Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and...
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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 V2, Koleva M2, Dimova P4, Bojidarova M5, Litvinenko I5, Todorov T3,
Iluca E6, Calusaru C6, Neagu E8, Craiu D6, 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
Maya Koleva
Petia Dimova
Maria Bojidarova
Ivan Litvinenko
Tihomir Todorov
Emanuela Iluca
Cristina Calusaru
Elena Neagu
Dana Craiu
Vanyo Mitev
Albena Todorova
Corresponding author:
Maria Glushkova
Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2 “Zdrave” str.,
Sofia 1431, Bulgaria
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.
REFERENCES
Astrinidis A. and Henske E. P. 2005 Tuberous sclerosis complex: linking growth and energy
signaling pathways with human disease. Oncogene 24, 7475-81.
Astrinidis A., Cash T. P., Hunter D. S., Walker C. L., Chernoff J. and Henske E.P. 2002 Tuberin, the
tuberous sclerosis complex 2 tumor suppressor gene product, regulates Rho activation, cell
adhesion and migration. Oncogene 21, 8470–8476.
Au K. S., Williams A. T., Gambello M. J. and Northrup H. 2004 Molecular genetic basis of tuberous
sclerosis complex: from bench to bedside. J. Child. Neurol. 19, 699-709.
Benvenuto G., Li S., Brown S. J., Braverman R., Vass W. C. and Cheadle J. P. 2000 The tuberous
sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression
of the TSC2 product tuberin by inhibiting its ubiquitination. Oncogene 19, 6306-6316
Chong-Kopera H., Inoki K., Li Y., Zhu T., Garcia-Gonzalo F. R., Rosa J. L. et al. 2006 TSC1
stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase. J.
Biol. Chem. 281, 8313-8316.
Costa-Mattioli M. and Monteggia L. M. 2013 mTOR complexes in neurodevelopmental and
neuropsychiatric disorders. Nat. Neurosci. 16, 1537-43.
Crino P. B., Nathanson K. L. and Henske E. P. 2006 The Tuberous Sclerosis Complex. N. Engl. J.
Med. 355, 1345-1356.
Curatolo P., Bombardieri R. and Jozwiak S. 2008 Tuberous Sclerosis. Lancet. 372, 657-668.
Dabora S. L., Jozwiak S., Franz D. N., Roberts P. S., Nieto A., Chung J. 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.
European Chromosome 16 Tuberous Sclerosis Consortium. 1993 Identification and characterization
of the tuberous sclerosis gene on chromosome-16. Cell 75, 1305-1315.
Hodges A. K., Li S., Maynard J., Parry L., Braverman R., Cheadle J. P. et al. 2001 Pathological
mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin. Hum. Mol.
Genet. 10, 2899-905.
Huang J. and Manning B. D. 2008 The TSC1-TSC2 complex: a molecular switchboard controlling
cell growth. Biochem. J. 412, 179-90.
Hug N., Longman D. and Cáceres J. F. 2016 Mechanism and regulation of the nonsense-mediated
decay pathway. Nucleic Acids Res. 44, 1483-95.
Jin F., Wienecke R., Xiao G. H., Maize Jr. J. C., DeClue J. E. and Yeung R. S. 1996 Suppression of
tumourigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region.
Proc. Natl. Acad. Sci. U.S.A. 93, 9154-9159.
Jones A. C., Daniells C. E., Snell R. G., Tachataki M., Idziaszczyk S. A., Krawczak M. et al. 1997
Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2
associated familial and sporadic tuberous sclerosis. Hum. Mol. Genet. 6, 2155-61.
Jones A. C., Shyamsundar M. M., Thomas M. W., Maynard J., Idziaszczyk S., Tomkins S. et al.
1999 Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150
families with tuberous sclerosis. Am. J. Hum. Genet. 64, 1305-15.
Kozlowski P., Roberts P., Dabora S., Franz D., Bissler J., Northrup H. et al. 2007 Identification of 54
large deletions/duplications in TSC1 and TSC2 using MLPA, and genotype-phenotype
correlations. Hum. Genet. 121, 389-400.
Kwiatkowski D. J., Whittemore V. H. and Thiele E. A. 2010 Tuberous Sclerosis Complex: Genes,
Clinical Features, and Therapeutics. Wiley-Blackwell. pp 29-31.
Lamb R. F., Roy C., Diefenbach T. J., Vinters H. V., Johnson M. W., Jay D. G. et al. 2000 The TSC1
tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase
Rho. Nat. Cell. Biol. 2, 281-287.
Langkau N., Martin N., Brandt R., Zugge K., Quast S., Wiegele G. et al. 2002 TSC1 and TSC2
mutations in tuberous sclerosis, the associated phenotypes and a model to explain observed
TSC1/TSC2 frequency ratios. Eur. J. Pediatr. 161, 393-402.
Luo R., Cai Q. and Mu D. 2015 A Chinese tuberous sclerosis complex family and a novel tuberous
sclerosis complex-2 mutation. Chin. Med. J. (Engl). 128, 128-30.
Mayer K., Goedbloed M., van Zijl K., Nellist M. and Rott H. D. 2004 Characterisation of a novel
TSC2 missense mutation in the GAP related domain associated with minimal clinical
manifestations of tuberous sclerosis. J. Med. Genet. 41, e64.
Nellist M., Verhaaf B., Goedbloed M. A., Reuser A. J., van den Ouweland A. M. and Halley D. J.
2001 TSC2 missense mutations inhibit tuberin phosphorylation and prevent formation of the
tuberin–hamartin complex. Hum. Mol. Genet. 10, 2889-2898.
Nellist M., Sancak O., Goedbloed M. A., Rohe C., van Netten D., Mayer K. et al. 2005b Distinct
effects of single amino-acid changes to tuberin on the function of the tuberin-hamartin
complex. Eur. J. Hum. Genet. 13, 59-68.
Northrup H. and Krueger D. A. 2013 International Tuberous Sclerosis Complex Consensus Group.
Tuberous sclerosis complex diagnostic criteria update: recommendations of the 2012
Iinternational Tuberous Sclerosis Complex Consensus Conference. Pediatr. Neurol. 49, 243-
54.
O’Callaghan F., Shiell A., Osborne J. and Martyn C. 1998 Prevalence of tuberous sclerosis estimated
by capture-recapture analysis. Lancet. 352, 318–319.
Plank T. L., Yeung R. S. and Henske E. P. 1998 Hamartin, the product of the tuberous sclerosis 1
(TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles. Cancer
Res. 58, 4766-70.
Qin J., Wang Z., Hoogeveen-Westerveld M., Shen G., Gong W., Nellist M. et al. 2016 Structural
Basis of the Interaction between Tuberous Sclerosis Complex 1 (TSC1) and Tre2-Bub2-Cdc16
Domain Family Member 7 (TBC1D7). J. Biol. Chem. 291, 8591-601.
Sampson J., Scahill S., Stephenson J., Mann L. and Connor J. 1989 Genetic aspects of tuberous
sclerosis in the west of Scotland. J. Med. Genet. 26, 28-31.
Sancak O., Nellist M., Goedbloed M., Elfferich P., Wouters C., Maat-Kievit A. et al. 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, 731-41.
Santiago Lima A. J., Hoogeveen-Westerveld M., Nakashima A., Maat-Kievit A., van den Ouweland
A., Halley D. et al. 2014 Identification of regions critical for the integrity of the TSC1-TSC2-
TBC1D7 complex. PLoS One 9, e93940.
Tee A. R., Sampson J. R., Pal D. K. and Bateman J. M. 2016 The role of mTOR signalling in
neurogenesis, insights from tuberous sclerosis complex. Semin. Cell. Dev. Biol. 52, 12-20.
Van Slegtenhorst M., de Hoogt R., Hermans C., Nellist M., Janssen B., Verhoef S. et al. 1997
Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277, 805-808.
Table 1. Clinical and genetic findings in 16 Bulgarian cases and one Romanian case with TSC
№ Clinical
Symptoms
TSC2
location
TSC2
mutations
TSC1
location
TSC1
mutation
Family
history
1 1 HM; 1 “café au lait macule”; focal epilepsy exon 34 c.4473delA, p.Val1492Cysfs*84 ¥
positive mother, 3 HMs
2
2 HMs; FAs; periventricular calcifications; early GTCE (1st month); West syndrome (from 7
month)
intron25 c.2838-122G>A
(splice-site)
De novo
3 HMs; cardiac rhabdomyoma; subependymal
nodules with calcifications; West syndrome exon 37 c. 4830G>A,
p.Trp1610* ¥
De novo
4 HMs; cortical dysplasias; subependymal nodules;
ID; West syndrome; TRE - - exon 15
c.1966G>T,
p.Gly656* #
na
5
> 5 HMs; FAs; ungual fibroma; SPs; cortical
dysplasias; SEGA - operated; subependymal
nodules; focal epilepsy with secondary generalization; ID with autism
exon 17 c.1769T>C, p.Leu590Pro
na
6
HMs; FAs; bilateral retinal hamartomas;
multiple subependymal nodules; subcortical tubers; focal epilepsy with secondary
generalization
exon 34 c.4051G>T, p.Glu1351* #
positive mother with renal
AML
7 HMs; multiple subcortical tubers; subependymal
nodules with calcification; TRE; ID with autism - - exon 5
c.325C>T, p.Gln109*
positive mother with 2 HMs; periventricular
nodules with calcifications
8
HMs; facial angiofibromas; multiple cortical tubers; renal angiomyolipomas; SEGA; focal
epilepsy with secondary generalization
Deletion of exon 1-16 # De novo
9 HMs; FAs; cardiac rhabdomyoma; symptomatic
epilepsy - - exon 22
c.2698_2699delCA,
p.(Gln900Glu*2) #
positive mother with HMs; FAs, SPs, subependimal
calcifications
10 HMs; 3 cardiac rhabdomyoma; West syndrome;
ID exon 38 c.4949A>G,
p.Tyr1650Cys
positive mother (mosaic mutation) with HMs, SPs,
renal AML, epilepsy, ID
11 HMs; FAs; SPs; SEGA; subcortical tubers; ID
with autism; West syndrome; GTCE exon 42 c.5228G>A,
p.Arg1743Gln
No family; positive brother with the same symptoms
without SEGA
12 5 HMs; FAs; subependymal noduls; cortical
dysplasias; GTCE - - exon 15
c. 1525C>T,
p.Arg509*
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 tonic-
clonic 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.