Megalencephalic leukoencephalopathy with subcortical cysts protein-1: A new calcium-sensitive protein functionally activated by endoplasmic reticulum calcium release and calmodulin binding in astrocytes.

BACKGROUND: MLC1 is a membrane protein highly expressed in brain perivascular astrocytes and whose mutations account for the rare leukodystrophy (LD) megalencephalic leukoencephalopathy with subcortical cysts disease (MLC). MLC is characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling which cause cognitive and motor dysfunctions and epilepsy. In cultured astrocytes, lack of functional MLC1 disturbs cell volume regulation by affecting anion channel (VRAC) currents and the consequent regulatory volume decrease (RVD) occurring in response to osmotic changes. Moreover, MLC1 represses intracellular signaling molecules (EGFR, ERK1/2, NF-kB) inducing astrocyte activation and swelling following brain insults. Nevertheless, to date, MLC1 proper function and MLC molecular pathogenesis are still elusive. We recently reported that in astrocytes MLC1 phosphorylation by the Ca2+/Calmodulin-dependent kinase II (CaMKII) in response to intracellular Ca2+ release potentiates MLC1 activation of VRAC. These results highlighted the importance of Ca2+ signaling in the regulation of MLC1 functions, prompting us to further investigate the relationships between intracellular Ca2+ and MLC1 properties. METHODS: We used U251 astrocytoma cells stably expressing wild-type (WT) or mutated MLC1, primary mouse astrocytes and mouse brain tissue, and applied biochemistry, molecular biology, video imaging and electrophysiology techniques. RESULTS: We revealed that WT but not mutant MLC1 oligomerization and trafficking to the astrocyte plasma membrane is favored by Ca2+ release from endoplasmic reticulum (ER) but not by capacitive Ca2+ entry in response to ER depletion. We also clarified the molecular events underlining MLC1 response to cytoplasmic Ca2+ increase, demonstrating that, following Ca2+ release, MLC1 binds the Ca2+ effector protein calmodulin (CaM) at the carboxyl terminal where a CaM binding sequence was identified. Using a CaM inhibitor and generating U251 cells expressing MLC1 with CaM binding site mutations, we found that CaM regulates MLC1 assembly, trafficking and function, being RVD and MLC-linked signaling molecules abnormally regulated in these latter cells. CONCLUSION: Overall, we qualified MLC1 as a Ca2+ sensitive protein involved in the control of volume changes in response to ER Ca2+ release and astrocyte activation. These findings provide new insights for the comprehension of the molecular mechanisms responsible for the myelin degeneration occurring in MLC and other LD where astrocytes have a primary role in the pathological process.

Phenotypic and molecular characterization of five patients with PIK3CA-related overgrowth spectrum (PROS).

Somatic and germline PI3K-AKT-mTOR pathway pathogenic variants are involved in several segmental overgrowth phenotypes such as the PIK3CA-related overgrowth spectrum (PROS), Proteus syndrome, and PTEN hamartoma tumor syndrome. In this study, we describe five patients with PROS. We identified by high-throughput sequencing four different somatic PIK3CA pathogenic variants in five individuals. The Glu726Lys variant, which was previously reported in megalencephaly-capillary malformation-polymicrogyria (MCAP) syndrome, was identified in two patients with unclassified PROS. The Cys420Arg substitution, which was previously reported in CLOVES, was found in a patient with fibroadipose hyperplasia. Additionally, relatively rare pathogenic variants, His1047Tyr and Tyr1021Cys, were detected in two patients with MCAP. Therefore, we suggest performing deep sequencing of PIK3CA in all patients with suspected PROS, instead of targeted polymerase chain reaction for hotspot pathogenic variants.

Endosomal trafficking defects alter neural progenitor proliferation and cause microcephaly.

Primary microcephaly and megalencephaly are severe brain malformations defined by reduced and increased brain size, respectively. Whether these two pathologies arise from related alterations at the molecular level is unclear. Microcephaly has been largely associated with centrosomal defects, leading to cell death. Here, we investigate the consequences of WDR81 loss of function, which causes severe microcephaly in patients. We show that WDR81 regulates endosomal trafficking of EGFR and that loss of function leads to reduced MAP kinase pathway activation. Mouse radial glial progenitor cells knocked-out for WDR81 exhibit reduced proliferation rate, subsequently leading to reduced brain size. These proliferation defects are rescued in vivo by expressing a megalencephaly-causing mutant form of Cyclin D2. Our results identify the endosomal machinery as an important regulator of proliferation rates and brain growth, demonstrating that microcephaly and megalencephaly can be caused by opposite effects on the proliferation rate of radial glial progenitors.

Co-Occurrence of Fragile X Syndrome with a Second Genetic Condition: Three Independent Cases of Double Diagnosis.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism caused by the instability of a CGG trinucleotide repeat in exon 1 of the FMR1 gene. The co-occurrence of FXS with other genetic disorders has only been occasionally reported. Here, we describe three independent cases of FXS co-segregation with three different genetic conditions, consisting of Duchenne muscular dystrophy (DMD), PPP2R5D--related neurodevelopmental disorder, and 2p25.3 deletion. The co-occurrence of DMD and FXS has been reported only once in a young boy, while in an independent family two affected boys were described, the elder diagnosed with FXS and the younger with DMD. This represents the second case in which both conditions coexist in a 5-year-old boy, inherited from his heterozygous mother. The next double diagnosis had never been reported before: through exome sequencing, a girl with FXS who was of 7 years of age with macrocephaly and severe psychomotor delay was found to carry a de novo variant in the PPP2R5D gene. Finally, a maternally inherited 2p25.3 deletion associated with a decreased level of the MYT1L transcript, only in the patient, was observed in a 33-year-old FXS male with severe seizures compared to his mother and two sex- and age-matched controls. All of these patients represent very rare instances of genetic conditions with clinical features that can be modified by FXS and vice versa.

Red flags for early recognition of adult patients with PTEN Hamartoma Tumour Syndrome.

Patients with PTEN Hamartoma Tumour Syndrome (PHTS) are at increased risk of developing cancer. Many adult PHTS patients are not recognized as such and do not receive the cancer surveillance they need. Our aim was to define phenotypic characteristics that can easily be assessed and manifest by early adulthood, and hence could serve as red flags (i.e. alerting signals) for early recognition of adult patients at high risk of PHTS. Phenotypic characteristics including macrocephaly, multinodular goitre (MNG), and oral features were examined in 81 paediatric and 86 adult PHTS patients by one of two medical experts during yearly surveillance visits at our Dutch PHTS expert centre between 1997 and 2020. MNG was defined as signs of thyroid nodules and/or goitre. Oral features included gingival hypertrophy, high palate (adults only) and oral papillomas. Based on the characteristics' prevalence in different age groups, combinations of phenotypic characteristics were defined and evaluated on their potential to recognize individuals with PHTS. Macrocephaly was present in 100% of paediatric and 67% of adult patients. The prevalence of MNG was ∼50% in paediatric and gradually increased to >90% in adult patients. Similar percentages were observed for any of the oral features. Scoring two out of three of these characteristics yielded a sensitivity of 100% (95%CI 94-100%) in adults. The presence of the combination macrocephaly, MNG, or multiple oral features could serve as a red flag for general practitioners, medical specialists, and dentists to consider further assessment of the diagnosis PHTS in adults. In this way, recognition of adult PHTS patients might be improved and cancer surveillance can be offered timely.

Cerebral organoids to unravel the mechanisms underlying malformations of human cortical development.

Genetic studies identified multiple mutations associated with malformations of cortical development (MCD) in humans. When analyzing the underlying mechanisms in non-human experimental models it became increasingly evident, that these mutations accumulate in genes, which functions evolutionary progressed from rodents to humans resulting in an incomplete reflection of the molecular and cellular alterations in these models. Human brain organoids derived from human pluripotent stem cells resemble early aspects of human brain development to a remarkable extent making them an attractive model to investigate MCD. Here we review how human brain organoids enable the generation of fundamental new insight about the underlying pathomechanisms of MCD. We show how phenotypic features of these diseases are reflected in human brain organoids and discuss challenges and future considerations but also limitations for the use of human brain organoids to model human brain development and associated disorders.

Diphthamide-deficiency syndrome: a novel human developmental disorder and ribosomopathy.

We describe a novel type of ribosomopathy that is defined by deficiency in diphthamidylation of translation elongation factor 2. The ribosomopathy was identified by correlating phenotypes and biochemical properties of previously described patients with diphthamide biosynthesis gene 1 (DPH1) deficiencies with a new patient that carried inactivating mutations in both alleles of the human diphthamide biosynthesis gene 2 (DPH2). The human DPH1 syndrome is an autosomal recessive disorder associated with developmental delay, abnormal head circumference (microcephaly or macrocephaly), short stature, and congenital heart disease. It is defined by variants with reduced functionality of the DPH1 gene observed so far predominantly in consanguineous homozygous patients carrying identical mutant alleles of DPH1. Here we report a child with a very similar phenotype carrying biallelic variants of the human DPH2. The gene products DPH1 and DPH2 are components of a heterodimeric enzyme complex that mediates the first step of the posttranslational diphthamide modification on the nonredundant eukaryotic translation elongation factor 2 (eEF2). Diphthamide deficiency was shown to reduce the accuracy of ribosomal protein biosynthesis. Both DPH2 variants described here severely impair diphthamide biosynthesis as demonstrated in human and yeast cells. This is the first report of a patient carrying compound heterozygous DPH2 loss-of-function variants with a DPH1 syndrome-like phenotype and implicates diphthamide deficiency as the root cause of this patient's clinical phenotype as well as of DPH1-syndrome. These findings define "diphthamide-deficiency syndrome" as a special ribosomopathy due to reduced functionality of components of the cellular machinery for eEF2-diphthamide synthesis.

Tau Reduction Prevents Key Features of Autism in Mouse Models.

Autism is characterized by repetitive behaviors, impaired social interactions, and communication deficits. It is a prevalent neurodevelopmental disorder, and available treatments offer little benefit. Here, we show that genetically reducing the protein tau prevents behavioral signs of autism in two mouse models simulating distinct causes of this condition. Similar to a proportion of people with autism, both models have epilepsy, abnormally enlarged brains, and overactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B)/ mammalian target of rapamycin (mTOR) signaling pathway. All of these abnormalities were prevented or markedly diminished by partial or complete genetic removal of tau. We identify disinhibition of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative PI3K regulator that tau controls, as a plausible mechanism and demonstrate that tau interacts with PTEN via tau's proline-rich domain. Our findings suggest an enabling role of tau in the pathogenesis of autism and identify tau reduction as a potential therapeutic strategy for some of the disorders that cause this condition.

Detection of a familial 1q21.1 microdeletion and concomitant CHD1L mutation in a fetus with oligohydramnios and bilateral renal dysplasia on prenatal ultrasound.

OBJECTIVE: We present detection of a familial 1q21.1 microdeletion and concomitant CHD1L mutation in a fetus with oligohydramnios and bilateral renal dysplasia on prenatal ultrasound. CASE REPORT: A 37-year-old, primigravid woman was referred for level II ultrasound examination at 16 weeks of gestation because of oligohydramnios. The parents were phenotypically normal, and there were no congenital malformations in the family. Prenatal ultrasound at 17 weeks of gestation revealed a fetus with fetal growth biometry equivalent to 16 weeks, oligohydramnios with an amniotic fluid index (AFI) of 1.4 cm and bilateral renal dysplasia without sonographic demonstration of bilateral renal arteries. The pregnancy was subsequently terminated, and a 137-g fetus was delivered without characteristic facial dysmorphism. Postnatal cytogenetic analysis of the umbilical cord and parental bloods revealed normal karyotypes. However, array comparative genomic hybridization (aCGH) analysis on the DNA extracted from the umbilical cord revealed a 2.038-Mb microdeletion of 1q21.1-q21.2 encompassing 11 [Online Mendelian Inheritance in Man (OMIM)] genes of PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, GJA8, GPR89B, NBPF14, TRN-GTT2-1 and NBPF20. The mother was found to carry the same microdeletion. A missense mutation of c.2353T > G, p.Ser785Ala in CHD1L was detected in the umbilical cord. The father was found to carry a heterozygous mutation of c.2353T > G, p.Ser785Ala in CHD1L. CONCLUSION: Fetuses with a 1q21.1 microdeletion and concomitant CHD1L mutation may present oligohydramnios and bilateral renal dysplasia on prenatal ultrasound.

Mutations in the sonic hedgehog pathway cause macrocephaly-associated conditions due to crosstalk to the PI3K/AKT/mTOR pathway.

The hedgehog (Hh) pathway is highly conserved and required for embryonic patterning and determination. Mutations in the Hh pathway are observed in sporadic tumors as well as under syndromic conditions. Common to these syndromes are the findings of polydactyly/syndactyly and brain overgrowth. The latter is also a finding most commonly observed in the cases of mutations in the PI3K/AKT/mTOR pathway. We have identified novel Hh pathway mutations and structural copy number variations in individuals with somatic overgrowth, macrocephaly, dysmorphic facial features, and developmental delay, which phenotypically closely resemble patients with phosphatase and tensin homolog (PTEN) mutations. We hypothesized that brain overgrowth and phenotypic overlap with syndromic overgrowth syndromes in these cases may be due to crosstalk between the Hh and PI3K/AKT/mTOR pathways. To test this, we modeled disease-associated variants by generating PTCH1 and Suppressor of Fused (SUFU) heterozygote cell lines using the CRISPR/Cas9 system. These cells demonstrate activation of PI3K signaling and increased phosphorylation of its downstream target p4EBP1 as well as a distinct cellular phenotype. To further investigate the mechanism underlying this crosstalk, we treated human neural stem cells with sonic hedgehog (SHH) ligand and performed transcriptional analysis of components of the mTOR pathway. These studies identified decreased expression of a set of mTOR negative regulators, leading to its activation. We conclude that there is a significant crosstalk between the SHH and PI3K/AKT/mTOR. We propose that this crosstalk is responsible for why mutations in PTCH1 and SUFU lead to macrocephaly phenotypes similar to those observed in PTEN hamartoma and other overgrowth syndromes associated with mutations in PI3K/AKT/mTOR pathway genes.

Megalencephaly syndromes associated with mutations of core components of the PI3K-AKT-MTOR pathway: PIK3CA, PIK3R2, AKT3, and MTOR.

Megalencephaly (MEG) is a developmental abnormality of brain growth characterized by early onset, often progressive, brain overgrowth. Focal forms of megalencephaly associated with cortical dysplasia, such as hemimegalencephaly and focal cortical dysplasia, are common causes of focal intractable epilepsy in children. The increasing use of high throughput sequencing methods, including high depth sequencing to more accurately detect and quantify mosaic mutations, has allowed us to identify the molecular etiologies of many MEG syndromes, including most notably the PI3K-AKT-MTOR related MEG disorders. Thorough molecular and clinical characterization of affected individuals further allow us to derive preliminary genotype-phenotype correlations depending on the gene, mutation, level of mosaicism, and tissue distribution. Our review of published data on these disorders so far shows that mildly activating variants (that are typically constitutional or germline) are associated with diffuse megalencephaly with intellectual disability and/or autism spectrum disorder; moderately activating variants (that are typically high-level mosaic) are associated with megalencephaly with pigmentary abnormalities of the skin; and strongly activating variants (that are usually very low-level mosaic) are associated with focal brain malformations including hemimegalencephaly and focal cortical dysplasia. Accurate molecular diagnosis of these disorders is undoubtedly crucial to more optimally treat children with these disorders using PI3K-AKT-MTOR pathway inhibitors.

MRX93 syndrome (BRWD3 gene): five new patients with novel mutations.

Overgrowth syndromes (OGS) comprise a heterogeneous group of disorders whose main characteristic is that either the weight, height, or head circumference are above the 97th centile or 2 to 3 SD above the mean for age and sex. Additional features, such as facial dysmorphism, developmental delay or intellectual disability (ID), congenital anomalies, neurological problems and an increased risk of neoplasia are usually associated with OGS. Genetic analysis in patients with overlapping clinical features is essential, to distinguish between two or more similar conditions, and to provide appropriate genetic counseling and recommendations for follow up. In the present paper, we report five new patients (from four unrelated families) with an X-linked mental retardation syndrome with overgrowth (XMR93 syndrome), also known as XLID-BRWD3-related syndrome. The main features of these patients include ID, macrocephaly and dysmorphic facial features. XMR93 syndrome is a recently described disorder caused by mutations in the Bromodomain and WD-repeat domain-containing protein 3 (BRWD3) gene. This article underscores the importance of genetic screening by exome sequencing for patients with OGS and ID with unclear clinical diagnosis, and expands the number of reported individuals with XMR93 syndrome, highlighting the clinical features of this unusual disease.

A mouse model of DEPDC5-related epilepsy: Neuronal loss of Depdc5 causes dysplastic and ectopic neurons, increased mTOR signaling, and seizure susceptibility.

DEPDC5 is a newly identified epilepsy-related gene implicated in focal epilepsy, brain malformations, and Sudden Unexplained Death in Epilepsy (SUDEP). In vitro, DEPDC5 negatively regulates amino acid sensing by the mTOR complex 1 (mTORC1) pathway, but the role of DEPDC5 in neurodevelopment and epilepsy has not been described. No animal model of DEPDC5-related epilepsy has recapitulated the neurological phenotypes seen in patients, and germline knockout rodent models are embryonic lethal. Here, we establish a neuron-specific Depdc5 conditional knockout mouse by cre-recombination under the Synapsin1 promotor. Depdc5flox/flox-Syn1Cre (Depdc5cc+) mice survive to adulthood with a progressive neurologic phenotype that includes motor abnormalities (i.e., hind limb clasping) and reduced survival compared to littermate control mice. Depdc5cc+ mice have larger brains with increased cortical neuron size and dysplastic neurons throughout the cortex, comparable to the abnormal neurons seen in human focal cortical dysplasia specimens. Depdc5 results in constitutive mTORC1 hyperactivation exclusively in neurons as measured by the increased phosphorylation of the downstream ribosomal protein S6. Despite a lack of increased mTORC1 signaling within astrocytes, Depdc5cc+ brains show reactive astrogliosis. We observed two Depdc5cc+ mice to have spontaneous seizures, including a terminal seizure. We demonstrate that as a group Depdc5cc+ mice have lowered seizure thresholds, as evidenced by decreased latency to seizures after chemoconvulsant injection and increased mortality from pentylenetetrazole-induced seizures. In summary, our neuron-specific Depdc5 knockout mouse model recapitulates clinical, pathological, and biochemical features of human DEPDC5-related epilepsy and brain malformations. We thereby present an important model in which to study targeted therapeutic strategies for DEPDC5-related conditions.

Cellular Phenotypes in Human iPSC-Derived Neurons from a Genetic Model of Autism Spectrum Disorder.

A deletion or duplication in the 16p11.2 region is associated with neurodevelopmental disorders, including autism spectrum disorder and schizophrenia. In addition to clinical characteristics, carriers of the 16p11.2 copy-number variant (CNV) manifest opposing neuroanatomical phenotypes-e.g., macrocephaly in deletion carriers (16pdel) and microcephaly in duplication carriers (16pdup). Using fibroblasts obtained from 16pdel and 16pdup carriers, we generated induced pluripotent stem cells (iPSCs) and differentiated them into neurons to identify causal cellular mechanisms underlying neurobiological phenotypes. Our study revealed increased soma size and dendrite length in 16pdel neurons and reduced neuronal size and dendrite length in 16pdup neurons. The functional properties of iPSC-derived neurons corroborated aspects of these contrasting morphological differences that may underlie brain size. Interestingly, both 16pdel and 16pdup neurons displayed reduced synaptic density, suggesting that distinct mechanisms may underlie brain size and neuronal connectivity at this locus.

A mouse model of the schizophrenia-associated 1q21.1 microdeletion syndrome exhibits altered mesolimbic dopamine transmission.

1q21.1 hemizygous microdeletion is a copy number variant leading to eightfold increased risk of schizophrenia. In order to investigate biological alterations induced by this microdeletion, we generated a novel mouse model (Df(h1q21)/+) and characterized it in a broad test battery focusing on schizophrenia-related assays. Df(h1q21)/+ mice displayed increased hyperactivity in response to amphetamine challenge and increased sensitivity to the disruptive effects of amphetamine and phencyclidine hydrochloride (PCP) on prepulse inhibition. Probing of the direct dopamine (DA) pathway using the DA D1 receptor agonist SKF-81297 revealed no differences in induced locomotor activity compared to wild-type mice, but Df(h1q21)/+ mice showed increased sensitivity to the DA D2 receptor agonist quinpirole and the D1/D2 agonist apomorphine. Electrophysiological characterization of DA neuron firing in the ventral tegmental area revealed more spontaneously active DA neurons and increased firing variability in Df(h1q21)/+ mice, and decreased feedback reduction of DA neuron firing in response to amphetamine. In a range of other assays, Df(h1q21)/+ mice showed no difference from wild-type mice: gross brain morphology and basic functions such as reflexes, ASR, thermal pain sensitivity, and motor performance were unaltered. Similarly, anxiety related measures, baseline prepulse inhibition, and seizure threshold were unaltered. In addition to the central nervous system-related phenotypes, Df(h1q21)/+ mice exhibited reduced head-to tail length, which is reminiscent of the short stature reported in humans with 1q21.1 deletion. With aspects of both construct and face validity, the Df(h1q21)/+ model may be used to gain insight into schizophrenia-relevant alterations in dopaminergic transmission.

A clinical review on megalencephaly: A large brain as a possible sign of cerebral impairment.

Megalencephaly and macrocephaly present with a head circumference measurement 2 standard deviations above the age-related mean. However, even if pathologic events resulting in both megalencephaly and macrocephaly may coexist, a distinction between these two entities is appropriate, as they represent clinical expression of different disorders with a different approach in clinical work-up, overall prognosis, and treatment. Megalencephaly defines an increased growth of cerebral structures related to dysfunctional anomalies during the various steps of brain development in the neuronal proliferation and/or migration phases or as a consequence of postnatal abnormal events. The disorders associated with megalencephaly are classically defined into 3 groups: idiopathic or benign, metabolic, and anatomic. In this article, we seek to underline the clinical aspect of megalencephaly, emphasizing the main disorders that manifest with this anomaly in an attempt to properly categorize these disorders within the megalencephaly group.

Hypoinsulinaemic, hypoketotic hypoglycaemia due to mosaic genetic activation of PI3-kinase.

OBJECTIVE: Genetic activation of the insulin signal-transducing kinase AKT2 causes syndromic hypoketotic hypoglycaemia without elevated insulin. Mosaic activating mutations in class 1A phospatidylinositol-3-kinase (PI3K), upstream from AKT2 in insulin signalling, are known to cause segmental overgrowth, but the metabolic consequences have not been systematically reported. We assess the metabolic phenotype of 22 patients with mosaic activating mutations affecting PI3K, thereby providing new insight into the metabolic function of this complex node in insulin signal transduction. METHODS: Three patients with megalencephaly, diffuse asymmetric overgrowth, hypoketotic, hypoinsulinaemic hypoglycaemia and no AKT2 mutation underwent further genetic, clinical and metabolic investigation. Signalling in dermal fibroblasts from one patient and efficacy of the mTOR inhibitor Sirolimus on pathway activation were examined. Finally, the metabolic profile of a cohort of 19 further patients with mosaic activating mutations in PI3K was assessed. RESULTS: In the first three patients, mosaic mutations in PIK3CA (p.Gly118Asp or p.Glu726Lys) or PIK3R2 (p.Gly373Arg) were found. In different tissue samples available from one patient, the PIK3CA p.Glu726Lys mutation was present at burdens from 24% to 42%, with the highest level in the liver. Dermal fibroblasts showed increased basal AKT phosphorylation which was potently suppressed by Sirolimus. Nineteen further patients with mosaic mutations in PIK3CA had neither clinical nor biochemical evidence of hypoglycaemia. CONCLUSIONS: Mosaic mutations activating class 1A PI3K cause severe non-ketotic hypoglycaemia in a subset of patients, with the metabolic phenotype presumably related to the extent of mosaicism within the liver. mTOR or PI3K inhibitors offer the prospect for future therapy.

A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly.

BACKGROUND: Constitutive activation of the PI3K-AKT-mTOR pathway (mTOR pathway) underlies megalencephaly in many patients. Yet, prevalence of the involvement of the PI3K-AKT-mTOR pathway in patients with megalencephaly remains to be elucidated, and molecular diagnosis is challenging. Here, we have successfully established a combination of genetic and biochemical methods for diagnosis of mTOR pathway-associated megalencephaly, and have attempted to delineate the clinical characteristics of the disorder. METHODS: Thirteen patients with an increased head circumference and neurological symptoms participated in the study. To evaluate the activation of the mTOR pathway, we performed western blot analysis to determine the expression levels of phosphorylated S6 ribosomal protein (phospho-S6 protein) in lymphoblastoid cell lines from 12 patients. Multiplex targeted sequencing analysis for 15 genes involved in the mTOR pathway was performed on 12 patients, and whole-exome sequencing was performed on one additional patient. Clinical features and MRI findings were also investigated. RESULTS: We identified pathogenic mutations in six (AKT3, 1 patient; PIK3R2, 2 patients; PTEN, 3 patients) of the 13 patients. Increased expression of phospho-S6 protein was demonstrated in all five mutation-positive patients in whom western blotting was performed, as well as in three mutation-negative patients. Developmental delay, dysmorphic facial features were observed in almost all patients. Syndactyly/polydactyly and capillary malformations were not observed, even in patients with AKT3 or PIK3R2 mutations. There were no common phenotypes or MRI findings among these patients. CONCLUSIONS: A combination of genetic and biochemical methods successfully identified mTOR pathway involvement in nine of 13 (approximately 70%) patients with megalencephaly, indicating a major contribution of the pathway to the pathogenesis of megalencephaly. Our combined approach could be useful to identify patients who are suitable for future clinical trials using an mTOR inhibitor.

PI3K/AKT pathway mutations cause a spectrum of brain malformations from megalencephaly to focal cortical dysplasia.

Malformations of cortical development containing dysplastic neuronal and glial elements, including hemimegalencephaly and focal cortical dysplasia, are common causes of intractable paediatric epilepsy. In this study we performed multiplex targeted sequencing of 10 genes in the PI3K/AKT pathway on brain tissue from 33 children who underwent surgical resection of dysplastic cortex for the treatment of intractable epilepsy. Sequencing results were correlated with clinical, imaging, pathological and immunohistological phenotypes. We identified mosaic activating mutations in PIK3CA and AKT3 in this cohort, including cancer-associated hotspot PIK3CA mutations in dysplastic megalencephaly, hemimegalencephaly, and focal cortical dysplasia type IIa. In addition, a germline PTEN mutation was identified in a male with hemimegalencephaly but no peripheral manifestations of the PTEN hamartoma tumour syndrome. A spectrum of clinical, imaging and pathological abnormalities was found in this cohort. While patients with more severe brain imaging abnormalities and systemic manifestations were more likely to have detected mutations, routine histopathological studies did not predict mutation status. In addition, elevated levels of phosphorylated S6 ribosomal protein were identified in both neurons and astrocytes of all hemimegalencephaly and focal cortical dysplasia type II specimens, regardless of the presence or absence of detected PI3K/AKT pathway mutations. In contrast, expression patterns of the T308 and S473 phosphorylated forms of AKT and in vitro AKT kinase activities discriminated between mutation-positive dysplasia cortex, mutation-negative dysplasia cortex, and non-dysplasia epilepsy cortex. Our findings identify PI3K/AKT pathway mutations as an important cause of epileptogenic brain malformations and establish megalencephaly, hemimegalencephaly, and focal cortical dysplasia as part of a single pathogenic spectrum.

CHD8 regulates neurodevelopmental pathways associated with autism spectrum disorder in neural progenitors.

Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10(-8)) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10(-10)). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.