CNV have long been known to be significant contributors to the aetiology of neurodevelopmental disorders.17 Similar to other neurodevelopmental disorders such as epilepsy, ID or ASD, CNV contribute to the pathogenesis of CP.9,10,11,12 For the purpose of molecular diagnosis, chromosomal microarrays (with SNP and/or oligonucleotide probes) are usually selected for detection of CNV because of their high sensitivity and specificity. In this study, we elected to take advantage of our unique, existing exome sequencing data of 186 individuals with CP to make an efficient and cost-effective survey of CNV burden in the largest unselected CP cohort to date, rather than reanalyse the entire cohort with arrays. The proportion of this cohort with pathogenic and likely pathogenic CNV in CP was lower compared with a previous study of a similar unselected cohort which could be due in part to the lower resolution of detection of exome CNV analysis compared to arrays.10 It is interesting to note that in our previous CNV study of 50 cases that were also reanalysed in this study (finding no additional CNV) that we did not find any de novo CNV. It is possible that the resolutions of the arrays we used previously, as well as the resolution for detection by exomes has contributed to this and smaller de novo CNV may yet be found.

RNA-Seq data identified genes within or adjacent to each CNV that were affected in expression and in one case, this confirmed a suspected deletion that was detected by the exome but not by SNP array. Based on the genotype tissue expression project (GTEx) median tissue expression data, 84.8% of transcripts expressed in cortex are also expressed in LCL (transcripts per million reads, TPM > 0) and expression levels are highly correlated, Pearson r = 0.83 therefore, this approach is useful for assessing the effects of CNV encompassing genes expressed in both tissues even in the context of neurodevelopmental disorders.18

Pathogenic CNV loci implicated in CP

We identified deletions of 2p25.3, Xp and 22q11.21 that overlap with deletions identified in previous studies of CNV in CP (Table 1).9,10,11 Deletions of 2p25.3 [OMIM: 616521] encompass a syndrome of ID, obesity, ASD, attention deficit hyperactivity disorder (ADHD) and delayed psychomotor development.19 The neurological phenotypes associated with 2p25.3 deletions have been linked with haploinsufficiency of MYT1L and that gene was also deleted in the individual in this study, however it was not expressed in LCL19 (Table S4).

Full or partial monosomy of the X chromosome in females causes Turner Syndrome. While not reported frequently, impaired movement that is not associated with intelligence scores has been observed in Turner Syndrome.20 There are two previously reported females with CP attributed to partial chromosome X monosomy.10,11 In this study, we discovered an individual with Xp monosomy and a terminal 1q43q44 trisomy due to translocation t(X;1), (p11.22; q43) (Table 1; Tables S2 and S3). This individual had a complex phenotype with dysmorphic features, short stature, microcephaly, global developmental delay and CP. By 11 years 4 months she was wheelchair bound. A review of 112 deletions and 47 duplications encompassing 1q43q44 noted problems with muscle tone and muscle control in 27 of the 47 individuals with pure or complex duplications.21 We concluded that, this translocation was pathogenic and there may be contributions from both the deleted Xp and the duplicated 1q43q44 to CP in this case.

Both deletions and duplications of 22q11.2 have been previously reported in CP.9,11

22q11.2 deletion syndrome [OMIM: 192430], also known as velocardiofacial or DiGeorge syndrome is clinically heterogeneous and the most common, recurrent, pathogenic microdeletion that occurs in humans.22 CP and other movement phenotypes are infrequently reported in this multisystemic disorder that affects the heart, immune system, parathyroid, craniofacial and central nervous system development.23 In one report, mild left hemiparesis was noted in a male with 22q11.2 deletion and cerebellar atrophy on brain MRI.24 Duplications of 22q11.2 are reported less frequently than deletions [OMIM: 608363] and penetrance is variable. A clinical review of multiple individuals affected by this duplication found that motor delay and hypotonia were frequent features.25

Pathogenic loci not previously implicated in CP

We identified two de novo CNV that are known to be pathogenic for neurodevelopmental disorders, also with incomplete penetrance and variable expressivity; a 16p11.2-p12.2 deletion [OMIM: 611913]26 and a 1q21.1 duplication. A review of clinical observations in 136 individuals with 16p11.2 deletions noted abnormal agility in 50%26 while duplications of 1q21.1 overlapping with the individual in this study have abnormal agility in 39%.27 While the known pathogenic CNV loci described above are associated with distinct neurodevelopmental syndromes, our clinical and genetic data, combined with previous observations of significant effects on movement, strongly suggest them as causing CP in these individuals.

At the time of writing, there were 33 individuals in the DECIPHER (DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources) database with open access phenotype information that includes the term “cerebral palsy”. We did not find any CNV or SNV in that cohort that overlapped with new CNV reported in this study.

Novel loci and genes

A de novo microdeletion at 3p22.3 encompassing only the gene for programmed cell death 6 interacting protein PDCD6IP (also commonly referred to as ALIX) that segregated with CP in monozygotic twin brothers was associated with significant down regulation of that gene in LCL derived from one of the affected individuals (Fig. 1a, Table S6). PDCD6IP has critical roles in cytokinesis and also interacts with the endosomal sorting complex required for transport (ESCRT) multiprotein complexes which, in turn are required for trafficking of multi vesicular bodies in the cell. Mice null for Pdcd6ip develop hydrocephalus resulting in bilateral enlargement of the lateral ventricles, thinning of the cerebral cortex and atrophy of the hippocampus.28 The loss of Pdcd6ip caused disorganisation of alignment of cilia and a loss of integrity of epithelial cell barriers in the brain and other tissues.28 These mouse data combined with our observations from zebrafish strongly implicate PDCD6IP as a disease locus in neurodevelopmental disorders.

Variants of uncertain significance

We identified five deletions and nine duplications that passed our filtering criteria and occurred at loci that are not yet implicated in CP or other neurodevelopmental syndromes. Based on available clinical data and functional annotation of the genes within these regions, we predict that six of these events have potential to be involved in CP (Table 1). We singled out these six CNV found on 6p22.3, 9p24.3-p24.2, 9q34.1, 10q22.1-q22.2, 19p13.3 and Xq22.1 (Table 1 and Tables S15-S18, S21 and S23) because in each case they have either multiple reported individuals in DECIPHER with similar but not identical phenotypes and/or encompass genes known to be involved in delayed motor development. Noteworthy among these CNV, was a duplication we identified located within Xq22.1 and inherited from an unaffected mother (Table 1, Table S23). Within this X-linked duplication, the mRNA polyadenylation factor CSTF2 was found to be significantly up regulated (Table S23) while other genes with detectable levels of expression were unaffected. This male also had a male second cousin related through possible female carriers that was also affected by CP, however a DNA sample was not available for segregation testing. Individuals with similar duplications in this region in DECIPHER have phenotypes consistent with those in the individual in this study including ID, ASD and epilepsy (Table 1).

For each of these variants of uncertain significance, additional patients and further cell molecular and animal modelling will be required to confirm or deny their pathogenicity.