An mTORC1-GRASP55 signaling axis controls unconventional secretion to reshape the extracellular proteome upon stress.

Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.

Severe hypertension in tuberous sclerosis complex caused by renal artery stenosis: A case report.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant neurocutaneous disease. Arterial hypertension is one of its uncommon complications, which is supposed to be caused by renal cysts or angiomyolipomas. Few studies have been reported in the literature on renal artery stenosis (RAS) as the cause of hypertension in TSC. Hence, we reported a boy who presented with uncontrolled hypertension under five anti-hypertension drugs and was diagnosed with TSC complicated with left RAS. His high blood pressure was relieved by percutaneous transluminal renal angioplasty (PTRA). In one and a half years follow-up, his blood pressure was normal whilst he took four anti-hypertensive drugs. In conclusion, children with TCS complicated with hypertension should be carefully screened for RAS, which might be relieved by percutaneous balloon dilatation.

Pathology of hereditary renal cell carcinoma syndromes: Tuberous sclerosis complex (TSC).

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disease characterized by hamartomatous tumors involving multiple organs such as the brain, skin, heart, lung and kidney. TSC is caused by inactivating mutations in TSC1/TSC2, which encodes hamartin and tuberin, respectively, and forms a complex that regulates mechanistic target of rapamycin complex 1 (mTORC1), resulting in cell overgrowth and oncogenesis. Since a leading cause of morbidity and mortality in TSC relates to chronic kidney disease and the ability to preserve renal function, this review describes the important pathologic findings in TSC-associated renal neoplasms and their correlating sporadic counterparts. The most common renal tumor in TSC patients are AMLs, followed by a heterogeneous spectrum of renal epithelial tumors, which may provide clues to establishing a diagnosis of TSC.

Isolated subependymal giant cell astrocytoma (SEGA) in the absence of clinical tuberous sclerosis: two case reports and literature review.

PURPOSE: Subependymal giant cell astrocytoma (SEGA) is a WHO grade I pediatric glioma arising in 5-15% of patients with tuberous sclerosis (TSC). Rare cases of isolated SEGA without TSC have been described. The etiology, genetic mechanisms, natural history, and response to treatment of these lesions are currently unknown. We describe two such cases of isolated SEGA with follow-up. METHODS: Retrospective review was performed at a single institution to describe the clinical course of pathology-confirmed SEGA in patients with germline testing negative for TSC mutations. RESULTS: Two cases of isolated SEGA were identified. Genetic analysis of the tumor specimen was available for one, which revealed an 18 base pair deletion in TSC1. Both cases were managed with surgical resection, one with preoperative embolization. In spite of a gross total resection, one patient experienced recurrence after three years. Treatment with an mTOR inhibitor led to a significant interval reduction of the mass on follow-up MRI. The patient tolerated the medication well for 6 years and is now off of treatment for 2 years with a stable lesion. CONCLUSION: Cases of SEGA outside of the context of TSC are exceedingly rare, with only 48 cases previously described. The genetic mechanisms and treatment response of these lesions are poorly understood. To date, these lesions appear to respond well to mTOR inhibitors and may behave similarly to SEGAs associated with TSC. However, given that experience is extremely limited, these cases should be followed long term to better understand their natural history and treatment response.

Tuberous sclerosis complex is associated with a novel human tauopathy.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder leading to epilepsy, developmental delay, and neurobehavioral dysfunction. The syndrome is caused by pathogenic variants in TSC1 (coding for hamartin) or TSC2 (coding for tuberin). Recently, we reported a progressive frontotemporal dementia-like clinical syndrome in a patient with a mutation in TSC1, but the neuropathological changes seen in adults with TSC with or without dementia have yet to be systematically explored. Here, we examined neuropathological findings in adults with TSC (n = 11) aged 30-58 years and compared them to age-matched patients with epilepsy unrelated to TSC (n = 9) and non-neurological controls (n = 10). In 3 of 11 subjects with TSC, we observed a neurofibrillary tangle-predominant "TSC tauopathy" not seen in epilepsy or non-neurological controls. This tauopathy was observed in the absence of pathological amyloid beta, TDP-43, or alpha-synuclein deposition. The neurofibrillary tangles in TSC tauopathy showed a unique pattern of post-translational modifications, with apparent differences between TSC1 and TSC2 mutation carriers. Tau acetylation (K274, K343) was prominent in both TSC1 and TSC2, whereas tau phosphorylation at a common phospho-epitope (S202) was observed only in TSC2. TSC tauopathy was observed in selected neocortical, limbic, subcortical, and brainstem sites and showed a 3-repeat greater than 4-repeat tau isoform pattern in both TSC1 and TSC2 mutation carriers, but no tangles were immunolabeled with MC1 or p62 antibodies. The findings suggest that individuals with TSC are at risk for a unique tauopathy in mid-life and that tauopathy pathogenesis may involve TSC1, TSC2, and related molecular pathways.

Three-Year Follow-Up after Intrauterine mTOR Inhibitor Administration for Fetus with TSC-Associated Rhabdomyoma.

Tuberous sclerosis complex (TSC) is a multisystem disorder characterized by seizures, neuropsychiatric disorders, and tumors of the heart, brain, skin, lungs, and kidneys. We present a three-year follow-up of a patient with TSC-associated rhabdomyoma detected in utero. Genetic examination of the fetus and the parents revealed a de novo variant in the TSC2 gene (c.3037delG, p.Asp1013IlefsTer3). Oral everolimus was initiated in the pregnant mother to regress the fetal tumor, which was successful. To the best of our knowledge, there is very little information regarding the use of everolimus therapy during pregnancy. West-syndrome was diagnosed when the proband was four months old. The symptoms were well-manageable, however temporarily. Therapy-resistant focal seizures were frequent. The patient had good vitals and was under regular cardiological control, showed a balanced circulation, and did not require any medication. Subependymal giant cell astrocytoma (SEGA) identified by regular neuroimaging examinations remained unchanged, which may be a consequence of early intrauterine treatment. Early detection of the pathogenic TSC2 variant, followed by in utero administration of everolimus and early vigabatrin therapy, allowed the detection of a milder developmental delay of the proband. Our study emphasizes how early genetic testing and management of epilepsy are pivotal for proper neurodevelopmental impacts and therapeutic strategies.

Progress of clinical research studies on tuberous sclerosis complex-related epilepsy in China.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome, with 75.6%-83.5% and 54.1% patients presenting with epilepsy and drug-resistant epilepsy (DRE), respectively. Clinical studies on TSC, particularly surgical interventions, have achieved rapid and substantial progress. The TSC-Task Force Committee of the China Association Against Epilepsy (CAAE-TFTSC) was founded in 2012, and annual academic conferences on the surgical treatment of TSC-related epilepsy have been held since 2013. 'China experts' consensus on surgical treatment of TSC-related epilepsy' was published in 2019. This review focuses on surgical treatment, including resective surgery, neuromodulations, corpus callosotomy and mini-invasive ablations, as well as studies on phenotype, genotype and anti-seizure therapies of mammalian target of rapamycin inhibitor, vigabatrin and ketogenic diet in patients with TSC-related DRE in China.

Malignant perivascular epithelioid cell tumor (PEComa) of the uterus.

BACKGROUND: Perivascular epithelioid cell tumors (PEComas) of the uterus is a rare type of mesenchymal tumors associated with myelomelanocytic differentiation and distinctive histological appearances. So far, the reported cases of uterine PEComas are usually benign. Documented malignant cases with aggressive behavior appear to be less common. CASE PRESENTATION: We report a 37-year-old female who received abdominal hysterectomy for uterine tumor in a local hospital. She was diagnosed with uterine leiomyosarcoma and referred to Hubei Cancer Hospital. Her histological slides were reviewed and immunohistochemical staining for specific markers of epithelial, melanocytic, myoid and some others were analyzed. The pathologic diagnosis was malignant uterine PEComa. Systematic imaging of the patient further revealed an abdominal para-aortic mass. She received pelvic and para-aortic lymph node dissection. Postoperative histology revealed para-aortic lymph nodal metastasis of malignant uterine PEComa. She received 8 cycles of chemotherapy after surgery. The chemotherapy regiment was epirubicin plus ifosfamide The patient is free of recurrence and metastasis 6 years after surgical resection. CONCLUSION: Uterine PEComas are indistinguishable from other uterine tumors such as leiomyoma and leiomyosarcoma before pathologic diagnosis could be made. For patients with malignant uterine PEComas, removal of both primary lesions and metastatic foci, if any, needs to be attempted. Postoperative chemotherapy or radiotherapy should also be considered in patients with distant metastases or positive lymph nodes.

Global and intertuberal epileptic networks in tuberous sclerosis based on stereoelectroencephalographic (sEEG) findings: a quantitative EEG analysis in pediatric subjects and surgical implications.

OBJECTIVE: Recent evidence favors a network concept in tuberous sclerosis (TSC) with seizure generation and propagation related to changes in global and regional connectivity between multiple, anatomically distant tubers. Direct exploration of network dynamics in TSC has been made possible through intracranial sampling with stereoelectroencephalography (sEEG). The objective of this study is to define epileptic networks in TSC using quantitative analysis of sEEG recordings. We also discuss the impact of the definition of these epileptic networks on surgical decision-making. METHODS: Intracranial sEEG recordings were obtained from four pediatric patients who presented with medically refractory epilepsy secondary to TSC and subjected to quantitative signal analysis methods. Cortical connectivity was quantified by calculating pairwise coherence between all contacts and constructing an association matrix. The global coherence, defined as the ratio of the largest eigenvalue to the sum of all the eigenvalues, was calculated for each frequency band (delta, theta, alpha, beta, gamma). Spatial distribution of the connectivity was identified by plotting the leading principal component (product of the largest eigenvalue and its corresponding eigenvector). RESULTS: Four pediatric subjects with TSC underwent invasive intracranial monitoring with sEEG, comprising 31 depth electrodes and 250 contacts, for localization of the epileptogenic focus and guidance of subsequent surgical intervention. Quantitative connectivity analysis revealed a change in global coherence during the ictal period in the beta/low gamma (14-30 Hz) and high gamma (31-80 Hz) bands. Our results corroborate findings from existing literature, which implicate higher frequencies as a driver of synchrony and desynchrony. CONCLUSIONS: Coordinated high-frequency activity in the beta/low gamma and high gamma bands among spatially distant sEEG define the ictal period in TSC. This time-dependent change in global coherence demonstrates evidence for intra-tuberal and inter-tuberal connectivity in TSC. This observation has surgical implications. It suggests that targeting multiple tubers has a higher chance of seizure control as there is a higher chance of disrupting the epileptic network. The use of laser interstitial thermal therapy (LITT) allowed us to target multiple disparately located tubers in a minimally invasive manner with good seizure control outcomes.

Fibroma-like PEComa: a newly recognized soft tissue neoplasm in tuberous sclerosis patients-imaging features and review of literature.

Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal neoplasms composed of spindled to epithelioid cells that co-express both melanocytic and myogenic markers. Recently, in 2018, a distinctive variant of PEComa has been described that arises in association with tuberous sclerosis complex (TSC) and resembles a fibroma by conventional morphology (called fibroma-like PEComa). Herein, we describe a case of a fibroma-like PEComa in a 4-year-old male child with a known diagnosis of tuberous sclerosis who presented with a firm mass along the anteromedial aspect of the right knee. The mass was excised, and microscopic examination showed bland spindled to stellate cells embedded in a dense collagenous stroma, morphologically resembling a fibroma. Immunohistochemistry analysis showed positivity for desmin (a myogenic marker) and HMB45 (a melanocytic marker), a hallmark for PEComas. To our knowledge, only six cases of fibroma-like PEComa have been described in the literature so far and this is the first report of such a tumor in the medial retinaculum of the knee joint with illustrations of conventional and diffusion imaging features. This case highlights the unique association of fibroma-like PEComa lesions with TSC. This should be considered a differential diagnosis for T2 hypointense masses in tuberous sclerosis patients. In addition, a diagnosis of fibroma-like PEComa should prompt further evaluation for associated TSC.

Experience using mTOR inhibitors for subependymal giant cell astrocytoma in tuberous sclerosis complex at a single facility.

BACKGROUND: Subependymal giant cell astrocytoma (SEGA) is occasionally seen in tuberous sclerosis complex (TSC). Two main options are currently available for treating SEGA: surgical resection or pharmacotherapy using mammalian target of rapamycin inhibitors (mTORi). We hypothesized that opportunities for surgical resection of SEGA would have reduced with the advent of mTORi. METHODS: We retrospectively reviewed the charts of patients treated between August 1979 and July 2020, divided into a pre-mTORi era group (Pre-group) of patients treated before November 2012, and a post-mTORi era group (Post-group) comprising patients treated from November 2012, when mTORi became available in Japan for SEGA. We compared groups in terms of treatment with surgery or mTORi. We also reviewed SEGA size, rate of acute hydrocephalus, recurrence of SEGA, malignant transformation and adverse effects of mTORi. RESULTS: In total, 120 patients with TSC visited our facility, including 24 patients with SEGA. Surgical resection was significantly more frequent in the Pre-group (6 of 7 patients, 86 %) than in the Post-group (2 of 17 patients, 12 %; p = 0.001). Acute hydrocephalus was seen in 1 patient (4 %), and no patients showed malignant transformation of SEGA. The group treated using mTORi showed significantly smaller SEGA compared with the group treated under a wait-and-see policy (p = 0.012). Adverse effects of pharmacotherapy were identified in seven (64 %; 6 oral ulcers, 1 irregular menstruation) of the 11 patients receiving mTORi. CONCLUSIONS: The Post-group underwent surgery significantly less often than the Pre-group. Since the treatment option to use mTORi in the treatment of SEGA in TSC became available, opportunities for surgical resection have decreased in our facility.

A bistable, multiport valve enables microformulators creating microclinical analyzers that reveal aberrant glutamate metabolism in astrocytes derived from a tuberous sclerosis patient.

There is a need for valves and pumps that operate at the microscale with precision and accuracy, are versatile in their application, and are easily fabricated. To that end, we developed a new rotary planar multiport valve to faithfully select solutions (contamination = 5.22 ± 0.06 ppb) and a rotary planar peristaltic pump to precisely control fluid delivery (flow rate = 2.4 ± 1.7 to 890 ± 77 µL/min). Both the valve and pump were implemented in a planar format amenable to single-layer soft lithographic fabrication. These planar microfluidics were actuated by a rotary motor controlled remotely by custom software. Together, these two devices constitute an innovative microformulator that was used to prepare precise, high-fidelity mixtures of up to five solutions (deviation from prescribed mixture = ±|0.02 ± 0.02| %). This system weighed less than a kilogram, occupied around 500 cm3, and generated pressures of 255 ± 47 kPa. This microformulator was then combined with an electrochemical sensor creating a microclinical analyzer (µCA) for detecting glutamate in real time. Using the chamber of the µCA as an in-line bioreactor, we compared glutamate homeostasis in human astrocytes differentiated from human-induced pluripotent stem cells (hiPSCs) from a control subject (CC-3) and a Tuberous Sclerosis Complex (TSC) patient carrying a pathogenic TSC2 mutation. When challenged with glutamate, TSC astrocytes took up less glutamate than control cells. These data validate the analytical power of the µCA and the utility of the microformulator by leveraging it to assess disease-related alterations in cellular homeostasis.

Disrupted white matter connectivity and organization of brain structural connectomes in tuberous sclerosis complex patients with neuropsychiatric disorders using diffusion tensor imaging.

OBJECTIVE: Tuberous sclerosis complex (TSC) is a genetic neurocutaneous syndrome with variable and unpredictable neurological comorbidity that includes epilepsy, intellectual disability (ID), autism spectrum disorder, and neurobehavioral abnormalities. The degree of white matter involvement is believed to be associated with the severity of neurological impairment. The goal of the present study was to evaluate diffusion characteristics of tubers, white matter lesions, and brain structural network alterations in TSC patients using diffusion tensor imaging (DTI), graph theoretical analysis (GTA), and network-based statistical (NBS) analysis. MATERIALS AND METHODS: Forty-two patients with a definitive diagnosis of TSC were recruited for this study. All patients underwent brain DTI examination using a 3 T magnetic resonance imaging system. Mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD) values, and fractional anisotropy (FA) mapping in 52 tubers and white matter lesions were measured and compared with those of contralateral normal regions. GTA was performed on the inter-regional connectivity matrix, and NBS analysis was used to identify the significance of any connected subnetworks evident in the set of altered connections. For neurological severity subgrouping, a neurological severity score was assigned to TSC patients including those with ID, seizure, autism, and other neuropsychiatric disorders (NPDs). RESULTS: Significantly higher MD, AD, and RD, and lower FA values, were found in TSC lesions compared with those measured in contralateral normal regions for tubers (P < 0.05). GTA and NBS analysis provided better local segregation but worse global integration of the structural network (regular-like network) in TSC patients with ID, seizure, and higher Neurological Severity Score. Disrupted subnetworks in TSC patients with severe status included connections from the frontal lobe to the parietal lobe, temporal lobe to the caudate, and temporal lobe to the insula. DISCUSSION: DTI has the potential to provide valuable information about cytoarchitectural changes in TSC lesions beyond morphological MRI findings alone. Using GTA and NBS, current results provide the information of disrupted white matter connectivity and organization in TSC patients with different neuropsychological impairments.

TSC2 pathogenic variants are predictive of severe clinical manifestations in TSC infants: results of the EPISTOP study.

PURPOSE: To perform comprehensive genotyping of TSC1 and TSC2 in a cohort of 94 infants with tuberous sclerosis complex (TSC) and correlate with clinical manifestations. METHODS: Infants were enrolled at age <4 months, and subject to intensive clinical monitoring including electroencephalography (EEG), brain magnetic resonance imaging (MRI), and neuropsychological assessment. Targeted massively parallel sequencing (MPS), genome sequencing, and multiplex ligation-dependent probe amplification (MLPA) were used for variant detection in TSC1/TSC2. RESULTS: Pathogenic variants in TSC1 or TSC2 were identified in 93 of 94 (99%) subjects, with 23 in TSC1 and 70 in TSC2. Nine (10%) subjects had mosaicism. Eight of 24 clinical features assessed at age 2 years were significantly less frequent in those with TSC1 versus TSC2 variants including cortical tubers, hypomelanotic macules, facial angiofibroma, renal cysts, drug-resistant epilepsy, developmental delay, subependymal giant cell astrocytoma, and median seizure-free survival. Additionally, quantitative brain MRI analysis showed a marked difference in tuber and subependymal nodule/giant cell astrocytoma volume for TSC1 versus TSC2. CONCLUSION: TSC2 pathogenic variants are associated with a more severe clinical phenotype than mosaic TSC2 or TSC1 variants in TSC infants. Early assessment of gene variant status and mosaicism might have benefit for clinical management in infants and young children with TSC.

The effect of mTOR inhibition on obstructive hydrocephalus in patients with tuberous sclerosis complex (TSC) related subependymal giant cell astrocytoma (SEGA).

PURPOSE: Mammalian target of rapamycin inhibitors (mTORi) are known to effectively reduce the size of subependymal giant cell astrocytomas (SEGAs), which are benign brain lesions associated with Tuberous Sclerosis Complex (TSC) that commonly cause obstructive hydrocephalus (OH). This retrospective case series reviews an institutional experience of the effect of mTORi on OH in patients with TSC-related SEGA. METHODS: Thirteen of 16 identified patients with TSC-related SEGA treated with mTORi from October 2007 to December 2018 were included. Serial magnetic resonance imaging (MRI) and clinical charts were reviewed to correlate symptoms and signs of increased intracranial pressure (iICP) with ventriculomegaly on MRI. A proposed ventriculomegaly scale was used: none (< 7 mm), mild (7-10 mm), moderate (11-30 mm), and severe (> 30 mm). OH was defined as moderate or severe ventriculomegaly, based on the largest measurement. RESULTS: Patients' median age at start of mTORi was 13 (6-17) years and five (38%) patients were female. Eight patients had OH at the time of mTORi initiation, five of whom were asymptomatic. Six patients had improvement of hydrocephalus on serial MRI imaging with mTORi therapy, while seven patients had no change based on the ventriculomegaly scale used. All three patients who presented with symptoms of iICP and had OH also had papilledema. None had worsening of hydrocephalus or required shunt placement. Out of five patients with symptoms of iICP, four avoided surgery. CONCLUSION: Most patients had asymptomatic OH at the time of diagnosis, and ventricular enlargement was not correlated with iICP symptoms. mTORi was successful for treatment of OH from TSC-related SEGA, even in the setting of acute symptoms of iICP.

Developmental outcomes in children/adolescents and one adult with tuberous sclerosis complex (TSC) and refractory epilepsy treated with everolimus.

This prospective observational study focuses on developmental outcomes in the treatment of tuberous sclerosis complex (TSC) with everolimus (EVO). Fourteen children/adolescents aged 1.7-13.07 and one adult aged 31 years, all with TSC and refractory epilepsy participated. All were treated with EVO for 3-70 months (md: 37). Development/adaptive functioning were evaluated at baseline with follow-up in 11 patients; all patients were assessed during the course of treatment. Our exploratory analyses included factors contributing to developmental impairment and change from baseline to last evaluation. The majority of patients showed severe developmental impairment (86%). Patients with a higher age at inclusion, duration of epilepsy, and number of previous antiepileptic drugs (AEDs) showed lower developmental levels. Earlier onset of epilepsy and a higher number of current AEDs were associated with worse adaptive functioning. At their last examination, four patients were seizure-free (27%), and four experienced a reduction of seizures >50% (27%). With treatment, (slight) increase was seen in absolute values of developmental age (DA) regarding both development and adaptive functioning. Yet, when accounting for age, decrease was seen in both assessments. While developmental disorders were prominent, we observed an overall progression at a slower pace. Despite a positive effect on seizure occurrence, treatment with EVO did not reverse developmental problems in the observation period of this study.

Neurosurgical treatment of subependymal giant cell astrocytomas in tuberous sclerosis complex: a series of 44 surgical procedures in 31 patients.

BACKGROUND: Subependymal giant cell astrocytomas (SEGA) are benign tumors characteristic of tuberous sclerosis complex (TSC) that may cause hydrocephalus. Various treatments are nowadays available as mTOR inhibitors or surgery. Surgery is still a valid option especially for symptomatic and larger tumors. METHODS: From January 1994 to December 2015, 31 TSC patients harboring SEGA underwent surgery at the Department of Neurosurgery of the Meyer Pediatric Hospital, Florence. Indications for surgery were tumor size and location, growth and cystization/hemorrhage, and hydrocephalus. Clinical data, preoperative and postoperative MRI, recurrence rate, further surgical procedures, and related complications were analyzed. RESULTS: A total of 44 surgeries were performed in 31 TSC patients affected by SEGA, achieving gross total removal (GTR) and subtotal removal (STR), respectively, in 36 and 8 patients. Recurrences occurred in 11 patients; 9 of them underwent further surgical procedures and 2 were treated with mTOR pathway inhibitors. Surgical morbidity and mortality were, respectively, 22.7% and 2.3%. After a mean follow-up of 4.9 years, 90% of patients were tumor-free with good neurological status in 93.3%; twelve (40%) had a ventriculo-peritoneal shunt (VPS) for hydrocephalus. CONCLUSIONS: The present series confirms that the surgical approach, combined with mTOR inhibitors, is still a valid option for the treatment of SEGAs.

Bilateral Renal Angiomyolipomas and Subependymal Giant Cell Astrocytoma Associated with Tuberous Sclerosis Complex: a Case Report and Review of The Literature.

Tuberous sclerosis complex (TSC) is an autosomal-dominant multi system disorder. The genetic basis of the disorder is mutations in the TSC1 or TSC2 gene, which leads to over activation of the mammalian target of rapamycin (mTOR) protein complex and results in development of benign tumors in different body systems such as brain, skin, lungs and kidney. The mTOR inhibitors are presently the main treatment option for patients with TSC. We here report a 21-year female patient with large bilateral angiomyolipoma (AML) in both kidneys with longest diameter more than 12.3 cm and subependymal giant cell astrocytoma (SEGA). Treatment with everolimus (EVE) was initiated at a dose of 10.0 mg/day and continued during the following 3 years. Magnetic resonance imaging (MRI) was performed before treatment with everolimus was initiated, and consequently at 12 and 36 months for follow-up of the efficacy of the treatment. After 3 years, the total size of largest AML decreased by ~24.0% in the longest diameter. A reduction of the total size of SEGA was also observed. The most common adverse effect of treatment was stomatitis grades 3 to 4 and one febrile episode associated with skin rash that required a reduced dose of EVE. In conclusion, the everolimus treatment improved even such a large renal AML and the effect persisted during the long-term administration with a small number of adverse effects. A positive effect was observed on the brain tumor as well.

The tuberous sclerosis complex subunit TBC1D7 is stabilized by Akt phosphorylation-mediated 14-3-3 binding.

The tuberous sclerosis complex (TSC) is a negative regulator of mTOR complex 1, a signaling node promoting cellular growth in response to various nutrients and growth factors. However, several regulators in TSC signaling still await discovery and characterization. Using pulldown and MS approaches, here we identified the TSC complex member, TBC1 domain family member 7 (TBC1D7), as a binding partner for PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), a negative regulator of Akt kinase signaling. Most TBC domain-containing proteins function as Rab GTPase-activating proteins (RabGAPs), but the crystal structure of TBC1D7 revealed that it lacks residues critical for RabGAP activity. Sequence analysis identified a putative site for both Akt-mediated phosphorylation and 14-3-3 binding at Ser-124, and we found that Akt phosphorylates TBC1D7 at Ser-124. However, this phosphorylation had no effect on the binding of TBC1D7 to TSC1, but stabilized TBC1D7. Moreover, 14-3-3 protein both bound and stabilized TBC1D7 in a growth factor-dependent manner, and a phospho-deficient substitution, S124A, prevented this interaction. The crystal structure of 14-3-3ζ in complex with a phospho-Ser-124 TBC1D7 peptide confirmed the direct interaction between 14-3-3 and TBC1D7. The sequence immediately upstream of Ser-124 aligned with a canonical ß-TrCP degron, and we found that the E3 ubiquitin ligase ß-TrCP2 ubiquitinates TBC1D7 and decreases its stability. Our findings reveal that Akt activity determines the phosphorylation status of TBC1D7 at the phospho-switch Ser-124, which governs binding to either 14-3-3 or ß-TrCP2, resulting in increased or decreased stability of TBC1D7, respectively.

A novel de novo splicing mutation c.1444-2A>T in the TSC2 gene causes exon skipping and premature termination in a patient with tuberous sclerosis syndrome.

Tuberous sclerosis complex (TSC) syndrome is a neurocutaneous syndrome that affects the brain, skin, and kidneys that has an adverse impact on the patient's health and quality of life. There have been several recent advances that elucidate the genetic complex of this disorder that will help understand the basic neurobiology of this disorder. We report a Tunisian patient with clinical manifestations of TSC syndrome. We investigated the causative molecular defect in this patient using PCR followed by direct sequencing. Subsequently, in silico studies and mRNA analysis were performed to study the pathogenicity of the new variation found in the TSC2. Bioinformatics tools predicted that the novel mutation c.1444-2A>T have pathogenic effects on splicing machinery. RT-PCR followed by sequencing revealed that the mutation c.1444-2A>T generates two aberrant transcripts. The first, with exon 15 skipping, is responsible for the loss of 52 amino acids, which causes the production of an aberrant protein isoform. The second, with the inclusion of 122 nucleotides of intron 14, is responsible for the creation of new premature termination codons (TGA), which causes the production of a truncated TSC2 protein. This study highlighted the clinical features of a Tunisian patient with TSC syndrome and revealed a splicing mutation c.1444-2A>T within intron 14 of TSC2 gene, which is present for the first time using Sanger sequencing approach, as a disease-causing mutation in a Tunisian patient with TSC syndrome.