Abstract

Tuberous Sclerosis Complex (TSC) is caused by loss of function germline variants in the TSC1 or TSC2 tumor suppressor genes. Genetic testing for the detection of pathogenic variants in either TSC1 or TSC2 was implemented as a diagnostic criterion for TSC. However, TSC molecular diagnosis can be challenging due to the absence of variant hotspots and the high number of variants described. This review aimed to perform an overview of TSC1/2 variants submitted in the ClinVar database. Variants of uncertain significance (VUS), missense and single nucleotide variants were the most frequent in clinical significance (37-40%), molecular consequence (37%-39%) and variation type (82%-83%) categories in ClinVar in TSC1 and TSC2 variants, respectively. Frameshift and nonsense VUS have potential for pathogenic reclassification if further functional and segregation studies were performed. Indeed, there were few functional assays deposited in the database and literature. In addition, we did not observe hotspots for variation and many variants presented conflicting submissions regarding clinical significance. This study underscored the importance of disseminating molecular diagnostic results in a public database to render the information largely accessible and promote accurate diagnosis. We encourage the performance of functional studies evaluating the pathogenicity of TSC1/2 variants.

Keywords:
ClinVar database; conflicting variants; TSC1; TSC2; variants of uncertain significance

Tuberous sclerosis: Epidemiology and symptomatology

Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic disorder with multisystemic manifestations associated with pathogenic germline variants in TSC1 (OMIM ID: 605284) or TSC2 genes (OMIM ID: 191092) (Curatolo et al., 2008Curatolo P, BombardierI R and Jozwiak S (2008) Tuberous sclerosis. Lancet 372:657-668.). The estimated incidence of the disease varies from 1:6,000 to 1:10,000 (Osborne et al., 1991Osborne JP, Fryer A and Webb D (1991) Epidemiology of tuberous sclerosis. Ann N Y Acad Sci 615:125-127.; O’Callaghan et al., 1998O’Callaghan FJ, ShielL AW, Osborne JP and Martyn CN (1998) Prevalence of tuberous sclerosis estimated by capture-recapture analysis. Lancet 351:1490.; Ebrahimi-Fakhari et al., 2018Ebrahimi-Fakhari D, Mann LL, Poryo M, Graf N, Von Kries R, Heinrich B, Ebrahimi-Fakhari D, Flotats-Bastardas M, Gortner L, Zemlin M et al. (2018) Incidence of tuberous sclerosis and age at first diagnosis: new data and emerging trends from a national, prospective surveillance study. Orphanet J Rare Dis 13:117.; Northrup et al., 2021Northrup H, Aronow ME, Bebin EM, Bissler J, Darling TN, De Vries PJ, Frost MD, Fuchs Z, Gosnell ES, Gupta N et al. (2021) Updated international Tuberous Sclerosis Complex diagnostic criteria and surveillance and management recommendations. Pediatr Neurol 123:50-66.). Patients with TSC have a broad spectrum of clinical manifestations, including hamartoma formation in different organs, commonly in the skin, kidney, and central nervous system (Northrup et al., 2021Northrup H, Aronow ME, Bebin EM, Bissler J, Darling TN, De Vries PJ, Frost MD, Fuchs Z, Gosnell ES, Gupta N et al. (2021) Updated international Tuberous Sclerosis Complex diagnostic criteria and surveillance and management recommendations. Pediatr Neurol 123:50-66.). In addition to hamartomas, central nervous system manifestations include autism, epilepsy, and cognitive impairment (Crino et al., 2006Crino PB, Nathanson KL and Henske EP (2006) The Tuberous Sclerosis Complex. N Engl J Med 355:1345-1356.) and cutaneous manifestations include angiofibromas, hypopigmented macules, shagreen patches, and confetti lesions (Northrup et al., 2013Northrup H, Aronow ME, Bebin EM, Bissler J, Darling TN, De Vries PJ, Frost MD, Fuchs Z, Gosnell ES, Gupta N et al. (2021) Updated international Tuberous Sclerosis Complex diagnostic criteria and surveillance and management recommendations. Pediatr Neurol 123:50-66.; Dimario et al., 2015Dimario FJ Jr, Sahin M and Ebrahimi-Fakhari D (2015) Tuberous Sclerosis Complex. Pediatr Clin North Am 62:633-648.). These symptoms appear in different lifetime periods. In childhood, the development of central nervous system tumors and renal tumors is not uncommon, and typical tumors are subependymal giant cell astrocytomas (SEGA) (Kotulska et al., 2014Kotulska K, Borkowska J, Mandera M, Roszkowski M, Jurkiewicz E, Grajkowska W, Bilska M and Jóźwiak S (2014) Congenital subependymal giant cell astrocytomas in patients with Tuberous Sclerosis Complex. Childs Nerv Syst 30:2037-2042.; Tsai et al., 2016Tsai JD, Wei CC, Tsao TF, Hsiao YP, Tsai HJ, Yang SH, Tsai ML and Sheu JN (2016) Association between the growth rate of subependymal giant cell astrocytoma and age in patients with Tuberous Sclerosis Complex. Childs Nerv Syst 32:89-95.) and angiomyolipomas (Warncke et al., 2017Warncke JC, Brodie KE, Grantham EC, Catarinicchia SP, Tong S, Kondo KL and Cost NG (2017) Pediatric Renal Angiomyolipomas in Tuberous Sclerosis Complex. J Urol 197:500-506.; Kingswood et al., 2019Kingswood JC, Belousova E, Benedik MP, Carter T, Cottin V, Curatolo P, Dahlin M, D’ Amato L, D’Augères GB, De Vries PJ et al. (2019) Renal angiomyolipoma in patients with Tuberous Sclerosis Complex: Findings from the TuberOus SClerosis registry to increase disease Awareness. Nephrol Dial Transplant 34:502-508. ). In adolescence, angiofibromas are prevalent (Liu et al., 2015Liu Z, Wang J, Wang H, Wang D, Hu L, Liu Q and Sun X (2015) Hormonal receptors and vascular endothelial growth factor in juvenile nasopharyngeal angiofibroma: Immunohistochemical and tissue microarray analysis. Acta Otolaryngol 135:51-57.) and renal cell carcinoma is more frequently diagnosed in adulthood (Yang et al., 2014Yang P, Cornejo KM, Sadow PM, Cheng L, Wang M, Xiao Y, Jiang Z, Oliva E, Jozwiak S, Nussbaum Rl et al. (2014) Renal cell carcinoma in Tuberous Sclerosis Complex. Am J Surg Pathol 38:895-909.; Henske et al., 2021Henske EP, Cornejo KM and Wu CL (2021) Renal Cell Carcinoma in Tuberous Sclerosis Complex. Genes (Basel) 12:1585.). Loss of heterozygosity (LOH) is usually required for TSC tumorigenesis following the Knudson theory of two events (Knudson, 1971Knudson AG Jr (1971) Mutation and cancer: Statistical study of retinoblastoma. Proc Natl Acad Sci U S A 68:820-823.).

Hamartin-tuberin Complex

The TSC1 gene (NG_012386.1) at chromosome 9q.34.4 comprises 23 exons and 60,286 base pairs (bp). The first two exons are not transcribed. The larger gene transcript contains 8,598 bp (NM_000368.5) and a long 3’-untranslated region (4,887 bp). Alternative splicing is common and there are many alternative isoforms (Carbonara et al., 1994Carbonara C, Longa L, Grosso E, Borrone C, Garrè MG, Brisigotti M and Migone N (1994) 9q34 loss of heterozygosity in a tuberous sclerosis astrocytoma suggests a growth suppressor-like activity also for the TSC1 gene. Hum Mol Genet 3:1829-32.; Van Slegtenhorst et al., 1997Van Slegtenhorst M, De Hoogt R, Hermans C, Nellist M, Janssen B, Verhoef S, Lindhout D, Van Den Ouweland A, Halley D, Young J et al. (1997) Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277:805-808. ; NCBINational Center for Biotechnology Information (NCBI),National Center for Biotechnology Information (NCBI),https://www.ncbi.nlm.nih.gov/ (accessed 09 December 2022)
https://www.ncbi.nlm.nih.gov/... ). The protein product is called hamartin, with 1,164 amino acids (NP_000359.1) and a well-characterized coiled-coil functional domain (exons 17 to 23) in the C-terminal region (Santiago Lima et al., 2014Santiago Lima AJ, Hoogeveen-Westerveld M, Nakashima A, Maat-Kievit A, Van Den Ouweland A, Halley D, Kikkawa U and Nellist M (2014) Identification of regions critical for the integrity of the TSC1-TSC2-TBC1D7 complex. PLoS One 9:e93940.; Gai et al., 2016Gai Z, Chu W, Deng W, Li W, Li H, He A, Nellist M and Wu G (2016) Structure of the TBC1D7-TSC1 complex reveals that TBC1D7 stabilizes dimerization of the TSC1 C-terminal coiled coil region. J Mol Cell Biol 8:411-425.). The N-terminal domain contains 265 amino acids forming a potential transmembrane domain (TMD) encoded by a conserved region of the gene (exon 6) (Ali et al., 2005Ali M, Girimaji SC, Markandaya M, Shukla AK, Sacchidanand S and Kumar A (2005) Mutation and polymorphism analysis of TSC1 and TSC2 genes in Indian patients with Tuberous Sclerosis Complex. Acta Neurol Scand 111:54-63.).

The TSC2 gene (NG_005895.1) is localized at chromosome position 16:q13, and consists of 42 exons and 46,814 bp. Like TSC1, it exhibits alternative splicing, producing many different isoforms, of which NM_000548.5 is the largest mRNA reference sequence (5,632 bp). It also presents several shorter transcripts with alterations in the 5’UTR (NM_001318829.2) and 3’UTR (NM_001114382.3). Exons 2-42 of the largest isoform encode the tuberin protein of 1,807 amino acids (NP_000539.2). The C-terminal region is the most conserved (Maheshwar et al., 1997Maheshwar MM, Cheadle JP, Jones AC, Myring J, Fryer AE, Harris PC and Sampson JR (1997) The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis. Hum Mol Genet 6:1991-1996. ), but variations in the C-terminal or N-terminal regions may occur due to previous alternative splicing (NM_001318832.2), representing the complexity and diversity of the TSC2 gene transcripts (Duan et al., 2022Duan J, Ye Y, Hu Z, Zhao X, Liao J and Chen L (2022) Identification of a novel canonical splice site variant. Front Genet 13:904224.; Liu et al., 2022Liu L, Yu C and Yan G (2022) Identification of a novel heterozygous TSC2 splicing variant in a patient with Tuberous Sclerosis Complex: A case report. Medicine (Baltimore) 101:e28666.; NCBI).

Hamartin, tuberin, and Tre2-Bub2-Cdc16 (TBC) 1 domain family member 7 (TBC1D7) form the TSC1-TSC2 (hamartin-tuberin) protein complex, which negatively regulates the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), providing the proper control of different cell processes, such as cell growth and proliferation, protein synthesis and autophagy (Santiago Lima et al., 2014Santiago Lima AJ, Hoogeveen-Westerveld M, Nakashima A, Maat-Kievit A, Van Den Ouweland A, Halley D, Kikkawa U and Nellist M (2014) Identification of regions critical for the integrity of the TSC1-TSC2-TBC1D7 complex. PLoS One 9:e93940.; Saxton and Sabatini, 2017Saxton RA and Sabatini DM (2017) mTOR Signaling in growth, metabolism, and disease. Cell 168:960-976. ; Zou et al., 2020Zou Z, Tao T, Li H and Zhu X (2020) mTOR signaling pathway and mTOR inhibitors in cancer: Progress and challenges. Cell Biosci 10:31. ). Activation of the mTORC1 pathway leads to a phosphorylation cascade in different proteins and positively regulates cell growth and proliferation. Through mTORC1 regulation, the hamartin-tuberin complex has a tumor suppressor function (Jozwiak, 2006Jozwiak J (2006) Hamartin and tuberin: Working together for tumour suppression. Int J Cancer 118:1-5.). The hamartin coiled-coil domain is the binding region of hamartin with tuberin, and stabilizes the complex by interacting with the third member of the complex, TBC1D7 (Santiago Lima et al., 2014Santiago Lima AJ, Hoogeveen-Westerveld M, Nakashima A, Maat-Kievit A, Van Den Ouweland A, Halley D, Kikkawa U and Nellist M (2014) Identification of regions critical for the integrity of the TSC1-TSC2-TBC1D7 complex. PLoS One 9:e93940.; Gai et al., 2016Gai Z, Chu W, Deng W, Li W, Li H, He A, Nellist M and Wu G (2016) Structure of the TBC1D7-TSC1 complex reveals that TBC1D7 stabilizes dimerization of the TSC1 C-terminal coiled coil region. J Mol Cell Biol 8:411-425.). The potential TMD is located in the N-terminal region of hamartin, which also appears to regulate complex stability and subcellular translocation (Hoogeveen-Westerveld et al., 2010Hoogeveen-Westerveld M, Exalto C, Maat-Kievit A, Van Den Ouweland A, Halley D and Nellist M (2010) Analysis of TSC1 truncations defines regions involved in TSC1 stability, aggregation and interaction. Biochim Biophys Acta 1802:774-781.), but the exact function of this domain is still unknown. The C-terminal region of tuberin contains the GTPase-activating protein (GAP) domain (encoded by exons 34-38, amino acids 1,531 to 1,758) (Yang et al., 2021Yang H, Yu Z, Chen X, Li J, Li N, Cheng J, Gao N, Yuan HX, Ye D, Guan KL et al. (2021) Structural insights into TSC complex assembly and GAP activity on Rheb. Nat Commun 12:339.) and the N-terminal region of tuberin (exons 2 to 12, amino acids 1 to 420) interacts with hamartin (Zech et al., 2016Zech R, Kiontke S, Mueller U, Oeckinghau SA and Kümmel D (2016) Structure of the Tuberous Sclerosis Complex 2 (TSC2) N Terminus provides insight into complex assembly and tuberous sclerosis pathogenesis. J Biol Chem 291:20008-20.; Hansmann et al., 2020Hansmann P, Brückner A, Kiontke S, Berkenfeld B, Seebohm G, Brouillard P, Vikkula M, Jansen FE, Nellist M, Oeckinghaus A et al. (2020) Structure of the TSC2 GAP Domain: Mechanistic insight into catalysis and pathogenic mutations. Structure 28:933-942.e4. ).

The GAP domain in tuberin has an important GTPase activity as it hydrolyses the GTP molecule associated with the Rheb protein. When Rheb associates with GTP, it stimulates the mTORC1 pathway, whereas when GTP is hydrolysed by the tuberin GTPase activity, the mTOR pathway is inhibited. The balance between mTORC1 activation and inhibition requires fine regulation, and thus patients with pathogenic variants in the TSC1 or TSC2 genes have a loss of function (LOF) of the hamartin-tuberin complex, leading to hyperactivity of the mTORC1 pathway. Hyperactivation of the mTORC1 pathway leads to deregulation of various cell functions, resulting in continuous cell proliferation, prolonged cell survival, and also inhibition of autophagy, which plays a central role in tumorigenesis and cancer metabolism (Kohrman, 2012Kohrman MH (2012) Emerging treatments in the management of Tuberous Sclerosis Complex. Pediatr Neurol 46:267-275.; Deleyto-Seldas and Efeyan, 2021Deleyto-Seldas N and Efeyan A (2021) The mTOR-autophagy axis and the control of metabolism. Front Cell Dev Biol 9:655731.).

Genotype-phenotype correlations

Genotype-phenotype correlations are not well established in TSC. Some examples that have been described in the literature include the occurrence of TSC2 variants and earlier onset of epilepsy (Alsowat et al., 2021Alsowat D, Whitney R, Hewson S, Jain P, Chan V, Kabir N, Amburgey K, Noone D, Lemaire M, Mccoy B et al. (2021) The phenotypic spectrum of Tuberous Sclerosis Complex: A Canadian cohort. Child Neurol Open 8:2329048X211012817.), and epilepsy with an intellectual deficit (Dabora et al., 2001Dabora SL, Jozwia KS, Franz DN, Roberts PS, Nieto A, Chung J, Choy YS, Reeve MP, Thiele E, Egelhoff JC et al. (2001) Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am J Hum Genet 68:64-80. ; Sancak et al., 2005Sancak O, Nellist M, Goedbloed M, Elfferich P, Wouters C, Maat-Kievit A, Zonnenberg B, Verhoef S, Halley D and Van Den Ouweland A (2005) Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting: Genotype--phenotype correlations and comparison of diagnostic DNA techniques in Tuberous Sclerosis Complex. Eur J Hum Genet 13:731-741.; Au et al., 2007Au KS, Williams AT, Roach ES, Batchelor L, Sparagana SP, Delgado MR, Wheless JW, Baumgartner JE, Roa BB, Wilson CM et al. (2007) Genotype/phenotype correlation in 325 individuals referred for a diagnosis of Tuberous Sclerosis Complex in the United States. Genet Med 9:88-100.; Farach et al., 2019Farach LS, Pearson DA, Woodhouse JP, Schraw JM, Sahin M, Krueger DA, Wu JY, Bebin EM, Lupo PJ, Au KS et al. (2019) Tuberous Sclerosis Complex genotypes and developmental phenotype. Pediatr Neurol 96:58-63.). Interestingly, patients with the TSC phenotype but no identifiable germline pathogenic variant often have milder systemic or neurological manifestations (Alsowat et al., 2021Alsowat D, Whitney R, Hewson S, Jain P, Chan V, Kabir N, Amburgey K, Noone D, Lemaire M, Mccoy B et al. (2021) The phenotypic spectrum of Tuberous Sclerosis Complex: A Canadian cohort. Child Neurol Open 8:2329048X211012817.). In general, alterations that result in decreased tuberin function are related to more severe symptoms (Dabora et al., 2001Dabora SL, Jozwia KS, Franz DN, Roberts PS, Nieto A, Chung J, Choy YS, Reeve MP, Thiele E, Egelhoff JC et al. (2001) Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs. Am J Hum Genet 68:64-80. ; Sancak et al., 2005Sancak O, Nellist M, Goedbloed M, Elfferich P, Wouters C, Maat-Kievit A, Zonnenberg B, Verhoef S, Halley D and Van Den Ouweland A (2005) Mutational analysis of the TSC1 and TSC2 genes in a diagnostic setting: Genotype--phenotype correlations and comparison of diagnostic DNA techniques in Tuberous Sclerosis Complex. Eur J Hum Genet 13:731-741.; Alsowat et al., 2021Alsowat D, Whitney R, Hewson S, Jain P, Chan V, Kabir N, Amburgey K, Noone D, Lemaire M, Mccoy B et al. (2021) The phenotypic spectrum of Tuberous Sclerosis Complex: A Canadian cohort. Child Neurol Open 8:2329048X211012817.). Furthermore, variants detected in the flanking regions of the TSC2 gene and not in middle regions (exons 22-33) are related to a high risk of infantile spasms (Van Eeghen et al., 2013Van Eeghen AM, Nellist M, Van Eeghen EE and Thiele EA (2013) Central TSC2 missense mutations are associated with a reduced risk of infantile spasms. Epilepsy Res 103:83-87.), and patients with TSC1 alterations tend to show more symptoms of an anxiety disorder and minor autism manifestation (Muzykewicz et al., 2007Muzykewicz DA, Newberry P, Danforth N, Halpern EF and Thiele EA (2007) Psychiatric comorbid conditions in a clinic population of 241 patients with Tuberous Sclerosis Complex. Epilepsy Behav 11:506-513.). Regarding kidney manifestations, large deletions encompassing TSC2 and the adjacent gene PKD1 result in polycystic kidney disease (Oyazato et al., 2011Oyazato Y, Iijima K, Emi M, Sekine T, Kamei K, Takanashi J, Nakao H, Namai Y, Nozu K and Matsuo M (2011) Molecular analysis of TSC2/PKD1 contiguous gene deletion syndrome. Kobe J Med Sci 57:E1-10.; Boronat et al., 2014BoronaT S, Caruso P, AuladelL M, Van Eeghen A and Thiele EA. (2014) Arachnoid cysts in Tuberous Sclerosis Complex. Brain Dev 36:801-806.).

Considering specific TSC1 and TSC2 (TSC1/2) variants, genotype-phenotype correlations have been described for only a few, such as TSC2 c.3106T>C and TSC2 c.2714G>A, which have been previously associated with seizures (O’Connor et al., 2003O’Connor SE, Kwiatkowski DJ, Roberts PS, Wollmann RL and Huttenlocher PR (2003) A family with seizures and minor features of Tuberous Sclerosis and a novel TSC2 mutation. Neurology 61:409-412.) and with mild disease (Jansen et al., 2006Jansen AC, Sancak O, D’agostino MD, Badhwar A, Roberts P, Gobbi G, Wilkinson R, Melanson D, Tampieri D, Koenekoop R et al. (2006) Unusually mild tuberous sclerosis phenotype is associated with TSC2 R905Q mutation. Ann Neurol 60:528-539.), respectively. Further studies are needed to make these correlations more robust and to identify other genotype-phenotype correlations.

Molecular diagnosis in tuberous sclerosis complex

The clinical diagnosis of TSC has been established by the International Tuberous Sclerosis Complex Consensus Group (Northrup et al., 2013Northrup H, Krueger DA and International Tuberous Sclerosis Complex Consensus Group (2013) Tuberous sclerosis complex diagnostic criteria update: Recommendations of the 2012 Iinternational Tuberous Sclerosis Complex Consensus Conference. Pediatr Neurol 49:243-254.). As TSC is a condition with high phenotypic variability, and symptoms develop at different stages of life, some patients may not fulfill the clinical criteria for a definitive diagnosis at any given time (Northrup et al., 1993Northrup H, Wheless JW, Bertin TK and Lewis RA (1993) Variability of expression in tuberous sclerosis. J Med Genet 30:41-43.; Northrup et al., 2013Northrup H, Krueger DA and International Tuberous Sclerosis Complex Consensus Group (2013) Tuberous sclerosis complex diagnostic criteria update: Recommendations of the 2012 Iinternational Tuberous Sclerosis Complex Consensus Conference. Pediatr Neurol 49:243-254.). Therefore, genetic testing is a crucial part of the diagnosis, as the presence of a pathogenic variant in the TSC1 and TSC2 genes results in a definitive diagnosis.

Molecular diagnosis of TSC is mainly performed by next-generation sequencing (NGS) using TSC1 and TSC2 panels, and although copy number variants (CNV) analysis used to be performed mainly by multiplex ligation-dependent probe amplification (MLPA) (Rosset et al., 2017Rosset C, Vairo F, Bandeira IC, Correia RL, de Goes FV, da Silva RTB, Bueno LSM, de Miranda Gomes MCS, Galvão HCR, Neri JICF et al. (2017) Molecular analysis of TSC1 and TSC2 genes and phenotypic correlations in Brazilian families with tuberous sclerosis. PLoS One 12:e0185713.), more recent protocols include CNV in the NGS analysis (Singh et al., 2021Singh AK, Olsen MF, Lavik LAS, Vold T, Drabløs F and Sjursen W (2021) Detecting copy number variation in next generation sequencing data from diagnostic gene panels. BMC Med Genomics 14:214.). In addition, whole exome sequencing (WES) is becoming more accessible. Its widespread use in the diagnosis of conditions including cognitive impairment has led to the detection of TSC1 and TSC2 variants in patients with less obvious clinical features and no family history of the disease (Kovesdi et al., 2021Kovesdi E, Ripszam R, Postyeni E, Horvath EB, Kelemen A, Fabos B, Farkas V, Hadzsiev K, Sumegi K, Magyari L et al. (2021) Whole exome sequencing in a series of patients with a clinical diagnosis of Tuberous sclerosis not confirmed by Targeted TSC1/TSC2 Sequencing. Genes (Basel) 12:1401. ).

Germline genetic panel testing or WES results in the identification of multiple variants and therefore a careful interpretation of NGS findings is crucial to identify causal variants (Northrup et al., 2013Northrup H, Krueger DA and International Tuberous Sclerosis Complex Consensus Group (2013) Tuberous sclerosis complex diagnostic criteria update: Recommendations of the 2012 Iinternational Tuberous Sclerosis Complex Consensus Conference. Pediatr Neurol 49:243-254.). A variant classification guideline was proposed by the American College of Medical Genetics and Genomics (ACMG) in 2015 (Richards et al., 2015Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E et al. (2015) Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17:405-424.) and optimized in 2017, with the publication of the Sherloc classification criteria (Nykamp et al., 2017Nykamp K, Anderson M, Powers M, Garcia J, Herrera B, Ho YY, Kobayashi Y, Patil N, Thusberg J, Westbrook M et al. (2017) Sherloc: A comprehensive refinement of the ACMG-AMP variant classification criteria. Genet Med 19:1105-1117.). Both guidelines provide a set of criteria to classify germline variants as pathogenic (P), likely pathogenic (LP), benign (B), likely benign (LB), and of uncertain significance (VUS). Broad evidence categories used to assess the pathogenicity of a variant are the type of variant, frequency in general genetic population databases, segregation analysis, in silico prediction tools, functional assays, and general clinical data (Richards et al., 2015Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E et al. (2015) Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17:405-424.). If the available evidence is insufficient to accurately determine the pathogenicity of the variant, it remains as a VUS. Each specific guideline differs in the weight of evidence, thresholds, and methods, such as semi-quantitative scores or Bayesian frameworks (Nykamp et al., 2017Nykamp K, Anderson M, Powers M, Garcia J, Herrera B, Ho YY, Kobayashi Y, Patil N, Thusberg J, Westbrook M et al. (2017) Sherloc: A comprehensive refinement of the ACMG-AMP variant classification criteria. Genet Med 19:1105-1117.; Tavtigian et al., 2018Tavtigian SV, Greenblatt MS, Harrison SM, Nussbaum RL, Prabhu SA, Boucher KM, Biesecker LG and Clingen Sequence Variant Interpretation Working Group (CLINGEN SVI) (2018) Modeling the ACMG/AMP variant classification guidelines as a Bayesian classification framework. Genet Med 20:1054-1060.). Currently, there is no specific variant classification guideline for TSC1 and TSC2 genes.

Challenges in molecular diagnosis in tuberous sclerosis complex

According to the literature, molecular testing for TSC1 and TSC2 in individuals with a clinical suspicion of TSC yields a pathogenic variant detection rate of 75-90% in different countries (Au et al., 2007Au KS, Williams AT, Roach ES, Batchelor L, Sparagana SP, Delgado MR, Wheless JW, Baumgartner JE, Roa BB, Wilson CM et al. (2007) Genotype/phenotype correlation in 325 individuals referred for a diagnosis of Tuberous Sclerosis Complex in the United States. Genet Med 9:88-100.; Northrup et al., 2013Northrup H, Krueger DA and International Tuberous Sclerosis Complex Consensus Group (2013) Tuberous sclerosis complex diagnostic criteria update: Recommendations of the 2012 Iinternational Tuberous Sclerosis Complex Consensus Conference. Pediatr Neurol 49:243-254.; Rosset et al., 2017Rosset C, Vairo F, Bandeira IC, Correia RL, de Goes FV, da Silva RTB, Bueno LSM, de Miranda Gomes MCS, Galvão HCR, Neri JICF et al. (2017) Molecular analysis of TSC1 and TSC2 genes and phenotypic correlations in Brazilian families with tuberous sclerosis. PLoS One 12:e0185713.; Reyna-Fabián et al., 2020Reyna-Fabián ME, Hernández-Martínez NL, Alcántara-Ortigoza MA, Ayala-Sumuano JT, Enríquez-Flores S, Velázquez-Aragón JA, Varela-Echavarría A, Todd-Quiñones CG and González-Del Angel A (2020) First comprehensive TSC1/TSC2 mutational analysis in Mexican patients with Tuberous Sclerosis Complex reveals numerous novel pathogenic variants. Sci Rep 10:6589.; Rosengren et al., 2020Rosengren T, Nanhoe S, De Almeida LGD, Schönewolf-Greulich B, Larsen LJ, Hey CAB, Dunø M, Ek J, Risom L, Nellist M et al. (2020) Mutational analysis of TSC1 and TSC2 in Danish patients with Tuberous Sclerosis Complex. Sci Rep 10:9909.; Alsowat et al., 2021Alsowat D, Whitney R, Hewson S, Jain P, Chan V, Kabir N, Amburgey K, Noone D, Lemaire M, Mccoy B et al. (2021) The phenotypic spectrum of Tuberous Sclerosis Complex: A Canadian cohort. Child Neurol Open 8:2329048X211012817.; Meng et al., 2021Meng Y, YU C, Chen M, Yu X, Sun M, Yan H, Zhao W and YU S (2021) Mutation landscape of TSC1/TSC2 in Chinese patients with Tuberous Sclerosis Complex. J Hum Genet 66:227-236.). Among the 10-25% of patients with no pathogenic variant identified, there are a few possibilities to explain the presence of clinical symptoms, including the presence of functional variants in non-coding sequences, mosaicism, and the presence of VUS with potential to be reclassified as pathogenic (Tyburczy et al., 2015Tyburczy ME, Dies KA, Glass J, Camposano S, Chekaluk Y, Thorner AR, Lin L, Krueger D, Franz DN, Thiele EA et al. (2015) Mosaic and intronic mutations in TSC1/TSC2 explain the majority of TSC patients with no mutation identified by conventional testing. PLoS Genet 11:e1005637.). The global median rate of VUS detection in mutation analysis studies of TSC patients is 5%. However, rates vary widely between countries: 8.0% in Brazil (Rosset et al., 2017Rosset C, Vairo F, Bandeira IC, Correia RL, de Goes FV, da Silva RTB, Bueno LSM, de Miranda Gomes MCS, Galvão HCR, Neri JICF et al. (2017) Molecular analysis of TSC1 and TSC2 genes and phenotypic correlations in Brazilian families with tuberous sclerosis. PLoS One 12:e0185713.), 6.5% in United States (Au et al., 2007Au KS, Williams AT, Roach ES, Batchelor L, Sparagana SP, Delgado MR, Wheless JW, Baumgartner JE, Roa BB, Wilson CM et al. (2007) Genotype/phenotype correlation in 325 individuals referred for a diagnosis of Tuberous Sclerosis Complex in the United States. Genet Med 9:88-100.), 9.8% in Canada (Alsowat et al., 2021Alsowat D, Whitney R, Hewson S, Jain P, Chan V, Kabir N, Amburgey K, Noone D, Lemaire M, Mccoy B et al. (2021) The phenotypic spectrum of Tuberous Sclerosis Complex: A Canadian cohort. Child Neurol Open 8:2329048X211012817.), 1.6% in Mexico (Reyna-Fabián et al., 2020Reyna-Fabián ME, Hernández-Martínez NL, Alcántara-Ortigoza MA, Ayala-Sumuano JT, Enríquez-Flores S, Velázquez-Aragón JA, Varela-Echavarría A, Todd-Quiñones CG and González-Del Angel A (2020) First comprehensive TSC1/TSC2 mutational analysis in Mexican patients with Tuberous Sclerosis Complex reveals numerous novel pathogenic variants. Sci Rep 10:6589.), 3.6% in Denmark (Rosengren et al., 2020Rosengren T, Nanhoe S, De Almeida LGD, Schönewolf-Greulich B, Larsen LJ, Hey CAB, Dunø M, Ek J, Risom L, Nellist M et al. (2020) Mutational analysis of TSC1 and TSC2 in Danish patients with Tuberous Sclerosis Complex. Sci Rep 10:9909.) and 0.7% in China (Meng et al., 2021Meng Y, YU C, Chen M, Yu X, Sun M, Yan H, Zhao W and YU S (2021) Mutation landscape of TSC1/TSC2 in Chinese patients with Tuberous Sclerosis Complex. J Hum Genet 66:227-236.). Furthermore, alterations in regions that are not commonly covered by TSC1 and TSC2 NGS or CNV assays may be associated with disease, such as regulatory regions, promoters, and deep intronic sequences. Thus far, deep intronic mutations have already been identified in TSC2 with the potential for a more thorough evaluation (Mayer et al., 2000Mayer K, Ballhausen W, Leistner W and Rott H (2000) Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice consensus sequences. Biochim Biophys Acta 1502:495-507. ; Nellist et al., 2015Nellist M, Brouwer RW, Kockx CE, Van Veghel-Plandsoen M, Withagen-Hermans C, Prins-Bakker L, Hoogeveen-Westerveld M, Mrsic A, Van Den Berg MM, Koopmans AE et al. (2015) Targeted next generation sequencing reveals previously unidentified TSC1 and TSC2 mutations. BMC Med Genet 16:10.; Tyburczy et al., 2015Tyburczy ME, Dies KA, Glass J, Camposano S, Chekaluk Y, Thorner AR, Lin L, Krueger D, Franz DN, Thiele EA et al. (2015) Mosaic and intronic mutations in TSC1/TSC2 explain the majority of TSC patients with no mutation identified by conventional testing. PLoS Genet 11:e1005637.).

The high processivity and decreasing cost of sequencing by NGS led to an increase in the discovery and accumulation of novel variants in a variety of genes, including TSC1 and TSC2. However, the ability of many laboratories to conduct functional, segregation, populational, and in silico studies to evaluate the pathogenicity of these variants is still limited. Therefore, a great number of variants remain as VUS, with scarce clinical and functional information. VUS detection poses a significant challenge for molecular diagnosis and clinical management as it is not clear when a variant is actionable, often leading to misinterpretations and bringing distress to the carriers, their families, and even healthcare providers (Hoffman-Andrews, 2017Hoffman-Andrews L (2017) The known unknown: The challenges of genetic variants of uncertain significance in clinical practice. J Law Biosci 4:648-657.). For example, in a study with patients with Hereditary Breast and Ovarian Cancer (HBOC) syndrome, 79% of patients who received a VUS report misinterpreted the result as a definitive predisposition to cancer (Vos et al., 2008Vos J, Otten W, Van Asperen C, Jansen A, Menko F and Tibben A (2008) The counsellees’ view of an unclassified variant in BRCA1/2: Recall, interpretation, and impact on life. Psychooncology 17:822-830.).

One possible way to address the VUS problem is the constant performance of variant reanalysis, searching for novel evidence that could lead to variant reclassification, such as the variant frequency in updated population databases, current patient and family history data, and novel in silico predictions and functional assays. Variant reclassification has been reported in numerous genes with several examples in the literature (Ha et al., 2020Ha HI, Ryu JS, Shim H, Kong SY and Lim MC (2020) Reclassification of BRCA1 and BRCA2 variants found in ovarian epithelial, fallopian tube, and primary peritoneal cancers. J Gynecol Oncol 31:e83.; Iancu et al., 2021Iancu IF, Avila-Fernandez A, Arteche A, Trujillo-Tiebas MJ, Riveiro-Alvarez R, Almoguera B, Martin-Merida I, Del Pozo-Valero M, Perea-Romero I, Corton M et al. (2021) Prioritizing variants of uncertain significance for reclassification using a rule-based algorithm in inherited retinal dystrophies. NPJ Genom Med 6:18.). Reanalysing VUS variants involves distinct challenges, such as: the overrepresentation of European patients in reference population databases often undermines evidence strength when evaluating variant frequency in sub-represented communities, such as Brazilian population (Gudmundsson et al., 2022Gudmundsson S, Singer-Berk M, Watts NA, Phu W, Goodrich JK, Solomonson M; Genome Aggregation Database Consortium; Rehm HL, Macarthur DG and O’donnell-Luria A (2022) Variant interpretation using population databases: Lessons from gnomAD. Hum Mutat 43:1012-1030.); most of the reported TSC1 and TSC2 variants are considered rare, and detailed clinical information is often lacking (Karczewski et al., 2020Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, Collins RL, Laricchia KM, Ganna A, Birnbaum DP et al. (2020) The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 581:434-443.); in silico prediction tools are a major source of discordance in variant classification, especially when using ACMG-AMP criteria (Amendola et al., 2016Amendola LM, Jarvik GP, Leo MC, Mclaughlin HM, Akkari Y, Amaral MD, Berg JS, Biswas S, Bowling KM, Conlin LK et al. (2016) Performance of ACMG-AMP variant-interpretation guidelines among nine laboratories in the clinical sequencing exploratory research consortium. Am J Hum Genet 99:247.) and the lack of robust functional studies. Well-established functional assays are an excellent solution in variant classification and clinical molecular diagnosis, even if they require a lot of effort and have method-specific limitations.

Most of the TSC1 and TSC2 variants detected by genetic testing are deposited in public databases. The database with the highest number of submitted variants is ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/). ClinVar is also the most used database for assessing the clinical interpretation of variants. However, the information related to the variants are controversial (carrier clinical information, variant details) and a high percentage of variants remain missing from public databases.

Aims

The present review explores the TSC1 and TSC2 variants in ClinVar, a main online variant database, with three major aims: 1) to review the distribution of the reported variants; 2) to describe the level of evidence used to classify variants as pathogenic, and 3) to assess the reclassification potential of current VUS. Additionally, we provide a review of the scientific literature to describe the distinct strategies used for functional assays to analyze TSC1 and TSC2 variants. We intended to summarize the TSC1 and TSC2 variant spectrum in ClinVar, as well as point out the provided and missing evidence in variant classification details, to identify bottlenecks in this process and help in its improvement.

Landscape of TSC1 and TSC2 variants in the ClinVar database

Up until January 04th, 2023, ClinVar had reported 3,690 variants in TSC1 and 8,500 variants in TSC2. ClinVar assorts variants into the following categories: clinical significance, types of conflicts, molecular consequence, variation type, variation size, variant length and review status. In each category, there are specific filters to analyze variants. We evaluated actionable and possibly actionable variants, conflicting variants, variation type and molecular consequence.

To explore the spectrum of these data, we first downloaded the complete list of variants of both genes in Excel format. The complete genomic range of both genes was analyzed. After that, we selected all filters (conflicting, B, LB, VUS, LP, P) in the clinical significance category in ClinVar for each gene, downloaded the correspondent Excel format and compared it with the respective complete variant list. The comparisons were performed using Excel. We found that 312 alterations in TSC1 and 877 in TSC2 do not have their clinical significance recorded in the database (not provided - NP), and were excluded in further analysis. For the variants with a clinical significance (TSC1=3,378 and TSC2=7,623), we used the ClinVar filters B and LB simultaneously to obtain the number of unique variants, since a few alterations are submitted both as B and LB (Table 1). We repeated this process with P and LP variants. We also checked for the number of VUS and variants with conflicting submissions. We used the same strategies to evaluate the molecular consequences and variation types of the alterations reported for each gene. The molecular consequences are described as frameshift, missense, nonsense, splice site, and untranslated region (UTR). We carefully analyzed the variants that have multiple molecular consequences reported and gathered them accordingly. The variation types are described as indel, deletion, duplication, insertion, and single nucleotide variants (SNV). Variants with multiple submissions were grouped accordingly.

Table 1 summarizes the data obtained from the aforementioned analyses. In both genes, VUS is the most reported category. This reinforces the need for functional and/or clinical segregation studies to understand the role of these variants (Hoogeveen-Westerveld et al., 2011Hoogeveen-Westerveld M, Wentink M, Van Den Heuvel D, Mozaffari M, Ekong R, Povey S, Den Dunnen JT, Metcalfe K, Vallee S, Krueger S et al. (2011) Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex. Hum Mutat 32:424-435.; Millot et al., 2012Millot GA, Carvalho MA, Caputo SM, Vreeswijk MP, Brown MA, Webb M, Rouleau E, Neuhausen SL, Hansen TV, Galli A et al. (2012) A guide for functional analysis of BRCA1 variants of uncertain significance. Hum Mutat 33:1526-1537.; Guidugli et al., 2014Guidugli L, Carreira A, Caputo SM, Ehlen A, Galli A, Monteiro AN, Neuhausen SL, Hansen TV, Couch FJ, Vreeswijk MP et al. (2014) Functional assays for analysis of variants of uncertain significance in BRCA2. Hum Mutat 35:151-164.; Nix et al., 2020Nix P, Mundt E, Manley S, Coffee B and Roa B (2020) Functional RNA studies are a useful tool in variant classification but must be used with caution: A case study of one BRCA2 variant. JCO Precis Oncol 4:730-735.). Regarding molecular consequence and variation type categories, missense and SNVs are the majority, respectively. Notably, we found a large number of variants with no information. For example, of the total variants in TSC1 and TSC2, 34.17% and 41.60%, respectively, do not have their molecular consequence reported. We analyzed the variants with missing clinical significance, molecular consequence and variation type in Excel. The terms used for excel filtering were not available in ClinVar as filters (e.g. synonymous, microsatellite and others). The available details about these variants are shown in ^{Table S1} Table S1 - All variants with not provided (NP) information in TSC1 and TSC2 for each category. . The majority of TSC1 variants without clinical significance are deletions (32.69%), duplications (15.71%) and nonsense (14.74%) variants. These variants in TSC2 are mostly deletions (27.14%), splice site (25.43%) and missense (16.65%) alterations. Moreover, many TSC2 variants with no variant type information are represented by deletions (29.82%), microsatellite (28.95%) and splice site variants (22.81%). In TSC1, microsatellites are 96.5% of these variants with no variant type information.

Next, we analyzed the molecular consequences and variation types according to their clinical significance. We first applied the clinical significance filter in ClinVar and divided variants in four groups: B/LB, P/LP, VUS and conflicting variants. Variants without a description of clinical significance were excluded from this analysis. A second simultaneous filter was applied, dividing the variants of each group by molecular consequence or by variation type. The results are summarized in Table S2. We analyzed the variants with clinical significance and no molecular consequence and/or variation type information using Excel. The terms used for excel filtering were not available in ClinVar as filters (e.g. synonymous, microsatellite and others). The results are described by group (B/LB, P/LP, VUS and conflicting variants) in ^{Tables S3} Table S3 - TSC1 and TSC2 variants with clinical significance and no molecular consequence submitted in ClinVar. and ^S4 Table S4 - TSC1 and TSC2 variants with clinical significance and no variation type submitter in ClinVar. .

Finally, to visually demonstrate the spectrum of variants with clinical significance relating their effects on protein, we constructed a sunburst chart using the total variants with reported clinical significance and molecular consequence as input (TSC1=2167 and TSC2=4282) (Figure 1). The figure shows that B/LB variants have specific profiles of molecular consequence. For example, missense and UTR alterations generally categorize B/LB variants. Most 3’ and 5’ UTR alterations were described as B/LB in TSC1 (10.30%, n=115/1116) and TSC2 (2.90%, n=80/2756). On the other hand, missense and UTR alterations represent the minority of P/LP variants in both genes. P/LP variants also have specific profiles of molecular consequences. Nonsense, frameshift and splice site alterations represent most of these variants. Nonsense variants have strong evidence for pathogenicity, since they induce the formation of a premature stop-codon, synthesizing a truncated protein. The damage level of a nonsense variant depends on the protein lacking region. Transcripts with nonsense variants can also be a target for the nonsense-mediated decay (NMD) pathway. This pathway degrades defective mRNA, displaying a pathogenic outcome due to haploinsufficiency (Kervestin and Jacobson, 2012Kervestin S and Jacobson A (2012) NMD: A multifaceted response to premature translational termination. Nat Rev Mol Cell Biol 13:700-712. ). Frameshift variants are also mostly pathogenic and were not detected in the B/LB spectrum, as expected. (^{Supplementary Table S2} Table S2 - TSC1 and TSC2 variants with clinical significance in ClinVar divided by molecular consequence and variation type. , Figure 1).

Figure 1 -
Sunburst charts representing all variants with clinical significance and molecular consequence reported in the ClinVar Database. A) TSC1 variants distribution B) TSC2 variants distribution. The external layer represents the variant type and the internal layer represents the clinical significance. Percentages in the internal layer are represented in relation to the total variants. Percentages in the external layer are represented in relation to clinical significance.

Missense variants represent 72.47% (n=1,111) and 80.2% (n=2,457) of total VUS on TSC1 and TSC2, respectively. Also, missense variants present a high number of conflicting interpretations. Likewise, UTR alterations were frequently described as VUS. In general, VUS have similar molecular consequences as B/LB alterations, like missenses or UTR variations. On the other hand, a few VUS are described as having similar molecular consequences as P/LP variants: nonsense (TSC1=2 and TSC2=11), frameshift (TSC1=11 and TSC2=15), and splice sites (TSC1=1 and TSC2=36) (Supplementary Table S2, Figure 1). Therefore, VUS with these consequences need more attention, as they have the potential for reclassification as pathogenic or likely pathogenic, representing 1.3% of VUS in TSC1 and 1.95% in TSC2.

Distribution of ClinVar variants in TSC1 and TSC2 genes

SNVs represent the great majority of variants in TSC1 (n=3,032/3,690) and TSC2 (n=7,090/8,500) (Table 1). Thus, we analyzed the distribution of SNVs along the two genes. This approach can help to identify putative hotspots for variation and clusters of variants depending on their clinical significance, such as pathogenic variants in regions that have a direct influence on protein function. Genomic localization and distribution of all SNVs (NM_000368.5 for TSC1; NM_000548.5 for TSC2) was performed using the ‘’Mutation Mapper’’ genomic tool provided by cBioPortal v5.2.0cBioPortal for Cancer Genomics (cBioPortal), cBioPortal for Cancer Genomics (cBioPortal), https://www.cbioportal.org/mutation_mapper (accessed 04 January 2023)
https://www.cbioportal.org/mutation_mapp... . The variant distribution is shown in Figures 2 and 3. Apparently, B/LB variants and VUS are even distributed in both genes. In TSC2, P/LP variants also seem to be evenly distributed. On the other hand, there are modest clusters of P/LP variants in the TSC1 gene portion that codes for the N-terminal region and coiled-coil domain of hamartin. In additional molecular studies, most of the pathogenic variants are detected in the TSC1 gene portion that codes for the N-terminal hamartin region, which includes the putative TSC1 TMD domain (Mozaffari et al., 2009Mozaffari M, Hoogeveen-Westerveld M, Kwiatkowski D, Sampson J, Ekong R, Povey S, Den Dunnen JT, Van Den Ouweland A, Halley D and Nellist M (2009) Identification of a region required for TSC1 stability by functional analysis of TSC1 missense mutations found in individuals with Tuberous Sclerosis Complex. BMC Med Genet 10:88.). In other proteins, TMDs have already been associated with the transport and sorting of transmembrane proteins (Cosson et al., 2013Cosson P, Perrin J and Bonifacino JS (2013) Anchors aweigh: Protein localization and transport mediated by transmembrane domains. Trends Cell Biol 23:511-517.). Menon et al. (2014Menon S, Dibble CC, Talbott G, Hoxhaj G, Valvezan AJ, Takahashi H, Cantley LC and Manning BD (2014) Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 156:771-785.) demonstrated that the TSC complex binds the lysosomal membrane in a dependent manner of the interaction with Rheb protein. However, further studies are essential to understand the functionality of this putative domain in hamartin.

Figure 2 -
Gene distribution of TSC1 single nucleotide variants (SNV) for benign or likely benign variants (B/LB), variants of uncertain significance (VUS), and pathogenic or likely pathogenic variants (P/LP). Intronic variants are not represented. Darker green dots represent missense variants. Yellow dots represent splice site variants. Black dots represent truncated variants (nonsense or frameshift deletions). Pink dots represent other variant types (synonymous, UTR and intronic variants). The TSC1 gene region that codes for TMD (predicted) and coiled coil domains are represented. The post translational modifications (PTM) are shown below the graphics (lighter green represents phosphorylation sites).

Figure 3 -
Gene distribution of TSC2 single nucleotide variants (SNV) for benign or likely benign variants (B/LB), variants of uncertain significance (VUS), and pathogenic or likely pathogenic variants (P/LP). Intronic variants are not represented. Darker green dots represent missense variants. Yellow dots represent splice site variants. Black dots represent truncated variants (nonsense or frameshift deletions). Pink dots represent other variant types (synonymous, UTR and intronic variants). The TSC2 gene regions that code for the Hamartin interaction region and GAP domain are demonstrated. The post translational modifications (PTM) are shown below the graphics (lighter green represents phosphorylation sites).

In TSC1, exons 9-15 code the main phosphorylation sites of hamartin (Figure 2). Therefore, variants detected in these exons may alter phosphorylation in the corresponding protein and potentially alter protein function. In addition, the hamartin C-terminal region contains the coiled-coil domain involved in hamartin-tuberin binding (Santiago Lima et al., 2014Santiago Lima AJ, Hoogeveen-Westerveld M, Nakashima A, Maat-Kievit A, Van Den Ouweland A, Halley D, Kikkawa U and Nellist M (2014) Identification of regions critical for the integrity of the TSC1-TSC2-TBC1D7 complex. PLoS One 9:e93940.). Many pathogenic SNVs are described in this region, clustered in exons 18-21. A pathogenic variant in this location could prevent hamartin-tuberin interaction, and consequently affect the GTPase activity of tuberin (Huang and Manning, 2008Huang J and Manning BD (2008) The TSC1-TSC2 complex: A molecular switchboard controlling cell growth. Biochem J 412:179-190.). Therefore, VUS in the coiled-coil domain requires greater attention, especially the frameshift, nonsense, and splice site variants.

Pathogenic variants are widespread in the most important functional regions in tuberin: the GAP domain and the sequence that interacts with hamartin. VUS detected in these regions need careful attention, as they are highly conserved domains (Maheshwar et al., 1997Maheshwar MM, Cheadle JP, Jones AC, Myring J, Fryer AE, Harris PC and Sampson JR (1997) The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis. Hum Mol Genet 6:1991-1996. ). Moreover, pathogenic variants that affect tuberin phosphorylation sites, such as in hamartin, occur mostly in the region between exons 33-34 (Figure 3).

Figure 4 -
An overview of TSC1 and TSC2 VUS, conflicting and pathogenic variants submitted in ClinVar. A,B) Distribution of VUS with molecular consequence similar to pathogenic variants: frameshift, nonsense, splice site in TSC1 and TSC2, respectively. C) TSC1 and TSC2 B/LB vs VUS conflicting variants were separated in four groups: VUS>B/LB, B/LB>VUS, B/LB > VUS > B/LB, VUS > B/LB > VUS; D) TSC2 P/LP vs VUS conflicting variants were separated in four groups: VUS>P/LP, P/LP>VUS, P/LP > VUS > P/LP, VUS > P/LP > VUS; E) Representation of the time (in years) between a VUS submission and its reclassification as B/LB. F) Review status of TSC1 and TSC2 pathogenic variants; G) Number of submissions per TSC1 and TSC2 pathogenic variants in ClinVar; H) Year of the most recent submission of TSC1 and TSC2 pathogenic variants; I) Number of citations of TSC1 and TSC2 pathogenic variants.

Variants of uncertain significance (VUS) in ClinVar: Spectrum and potential for reclassification

As previously mentioned, when the available evidence does not support a variant classification, the variant pathogenicity cannot be ascertained, and it is categorized as VUS. VUS are not informative in the prediction of disease occurrence and/or risk and are often referred to as not actionable, demanding special attention and periodic reanalysis (Chern et al., 2019Chern JY, Lee SS, Frey MK, Lee J and Blank SV (2019) The influence of BRCA variants of unknown significance on cancer risk management decision-making. J Gynecol Oncol 30:e60.). Therefore, we evaluated their review status in the ClinVar database. ClinVar reports the level of review supporting the assertion of clinical significance for the variation as review status. Stars provide a graphical representation of the variant aggregate review status. In this classification, one star is given for variants with a single submitter or conflicting interpretations (with multiple submitters); two stars when there are multiple submitters with no conflicts in interpretation; three for variants reviewed by an expert panel; and four for variants classified by specific guidelines. Variants with no stars are those not reviewed and/or with no information registered. Review statuses of TSC1 and TSC2 VUS and variants with conflicting interpretations (B/LB vs VUS or P/LP vs VUS) are summarized in Table 2. None of the variants described in ClinVar have three or four stars and most of the VUS have a single star. This is an important finding that highlights the lack of VUS review by ClinVar submitters and/or the lack of information registered in ClinVar.

Since VUS represent a challenge in molecular diagnostics and they are poorly reviewed in Clinvar, we analyzed the distribution of frameshift, nonsense, and splice site VUS in more detail due to their potential to be reclassified as pathogenic. The distribution of these types of VUS is shown in Figure 4A,B. In TSC1, a total of 95% of the analyzed VUS (19/20) are located in exons that code for the coiled-coil domain. Of these VUS, 15 are located between exons 22-23 (Figure 4A). In our previous analysis of pathogenic variant distribution, (Figure 2C), exon 22 showed few alterations, and exon 23 did not show pathogenic variants. Hence, these exons have a high VUS description and few pathogenic variants. Perhaps, this region lacks functional analyses and deserves to be explored for VUS reclassification.

Furthermore, we investigated VUS detected in other exons that code for the hamartin C-terminal domain, i.e. exons 15 to 21. This region has a high number of pathogenic variants detected. A few VUS described in this region are predicted to affect splice sites, such as c.2209-2A>G. On the other hand, the variant c.2624_2625+3dup does not have a prediction of effect on splicing, but it is positioned at a conserved region. The variant c.2391+1G>A detected in intron 17 has no available information in ClinVar, but Varsome (version 11.8.0) classified it as likely pathogenic (Kopanos et al., 2019Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Albarca Aguilera M, Meyer R and Massouras A (2019) VarSome: The human genomic variant search engine. Bioinformatics 35:1978-1980.; VarSomeVarSome The Human Genomics Community (VarSome),VarSome The Human Genomics Community (VarSome),https://varsome.com/variant/hg38/chr9%3A132902604%3AC%3AT (accessed 26 January 2023)
https://varsome.com/variant/hg38/chr9%3A... ).

Regarding TSC2, a total of 15 VUS are located in exon 26 (E26) or exon 32 (E32), and six in intron 26 or intron 32 (Figure 4B). Six of these VUS predict nonsense alterations (E26=3; E32=3), eight frameshift (E26=3; E32=5), and three are splice site alterations (E32=3). Exon 34 is the largest TSC2 exon and has many pathogenic variants detected (Figure 3). The variants c.4081C>T (nonsense) and c.4008_4010del (in frame deletion) are detected in this exon. The variant c.4008_4010del has three VUS submissions in ClinVar, with an inconclusive in silico study (Choi et al., 2012Choi Y, Sims GE, Murphy S, Miller JR and Chan AP (2012) Predicting the functional effect of amino acid substitutions and indels. PLoS One 7:46688.). Additionally, two splice site VUS have been identified in the GAP domain (exon 34-38) and five are located in introns 34-38. Fifty variants present only one submission and 11 have two submissions, highlighting again the lack of information about certain submitted variants.

Variants with conflict interpretation in TSC1 and TSC2 in ClinVar

ACMG and Sherloc guidelines present standardized criteria for variant evaluation (Richards et al., 2015Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E et al. (2015) Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17:405-424.; Nykamp et al., 2017Nykamp K, Anderson M, Powers M, Garcia J, Herrera B, Ho YY, Kobayashi Y, Patil N, Thusberg J, Westbrook M et al. (2017) Sherloc: A comprehensive refinement of the ACMG-AMP variant classification criteria. Genet Med 19:1105-1117.). Even though they are standardized, criteria interpretation may be subjective, which leads to variable classifications of the same variant by distinct groups. A few groups/laboratories that submit variants in ClinVar adopt standard or modified versions of ACMG-AMP and Sherloc criteria. Most of the submitters develop their own classification criteria, some of them not publicly available (Niehaus et al., 2019Niehaus A, Azzariti DR, Harrison SM, Distefano MT, Hemphill SE, Senol-Cosar O and Rehm HL (2019) A survey assessing adoption of the ACMG-AMP guidelines for interpreting sequence variants and identification of areas for continued improvement. Genet Med 21:1699-1701.). The use of numerous distinct guidelines, unavailable data and methodology can undermine variant classifications and result in multiple conflicting submissions in ClinVar (Yang et al., 2017Yang S, Lincoln SE, Kobayashi Y, Nykamp K, Nussbaum RL and Topper S (2017) Sources of discordance among germ-line variant classifications in ClinVar. Genet Med 19:1118-1126.).

To analyze variants with conflicting interpretations, we filtered the ClinVar variants with the conflicts P/LP versus VUS and B/LB versus VUS (TSC1=230, TSC2=664). A total of 227 and 627 variants presents the conflict B/LB versus VUS in TSC1 and TSC2. Three and 29 are conflicting P/LP versus VUS in TSC1 and TSC2 variants. Eight variants present multiple conflicts (B/LB vs P/LP vs VUS) in TSC2. Single nucleotide variation represents 222 and 646 in TSC1 and TSC2, respectively.

Further, we separated the variants with conflicting interpretations into four groups: Group 1, variants with a first submission as VUS, which were later classified as B/LB; Group 2, variants with a first submission as B/LB, which were later classified as VUS; Group 3, variants with a first submission as B/LB, second submission as VUS and a third submission as B/LB again; and Group 4, variants with a first submission as VUS, second submission as B/LB and a third submission as VUS again. We excluded only one VUS once its year of submission was not demonstrated. Variants in group 1 were possibly reclassified as B/LB in a second evaluation, depending on the employed criteria. Groups 3 and 4 represent extensive conflicting interpretations. All variants in each group are shown in Figure 4C.

Additionally, we used the same grouping strategies to evaluate P/LP versus VUS conflicts in TSC2 (n=28). In TSC1, only three variants present P/LP variants versus VUS conflicts, thus were not included in further analysis. Group 1 (n=7/664) and group 2 (n=15/664) represent the majority of TSC2 conflicting variants (Figure 4D). Curiously, a few variants were submitted as P/LP in the first submission even without presenting the sufficient information for this classification. These variants were later submitted as VUS by other laboratories. This issue raises concern about the variant classification process by different users. The criteria for variant classification are subjective, and errors or misinterpretations might occur. Moreover, a few groups use their own criteria, many times not publicly available. Thus, ClinVar consultants should be cautious about variant classification submissions. On other hand, a few B/LB variants were submitted firstly as VUS over a lack of information and posteriorly classified as B/LB, with the rise of functional studies or other additional information to classify the variant accordingly.

For variants with the conflict B/LB versus VUS, group 1 variants were filtered to examine how many years it took for VUS reclassification as B/LB. We examined the year of VUS submissions that were later submitted as B/LB (Figure 4E). It seems that TSC2 is more often reviewed since most of its VUS (n=94/271) were reclassified as B/LB in less than a year. In TSC1, a significant number of variants were also reclassified in less than one year (n=29/100), but most variants took more than four years for a reclassification (n=31/100). Finally, for variants with the conflict P/LP versus VUS, seven TSC2 conflicting variants were later submitted as P/LP. Five of these variants were given a second classification in less than one year and two in 2-4 years.

Pathogenic variants in TSC1 and TSC2 in ClinVar

We subsequently collected information from TSC1 and TSC2 variants with at least one submission as pathogenic in Clinvar. Information gathered for the selected variants were review status, number of submissions, last submission date, functional evidence, and number of citations in literature. A total of 434 and 883 pathogenic variants were analyzed for TSC1 and TSC2, respectively. Review status of the variants was mainly one star (TSC1=311; TSC2=654), followed by two stars (TSC1=88; TSC2=170), and an absence of variants with three or four stars (Figure 4F). Most of the variants had one submission, accounting for 64.8% of variants in TSC1 and 61.4% in TSC2 (Figure 4G). A high number of variants had their most recent submission in the year of 2022 (TSC1=290; TSC2=563). Except for four TSC2 variants classified in 2013, no variants had their last submission date before 2015 (Figure 4H). There were a significant number of variants with no literature citations identified (TSC1=166; TSC2=406) (Figure 4I). Only seven variants had functional evidence available in ClinVar (TSC1 = 2 and TSC2 = 5).

Strategies for functional assessment of TSC1 and TSC2 variants

Functional studies are important to understand the role of variants in protein function and consequently in disease. In this sense, they are crucial for VUS reclassification and/or to reinforce the classification of B/LB, P, and LP variants. To analyze the strategies used to functionally evaluate TSC variants, we searched the scientific literature in PubMed using the words “functional assessment”, “TSC1” OR “TSC2”, and found 431 manuscripts. Of these, only twelve studies had performed a functional assessment.

The majority (9/12) of the functional studies found in our search used similar strategies applied by the Nellist group in 2001Nellist M, Verhaaf B, Goedbloed MA, Reuser AJ, Van Den Ouweland AM and Halley DJ (2001) TSC2 missense mutations inhibit tuberin phosphorylation and prevent formation of the tuberin-hamartin complex. Hum Mol Genet 10:2889-2898. and 2005 (Nellist et al., 2001Nellist M, Verhaaf B, Goedbloed MA, Reuser AJ, Van Den Ouweland AM and Halley DJ (2001) TSC2 missense mutations inhibit tuberin phosphorylation and prevent formation of the tuberin-hamartin complex. Hum Mol Genet 10:2889-2898., 2005Nellist M, Sancak O, Goedbloed MA, Rohe C, Van Netten D, Mayer K, Tucker-Williams A, Van Den Ouweland AM and Halley DJ (2005) Distinct effects of single amino-acid changes to tuberin on the function of the tuberin-hamartin complex. Eur J Hum Genet 13:59-68.). These strategies are the transfection of TSC1 and/or TSC2 defective transcripts via lipofectamine or plasmid in HEK-293 cells, followed by immunoprecipitation by immunoblotting. To confirm whether the variant in question decreases the TSC1-TSC2 interaction and decreases the stability of the TSC2 protein, studies evaluated the TSC1 signal and the TSC2 signal in immunoblotting, respectively. Furthermore, as the TSC2-TSC1 complex has the function of inhibiting mTOR, the mTORC1 activity was analyzed by the ratio of phosphorylation of downstream proteins by immunoblot, such as T389-phosphorylation at p70 S6 kinase (S6K) (Hodges et al., 2001Hodges AK, Li S, Maynard J, Parry L, Braverman R, Cheadle JP, Declue JE and Sampson JR (2001) Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin. Hum Mol Genet 10:2899-2905.; Jansen et al., 2008Jansen FE, Braams O, Vincken KL, Algra A, Anbeek P, Jennekens-Schinkel A, Halley D, Zonnenberg BA, Van Den Ouweland A, Van Huffelen AC et al. (2008) Overlapping neurologic and cognitive phenotypes in patients with TSC1 or TSC2 mutations. Neurology 70:908-915.; Nellist et al., 2008Nellist M, Sancak O, Goedbloed M, Adriaans A, Wessels M, Maat-Kievit A, Baars M, Dommering C, Van Den Ouweland A and Halley D (2008) Functional characterisation of the TSC1-TSC2 complex to assess multiple TSC2 variants identified in single families affected by Tuberous Sclerosis Complex. BMC Med Genet 9:10.; Dunlop et al., 2011Dunlop EA, Dodd KM, Land SC, Davies PA, Martins N, Stuart H, Mckee S, Kingswood C, Saggar A, Corderio I et al. (2011) Determining the pathogenicity of patient-derived TSC2 mutations by functional characterization and clinical evidence. Eur J Hum Genet 19:789-795.; Hoogeveen-Westerveld et al., 2011Hoogeveen-Westerveld M, Wentink M, Van Den Heuvel D, Mozaffari M, Ekong R, Povey S, Den Dunnen JT, Metcalfe K, Vallee S, Krueger S et al. (2011) Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex. Hum Mutat 32:424-435., 2012Hoogeveen-Westerveld M, Ekong R, Povey S, Karbassi I, Batish SD, Den Dunnen JT, Van Eeghen A, Thiele E, Mayer K, Dies K et al. (2012) Functional assessment of TSC1 missense variants identified in individuals with Tuberous Sclerosis Complex. Hum Mutat 33:476-479., 2013Hoogeveen-Westerveld M, Ekong R, Povey S, Mayer K, Lannoy N, Elmslie F, Bebin M, Dies K, Thompson C, Sparagana SP et al. (2013) Functional assessment of TSC2 variants identified in individuals with Tuberous Sclerosis Complex. Hum Mutat 34:167-175.; Overwater et al., 2016Overwater IE, Swenker R, Van Der Ende EL, Hanemaayer KB, Hoogeveen-Westerveld M, Van Eeghen AM, Lequin MH, Van Den Ouweland AM, Moll HA, Nellist M et al. (2016) Genotype and brain pathology phenotype in children with Tuberous Sclerosis Complex. Eur J Hum Genet 24:1688-1695.; Živčić-Ćosić et al., 2017Živčić-Ćosić S, Mayer K, Đorđević G, Nellist M, Hoogeveen-Westerveld M, Miletić D, Rački S, Klein HG and Trobonjača Z (2017) Severe bleeding complications and multiple kidney transplants in a patient with Tuberous Sclerosis Complex caused by a novel TSC2 missense variant. Croat Med J 58:416-423.).

In addition, intronic and exonic variants may cause mRNA processing errors. For this, some studies used the cDNA analysis strategy to verify the presence of all exons or the inclusion of introns in the transcripts (5/12). This cDNA analysis consists of a RT-PCR, a method to analyze the difference of amplicon sizes of TSC1 and TSC2 specific primers by electrophoresis. Therefore, differences in amplicon size can show the exon absence or intron inclusion, indicating an error in mRNA processing (Kobayashi et al., 1995KobayashI T, Nishizawa M, Hirayama Y, Kobayashi E and Hino O (1995) cDNA structure, alternative splicing and exon-intron organization of the predisposing tuberous sclerosis (Tsc2) gene of the Eker rat model. Nucleic Acids Res 23:2608-2613.; Jeganathan et al., 2002Jeganathan D, Fox MF, Young JM, Yates JR, Osborne JP, Povey S (2002) Nonsense-mediated RNA decay in the TSC1 gene suggests a useful tool pre- and post-positional cloning. Hum Genet 111:555-565. ; Jansen et al., 2008Jansen FE, Braams O, Vincken KL, Algra A, Anbeek P, Jennekens-Schinkel A, Halley D, Zonnenberg BA, Van Den Ouweland A, Van Huffelen AC et al. (2008) Overlapping neurologic and cognitive phenotypes in patients with TSC1 or TSC2 mutations. Neurology 70:908-915.; Tyburczy et al., 2015Tyburczy ME, Dies KA, Glass J, Camposano S, Chekaluk Y, Thorner AR, Lin L, Krueger D, Franz DN, Thiele EA et al. (2015) Mosaic and intronic mutations in TSC1/TSC2 explain the majority of TSC patients with no mutation identified by conventional testing. PLoS Genet 11:e1005637.; Overwater et al., 2016Overwater IE, Swenker R, Van Der Ende EL, Hanemaayer KB, Hoogeveen-Westerveld M, Van Eeghen AM, Lequin MH, Van Den Ouweland AM, Moll HA, Nellist M et al. (2016) Genotype and brain pathology phenotype in children with Tuberous Sclerosis Complex. Eur J Hum Genet 24:1688-1695.). Additionally, three studies (3/12) accomplished the same technique for cDNA analysis but did not perform additional functional analysis (Mayer et al., 2000Mayer K, Ballhausen W, Leistner W and Rott H (2000) Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice consensus sequences. Biochim Biophys Acta 1502:495-507. ; Tyburczy et al., 2015Tyburczy ME, Dies KA, Glass J, Camposano S, Chekaluk Y, Thorner AR, Lin L, Krueger D, Franz DN, Thiele EA et al. (2015) Mosaic and intronic mutations in TSC1/TSC2 explain the majority of TSC patients with no mutation identified by conventional testing. PLoS Genet 11:e1005637.; Qiu et al., 2020Qiu C, Li C, Tong X, Dai L, Liu W, Xie Y, Zhang Q, Yang G and Li T (2020) A novel TSC1 frameshift mutation c.1550_1551del causes Tuberous Sclerosis Complex by aberrant splicing and nonsense-mediated mRNA degradation (NMD) simultaneously in a Chinese family. Mol Genet Genomic Med 8:e1410.).

The functional assessments using cDNA could reveal alterations outside the scope of NGS and MLPA. For example, we can obtain information about RNA processing attributed to variants in cDNA analysis. Furthermore, by analyzing proteins, we can verify the presence or absence of parts of the protein, the stability and functionality of the TSC1/TSC2 complex, and the activation of downstream proteins in the mTOR pathway. Functional assays are arduous and require a long time of laboratory activity to standardize the methodology and analysis. However, the existing TSC1 and TSC2 functional assays often don’t meet the requirements to be considered well-established and strong evidence (Gelman et al., 2019Gelman H, Dines JN, Berg J, Berger AH, Brnich S, Hisama FM, James RG, Rubin AF, Shendure J, Shirts B et al. (2019) Recommendations for the collection and use of multiplexed functional data for clinical variant interpretation. Genome Med 11:85). Nonetheless, if a well-established in vitro or in vivo functional assay shows a variant with deleterious effect that fits in PS3 ACMG criteria, it could be supported as pathogenic evidence (Richards et al., 2015Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E et al. (2015) Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17:405-424.). Studies should be benchmarked with well-known pathogenic and benign variants that can fully demonstrate the dynamic range of the assay and the whole spectrum of pathogenicity in a given gene. Additionally, functional assays based on cDNA constructs lack regulatory regions and might not fully represent the endogenous situation (Brnich et al., 2019Brnich SE, Abou Tayoun AN, Couch FJ, Cutting GR, Greenblatt MS, Heinen CD, Kanavy DM, Luo X, Mcnulty SM, Starita LM et al. (2019) Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework. Genome Med 12:3.). In spite of functional assessment limitations, it is very important to perform and report these assays to better understand the role of these variants.

Conclusions

In this review, we explored the TSC1 and TSC2 ClinVar database and evaluated variant distribution, the level of evidence used to classify variants as pathogenic, and the potential of reclassification of current VUS. Up until January 4th, 2023, ClinVar had reported 3,690 variants in TSC1 and 8,500 variants in TSC2. We did not observe hotspots for variation in both genes, and missense and single nucleotide variants were the majority. In addition, VUS is the most frequently reported category. This reinforces the need for functional and/or clinical segregation studies to understand the role of these variants. Our analyses revealed that in general VUS have similar molecular consequences as B/LB alterations. However, VUS described as having similar molecular consequences as P/LP variants need more attention, as they have the potential for reclassification as pathogenic or likely pathogenic. We also observed that most of the VUS lack information in their ClinVar submissions and have poor review status. This highlights the importance of complete submissions in online databases, including criteria used for variant classification, clinical, and segregation data. In addition to these issues, the use of numerous distinct guidelines, unavailable data and methodology can undermine variant classifications and result in multiple conflicting submissions in ClinVar. Indeed, we found a high number of variants with conflict interpretations: 230 in TSC1 and 664 in TSC2. Of these variants, 43.4% in TSC1 were VUS in the first submission and were later classified as B/LB (20.4% in less than two years). For TSC2, 40.8% were VUS in the first submission and were later classified as B/LB (19.7% in less than two years). For VUS that were later classified as P/LP, 2/230 (0.8%) were found in TSC1 (0.4% in less than two years) and 7/664 (1.05%) in TSC2 (0.75% in less than two years). These numbers reinforce the need for further studies to evaluate VUS with the potential for pathogenic reclassification. Functional studies are crucial for VUS reclassification and/or to reinforce the classification of B/LB, P, and LP variants. We observed a lack of these types of studies in TSC1/2 genes. Considering all pathogenic variants, only six of the 1,211 variants submitted for both genes have functional assays to confirm pathogenicity. We found only 12 functional studies for TSC1 and TSC2 variants in the scientific literature. This encourages the performance of further functional studies evaluating the role of TSC1 and TSC2 variants in protein function. In summary, the critical analysis of the ClinVar database and functional variants in literature could be reapplied for other genes related to other diseases, helping in early diagnosis, the prognosis of affected patients, and genetic counseling for affected families.

Acknowledgements

We gratefully thank Fundo de Incentivo à Pesquisa e Eventos (FIPE) of Hospital de Clínicas de Porto Alegre, Fundação de Apoio à Pesquisa do Rio Grande do Sul (FAPERGS) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for their financial support.

References

Supplementary material

The following online material is available for this article:

Table S1 - All variants with not provided (NP) information in TSC1 and TSC2 for each category.

Table S2 - TSC1 and TSC2 variants with clinical significance in ClinVar divided by molecular consequence and variation type.

Table S3 - TSC1 and TSC2 variants with clinical significance and no molecular consequence submitted in ClinVar.

Table S4 - TSC1 and TSC2 variants with clinical significance and no variation type submitter in ClinVar.

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