TGFB3 mutations cause aortic aneurysms and dissections
SUPPLEMENTAL DATA
Online Figure 1: Plots of the genome wide linkage analysis (upper graph) and chromosome 14 and 15
(lower graph) displaying the largest genomic regions identified. LOD scores appear in the Y-axis and
chromosome numbers are on the X-axis. Vertical red lines indicate SNP coverage.
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TGFB3 mutations cause aortic aneurysms and dissections
Online Table 1. List of PCR primers used for Sanger sequencing
Name
Sense primer
Antisense primer
TGFB3 ex1
5’-CCCTCCTTCTGCACGTCTGC-3’
5’-TCCCAGCTCCAGTTCAGACC-3’
487
TGFB3 ex2
5’-GAGGCCACACTGCTCCTTGC-3’
5’-AAACCCAGGTCTGCCAAACG-3’
469
TGFB3 ex3
5’-GAGGGCCATGGCATTCAGG-3’
5’-TGGAGGATACTCAGTGGCAAAGC-3’
391
TGFB3 ex4
5’-GCAACGTGCGCTTGAAGG-3’
5’-GGAGCTTGGTTTCTTTTCTTGAGG-3’
402
TGFB3 ex5
5’-CACGGGGCATCTTTCAGTGG-3’
5’-TGAGTGTGGCTTGGCTCTGG-3’
502
TGFB3 ex6
5’-TGGCATGAAAGGACTCCAAGG3’
5’-GGACTGCCCCTGGAACTGG-3’
5’-TCACCCCAGACCATCATTTGC-3’
365
5’-TCCCTTTCCTCTATCCCCATCC-3’
532
TGFB3 ex7
Amplicon size
Detailed Methods:
Immunohistochemistry: Protocol for staining of FFPE sections was adapted from Baschong et al. with
the following modifications. Slides were stained overnight at 4°C with anti-pSmad2 antibody (clone A5S,
Millipore, Cat. # 04-953, 1:100) and anti-pERK1/2 (clone D13.14.4E, Cell Signaling, Cat. # 4370, 1:100)
in 0.1% Triton/TBS buffer, washed 3x10min in Perm/Staining buffer, and then stained with anti-rabbit
Alexa594 (Molecular Probes) at 1:200 for 1h at room temperature (RT). Slides were then washed
3x10min in Perm/Staining buffer and mounted with Hard Set VECTASHIELD® Mounting Media with
DAPI. Images were acquired on Zeiss AxioExaminer with 710NLO-Meta multiphoton confocal
microscope at a 25x magnification.
For removal of paraffin, slides were immersed in xylene (3x5 min), followed by graded ethanol treatment
(3x5’ 100%, 3x5’ 95%, 3x5’ 80%, 3x5’ 70%), rinsed in water and immersed in Tris buffered saline (TBS)
buffer (50 mM Tris-Cl, pH 7.6, 150 mM NaCl). Antigen retrieval was performed in a rice steamer using
1M Sodium Citrate Buffer/0.1% Tween for 30 min. Slides were then cooled to RT for 20 min, rinsed in
water, and transferred to TBS buffer. In order to minimize autofluorescence background, slides were then
treated with freshly prepared “bubbling” 10mg/ml of sodium borohydride in TBS for 30 minutes and then
rinsed 3 times in TBS. Slides were then permeabilized with Perm/Staining buffer (0.1% Triton in TBS
buffer) for 20 min, followed by a 20 min treatment with Fc block Reagent (Innovex Biosciences) and a 20
min treatment with Background Buster Reagent (Innovex Biosciences).
In situ RNA with ACD® RNAscope probes: The ACD® RNAscope probe Hs-TGFB1 probe
(cat#400881) was used to detect human TGFB1 transcript in conjunction with the RNAscope® 2.0 HD
Reagent Kit (RED) from ACD®. FFPE sections were stained following the manufacturer recommended
protocol, except that slides were counterstained with DAPI and not with hematoxylin. Red fluorescent
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TGFB3 mutations cause aortic aneurysms and dissections
signal was detected on Zeiss AxioExaminer with 710NLO-Meta multiphoton confocal microscope at a
25x magnification.
Online Figure 2: To investigate the effect of the TGFB3 c.754+2T>C variant on splicing, we designed
three different PCR experiments using blood complementary DNA (cDNA) from two patients from
family 1 (II:12 and III:11). The investigated fragments covered TGFB3 exons 2-5, 3-6 and 2-6. PCR
fragments corresponding to exons 2 to 5 are shown in the gel picture (in duplicate, patients in lanes 2 to 5
and normal controls in lanes 6-7). Normal expected size was 466 bp. An abnormal, smaller fragment,
probably with a deletion of exon 4 was observed in the patients.
Sanger sequencing using the smaller PCR band as template confirmed that the TGFB3 c.754+2T>C
mutation is leading to a deletion of exon 4 with an in-frame deletion of 108 nucleotides.
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TGFB3 mutations cause aortic aneurysms and dissections
Online Table 2. Clinical characteristics of family 1 (c.754+2T>C, p.Glu216_Lys251del)
Person
1-II:4
1-II:5*
1-II:7
1-II:10
1-III:6*
1-III:8
1-IV:1*
1-IV:2*
1-II:12*
1-II:14*
1-III:11*
1-II:16*
1-III:13*
Age
Sex
Height
Vascular
57†
M
Ruptured
descending
TAA at age
57
AAD at age
57
56†
M
Carotid artery
surgery
Descending
TAA at age
50
Infrarenal AA
at age 52
40†
M
Aortic
rupture at
age 40
74
F
168 cm
No
imaging
51
M
183 cm
Aortic sinus:
38 mm
49
M
190 cm
Aortic sinus:
36 mm
Stroke at age
48
17
F
181 cm
Aortic sinus:
32 mm
24
M
198 cm
No
imaging
72
F
162 cm
Dissection
aortic arch
at age 45
Aneurysm
left
subclavian
artery at
age 45
72
F
?
Infrarenal
AA at age
68
47
M
185 cm
TAA
69
F
168 cm
Aortic sinus:
32 mm
Ascending
aorta: 35 mm
44
M
187 cm
Aortic sinus:
39 mm
Aneurysm right
common iliac
artery at age 44
Mild MVP
MI with
chorda
rupture
surgery at age
49
Mitral
valve
Skeletal
Long face
Pectus
excavatum
High-arched
palate
Retrognathia
Mild scoliosis
Mild joint
laxity
Pectus
carinatum
surgery at
age 14
Higharched
palate
Retrognathia
Soft skin
Easy bruising
LDS
features
Other
MVP with
MI grade 3
TI grade 3
Varices
Long face
Mild MVP
Long face
Pes plani
Mild scoliosis
Total hip
replacement
Hiatal
hernia
Pelvic organ
prolapse
Hiatal hernia
Varices
Severe scoliosis
Spondylodesis
Th4-L1
Long face
Retrognathia
Thin skin
Easy
bruising
Varices
Hiatal
hernia
AA: aortic aneurysm; AAD: abdominal aortic dissection; TAA(D): thoracic aortic aneurysm and (dissection); MVP: mitral valve prolapse; MI: mitral insufficiency, TI: tricuspid insufficiency; Th:
thoracic vertebra; L: lumbar vertebra; †: deceased. * Mutation present. No DNA was available from the other patients.
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TGFB3 mutations cause aortic aneurysms and dissections
Online Table 3. Clinical characteristics of family 2 (c.898C>T, p.Arg300Trp)
Person
Age
Sex
Aorta
2-II:1
55†
M
Cerebral
aneurysm
dissection
2-II:4
60
F
Normal echo
2-III:3
26
F
Normal echo
Skeletal
Arachnodactyly
Pectus excavatum
Joint
hypermobility
Arm-span to
height ratio >
1.05
Bifid uvula
Bifid uvula
Hypertelorism
Blue sclerae
LDS features
2-IV:1
6
M
Aortic dilatation
Cleft palate
Hypertelorism
Other
2-III:7*
31
F
Aortic sinus: 28 mm
at age 29 years
(Z score = -0.25)
2-IV:2*
7
F
Normal echo
at age 4 years
2-IV:3
6
M
Normal echo
at age 5 years
Pectus carinatum
Cervical rib
Tall stature
Cervical spine
instability C2-C4
Club feet
Joint hypermobility
Hip dysplasia
Arachnodactyly
Joint hypermobility
Bifid uvula
Hypertelorism
Translucent skin
Bifid uvula
Hypertelorism
Bifid uvula
Hypertelorism
Hemiparesis – cause
unknown
Learning difficulties
* Mutation present. No DNA was available from the other patients; C: cervical vertebra; †: deceased
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TGFB3 mutations cause aortic aneurysms and dissections
Online Table 4. Clinical characteristics of families 3-6 (c.787G>C, p.Asp263His in family 3, c.898C>T, p.Arg300Trp in families 4 and 5, c.1095C>A, p.Tyr365* in family 6).
Person
Age
Sex
Aorta
3-II:2*
64
F
Aortic sinus:
34 mm
Mild MI
3-II:3*
70
F
Aortic sinus:
38 mm
Mild MI
3-III:1*
31
M
TAAD at age 30
with root = 70 mm
Skeletal
Dolichocephaly
High-arched palate
LDS
features
Retrognathia with
surgery at age 22
Other
Delayed puberty
Short stature:
growth hormone
therapy
3-III:2*
34
M
Aortic sinus:
32 mm
4-II:2*
42
M
Aortic sinus: 42
mm
4-III:1
13
F
Normal echo
Arachnodactyly
Increased armspan to height
ratio
Retrognathia
Scoliosis
Pes plani
Bifid uvula
Hypertelorism
Soft skin
Arachnodactyly
Asymmetric chest
Narrow palate
with dental
crowding
Inguinal hernia
surgery at age 6
years
Inguinal hernia
Cleft palate
4-III:2
10
F
Normal
echo
Cleft palate
5-II:1*
24
M
Aortic sinus: 40
mm (Z=3.6)
MVP, PFO
Pectus carinatum
Pes plani
6-II:1*
42
F
Normal echo
Bifid uvula
Cleft palate
Hypertelorism
Club foot
Bifid uvula
Hypertelorism
Easy bruising
Arachnodactyly
Joint hyperlaxity
Camptodactyly toes
Pes plani
Delayed motor
development
Varices
Cataract
MI: mitral insufficiency; MVP: mitral valve prolapse; PFO: patent foramen ovale; *Mutation present. No DNA was available from the other patients
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TGFB3 mutations cause aortic aneurysms and dissections
Online Table 5. Clinical characteristics of families 7-8 (c.1157delT, p.Leu386Argfs*21 in family 7; c.1202T>C, p.Leu401Pro in family 8).
Person
7-I:1
7-II:1*
7-II:4
7-III:4
8-II:1*
8-III:1*
Age
Sex
Aorta
70†
M
TAA at age 61 with
TAAD at age 70
43
M
No aneurysm
MVP
?
F
14
F
Normal
echocardiography
30
F
Normal
echocardiography
Skeletal
Tall stature
Dolichocephaly
Tall stature
Arachnodactyly
Scoliosis
Pes plani
Tall stature
Arachnodactyly
Tall stature
High arched palate
Arachnodactyly
Scoliosis
50†
M
Age 42: ascending
aorta of 60 mm
At age 43: Type A
dissection with
supracoronary
replacement
At age 44: TEVAR
for type B dissection
Age 50: abdominal
aortic dissection
Enlarged arm span
Arachnodactyly
Dolichocephaly
Mild scoliosis
LDS features
Cleft palate and
bifid uvula
Bifid uvula
Hypertelorism
Other
Enlarged arm span
Arachnodactyly
Scoliosis
Joint hypermobility
Metatarsus adductus
Camptodactyly of
4th/5th toes
Hypertelorism
Spondylosis C3-C4,
C5-C6 and C6-C7
Bilateral inguinal
hernia surgery at
age 39
TAA(D): thoracic aortic aneurysm and (dissection); MVP: mitral valve prolapse; PFO: patent foramen ovale; C: cervical vertebra; TEVAR: Thoracic EndoVascular Aortic Repair; *Mutation present.
No DNA was available from the other patients; †: deceased
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TGFB3 mutations cause aortic aneurysms and dissections
Online Table 6. Clinical characteristics of families 9-10 (c.704delA, p.Asn235Metfs*11 in family 9; c.965T>C, p.Ile322Thr in family 10;
c.898C>T,p.Arg300Trp in family 11).
Person
9-I:1
9-II:2*
9-III:1*
10-II:1*
10-III:1*
10-III:2*
11-I:1*
11-II:1*
Age
Sex
Aorta
80†
M
TAAD at
age 80
67
F
TAAD type A
at age 59
43
M
TAAD type A
at age 40
24
M
Aortic root 32
mm
22
F
Aortic root 34 mm
27
M
3
F
VSD+ASD
Aortic root
aneurysm: 19.5
mm (Z>2)
Thin and tall
habitus
Arachnodactyly
Tall stature
Kyphoscoliosis
Pectus
deformity
Retrognathia
Flat occiput
56
M
AAA with Ygraft at age 43
TAA (47 mm)
with severe AR:
Bentall surgery
Tall stature
Arachnodactyly
Kyphoscoliosis
Pectus deformity
Retrognathia
Down-slanting
palp fissures
Tall stature
Arachnodactyly
Scoliosis
Tall stature
Arachnodactyly
Joint hypermobility
Kyphoscoliosis
Pes plani
Retrognathia
Down-slanting palp
fissures
Tall stature
Arachnodactyly
Joint
hypermobility
High arched
palate
Kyphosis
Pes plani
Tall stature
Arachnodactyly
Joint
hypermobility
Retrognathia
Down-slanting
palpebral fissures
Hypertelorism
Bifid uvula
Hypertelorism
Bifid uvula
Spondylolisthesis
Hypertelorism
Bifid uvula
Osteoarthritis
Translucent skin
Easy bruising
Skeletal
Hypertelorism
Osteoarthritis
LDS
features
Other
Inguinal hernia
Myopia
Striae
TAA(D): thoracic aortic aneurysm and (dissection); AAA: abdominal aortic aneurysm; ASD, atrial septal defect; VSD, ventricular septal defect; *Mutation present, No DNA available on other patients;
†: decease
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TGFB3 mutations cause aortic aneurysms and dissections
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