Family 1 c.1463T>C p.Leu488Pro Family 2 c.248T>C p.Leu83Pro Family 3 c.1444G>T p.Gly482Trp Family 4 c.1238G>A p.Arg413Glu b (a) Affected individuals of the four families from this study are in bold (b). The increased bone density in GHDD is characterised by diaphyseal involvement, abnormal long bone modelling and cortical hyperostosis. a Supplementary figure 1: families pedigree and skeletal x-rays of affected individuals Thromboxane synthase mutations in an increased bone density disorder (Ghosal syndrome). David Geneviève, Valérie Proulle, Bertrand Isidor, Samuel Bellais, Valérie Serre, Fatima Djouadi, Capucine Picard, Capucine Vignon-Savoye, Brigitte Bader-Meunier, Stéphane Blanche, Marie- Christine de Vernejoul, Laurence Legeai-Mallet, Anne-Marie Fischer, Martine Le Merrer, Marie Dreyfus, Pascale Gaussem, Arnold Munnich and Valérie Cormier-Daire.
Transcript
Family 1
c.1463T>C
p.Leu488Pro
Family 2
c.248T>C
p.Leu83Pro
Family 3
c.1444G>T
p.Gly482Trp
Family 4
c.1238G>A
p.Arg413Glu
b
(a) Affected individuals of the four families from this study are in bold
(b). The increased bone density in GHDD is characterised by diaphyseal involvement, abnormal
long bone modelling and cortical hyperostosis.
a
Supplementary figure 1: families pedigree and skeletal x-rays of affected individuals
Thromboxane synthase mutations in an increased bone density disorder (Ghosal syndrome).
David Geneviève, Valérie Proulle, Bertrand Isidor, Samuel Bellais, Valérie Serre, Fatima Djouadi,
Conservation across species of the four amino acids changes found in this study. * indicate amino acids with hydrogen bonds to the A-ring propionate of the heme (N110; R413 and R478); indicate amino acids with hydrogen bonds to the D-ring propionate of the heme (W133; R137). In bold, the amino acid changes observed in patients.
Supplementary Figure 3:
OB OC Ch M F Ctrl ΨΨ Ly
TBXAS1
GAPDH
RT-PCR in human osteoblasts (OB), osteoclasts (OC), chondrocytes, muscle,
human lymphoblastoïd cell line and skin fibroblasts. Ψ = DNA ladder
Supplementary Figure 4: Expression profile of TBXAS1
Su
pp
lem
en
tary T
ab
le 1
: C
linic
al, bio
logic
al a
nd r
adio
logic
al d
ata
in f
ou
r G
HD
D f
amil
ies.
F
amil
y 1
F
amil
y 2
F
amil
y 3
F
amil
y 4
Pat
ient
II1
II
2
III1
II
1
II3
II
2
II6
II
1
II2
II
5
Geo
gra
phic
ori
gin
A
lger
ia
Tunis
ia
Tunis
ia
Pak
ista
n
Consa
nguin
ity
1/1
6
? 1/3
2
1/1
6
Age
at d
iagnosi
s 34 y
ears
24 y
ears
16 m
onth
s 25 m
onth
s 18 m
onth
s 16 y
ears
2 y
ears
13 y
ears
13 y
ears
5 m
onth
s
Gen
der
M
M
M
M
M
F
M
F
F
F
“Asy
mpto
mat
ic”
- +
-
- -
+
- +
+
-
Cort
icost
eroid
ther
apy
+
- +
-
+
- +
-
- +
Anem
ia
+
+ (
mil
d)
+
+*
+*
- +
-
- +
Thro
mbocyto
pen
ia
+
- +
+
+
-
+
- -
+
Thro
mbocyto
sis
epis
odes
-
- -
- -
- -
- -
+
Leu
copen
ia
- -
- -
- -
- -
- -
Bio
logic
al i
nfl
amm
atio
n
+
- +
-
- -
+
- -
+
Dia
ph
yse
al d
ysp
lasi
a +
+
+
+
+
+
+
+
+
+
Blo
od P
ress
ure
142/7
3a
(35 y
ears
) N
D
100/4
5
(6 y
ears
)
105/6
2
(6 y
ears
)
88/5
6
(1 y
ear)
N
D
110/7
0
(7 y
ears
) N
D
115/6
2
14 y
ears
122/6
5
(6 y
ears
)
IVY
tes
t (n
orm
al a
dult
: 4-8
min
, C
hil
d:
2 t
o 4
min
) N
D
4.5
min
N
D
ND
3 m
in
ND
2.5
min
N
D
ND
N
D
Bone
den
sity
(adult
: T
sco
re, S
D;
ahil
d:
Z
score
, S
D)
+ 2
.37
ND
N
D
+ 2
.4
+2.3
N
D
+3.3
N
D
ND
+
3.9
TB
XA
S1 m
uta
tion
c.1463T
>C
, p.L
eu488P
ro
c.248T
>C
, p.L
eu83P
ro
c.1444G
>T
, p.G
ly482T
rp
c.1238G
>A
, p.A
rg413G
lu
* S
ponta
neo
us
rem
issi
on. N
D:
not
done.
a this
pat
ient
pre
sent
a re
nal
am
ylo
idosi
s (p
robab
ly d
ue
to c
hro
nic
infl
amm
atio
n)
wh
ich e
xpla
in t
he
arte
rial
hyper
tensi
on.
Supplementary Table 2: TXB2 and PGE2 levels in plasma rich platelet from GHDD patient
compared to control.
TXB2 levels (ng/ml) Before AA induction AA 0.25 mg/ml AA 0.5 mg/ml
Patient II1 (Family 1) 1.9 4.2 5.8
Patient II2 (Family 1) 2.27 3.5 4.3
Control 325 450 870
PGE2 levels (ng/ml) Before AA induction AA 0.25 mg/ml AA 0.5 mg/ml
Patient II1 (Family 1) 182.5 342 620
Patient II2 (Family 1) 50 580 625
Control 9.8 30.5 52.5
AA: arachidonic acid
Online Materials and Methods
Affected individuals. We studied 10 affected individuals belonging to 4 inbred families.
Criteria diagnostic for inclusion were: i) severe anemia requiring blood infusions and ii)
skeletal changes such as diaphyseal dysplasia with increase bone density, abnormal long bone
modeling, cortical hyperostosis and normal metaphyses and epiphyses. DNA samples were
obtained after signed informed consent;
Mutation detection. A series of 17 intronic primers was designed to amplify the 4 non-
coding and 13 coding exons of the TBXAS1 gene (primer sequences available on request). The
amplification products were purified and sequenced using the fluorescent dideoxy-terminator
method on an automatic sequencer (ABI 3100)
RT-PCR. Total RNAs were extracted from human muscle, primary culture cells (osteoblasts,
chondrocytes and fibroblasts) and from cord blood cells transdifferentiation into osteoclasts
using the RNeasy Mini Kit (Qiagen). cDNA were synthetized by priming with random
hexamers in the presence of MuLV reverse transcriptase using the manufacturer’s protocol
(GeneAmp RNA PCR Core Kit, Roche). A total of 30 PCR cycles were performed at an
annealing temperature of 60°C to amplify a 772-bp fragment specific for TBXAS1 including
exon 12 known to be spliced (primer sequences available on request). Sequence of sense and
antisense primers used for GAPDH amplification are available on request.
The three dimensional structure of the human Thromboxane synthase (isoform TXS-I) was
modeled by comparative modeling methods and energy minimization using the program
Swiss-Model in the optimized mode1. The degree of identity between TXAS and five
templates (41.31 %, 40.01 %, 37.52 %, 27.72 % and 33.24 % for 1W0F, 1TQN, 2j0C, 1OG5
and 1BVY respectively) allowed us to model the structure of human TXAS using the X-ray
coordinates of these templates. The 2.65 Å (PDB code 1W0F), 2.05 Å (1TQN), 2.80 Å
(2J0C), 2.55 Å (1OG5) and 2.03 Å (1BVY) coordinate sets were used as templates for the
structural model. The overall folding was quite similar to structurally established P450
proteins, except for two regions (amino acids 260-277 and 295-330) due to a lack of structural
informations. Residues 32-534 of the submitted sequence were used to build the model.
Swiss-Pdb Viewer 3.7 was used to visualize the structures and to analyze the structural insight
into TSAS-I mutations.
Thromboxane and Prostaglandin E2 Levels.
De novo levels of TXB2 and PGE2 production in plasma rich platelet before and after AA
induction were directly determined in patients II1 and II2 from family 1 by Enzyme-linked
immunosorbent assay (ELISA) (Cayman chemical ref 519031.1 and 514010.1, Ann Arbor,
MI) and compared to sex and age match control. ELISA data were analysed using Revelation
software V4.22 (Dynec technology).
Platelet aggregation tests. Aggregation studies were performed within 2 hours of blood
collection, at 37°C, by using a photometric method on a 4-channel aggregometer (Regulest,
Amneville, France)2. Briefly, a
280-µL aliquot of platelet-rich plasma was incubated for 3
minutes at 37°C and was then stirred at 1100 rpm for 2 minutes before adding
20 µL of saline
or following agonists: arachidonic acid 1.5, 0.75 and 0.375 mmol/L (Helena), ADP 2, 5 and
![program-divadelni-predstaveni [režim kompatibility] · family" was written as a story of Czech Slovakian family pech. All Pechs grew abit older and it was Grandpa Pech Jubilee](https://static.dokumenty.site/doc/80x56/6038b98d6e0ea366ba711ec3/program-divadelni-predstaveni-reim-kompatibility-family-was-written.jpg)
