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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 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 [email protected]

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Page 1: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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

[email protected]

Page 3: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

Corresponding author:

Maria Glushkova

Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2 “Zdrave” str.,

Sofia 1431, Bulgaria

[email protected]

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.

Page 4: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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

Page 5: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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,

Page 6: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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

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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

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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).

Page 9: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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

Page 10: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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

Page 11: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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.

Page 12: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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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

Page 17: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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;

Page 18: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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

Page 19: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria

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.

Page 20: Molecular-genetic diagnostics of Tuberous sclerosis ... · Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2“Zdrave” str., 1431Sofia, Bulgaria