Mutations disrupting neuritogenesis genes confer risk for cerebral palsy
AbstractIn addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent–offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1A and CTNNB1) met genome-wide significance. We identified two novel monogenic etiologies, FBXO31 and RHOB, and showed that the RHOB mutation enhances active-state Rho effector binding while the FBXO31 mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in a Drosophila reverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.
Data availabilitySequencing data from University of Adelaide Robinson Research Institute (n = 154 trios) are available from the corresponding author on request, subject to human research ethics approval and patient consent. Data from PCH (n = 52 trios) are available from the corresponding author on request, subject to patient consent. Data from Zhengzhou City Children’s Hospital (n = 44 trios) are available in the CNSA of China National GeneBank DataBase repository (https://db.cngb.org/cnsa/). Source data are provided with this paper.References1.Christensen, D. et al. Prevalence of cerebral palsy, co-occurring autism spectrum disorders, and motor functioning – Autism and Developmental Disabilities Monitoring Network, USA, 2008. Dev. Med. Child Neurol. 56, 59–65 (2014).PubMed
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Download referencesAcknowledgementsWe gratefully acknowledge the support of the patients and families who have graciously and patiently supported this work from its inception. Without their partnership, these studies would not have been possible. We acknowledge the support of the clinicians who generously provided their expertise in support of this study, including M.-C. Waugh, M. Axt and V. Roberts of the Children’s Hospital Westmead; K. Lowe of Sydney Children’s Hospital; R. Russo, J. Rice and A. Tidemann of the Women’s and Children’s Hospital, Adelaide; T. Carroll and L. Copeland of the Lady Cilento Children’s Hospital, Brisbane; and J. Valentine of Perth Children’s Hospital. We appreciate the collaboration of S. Knoblach and E. Hoffman (Children’s National Medical Center). This work was supported in part by the Cerebral Palsy Alliance Research Foundation (M.C.K.), the Yale-NIH Center for Mendelian Genomics (U54 HG006504-01), Doris Duke Charitable Foundation CSDA 2014112 (M.C.K.), the Scott Family Foundation (M.C.K.), Cure CP (M.C.K.), NHMRC grant 1099163 (A.H.M., C.L.v.E., J.G. and M.A.C.), NHMRC Senior Principal Research Fellowship 1155224 (J.G.), Channel 7 Children’s Research Foundation (J.G.), a Cerebral Palsy Alliance Research Foundation Career Development Award (M.A.C.), the Tenix Foundation (A.H.M., J.G., C.L.v.E. and M.A.C.), the National Natural Science Foundation of China (U1604165, X.W.), Henan Key Research Program of China (171100310200, C. Zhu), VINNOVA (2015-04780, C. Zhu), the James Hudson Brown–Alexander Brown Coxe Postdoctoral Fellowship at the Yale University School of Medicine (S.C.J.), an American Heart Association Postdoctoral Fellowship (18POST34060008 to S.C.J.), the NIH K99/R00 Pathway to Independence Award (R00HL143036-02 to S.C.J.) and NIH grants R01NS091299 (D.C.Z.) and NIH R01NS106298 (M.C.K.).Author informationAuthor notesThese authors contributed equally: Sheng Chih Jin, Sara A. Lewis, Somayeh Bakhtiari, Xue Zeng.These authors jointly supervised this work: Qinghe Xing, Changlian Zhu, Kaya Bilguvar, Sergio Padilla-Lopez, Richard P. Lifton, Jozef Gecz, Alastair H. MacLennan, Michael C. Kruer.AffiliationsDepartment of Genetics, Yale University School of Medicine, New Haven, CT, USASheng Chih Jin, Xue Zeng, Michael C. Sierant, Junhui Zhang, Amar H. Sheth, Kaya Bilguvar & Richard P. LiftonLaboratory of Human Genetics and Genomics, Rockefeller University, New York, NY, USASheng Chih Jin, Xue Zeng, Michael C. Sierant & Richard P. LiftonDepartment of Genetics, Washington University School of Medicine, St Louis, MO, USASheng Chih JinPediatric Movement Disorders Program, Division of Pediatric Neurology, Barrow Neurological Institute, Phoenix Children’s Hospital, Phoenix, AZ, USASara A. Lewis, Somayeh Bakhtiari, Sheetal Shetty, Sandra M. Nordlie, Aureliane Elie, Bethany Y. Norton, Brandon S. Guida, Helen Magee, James Liu, Jennifer Heim, Sergio Padilla-Lopez & Michael C. KruerDepartments of Child Health, Neurology, and Cellular & Molecular Medicine, and Program in Genetics, University of Arizona College of Medicine–Phoenix, Phoenix, AZ, USASara A. Lewis, Somayeh Bakhtiari, Sheetal Shetty, Sandra M. Nordlie, Aureliane Elie, Bethany Y. Norton, Brandon S. Guida, Helen Magee, James Liu, Sergio Padilla-Lopez & Michael C. KruerRobinson Research Institute, The University of Adelaide, Adelaide, South Australia, AustraliaMark A. Corbett, Clare L. van Eyk, Jesia G. Berry, Kelly Harper, Dani L. Webber, Mahalia S. B. Frank, Jozef Gecz & Alastair H. MacLennanDepartment of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, London, UKShozeb HaiderMolecular Brain Sciences, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, ON, CanadaStephen Pastore & John B. VincentOne CP Place, Plano, TX, USAJanice Brunstrom-HernandezDivision of Paediatric Neurology, Iaso Children’s Hospital, Athens, GreeceAntigone PapavasileiouDepartment of Pediatrics, Monash University, Melbourne, Victoria, AustraliaMichael C. FaheyHenan Key Laboratory of Child Genetics and Metabolism, Rehabilitation Department, Children’s Hospital of Zhengzhou University, Zhengzhou, ChinaChongchen Zhou & Qing ShangDepartment of Biostatistics, Yale School of Public Health, New Haven, CT, USABoyang LiHenan Key Laboratory of Child Brain Injury, Third Affiliated Hospital of Zhengzhou University, Zhengzhou, ChinaLei Xia, Yiran Xu, Dengna Zhu, Bohao Zhang, Xiaoyang Wang & Changlian ZhuYale Center for Genome Analysis, Yale University, New Haven, CT, USAJames R. Knight, Christopher Castaldi, Irina R. Tikhonova, Francesc López-Giráldez, Shrikant M. Mane & Kaya BilguvarDepartment of Genetics, Pitié-Salpêtrière Hospital, APHP.Sorbonne Université, Paris, FranceBoris Keren & Julien BurattiUF de Génétique Clinique et Centre de Référence Anomalies du Développement et Syndromes Malformatifs, APHP.Sorbonne Université, Hôpital Armand Trousseau, Paris, FranceSandra WhalenSorbonne Université, APHP, Service de Neurologie Pédiatrique et Centre de Référence Neurogénétique, Hôpital Armand Trousseau, Paris, FranceDiane DoummarGeneDx, Gaithersburg, MD, USAMegan Cho, Kyle Retterer & Francisca MillanInstitute of Biomedical Science and Children’s Hospital, and Key Laboratory of Reproduction Regulation of the National Population and Family Planning Commission (NPFPC), Shanghai Institute of Planned Parenthood Research (SIPPR), IRD, Fudan University, Shanghai, ChinaYangong Wang & Qinghe XingDepartments of Pediatrics & Neurology, University of Texas Southwestern and Children’s Medical Center of Dallas, Dallas, TX, USAJeff L. WaughDepartments of Genetics & Genomics and Neurology, Boston Children’s Hospital, Boston, MA, USALance RodanDivision of Neurogenetics and Hugo W. Moser Research Institute, Kennedy Krieger Institute, Baltimore, MD, USAJulie S. Cohen & Ali FatemiMedical Genetics, Department of Pediatrics, MassGeneral Hospital for Children, Boston, MA, USAAngela E. LinDepartments of Pediatrics and Neurology, University of New Mexico, Albuquerque, NM, USAJohn P. PhillipsDivision of Pediatric Neurology, Gillette Children’s Hospital, St Paul, MN, USATimothy FeymaDepartment of Paediatric Neurology, Women’s & Children’s Hospital, Adelaide, South Australia, AustraliaSuzanna C. MacLennanDepartments of Molecular & Cellular Biology and Neuroscience, University of Arizona, Tucson, AZ, USASpencer Vaughan & Daniela C. ZarnescuMurdoch Children’s Research Institute and University of Melbourne Department of Paediatrics, Royal Children’s Hospital, Melbourne, Victoria, AustraliaKylie E. Crompton, Susan M. Reid, Dinah S. Reddihough & David J. AmorRehabilitation Department, Children’s Hospital of Zhengzhou University/Henan Children’s Hospital, Zhengzhou, ChinaChao GaoCerebral Palsy Alliance Research Institute, University of Sydney, Sydney, New South Wales, AustraliaIona Novak, Nadia Badawi, Yana A. Wilson & Sarah J. McIntyreInstitute of Neuroscience and Physiology, Sahlgrenska Academy, Gothenburg University, Gothenburg, SwedenXiaoyang Wang & Changlian ZhuDepartment of Biostatistics & Medical Informatics, University of Wisconsin-Madison, Madison, WI, USAQiongshi LuContributionsK.B., S.P.-L., Q.X., C. Zhu, R.P.L., A.H.M., J.G. and M.C.K. contributed to study design, data interpretation and oversight. B.Y.N., J.G.B., K.H., C. Zhou, D.Z., B.Z., B.K., S.W., J.B., S.P., J.B.V., J.B.-H., A.P., M.C.F., L.X., Y.X., M.C., K.R., F.M., Y.W., J.L.W., L.R., J.S.C., A.F., A.E.L., J.P.P., T.F., S.J.M., K.E.C., S.M.R., D.S.R., Q.S., C.G., Y.A.W., N.B., I.N., S.C.M., X.W., D.J.A., J.H. and M.C.K. provided cohort ascertainment, recruitment and phenotypic characterization. K.B., C.C., A.E., J.L., C.L.v.E., H.M., S.M.M., I.R.T., F.L.-G., Y.A.W., B.S.G., J.Z., D.L.W., M.S.B.F., C. Zhou and M.A.C. performed exome sequencing production and validation. S.B., S.C.J., M.A.C., M.C.S., X.Z., J.R.K. and A.H.S. performed WES analysis. A.E., H.M., J.L., B.S.G. and S.P.-L. performed RHOB validation. S.M.N., S.P.-L., S.P., J.B.V., D.D. and S.A.L. performed FBXO31 validation. S.A.L., S.V. and D.C.Z. performed Drosophila locomotor experiments. S.C.J., S.A.L., S.B., S.S., B.L., Q.L., M.C.S. and X.Z. conducted statistical analysis. S.H. performed biophysical simulation for RHOB and FBXO31. S.C.J., S.A.L., J.G., Q.L., S.P.-L., R.P.L., A.H.M., S.M., B.Y.N., M.C.S., X.Z., C.L.v.E., X.W., Q.X., C. Zhu and M.C.K. wrote and reviewed the manuscript. K.B., R.P.L., Q.X., C. Zhu, A.H.M., J.G., S.P.-L. and M.C.K. acquired funding and supervised the project and were considered co-senior authors. All authors have read and approved the final manuscript.Corresponding authorCorrespondence to
Michael C. Kruer.Ethics declarations
Competing interests
The authors declare no competing interests.
Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Brain MRI features of idiopathic cerebral palsy.F050: bilateral periventricular leukomalacia; F055: right sided porencephaly; F057: normal (equivocal putaminal rim hyperintensity); F063: mildly globally diminished cerebral volume; F066: normal; F068, bilateral mild periventricular leukomalacia, white matter thinning and colpocephaly; F069: diminished cortical more than cerebellar volumes; F074: normal; F076: ex vacuo ventriculomegaly; bilateral periventricular leukomalacia, and bilateral perisylvian pachygyria; F077: mild periventricular leukomalacia; F082: scattered subcortical T2 hyperintensities; F084: normal; F085: colpocephaly, thinning of periventricular white matter, hypoplastic corpus callosum, diminished left cerebellar hemispheric volume; F093: normal; F124: normal; F162: normal; F217: equivocal ex vacuo ventriculomegaly; F218: normal; F300: bilateral periventricular leukomalacia with thin corpus callosum; F306: scattered bilateral subcortical punctate T2/FLAIR hyperintensities; F309: simplified gyral pattern; F311: normal; F312: normal; F313: normal; F342: diminished cortical volume, thinning and T2/FLAIR signal hyperintensity of periventricular white matter, thin corpus callosum; F356: bilateral perisylvian polymicrogyria; F357: thin corpus callosum; F377: equivocally simplified gyri with ‘open opercula’; F383: bilateral occipital horn heterotopias; F385: hydrocephalus and periventricular leukomalacia; F393: periventricular leukomalacia; F433: normal; F439: increased frontotemporal extra-axial fluid spaces and thin corpus callosum; F444: normal (equivocally thickened corpus callosum); F468: slight ex vacuo ventriculomegaly; F470: equivocally diminished cortical volume; F606: bilateral perislyvian pachygyria; F609: bi hemispheric periventricular leukomalacia; F617: ex vacuo ventriculomegaly; F623: dysplastic corpus callosum, bitemporal diminished cortical volumes; F629: thin corpus callosum, colpocephaly, with periventricular leukomalacia; F648: periventricular leukomalacia; F658: right sided encephalomalacia affecting putamen and thalamus.Extended Data Fig. 2 De novo mutation rate closely approximates Poisson distribution in cases and controls.Observed number of de novo mutations per subject (bars) compared to the numbers expected (line) from the Poisson distribution in the case (red) and control (blue) cohorts. Here, ‘P’ denotes chi-squared P-value.Extended Data Fig. 3 De novo mutation in TUBA1A encoding α-tubulin.a, TUBA1A functional domains schematic with locations of previously-described pathogenic variants (red) compared to those from this work (black). b, Phylogenetic conservation of reference amino acid at each mutated position described in this work. c, Sanger-verified mutated base (red arrow) with the corresponding reference bases. d, MRI of the brain (F356) demonstrates evidence of bilateral perisylvian pachygyria (blue arrows). Conserved Domain Annotations: TNBDL (AA 1-244) as IPro36525; SD (AA 418-451) annotated as per39.Extended Data Fig. 4 De novo mutations in CTNNB1 encoding β-catenin.a, CTNNB1 functional domain with location of previously reported pathogenic variants (red) and those identified in this work (black). (Given the loss-of-function nature of the identified variants, phylogenetic alignments were not performed; however, 100% identify is seen at these loci (p.E54, p.F99, and p.R449) in primates). b, Sanger-verified mutated base (red arrow) with corresponding reference bases. c, Brain MRI (F066) was unremarkable. Conserved Domain Annotations: ARM, Armadillo/beta-catenin-like repeats from UniProtKB/Swiss-Prot (P35222.1); SCRIB, interaction with SCRIB (AA 772-781, by similarity, experimental evidence); BCL9, interaction with BCL9 (AA 156-178, by similarity, experimental evidence); VCL, interaction with VCL (AA 2-23, by similarity, experimental evidence).Extended Data Fig. 5 De novo mutations in ATL1 encoding atlastin-1.a, ATL1 functional domain with location of previously reported variants (red) as well as those identified in this work (black). b, Phylogenetic conservation of reference amino acid at each affected position. c, Sanger-verified mutated base (red arrow) with the corresponding reference bases. d, Brain MRI images from F050 and F609 demonstrate mild periventricular T2 hyperintensity (blue arrows). Conserved Domain Annotations: GBP (AA 43-314) as pfam02263; Membrane localization domain (AA 448-558) from UniProtKB (Q8WXF7.1).Extended Data Fig. 6 De novo mutations in SPAST encoding spastin.a, SPAST functional domains with location of CP-associated damaging variants identified in this study (black); 277 pathological mutations58 have previously been identified in SPAST with the majority (82%) located within the conserved domains (red). b, Phylogenetic conservation of wild-type amino acid at each mutated position. c, Sanger-verified mutated base indicated by red arrow with corresponding reference bases. d, Brain MRI (F082) showed mild subcortical T2 hyperintensities (blue arrows). Conserved Domain Annotations: MIT (AA 116-196) as CDD:239142; Microtubule Binding domain (AA 270-328) from UniProtKB/Swiss-Prot (Q9UBP0.1); ATPase AAA Core and Lid domains (378-567) from IPR003959 and IPR041569, respectively.Extended Data Fig. 7 De novo mutations in DHX32 encoding the DEAH box polypeptide 32.a, DHX32 functional domains with location of CP-associated damaging variants from this work (black). Germline DHX32 variants have not been previously associated with human disease although somatic variants (>40) have been associated with variants cancers (COSMIC). b, Phylogenetic conservation of wild-type amino acid at each mutated position. c, Sanger-verified mutated base indicated by red arrow with corresponding reference bases. d, Brain MRI (F063) showed diffusely diminished cortical volume. Conserved Domain Annotations: Helicase and DEAD domains overlap (72-378 and 146-403) from IPR014001 and cd17912, respectively; HA2 domain (AA 458-547) as IPR007502; Helicase associated domain of unknown function (AA 616-696) from IPR011709.Extended Data Fig. 8 De novo mutations in ALK encoding the anaplastic lymphoma kinase.a, ALK functional domain with location of previously reported pathogenic variants associated with susceptibility to neuroblastoma (OMIM# 613014) (red) as well as CP-associated damaging variants identified in this work (black). b, Phylogenetic conservation of wild-type amino acid at each mutated position. c, Sanger-verified mutated base indicated by red arrow with corresponding reference bases. d, Brain MRI (F306) demonstrates punctate subcortical T2 hyperintensities of both hemispheres. Conserved Domain Annotations: Signal Peptide (AA 1-18) by SignalP 4.0; MAM (AA 266-427, 480-636) as pfam #00629; LDLa (AA 441-467) as smart#00192; Fxa (AA 987-1021) as pfam#14670; PTKc ALK LTK (AA 1109-1385) as CDD#05036.Extended Data Fig. 9 Additional locomotor phenotypes of loss of function mutations in Drosophila orthologs of candidate cerebral palsy risk genes.Drosophila mutant and control genotypes are shown in Supplementary Table 9. a, Turning time, a measure of coordinated movements, is increased in larva with mutations in AKT3 and PNPLA7 orthologs, but not in MAP2K4. b-o, Distance threshold assay examining negative geotaxis climbing defects in for 14 day-old adult flies with mutations in orthologs of AGAP1 (b), AKT3 (c), ANKS1A (d), ARHGEF17 (e), DIAPH2 (f), HSPG2 (g), KIDINS220 (h), MAP2K4 (i), MPP1 (j), PNPLA7 (k), PRICKLE1 (l), SYNGAP1 (m), TBC1D17 (n), and TENM1 (o). Impairments in the climbing assay was detected for males with mutations in AKT3 and PRICKLE1 (c,l) and for both sexes with mutations in MAP2K4 and MPP1 (i,j) orthologs. Climbing phenotype mapped to gene using deficiency chromosome for AGAP1 (b), but did not map for TENM1 (o). There was no locomotor impairment in the two negative control genotypes, ARHGEF15 and ANKS1A, where the patient variant did not pass our deleteriousness filters (d). For larval turning, box indicates 75th and 25th percentile with median line; whiskers indicate 10th and 90th percentile (n = 50 larvae). Locomotor curve represents average of all trials and bars indicate standard error (n = 10-21 trials). Statistics between larval turning times determined using unpaired 2-tailed t-test. Locomotor curves considered to be significantly different from each other if P
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