Comparison of the functional and structural characteristics of rare
            <i>TSC2</i>
            variants with clinical and genetic findings

Almeida, Luiz Gustavo Dufner de; Nanhoe, Santoesha; Zonta, Andrea; Hosseinzadeh, Mitra; Kom-Gortat, Regina; Elfferich, Peter; Schaaf, Gerben; Kenter, Annegien; Kümmel, Daniel; Migone, Nicola; Povey, Sue; Ekong, Rosemary; Nellist, Mark

doi:10.1002/humu.23963

Cited by 22 publications

(14 citation statements)

References 30 publications

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“…All RHEB gene variants were cloned in our dual promoter expression vector using AscI and PacI restriction sites [ 23 ], and the empty vector used as control refers to the dual promoter expression vector without a gene inserted and expressing either tdTOMATO or EGFP (specified in the figures or in the figures’ legends). Expression constructs for TSC1 , TSC2 , and a myc-tagged S6K reporter were as described previously [ 65 ]. The following DNA plasmids were obtained from Addgene: pGEMTEZ-TeTxLC (Addgene plasmid #32640; http://n2t.net/addgene:32640 ; RRID:Addgene_32640) [ 66 ]; RV-CAG-DIO-EGFP (Addgene plasmid #87662; http://n2t.net/addgene:87662 ; RRID:Addgene_87662) [ 67 ]; pCAG-ERT2CreERT2 (Addgene plasmid #13777; http://n2t.net/addgene:13777 ; RRID:Addgene_13777) [ 61 ]; pCAGGS-ChR2-Venus (Addgene plasmid #15753; http://n2t.net/addgene:15753 ; RRID:Addgene_15753) [ 32 ].…”

Section: Methodsmentioning

confidence: 99%

RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity

et al. 2021

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Hyperactivation of the mammalian target of rapamycin (mTOR) pathway can cause malformation of cortical development (MCD) with associated epilepsy and intellectual disability (ID) through a yet unknown mechanism. Here, we made use of the recently identified dominant-active mutation in Ras Homolog Enriched in Brain 1 (RHEB), RHEBp.P37L, to gain insight in the mechanism underlying the epilepsy caused by hyperactivation of the mTOR pathway. Focal expression of RHEBp.P37L in mouse somatosensory cortex (SScx) results in an MCD-like phenotype, with increased mTOR signaling, ectopic localization of neurons, and reliable generalized seizures. We show that in this model, the mTOR-dependent seizures are caused by enhanced axonal connectivity, causing hyperexcitability of distally connected neurons. Indeed, blocking axonal vesicle release from the RHEBp.P37L neurons alone completely stopped the seizures and normalized the hyperexcitability of the distally connected neurons. These results provide new evidence of the extent of anatomical and physiological abnormalities caused by mTOR hyperactivity, beyond local malformations, which can lead to generalized epilepsy.

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Section: Methodsmentioning

confidence: 99%

RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity

et al. 2021

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“…TSC1 À/À knock-out MEFs were described and characterized previously ( (Demetriades et al, 2014;Kwiatkowski et al, 2002;Zhang et al, 2003). HEK293T wild-type and the respective TSC1/2 double-deficient HEK293T cells were described previously (Dufner Almeida et al, 2020). MCF-7 cells were a kind gift from Michael Meisterernst and obtained from DSMZ (Braunschweig, Germany).…”

Section: Cell Culture and Treatmentsmentioning

confidence: 99%

TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation

et al. 2021

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“…All constructs were verified by sequencing of the complete TSC2 open reading frame. Each variant was tested in at least 3 separate transfection experiments in 3H9-1B1 (TSC2:/TSC1 double knockout HEK 293 T) cells 19 . Cells expressing the variants were compared to cells expressing wild-type TSC2, the pathogenic TSC2 p.Arg611Gln variant, and cells not expressing TSC2 (TSC1/S6K only).…”

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Mutational analysis of TSC1 and TSC2 in Danish patients with tuberous sclerosis complex

Rosengren

Nanhoe

Almeida

et al. 2020

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tuberous sclerosis complex (tSc) is an autosomal dominant disorder characterized by hamartomas in the skin and other organs, including brain, heart, lung, kidney and bones. tSc is caused by mutations in TSC1 and TSC2. Here, we present the TSC1 and TSC2 variants identified in 168 Danish individuals out of a cohort of 327 individuals suspected of TSC. A total of 137 predicted pathogenic or likely pathogenic variants were identified: 33 different TSC1 variants in 42 patients, and 104 different TSC2 variants in 126 patients. In 40 cases (24%), the identified predicted pathogenic variant had not been described previously. In total, 33 novel variants in TSC2 and 7 novel variants in TSC1 were identified. To assist in the classification of 11 TSC2 variants, we investigated the effects of these variants in an in vitro functional assay. Based on the functional results, as well as population and genetic data, we classified 8 variants as likely to be pathogenic and 3 as likely to be benign. Tuberous sclerosis complex (TSC) is an autosomal dominant disorder of high penetrance with an incidence of 1:6,000-1:10,000 and an estimated prevalence of 1:14,000-1:25,000 1,2. TSC is characterized by the presence of mainly benign tumors that can affect multiple organ systems e.g. the central nervous system, heart, kidney, lung, bone and skin. TSC patients are phenotypically and genetically heterogeneous and there is considerable variation in the number, location and size of the different TSC-associated lesions. Mutations in one of two genes, TSC1 (OMIM#191100) and TSC2 (OMIM#191092), cause TSC 3,4. TSC1 is located on chromosome 9q34 and consists of 23 exons, which encode the 130 kDa TSC1 protein, hamartin. TSC2 is located on chromosome 16p13.3 and consists of 42 exons which encode the 200 kDa TSC2 protein, tuberin. TSC1 and TSC2, together with a third subunit, TBC1D7 5 , form a stable protein complex, the TSC complex. The TSC complex is a GTPase-activating protein (GAP) specific for the small GTPase, Ras homologue enriched in brain (RHEB) 6. Active RHEB is involved in the activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a critical regulator of anabolic processes such as protein and lipid synthesis 7. The TSC complex inactivates RHEB to down-regulate mTORC1 signaling and inhibit cell growth. TSC-associated tumors are characterized by increased phosphorylation of S6, elongation factor 4E binding protein 1 (4E-BP1), p70 S6 kinase (S6K) and other downstream targets of mTORC1 (Fig. 1). Approximately 2/3 of TSC cases are due to sporadic de novo germline mutations 2. TSC2 mutations are identified in the majority of TSC patients and, in general, cause a more severe phenotype than TSC1 mutations 8,9. Exceptions to this rule are however observed 10,11. Large genomic deletions that affect both TSC2 and the adjacent PKD1 (OMIM# 601313) locus are associated with a subset of patients with TSC and severe, early-onset autosomal dominant polycystic kidney disease. While a pathogenic TSC1 or TSC2 variant can be identified in m...

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Comparison of the functional and structural characteristics of rare TSC2 variants with clinical and genetic findings

Cited by 22 publications

References 30 publications

RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity

RHEB/mTOR hyperactivity causes cortical malformations and epileptic seizures through increased axonal connectivity

TSC1 binding to lysosomal PIPs is required for TSC complex translocation and mTORC1 regulation

Mutational analysis of TSC1 and TSC2 in Danish patients with tuberous sclerosis complex

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