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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.
p.Leu488Prop.Leu488Pro
p.Leu83Prop.Leu83Prop.Arg413Glup.Arg413Glu
p.Gly482Trpp.Gly482Trp
b
COOH
Family 2 Family 4 Family 3Family 1
c.1463T>C
p.Leu488Pro
c.248T>C
p.Leu83Pro
c.1444G>T
p.Gly482Trp
c.1238G>A
p.Arg413Glu
a
1 1131 533
NH2
L83P
R413Q
G482W
L488P
p.Gly482Trpp.Gly482Trp
c
(a) The location of the mutations detected in GHDD families is indicated in red and green.
(b) Three dimensional Swiss-Pdb Viewer representation of TXAS. The four mutated residues were
located in one of the seven β-sheet (amino acid 83, in green), in the vicinity of the heme and the
enzymatic pouch (amino acids 413 and 482, in red) and in an helix α (amino acid 488, in green).
(c) Swiss-Pdb Viewer representation of the van der Waals volumes of the amino acid in position 482.
The glycine (left) is changed into tryptophan in family 3 (right). Note the modification of the steric
hindrance at the vicinity of the catalytic site (heme in blue).
Supplementary Figure 2: Schematic representation of the human Thromboxane synthase (TXAS).
10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| TBXAS1 Human 1 MMEALGFLKLEVNGPMVTVALSVALLALLKWYSTSAFSRLEKLGLRHPKPSPFIGNLTFFRQGFWESQME 70 TBXAS1 Pan Troglodyt 1 -..V..LFR...S..........VF...................I............A.........H.. 69 TBXAS1 Canis familia 1 -..V.S.................V..........A.........I............P..C......... 69 TBXAS1 Mus musculus 1 -..V..L..F..S.TI...T.L...........M..........I........V...M..........L. 69 TBXAS1 Rattus norveg 1 -..V..L..F..S.TV...T...V................R...I...E....V...M.........HL. 69 TBXAS1 C. elegans 1 -----------MSLGF.LAVTFSIF.GI.TY.-LWIWTYWMRK.VKG.RGR..V.V.DVLLEHETPGLIK 58
80 90 100 110 120 130 140 ....|....|....|P...|....|....|....|....|..*.|....|....|....|....|... | TBXAS1 Human 71 L---RKLYGPLCGYYLGRRMFIVISEPDMIKQVLVENFSNFTNRMASGLEFKSVADS---VLFLRDKRWE 134 TBXAS1 Pan Troglodyt 70 .---..Q....S......L...........E.....K.........T...P.P....---.......... 133 TBXAS1 Canis familia 70 .---..................................H......V....S.P.M..---.......... 133 TBXAS1 Mus musculus 70 .---.ER.............HV...................S........P.M....---..L...R... 133 TBXAS1 Rattus norveg 70 .---.ER.............Y....D.....E.........S........P.LI...---..M...R... 133 TBXAS1 C. elegans 59 .GEWT.K..KVY..TD.TQRTL.VAD.A.VHEIF.KQ.D..YG.KLNPIQGNPEKEQRVHL.AAQGY..K 128
150 160 170 180 190 200 210 .. .|....|....|....|....|....|....|....|....|....|....|....|....|....| TBXAS1 Human 135 EVRGALMSAFSPEKLNEMVPLISQACELLLAHLKRYAESGDAFDIQRCYCNYTTDVVASVAFGTPVDSWQ 204 TBXAS1 Pan Troglodyt 134 ...SV.TV.......S..T....R..DV.....E.H.Q..E......T..C.............E.N.QE 203 TBXAS1 Canis familia 134 ...SV.T...........T.......D.......H..............SC.............Q...RR 203 TBXAS1 Mus musculus 134 ........S......D..T..........V.......A.R...N......C..I..........Q...QN 203 TBXAS1 Rattus norveg 134 ..................T...........S...HS.A............CF..N........IE.N.QD 203 TBXAS1 C. elegans 129 RL.TISSQS..NAS.KK.KRTVEDSALE..R.IEKQTAG.EQI.ML.F.QE..M..IGRF.M.QTDS--M 196
220 230 240 250 260 270 280 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| TBXAS1 Human 205 APEDPFVKHCKRFFEFCIPRPILVLLLSFPS--IMVPLARILPNKNRDELNGFFNKLIRNVIALRDQQAA 272 TBXAS1 Pan Troglodyt 204 ...H...E..R...ASS..K.L.........--..................................... 271 TBXAS1 Canis familia 204 ..G.......R...AYS........I.....--..................................... 271 TBXAS1 Mus musculus 204 S......Q..R.AST......L...I.....--......................T.............. 271 TBXAS1 Rattus norveg 204 .......Q..Q.V.A.ST...L.A.I.....--......................T..........K.T. 271 TBXAS1 C. elegans 197 MFKN.I.NVVREI.CGSRKNLM.ICQVFP.IGQFIRD.TFKF.RIPAFK.YSIMQDVVAAR..Q.EREKG 266
290 300 310 320 330 340 350 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| TBXAS1 Human 273 EE--RRRDFLQMVLDARHSASPMGVQDFDIVRDVFSSTGCKPNPSRQHQPSPMARPLTVDEIVGQAFIFL 340 TBXAS1 Pan Troglodyt 272 ..--.........Q.V....ATV..EN.....Q...A.K.PA..P.R.L.R.LSK..S...V........ 339 TBXAS1 Canis familia 272 ..--.......LI.....L.TSL..DS..M..Q.....D.TVG...P...RHLSQ...L........... 339 TBXAS1 Mus musculus 272 ..--............Q..MNSV..EG..M.PESL..SE.TKE.PQRCH.TSTSK.F..........L.. 339 TBXAS1 Rattus norveg 272 ..--..G.........QR.M.SV..EA..M.TEAL..AE.MGD.PQRCH.TST.K.......A....L.. 339 TBXAS1 C. elegans 267 A.SGEPQ..IDLF....SDDVDFSAEAREDFSKRN---------------LKITKE.SA..V...C.L.. 321
360 370 380 390 400 410 420 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| TBXAS1 Human 341 IAGYEIITNTLSFATYLLATNPDCQEKLLREVDVFKEKHMAPEFCSLEEGLPYLDMVIAETLRMYPPAFR 410 TBXAS1 Pan Troglodyt 340 ......V...............E......E...C.SKE.L...Y...Q..........K........... 409 TBXAS1 Canis familia 340 .............................A...S....YT.LDY...Q...............I...... 409 TBXAS1 Mus musculus 340 ...H.V.......I......H.....R..K...L.MG..P...YH..Q..........S........... 409 TBXAS1 Rattus norveg 340 ...H..T......I......H.E...R..K...L.M...P...Y.N.Q.........V............ 409 TBXAS1 C. elegans 322 .G.FDTTALS..YV.....V..KI...VIE.IAREFG-TSEV..EK.GR-.K.M.C..K.A..L..L.SI 389
* 430 440 450 460 470 480 490 ...Q|....|....|....|....|....|....|....|....|....|....|....|....|....*TBXAS1 Human 410 -FTREAAQDCEVLGQRIPAGAVLEMAVGALHHDPEHWPS-PETFNPERFTAEARQQHRPFTYLPFGAGPR 478 TBXAS1 Pan Troglodyt 409 -...V...................T......Y......N-..N..........Q.RR..Y.......... 477 TBXAS1 Canis familia 409 -......R....R.........V.V......R...Y..Q-.........K...QRRQQ............ 477 TBXAS1 Mus musculus 409 -...................T...I.............N-....D.........L.R............. 477 TBXAS1 Rattus norveg 409 -..............H....S...I.............N-....D.........L.QK............ 477 TBXAS1 C. elegans 390 SNS.KCMKTTT.N.VK.E..VYVQ.DTWS..Y...L.GEDVKE.K...WSTDEPLE.KG-A.....L... 458
500 510 520 530 540 ...W|....P....|....|....|....|....|....|....|....|....|... TBXAS1 Human 479 SCLGVRLGLLEVKLTLLHVLHKFRFQACPETQVPLQLESKSALGPKNGVYIKIVSR-- 534 TBXAS1 Pan Troglodyt 478 ...........L......I.R....E.........................R....-- 533 TBXAS1 Canis familia 478 .................Q...Q...E...........D..........I.......-- 533 TBXAS1 Mus musculus 478 ..........V....I.Q.......E.S............................-- 533 TBXAS1 Rattus norveg 478 ..........V......Q.......E.................C......V.....-- 533 TBXAS1 C. elegans 459 Q.I.M..AIM.Q.IL.T.L.KNYT.ETGNK.RI..K.VGSATTS.ED-.FVHLRP.IW 515
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
10 µmol/L (Sigma Aldrich), U46619 1.5 µmol/L (Calbiochem) and collagen 1 µg/mL
(Horm). Platelets aggregate when the agonist is added, thereby leading to an increase in light
transmission, which is recorded for 5 min. Aggregation was expressed as the maximal percent
change in light transmission from baseline, using platelet-poor plasma as reference (arbitrarily
100%).
Analysis of platelet glycoproteins and platelet activation by flow cytometry. Surface
expression of platelet glycoproteins GPIb (CD42b), GPIIIa (CD61) and P-selectin (CD62P)
before and after activation was performed in plasma rich platelet by flow cytometry using a
FACSCalibur cytometer (Becton Dickinson) and using Platelet GP Receptors (Biocytex)
according to the manufacturer instructions3. Activation was achieved using TRAP (50 µM ,
25 µM, 12.5 µM, NeoMPS), arachidonic acid (0.5 mg/mL, Sigma), U46619 (5 µM, VWR
Calbiochem). Results are expressed in percentage of the mean fluorescence intensity (MFI) at
the surface of patient’s platelets compared to the MFI at the surface of a control studied in the
same conditions. Change in P-selectin expression induced by agonistS is expressed in MFI.
Osteoblasts culture and real-time quantitative PCR. Human normal cranial suture were
obtained after signed informed consent during routine surgery for craniosynostosis.
Osteoblasts were cultured in Dulbecco's modified Eagle medium (with
10% fetal bovine
serum) as described elsewhere4. Ozagrel (Cayman Chemical) was daily added at 0.25 µM
(IC50 of 11nM) in culture media during 3 days and CTA2 (Cayman Chemical) was added at
day one in culture media at two concentrations (5 µM and 15 µM). Cells were harvested at
day 3. Real-time quantitative PCR using Light Cycler
technology (Roche Manheim) was
performed for OPG and RANKL mRNA in each sample. The experiments were performed five
times. In each experiment, samples were run in
triplicate. The amounts of
OPG and RANKL
mRNA were normalized to the amount of β-actin mRNA. Student test was used for p values
determination.
1. Schwede, T. Kopp, J. Guex, N. Peitsch, M.C. SWISS-MODEL: An automated protein
homology-modeling server. Nucleic Acids Res 31, 3381-3385 (2003).
2. Dupont, A. et al. An intronic polymorphism in the PAR-1 gene is associated with
platelet receptor density and the response to SFLLRN. Blood. 101, 1833-1840 (2003).
3. Hezard, N. et al. Unexpected persistence of platelet hyporeactivity beyond the neonatal
period: a flow cytometric study in neonates, infants and older children. Thromb Haemost.
90, 116-123 (2003).
4. De Pollack, C. Renier, D. Hott, M. & Marie, P.J. Increased bone formation and
osteoblastic cell phenotype in premature cranial suture ossification (craniosynostosis). J.
Bone. Miner. Res. 11:401-407 (1996).