De novo variants in DENND5B cause a neurodevelopmental disorder.

The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement.

Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles.

Background: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney,caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative (DN) mode-of-action, wherein an increased level of AFF3 resulted in pathological effects. Methods: Evolutionary constraints suggest that other mode-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be deleterious variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. Results: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous LoF or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not complement. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring +/+, DN/DN, LoF/+, LoF/LoF or DN/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the DN/DN or the LoF/LoF lines. While the same pathways are affected, only about one-third of the differentially expressed genes are common to these homozygote datasets, indicating that AFF3 LoF and DN variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. Conclusions: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.

DAG1 haploinsufficiency is associated with sporadic and familial isolated or pauci-symptomatic hyperCKemia.

DAG1 encodes for dystroglycan, a key component of the dystrophin-glycoprotein complex (DGC) with a pivotal role in skeletal muscle function and maintenance. Biallelic loss-of-function DAG1 variants cause severe muscular dystrophy and muscle-eye-brain disease. A possible contribution of DAG1 deficiency to milder muscular phenotypes has been suggested. We investigated the genetic background of twelve subjects with persistent mild-to-severe hyperCKemia to dissect the role of DAG1 in this condition. Genetic testing was performed through exome sequencing (ES) or custom NGS panels including various genes involved in a spectrum of muscular disorders. Histopathological and Western blot analyses were performed on muscle biopsy samples obtained from three patients. We identified seven novel heterozygous truncating variants in DAG1 segregating with isolated or pauci-symptomatic hyperCKemia in all families. The variants were rare and predicted to lead to nonsense-mediated mRNA decay or the formation of a truncated transcript. In four cases, DAG1 variants were inherited from similarly affected parents. Histopathological analysis revealed a decreased expression of dystroglycan subunits and Western blot confirmed a significantly reduced expression of beta-dystroglycan in muscle samples. This study supports the pathogenic role of DAG1 haploinsufficiency in isolated or pauci-symptomatic hyperCKemia, with implications for clinical management and genetic counseling.

Spliceosome malfunction causes neurodevelopmental disorders with overlapping features.

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

Dissecting genetics of spectrum of epilepsies with eyelid myoclonia by exome sequencing.

OBJECTIVE: Epilepsy with eyelid myoclonia (EEM) spectrum is a generalized form of epilepsy characterized by eyelid myoclonia with or without absences, eye closure-induced seizures with electroencephalographic paroxysms, and photosensitivity. Based on the specific clinical features, age at onset, and familial occurrence, a genetic cause has been postulated. Pathogenic variants in CHD2, SYNGAP1, NEXMIF, RORB, and GABRA1 have been reported in individuals with photosensitivity and eyelid myoclonia, but whether other genes are also involved, or a single gene is uniquely linked with EEM, or its subtypes, is not yet known. We aimed to dissect the genetic etiology of EEM. METHODS: We studied a cohort of 105 individuals by using whole exome sequencing. Individuals were divided into two groups: EEM- (isolated EEM) and EEM+ (EEM accompanied by intellectual disability [ID] or any other neurodevelopmental/psychiatric disorder). RESULTS: We identified nine variants classified as pathogenic/likely pathogenic in the entire cohort (8.57%); among these, eight (five in CHD2, one in NEXMIF, one in SYNGAP1, and one in TRIM8) were found in the EEM+ subcohort (28.57%). Only one variant (IFIH1) was found in the EEM- subcohort (1.29%); however, because the phenotype of the proband did not fit with published data, additional evidence is needed before considering IFIH1 variants and EEM- an established association. Burden analysis did not identify any single burdened gene or gene set. SIGNIFICANCE: Our results suggest that for EEM, as for many other epilepsies, the identification of a genetic cause is more likely with comorbid ID and/or other neurodevelopmental disorders. Pathogenic variants were mostly found in CHD2, and the association of CHD2 with EEM+ can now be considered a reasonable gene-disease association. We provide further evidence to strengthen the association of EEM+ with NEXMIF and SYNGAP1. Possible new associations between EEM+ and TRIM8, and EEM- and IFIH1, are also reported. Although we provide robust evidence for gene variants associated with EEM+, the core genetic etiology of EEM- remains to be elucidated.

A PAK1 Mutational Hotspot Within the Regulatory CRIPaK Domain is Associated With Severe Neurodevelopmental Disorders in Children.

BACKGROUND: P-21-activated kinases (PAKs) are protein serine/threonine kinases, part of the RAS/mitogen-activated protein kinase pathway. PAK1 is highly expressed in the central nervous system and crucially involved in neuronal migration and brain developmental processes. Recently, de novo heterozygous missense variants in PAK1 have been identified as an ultrarare cause of pediatric neurodevelopmental disorders. METHODS: We report a series of children affected with postnatal macrocephaly, neurodevelopmental impairment, and drug-resistant epilepsy. Repeated electroencephalographic (EEG) and video-EEG evaluations were performed over a two- to 10-year period during follow-up to delineate electroclinical histories. Genetic sequencing studies and computational evaluation of the identified variants were performed in our patient cohort. RESULTS: We identified by whole-exome sequencing three novel de novo variants in PAK1 (NM_001128620: c.427A>G, p.Met143Val; c.428T>C, p.Met143Thr; c.428T>A, p.Met143Lys) as the underlying cause of the disease in our families. The three variants affected the same highly conserved Met143 residue within the cysteine-rich inhibitor of PAK1 (CRIPaK) domain, which was identified before as a PAK1 inhibitor target. Computational studies suggested a defective autoinhibition presumably due to impaired PAK1 autoregulation as a result of the recurrent substitution. CONCLUSIONS: We delineated the electroclinical phenotypes of PAK1-related neurological disorders and highlight a novel mutational hotspot that may involve defective autoinhibition of the PAK1 protein. The three novel variants affecting the same hotspot residue within the CRIPaK domain highlight potentially impaired PAK1-CRIPaK interaction as a novel disease mechanism. These findings shed light on possible future treatments targeted at the CRIPaK domain, to modulate PAK1 activity and function.

Refining the electroclinical spectrum of NPRL3-related epilepsy: A novel multiplex family and literature review.

OBJECTIVE: NPRL3-related epilepsy (NRE) is an emerging condition set within the wide GATOR-1 spectrum with a particularly heterogeneous and elusive phenotypic expression. Here, we delineated the genotype-phenotype spectrum of NRE, reporting an illustrative familial case and reviewing pertinent literature. METHODS: Through exome sequencing (ES), we investigated a 12-year-old girl with recurrent focal motor seizures during sleep, suggestive of sleep-related hypermotor epilepsy (SHE), and a family history of epilepsy in siblings. Variant segregation analysis was performed by Sanger sequencing. All previously published NRE patients were thoroughly reviewed and their electroclinical features were analyzed and compared with the reported subjects. RESULTS: In the proband, ES detected the novel NPRL3 frameshift variant (NM_001077350.3): c.151_152del (p.Thr51Glyfs*5). This variant is predicted to cause a loss of function and segregated in one affected brother. The review of 76 patients from 18 publications revealed the predominance of focal-onset seizures (67/74-90%), with mainly frontal and frontotemporal (32/67-47.7%), unspecified (19/67-28%), or temporal (9/67-13%) onset. Epileptic syndromes included familial focal epilepsy with variable foci (FFEVF) (29/74-39%) and SHE (11/74-14.9%). Fifteen patients out of 60 (25%) underwent epilepsy surgery, 11 of whom achieved complete seizure remission (11/15-73%). Focal cortical dysplasia (FCD) type 2A was the most frequent histopathological finding. SIGNIFICANCE: We reported an illustrative NPRL3-related epilepsy (NRE) family with incomplete penetrance. This condition consists of a heterogeneous spectrum of clinical and neuroradiological features. Focal-onset motor seizures are predominant, and almost half of the cases fulfill the criteria for SHE or FFEVF. MRI-negative cases are prevalent, but the association with malformations of cortical developments (MCDs) is significant, especially FCD type 2a. The beneficial impact of epilepsy surgery in patients with MCD-related epilepsy further supports the inclusion of brain MRI in the workup of NRE patients.

Exome sequencing data screening to identify undiagnosed Aromatic l-amino acid decarboxylase deficiency in neurodevelopmental disorders.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive neurometabolic disorder caused by biallelic pathogenic variants in the DDC gene and mainly characterized by developmental delay, hypotonia, and oculogyric crises. Early diagnosis is crucial for correct patient management; however, many patients remain misdiagnosed or undiagnosed due to the rarity and clinical heterogeneity of the disorder especially in the milder forms. Here, we applied exome sequencing approach by screening 2000 paediatric patients with neurodevelopmental disorders to identify possible new AADC variants and AADC deficiency patients. We identified five distinct DDC variants in two unrelated individuals. Patient #1 harboured two compound heterozygous DDC variants: c.436-12T > C and c.435 + 24A>C and presented with psychomotor delay, tonic spasms, and hyperreactivity. Patient #2 had three homozygous AADC variants: c.1385G > A; p.Arg462Gln, c.234C > T; p.Ala78 = , and c.201 + 37A > G and presented with developmental delay and myoclonic seizures. The variants were classified as benign class I variants and therefore non-causative according to the ACMG/AMP guidelines. Since the AADC protein is a structural and functional obligate homodimer, we evaluated the possible AADC polypeptide chain combinations in the two patients and determined the effects resulting from the amino acid substitution Arg462Gln. Our patients carrying DDC variants presented clinical manifestations not precisely overlapped to the classical symptoms exhibited by the most severe AADC deficiency cases. However, screening data derived from exome sequencing in patients featuring wide-range symptoms related to neurodevelopmental disorders may help to identify AADC deficiency patients, especially when applied to larger cohorts.

BRAT1-related disorders: phenotypic spectrum and phenotype-genotype correlations from 97 patients.

BRAT1 biallelic variants are associated with rigidity and multifocal seizure syndrome, lethal neonatal (RMFSL), and neurodevelopmental disorder associating cerebellar atrophy with or without seizures syndrome (NEDCAS). To date, forty individuals have been reported in the literature. We collected clinical and molecular data from 57 additional cases allowing us to study a large cohort of 97 individuals and draw phenotype-genotype correlations. Fifty-nine individuals presented with BRAT1-related RMFSL phenotype. Most of them had no psychomotor acquisition (100%), epilepsy (100%), microcephaly (91%), limb rigidity (93%), and died prematurely (93%). Thirty-eight individuals presented a non-lethal phenotype of BRAT1-related NEDCAS phenotype. Seventy-six percent of the patients in this group were able to walk and 68% were able to say at least a few words. Most of them had cerebellar ataxia (82%), axial hypotonia (79%) and cerebellar atrophy (100%). Genotype-phenotype correlations in our cohort revealed that biallelic nonsense, frameshift or inframe deletion/insertion variants result in the severe BRAT1-related RMFSL phenotype (46/46; 100%). In contrast, genotypes with at least one missense were more likely associated with NEDCAS (28/34; 82%). The phenotype of patients carrying splice variants was variable: 41% presented with RMFSL (7/17) and 59% with NEDCAS (10/17).

Novel biallelic variants expand the phenotype of NAA20-related syndrome.

NAA20 is the catalytic subunit of the NatB complex, which is responsible for N-terminal acetylation of approximately 20% of the human proteome. Recently, pathogenic biallelic variants in NAA20 were associated with a novel neurodevelopmental disorder in five individuals with limited clinical information. We report two sisters harboring compound heterozygous variant (c.100C>T (p.Gln34Ter) and c.11T>C p.(Leu4Pro)) in the NAA20 gene, identified by exome sequencing. In vitro studies showed that the missense variant p.Leu4Pro resulted in a reduction of NAA20 catalytic activity due to weak coupling with the NatB auxiliary subunit. In addition, unpublished data of the previous families were reported, outlining the core phenotype of the NAA20-related disorder mostly characterized by cognitive impairment, microcephaly, ataxia, brain malformations, dysmorphism and variable occurrence of cardiac defect and epilepsy. Remarkably, our two patients featured epilepsy onset in adolescence suggesting this may be a part of syndrome evolution. Functional studies are needed to better understand the complexity of NAA20 variants pathogenesis as well as of other genes linked to N-terminal acetylation.

Genotype-phenotype correlation in contactin-associated protein-like 2 (CNTNAP-2) developmental disorder.

Contactin-associated protein-like 2 (CNTNAP2) gene encodes for CASPR2, a presynaptic type 1 transmembrane protein, involved in cell-cell adhesion and synaptic interactions. Biallelic CNTNAP2 loss has been associated with "Pitt-Hopkins-like syndrome-1" (MIM#610042), while the pathogenic role of heterozygous variants remains controversial. We report 22 novel patients harboring mono- (n = 2) and bi-allelic (n = 20) CNTNAP2 variants and carried out a literature review to characterize the genotype-phenotype correlation. Patients (M:F 14:8) were aged between 3 and 19 years and affected by global developmental delay (GDD) (n = 21), moderate to profound intellectual disability (n = 17) and epilepsy (n = 21). Seizures mainly started in the first two years of life (median 22.5 months). Antiseizure medications were successful in controlling the seizures in about two-thirds of the patients. Autism spectrum disorder (ASD) and/or other neuropsychiatric comorbidities were present in nine patients (40.9%). Nonspecific midline brain anomalies were noted in most patients while focal signal abnormalities in the temporal lobes were noted in three subjects. Genotype-phenotype correlation was performed by also including 50 previously published patients (15 mono- and 35 bi-allelic variants). Overall, GDD (p < 0.0001), epilepsy (p < 0.0001), hyporeflexia (p = 0.012), ASD (p = 0.009), language impairment (p = 0.020) and severe cognitive impairment (p = 0.031) were significantly associated with the presence of biallelic versus monoallelic variants. We have defined the main features associated with biallelic CNTNAP2 variants, as severe cognitive impairment, epilepsy and behavioral abnormalities. We propose CASPR2-deficiency neurodevelopmental disorder as an exclusively recessive disease while the contribution of heterozygous variants is less likely to follow an autosomal dominant inheritance pattern.

Moyamoya Vasculopathy in Neurofibromatosis Type 1 Pediatric Patients: The Role of Rare Variants of RNF213.

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder caused by mutations in NF1 gene, coding for neurofibromin 1. NF1 can be associated with Moyamoya disease (MMD), and this association, typical of paediatric patients, is referred to as Moyamoya syndrome (MMS). MMD is a cerebral arteriopathy characterized by the occlusion of intracranial arteries and collateral vessel formation, which increase the risk of ischemic and hemorrhagic events. RNF213 gene mutations have been associated with MMD, so we investigated whether rare variants of RNF213 could act as genetic modifiers of MMS phenotype in a pediatric cohort of 20 MMS children, 25 children affected by isolated MMD and 47 affected only by isolated NF1. By next-generation re-sequencing (NGS) of patients' DNA and gene burden tests, we found that RNF213 seems to play a role only for MMD occurrence, while it does not appear to be involved in the increased risk of Moyamoya for MMS patients. We postulated that the loss of neurofibromin 1 can be enough for the excessive proliferation of vascular smooth muscle cells, causing Moyamoya arteriopathy associated with NF1. Further studies will be crucial to support these findings and to elucidate the possible role of other genes, enhancing our knowledge about pathogenesis and treatment of MMS.

Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development.

Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. In this study, we identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, we provide evidence linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, we show that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, our results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.

Neuroimaging features of WOREE syndrome: a mini-review of the literature.

The WWOX gene encodes a 414-amino-acid protein composed of two N-terminal WW domains and a C-terminal short-chain dehydrogenase/reductase (SDR) domain. WWOX protein is highly conserved among species and mainly expressed in the cerebellum, cerebral cortex, brain stem, thyroid, hypophysis, and reproductive organs. It plays a crucial role in the biology of the central nervous system, and it is involved in neuronal development, migration, and proliferation. Biallelic pathogenic variants in WWOX have been associated with an early infantile epileptic encephalopathy known as WOREE syndrome. Both missense and null variants have been described in affected patients, leading to a reduction in protein function and stability. The most severe WOREE phenotypes have been related to biallelic null/null variants, associated with the complete loss of function of the protein. All affected patients showed brain anomalies on magnetic resonance imaging (MRI), suggesting the pivotal role of WWOX protein in brain homeostasis and developmental processes. We provided a literature review, exploring both the clinical and radiological spectrum related to WWOX pathogenic variants, described to date. We focused on neuroradiological findings to better delineate the WOREE phenotype with diagnostic and prognostic implications.

Congenital myopathy associated with a novel mutation in MEGF10 gene, myofibrillar alteration and progressive course.

Early-onset myopathy, areflexia, respiratory distress, and dysphagia (EMARDD) is caused by homozygous or compound heterozygous mutation in the MEGF10 gene (OMIM #614399). Phenotypic spectrum of EMARDD is variable, ranging from severe infantile forms in which patients are ventilator-dependent and die in childhood, to milder chronic disorders with a more favorable course (mild variant, mvEMARDD). Here we describe a 22 years old boy, offspring of consanguineous parents, presenting a congenital myopathic phenotype since infancy with elbow contractures and scoliosis. The patient developed a slowly progressive muscle weakness with impaired walking, rhinolalia, dysphagia, and respiratory involvement, which required noninvasive ventilation therapy since the age of 16 years. First muscle biopsy revealed unspecific muscle damage, with fiber size variation, internal nuclei and fibrosis. Myofibrillar alterations were noted at a second muscle biopsy including whorled fibres, cytoplasmic inclusion and minicores. Exome sequencing identified a homozygous mutation in MEGF10 gene, c.2096G > C (p.Cys699Ser), inherited by both parents. This variant, not reported in public databases of mutations, is expected to alter the structure of the protein and is therefore predicted to be probably damaging according to ACMG classification. In conclusion, we found a new likely pathogenic mutation in MEGF10, which is responsible for a progressive form of mvEMARDD with myofibrillar alterations at muscle biopsy. Interestingly, the presence of MEGF10 mutations has not been reported in Italian population. Early diagnosis of MEGF10 myopathy is essential in light of recent results from in vivo testing demonstrating a potential therapeutic effect of SSRIs compounds.

Clinical and genetic analysis of patients with segmental overgrowth features and somatic mammalian target of rapamycin (mTOR) pathway disruption: Possible novel clinical issues.

Segmental overgrowth syndromes include a group of clinical entities, all characterized by the abundant proliferation of tissues or organs in association with vascular abnormalities. These syndromes show a wide spectrum of severity ranging from limited involvement of only small areas of the body to complex cases with impressive distortions of multiple tissues and organs. It is now clear that somatic mutations in genes of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway (in brief "mTOR pathway") are responsible for such entities. Not all the cells of the body carry the same causative mutation, which is mosaic, appearing from two (or more) distinct cell lineages after fertilization. In this article, we reconsider the clinical spectrum and surveillance programs of patients with segmental overgrowth syndromes, based on the features of six patients with diverse clinical forms of overgrowth and pathogenic variants in genes of the mTOR pathway.

Hydranencephaly in CENPJ-related Seckel syndrome.

Pathogenic variants in CENPJ have been first identified in consanguineous Pakistani families with Hereditary Primary Microcephaly type 6 (MCPH6). In addition to primary microcephaly, the CENPJ-related phenotypic spectrum lately included also distinctive and peculiar 'bird-like' craniofacial dysmorphisms, intrauterine and/or postnatal growth retardation, and moderate to severe intellectual disability (ID). These features are also part of the clinical spectrum of Seckel syndrome (SCKL) a genetically heterogeneous neurodevelopmental condition caused by mutations in different genes involved in cell cycle progression. Among these, CENPJ is responsible for type 4 Seckel syndrome (SCKL4). The literature reports two individuals affected by SCKL4 suffering from seizures and other two individuals with other brain malformations in addition to microcephaly. However, neither epilepsy nor brain malformations are described in detail and genotype-phenotype information remains limited. We describe the first Caucasian affected with SCKL4 and harboring a novel, homozygous mutation in CENPJ. We detail the clinical and neuroradiological findings including structural focal epilepsy and a severe brain malformation (i.e., hydranencephaly) that was never associated with SCKL4 to date.

Vesicular Glutamate Release from Feeder-FreehiPSC-Derived Neurons.

Human-induced pluripotent stem cells (hiPSCs) represent one of the main and powerful tools for the in vitro modeling of neurological diseases. Standard hiPSC-based protocols make use of animal-derived feeder systems to better support the neuronal differentiation process. Despite their efficiency, such protocols may not be appropriate to dissect neuronal specific properties or to avoid interspecies contaminations, hindering their future translation into clinical and drug discovery approaches. In this work, we focused on the optimization of a reproducible protocol in feeder-free conditions able to generate functional glutamatergic neurons. This protocol is based on a generation of neuroprecursor cells differentiated into human neurons with the administration in the culture medium of specific neurotrophins in a Geltrex-coated substrate. We confirmed the efficiency of this protocol through molecular analysis (upregulation of neuronal markers and neurotransmitter receptors assessed by gene expression profiling and expression of the neuronal markers at the protein level), morphological analysis, and immunfluorescence detection of pre-synaptic and post-synaptic markers at synaptic boutons. The hiPSC-derived neurons acquired Ca2+-dependent glutamate release properties as a hallmark of neuronal maturation. In conclusion, our study describes a new methodological approach to achieve feeder-free neuronal differentiation from hiPSC and adds a new tool for functional characterization of hiPSC-derived neurons.