The genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple disease-causing missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C. We find a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus implicated in unexplained MCD. The mutations in KIF5C, KIF2A and DYNC1H1 drastically affect ATP hydrolysis, productive protein folding or microtubule binding, while suppression of Tubg1 expression in vivo interferes with proper neuronal migration and expression of Tubg1 mutations in S. cerevisiae results in disruption of normal microtubule behaviour. Our data reinforce the importance of centrosome- and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and post-mitotic processes are major contributors to the pathogenesis of MCD.

Complex cortical malformations associated with mutations in tubulin genes: TUBA1A, TUBA8, TUBB2B, TUBB3, TUBB5 and TUBG1 commonly referred to as tubulinopathies, are a heterogeneous group of conditions with a wide spectrum of clinical severity. Among the 106 patients selected as having complex cortical malformations, 45 were found to carry mutations in TUBA1A (42.5%), 18 in TUBB2B (16.9%), 11 in TUBB3 (10.4%), three in TUBB5 (2.8%), and three in TUBG1 (2.8%). No mutations were identified in TUBA8. Systematic review of patients' neuroimaging and neuropathological data allowed us to distinguish at least five cortical malformation syndromes: (i) microlissencephaly (n = 12); (ii) lissencephaly (n = 19); (iii) central pachygyria and polymicrogyria-like cortical dysplasia (n = 24); (iv) generalized polymicrogyria-like cortical dysplasia (n = 6); and (v) a 'simplified' gyral pattern with area of focal polymicrogyria (n = 19). Dysmorphic basal ganglia are the hallmark of tubulinopathies (found in 75% of cases) and are present in 100% of central pachygyria and polymicrogyria-like cortical dysplasia and simplified gyral malformation syndromes. Tubulinopathies are also characterized by a high prevalence of corpus callosum agenesis (32/80; 40%), and mild to severe cerebellar hypoplasia and dysplasia (63/80; 78.7%). Foetal cases (n = 25) represent the severe end of the spectrum and show specific abnormalities that provide insights into the underlying pathophysiology. The overall complexity of tubulinopathies reflects the pleiotropic effects of tubulins and their specific spatio-temporal profiles of expression. In line with previous reports, this large cohort further clarifies overlapping phenotypes between tubulinopathies and although current structural data do not allow prediction of mutation-related phenotypes, within each mutated gene there is an associated predominant pattern of cortical dysgenesis allowing some phenotype-genotype correlation. The core phenotype of TUBA1A and TUBG1 tubulinopathies are lissencephalies and microlissencephalies, whereas TUBB2B tubulinopathies show in the majority, centrally predominant polymicrogyria-like cortical dysplasia. By contrast, TUBB3 and TUBB5 mutations cause milder malformations with focal or multifocal polymicrogyria-like cortical dysplasia with abnormal and simplified gyral pattern.

AMPA receptors (AMPARs) are tetrameric ligand-gated channels made up of combinations of GluA1-4 subunits encoded by GRIA1-4 genes. GluA2 has an especially important role because, following post-transcriptional editing at the Q607 site, it renders heteromultimeric AMPARs Ca 2+ -impermeable, with a linear relationship between current and trans-membrane voltage. Here, we report heterozygous de novo GRIA2 mutations in 28 unrelated patients with intellectual disability (ID) and neurodevelopmental abnormalities including autism spectrum disorder (ASD), Rett syndrome-like features, and seizures or developmental epileptic encephalopathy (DEE). In functional expression studies, mutations lead to a decrease in agonist-evoked current mediated by mutant subunits compared to wild-type channels. When GluA2 subunits are co-expressed with GluA1, most GRIA2 mutations cause a decreased current amplitude and some also affect voltage rectification. Our results show that de-novo variants in GRIA2 can cause neurodevelopmental disorders, complementing evidence that other genetic causes of ID, ASD and DEE also disrupt glutamatergic synaptic transmission.

Deficiency of adenosine deaminase 2 (DADA2) is a recently described autoinflammatory disorder. Genetic analysis is required to confirm the diagnosis. We aimed to describe the identifying symptoms and genotypes of patients referred to our reference centres and to improve the indications for genetic testing. DNA from 66 patients with clinically suspected DADA2 were sequenced by Sanger or next-generation sequencing. Detailed epidemiological, clinical and biological features were collected by use of a questionnaire and were compared between patients with and without genetic confirmation of DADA2. We identified 13 patients (19.6%) carrying recessively inherited mutations in ADA2 that were predicted to be deleterious. Eight patients were compound heterozygous for mutations. Seven mutations were novel (4 missense variants, 2 predicted to affect mRNA splicing and 1 frameshift). The mean age of the 13 patients with genetic confirmation was 12.7 years at disease onset and 20.8 years at diagnosis. Phenotypic manifestations included fever (85%), vasculitis (85%) and neurological disorders (54%). Features best associated with a confirmatory genotype included fever with neurologic or cutaneous attacks (odds ratio [OR] 10.71, p = 0.003 and OR 10.9, p < 0.001), fever alone (OR 8.1, p = 0.01), and elevated C-reactive protein (CRP) level with neurologic involvement (OR 6.63, p = 0.017). Our proposed decision tree may help improve obtaining genetic confirmation of DADA2 in the context of autoinflammatory symptoms. Prerequisites for quick and low-cost Sanger analysis include one typical cutaneous or neurological sign, one marker of inflammation (fever or elevated CRP level), and recurrent or chronic attacks in adults.

ObjectiveTo provide new insights into the FOXG1-related clinical and imaging phenotypes and refine the phenotype-genotype correlation in FOXG1 syndrome.MethodsWe analyzed the clinical and imaging phenotypes of a cohort of 45 patients with a pathogenic or likely pathogenic FOXG1 variant and performed phenotype-genotype correlations.ResultsA total of 37 FOXG1 different heterozygous mutations were identified, of which 18 are novel. We described a broad spectrum of neurodevelopmental phenotypes, characterized by severe postnatal microcephaly and developmental delay accompanied by a hyperkinetic movement disorder, stereotypes and sleep disorders, and epileptic seizures. Our data highlighted 3 patterns of gyration, including frontal pachygyria in younger patients (26.7%), moderate simplified gyration (24.4%) and mildly simplified or normal gyration (48.9%), corpus callosum hypogenesis mostly in its frontal part, combined with moderate-to-severe myelination delay that improved and normalized with age. Frameshift and nonsense mutations in the N-terminus of FOXG1, which are the most common mutation types, show the most severe clinical features and MRI anomalies. However, patients with recurrent frameshift mutations c.460dupG and c.256dupC had variable clinical and imaging presentations.ConclusionsThese findings have implications for genetic counseling, providing evidence that N-terminal mutations and large deletions lead to more severe FOXG1 syndrome, although genotype-phenotype correlations are not necessarily straightforward in recurrent mutations. Together, these analyses support the view that FOXG1 syndrome is a specific disorder characterized by frontal pachygyria and delayed myelination in its most severe form and hypogenetic corpus callosum in its milder form.

BackgroundBalanced chromosomal rearrangements associated with abnormal phenotype are rare events, but may be challenging for genetic counselling, since molecular characterisation of breakpoints is not performed routinely. We used next-generation sequencing to characterise breakpoints of balanced chromosomal rearrangements at the molecular level in patients with intellectual disability and/or congenital anomalies.MethodsBreakpoints were characterised by a paired-end low depth whole genome sequencing (WGS) strategy and validated by Sanger sequencing. Expression study of disrupted and neighbouring genes was performed by RT-qPCR from blood or lymphoblastoid cell line RNA.ResultsAmong the 55 patients included (41 reciprocal translocations, 4 inversions, 2 insertions and 8 complex chromosomal rearrangements), we were able to detect 89% of chromosomal rearrangements (49/55). Molecular signatures at the breakpoints suggested that DNA breaks arose randomly and that there was no major influence of repeated elements. Non-homologous end-joining appeared as the main mechanism of repair (55% of rearrangements). A diagnosis could be established in 22/49 patients (44.8%), 15 by gene disruption (KANSL1, FOXP1, SPRED1, TLK2, MBD5, DMD, AUTS2, MEIS2, MEF2C, NRXN1, NFIX, SYNGAP1, GHR, ZMIZ1) and 7 by position effect (DLX5, MEF2C, BCL11B, SATB2, ZMIZ1). In addition, 16 new candidate genes were identified. Systematic gene expression studies further supported these results. We also showed the contribution of topologically associated domain maps to WGS data interpretation.ConclusionPaired-end WGS is a valid strategy and may be used for structural variation characterisation in a clinical setting.

Summary Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule associated protein, that is predominantly expressed in post-mitotic neurons, and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.

De novo mutations in the TUBA1A gene are responsible for a wide spectrum of neuronal migration disorders, ranging from lissencephaly to perisylvian pachygyria. Recently, one family with polymicrogyria (PMG) and mutation in TUBA1A was reported. Hence, the purpose of our study was to determine the frequency of TUBA1A mutations in patients with PMG and better define clinical and imaging characteristics for TUBA1A-related PMG. We collected 95 sporadic patients with non-syndromic bilateral PMG, including 54 with perisylvian PMG and 30 PMG with additional brain abnormalities. Mutation analysis of the TUBA1A gene was performed by sequencing of PCR fragments corresponding to TUBA1A-coding sequences. Three de novo missense TUBA1A mutations were identified in three unrelated patients with PMG representing 3.1% of PMG and 10% of PMGs with complex cerebral malformations. These patients had bilateral perisylvian asymmetrical PMG with dysmorphic basal ganglia cerebellar vermian dysplasia and pontine hypoplasia. These mutations (p.Tyr161His; p.Val235Leu; p.Arg390Cys) appear distributed throughout the primary structure of the alpha-tubulin polypeptide, but their localization within the tertiary structure suggests that PMG-related mutations are likely to impact microtubule dynamics, stability and/or local interactions with partner proteins. These findings broaden the phenotypic spectrum associated with TUBA1A mutations to PMG and further emphasize that additional brain abnormalities, that is, dysmorphic basal ganglia, hypoplastic pons and cerebellar dysplasia are key features for the diagnosis of TUBA1A-related PMG.