RESEARCH ARTICLE Molecular-genetic diagnostics of Tuberous sclerosis complex (TSC) in Bulgaria: six novel mutations in the TSC1 and TSC2 genes Running title: Six novel TSC mutations in Bulgaria Glushkova M 1,3, Bojinova V 2, Koleva M 2, Dimova P 4, Bojidarova M 5, Litvinenko I 5, Todorov T 3, Iluca E 6, Calusaru C 6, Neagu E 8, Craiu D 6, 7, Mitev V 1,*, Todorova A 1,3,* 1. Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2 Zdrave str., 1431Sofia, Bulgaria 2. Clinic of Child Neurology, University Hospital Sv. Naum, Medical University Sofia, 1 Louben Roussev Str., 1113 Sofia, Bulgaria 3. Genetic Medico-Diagnostic Laboratory Genica, 84 Ami Bue Str., 1612 Sofia, Bulgaria 4. Epilepsy Center, Department of Neurosurgery, University Hospital St. Ivan Rilski, 15 "Akad. Ivan Geshov " Blvd., Sofia 1431, Bulgaria 5. Department of Neurology, University Pediatric Hospital, Medical University Sofia, 11 Acad. Ivan Evstatiev Geshov Str., 1612 Sofia, Bulgaria 6. Pediatric Neurology Clinic, Al Obregia Hospital, 10 Sos. Berceni, 041914 Bucharest, Romania 7. Department of Clinical Neurosciences, "Carol Davila" University of Medicine and Pharmacy, 37Strada Dionisie Lupu, 030167 Bucharest, Romania 8. Human Genetics Laboratory, National Institute of Forensic Medicine "Mina Minovici", 9 Vitan Barzesti, 42122 Bucharest, Romania * - equally contributing authors Veneta Bojinova vsbojinova@abv.bg
Maya Koleva maya_m_koleva@abv.bg Petia Dimova psdimova@gmail.com Maria Bojidarova drvanmar@yahoo.com Ivan Litvinenko ilitvinenko@excite.com Tihomir Todorov tisho.todorov@abv.bg Emanuela Iluca ema.adelina.iluca@gmail.com Cristina Calusaru cristina_calusaru@yahoo.com Elena Neagu elenaneagu1@yahoo.com Dana Craiu dcraiu@yahoo.com Vanyo Mitev mitev@medfac.acad.bg Albena Todorova todorova_albena@abv.bg
Corresponding author: Maria Glushkova Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2 Zdrave str., Sofia 1431, Bulgaria glushkova.mariq@gmail.com Key words: TSC, TSC2 gene, TSC1 gene, tubers ABSTRACT Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder characterized by development of hamartomas localized in various tissues which can occur in the skin, brain, kidneys and other organs. TSC is caused by mutations in the TSC1 and TSC2 genes. Here we report on the results from the first molecular testing of 16 Bulgarian patients and one Romanian patient in which we found six novel mutations: four of them in the TSC2 gene, which are one nonsense, two frameshift and one large deletion of sixteen exons and two in the TSC1 gene, one nonsense and one frameshift, respectively. In addition, we detected 10 previously reported mutations; some of them described only ones in the literature. Our data is similar to previous studies with exception of the larger number of TSC1 mutations than the reported in the literature data. In total 40% (4/10) from the mutation in the TSC2 gene are located in the GAP domain, while 50% (3/6) from the mutation in the TSC1 gene are clustered in exon 15. All the cases represent the typical clinical symptoms of TSC and met the clinical criteria for TSC diagnosis. In 35% of our cases the family history was positive.
Our results add novel findings in the genetic heterogeneity and pathogenesis of TSC. The phenomenon of genetic heterogeneity might further play a role in clinical variability among families with the same diagnosis and within a single family which makes the genetic testing and genetic counseling in TSC affected families very important task. INTRODUCTION Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder caused by inactivating TSC1 or TSC2 gene variants (Nellist et al., 1993; van Slegtenhorst et al., 1997). The disease is characterized by development of hamartomas localized in various tissues which can occur in the skin, brain, kidneys and other organs. The tumours are usually benign, but their specific manifestation as well as localization in the body can lead to the development of severe complications (Crino et al., 2006; Curaloto et al., 2008). The disease frequency is 1 in 6000 to 1 in 10,000 live births (O Callaghan et al., 1998; Sampson et al., 1989). The TSC1 (MIM# 605284) and TSC2 (MIM#191092) genes are located on chromosome 9q34.13 and 16p13.3. Both genes are tumour suppressor genes and encode the proteins hamartin and tuberin, respectively. The mutations in the TSC1 gene are more often associated with less severe disease than those in the TSC2 gene (Dabora et al., 2001; Jones et al., 1997; Langkau et al., 2002; Mayer et al., 2004). About 75-90% of the patients with definite diagnosis of TSC have identifiable mutations (Northrup and Krueger, 2013; van Slegtenhorst et al., 1997; European Chromosome 16 Tuberous Sclerosis, Consortium, 1993). Larg genomic rearrangements are more common in the TSC2 gene (about 6%), while in the TSC1 gene such mutations are very rare (Kozlowski et al., 2007). The TSC1 mutations commonly involve deletions or nonsense mutations (37 and 36%, respectively), causing premature protein truncation, while missense mutations are rare (3.1%) (Kwiatkowski, 2010). Deletions, nonsense, and missense mutations all occur at similar frequencies in the TSC2 gene (22-27%), while splice-site changes and insertions are less common (16
and 9%, respectively) (Kwiatkowski, 2010). Approximately 20% of the patients have positive family history of TSC and the remaining 80% represent de novo mutations in either TSC1 or TSC2 gene (Astrinidis and Henske, 2005). Here we report on the results from the first molecular testing of 16 Bulgarian patients and one Romanian patient with clinically suspected TSC. 59% of them were positive for mutations either in the TSC2 or TSC1 genes (35%). We found six novel mutations: four of them in the TSC2 gene, which are one nonsense, two frameshift and one large deletion of sixteen exons and two in the TSC1 gene, one nonsense and one frameshift, respectively. In addition we detected 10 previously reported mutations; some of them described only ones in the literature. MATERIALS AND METHODS Sixteen unrelated Bulgarian patients and one Romanian patient were clinically suspected of having TSC and were referred for genetic testing for mutations in the TSC1 or TSC2 genes. All patients or their parents signed an informed consent for genetic examinations. The patients genomic DNA was extracted from peripheral blood by standard salting-out procedure. All patients included in the study, meet the clinical diagnostics criteria, which involve two major symptoms or two minor and one major (see Table 1) (Northrup and Krueger, 2013). Both genes were subjected to direct Sanger sequencing (BigDye terminator v.3.1, Applied Biosystems, Foster City CA), followed by MLPA in the Sanger sequencing-negative patients for both genes. All the exons and exon/intron boundaries were covered by primers designed on the reference sequence NM_000368.4 for the TSC1 genе and NM_000548.4 for the TSC2 gene (the primers sequence is available upon request). The MLPA analysis for large deletions was performed with standard MLPA kits for both genes (SALSA MLPA P124 TSC1 probemix and SALSA MLPA P046 TSC2 probemix,
respectively) (http://www.mlpa.com). The detected TSC1 and TSC2 mutations were compared with the known aberrations listed in publically available databases, such as: ENSEMBL (http://www.ensembl.org), the TSC1, TSC2 Gene Variant Database (http://databases.lovd.nl/shared/genes/tsc1; http://databases.lovd.nl/shared/genes/tsc2) and National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/clinvar/). RESULTS In fifteen Bulgarian cases and one Romanian case the clinical diagnosis of TSC was confirmed at molecular level (94%). The remaining 6% (patient #14, table 1) are TSC1 and TSC2 negative and at present they are genetically not clarified. The clinical symptoms and the family history are listed in table 1. The detected disease-causing genetic variants in the TSC2 gene are: three missense, two nonsense, three frameshift mutations (an indel), one splice-site and a large deletion of exons 1 to 16 (table 1, figure 1b). In the TSC1 gene we detected four nonsense mutations and two frameshift mutations (table 1, figure 1a). The reported here six novel mutations are one nonsense, one frameshift, an indel and the deletion of sixteen exons in the TSC2 gene, and one nonsense and one frameshift mutataions in the TSC1 gene (table 1). The sequencing chromatograms of the four novel mutations in the TSC2 gene are presented in figure 2 a, b, c, d and both novel mutations in the TSC1 gene are presented in figure 3a and b. Some of the remaining mutations are described only once in the literature. The mutations found in the present study are illustrated on figure 1a and b. DISCUSSION More than three hundred germline mutations are described in the TSC2 gene, which includes missense and nonsense (20%), frameshift and splice site mutations (Jones et al., 1999; Dabora et al., 2001; Sancak et al., 2005). In contrast, most of the described mutations in the TSC1 gene are either
nonsense or frameshift, causing premature protein truncation (van Slegtenhorst et al., 1997; Kwiatkowski, 2010; Astrinidis and Henske, 2005). The TSC1 and TSC2 protein products, hamartin (TSC1) with 1164 amino acids and tuberin (TSC2) with 1807 amino acids, interact with each other to form heterodimers and together they function as a tumour suppressor complex. They are expressed by the same cell types within multiple organs including the brain, lung, kidney and pancreas (Jones et al., 1999; Jin et al., 1996; Plank et al., 1998). The GAP activity of tuberin is of obvious functional importance to the complex and is essential for the tumour suppressive function, while hamartin is required to stabilize tuberin and to prevent its ubiquitin-mediated degradation (Jin et al., 1996; Benvenuto et al., 2000; Chong-Kopera et al., 2006). Apart from the genetic changes affecting the tuberin GAP domain localized in the C- terminal part of the gene, missense mutations in both genes commonly destabilize the complex and leading to degradation of tuberin (Nellist et al., 2001). Our results adds some new data to the TSC mutation spectra in both genes by presenting six cases with novel mutations and ten previously reported in the literature (figure 1a, b). The study represents first genetically verified cases with TSC in Bulgaria. The detected TSC2 variants in our cohort are missense, frameshift and nonsense mutations, while in the TSC1 gene most of them are nonsense mutations as it has been previously described in the literature (Astrinidis and Henske, 2005). The mutations in the TSC2 gene are spread through the whole gene with rough concentration of four of them within the GAP domain of the TSC2 gene (figure 1b), while three from the four TSC1 gene nonsense mutations are localized in exon 15, one of the largest gene exons (figure 1a). In our study we detected larger number of TSC1 mutations than the reported in the literature (Dabora et al., 2001; Sancak et al., 2005). The TSC1-TSC2 complex functions in several cell-signaling pathways such as a growth and translation regulatory pathway (PI3K/PKB pathway), a cell adhesion/migration/protein transportation
pathway (GSK3/FAK/Rho pathway), and a cell growth and proliferation pathway MAPK pathway (Au et al., 2004). The TSC1-TSC2 complex was a critical negative regulator of signalling downstream of PI3K/PKB pathway (Langkau et al., 2002). PI3K/PKB pathway loss-of-function mutants could be partially rescued by loss of one functional copy of either TSC1 or TSC2. PKB activated by PI3K directly phosphorylates TSC2, rendering it inactivate, which results in activation of Rheb and consequently of mtor complex 1 (mtorc1) (Costa-Mattioli and Monteggia, 2013; Tee et al., 2016; Huang and Manning, 2008). It is found that the TSC1 TSC2 complex acts upstream of the mtorc1. It is a critical negative regulator of mtorc1 activation and the loss of the TSC tumour suppressors gives rise to abnormally increased mtorc1 mediated translation which is responsible for the ASD (autism spectrum disorder) phenotype as reported in three of our cases (patients #5, 7, 11, presented in table 1). Defects in the regulation of the TSC1 TSC2 complex are likely to contribute to tumorigenesis and cancer (Costa-Mattioli and Monteggia, 2013). Gene mutations often cause essential alterations of TSC1/TSC2 encoded proteins. The product of TSC2 gene, tuberin, is known to have 7 domains: a leucine zipper region, two small coiled-coil domains (CCD1, CCD2), a small region of similarity with GTPase-activating protein (GAPD), two transcriptional activation domains (TAD1, TAD2), and a calmodulin-binding site (CaMD) (see figure 1b) (Luo et al., 2015; Hodges et al., 2001). Four of the reported here genetic variants are localized in the GAPD (1336-1617 amino acids) of tuberin and all of them include typical clinical symptoms of TSC (patients # 1, 3, 6, 10, presented in table 1; figure 1b). The first novel nonsense mutation c.4051g>t, p.glu1351* in the TSC2 gene is detected in patient #6 which is the only case from our cohort who developed bilateral retinal hamartomas (table 1, figure 2a). It is known that patients with mutations in the GAPD of the TSC2 have low GAP activity toward Rheb which gives rise to the highest levels of mtorc1 signalling (Jones et al., 1999; Costa-Mattioli and Monteggia, 2013).
The second novel mutation c.2954_2957dupatgt, p.val987cysfs*19 in exon 26 of the TSC2 gene detected in patient #13 is a frameshift and it is localized in the CCD2 domain (947-988 amino acids) of the tuberin (table 1, figure 2b). It is predicted to truncate the TSC2 protein by nonsense-mediated mrna decay (NMD) pathway which selectively degrades mrnas harboring premature termination codons (PTCs) (Hug et al., 2016). This genetic alteration most probably leads to uncontrolled cell growth and tumorigenesis manifested in our patient with subependymal nodules with calcifications (see patient #12, table 1). The third novel mutation representing another frameshift mutation c.2066_2073del8, insacgggcagggacctcgctgggfs*18, p.(leu689hisfs*17) in exon 18 of the TSC2 gene was found in patient #16 (table 1, figure 2c). It is unclear in which protein domain falls exon 18 of the TSC2 gene, but it is obvious that such complicated indels lead to premature protein termination. Moreover, TSC2 mutations which disrupt the connection between tuberin and hamartin have decreased GAP activity, indicating that interaction with hamartin is essential for tuberin s function as a GAP (Astrinidis and Henske, 2005; Jones et al., 1999; Nellist et al., 2005b). In this patient the disease is manifested by severe skin and brain features as SEGA, subependymal noduls, subcortical tubers, ID and GTCE (table 1, patient #16). The fourth novel mutation is large deletion of exons 1 to 16 of the TSC2 gene which was found in patient #8, who manifests severe clinical features (table 1, figure 2d). The first exons of tuberin are important for the interaction with hamartin. The mutation is de novo. It is well known that de novo mutations in the TSC2 are found in higher percentage of the patients with very severe clinical picture of TSC than de novo TSC1 pathogenic variants (Astrinidis and Henske, 2005). Moreover, we found two novel mutations in the TSC1 gene. The first case is the novel nonsense mutation c.1966g>t, p.gly656* in exon 15, which causes the disease in patient #4 (table 1, figure 3a). The nonsense mutations lead to truncated protein. The lack of C-terminal part of hamartin
where the ezrin, radixin, and moesin domain (ERM) is localized, most probably disturbs the connection of integral membrane proteins with cytoskeletal proteins (Lamb et al., 2000; Astrinidis et al., 2002). Destruction of this interaction is proposed to cause cells to lose adhesion to the extracellular matrix, leading to abnormal cell migration and hamartoma formation as in our patient #4 who has cortical dysplasias and subependymal noduls (table 1) (Lamb et al., 2000; Astrinidis et al., 2002). The second novel TSC1 mutation c.2698_2699delca, p.gln900glu*2, again frameshift was detected in patient #9 (table 1, figure 2b). The mutation falls in exon 22 of the TSC1 gene, localized in the coiled coil domain (CCD) of the hamartin (exons 17-23) (Astrinidis et al., 2002). This domain is involved in protein-protein interactions with hamartin. The TBC1D7 binding site is encoded by TSC1 exon 22 (Santiago et al., 2014). It is important for TSC1-TBC1D7 interaction, where TBC1D7 is the third subunit of the TSC complex, and helps to stabilize the TSC1-TSC2 complex (Qin et al., 2016). These functions are most probably impaired by the disease-causing mutation, detected in patient #9. This patient is among those manifesting cardiac rhabdomyoma. In all but one of the cases (94%) the diagnosis was genetically confirmed. This single case represents a family case of female with classical clinical picture of TSC included facial angiofibromas, ungual fibromas, subependymal and periventricular nodules (see patient# 14, table 1). Her son was severely affected by ID with ASD (as reported by the family members). The index patient has also a healthy daughter, but the grandson also manifests some symptoms of ASD. This family case is interesting to be further genetically investigated. CONCLUSION In the present study we describe six novel mutations: four in the TSC2 and two in the TSC1 genes. In respect to the percentage and the type of detected by us mutations, our data is similar to the
previously reported with exception of the larger number of the TSC1 mutations than the literature data. In total 40% (4/10) from the mutation in the TSC2 gene are located in the GAP domain, while 50% (3/6) from the mutation in the TSC1 gene are clustered in exon 15. All the cases in our study represent the typical clinical features of TSC and met the clinical criteria for TSC diagnosis. In all but one of the cases the diagnosis was genetically confirmed. In 35% (6/17) of our cases the family history was positive, in 24% (4/17) de novo mutation was detected and in 29% (5/17) of the cases the parents were not available for genetic testing. Our results add novel findings in the genetic heterogeneity and pathogenesis of TSC. The phenomenon of genetic heterogeneity might further play a role in clinical variability among families with the same diagnosis and within a single family which makes the genetic testing and genetic counseling in TSC affected families very important task. ACKNOWLEDGMENTS The authors thank the following foundations for financial support by the Medical University Sofia, Bulgaria (grant No D-131/ 2017). ETHICAL APPROVAL The work was approved by the Ethics Committee of Medical University Sofia. CONFLICT OF INTEREST The authors declare that they have no conflict of interest.
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Table 1. Clinical and genetic findings in 16 Bulgarian cases and one Romanian case with TSC Clinical TSC2 TSC2 Symptoms location mutations 1 1 HM; 1 café au lait macule ; focal epilepsy exon 34 c.4473dela, p.val1492cysfs*84 2 3 4 5 6 7 8 9 10 11 12 2 HMs; FAs; periventricular calcifications; early GTCE (1st month); West syndrome (from 7 month) HMs; cardiac rhabdomyoma; subependymal nodules with calcifications; West syndrome HMs; cortical dysplasias; subependymal nodules; ID; West syndrome; TRE > 5 HMs; FAs; ungual fibroma; SPs; cortical dysplasias; SEGA - operated; subependymal nodules; focal epilepsy with secondary generalization; ID with autism HMs; FAs; bilateral retinal hamartomas; multiple subependymal nodules; subcortical tubers; focal epilepsy with secondary generalization HMs; multiple subcortical tubers; subependymal nodules with calcification; TRE; ID with autism HMs; facial angiofibromas; multiple cortical tubers; renal angiomyolipomas; SEGA; focal epilepsy with secondary generalization HMs; FAs; cardiac rhabdomyoma; symptomatic epilepsy HMs; 3 cardiac rhabdomyoma; West syndrome; ID HMs; FAs; SPs; SEGA; subcortical tubers; ID with autism; West syndrome; GTCE 5 HMs; FAs; subependymal noduls; cortical dysplasias; GTCE intron25 exon 37 exon 17 exon 34 exon 38 exon 42 c.2838-122g>a (splice-site) c. 4830G>A, p.trp1610* TSC1 location - - exon 15 c.1769t>c, p.leu590pro c.4051g>t, p.glu1351* # - - exon 5 Deletion of exon 1-16 # - - exon 22 c.4949a>g, p.tyr1650cys c.5228g>a, p.arg1743gln - - exon 15 TSC1 mutation c.1966g>t, p.gly656* # c.325c>t, p.gln109* c.2698_2699delca, p.(gln900glu*2) # c. 1525C>T, p.arg509* Family history positive mother, 3 HMs De novo De novo na na positive mother with renal AML positive mother with 2 HMs; periventricular nodules with calcifications De novo positive mother with HMs; FAs, SPs, subependimal calcifications positive mother (mosaic mutation) with HMs, SPs, renal AML, epilepsy, ID No family; positive brother with the same symptoms without SEGA na 13 HMs; FAs; subependymal nodules with calcifications; focal epilepsy exon 26 c.2954_2957dupatgt, p.(val987cysfs*19) # positive father with HMs, FAs; brother with multiple HMs; FAs; focal epilepsy 14 FAs; ungual fibromas; multiple renal cysts (bilateral); subependymal and periventricular nodules TSC2 / MLPA - TSC1 / MLPA - This proband has a male with symptoms of TSC and healthy daughter 15 HMs; periventricular calcificate; West syndrome; intellectual disability - - exon 15 c. 1453G>T, p.glu485* na - mother with HMs; FAs; calcificate; grandfather with HMs 16 HMs; FAs; SPs; multiple ungual fibromas; subependymal noduls; subcortical tubers; SEGA; ID; GTCE; fibroma of left eyelid exon 18 c.2066_2073del8, insacgggcaggga CCTCGCTGGGfs*18, p.(leu689hisfs*17) # De novo
17 Romanian case HMs; FAs; 1SP; 1 café au lait macule ; periventricular calcificate (CT scan) exon 6 c.433_434delca, p.gln145valfs*7 This proband has a child with TSC: HMs; FAs; 1SP; multiple tubers; calcified periventricular nodules; AMLs; focal epilepsy # novel mutations; submitted only once; HMs hypomelanotic macules; FAs facial angiofibromas; SPs Shagreen patches; TRE therapeutic resistant epilepsy; SEGA - subependymal giant cell astrocytoma; AMLs angiomyolipomas; ID - intellectual disability; GTCE - generalized tonicclonic epilepsy; na not available LEGENDS OF FIGURES: Figure 1. Distribution of the disease-causing mutations along the TSC1 and TSC2 genes and their localization along the protein domains of the hamartin and tuberin, respectively. (a) Schematic representation of the TSC1 gene exons and the domains of the protein hamartin: TMD - transmembrane domain; CCD - coil coil domain; ERM domain - ezrin radixin moesin. (b) Schematic representation of the TSC2 gene exons and the domains of the protein tuberin: LZD - leucine zipper domain; CCD1/CCD2 - coil coil domain 1/2; GAP - GTPase-activating protein; TAD2 - transcription-activating domain; CaMD - calmodulin-binding domain;
Figure 2. Sequencing chromatograms of 4 novel mutations in the TSC2 gene. (a) Case 6 showing nonsense mutation c.4051g>t, p.glu1351*. (b) Case 13 showing frameshift mutation c.2954_2957dupatgt, p.val987cysfs*19. (c) Case 16 showing frameshift mutation c.2066_2073del8, insacgggcagggacctcgctgggfs*18, p.(leu689hisfs*17) (d) Case 8 showing deletion of exon 1-16
Figure 3. Sequencing chromatograms of 4 novel mutations in the TSC1 gene. (a) Case 4 showing nonsense mutation c.1966g>t, p.gly656*. (b) Case 9 showing frameshift mutation c.2698_2699delca, p.gln900glu*2.