Providence, RI, USA.
⁷ Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, Netherlands.
⁸ Department of Pediatrics, University of Cincinnati College of Medicine, Division of Human Genetics, Cincinnati Children's Hospital, Cincinnati, OH, USA.
⁹ Institute for Human Genetics and Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA.
¹⁰ GeneDx, Gaithersburg, MD, USA.
¹¹ University Medical Center Utrecht, Utrecht, Netherlands.
¹² Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, China.
¹³ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
¹⁴ Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA.
¹⁵ Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam, Netherlands.
¹⁶ Goryeb Children's Hospital, Atlantic Health System, Morristown, NJ, USA.
¹⁷ Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden.
¹⁸ Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden.
¹⁹ Department of Neurology, Kennedy Krieger Institute, Baltimore, MD, USA.
²⁰ Program in Human Genetics, Johns Hopkins School of Medicine, Baltimore, MD, USA.
²¹ Oasi Research Institute-IRCCS, Troina, Italy.
²² School of Medicine and the Robinson Research Institute, University of Adelaide at the Women's and Children's Hospital, Adelaide, South Australia, Australia.
²³ Adult Genetics Unit, Royal Adelaide Hospital, and School of Medicine, University of Adelaide, Adelaide, South Australia, Australia.
²⁴ Department of Pediatrics, University of South Florida, Tampa, FL, USA.
²⁵ Division of Medical Genetics, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA.
²⁶ Central Washington Genetics Program, Virginia Mason Memorial, Yakima, WA, USA.
²⁷ Children Development Behavior Center of the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China.
²⁸ Mental Health Institute of the Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
²⁹ Baylor Genetics, Houston, TX, USA.
³⁰ Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands.
³¹ Department of Psychiatry, University of Washington, Seattle, WA, USA.
³² Ophthalmic Genetics and Visual Function Branch, National Eye Institute, NIH, Bethesda, MD, USA.
³³ Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA.
³⁴ Key Laboratory of Medical Information Research, Central South University, Changsha, Hunan, China.
³⁵ CAS Center for Excellence in Brain Science and Intelligences Technology (CEBSIT), Chinese Academy of Sciences, Shanghai 200030, China.
³⁶ Hunan Key Laboratory of Animal Models for Human Diseases, Central South University, Changsha, Hunan 410078, China.

PMID: 31579823
PMCID: PMC6760934
DOI: 10.1126/sciadv.aax2166

Abstract

RNA binding proteins are key players in posttranscriptional regulation and have been implicated in neurodevelopmental and neuropsychiatric disorders. Here, we report a significant burden of heterozygous, likely gene-disrupting variants in CSDE1 (encoding a highly constrained RNA binding protein) among patients with autism and related neurodevelopmental disabilities. Analysis of 17 patients identifies common phenotypes including autism, intellectual disability, language and motor delay, seizures, macrocephaly, and variable ocular abnormalities. HITS-CLIP revealed that Csde1-binding targets are enriched in autism-associated gene sets, especially FMRP targets, and in neuronal development and synaptic plasticity-related pathways. Csde1 knockdown in primary mouse cortical neurons leads to an overgrowth of the neurites and abnormal dendritic spine morphology/synapse formation and impaired synaptic transmission, whereas mutant and knockdown experiments in Drosophila result in defects in synapse growth and synaptic transmission. Our study defines a new autism-related syndrome and highlights the functional role of CSDE1 in synapse development and synaptic transmission.

Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

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Figures

**Fig. 1. Spectrum of *CSDE1* LGD variants and patient facial features.**
(A) Diagram of the canonical *CSDE1* isoform (NM_001242891.1 and NP_001229820.1). The locations of LGD variants are indicated. (B) Pedigrees of eight families with de novo LGD variants (above) and eight families with transmitted LGD variants (below). Carrier parents or sibling in at least four families (PU2, BU2, SU1, and PU1) are affected or show substantial family history.

**Fig. 2. Enrichment analyses of Csde1 RNA binding targets.**
(A) Bar plot shows Csde1-binding targets significantly enriched in seven ASD-related gene sets, especially the FMRP RNA binding targets. (B) Venn diagram shows the overlap of the RNA binding targets of CSDE1 and other ASD-associated RBPs (FMRP and RBFOX). (C) Venn diagram shows the overlap of the SFARI genes that are RNA binding targets of CSDE1, FMRP, and RBFOX, with gene names indicated. (D) Bar plot shows Csde1-binding targets enriched in neuronal development– and synapse development–related cell components [Fisher’s exact test, false discovery rate (FDR)–corrected]. Top 15 significant cell components are shown.

**Fig. 3. Disruption of Csde1 interferes with neuronal development.**
(A) Csde1 KD promoted neurite and axon growth (NC, 73 neurons; Csde1 KD1, 60 neurons; Csde1 KD2, 53 neurons; Csde1 KD1 + WT, 82 neurons). Neurons were colabeled with 4′,6-diamidino-2-phenylindole (DAPI) (nuclei), green fluorescent protein (GFP) (overall neuronal morphology), and SMI 312 (axon). Scale bar, 100 μm. (B) Csde1 KD reduced the complexity of dendritic arborization (NC, 32 neurons; Csde1 KD1, 21 neurons; Csde1 KD2, 42 neurons; Csde1 KD2 + WT, 27 neurons; Csde1 KD2 + Ctnnb1, 26 neurons). (C) Csde1 KD disrupted dendritic spine morphogenesis and maturation (NC, 27 neurons; Csde1 KD1, 41 neurons; Csde1 KD2, 23 neurons; Csde1 KD2 + WT, 40 neurons; Csde1 KD2 + Ctnnb1, 28 neurons; Csde1 KD2 + Licl, 23 neurons). Scale bar, 10 μm. (D) Csde1 KD reduced the number of excitatory (vGlut) and inhibitory (vGAT) synapses (NC, 41 neurons; Csde1 KD2, 46 neurons; Csde1 KD2 + WT, 30 neurons; Csde1 KD2 + Ctnnb1, 39 neurons; Csde1 KD2 + Licl, 28 neurons). Scale bar, 10 μm. (E and F) Voltage-clamp whole-cell recordings showed that both frequency and amplitude of mEPSCs and mIPSCs were decreased in Csde1 KD neurons (three neurons for each condition). (G) Immunoblot showed that β-catenin expression was markedly decreased in Csde1 KD neurons. Statistical data were presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

**Fig. 4. Disruption of dUnr interferes with synapse development and transmission in *Drosophila*.**
(A) Left: Representative NMJ4 synapses of dUnr-related lines (WT, *dunr*/+, *dunr*, *dunr/Df1*, and *dunr/Df2*). Right: Both bouton number and satellite bouton number were increased in *dunr/+* line (n = 22), *dunr* line (n = 58), *dunr/Df1* line (n = 23), and *dunr/Df2* line (n = 25) compared to control (n = 61). The boutons were costained with anti-HRP labeling the neuronal plasma membrane (red) and anti-DLG (green) labeling a postsynaptic scaffold protein. Magnified image of the boxed region at left bottom shows the terminal bouton or the satellite bouton. Scale bar, 5 μm. (B) Left: Representative NMJ4 morphology of the Elav/+ line, a pan-neuronal dUnr KD line (Elav-RNAi), a *Drosophila* UNR rescue line (dUnr-res), and a human CSDE1 rescue line (hCSDE1-res). Right: Both bouton number and satellite bouton number were increased in Elav-RNAi line (n = 45) compared to Elav/+ line (n = 45). In dUnr-res line (n = 34) and hCSDE1-res line (n = 28), the numbers were decreased compared to Elav-RNAi line (n = 45). Scale bar, 5 μm. (C) Left: Representative traces of EJPs and mEJPs in the indicated genotypes. Right: The amplitudes of EJPs were mildly decreased in both *dunr/Df1* line (WT: n = 9; *dunr/Df1*: n = 13) and pan-neuron KD line (Elav/+: n = 29; Elav-RNAi: n = 17). Slightly but not significantly decreased mEJP amplitude and no significant change of quantal content were observed on both KD and *dunr/Df1* lines. NS, not significant. (D) The normalized fluorescent intensity of BRP was slightly increased in *dunr* (n = 76) and *dunr/Df1* (n = 63) lines compared to controls. Scale bar, 5 μm. (E) The normalized fluorescent intensity of postsynaptic GluRIIA was markedly reduced in *dunr/Df1* (n = 18), *dunr/Df2* (n = 23), and KD lines (n = 25) compared to controls. Scale bar, 5 μm. (F) Decreased normalized fluorescent intensity of FM 1-43 dye was detected in *dunr* (n = 14), *dunr/Df1* (n = 40), and KD (n = 61) lines compared to controls. Scale bar, 5 μm. Statistical data were presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

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Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission

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Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission

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