Brain Somatic Mutations in MTOR Disrupt Neuronal Ciliogenesis, Leading to Focal Cortical Dyslamination.

Focal malformations of cortical development (FMCDs), including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are major etiologies of pediatric intractable epilepsies exhibiting cortical dyslamination. Brain somatic mutations in MTOR have recently been identified as a major genetic cause of FMCDs. However, the molecular mechanism by which these mutations lead to cortical dyslamination remains poorly understood. Here, using patient tissue, genome-edited cells, and mouse models with brain somatic mutations in MTOR, we discovered that disruption of neuronal ciliogenesis by the mutations underlies cortical dyslamination in FMCDs. We found that abnormal accumulation of OFD1 at centriolar satellites due to perturbed autophagy was responsible for the defective neuronal ciliogenesis. Additionally, we found that disrupted neuronal ciliogenesis accounted for cortical dyslamination in FMCDs by compromising Wnt signals essential for neuronal polarization. Altogether, this study describes a molecular mechanism by which brain somatic mutations in MTOR contribute to the pathogenesis of cortical dyslamination in FMCDs.

[Detection of TSC1/TSC2 gene mutations among patients with tuberous sclerosis complex by Ion Torrent semiconductor sequencing].

OBJECTIVE: To develop and validate a method for mutation screening and prenatal diagnosis of TSC1/TSC2 mutations among patients with tuberous sclerosis complex (TSC) by Ion Torrent semiconductor sequencing. METHODS: Potential mutations of SC1/TSC2 gene was detected in 2 TSC families and 1 sporadic TSC patient using an Ion Torrent PGM sequencer. Candidate variants were validated by Sanger sequencing. The corresponding site of TSC2 in the fetus of family 2 was also detected with Sanger sequencing. RESULTS: Ion Torrent semiconductor sequencing has identified a probably pathogenic TSC2 mutation (c.311-312insGCTG) in the patient from family 1, and a probably pathogenic TSC2 mutation (c.1790A>G) in the patient of family 2. CONCLUSION: Targeted Ion Torrent PGM sequencing is an accurate and efficient method to detect TSC1/TSC2 mutations in TSC.

Prenatal diagnosis of giant cardiac rhabdomyoma in tuberous sclerosis complex: a new therapeutic option with everolimus.

Tuberous sclerosis complex (TSC) is a genetic disorder characterized by abnormal cell proliferation and tumor growth in a number of organ systems, primarily the brain, kidneys, eyes and heart. Clinical symptoms vary according to the location of the tumor. The most common disorders are seizures, neurodevelopmental disorders, renal failure and arrhythmias. TSC was found to be influenced by inhibitors of the protein kinase mammalian target of rapamycin (mTOR), which regulates abnormal cellular proliferation. mTOR inhibitors have been studied effectively in patients with subependymal giant-cell astrocytomas and renal angiolipomas in the context of TSC. We describe a prenatally diagnosed case of giant rhabdomyoma, due to right ventricular outflow tract obstruction, which presented as a duct-dependent lesion. Postnatal treatment with the mTOR inhibitor everolimus initiated significant regression of the cardiac tumor. This finding suggests that mTOR inhibitor therapy is an option for giant rhabdomyomas that develop in the neonatal period.

PGD for germline mosaicism.

The aim of this study was to develop and perform a preimplantation genetic diagnosis (PGD) assay discriminating between wild-type and mutant alleles in two families with germline mosaicism. Family 1 had two children affected with severe myoclonic epilepsy (SCNA1A del exons 1-22). Family 2 had two children with tuberous sclerosis (TSC2 C1327T) and two healthy children. Neither mutation was detected in genomic DNA derived from the parents in either family. Informative microsatellite markers flanking SCNA1A and TSC2 along with the identified mutations were used to construct haplotypes. For tuberous sclerosis, single spermatozoa were analysed using a multiplex assay that included six informative markers and the TSC2 mutation. In family 1, deletion in the maternal allele was detected in the affected child. In family 2, both affected children and one healthy child shared the same paternal allele. To confirm mutant paternal transmission, single spermatozoa were analysed for the mutation along with six markers. Of 44 single spermatozoa, four showed the mutant T allele, allowing linkage between the mutation and the genetic markers. Both families delivered healthy children following IVF/PGD. In conclusion, germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm analysis is a useful tool for verifying allelic linkage.

The differential effects of prenatal and/or postnatal rapamycin on neurodevelopmental defects and cognition in a neuroglial mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is caused by heterozygous mutations in either the TSC1 (hamartin) or the TSC2 (tuberin) gene. Among the multisystemic manifestations of TSC, the neurodevelopmental features cause the most morbidity and mortality, presenting a considerable clinical challenge. Hamartin and tuberin form a heterodimer that inhibits the mammalian target of rapamycin complex 1 (mTORC1) kinase, a major cellular regulator of protein translation, cell growth and proliferation. Hyperactivated mTORC1 signaling, an important feature of TSC, has prompted a number of preclinical and clinical studies with the mTORC1 inhibitor rapamycin. Equally exciting is the prospect of treating TSC in the perinatal period to block the progression of brain pathologies and allow normal brain development to proceed. We hypothesized that low-dose rapamycin given prenatally and/or postnatally in a well-established neuroglial (Tsc2-hGFAP) model of TSC would rescue brain developmental defects. We developed three treatment regimens with low-dose intraperitoneal rapamycin (0.1 mg/kg): prenatal, postnatal and pre/postnatal (combined). Combined rapamycin treatment resulted in almost complete histologic rescue, with a well-organized cortex and hippocampus almost identical to control animals. Other treatment regimens yielded less complete, but significant improvements in brain histology. To assess how treatment regimens affected cognitive function, we continued rapamycin treatment after weaning and performed behavioral testing. Surprisingly, the animals treated with the combined therapy did not perform as well as postnatally-treated animals in learning and memory tasks. These results have important translational implications in the optimization of the timing and dosage of rapamycin treatment in TSC affected children.

Single-cell Tsc1 knockout during corticogenesis generates tuber-like lesions and reduces seizure threshold in mice.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by mutations in Tsc1 or Tsc2 that lead to mammalian target of rapamycin (mTOR) hyperactivity. Patients with TSC suffer from intractable seizures resulting from cortical malformations known as tubers, but research into how these tubers form has been limited because of the lack of an animal model. To address this limitation, we used in utero electroporation to knock out Tsc1 in selected neuronal populations in mice heterozygous for a mutant Tsc1 allele that eliminates the Tsc1 gene product at a precise developmental time point. Knockout of Tsc1 in single cells led to increased mTOR activity and soma size in the affected neurons. The mice exhibited white matter heterotopic nodules and discrete cortical tuber-like lesions containing cytomegalic and multinucleated neurons with abnormal dendritic trees resembling giant cells. Cortical tubers in the mutant mice did not exhibit signs of gliosis. Furthermore, phospho-S6 immunoreactivity was not upregulated in Tsc1-null astrocytes despite a lower seizure threshold. Collectively, these data suggest that a double-hit strategy to eliminate Tsc1 in discrete neuronal populations generates TSC-associated cortical lesions, providing a model to uncover the mechanisms of lesion formation and cortical hyperexcitability. In addition, the absence of glial reactivity argues against a contribution of astrocytes to lesion-associated hyperexcitability.

Considerations for prenatal counselling of patients with cardiac rhabdomyomas based on their cardiac and neurologic outcomes.

Cardiac rhabdomyomas are benign cardiac tumours with few cardiac complications, but with a known association to tuberous sclerosis that affects the neurologic outcome of the patients. We have analysed the long-term cardiac and neurological outcomes of patients with cardiac rhabdomyomas in order to allow comprehensive prenatal counselling, basing our findings on the records of all patients seen prenatally and postnatally with an echocardiographic diagnosis of cardiac rhabdomyoma encountered from August, 1982, to September, 2007. We analysed factors such as the number and the location of the tumours to establish their association with a diagnosis of tuberous sclerosis, predicting the cardiac and neurologic outcomes for the patients.Cardiac complications include arrhythmias, obstruction of the ventricular outflow tracts, and secondary cardiogenic shock. Arrhythmias were encountered most often during the neonatal period, with supraventricular tachycardia being the commonest rhythm disturbance identified. No specific dimension or location of the cardiac rhabdomyomas predicted the disturbances of rhythm.The importance of the diagnosis of tuberous sclerosis is exemplified by the neurodevelopmental complications, with four-fifths of the patients showing epilepsy, and two-thirds having delayed development. The presence of multiple cardiac tumours suggested a higher risk of being affected by tuberous sclerosis. The tumours generally regress after birth, and cardiac-related problems are rare after the perinatal period. Tuberous sclerosis and the associated neurodevelopmental complications dominate the clinical picture, and should form an important aspect of the prenatal counselling of parents.

[Imaging of the brain malformations].

Cortical dysplasia (CD) is a malformation predominantly affected cerebral neocortex, resulting in disorganized brain cytoarchitecture. Normal cortical lamination is disturbed and neurons are abnormally located. Adjacent white matter is often involved. Chronic seizures are a clinical feature of developmental disorders of the brain, including CD and agenesis of the corpus callosum. A morphologically distinct form of CD is focal cortical dysplasia (FCD) characterized by the presence of cytomegalic neurons, seen in specimens resected from patients with partial epilepsy. The abnormal area usually appears normal externally, but may on occasion be represented by a wilder than normal gyrus, while the cut surface shows blurred grey-and white-matter demarcation. MR images of FCD show blurred grey-and white-matter demarcation and T2 elongation in the white matter. In our cases of FCD, no calcifications or mass effects were observed. The lesions of FCD also bear some resemblance to those of a forme fruste of tuberous sclerosis (solitary cortical tuber). The cytoarchitectual abnormalities in cortical tubers are, however, much more extensive and a characteristic feature of cortical tubers is the presence of subpial clusters of giant astrocytes and sheaves of astrocytic processes. CT images of a solitary cortical tuber show abnormal cortex with high density and frequently associated with calcifications and mass effect. An abnormal dilatated and oriented cerebral fissure is often observed around the lesion. White matter bands and blurred grey-and white-matter discrimination are occasionally seen in the white matter near the lesion. FCD is a different lesion from a solitary cortical tuber.

Ultrafast MRI in the prenatal diagnosis of Bourneville's tuberous sclerosis.

The purpose of this report is to highlight the utility of prenatal MRI as an adjunctive imaging modality in the diagnosis and prognosis of Tuberous Sclerosis (TS) (Bourneville's disease). We report a case of TS detected in utero at 30 weeks gestation. A routine ultrasonography at 26 weeks in a 28-year-old primigravida was followed by an ultrafast MRI examination at 30 weeks gestation. Ultrasound raised the possibility of TS based on the detection of multiple cardiac rhabdomyomas. Fetal MRI, subsequently performed, showed the presence of cortical tubers and subependymal nodules establishing the diagnosis. Fetal MRI in the appropriate clinical setting can be potentially invaluable and can have important prognostic implications.

Cardiac rhabdomyoma with tuberous sclerosis: a case report.

BACKGROUND: Rhabdomyomas are the most common benign cardiac neoplasms occurring in the fetus and neonate, with most of them identified within the first year of life. Cardiac rhabdomyomas are frequently associated with tuberous sclerorosis. CASE: A 25-year-old, pregnant woman with no remarkable personal or family history was referred to us for a suspected fetal cardiac anomaly. Ultrasonographic examination of the fetus revealed multiple solid masses consistent with rhabdomyoma in the ventricular septum and ventricular wall. No other anomalies could be detected. Postnatal echocardiography confirmed the presence of cardiac rhabdomyoma, and periventricular subependymal multiple hamartomas were diagnosed by postnatal magnetic resonance imaging. CONCLUSION: When fetal cardiac rhabdomyoma is diagnosed, careful evaluation of other fetal structures, including brain and renal parenchyma, should be performed to search for signs of tuberous sclerosis.

Precise prenatal diagnosis of tuberous sclerosis by sequencing the TSC2 gene.

BACKGROUND: The presumptive prenatal diagnosis of tuberous sclerosis (TSC) previously depended upon fetal imaging. Cloning of the two TSC genes (TSC1 and TSC2) now enables precise molecular diagnosis by gene sequencing. We used this approach for the prenatal diagnosis of a fetus showing multiple intracardiac tumors. METHODS: DNA extracted from cultivated amniotic fluid cells underwent sequencing of all coding regions and exon-intron boundaries of the TSC1 and TSC2 genes. RESULTS: A mutation (R611Q) was found in exon 16 of the TSC2 gene. Thus far, neither clinically unaffected parents has provided blood samples for mutation analysis. CONCLUSION: For the first time, mutation analysis of a TSC gene enabled a precise prenatal diagnosis.

Tuberous sclerosis in a 20-week gestation fetus: immunohistochemical study.

We report an autopsy case of tuberous sclerosis complex (TSC) in a 20-week gestational age female fetus. The brain showed lesions suggestive of early cortical tubers and subependymal hamartomatous nodules. The large cells within these nodular clusters were variably immunoreactive for glial fibrillary acidic protein (GFAP) and vimentin and negative for synaptophysin and neurofilament. Subependymal radial glia expressed both vimentin and GFAP, but subpial radial glia either did not express these markers (in contrast to an age-matched control) or were absent. Tuberin expression was noted in heterotopic neurons in the white matter and brain cells consistent with Cajal Retzius cells in the neocortical molecular layer, very weakly in superficial cortical neurons, neurons in the basal ganglia, Purkinje cells and external granular cells of cerebellum, cranial nerve nuclei neurons, occasional germinal matrix cells, ependymal cells, choroid plexus epithelium, and pituitary gland neuroendocrine cells; it was not seen within the cells of subependymal nodules. The pattern of tuberin immunoreactivity was similar to that which we have observed in older TSC patients. Proliferating cell labeling indexes were comparable in the germinal matrix of the TSC patient and an age-matched control. Abnormal subpial radial glia may be responsible for some of the neuronal migration abnormalities that appear to result in neocortical tubers.

Cell lineage and cell migration in the neural crest.

The neural crest is a transient embryonic structure whose cells migrate extensively before giving rise to a variety of differentiated cell types. Both intrinsic cell lineage information and environmental cues are thought to play a role in determining the fate of these cells. Early in development, these cells can be divided into distinct populations based on their axial level of origin. Cranial neural crest cells differentiate into facial cartilage and cranial ganglia, whereas vagal crest cells give rise to the enteric ganglia. Trunk neural crest cells normally give rise to melanocytes, neurons, and glia of the peripheral nervous system and chromaffin cells of the adrenal medulla. Cell lineage studies of premigratory trunk neural crest cells using single cell injection of a vital dye have shown that single cells can give rise to a number of differentiated cell types. A host of extracellular matrix (ECM) molecules have been tested for their ability to support neural crest cell migration in vitro and in vivo. In general, the large glycoproteins (i.e., fibronectin and laminin) can support migration, whereas proteoglycans seem to modulate neural crest cell migration on other ECM molecules. However, no single molecule has been identified as the sole regulator of the complex pattern of neural crest cell migration.