Profiling PI3K-AKT-MTOR variants in focal brain malformations reveals new insights for diagnostic care.

Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.

Generation of an induced pluripotent stem cell line (UCSCi001-A) from a patient with early-onset amyotrophic lateral sclerosis carrying a FUS variant.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting upper and lower motor neurons. We generated patient-derived-induced Pluripotent Stem Cells (iPSCs), from an ALS patient affected by an early-onset and aggressive form of the disease, carrying a missense pathogenic variant in FUS gene. We reprogrammed somatic cells using an established Sendai virus protocol and we obtained clones of iPSC. We confirmed their stemness and further generated embryoid bodies, showing their potential of differentiating in all three germ layers. This iPSC line, carrying a pathogenic FUS variant, is a valuable tool to deeply investigate pathogenic mechanisms leading to ALS.

Proximal variants in CCND2 associated with microcephaly, short stature, and developmental delay: A case series and review of inverse brain growth phenotypes.

Cyclin D2 (CCND2) is a critical cell cycle regulator and key member of the cyclin D2-CDK4 (DC) complex. De novo variants of CCND2 clustering in the distal part of the protein have been identified as pathogenic causes of brain overgrowth (megalencephaly, MEG) and severe cortical malformations in children including the megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndrome. Megalencephaly-associated CCND2 variants are localized to the terminal exon and result in accumulation of degradation-resistant protein. We identified five individuals from three unrelated families with novel variants in the proximal region of CCND2 associated with microcephaly, mildly simplified cortical gyral pattern, symmetric short stature, and mild developmental delay. Identified variants include de novo frameshift variants and a dominantly inherited stop-gain variant segregating with the phenotype. This is the first reported association between proximal CCND2 variants and microcephaly, to our knowledge. This series expands the phenotypic spectrum of CCND2-related disorders and suggests that distinct classes of CCND2 variants are associated with reciprocal effects on human brain growth (microcephaly and megalencephaly due to possible loss or gain of protein function, respectively), adding to the growing paradigm of inverse phenotypes due to dysregulation of key brain growth genes.

Biallelic variants in TMEM222 cause a new autosomal recessive neurodevelopmental disorder.

PURPOSE: To elucidate the novel molecular cause in families with a new autosomal recessive neurodevelopmental disorder. METHODS: A combination of exome sequencing and gene matching tools was used to identify pathogenic variants in 17 individuals. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and subcellular localization studies were used to characterize gene expression profile and localization. RESULTS: Biallelic variants in the TMEM222 gene were identified in 17 individuals from nine unrelated families, presenting with intellectual disability and variable other features, such as aggressive behavior, shy character, body tremors, decreased muscle mass in the lower extremities, and mild hypotonia. We found relatively high TMEM222 expression levels in the human brain, especially in the parietal and occipital cortex. Additionally, subcellular localization analysis in human neurons derived from induced pluripotent stem cells (iPSCs) revealed that TMEM222 localizes to early endosomes in the synapses of mature iPSC-derived neurons. CONCLUSION: Our findings support a role for TMEM222 in brain development and function and adds variants in the gene TMEM222 as a novel underlying cause of an autosomal recessive neurodevelopmental disorder.

FXS-Like Phenotype in Two Unrelated Patients Carrying a Methylated Premutation of the FMR1 Gene.

Fragile X syndrome (FXS) is mostly caused by two distinct events that occur in the FMR1 gene (Xq27.3): an expansion above 200 repeats of a CGG triplet located in the 5'UTR of the gene, and methylation of the cytosines located in the CpG islands upstream of the CGG repeats. Here, we describe two unrelated families with one FXS child and another sibling presenting mild intellectual disability and behavioral features evocative of FXS. Genetic characterization of the undiagnosed sibling revealed mosaicism in both the CGG expansion size and the methylation levels in the different tissues analyzed. This report shows that in the same family, two siblings carrying different CGG repeats, one in the full-mutation range and the other in the premutation range, present methylation mosaicism and consequent decreased FMRP production leading to FXS and FXS-like features, respectively. Decreased FMRP levels, more than the number of repeats seem to correlate with the severity of FXS clinical phenotypes.

Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

BACKGROUND: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. METHODS: We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. RESULTS: Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically significant differences in the variation of PAX6 and SOX1-positive NPCs between the two human pluripotent cell-derived methods; therefore, both methods are suitable for producing stable dorsal NPCs. When further differentiated into mature neurons, NPCs gave rise to a population of almost exclusively forebrain cortical neurons, confirming the dorsal fate commitment of the progenitors. CONCLUSIONS: The methods described in this study show improvements over previously published studies and are highly efficient at differentiating human and mouse pluripotent cell types into dorsal PAX6-positive NPCs and eventually into forebrain cortical neurons.

From microcephaly to megalencephaly: determinants of brain size.

Expansion of the human brain, and specifically the neocortex, is among the most remarkable evolutionary processes that correlates with cognitive, emotional, and social abilities. Cortical expansion is determined through a tightly orchestrated process of neural stem cell proliferation, migration, and ongoing organization, synaptogenesis, and apoptosis. Perturbations of each of these intricate steps can lead to abnormalities of brain size in humans, whether small (microcephaly) or large (megalencephaly). Abnormalities of brain growth can be clinically isolated or occur as part of complex syndromes associated with other neurodevelopmental problems (eg, epilepsy, autism, intellectual disability), brain malformations, and body growth abnormalities. Thorough review of the genetic literature reveals that human microcephaly and megalencephaly are caused by mutations of a rapidly growing number of genes linked within critical cellular pathways that impact early brain development, with important pathomechanistic links to cancer, body growth, and epilepsy. Given the rapid rate of causal gene identification for microcephaly and megalencephaly understanding the roles and interplay of these important signaling pathways is crucial to further unravel the mechanisms underlying brain growth disorders and, more fundamentally, normal brain growth and development in humans. In this review, we will (a) overview the definitions of microcephaly and megalencephaly, highlighting their classifications in clinical practice; (b) overview the most common genes and pathways underlying microcephaly and megalencephaly based on the fundamental cellular processes that are perturbed during cortical development; and (c) outline general clinical molecular diagnostic workflows for children and adults presenting with microcephaly and megalencephaly.

El crecimiento del cerebro humano, específicamente del neocórtex, está entre los procesos evolutivos más remarcables que se correlacionan con habilidades cognitivas, emocionales y sociales. El crecimiento cortical está determinado por un proceso estrictamente coordinado de la proliferación, migración, organización, sinaptogénesis y apoptosis de las células madre neurales. La alteración de cada una de estas intrincadas etapas puede llevar a anormalidades del tamaño cerebral en los humanos, sea pequeño (microcefalia) o grande (megalencefalia). Las anormalidades del crecimiento cerebral puedan estar clínicamente aisladas o constituir parte de síndromes complejos asociados con otros problemas del neurodesarrollo (como epilepsia, autismo, incapacidad intelectual), malformaciones cerebrales y anormalidades del crecimiento corporal. La revisión de la literatura genética revela que la microcefalia y la megalencefalia son causadas por mutaciones de un número rápidamente creciente de genes relacionados con las vías celulares esenciales que influyen sobre el desarrollo precoz del cerebro, con importantes mecanismos patológicos vinculados con cáncer, crecimiento corporal y epilepsia. Dada la rápida tasa de identificación de genes que causan microcefalia y megalencefalia, la comprensión del papel y de la interacción de estas importantes vías de señalización es crucial para desentrañar los mecanismos subyacentes a los trastornos del crecimiento cerebral y, más fundamentalmente, al crecimiento y desarrollo normal del cerebro en los seres humanos. En esta revisión se presenta: a) una panorámica de las definiciones de microcefalia y megalencefalia, destacando sus clasificaciones en la práctica clínica, b) una panorámica de los genes y vías más comunes que subyacen a la microcefalia y la megalencefalia basada en los procesos celulares fundamentales que están alterados durante el desarrollo cerebral y c) un resumen del plan de trabajo general para el diagnóstico clínico molecular de niños y adultos con microcefalia y megalencefalia.

L'expansion du cerveau humain, et surtout celle du néocortex, est l'un des processus les plus remarquables de l'évolution, corrélé avec les capacités cognitives, émotionnelles et sociales. L'expansion corticale est déterminée au travers d'un processus étroitement orchestré de prolifération, de migration, d'organisation, de synaptogenèse et d'apoptose des cellules souches neurales. Toute perturbation de chacune de ces étapes intriquées peut générer des anomalies de la taille du cerveau humain, qu'elle soit petite (microcéphalie) ou grande (macrocéphalie). Les anomalies de la croissance du cerveau peuvent être isolées cliniquement ou participer à des syndromes complexes associés à d'autres problèmes du neurodéveloppement (par exemple, épilepsie, autisme, déficit intellectuel), à des malformations cérébrales et à des anomalies de la croissance corporelle. Une revue de la littérature génétique montre que la microcéphalie et la macrocéphalie humaines sont dues à des mutations d'un nombre de gènes augmentant rapidement, liées à des voies cellulaires essentielles qui influent sur le développement précoce du cerveau avec des liens mécanistiques pathologiques importants aux cancers, à la croissance corporelle et a l'épilepsie. Compte tenu du taux d'identification rapide de gène causal pour la micro et la macrocéphalie, il est essentiel de comprendre les rôles et l'interaction de ces importantes voies de signalisation pour mieux découvrir les mécanismes sous-tendant les troubles de la croissance cérébrale et, plus fondamentalement, ceux de la croissance cérébrale et du développement normaux chez l'homme. Nous présentons dans cet article 1) une vue d'ensemble des définitions de la micro et de la macrocéphalie, en soulignant leurs classifications en pratique clinique 2) une vue d'ensemble des voies et des gènes les plus courants sous-tendant la micro- et la macrocéphalie d'après les processus cellulaires fondamentaux perturbés au cours du développement cortical et 3) un aperçu des plans de travail généraux du diagnostic clinique moléculaire pour les enfants et les adultes micro- et macrocéphales.

Advances in understanding - genetic basis of intellectual disability.

Intellectual disability is the most common developmental disorder characterized by a congenital limitation in intellectual functioning and adaptive behavior. It often co-occurs with other mental conditions like attention deficit/hyperactivity disorder and autism spectrum disorder, and can be part of a malformation syndrome that affects other organs. Considering the heterogeneity of its causes (environmental and genetic), its frequency worldwide varies greatly. This review focuses on known genes underlying (syndromic and non-syndromic) intellectual disability, it provides a succinct analysis of their Gene Ontology, and it suggests the use of transcriptional profiling for the prioritization of candidate genes.

Lim mineralization protein 3 induces the osteogenic differentiation of human amniotic fluid stromal cells through Kruppel-like factor-4 downregulation and further bone-specific gene expression.

Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs), can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the Kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.

Bradeion (SEPT4) as a urinary marker of transitional cell bladder cancer: a real-time polymerase chain reaction study of gene expression.

PURPOSE: We evaluated whether Bradeion/SEPT4 gene expression could be used as a potential urinary marker to diagnose bladder transitional cell carcinoma. MATERIALS AND METHODS: From 2005 to 2007 we collected urine samples from 58 individuals, 17 healthy controls and 41 patients in whom bladder tumors were previously diagnosed by cystoscopy. Urine was collected from all patients before transurethral resection of bladder tumor. We performed real-time reverse transcriptase-polymerase chain reaction to evaluate Bradeion/SEPT4 transcript levels using urine sample mRNA. Statistical analysis was done with the Mann-Whitney test and ROC curves. RESULTS: Pathological examination of bladder tumor specimens revealed transitional cell bladder cancer. According to the 2002 TNM classification stage was Ta in 11 patients, T1 in 18 and T2/T3 in 12. All patients had G2 or G3 tumors according to the 1973 WHO grade classification. Relative quantification analysis of Bradeion transcript showed significantly increased levels compared to controls, namely 21.85 times higher in Ta stage tumors, 7.21 times higher in T1 tumors and 4.36 times higher in grade T2/T3 tumors. We compared each tumor stage group with the control group using the Mann-Whitney test to verify the statistical significance of observed differences. The ROC curve built on the change in threshold cycle revealed that with this method we attained 92.68% sensitivity and 64.71% specificity (AUC 0.798, p=0.0001). CONCLUSIONS: Bradeion/SEPT4 transcript levels are significantly increased in patients with transitional cell bladder cancer compared to healthy controls. Our preliminary study supports the possible usefulness of Bradeion as a urinary marker of urothelial disease.

The mGluR5 antagonist AFQ056 does not affect methylation and transcription of the mutant FMR1 gene in vitro.

BACKGROUND: Fragile X syndrome (FXS), the leading cause of inherited mental retardation, is due to expansion and methylation of a CGG sequence in the FMR1 gene, which result in its silencing and consequent absence of FMRP protein. This absence causes loss of repression of metabotropic glutamate receptor 5 (mGluR5)-mediated pathways resulting in the behavioral and cognitive impairments associated with FXS. In a randomized, double-blind trial it was recently demonstrated a beneficial effect of AFQ056, a selective inhibitor of metabotrobic glutamate receptor type 5 (mGluR5), on fully methylated FXS patients respect to partially methylated FXS ones. METHODS: To determine whether AFQ056 may have secondary effects on the methylation and transcription of FMR1, here we treated three FXS lymphoblastoid cell lines and one normal control male line. A quantitative RT-PCR was performed to assess transcriptional reactivation of the FMR1 gene. To assess the methylation status of the FMR1 gene promoter it was carried out a bisulphite sequencing analysis. RESULTS: Both FMR1-mRNA levels and DNA methylation were unmodified with respect to untreated controls. CONCLUSIONS: These results demonstrate that the AFQ056 effect on fully methylated FXS patients is not due to a secondary effect on DNA methylation and consequent transcriptional activation of FMR1.

The FRAXopathies: definition, overview, and update.

The fragile X syndrome, fragile X tremor ataxia syndrome, and premature ovarian insufficiency are conditions related to the X chromosome folate-sensitive fragile site FRAXA. Therefore, we propose that they are considered as a family of disorders under the general designation of FRAXopathies. The present review will outline the main clinical and molecular features of these disorders, with special emphasis on the pathogenic mechanisms that lead to distinct phenotypes, starting from related mutations. The understanding of these mechanisms is already generating promising therapeutic approaches.

Insertion of 16 amino acids in the BAR domain of the oligophrenin 1 protein causes mental retardation and cerebellar hypoplasia in an Italian family.

We observed a three-generation family with two maternal cousins and an uncle affected by mental retardation (MR) with cerebellar hypoplasia. X-linked inheritance and the presence of cerebellar malformation suggested a mutation in the OPHN1 gene. In fact, mutational screening revealed a 2-bp deletion that abolishes a donor splicing site, resulting in the inclusion of the initial 48 nucleotides of intron 7 in the mRNA. This mutation determines the production of a mutant oligophrenin 1 protein with 16 extra amino acids inserted in-frame in the N-terminal BAR (Bin1/amphiphysin/Rvs167) domain. This is the first case of a mutation in OPHN1 that does not result in the production of a truncated protein or in its complete loss. OPHN1 (ARHGAP41) encodes a GTPase-activating (GAP) protein belonging to the GRAF subfamily characterized by an N-terminal BAR domain, followed by a pleckstrin-homology (PH) domain and the GAP domain. GRAF proteins play a role in endocytosis and are supposed to dimerize via their BAR domain, that induces membrane curvature. The extra 16 amino acids cause the insertion of 4.4 turns in the third alpha-helix of the BAR domain and apparently impair the protein function. In fact, the clinical phenotype of these patients is identical to that of patients with loss-of-function mutations.