Antonio Falace

TBC1D24, an ARF6-Interacting Protein, Is Mutated in Familial Infantile Myoclonic Epilepsy

Idiopathic epilepsies (IEs) are a group of disorders characterized by recurrent seizures in the absence of detectable brain lesions or metabolic abnormalities. IEs include common disorders with a complex mode of inheritance and rare Mendelian traits suggesting the occurrence of several alleles with variable penetrance. We previously described a large family with a recessive form of idiopathic epilepsy, named familial infantile myoclonic epilepsy (FIME), and mapped the disease locus on chromosome 16p13.3 by linkage analysis. In the present study, we found that two compound heterozygous missense mutations (D147H and A509V) in TBC1D24, a gene of unknown function, are responsible for FIME. In situ hybridization analysis revealed that Tbc1d24 is mainly expressed at the level of the cerebral cortex and the hippocampus. By coimmunoprecipitation assay we found that TBC1D24 binds ARF6, a Ras-related family of small GTPases regulating exo-endocytosis dynamics. The main recognized function of ARF6 in the nervous system is the regulation of dendritic branching, spine formation, and axonal extension. TBC1D24 overexpression resulted in a significant increase in neurite length and arborization and the FIME mutations significantly reverted this phenotype. In this study we identified a gene mutation involved in autosomal-recessive idiopathic epilepsy, unveiled the involvement of ARF6-dependent molecular pathway in brain hyperexcitability and seizures, and confirmed the emerging role of subtle cytoarchitectural alterations in the etiology of this group of common epileptic disorders.

Novel Compound Heterozygous Mutations in TBC1D24 Cause Familial Malignant Migrating Partial Seizures of Infancy

Early‐onset epileptic encephalopathies (EOEEs) are a group of rare devastating epileptic syndromes of infancy characterized by severe drug‐resistant seizures and electroencephalographic abnormalities. The current study aims to determine the genetic etiology of a familial form of EOEE fulfilling the diagnosis criteria for malignant migrating partial seizures of infancy (MMPSI). We identified two inherited novel mutations in TBC1D24 in two affected siblings. Mutations severely impaired TBC1D24 expression and function, which is critical for maturation of neuronal circuits. The screening of TBC1D24 in an additional set of eight MMPSI patients was negative. TBC1D24 loss of function has been associated to idiopathic infantile myoclonic epilepsy, as well as to drug‐resistant early‐onset epilepsy with intellectual disability. Here, we describe a familial form of MMPSI due to mutation in TBC1D24, revealing a devastating epileptic phenotype associated with TBC1D24 dysfunction.

Clinical Significance of Rare Copy Number Variations in Epilepsy A Case-Control Survey Using Microarray-Based Comparative Genomic Hybridization

OBJECTIVE To perform an extensive search for genomic rearrangements by microarray-based comparative genomic hybridization in patients with epilepsy. DESIGN Prospective cohort study. SETTING Epilepsy centers in Italy. PATIENTS Two hundred seventy-nine patients with unexplained epilepsy, 265 individuals with nonsyndromic mental retardation but no epilepsy, and 246 healthy control subjects were screened by microarray-based comparative genomic hybridization. MAIN OUTCOME MEASURES Identification of copy number variations (CNVs) and gene enrichment. RESULTS Rare CNVs occurred in 26 patients (9.3%) and 16 healthy control subjects (6.5%) (P = .26). The CNVs identified in patients were larger (P = .03) and showed higher gene content (P = .02) than those in control subjects. The CNVs larger than 1 megabase (P = .002) and including more than 10 genes (P = .005) occurred more frequently in patients than in control subjects. Nine patients (34.6%) among those harboring rare CNVs showed rearrangements associated with emerging microdeletion or microduplication syndromes. Mental retardation and neuropsychiatric features were associated with rare CNVs (P = .004), whereas epilepsy type was not. The CNV rate in patients with epilepsy and mental retardation or neuropsychiatric features is not different from that observed in patients with mental retardation only. Moreover, significant enrichment of genes involved in ion transport was observed within CNVs identified in patients with epilepsy. CONCLUSIONS Patients with epilepsy show a significantly increased burden of large, rare, gene-rich CNVs, particularly when associated with mental retardation and neuropsychiatric features. The limited overlap between CNVs observed in the epilepsy group and those observed in the group with mental retardation only as well as the involvement of specific (ion channel) genes indicate a specific association between the identified CNVs and epilepsy. Screening for CNVs should be performed for diagnostic purposes preferentially in patients with epilepsy and mental retardation or neuropsychiatric features.

TBC1D24 regulates neuronal migration and maturation through modulation of the ARF6-dependent pathway

Significance The six-layered cerebral cortex forms through tightly regulated steps including neuronal proliferation, migration, and circuitry formation. Alterations of cortical development are associated with several neurological conditions, but the underlying pathogenetic mechanisms remain largely unknown. Here, we describe the role of TBC1 domain family member 24 (TBC1D24), a gene associated with syndromes combining epilepsy and cognitive deficits, in cortical development. Using an in vivo approach, we found that TBC1D24 regulates neuronal polarity, thus promoting neuronal migration and maturation. We further show that TBC1D24 exerts its function through the modulation of the activity of ADP ribosylation factor 6, a GTPase involved in membrane trafficking. Collectively, our data disclose a previously uncharacterized molecular mechanism involved in cortical development and underline how appropriate and timely neuronal positioning is essential for brain function. Alterations in the formation of brain networks are associated with several neurodevelopmental disorders. Mutations in TBC1 domain family member 24 (TBC1D24) are responsible for syndromes that combine cortical malformations, intellectual disability, and epilepsy, but the function of TBC1D24 in the brain remains unknown. We report here that in utero TBC1D24 knockdown in the rat developing neocortex affects the multipolar-bipolar transition of neurons leading to delayed radial migration. Furthermore, we find that TBC1D24-knockdown neurons display an abnormal maturation and retain immature morphofunctional properties. TBC1D24 interacts with ADP ribosylation factor (ARF)6, a small GTPase crucial for membrane trafficking. We show that in vivo, overexpression of the dominant-negative form of ARF6 rescues the neuronal migration and dendritic outgrowth defects induced by TBC1D24 knockdown, suggesting that TBC1D24 prevents ARF6 activation. Overall, our findings demonstrate an essential role of TBC1D24 in neuronal migration and maturation and highlight the physiological relevance of the ARF6-dependent membrane-trafficking pathway in brain development.

Impairment of ceramide synthesis causes a novel progressive myoclonus epilepsy

Alterations of sphingolipid metabolism are implicated in the pathogenesis of many neurodegenerative disorders.

TBC1D24 genotype–phenotype correlation: Epilepsies and other neurologic features

Objective: To evaluate the phenotypic spectrum associated with mutations in TBC1D24. Methods: We acquired new clinical, EEG, and neuroimaging data of 11 previously unreported and 37 published patients. TBC1D24 mutations, identified through various sequencing methods, can be found online (http://lovd.nl/TBC1D24). Results: Forty-eight patients were included (28 men, 20 women, average age 21 years) from 30 independent families. Eighteen patients (38%) had myoclonic epilepsies. The other patients carried diagnoses of focal (25%), multifocal (2%), generalized (4%), and unclassified epilepsy (6%), and early-onset epileptic encephalopathy (25%). Most patients had drug-resistant epilepsy. We detail EEG, neuroimaging, developmental, and cognitive features, treatment responsiveness, and physical examination. In silico evaluation revealed 7 different highly conserved motifs, with the most common pathogenic mutation located in the first. Neuronal outgrowth assays showed that some TBC1D24 mutations, associated with the most severe TBC1D24-associated disorders, are not necessarily the most disruptive to this gene function. Conclusions: TBC1D24-related epilepsy syndromes show marked phenotypic pleiotropy, with multisystem involvement and severity spectrum ranging from isolated deafness (not studied here), benign myoclonic epilepsy restricted to childhood with complete seizure control and normal intellect, to early-onset epileptic encephalopathy with severe developmental delay and early death. There is no distinct correlation with mutation type or location yet, but patterns are emerging. Given the phenotypic breadth observed, TBC1D24 mutation screening is indicated in a wide variety of epilepsies. A TBC1D24 consortium was formed to develop further research on this gene and its associated phenotypes.

Inherited neuromyotonia: a clinical and genetic study of a family.

Neuromyotonia is a disorder of peripheral nerve hyperexcitability characterized by myokymia, muscle cramps and stiffness, delayed muscle relaxation after contraction (pseudomyotonia), and hyperhidrosis, associated with well described spontaneous electromyographic features. It is usually an acquired disorder associated with autoantibodies against neuronal voltage-gated potassium channels. However, mutations of KCNA1, encoding the K(+) channel subunit hKv1.1, have been reported in rare families with neuromyotonia, and mutations in KCNQ2, encoding voltage-gated potassium M channel subunit, in families with benign neonatal seizures and myokymia. We report a three-generation family with inherited neuromyotonia without evidence of immunological involvement. Genetic study excluded mutations in KCNA1, KCNA2, KCNA6 and KCNQ2 genes. Our study does not completely exclude the involvement of other genes encoding ion channels subunits in the pathogenesis of this disorder. Further studies of familial cases will shed light on the molecular basis of inherited neuromyotonia.

A de novo microdeletion of SEMA5A in a boy with autism spectrum disorder and intellectual disability

Semaphorins are a large family of secreted and membrane-associated proteins necessary for wiring of the brain. Semaphorin 5A (SEMA5A) acts as a bifunctional guidance cue, exerting both attractive and inhibitory effects on developing axons. Previous studies have suggested that SEMA5A could be a susceptibility gene for autism spectrum disorders (ASDs). We first identified a de novo translocation t(5;22)(p15.3;q11.21) in a patient with ASD and intellectual disability (ID). At the translocation breakpoint on chromosome 5, we observed a 861-kb deletion encompassing the end of the SEMA5A gene. We delineated the breakpoint by NGS and observed that no gene was disrupted on chromosome 22. We then used Sanger sequencing to search for deleterious variants affecting SEMA5A in 142 patients with ASD. We also identified two independent heterozygous variants located in a conserved functional domain of the protein. Both variants were maternally inherited and predicted as deleterious. Our genetic screens identified the first case of a de novo SEMA5A microdeletion in a patient with ASD and ID. Although our study alone cannot formally associate SEMA5A with susceptibility to ASD, it provides additional evidence that Semaphorin dysfunction could lead to ASD and ID. Further studies on Semaphorins are warranted to better understand the role of this family of genes in susceptibility to neurodevelopmental disorders.

Do regulatory regions matter in FOXG1 duplications

Duplications of FOXG1 gene at 14q12 have been reported in patients with infantile spasms and developmental delay of variable severity.1, 2, 3 FOXG1 encodes the forkhead protein G1, a brain-specific transcriptional repressor, regulating corticogenesis in the developing brain and neuronal stem cell self-renewal in the postnatal brain.4 Recently, Amor et al.5 reported on this journal an interstitial duplication of ∼88 kb at 14q12 in a father–son pair with hemifacial microsomia and normal neurocognitive phenotype. The duplication contains only two polypeptide-encoding genes, FOXG1 and C14orf23, suggesting that FOXG1 duplication may be benign or at least incompletely penetrant. That makes the involvement of FOXG1 duplication in the pathogenesis of the neurocognitive impairment and epilepsy controversial. As also discussed by Brunetti-Pierri et al,6 we feel that this statement needs special caution. Functional consequences of chromosomal microduplication and microdeletion rely on the final gene dosage, which is strongly influenced by the location of the breakpoint. In this context, the understanding of the contribution of regulatory sequences in gene transcription is critical to understand the relationship between CNVs and human diseases. With this purpose, the Encyclopedia of DNA Elements (ENCODE) project has recently performed a systematic analysis of transcriptional regulation in different human cell lines, providing new understanding about transcription start sites, including their relationship with specific regulatory sequences and histone modification and features of chromatin accessibility.7, 8 Interestingly, analysis of histone modifications from the ENCODE project revealed the presence of a putative regulatory element upstream FOXG1 gene between 28 188 and 28 217 kb (UCSC genome browser, NCBI Build 36/hg18) (Figure 1). This conserved region localizes about 130 kb upstream FOXG1 gene and contains histone modifications typical of enhancers of gene transcription (eg, histone H3 and Lysine 4 monomethylation) in eight different human cells lines. Analysis of regulatory potential scores, comparing frequencies of short alignment patterns between known regulatory elements and neutral DNA,9 also disclose two additional putative elements typical of cis-regulatory modules within this region (Figure 1). Moreover, it contains a DNaseI hypersensitive site (DHS). DHSs reflect genomic regions thought to be enriched for regulatory information and many DHSs reside at or near transcription start site. Notably, no other polypeptide-encoding genes or non-coding RNAs and pseudogenes are present in the region, suggesting that this regulatory element might regulate FOXG1 transcription. Analysis of duplication breakpoints previously reported on 14q12 revealed that duplications associated with an epileptic phenotype localizes uniquely upstream this regulatory element, whereas downstream duplications were identified only in the cases without seizures (Figure 1). On the basis of this finding, we suggest that FOXG1 duplication including this putative regulatory region allows the efficient transcription of the supernumerary copy of FOXG1 gene, resulting in an effective increase in FOXG1 expression and, thereby, in brain hyperexcitability. In contrast, duplications starting downstream this putative regulatory site do not allow efficient transcription of FOXG1, which may underlie the lack of neurological phenotype in the case reported by Amor et al5. Figure 1 UCSC genome browser (NCBI36/hg18) schematic view of reported 14q12 duplications encompassing FOXG1 gene. Histone modifications from the ENCODE project7 in eight cells lines (GM12878, H1-hESC, HMEC, HSMM, HUVEC, K562, NHEK and NHLF) indicate the presence ... Even if the functional relevance of this putative long-range regulatory element on FOXG1 transcription deserves to be experimentally verified, it provides an interesting clue to dissect genotype–phenotype correlation in FOXG1 microduplication and to uncover the real actual contribution of FOXG1 in the neurodevelopmental phenotype associated with 14q12 duplication. Notably, chromosome rearrangements disrupting or displacing putative cis-regulatory elements distal to FOXG1 gene in patients with severe cognitive disabilities has been also reported,10, 11 pointing out the relevance of regulatory sequences in the expression of FOXG1 gene.

A novel strategy combining array-CGH, whole-exome sequencing and in utero electroporation in rodents to identify causative genes for brain malformations

Birth defects that involve the cerebral cortex - also known as malformations of cortical development (MCD) - are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.