In 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.
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Sequencing 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.
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We 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.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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).
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).
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.
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.
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 < 0.05 for Kolomogrov-Smirnov test in addition to a significant difference at one or more time bins by Mann-Whitney rank sum 2-tailed test. *P < 0.05, ****P < 1 ×10−6. Exact genotypes, n, and P values are provided in Supplementary Table 9.
a, Estimation of the number of cerebral palsy risk genes via de novo mechanism. Monte Carlo simulation performed was performed based on observed damaging de novo mutations in 3,049 loss-of-function intolerant genes (pLI ≥ 0.9 in gnomAD (v2.1.1)) using 20,000 iterations. We estimate that the number of risk genes via de novo events to be ~75 (95% confidence interval = (26.5, 123.5)). b, Estimation of the number of recurrent genes. The number of trios and the number of genes with more than one damaging de novo mutation are specified on the x and y-axis, respectively. We modeled the expected rate of damaging de novo mutations given an increasing sample size. A total of 10,000 iterations were performed to estimate the number of genes with more than one damaging de novo mutations taking into account of the damaging de novo mutation probability. WES of 2,500 and 7,500 trios are expected to yield a 65.3% and 91.8% saturation rate, respectively, for all cerebral palsy risk genes.
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Jin, S.C., Lewis, S.A., Bakhtiari, S. et al. Mutations disrupting neuritogenesis genes confer risk for cerebral palsy. Nat Genet 52, 1046–1056 (2020). https://doi.org/10.1038/s41588-020-0695-1