Cerebral palsy (CP), a neurodevelopmental disorder characterized by irreversible, nonprogressive central motor dysfunction, is commonly associated with prematurity or perinatal brain injury. However, accumulating evidence suggests deleterious genomic variants may contribute to CP in addition to environmental insults. To identify genes contributing to risk for CP, we performed whole-exome sequencing on 250 parent-offspring CP trios. We identified a significant contribution of damaging de novo mutations (DNMs), especially in genes that are intolerant to loss of function mutations. Eight genes had multiple, independently-arising damaging DNMs, including two novel CP-associated genes, FBXO31 and RHOB, and four genes previously implicated in cerebral palsy phenotypes, TUBA1A, CTNNB1, SPAST, and ATL1. Functional experiments, including molecular and biochemical assays and patient fibroblast studies indicate that the recurrent RHOB mutation identified in patients enhances Rho effector binding in the active state and that the FBXO31 mutation leads to elevated levels of cyclin D. Analysis of candidate CP risk genes highlighted genetic overlap with hereditary spastic paraplegia as well as intellectual disability, autism, and epilepsy, converging with epidemiologic findings. Computational network analysis of risk genes identified significant enrichment of Rho GTPase, extracellular matrix, focal adhesions, cytoskeleton, and cell projection pathways. CP risk genes in Rho GTPase, cytoskeleton and cell projection pathways were found to play an important role in neuromotor development via a Drosophila reverse genetics screen. Based on enrichment analysis, we estimate that an excess of damaging de novo and inherited recessive variants collectively account for ~14% of the cases in our cohort, whereas perinatal asphyxia is currently estimated to occur in 8-10% of CP cases. Together, these findings provide evidence for the role of genetically-mediated dysregulation of early brain connectivity in CP.

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Cerebral palsy (CP) is the most frequent movement disorder of childhood affecting 1 in 500 live births in developed countries. We previously identified likely pathogenic de novo or inherited single nucleotide variants (SNV) in 14% (14/98) of trios by exome sequencing and a further 5% (9/182) from evidence of outlier gene expression using RNA sequencing. Here, we detected copy number variants (CNV) from exomes of 186 unrelated individuals with CP (including our original 98 trios) using the CoNIFER algorithm. CNV were validated with Illumina 850 K SNP arrays and compared with RNA-Seq outlier gene expression analysis from lymphoblastoid cell lines (LCL). Gene expression was highly correlated with gene dosage effect. We resolved an additional 3.7% (7/186) of this cohort with pathogenic or likely pathogenic CNV while a further 7.7% (14/186) had CNV of uncertain significance. We identified recurrent genomic rearrangements previously associated with CP due to 2p25.3 deletion, 22q11.2 deletions and duplications and Xp monosomy. We also discovered a deletion of a single gene, PDCD6IP, and performed additional zebrafish model studies to support its single allele loss in CP aetiology. Combined SNV and CNV analysis revealed pathogenic and likely pathogenic variants in 22.7% of unselected individuals with CP.

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A growing body of evidence points to a considerable and heterogeneous genetic aetiology of cerebral palsy (CP). To identify recurrently variant CP genes, we designed a custom gene panel of 112 candidate genes. We tested 366 clinically unselected singleton cases with CP, including 271 cases not previously examined using next-generation sequencing technologies. Overall, 5.2% of the naïve cases (14/271) harboured a genetic variant of clinical significance in a known disease gene, with a further 4.8% of individuals (13/271) having a variant in a candidate gene classified as intolerant to variation. In the aggregate cohort of individuals from this study and our previous genomic investigations, six recurrently hit genes contributed at least 4% of disease burden to CP: COL4A1, TUBA1A, AGAP1, L1CAM, MAOB and KIF1A. Significance of Rare VAriants (SORVA) burden analysis identified four genes with a genome-wide significant burden of variants, AGAP1, ERLIN1, ZDHHC9 and PROC, of which we functionally assessed AGAP1 using a zebrafish model. Our investigations reinforce that CP is a heterogeneous neurodevelopmental disorder with known as well as novel genetic determinants.