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Focal Cortical Dysplasia (FCD) is one of the most common brain malformations and a frequent cause of intractable epilepsy. Affected individuals often require surgical removal of the affected cortex for seizure control. The European Epilepsy Brain Bank estimates that different subtypes of FCD are the reason for up to 70.6% of epilepsy surgery related to brain malformations. Given the clinical importance of this disorder, there is an urgent need to further understand its biology and disease mechanism. Although FCD was first described 50 years ago, the genetic and pathogenic mechanisms of the disorder remain incompletely understood. We hypothesise that the delineation of disease mechanism underlying FCD will not only improve diagnosis and clinical management, but will also provide novel insights into the mechanisms underpinning human brain development and function. This project applied modern genomic technologies to comprehensively study a unique archive of surgical brain tissues derived from individuals affected by FCD. The first part of this study focussed on understanding the genetic basis of a subtype of FCD, known as bottom-of-sulcus dysplasia (BOSD). BOSD is a highly localised subtype of FCD with the dysplastic features being maximal at the bottom of sulcus. Using deep sequencing, we analysed blood and brain derived gDNA from 20 BOSD brain specimens. We found that brain-specific, pathogenic somatic MTOR variants accounted for 50% of the cases, with an additional two cases caused by heterozygous germline variant in DEPDC5 or NPRL3. These genes are all involved in the mTOR signalling pathway. Further characterisation of four somatic MTOR cases found that the somatic variants were more abundant in the bottom of sulcus compared to the nearby cortex, with a positive correlation with the severity of histopathology. Our study shows that BOSD belongs to an expanding group of “mTORopathies” and highlights that BOSD lesion is highly localised, with well-defined genetic and

An inherited neurologic syndrome in a family of mixed-breed Oriental cats has been characterized as forebrain commissural malformation, concurrent with ventriculomegaly and interhemispheric cysts. However, the genetic basis for this autosomal recessive syndrome in cats is unknown. Forty-three cats were genotyped on the Illumina Infinium Feline 63K iSelect DNA Array and used for analyses. Genome-wide association studies, including a sib-transmission disequilibrium test and a case-control association analysis, and homozygosity mapping, identified a critical region on cat chromosome A3. Short-read whole genome sequencing was completed for a cat trio segregating with the syndrome. A homozygous 7 bp deletion in growth differentiation factor 7 (GDF7) (c.221_227delGCCGCGC [p.Arg74Profs]) was identified in affected cats, by comparison to the 99 Lives Cat variant dataset, validated using Sanger sequencing and genotyped by fragment analyses. This variant was not identified in 192 unaffected cats in the 99 Lives dataset. The variant segregated concordantly in an extended pedigree. In mice, GDF7 mRNA is expressed within the roof plate when commissural axons initiate ventrally-directed growth. This finding emphasized the importance of GDF7 in the neurodevelopmental process in the mammalian brain. A genetic test can be developed for use by cat breeders to eradicate this variant.

An inherited neurologic syndrome in a family of mixed-breed Oriental cats has been characterized as forebrain commissural malformation, concurrent with ventriculomegaly and interhemispheric cysts. However, the genetic basis for this autosomal recessive syndrome in cats is unknown. Forty-three cats were genotyped on the Illumina Infinium Feline 63K iSelect DNA Array and used for analyses. Genome-wide association studies, including a sib-transmission disequilibrium test and a case-control association analysis, and homozygosity mapping, identified a critical region on cat chromosome A3. Short-read whole genome sequencing was completed for a cat trio segregating with the syndrome. A homozygous 7 bp deletion in growth differentiation factor 7 (GDF7) (c.221_227delGCCGCGC [p.Arg74Profs]) was identified in affected cats, by comparison to the 99 Lives Cat variant dataset, validated using Sanger sequencing and genotyped by fragment analyses. This variant was not identified in 192 unaffected cats in the 99 Lives dataset. The variant segregated concordantly in an extended pedigree. In mice, GDF7 mRNA is expressed within the roof plate when commissural axons initiate ventrally-directed growth. This finding emphasized the importance of GDF7 in the neurodevelopmental process in the mammalian brain. A genetic test can be developed for use by cat breeders to eradicate this variant.

An inherited neurologic syndrome in a family of mixed-breed Oriental cats has been characterized as forebrain commissural malformation, concurrent with ventriculomegaly and interhemispheric cysts. However, the genetic basis for this autosomal recessive syndrome in cats is unknown. Forty-three cats were genotyped on the Illumina Infinium Feline 63K iSelect DNA Array and used for analyses. Genome-wide association studies, including a sib-transmission disequilibrium test and a case-control association analysis, and homozygosity mapping, identified a critical region on cat chromosome A3. Short-read whole genome sequencing was completed for a cat trio segregating with the syndrome. A homozygous 7 bp deletion in growth differentiation factor 7 (GDF7) (c.221_227delGCCGCGC [p.Arg74Profs]) was identified in affected cats, by comparison to the 99 Lives Cat variant dataset, validated using Sanger sequencing and genotyped by fragment analyses. This variant was not identified in 192 unaffected cats in the 99 Lives dataset. The variant segregated concordantly in an extended pedigree. In mice, GDF7 mRNA is expressed within the roof plate when commissural axons initiate ventrally-directed growth. This finding emphasized the importance of GDF7 in the neurodevelopmental process in the mammalian brain. A genetic test can be developed for use by cat breeders to eradicate this variant.

Previous studies have proposed that the early elucidation of brain injury from structural Magnetic Resonance Images (sMRI) is critical for the clinical assessment of children with cerebral palsy (CP). Although distinct aetiologies, including cortical maldevelopments, white and grey matter lesions and ventricular enlargement, have been categorised, these injuries are commonly only assessed in a qualitative fashion. As a result, sMRI remains relatively underexploited for clinical assessments, despite its widespread use. In this study, several automated and validated techniques to automatically quantify these three classes of injury were generated in a large cohort of children (n = 139) aged 5–17, including 95 children diagnosed with unilateral CP. Using a feature selection approach on a training data set (n = 97) to find severity of injury biomarkers predictive of clinical function (motor, cognitive, communicative and visual function), cortical shape and regional lesion burden were most often chosen associated with clinical function. Validating the best models on the unseen test data (n = 42), correlation values ranged between 0.545 and 0.795 (p<0.008), indicating significant associations with clinical function. The measured prevalence of injury, including ventricular enlargement (70%), white and grey matter lesions (55%) and cortical malformations (30%), were similar to the prevalence observed in other cohorts of children with unilateral CP. These findings support the early characterisation of injury from sMRI into previously defined aetiologies as part of standard clinical assessment. Furthermore, the strong and significant association between quantifications of injury observed on structural MRI and multiple clinical scores accord with empirically established structure-function relationships. © 2017 Pagnozzi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and

To gain a better understanding of the clinical and genetic features associated with agenesis of corpus callosum, we enrolled and characterized 162 patients with complete or partial agenesis of corpus callosum. Clinical and genetic protocols allowed us to categorize patients as syndromic subjects, affected by complex extra-brain malformations, and nonsyndromic subjects without any additional anomalies. We observed slight differences in sex ratio (56% males) and agenesis type (52% complete). Syndromic agenesis of corpus callosum subjects were prevalent (69%). We detected associated cerebral malformations in 48% of patients. Neuromotor impairment, cognitive and language disorders, and epilepsy were frequently present, regardless of the agenesis of corpus callosum subtype. Long-term follow-up allowed us to define additional indicators: syndromic agenesis of corpus callosum plus patients showed the most severe clinical features while isolated complete agenesis of corpus callosum patients had the mildest symptoms, although we observed intellectual disability (64%) and epilepsy (15%) in both categories. We achieved a definitive (clinical and/or genetic) diagnosis in 42% of subjects.

Genomic sequence interpretation can miss clinically relevant missense variants for several reasons. Rare missense variants are numerous in the exome and difficult to prioritise. Affected genes may also not have existing disease association. To improve variant prioritisation, we leverage population exome data to identify intragenic missense-depleted regions (MDRs) genome-wide that may be important in disease. We then use missense depletion analyses to help prioritise undiagnosed disease exome variants. We demonstrate application of this strategy to identify a novel gene association for human brain malformation. We identified de novo missense variants that affect the GDP/GTP-binding site of ARF1 in three unrelated patients. Corresponding functional analysis suggests ARF1 GDP/GTP-activation is affected by the specific missense mutations associated with heterotopia. These findings expand the genetic pathway underpinning neurologic disease that classically includes FLNA. ARF1 along with ARFGEF2 add further evidence implicating ARF/GEFs in the brain. Using functional ontology, top MDR-containing genes were highly enriched for nucleotide-binding function, suggesting these may be candidates for human disease. Routine consideration of MDR in the interpretation of exome data for rare diseases may help identify strong genetic factors for many severe conditions, infertility/reduction in reproductive capability, and embryonic conditions contributing to preterm loss.

Genomic sequence interpretation can miss clinically relevant missense variants for several reasons. Rare missense variants are numerous in the exome and difficult to prioritise. Affected genes may also not have existing disease association. To improve variant prioritisation, we leverage population exome data to identify intragenic missense-depleted regions (MDRs) genome-wide that may be important in disease. We then use missense depletion analyses to help prioritise undiagnosed disease exome variants. We demonstrate application of this strategy to identify a novel gene association for human brain malformation. We identified de novo missense variants that affect the GDP/GTP-binding site of ARF1 in three unrelated patients. Corresponding functional analysis suggests ARF1 GDP/GTP-activation is affected by the specific missense mutations associated with heterotopia. These findings expand the genetic pathway underpinning neurologic disease that classically includes FLNA. ARF1 along with ARFGEF2 add further evidence implicating ARF/GEFs in the brain. Using functional ontology, top MDR-containing genes were highly enriched for nucleotide-binding function, suggesting these may be candidates for human disease. Routine consideration of MDR in the interpretation of exome data for rare diseases may help identify strong genetic factors for many severe conditions, infertility/reduction in reproductive capability, and embryonic conditions contributing to preterm loss.

Objectives: Apert syndrome is characterized by craniosynostosis and complex hand and foot syndactyly, and an increased risk of brain, palate, heart, and visceral malformations, and intellectual disability. This study aims to describe the structural brain abnormalities detected by dedicated neuroimaging of fetuses with Apert syndrome. Method(s): Retrospective review of ultrasound and magnetic resonance imaging brain imaging obtained in six fetuses with a diagnosis of Apert syndrome. Result(s): Five fetuses had attenuation of the septal leaflets, and two had corpus callosum dysgenesis. All six had temporal lobe expansion and overconvolution and temporal lobe clefts. The temporal lobe abnormalities preceded the development of cranial deformity in two fetuses. Conclusion(s): Overexpansion and overconvolution of the temporal lobe is evident antenatally and is particularly conspicuous in the fetus when the normal brain is still relatively smooth (approximately 24 to 28weeks of gestation).Copyright © 2014 John Wiley & Sons, Ltd.

Export of mRNA from the cell nucleus to the cytoplasm is essential for protein synthesis, a process vital to all living eukaryotic cells. mRNA export is highly conserved and ubiquitous. Mutations affecting mRNA and mRNA processing or export factors, which cause aberrant retention of mRNAs in the nucleus, are thus emerging as contributors to an important class of human genetic disorders. Here, we report that variants in THOC2, which encodes a subunit of the highly conserved TREX mRNA-export complex, cause syndromic intellectual disability (ID). Affected individuals presented with variable degrees of ID and commonly observed features included speech delay, elevated BMI, short stature, seizure disorders, gait disturbance, and tremors. X chromosome exome sequencing revealed four missense variants in THOC2 in four families, including family MRX12, first ascertained in 1971. We show that two variants lead to decreased stability of THOC2 and its TREX-complex partners in cells derived from the affected individuals. Protein structural modeling showed that the altered amino acids are located in the RNA-binding domains of two complex THOC2 structures, potentially representing two different intermediate RNA-binding states of THOC2 during RNA transport. Our results show that disturbance of the canonical molecular pathway of mRNA export is compatible with life but results in altered neuronal development with other comorbidities.Copyright © 2015 The American Society of Human Genetics.

Objectives: Apert syndrome is characterized by craniosynostosis and complex hand and foot syndactyly, and an increased risk of brain, palate, heart, and visceral malformations, and intellectual disability. This study aims to describe the structural brain abnormalities detected by dedicated neuroimaging of fetuses with Apert syndrome. Method(s): Retrospective review of ultrasound and magnetic resonance imaging brain imaging obtained in six fetuses with a diagnosis of Apert syndrome. Result(s): Five fetuses had attenuation of the septal leaflets, and two had corpus callosum dysgenesis. All six had temporal lobe expansion and overconvolution and temporal lobe clefts. The temporal lobe abnormalities preceded the development of cranial deformity in two fetuses. Conclusion(s): Overexpansion and overconvolution of the temporal lobe is evident antenatally and is particularly conspicuous in the fetus when the normal brain is still relatively smooth (approximately 24 to 28weeks of gestation).Copyright © 2014 John Wiley & Sons, Ltd.

Export of mRNA from the cell nucleus to the cytoplasm is essential for protein synthesis, a process vital to all living eukaryotic cells. mRNA export is highly conserved and ubiquitous. Mutations affecting mRNA and mRNA processing or export factors, which cause aberrant retention of mRNAs in the nucleus, are thus emerging as contributors to an important class of human genetic disorders. Here, we report that variants in THOC2, which encodes a subunit of the highly conserved TREX mRNA-export complex, cause syndromic intellectual disability (ID). Affected individuals presented with variable degrees of ID and commonly observed features included speech delay, elevated BMI, short stature, seizure disorders, gait disturbance, and tremors. X chromosome exome sequencing revealed four missense variants in THOC2 in four families, including family MRX12, first ascertained in 1971. We show that two variants lead to decreased stability of THOC2 and its TREX-complex partners in cells derived from the affected individuals. Protein structural modeling showed that the altered amino acids are located in the RNA-binding domains of two complex THOC2 structures, potentially representing two different intermediate RNA-binding states of THOC2 during RNA transport. Our results show that disturbance of the canonical molecular pathway of mRNA export is compatible with life but results in altered neuronal development with other comorbidities.Copyright © 2015 The American Society of Human Genetics.

Aim: The aim of this study was to describe the distribution of magnetic resonance imaging (MRI) patterns in a large population sample of children with cerebral palsy (CP) and to examine associations between MRI patterns, and antenatal and perinatal variables. Method(s): Data were retrieved from the Victorian CP Register for 884 children (527 males, 357 females) born between 1999 and 2006. Postneonatal MRI was classified for 594 children. For 563 children (329 males, 234 females) for whom classification was to a single MRI pattern, the frequency of each variable was compared between patterns and with the population frequency. Result(s): White matter injury was the most common MRI pattern (45%), followed by grey matter injury (14%), normal imaging (13%), malformations (10%), focal vascular insults (9%), and miscellaneous patterns (7%). Parity, birth gestation, level of neonatal care, Apgar score, and time to established respiration varied between MRI patterns (p<0.01). Nulliparity was most strongly associated with focal vascular insults, whereas multiparity was associated only with malformations. Grey matter injury was not associated with birth in a tertiary unit, but was strongly associated with severe perinatal compromise. The frequency of neonatal seizures and of nursery admissions was lowest among children with malformations. Interpretation(s): As known risk factors for CP are differentially associated with specific MRI patterns, future exploration of causal pathways might be facilitated when performed in pathogenically defined groups. © 2013 Mac Keith Press.

Aim: To demonstrate abnormalities found on fetal MRI in complicated monochorionic gestations and to discuss the adjunctive role of fetal MRI in the imaging diagnosis and management of these pregnancies. Method(s): Retrospective audit of all women referred to the Monash Health Fetal MRI service for evaluation of monochorionic twin gestations at >20 weeks following either spontaneous co - twin death or post-selective fetoscopic laser photocoagulation (SFLP) of placental anastomoses. Result(s): Fourteen women were referred for fetal MRI. A variety of findings were identified some of which were undiagnosed or nonspecific on ultrasound. These included polymicrogyria, dural sinus malformation, intraventricular haemorrhage, cerebral infarction, isolated ventriculomegaly and normal findings. Conclusion(s): Fetal MRI can provide additional for prognostication, counselling and further management of women with complicated monochorionic gestations as well as surrogate clinical follow-up data about the short term outcome of fetuses from pregnancies treated with SFLP. Fetuses that were the surviving donor in the setting of twin twin transfusion or co-twin death were at highest risk for cerebral malformations.

Aim: The aim of this study was to review the distribution of neuroimaging findings from a contemporary population cohort of individuals with cerebral palsy (CP) and to facilitate standardization of imaging classification. Method(s): Publications from 1995 to 2012 reporting imaging findings in population cohorts were selected through a literature search, and review of the titles, abstracts, and content of studies. Relevant data were extracted, including unpublished data from Victoria, Australia. The proportions for each imaging pattern were tabulated, and heterogeneity was assessed for all individuals with CP, and for subgroups based on gestational age, CP subtype, and Gross Motor Function Classification System level. Result(s): Studies from three geographic regions met the inclusion criteria for individuals with CP, and two additional studies reported on specific CP subtypes. Brain abnormalities were observed in 86% of scans, but were observed least often in children with ataxia (24-57%). White matter injury was the most common imaging pattern (19-45%), although the proportions showed high heterogeneity. Additional patterns were grey matter injury (21%), focal vascular insults (10%), malformations (11%), and miscellaneous findings (4-22%). Interpretation(s): This review suggests areas where further dialogue will facilitate progress towards standardization of neuroimaging classification. Standardization will enable future collaborations aimed at exploring the relationships among magnetic resonance imaging patterns, risk factors, and clinical outcomes, and, ultimately, lead to better understanding of causal pathways and opportunities for prevention. © 2013 Mac Keith Press.

Aim: The aim of this study was to describe the distribution of magnetic resonance imaging (MRI) patterns in a large population sample of children with cerebral palsy (CP) and to examine associations between MRI patterns, and antenatal and perinatal variables. Method(s): Data were retrieved from the Victorian CP Register for 884 children (527 males, 357 females) born between 1999 and 2006. Postneonatal MRI was classified for 594 children. For 563 children (329 males, 234 females) for whom classification was to a single MRI pattern, the frequency of each variable was compared between patterns and with the population frequency. Result(s): White matter injury was the most common MRI pattern (45%), followed by grey matter injury (14%), normal imaging (13%), malformations (10%), focal vascular insults (9%), and miscellaneous patterns (7%). Parity, birth gestation, level of neonatal care, Apgar score, and time to established respiration varied between MRI patterns (p<0.01). Nulliparity was most strongly associated with focal vascular insults, whereas multiparity was associated only with malformations. Grey matter injury was not associated with birth in a tertiary unit, but was strongly associated with severe perinatal compromise. The frequency of neonatal seizures and of nursery admissions was lowest among children with malformations. Interpretation(s): As known risk factors for CP are differentially associated with specific MRI patterns, future exploration of causal pathways might be facilitated when performed in pathogenically defined groups. © 2013 Mac Keith Press.

Aim: To demonstrate abnormalities found on fetal MRI in complicated monochorionic gestations and to discuss the adjunctive role of fetal MRI in the imaging diagnosis and management of these pregnancies. Method(s): Retrospective audit of all women referred to the Monash Health Fetal MRI service for evaluation of monochorionic twin gestations at >20 weeks following either spontaneous co - twin death or post-selective fetoscopic laser photocoagulation (SFLP) of placental anastomoses. Result(s): Fourteen women were referred for fetal MRI. A variety of findings were identified some of which were undiagnosed or nonspecific on ultrasound. These included polymicrogyria, dural sinus malformation, intraventricular haemorrhage, cerebral infarction, isolated ventriculomegaly and normal findings. Conclusion(s): Fetal MRI can provide additional for prognostication, counselling and further management of women with complicated monochorionic gestations as well as surrogate clinical follow-up data about the short term outcome of fetuses from pregnancies treated with SFLP. Fetuses that were the surviving donor in the setting of twin twin transfusion or co-twin death were at highest risk for cerebral malformations.

Purpose: Malformations of cortical development (MCDs) are usually highly epileptogenic, and their hyperexcitability could facilitate the occurrence of reflex seizures. We sought to characterize reflex seizures in patients with MCDs and refractory epilepsy. Method(s): Clinical, electrographic, and neuroimaging data were reviewed in eight patients with MCDs who had reflex seizures reproduced during presurgical evaluation. Result(s): All eight patients had both reflex and spontaneous seizures. In six, however, drop attacks or axial myoclonic seizures occurred only upon specific sensory stimulation. Reflex seizures were induced by more than one type of stimulus in most patients, but anatomofunctional correlations could usually be invoked. Six patients had significant intellectual impairment. Surgical resection controlled seizures in two patients. Conclusion(s): Reflex seizures in patients with MCDs may be medically refractory and may often manifest as drop attacks or axial myoclonus. Surgical resection of focal lesions can bring reflex seizures under control. Putative mechanisms related to the relatively low frequency of reflex seizures in MCDs are discussed. © 2005 International League Against Epilepsy.

Purpose: Malformations of cortical development (MCDs) are usually highly epileptogenic, and their hyperexcitability could facilitate the occurrence of reflex seizures. We sought to characterize reflex seizures in patients with MCDs and refractory epilepsy. Method(s): Clinical, electrographic, and neuroimaging data were reviewed in eight patients with MCDs who had reflex seizures reproduced during presurgical evaluation. Result(s): All eight patients had both reflex and spontaneous seizures. In six, however, drop attacks or axial myoclonic seizures occurred only upon specific sensory stimulation. Reflex seizures were induced by more than one type of stimulus in most patients, but anatomofunctional correlations could usually be invoked. Six patients had significant intellectual impairment. Surgical resection controlled seizures in two patients. Conclusion(s): Reflex seizures in patients with MCDs may be medically refractory and may often manifest as drop attacks or axial myoclonus. Surgical resection of focal lesions can bring reflex seizures under control. Putative mechanisms related to the relatively low frequency of reflex seizures in MCDs are discussed. © 2005 International League Against Epilepsy.

Occipital epilepsies often elude diagnosis as they frequently masquerade as other seizure syndromes. Visual hallucinations are the key clinical symptoms indicating an occipital focus, but may be difficult to elicit on history, especially from children, and are not always present. When visual symptoms are not prominent, the seizure semiology and scalp EEG may lead the clinician away from considering an occipital focus, as they often reflect seizure propagation rather than seizure origin. Clinical and neuroimaging advances have led to the recognition of many new occipital epilepsy syndromes, which generally present in childhood or adolescence. Major groups include malformations of cortical development [focal cortical dysplasia, periventricular heterotopia (PVH), subcortical band heterotopia (SBH), polymicrogyria], vascular (including epilepsy with bilateral occipital calcifications often associated with coeliac disease), metabolic and the emerging idiopathic occipital epilepsies. The idiopathic occipital epilepsies now comprise three identifiable electroclinical syndromes of childhood and adolescence, the biological inter-relationships and overlap with idiopathic generalized epilepsies of which are discussed here. We emphasize the clues to recognition of specific occipital epilepsies, some of which now have specific treatments. Where medical therapy is ineffective, occipital corticectomy should be considered. Emerging evidence suggests that some syndromes have a good surgical outcome, and the consequences to visual function may be less severe than anticipated.

Occipital epilepsies often elude diagnosis as they frequently masquerade as other seizure syndromes. Visual hallucinations are the key clinical symptoms indicating an occipital focus, but may be difficult to elicit on history, especially from children, and are not always present. When visual symptoms are not prominent, the seizure semiology and scalp EEG may lead the clinician away from considering an occipital focus, as they often reflect seizure propagation rather than seizure origin. Clinical and neuroimaging advances have led to the recognition of many new occipital epilepsy syndromes, which generally present in childhood or adolescence. Major groups include malformations of cortical development [focal cortical dysplasia, periventricular heterotopia (PVH), subcortical band heterotopia (SBH), polymicrogyria], vascular (including epilepsy with bilateral occipital calcifications often associated with coeliac disease), metabolic and the emerging idiopathic occipital epilepsies. The idiopathic occipital epilepsies now comprise three identifiable electroclinical syndromes of childhood and adolescence, the biological inter-relationships and overlap with idiopathic generalized epilepsies of which are discussed here. We emphasize the clues to recognition of specific occipital epilepsies, some of which now have specific treatments. Where medical therapy is ineffective, occipital corticectomy should be considered. Emerging evidence suggests that some syndromes have a good surgical outcome, and the consequences to visual function may be less severe than anticipated.