Yonago Acta medica 2009;52:57–72

Variations in Clinical Findings ofPatients with Identical Tuberous Sclerosis Gene Mutations
Yuko Sakurai, Yoshiaki Saito*, Eiji Nanba†, Toshiyuki Yamamoto‡ and Kousaku
Ohno
Division of Child Neurology, Institute of Neurological Sciences, Tottori University Faculty of
Medicine, Yonago 683-8503, *Department of Child Neurology, National Center Hospital of Neurology and Psychiatry, Tokyo 187-8551, †Division of Functional Genomics, Research Center for
Bioscience and Technology, Tottori University, Yonago 683-8503 and ‡International Researchand Educational Institute for Integrated Medical Sciences, Tokyo Women’s Medical University,
Tokyo 162-8666 Japan

We herein report on 3 nonsense and 1 deletion mutations in TSC1 or TSC2 genes in
10 Japanese individuals with various phenotypes of tuberous sclerosis complex (TSC).
Even having identical mutations, some patients suffered from intractable epilepsy andshowed severe intellectual and behavioral disabilities, while others were intellectuallynormal and epilepsy was absent or easily controlled. Review of the data of these andother 196 cases in the literature revealed that certain missense mutations are characteristic in yielding mild phenotype, particularly at the GTPase-activating protein domain of
TSC2 gene. Non-truncating mutations in this functionally important domain may tend
to cause clinical symptoms, while those in the other regions may remain subclinical andinterpreted as polymorphism. On the other hand, many truncating and missense mutations of TSC genes could cause either mild or severe phenotypes. Somatic mosaicism, either in the initial or second-hit mutations, cannot explain the whole feature of this clinical variability. Mutation database with sufficient information of clinical manifestations
and family history is necessary to draw reliable conclusion for genetic counseling, as wellas to evaluate any modifying factors on the clinical severity other than the TSC gene mutations.
Key words: hamartin; haploinsufficiency; GTPase-activating protein domain; mammalian target of rapamycin; tuberin

Tuberous sclerosis complex (TSC) is a disorderwith autosomal dominant inheritance, characterized by development of hamartomatous lesions invarious organs and a wide range of neurologicalabnormalities. Two genes causing TSC have been
identified: TSC1 is located at chromosome 9q34
and encodes hamartin, and TSC2 is at chromosome 16p13.3 and encodes tuberin. These proteinsinteract and form a cytoplasmic heterodimer complex that inhibits the phosphorylation of mammalian target of rapamycin (mTOR) pathway (Swiech
et al., 2008), and play a cardinal role in the regulation of differentiation, growth, and proliferation ofvarious cell types.
The disruption of mTOR pathway could
explain the extracerebral complications; i.e., an

Abbreviations: CT, computed tomography; GAP, GTPase-activating protein; MR, magnetic resonance; mTOR, mammalian target of rapamycin; TSC, tuberous sclerosis complex

57

Y. Sakurai et al.

inherited mutation is carried in one allele, and thesecond mutation in the other allele of TSC genes
have been identified in the hamartomatous lesionsof TSC patients. However, this second hit has notbeen identified in the dysplastic cerebral tissuesof TSC patients, including cortical tubers (Niida
et al., 2001; Ramesh et al., 2003). In addition,
studies on the genotype-phenotype correlations
in TSC mutants have revealed that mutations in
TSC2 gene tend to cause more severe neurologicaloutcome than TSC1 gene mutations (Dabora et al.,
2001; Sancak et al., 2005; Au et al., 2007), resulting in lower intelligence quotient and higher prevalence of autistic traits and infantile spasms (Lewis
et al., 2004). Variation of severity can be seen
even between monozygotic twins with identical
TSC gene mutations (Gomez et al., 1982; Martin
et al., 2003; Humphrey et al., 2004). These facts
have made the pathogenesis of cerebral lesions difficult to understand and the prognosis of individual
TSC patients hard to predict.
In order to have a better insight into the clinical variability of TSC patients, particularly regarding neurological complications, we reviewed
our Japanese series of TSC gene mutations andcompared the clinical features of patients withidentical mutations. In addition, we reviewed
the around 400 TSC1 and 1,000 TSC2 mutations
that have been reported to date*, and identified
5 TSC1 mutations and 33 TSC2 mutations thathave been found in different members of a single
family or in individuals from different families,
for whom clinical data were also provided in theliterature (Smalley et al., 1994; Vrtel et al., 1996;
Wilson et al., 1996; Jobert et al., 1997; Maheshwar et al., 1997; Au et al., 1998; Beauchamp etal., 1998; Kwiatkowska et al., 1998; Verhoef et al,
1998, 1999; Niida et al., 1999; Zhang et al., 1999;
Yamashita et al., 2000; Dabora et al., 2001; Yamamoto et al., 2002; Martin et al., 2003; Feng et al.,
2004; Humphrey et al., 2004; Mayer et al., 2004;
Ali et al., 2005; Rok et al., 2005; Choi et al., 2006;
Hung et al., 2006; Jansen et al., 2006; Lyczkowski
et al., 2007). We found that certain mutations
were identified in patients with various severities,

or otherwise were common in mildly affectedpatients. Analysis on the mutation types, either
truncating or missense, also revealed that missense
mutations were clustered in the GTPase-activating
protein (GAP) domain of TSC2 gene. Significanceof these findings is discussed.

Subjects and Methods
In the series of 140 Japanese patients, clinicallysuspected with either definite, probable or possible TSC (Roach et al., 1998), mutation analysis
of TSC1 and TSC2 genes in blood samples was
performed by means of polymerase chain reactionsingle strand conformation polymorphism analysis
as described in previous reports (Zhang et al.,
1999; Yamamoto et al., 2002). We found TSC1
mutation in 20 patients, and TSC2 mutation in
49 patients. Out of the 20 TSC1 mutations, there
were 9 missense mutations, 4 nonsense mutations,4 insertions, 3 deletions. The 49 TSC2 mutations
included 25 missense mutations, 5 nonsense mutations, 3 insertions, 9 deletions, and 7 mutationsin the intron sequence that were considered toresult in splicing errors. Among these, there were
4 sets of patients with the same type of the mutations. These identical mutations were present in
3 patients having the same TSC1 mutations, and7 patients in 3 sets of TSC2 mutations. Clinicaldata of these 10 patients were collected in terms of
cutaneous, cardiac, renal, and liver involvement, as
well as the presence and nature of epilepsy, intellectual assessment, autistic traits, and findings on
neuroimaging (computed tomography (CT) in 9,
and magnetic resonance (MR) imaging in 6 patients). Some data of patients 1, 2, 5, 6 and 7 havebeen published previously (Zhang et al., 1999;
Feng et al., 2004).
Results
TSC1 c.1746C>T (p.R509X) mutation (Table 1)
Patient 1: This patient, a 41-year-old woman,suffered from infantile spasms when she was 2
months old. Adrenocorticotropin was effective* http://chromium.liacs.nl/LOVD2/TSC/home.php?select_db=TSC1 or db=TSC2

58

Variations in identical TSC mutations

Table 1. Clinical features of patients with TSC1 c.1746C>T (p.R509X) mutation
Symptom

Patient 1

Age examined (yr)Sex
Heredity

41
FemaleSporadic
Infantile spasms g complex partial seizures
2 mo
Good
Severe (DQ < 10)
–
–
–
–
–
–
–
+
+

Type of epileptic seizures
Onset of seizure
Control of seizure
Mental retardation
Autism
Impaired social interaction
Stereotypical behavior
Other autistic behaviors
Hypomelanotic macule
Facial angiofibroma
Shagreen patch
Renal angiomyolipoma
Periventricular calcificationon brain CT
Cortical tuber

Patient 2

Patient 3

27
Male
Familial

62
Male
Sporadic(No seizures)

Complex partial seizures

Not applicableNot applicable
–
–
–
–
–
+
–
Not described+

School age
Good
–
–
–
–
–
+
+
+
+

+

+

+

+
		
Other symptoms
Triplegia, mastoadenoma			+Liver cyst, adrenal nodule(1.5 mm), ungual fibroma–, absent; +, present; DQ, developmental quotient; mo, months; yr, years.

A

B

C

D

E

F

Fig. 1. Neuroimaging of patients with TSC1 p.R509X mutation. A to D: Patient 1. E and F: Patient 2. A and B:
computed tomography (CT). C to F: magnetic resonance (MR) imaging [C: T1 weighted image, D to F: Fluid-attenuation recovery images].

59

Y. Sakurai et al.

Table 2. Clinical features of patients with TSC2 c. 3355C>T (p.Q1119X) mutation
Symptom
Age examined (yr)HeredityType of epileptic seizures
Onset of seizure
Control of seizure
Mental retardation
Autism
Impaired social interaction
Stereotypical behavior
Other autistic behaviors
Hypomelanotic macule
Facial angiofibroma
Renal angiomyolipoma
Periventricular calcificationon brain CT
Cortical tuber
Other symptoms

Patient 4

Patient 5

8Sporadic
Infantile spasms
6 mo
Good
Severe (DQ 17)++ (disobedience)++
Panic, self injury
+
+
–

23
Sporadic
Right hemiconvulsion
13 mo (with fever)
Only once
Normal intelligence
–
–
–
–
–
–
2 (diameter 1 cm, 19 yr)

84+
Cardiac rhabdomyoma,
Wolff-Parkinson-White syndrome

Not available
Renal cystic dysplasia with cancer-like lesions(3 mo), angiomyolipoma of liver (15 yr)–, absent; +, present; ++, marked; DQ, developmental quotient; mo, months; yr, years.

Ain treatment of her epilepsy, and subsequent complex partial seizures
have been controlled well. Paraparesis and left hemiparesis have been
noted, and a large cortical tuber with calcification at the medial partof right frontal lobe has been revealed on neuroimaging (Figs. 1A to D).
Patient 2: The patient is a 27-year-old man, who has hypomelanotic macules, but has not shown intellectual disability or epileptic
seizures. A small number of subependymal nodules and cortical tubers are noted (Figs. 1E and 1F), but less evident compared to Patient 1.
Clinical data on other family members were not available.
Patient 3: This 62-year-old man is intellectually normal. He
suffered from epilepsy in his school years, which was well controlled
by antiepileptics thereafter.
TSC2 c.3355C>T (p.Q1119X) mutation (Table 2)

B
Fig. 2. Neuroimaging of Patient 4
with TSC2 p.Q1119X mutation.
A: CT image.
B: Fluid-attenuation recovery
MR image.

Patient 4: This 8-year-old girl suffered from infantile spasms
at age 6 months. Residual seizures are currently controlled by potassium bromide, but she shows severe intellectual disability and autistic
behavior including panic and self injury. Numerous cortical tubers
and subependymal nodules are noted on neuroimaging (Fig. 2).
Patient 5: A large renal tumor, histologically identified as cystic
dysplasia with renal cancer, was resected when this girl was 3 months
old. She suffered from febrile hemiconvulsion at age 13 months,
but seizure never recurred thereafter, and electroencephalography
remained normal. Intracranial calcifications and hepatic angiomyolipoma were revealed at the age of 15 years. She shows normal intelligence and has graduated from college.
60

Variations in identical TSC mutations

Table 3. Clinical features of patients with TSC2 c. 4393 C>T (p. R1459X) mutation
Symptom

Patient 6

Patient 7

Age of examined patient (yr)Sex
HeredityType of epileptic seizures
Onset of seizure
Focus in electroencephalography

18
Male
Familial
Complex partial seizures
3 yr and 2 moRight fronto-parietal, left frontal
Fair ( twice a year since 12 yr)
Control of seizure		Mental retardation
IQ 33
Autism
+Impaired social interaction+
Stereotypical behavior
Preoccupation with special events
Other autistic behaviors
Difficult to adapt to changes
Hypomelanotic maculeNot available
Facial angiofibroma
Not available
Renal angiomyolipoma+
Periventricular calcification+on brain CT
Cortical tubers,
Brain MRI
bilateral subependymal nodules

19
FemaleSporadic
Complex partial seizures
1 yr
Right frontal
Poor excision of right frontal
lobe (17 yr) goodIQ 41+
+ (likes to play alone)
Compulsive
Anxious, easily excited++many embolization
several, 1 in right frontal
Cortical tubers+, present; IQ: intelligence quotient; mo, months; yr, years.

TSC2 c.4393C>T (p.R1459X) mutation (Table 3)

A

Patient 6: This patient suffered from epilepsy with complex
partial seizures since age 3. He is now 18 years old, and seizures still
appear twice per year despite treatment. Moderate intellectual disability and autistic traits have been evident. Multiple subependymal
nodules (Fig. 3A) and cortical tubers are noted. Data on other family
members with TSC were not available.
Patient 7: This girl suffered from intractable, complex partial
seizures since 1 year of age, which disappeared after resection of theright frontal lobe at age 17. She has moderate intellectual disability,
likes to play on her own, and shows anxious and compulsive behaviors. Numerous cortical tubers (Fig. 3B) are noted on MR imaging.
Renal angiomyolipoma (Fig. 3C) were treated with arterial embolization at age 17 years.

Fig. 3. Radiological findings of patientswith TSC2 p.R1459X mutation.
A: brain CT of Patient 6.
B: brain MR imaging of Patient 7.
C: abdominal CT image of Patient 7.
Arrows in C indicate the angiomyolipoma
in bilateral kidneys.

B

C
61

Y. Sakurai et al.

Table 4. Clinical features of patients with TSC2 c.5238–5255del (p. del 1746 HIKRLR) mutation
Symptom

Patient 8

Patient 9

Age of examined patient (yr)
3
6
Sex
Male
FemaleHeredity
Sporadic
Sporadic
Type of epileptic seizures
Infantile spasms
CPS, infantile spasms
Onset of seizure
4 mo
6 mo
Focus in 		
Right front-parietal,
electroencephalography		right frontal, right central,		left parieto-temporal?
Control of seizure
Good (since 2 yr)
Fair (5–10 times/day)
Mental retardation
Moderate–severe
Severe (DQ 35 at 2 yr)
Autism
–+Impaired social interaction–+ (lack of social		reciprocity, failure		
to use eye-to-eye gaze)Communication disturbance–+ (nonverbal)
Stereotypical behavior–
Not available
Other autistic behaviors–
Hypomelanotic macule++
Facial angiofibroma+–
Renal angiomyolipoma––
Other renal lesions						Periventricular calcification++on brain CT
Brain MRI
Not available
+
			
			
Other symptoms
Cardiac rhabdomyoma,
fibroma on the occiput

Patient 10
13
Male
Sporadic
Infantile spasms
5 moOnce in 3 moModerate (IQ 45–50)–+ (until 2–3 yr) g– (since 5 yr)–+ (mild)+––
One renal cyst in the rightkidney, bilateral lipoma+
Subependymal nodules,
multiple subcortical
hypomyelination–, absent; +, present; CPS, complex partial seizures; DQ, developmental quotient; IQ, intelligence quotient; mo, months; yr, years.

TSC2 c.5238–5255del (p.del 1746HIKRLR)
mutation (Table 4)

TSC patients with identical mutations in
the literature (Table 5)

Patient 8: This patient, a severely retarded
but not autistic boy, suffered from infantile spasms
at age 4 months, which evolved into localizationrelated epilepsy and remained intractable.
Patient 9: This patient also developed infantile spasms and complex partial seizures at age 5months. The seizures have been intractable, andshe shows severe intellectual disability and autism.
Patient 10: This boy developed infantile
spasms when he was 5 months old. He gained theability to utter meaningful words since 2 years ofage. He was hyperactive and had difficulties in social relationships during early childhood. However,
his social skills improved since he was 5. Whenhe was 13, he was no longer autistic, but showedoccasional, compulsive behaviors.

By adding the 10 patients in this study, we could
identify 5 TSC1 mutations in 17 patients, and 33
TSC2 mutations found in 179 patients (Table 5),
which were found in 2 or more individuals. Mostof these groups with identical mutations, either
truncating or non-truncating, included both individuals with normal and disabled intelligence. In
contrast, individuals with L61R mutation in TSC1,
and K12X, E1542K, 1554Q, G1556S, and 1628X
mutations in TSC2, did not show intellectual disabilities, even having multiple cortical tubers.
R905Q mutation of TSC2 gene also showed resultant tendency of milder cognitive impairment.
Out of the 14 missense mutations in TSC2, 6 wereconcentrated at exons 34 to 38, which corresponds
to the GAP homology domain (Maheshwar et al.,1997).
62

Table 5. TSC patients with identical mutations in the literature [i]Reference
Patient

re d
He

ity

t
P ro

403T>G
403T>G
403T>G

L61R
L61R
L61R

ein

2105 del AAAG
2105 del AAAG
2105 del AAAGe
Agear)
(y

n
nce
atio
ige
Sex Muttype ntell
I

51
21
17

m MM IQ 100
f MM IQ 101
f MM IQ 72

27
7
3

f

FS
FS
FS

tism
Au

zu
Sei

re

ber
Tu

Controlled 14
Occasional 12
Intractable 18

Severe MR
MR (–)

+
–
+

+
+
+

+
+
+

+

+
–

+

+

+

+

+

–

–

–

–
+

–
–

–

+
+

63

3.5 m NMLD

1746 C>T1746 C>T

R509X
R509X

41
27

f NM
m NM

Severe MR
MR (–)––

IS, partial Sz +–+1746 C>T

R509X

62

m NM

MR (–)

–

+

+

+

+

+

+

+

+

Brain
image (+)
Brain
image (+)++

Var Var

–

–

–

–

+

+

NA

NM

MR

+

Family 38

F TSC1 exon 172295 C>T

R692X

NM

MR (–)/LD

+

F
F
F
F
F
F
F
F

2790insG
2790insG
2790insG
2790insG
2790insG
2790insG
2790insG
2790insG

88
55
52
31
30
28
27
23

FS
FS
FS
FS
FS
FS
FS
FS

NA
NA
NA
IQ 87
NA
Impaired
IQ 79
IQ 73

NA
NA
NA
NA
NA
NA
Intractable +Controlled +Intractable NA
Controlled 9Intractable 45

F TSC2 exon 1

52A>T

F TSC2 exon 1

52A>T

K12X
30 m IF?
(1–49 del?)K12X
1.5 m IF?
(1–49 del?)F TSC2 intron 1
F TSC2 intron 1

156+1G>A
156+1G>A

31X
31X

m
f
m
m
m
m
m
f

m FS
m FS

Table 5, continued to the next page. Footnotes, refer to Table 5 [vii], end of this table.

++
+

NA
NA
NA
+
+
NA
+
+

MR (–)

NA
NA
NA
+
+
NA

+
+ +
NA NA
+
+
+ +
+
+

–

+ +
+
NA NA
+
+

+

NA
NA
NA NA
–
–
–
–
–
–
+
–
–

Liver cyst
s/o, adrenal nodule

+
NA
NA
–
–
–

+
+

+
+

+
+

(retinal HM)

NA
NA–
Retinal HM–––

Retinal HM
Gingival
fibroma

+

IS
+

s
her

–

–

MR (–)
Mild MR

Ot

–
–

R509X

R692X

Verhoef (1999)
Family 4 sib 1
Family 4 sib 2+++

1525C>T2295 C>T

Son

+
+
+

al
L
F P
C en tPU Shg AM RC R cys CR+

F TSC1 exon 17

TSC1 exon 20
TSC1 exon 20
TSC1 exon 20
TSC1 exon 20
TSC1 exon 20
TSC1 exon 20
TSC1 exon 20
TSC1 exon 20

GA M
SE HP AF++

Niida (1999)
Family 26

Lyczkowski (2007)
C-I-1
C-II-2C-II-3
C-III-1
C-III-2
C-III-3
C-III-4
C-III-5
Vrtel (1996)
Father

N
SE

+

+
+

+

Gingival
fibroma

Variations in identical TSC mutations

Lyczkowski (2007)
A-I-1 (father)
F TSC1 exon 4
A-II-1 (sib)
F TSC1 exon 4
A-II-2 (sib)
F TSC1 exon 4
Kwiatkowska (1998)
MotherF TSC1 exon 15
Sib 1
F TSC1 exon 15
Sib 2
F TSC1 exon 15
Hung (2006)
61
F TSC1 exon 15
Zhang (1999), the present study
3; Patient 1
S TSC1 exon 15
26; Patient 2
F TSC1 exon 15
The present study
Patient 3
S TSC1 exon 15

nge
cha
A
DN

Table 5. TSC patients with identical mutations in the literature [ii]
tei
P ro

F TSC2 exon 12

1365G>C

M449I

MM MR(–)

TSC-028-2
TSC-028-3

F TSC2 exon 12
F TSC2 exon 12

1365G>C
1365G>C

M449I
M449I

MM MR
MM

+

TSC-028-4
Beauchamp (1998)
F17-01F TSC2 exon 12

1365G>C

M449I

MM MR

+

F TSC2 exon 12

1348G>T

E450X

3

NM

Mild MR+

F16-01F TSC2 exon 12

1348G>T

E450X

3

NM

MR(–)

+

Niida (1999)
326

S TSC2 exon 12

1348G>T

E450X

NM

MR

+

Brain
image (+)

Jobert (1997)
B17 II-1 (father)

F TSC2 exon 14

482–492 delm IF

MR (–)+MRI (+)

B17 III-2 (son)F TSC2 exon 14

1462del33mRNA
1462del33mRNA
1462del33mRNA
1462del33mRNA

482–492 del

m IF

MR

+

482–492 delf

MR (–)+MRI (+)

482–492 del

m IF

MR

+

+

re d
He

Wilson (1996)
TSC-028-1

ity

B95 I-1 (mother) F TSC2 exon 14
B95 II-1 (son)F TSC2 exon 14ne
Agear)
(y

n
nce
atio
ige
Sex Muttype ntell
I

IF

tism
Au

zu
Sei

Beh/
LD (–)

+

re

al
L
F P
C en tPU Shg AM RC R cys CR

Ot

–

+

–

Eye (+)

+
+

–
+

–

+

–

–

–

+

–

–

–

NA

–

+

–

–

–

NA

–

+

–

–

–

–

+

+

+

+

+

+

+

+

+

CT (+)–++–MRI (+)CT (+)

+

+

+

+

Renal
ab (+)
Renal
ab (–)
Renal
ab (+)
Renal
ab (+)

+

+

+

+

+

+

+

+
+

+
+

–

ber
Tu

N
SE

GA M
SE HP AFCT (+)++CT (–)CT (+)
Brain findings (+)
Brain findings (+)

CT (+)Eye (–)
Renal
ab (+)Eye (–)Eye (+)Eye (+)

TSC2 exon 14

1513C>T

R505X

NM

MR

Wilson (1996)
TSC-037-1
TSC-037-2

F TSC2 exon 14
F TSC2 exon 14

1531C>T
1531C>T

R505X
R505X

NM
NM

MR (–)MR (–)S TSC2 exon 14
S TSC2 exon 14

1513C>T
1513C>T

R505X
R505X

m NM
f NM

MR (–)
MR

+
+

+
+

+
+

+
+

–
–

–
+

(+)?
–

F TSC2 exon 16

1832G>AR611Q

MM MR (–)+

Brain
image (+)

+

+

–

–

NA

Au (1998)
TS94-31

TSC2 exon 16

1832G>A

R611Q

MM MR

+

+

+

+

+

–

–

–

–

+

TS93-29

TSC2 exon 16

1832G>A

R611Q

MM MR

+

+

+

+

–

–

–

–

–

–

Hung (2006)
20
57
Niida (1999)
53

Table 5, continued to the next page. Footnotes, refer to Table 5 [vii], end of this table.

Beh/
LD (+)Beh/
LD (+)++CT (+)

Retinal HM

Eye (–)Au (1998)
TS94-96Beh/
LD (+)sher+

Eye (–)+

Eye(+)

Y. Sakurai et al.

64

nge
cha
A
DN

Reference
Patient