Application of multiple mosaic callers improves post-zygotic mutation detection from exome sequencing data.

PURPOSE: The gold standard for identification of post-zygotic variants (PZVs) is droplet digital polymerase chain reaction or high-depth sequencing across multiple tissues types. These approaches are yet to be systematically implemented for monogenic disorders. We developed PZV detection pipelines for correct classification of de novo variants. METHOD: Our pipelines detect PZV in parents (gonosomal mosaicism [pGoM]) and children (somatic mosaicism, "M3"). We applied them to research exome sequencing (ES) data from the Australian Cerebral Palsy Biobank (n = 145 trios) and Simons Simplex Collection (n = 405 families). Candidate mosaic variants were validated using deep amplicon sequencing or droplet digital polymerase chain reaction. RESULTS: 69.2% (M3trio), 63.9% (M3single), and 92.7% (pGoM) of detected variants were validated, with 48.6%, 56.7%, and 26.2% of variants, respectively, meeting strict criteria for mosaicism. In the Australian Cerebral Palsy Biobank, 16.6% of probands and 20.7% of parents had at least 1 true-positive somatic or pGoM variant, respectively. A large proportion of PZVs detected in Simons Simplex Collection parents (79.8%) and child (94.5%) were not previously reported. We reclassified 3.7% to 8.0% of germline de novo variants as mosaic. CONCLUSION: Many PZVs were incorrectly classified as germline variants or missed by previous approaches. Systematic application of our pipelines could increase genetic diagnostic rate, improve estimates of recurrence risk in families, and benefit novel disease gene identification.

Exome sequencing reveals genetic heterogeneity and clinically actionable findings in children with cerebral palsy.

Cerebral palsy (CP) is the most common motor disability in children. To ascertain the role of major genetic variants in the etiology of CP, we conducted exome sequencing on a large-scale cohort with clinical manifestations of CP. The study cohort comprised 505 girls and 1,073 boys. Utilizing the current gold standard in genetic diagnostics, 387 of these 1,578 children (24.5%) received genetic diagnoses. We identified 412 pathogenic and likely pathogenic (P/LP) variants across 219 genes associated with neurodevelopmental disorders, and 59 P/LP copy number variants. The genetic diagnostic rate of children with CP labeled at birth with perinatal asphyxia was higher than the rate in children without asphyxia (P = 0.0033). Also, 33 children with CP manifestations (8.5%, 33 of 387) had findings that were clinically actionable. These results highlight the need for early genetic testing in children with CP, especially those with risk factors like perinatal asphyxia, to enable evidence-based medical decision-making.

Transgenerational adverse effects of valproate? A patient report from 90 affected families.

BACKGROUND: Valproate use during pregnancy increases risk in malformations and neurodevelopmental disorders. Data from the experimental setting in mice showed valproate is a direct inhibitor of histone deacetylase, inducing histone hyperacetylation, histone methylation, and DNA demethylation causing congenital malformations with an epigenetic inheritance. We investigated potential transgenerational adverse effects of valproate. METHODS: We questioned 108 individuals (from 90 families) suffering complications due to valproate exposure in utero who were parents themselves (85 women and 23 men) about the occurrence of malformations and neurodevelopmental disorders in their children. All were member of Aide aux Parents d'Enfants souffrants du Syndrome de l'AntiConvulsivant (APESAC), a charity created in 2011 to provide personal assistance and support to families suffering complications due to valproate exposure during pregnancy. RESULTS: Among their 187 children they reported 43 (23%) children with malformation(s) (26 hand or foot malformations; 15 dysmorphic facial features; 10 renal/urologic malformations; 6 spina bifida; 4 cardiac malformation; 2 craniosynostosis; 2 cleft lip and palate) and 82 (44%) children with neurodevelopmental disorders (63 problematic behaviors and autism; 41 psychomotor disorders; 16 language problems; 16 attention deficit; 5 mental retardation). Only 88 (47%) children had neither malformation nor developmental disorders. CONCLUSION: These data add to the need for funding pharmacoepidemiological investigations of epigenetic inheritance caused by drugs causing malformations or neurodevelopmental disorders. Individuals exposed in utero to valproate must be informed about the risk, so they can consider fertility options, antenatal diagnosis, and adequate early surveillance.

Definition and diagnosis of cerebral palsy in genetic studies: a systematic review.

AIM: To conduct a systematic review of phenotypic definition and case ascertainment in published genetic studies of cerebral palsy (CP) to inform guidelines for the reporting of such studies. METHOD: Inclusion criteria comprised genetic studies of candidate genes, with CP as the outcome, published between 1990 and 2019 in the PubMed, Embase, and BIOSIS Citation Index databases. RESULTS: Fifty-seven studies met the inclusion criteria. We appraised how CP was defined, the quality of information on case ascertainment, and compliance with international consensus guidelines. Seven studies (12%) were poorly described, 33 studies (58%) gave incomplete information, and 17 studies (30%) were well described. Missing key information precluded determining how many studies complied with the definition by Rosenbaum et al. Only 18 out of 57 studies (32%) were compliant with the Surveillance of Cerebral Palsy in Europe (SCPE) international guidelines on defining CP. INTERPRETATION: Limited compliance with international consensus guidelines on phenotypic definition and mediocre reporting of CP case ascertainment hinders the comparison of results among genetic studies of CP (including meta-analyses), thereby limiting the quality, interpretability, and generalizability of study findings. Compliance with the SCPE guidelines is important for ongoing gene discovery efforts in CP, given the potential for misclassification of unrelated neurological conditions as CP.

Erratum: Author Correction: Pathogenic copy number variants that affect gene expression contribute to genomic burden in cerebral palsy.

[This corrects the article DOI: 10.1038/s41525-018-0073-4.].

Genetic or Other Causation Should Not Change the Clinical Diagnosis of Cerebral Palsy.

High throughput sequencing is discovering many likely causative genetic variants in individuals with cerebral palsy. Some investigators have suggested that this changes the clinical diagnosis of cerebral palsy and that these individuals should be removed from this diagnostic category. Cerebral palsy is a neurodevelopmental disorder diagnosed on clinical signs, not etiology. All nonprogressive permanent disorders of movement and posture attributed to disturbances that occurred in the developing fetal and infant brain can be described as "cerebral palsy." This definition of cerebral palsy should not be changed, whatever the cause. Reasons include stability, utility and accuracy of cerebral palsy registers, direct access to services, financial and social support specifically offered to families with cerebral palsy, and community understanding of the clinical diagnosis. Other neurodevelopmental disorders, for example, epilepsy, have not changed the diagnosis when genomic causes are found. The clinical diagnosis of cerebral palsy should remain, should prompt appropriate genetic studies and can subsequently be subclassified by etiology.

Pathogenic copy number variants that affect gene expression contribute to genomic burden in cerebral palsy.

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.

Analysis of 182 cerebral palsy transcriptomes points to dysregulation of trophic signalling pathways and overlap with autism.

Cerebral palsy (CP) is the most common motor disability of childhood. It is characterised by permanent, non-progressive but not unchanging problems with movement, posture and motor function, with a highly heterogeneous clinical spectrum and frequent neurodevelopmental comorbidities. The aetiology of CP is poorly understood, despite recent reports of a genetic contribution in some cases. Here we demonstrate transcriptional dysregulation of trophic signalling pathways in patient-derived cell lines from an unselected cohort of 182 CP-affected individuals using both differential expression analysis and weighted gene co-expression network analysis (WGCNA). We also show that genes differentially expressed in CP, as well as network modules significantly correlated with CP status, are enriched for genes associated with ASD. Combining transcriptome and whole exome sequencing (WES) data for this CP cohort likely resolves an additional 5% of cases separated to the 14% we have previously reported as resolved by WES. Collectively, these results support a convergent molecular abnormality in CP and ASD.

The genetic basis of cerebral palsy.

Although prematurity and hypoxic-ischaemic injury are well-recognized contributors to the pathogenesis of cerebral palsy (CP), as many as one-third of children with CP may lack traditional risk factors. For many of these children, a genetic basis to their condition is suspected. Recent findings have implicated copy number variants and mutations in single genes in children with CP. Current studies are limited by relatively small patient numbers, the underlying genetic heterogeneity identified, and the paucity of validation studies that have been performed. However, several genes mapping to intersecting pathways controlling neurodevelopment and neuronal connectivity have been identified. Analogous to other neurodevelopmental disorders such as autism and intellectual disability, the genomic architecture of CP is likely to be highly complex. Although we are just beginning to understand genetic contributions to CP, new insights are anticipated to serve as a unique window into the neurobiology of CP and suggest new targets for intervention.

Cerebral palsy: causes, pathways, and the role of genetic variants.

Cerebral palsy (CP) is heterogeneous with different clinical types, comorbidities, brain imaging patterns, causes, and now also heterogeneous underlying genetic variants. Few are solely due to severe hypoxia or ischemia at birth. This common myth has held back research in causation. The cost of litigation has devastating effects on maternity services with unnecessarily high cesarean delivery rates and subsequent maternal morbidity and mortality. CP rates have remained the same for 50 years despite a 6-fold increase in cesarean birth. Epidemiological studies have shown that the origins of most CP are prior to labor. Increased risk is associated with preterm delivery, congenital malformations, intrauterine infection, fetal growth restriction, multiple pregnancy, and placental abnormalities. Hypoxia at birth may be primary or secondary to preexisting pathology and international criteria help to separate the few cases of CP due to acute intrapartum hypoxia. Until recently, 1-2% of CP (mostly familial) had been linked to causative mutations. Recent genetic studies of sporadic CP cases using new-generation exome sequencing show that 14% of cases have likely causative single-gene mutations and up to 31% have clinically relevant copy number variations. The genetic variants are heterogeneous and require function investigations to prove causation. Whole genome sequencing, fine scale copy number variant investigations, and gene expression studies may extend the percentage of cases with a genetic pathway. Clinical risk factors could act as triggers for CP where there is genetic susceptibility. These new findings should refocus research about the causes of these complex and varied neurodevelopmental disorders.

Rare copy number variation in cerebral palsy.

Recent studies have established the role of rare copy number variants (CNVs) in several neurological disorders but the contribution of rare CNVs to cerebral palsy (CP) is not known. Fifty Caucasian families having children with CP were studied using two microarray designs. Potentially pathogenic, rare (<1% population frequency) CNVs were identified, and their frequency determined, by comparing the CNVs found in cases with 8329 adult controls with no known neurological disorders. Ten of the 50 cases (20%) had rare CNVs of potential relevance to CP; there were a total of 14 CNVs, which were observed in <0.1% (<8/8329) of the control population. Eight inherited from an unaffected mother: a 751-kb deletion including FSCB, a 1.5-Mb duplication of 7q21.13, a 534-kb duplication of 15q11.2, a 446-kb duplication including CTNND2, a 219-kb duplication including MCPH1, a 169-kb duplication of 22q13.33, a 64-kb duplication of MC2R, and a 135-bp exonic deletion of SLC06A1. Three inherited from an unaffected father: a 386-kb deletion of 12p12.2-p12.1, a 234-kb duplication of 10q26.13, and a 4-kb exonic deletion of COPS3. The inheritance was unknown for three CNVs: a 157-bp exonic deletion of ACOX1, a 693-kb duplication of 17q25.3, and a 265-kb duplication of DAAM1. This is the first systematic study of CNVs in CP, and although it did not identify de novo mutations, has shown inherited, rare CNVs involving potentially pathogenic genes and pathways requiring further investigation.

Cesarean delivery and cerebral palsy: a systematic review and meta-analysis.

OBJECTIVE: To examine the association of cesarean delivery and cerebral palsy using a systematic literature review and meta-analysis. DATA SOURCES: MEDLINE, Embase, and ClinicalTrials.gov were systematically searched for articles relating to cerebral palsy and cesarean delivery from inception until December 2012. Only articles reporting confirmed cases of cerebral palsy were included. Meta-analysis was used to assess combined results and also the following subgroups: emergency cesarean; elective cesarean; term delivery; preterm delivery; and delivery of breech-presenting newborns. METHODS OF STUDY SELECTION: Literature searches returned 1,874 articles with 58 considered in full. Studies were selected if they reported an endpoint of cerebral palsy, an intervention or risk of cesarean delivery, were in English, and gave sufficient details to perform meta-analysis. TABULATION, INTEGRATION, AND RESULTS: Nine case-control and four cohort studies were included in the overall analysis. Meta-analysis showed no overall association of cesarean delivery with cerebral palsy (odds ratio [OR] 1.29; 95% confidence interval [CI] 0.92-1.79; 3,810 case group participants and 1,692,580 control group participants). Emergency cesarean delivery was associated with increased risk of cerebral palsy (OR 2.17; 95% CI 1.58-2.98), whereas there was no significant association between elective cesarean delivery and cerebral palsy (OR 0.81; 95% CI 0.41-1.58). Any type of cesarean delivery (elective or emergency) for term newborns was associated with cerebral palsy (OR 1.6; 95% CI 1.05-2.44), whereas there was no association between any type of cesarean delivery and cerebral palsy in preterm newborns (OR 0.81; 95% CI 0.47-1.40). Cesarean delivery did not significantly modify cerebral palsy risk for breech-presenting newborns (OR 0.51; 95% CI 0.13-2.05). CONCLUSION: A review of the literature does not support the use of elective or emergency cesarean delivery to prevent cerebral palsy.

NKX2-1 mutation in a family diagnosed with ataxic dyskinetic cerebral palsy.

Benign hereditary chorea caused by mutations in the NK2 homeobox 1 gene (NKX2-1), shares clinical features with ataxic and dyskinetic cerebral palsy (CP), resulting in the possibility of misdiagnosis. A father and his two children were considered to have ataxic CP until a possible diagnosis of benign familial chorea was made in the children in early teenage. The father's neurological condition had not been appreciated prior to examination of the affected son. Whole exome sequencing of blood derived DNA and bioinformatics analysis were performed. A 7 bp deletion in exon 1 of NKX2-1, resulting in a frame shift and creation of a premature termination codon, was identified in all affected individuals. Screening of 60 unrelated individuals with a diagnosis of dyskinetic or ataxic CP did not identify NKX2-1 mutations. BHC can be confused with ataxic and dyskinetic CP. Occasionally these children have a mutation in NKX2-1.