A new nonsense mutation of SMAD8 associated with pulmonary arterial hypertension
Pulmonary arterial hypertension (PAH) is a progressive disorder characterised by raised pulmonary artery pressures with pathological changes in small pulmonary arteries. Previous studies have shown that approximately 70% of familial PAH and also 11-40% of idiopathic PAH (IPAH) cases have mutations in the bone morphogenetic protein receptor type II (BMPR2) gene. In addition, mutations in the activin receptor-like kinase 1 (ALK1) gene have been reported in PAH patients. Since both the BMPR2 and ALK1 belonging to the transforming growth factor (TGF)-beta superfamily are known to predispose to PAH, mutations in other genes of the TGF-beta/BMP signalling pathways may also predispose to PAH. We screened for mutations in ENDOGLIN(ENG), SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6 and SMAD8 genes, which are involved in the TGF-beta/BMP signallings, in 23 patients with IPAH who had no mutations in BMPR2 or ALK1. A nonsense mutation in SMAD8 designated c.606 C>A, p.C202X was identified in one patient. The father of this patient was also identified as having the same mutation. Functional analysis showed the truncated form of the SMAD8 C202X protein was not phosphorylated by constitutively active ALK3 and ALK1. The SMAD8 mutant was also unable to interact with SMAD4. The response to BMP was analysed using promoter-reporter activities with SMAD4 and/or ca-ALK3. The transcriptional activation of the SMAD8 mutant was inefficient compared with the SMAD8 wild type. We describe the first mutation in SMAD8 in a patient with IPAH. Our findings suggest the involvement of SMAD8 in the pathogenesis of PAH.
A new nonsense mutation of SMAD8 associated
with pulmonary arterial hypertension
c Additional tables and figures
are published online only at
International Research and
Educational Institute for
Integrated Medical Sciences
(IREIIMS), Tokyo Women’s
Medical University, Tokyo,
Division of Genomic
Medicine, Institute of Advanced
Biomedical Engineering and
Science, Graduate School of
Medicine, Tokyo Women’s
Medical University, Tokyo,
Pediatrics, Toho University
Medical Center, Omori Hospital,
Pediatric Cardiology, Tokyo
Women’s Medical University,
Dr R Matsuoka, International
Research and Educational
Institute for Integrated Medical
Sciences (IREIIMS), Tokyo
Women’s Medical University,
8-1 Kawada-cho, Shinjuku-ku,
Tokyo, 162-8666, Japan;
Received 9 September 2008
Revised 13 December 2008
Accepted 21 January 2009
Published Online First
26 February 2009
Background: Pulmonary arterial hypertension (PAH) is a
progressive disorder characterised by raised pulmonary
artery pressures with pathological changes in small
pulmonary arteries. Previous studies have shown that
approximately 70% of familial PAH and also 11–40% of
idiopathic PAH (IPAH) cases have mutations in the bone
morphogenetic protein receptor type II (BMPR2) gene. In
addition, mutations in the activin receptor-like kinase 1
(ALK1) gene have been reported in PAH patients. Since
both the BMPR2 and ALK1 belonging to the transforming
growth factor (TGF)-b superfamily are known to predis-
pose to PAH, mutations in other genes of the TGF-b/BMP
signalling pathways may also predispose to PAH.
Methods: We screened for mutations in
ENDOGLIN(ENG), SMAD1, SMAD2, SMAD3, SMAD4,
SMAD5, SMAD6 and SMAD8 genes, which are involved
in the TGF-b/BMP signallings, in 23 patients with IPAH
who had no mutations in BMPR2 or ALK1.
Results: A nonsense mutation in SMAD8 designated
c.606 C.A, p.C202X was identified in one patient. The
father of this patient was also identified as having the
same mutation. Functional analysis showed the truncated
form of the SMAD8 C202X protein was not phosphory-
lated by constitutively active ALK3 and ALK1. The SMAD8
mutant was also unable to interact with SMAD4. The
response to BMP was analysed using promoter-reporter
activities with SMAD4 and/or ca-ALK3. The transcriptional
activation of the SMAD8 mutant was inefficient compared
with the SMAD8 wild type.
Conclusion: We describe the first mutation in SMAD8 in
a patient with IPAH. Our findings suggest the involvement
of SMAD8 in the pathogenesis of PAH.
Pulmonary arterial hypertension (PAH; MIM
178600) is a rare condition, with an annual
incidence of 1–2 cases per 1 million in the general
It is characterised by abnormal
proliferation of endothelial and smooth muscle
cells in the pulmonary arterioles, which leads to
sustained elevation of mean pulmonary artery
pressure >25 mm Hg at rest and/or >30 mm Hg
Although the disease may pre-
sent at any age, PAH is usually diagnosed in the
fourth decade of life, with a female-to-male ratio of
Without modern treatments the disorder
progresses rapidly, leading to right heart failure
within 3 years of diagnosis. It has been reported
that among all PAH patients, familial PAH
accounts for at least 6% of the cases, with 10–
20% of penetrance.
A recent classification study proposed five sub-
groups of PAH: idiopathic PAH (IPAH); familial
PAH (FPAH); PAH associated with other disease
(APAH) such as collagen vascular disease, congenital
systemic to pulmonary shunts, portal hypertension,
and HIV infection; PAH associated with significant
venous or capillary involvement; and persistent
pulmonary hypertension of the newborn (PPHN).
A study in France reported that FPAH and IPAH
accounted for prevalence rates of 4.2% and 38.9%
in 553 PAH cases, respectively.
FPAH and IPAH
share the same clinical feature, histopathology and
clinical course, and FPAH has a low penetrance.
Therefore, the true incidence and prevalence of
FPAH have not been fully elucidated.
Heterogeneous germline mutations in the bone
morphogenetic protein (BMP) receptor type II gene
(BMPR2), a receptor for the transforming growth
factor (TGF)-b superfamily on chromosome 2q33,
have been identified in FPAH.
have been identified in up to 70% of FPAH and
11–40% of patients with IPAH.
Mutations in the activin receptor-like kinase 1
(ALK1) gene, a member of the TGF-b superfamily,
on chromosome 12q13 have also been reported in
and in patients with
hereditary haemorrhagic telangiectasia (HHT)
Except for the association with
BMPR2 and ALK1 mutations, there is a paucity of
published studies reporting broad and systematic
mutation screening of PAH patients.
both BMPR2 and ALK1 genes, which belong to the
TGF-b superfamily, are known to predispose to
PAH, the question is raised as to whether muta-
tions in other genes of this signalling pathway may
also predispose to PAH. In this study, 23 patients
clinically diagnosed with IPAH, who had no
mutations in BMPR2 or ALK1, were screened for
mutations in eight genes involved in the TGF-b/
BMP signalling pathway—ENDOGLIN (ENG),
SMAD1, SMAD2, SMAD3, SMAD4, SMAD5,
SMAD6 and SMAD8 (which also known as
SMAD9)—to determine whether any of them are
involved in the pathogenesis of PAH.
All assessments were done with the approval of the
ethics committees of Tokyo Women’s Medical
University and Toho University, Tokyo, Japan.
Patients were recruited from Tokyo Women’s
Medical University and Toho University. Written
informed consent was obtained from all study
subjects. If patients were under 16 years of age, the
informed consent was given by their guardians. We
assessed each patient by clinical history, physical
examination and a review of their medical records.
The diagnosis of IPAH/FPAH was made through
clinical evaluation, chest radiography, electrocardio-
graphy, echocardiography and cardiac catheterisation
J Med Genet 2009;46:331–337. doi:10.1136/jmg.2008.062703 331
based on current international consensus criteria (Venice 2003).
Patients with APAH, PAH associated with significant venous or
capillary involvement, PPHN and other pulmonary hypertensions
were excluded from this study by trained cardiologists.
This study contains individuals clinically diagnosed with
IPAH who were derived from two cohorts. The first cohort of
12 patients with IPAH was derived from a previous study
these 12 patients were confirmed to be without the BMPR2 or
ALK1 mutation by direct sequencing and multiplex ligation
dependent probe amplification (MLPA) analysis.
cohort contained 21 patients with either IPAH or FPAH, and
mutation analysis revealed 10 patients, nine of whom had
mutations inclusive of three patients with exonic deletions in
BMPR2, and one had a mutation in ALK1 (unpublished data).
These 10 patients were excluded from this study. The remaining
11 patients with IPAH from the second cohort and the 12
patients with IPAH from the first cohort, 23 patients in total,
who were confirmed to have no mutation in BMPR2 or ALK1,
were included in this study.
Summary of baseline characteristics and haemodynamic
parameters of the 23 patients is presented in table 1. Available
data on characteristics and haemodynamic parameters of 23
patients with IPAH are provided in supplemental table 1.
Sequencing and mutation analysis
Genomic DNA was extracted from peripheral blood leucocytes
of the patients or from their lymphoblastoid cell lines
transformed by the Epstein–Barr virus as described previously.
In the 23 patients with no mutations in BMPR2 or ALK1, all
coding exons and adjacent intronic regions for ENG, SMAD1,
SMAD2, SMAD3, SMAD4, SMAD5, SMAD6 and SMAD8 were
amplified using polymerase chain reaction (PCR primer details
are available in supplemental table 2). PCR amplified products
were purified and directly sequenced as previously described.
In all cases, any sequence variations found were reamplified
and resequenced to confirm the observed changes. The observed
changes were also confirmed by amplification refractory
mutation system (ARMS) assay.
The sequences generated were compared with wild type ENG
(GenBank accession number NM_000118), SMAD1 (GenBank
NM_005900), SMAD2 (GenBank NM_005901), SMAD3
(GenBank NM_005902), SMAD4 (GenBank NM_005359),
SMAD5 (GenBank NM_005903), SMAD6 (GenBank
NM_005585), and SMAD8 (GenBank NM_005905).
When a mutation was detected, we confirmed that it was not
present in 150 healthy controls by direct sequencing.
Plasmids and antibodies
Human pcDNA3.0-6xMyc-SMAD8, human pcDNA3.0-Flag-
SMAD4, human pcDNA3.0-ALK1-haemagglutinin (HA),
human pcDNA3.0-ALK3-HA, BMP-responsive promoter repor-
ter construct 3GC2-Lux, and TGF-b responsive promoter
reporter construct 3TP-Lux were kindly provided by Dr K
Miyazono (Tokyo, Japan). 3GC2-Lux contains three repeats of a
GC-rich sequence derived from the proximal BMP response
element in the Smad6 promoter.
3TP-Lux is a TGF-b
responsive luciferase reporter gene that contains three con-
secutive tetradecanoylphorbol acetate (TPA) response elements
and a portion of the plasminogen activator inhibitor 1 (PAI-1)
Human constitutively active (ca) ALK1 and ALK3 were
generated by mutation of Glu-201 into aspartic acid and a
mutation of Glu-233 into aspartic acid, respectively.
Site directed mutagenesis was carried out by a PCR based
approach. The constructed plasmids were verified by sequen-
cing. The antibodies used were as follows: anti-Flag antibody
(F3165, Sigma, St Louis, Missouri, USA), anti-HA antibody
(11867423001, Roche, Mannheim, Baden-Wu¨rttemberg,
Germany), anti-Myc antibody (#2276, Cell Signaling
Technology, Danvers, Massachusetts, USA), anti-Myc antibody
(#06-549, Upstate, Lake Placid, New York, USA) and anti-
phospho-Smad1/Smad5/Smad8 antibody (#9511, Cell
Transfection, cell lysis, immunoblotting and immunoprecipitation
HEK293 and COS1 cells were grown in DMEM/F-12 (Sigma)
supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand
Island, New York, USA), 100 units/ml penicillin/streptomycin
(Gibco) and 250 ng/ml amphotericin B (Sigma). Transfection
was performed with Lipofectamine 2000 reagent (Invitrogen,
Carlsbad, California, USA) according to the manufacturer’s
instructions. Twenty-four hours after transfection, the cells
were lysed in lysis buffer (1M Tris-HCl (pH 8.0) 50 mM, 0.5 M
EDTA 1 mM (pH8.0), 5 M NaCl 120 mM, NP-40 0.25%). For
co-immunoprecipitation assays, the lysates were incubated
with monoclonal anti-Flag antibody (Sigma) and protein G-
Sepharose beads (GE Healthcare, Little Chalfont,
Buckinghamshire, UK), and immunoblotted with polyclonal
anti-Myc antibody (Upstate).
COS1 cells were co-transfected in Opti-MEM (Invitrogen) using
Lipofectamine 2000 reagent (Invitrogen) with 3GC2-Lux or
p3TP-Lux and wild type or mutant pcDNA3.0-SMAD8 and/or
pcDNA3.0-SMAD4 and/or pcDNA3.0-ca-ALK3 (total 0.9 mg).
Twenty-four hours after transfection, the cells were harvested.
Firefly and renilla luciferase activities were measured with the
Dual luciferase reporter assay (Promega, Madison, Wisconsin,
USA) following manufacturer’s instructions. Results were
expressed as the ratio of firefly luciferase activity to renilla
luciferase activities. All assays were performed in triplicate.
All results are expressed as mean (SD). For statistical compar-
ison of two samples, a two-tailed Student’s t test was used
where applicable. Values of p,0.05 were considered significant.
Table 1 Baseline characteristics and haemodynamic
parameters (mean (SD)) (n = 23)
Age (years) 9.8 (7.1)
Gender (male/female) 9/14
Familial/idiopathic (clinically) 0/23
mPAP (mm Hg) 71.7 (23.3)
RAP (mm Hg) 9.2 (3.9)
) 2.5 (0.9)
) 33.7 (12.6)
) 27.5 (12.3)
PAWP (mm Hg) 10.2 (3.6)
CI, cardiac index; mPAP, mean pulmonary arterial pressure; PAWP,
pulmonary artery wedge pressure; PVR, pulmonary vascular
resistance; RAP, right atrial pressure; TPR, total pulmonary
332 J Med Genet 2009;46:331–337. doi:10.1136/jmg.2008.062703
To identify mutations in genes involved in the TGF-b/BMP
signalling pathway, we screened mutations in ENG, SMAD1,
SMAD2, SMAD3, SMAD4, SMAD5, SMAD6 and SMAD8 genes
in 23 patients with IPAH who had no mutations in BMPR2 or
In this study, no mutations were identified in ENG, SMAD1,
SMAD2, SMAD3, SMAD4, SMAD5 or SMAD6, whereas
common polymorphisms were found in ENG, SMAD3 and
SMAD6 (supplemental table 3).
However, a nonsense mutation in SMAD8, c.606 C.A,
p.C202X was identified in one patient (proband 14) (fig 1A, B).
The SMAD8 mutant introduces a premature stop codon into
exon 2 and results in a truncated protein that lacks 228 carboxy-
terminal amino acids, including the MH2 domain and the SXS
phosphorylation site (fig 1C).
The patient harbouring a mutation in SMAD8 was diagnosed
with PAH based on an accentuated second heart sound in the
pulmonary region during hospitalisation with pneumonia at
8 years of age. His haemodynamic data at 8 years of age showed
a mean pulmonary arterial pressure (mPAP) of 53 mm Hg, right
atrial pressure (RAP) of 5 mm Hg, cardiac index (CI) of
, total pulmonary resistance (TPR) of
, pulmonary vascular resistance (PVR)
of 12.4 Wood?Unit
and pulmonary artery wedge pressure
(PAWP) of 6 mm Hg. His condition progressed to World Health
Organization functional class III with episodes of shortness of
breath during physical activity at 9 years of age. He has been
receiving epoprostenol, continuous intravenous prostacyclin,
since the age of 9.
His current condition is WHO functional class I–II at 16 years
of age. His recent haemodynamic data showed mPAP of
Figure 1 SMAD8 mutation in pulmonary
arterial hypertension. (A) DNA sequences
showing c.606 C.AinSMAD8. (B) The
panel shows confirmation of the mutation
by ARMS assay using a reverse primer
specific for the mutant allele yields a
330 bp product but no product in a
control individual. The upper band is of
the internal control. (C) Schematic
representation of SMAD8 wild type and
SMAD8 C202X mutant. (D) Pedigree of
the patient’s family. PAH, pulmonary
J Med Genet 2009;46:331–337. doi:10.1136/jmg.2008.062703 333
49 mm Hg, RAP of 7 mm Hg, CI of 4.98 litres?min
of 9.9 Wood?Unit
, PVR of 8.5 Wood?Unit
PAWP of 7 mm Hg.
The patient’s mother and his elder brother did not have the
same mutation. However, his 58-year-old father was identified
as having the same mutation although no clinical symptoms of
PAH were observed (supplemental fig 1). According to the
patient’s father, he was the sixth of seven children. His third
sister and fifth brother died of pulmonary diseases at 13 years of
age and at (2 years of age, respectively (fig 1D). The other four
siblings of the father have not been screened for SMAD8
mutations, because their blood samples were not obtainable at
this point in time.
Immunoblotting assay and co-immunoprecipitation
SMAD8 mutant was neither phosphorylated nor stimulated to
interact with SMAD4 by constitutively active ALK3 and ALK1
It has previously been reported that Smad8 is phosphorylated
by constitutively active TGF-b/BMP type I receptors, except ca-
ALK5, and is stimulated to interact with Smad4.
C-terminal truncated Smad8 lacking the SXS phosphorylation
site was shown to be unphosphorylated in the presence of ca-
ALK3 and failed to interact with Smad4.
We first examined the effects of ca-ALK3 and ca-ALK1 on the
phosphorylation of the SMAD8 C202X mutant. The SMAD8
mutant was not phosphorylated in the presence of ca-ALK3 and
ca-ALK1 (fig 2A). We further examined the ability of the
Figure 2 SMAD8 C202X mutant is not
phosphorylated by TGF-b/BMP type I
receptors, and does not interact with
SMAD4. (A) Phosphorylation of Myc-
SMAD8 wild type and SMAD8 C202X
mutant in COS1 cells were examined by
immunoblotting (Blot). Myc-SMAD8 wild
type or SMAD8 C202X mutant was
transiently co-expressed with
constitutively active HA-TGF-b/BMP type
I receptor ALK3 and ALK1. The type I
receptor dependent phosphorylation of
SMADs was shown by anti-phospho-
Smad1/5/8 antibody, which also reacts
with phosphorylation of SMAD8. (B) The
interactions of Myc-SMAD8 wild type and
SMAD8 C202X mutant with Flag-SMAD4
in HEK293 cells were examined by
immunoprecipitation (IP) followed by
immunoblotting (Blot). HEK293 cells were
transfected with SMAD8 wild type,
SMAD8 C202X mutant, SMAD4 and
constitutively active ALK3 and ALK1. Cell
lysates were subjected to
immunoprecipitation with anti-Flag
antibody and analysed by immunoblotting
with anti-Myc antibody.
334 J Med Genet 2009;46:331–337. doi:10.1136/jmg.2008.062703
SMAD8 mutant to interact with SMAD4 by co-immunopreci-
pitation. Interaction between SMAD8 wild type and SMAD4
was observed in the presence of ca-ALK3 or ca-ALK1. However,
the SMAD8 mutant failed to interact with SMAD4 (fig 2B).
SMAD8 mutant was not capable of activating the BMP/TGF-b
It has been reported that Smad8 alone or co-expressed with
Smad4 increases BMP responsive promoter-reporters in the
absence of either BMP ligands or constitutively active TGF-b/
BMP type I receptors.
Therefore, to determine if the SMAD8
C202X mutant is able to increase BMP and TGF-b responsive
promoter-reporter activity, we investigated the transcriptional
activity mediated by SMAD8 wild type and the mutant with or
without SMAD4 and/or ca-ALK3 by using the BMP responsive
promoter-reporter, 3GC2-Lux and the TGF-b responsive pro-
moter-reporter, 3TP-Lux in COS-1 cells.
SMAD8 wild type induced BMP responsive promoter activity.
The co-expression of SMAD8 wild type with either SMAD4 or
ca-ALK3, induced approximately two times higher activity than
SMAD8 wild type alone. The SMAD8 wild type co-expressed
both SMAD4 and ca-ALK3 induced approximately three times
higher activity than the wild type alone. However, the SMAD8
mutant, even when co-expressed with SMAD4 or/and ca-ALK3,
were inefficient in activating the BMP responsive promoter-
reporter compared with SMAD8 wild type (fig 3).
Similar results were obtained using the TGF-b responsive
promoter-reporter, 3TP-Lux (supplemental fig 2). These results
demonstrate that the SMAD8 C202X mutant is not capable of
activating BMP/TGF-b responsive promoter-reporters.
Smad8 is one of the receptor regulated Smads (R-Smads) for the
TGF-b superfamily of receptors. Smad8 as well as Smad1 and
Smad5 are direct substrates of TGF-b/BMP type I receptors and
are phosphorylated at the SXS motif in the C-terminal region.
Phosphorylated Smad8 then associates with Co-Smad or Smad4
and translocates into the nucleus, where they regulate the
transcription of target genes.
Previous reports have suggested that the loss of signals
mediated by SMAD1/5/8 plays an important role in pulmonary
vascular remodelling and the pathogenesis of PAH. Reduced
phosphorylation of SMAD1 within the media and intima cells
of small pulmonary arteries in patients with IPAH and FPAH
The expression of BMPR2 in pulmonary
vascular lesions of IPAH/FPAH patients, especially in those
having the BMPR2 mutation, was reduced.
on BMPR2 mutations have shown that BMPR2 mutations
disrupted or down regulated SMAD1/5/8 signalling.
expression of ALK3 was markedly reduced in the lung of IPAH
patients who had no mutations in BMPR2 or ALK1.
Furthermore, expression of phosphorylated Smad1/5/8 in lung
was markedly reduced in conditional knockout mice lacking
Alk3 in alveolar epithelium.
Functional analysis of ALK1
mutations in HHT have shown that ALK1 mutations disrupt or
down regulate SMAD1/5/8 signalling.
indicate that the down regulation of TGF-b/BMP signals via
SMAD1/5/8 plays an important role in the pathogenesis of
BMPR2 has been identified as the major causative gene of
Also, ALK1 mutations have been reported in
patients with IPAH/FPAH.
A ENG mutation was reported in
an infant patient with IPAH and was later diagnosed with HHT
at 8 years of age.
However, approximately 30% of FPAH and
60–89% of apparently IPAH patients do not harbour mutations
About 70% of paediatric patients with IPAH do
not harbour mutations in either BMPR2 or ALK1.
this study, we investigated whether mutations in other genes
involved in TGF-b/BMP signalling pathways occur in 23
patients with IPAH.
We identified a nonsense mutation in SMAD8, c.606 C.A,
p.C202X in one patient (fig 1A). To our knowledge, a germline
mutation of SMAD8 has not yet been reported in either PAH or
other diseases, whereas loss of SMAD8 expression in breast,
colon and prostate cancer has been reported.
As predicted by the truncated protein structure of the
SMAD8 C202X mutant (fig 1C), our immunoblotting and co-
immunoprecipitation assay showed that it was not phophory-
lated by TGF-b/BMP type I receptors, and that it did not
interact with SMAD4 (fig 2A, B). These results indicate the
SMAD8 mutant disturbs the downstream signalling of TGF-b/
It has previously been reported that Smad8 was able to
translocate into the nucleus in the absence of ca-ALKs, although
its translocation efficiency was much less than that observed in
the presence of the ca-ALKs.
Therefore, we investigated
whether the SMAD8 mutant could perform some of the
transcription functions of the wild-type protein by using a
BMP responsive promoter-reporter, the 3GC2-Lux construct, in
COS-1 cells. The luciferase assay showed that SMAD8 wild
type produced a highly significant increase in BMP responsive
promoter-reporter activity, particularly when it was transfected
with both SMAD4 and ca-ALK3. In contrast, the SMAD8
mutant was inefficient in activating the reporter expression
with either SMAD4 or ca-ALK3 (fig 3). This can be explained by
the truncated protein structure of the SMAD8 mutant which
lacks both the MH2 domain and the SXS phosphorylation site
in the C-terminal region. The MH2 domain is not only required
for SMAD4 interaction but also for binding to other nuclear
Figure 3 The effect of SMAD8 wild type and SMAD8 C202X mutant on
the transcriptional activation of 3GC2-Lux were determined in the
presence and absence of SMAD4 and/or ca-ALK3 in COS1 cells. Values
represent mean (SD). Statistical differences between groups were
assessed by Student’s t test. **p,0.01.
J Med Genet 2009;46:331–337. doi:10.1136/jmg.2008.062703 335
factors such as DNA binding cofactors, co-activators and co-
repressors for the assembly of transcriptional complexes.
The clinical phenotype of our patient with SMAD8 mutation
was not different from other IPAH patients. He has no other
symptoms other than those of PAH at 16 years of age. His
father has the same SMAD8 mutation, but clinical symptoms or
objective measurement diagnostics of PAH have not been
observed to date. This situation is not surprising as FPAH with
germline BMPR2 mutations has a low penetrance. Only 10–20%
of BMPR2 mutation carriers will manifest clinical PAH.
penetrance was also observed in our previous study; two fathers
of paediatric patients with FPAH had the same ALK1 mutation
as their children, but had no symptoms of PAH or HHT.
In this study, a SMAD8 mutation was found in only one
patient out of the 23 IPAH patients (1/23, 4.3%). Clearly,
additional IPAH/FPAH patients will have to be screened to
determine the prevalence, penetrance and precise role of SMAD8
Previous studies have suggested that the down regulation of
TGF-b/BMP signals via SMAD1/5/8 plays an important role in
the pathogenesis of PAH. Our finding of SMAD8 mutation in a
PAH patient provides one more piece of evidence to support it.
The 22 of the 23 patients with IPAH (22/23, 95.7%) were not
identified mutations in ENG, SMAD1, SMAD2, SMAD3,
SMAD4, SMAD5, SMAD6 and SMAD8 in this study. It suggests
that there are still unidentified genes in the TGF-b/BMP signal
pathways or other pathways predisposing to PAH. In addition,
the low penetrance of FPAH despite the mutation in broadly
expressed genes indicates that additional genetic and/or
environmental factors play a critical role in the development
of PAH in carriers of the SMAD8, BMPR2 and ALK1 mutations.
It has been reported that prolonged hypoxia induces pulmonary
vascular remodelling and PAH in mice.
mutant mice responded to inflammatory stress with a marked
increase in right ventricular systolic pressure.
Thus, it will be
interesting to determine whether Smad8 homozygous or
heterozygous mutant mice develop PAH in response to stimuli
such as chronic hypoxia or inflammatory stress. Furthermore, to
determine the functions of SMAD8 in the context of PAH,
further investigations using human pulmonary artery smooth
muscle cells and human pulmonary artery endothelial cells will
Acknowledgements: We are grateful to the patients and their family members. We
thank Dr Bernardo Nadal-Ginard for his valuable comments. We thank Dr Kohei
Miyazono for providing the plasmids. We also thank Dr Shin-ichiro Imamura, Dr Shoichi
Arai, Dr Yoshiyuki Furutani, Dr Maya Fujiwara, Dr Emiko Hayama and Ms Michiko
Furutani for their excellent technical assistance.
Funding: This work was supported by the Program for Promoting the Establishment of
Strategic Research Centers, Special Coordination Funds for Promoting Science and
Technology, Ministry of Education, Culture, Sports, Science and Technology (Japan).
Competing interests: None.
Patient consent: Obtained.
1. Runo J, Loyd J. Primary pulmonary hypertension. Lancet 2003;361:1533–44.
2. Farber H, Loscalzo J. Pulmonary arterial hypertension. N Engl J Med
3. Austin E, Loyd J. Genetics and mediators in pulmonary arterial hypertension. Clin
Chest Med 2007;28:43–57
4. Rosenkranz S. Pulmonary hypertension: current diagnosis and treatment. Clin Res
5. Simonneau G, Galie` N, Rubin LJ, Langleben D, Seeger W, Domenighetti G, Gibbs S,
Lebrec D, Speich R, Beghetti M, Rich S, Fishman A. Clinical classification of
pulmonary hypertension. J Am Coll Cardiol 2004;43(12 Suppl S):5S–12S
6. Humbert M, Sitbon O, Chaouat A, Bertocchi M, Habib G, Gressin V, Yaici A,
Weitzenblum E, Cordier J, Chabot F, Dromer C, Pison C, Reynaud-Gaubert M, Haloun
A, Lairent M, Hachulla E, Simonneau G. Pulmonary arterial hypertension in France:
results from a national registry. Am J Respir Crit Care Med 2006;173:1023–30
7. Thomson JR, Machado RD, Pauciulo MW, Morgan NV, Humbert M, Elliott GC, Ward
K, Yacoub M, Mikhail G, Rogers P, Newman J, Wheeler L, Higenbottam T, Gibbs JS,
Egan J, Crozier A, Peacock A, Allcock R, Corris P, Loyd JE, Trembath RC, Nichols WC.
Sporadic primary pulmonary hypertension is associated with germline mutations of
the gene encoding BMPR-II, a receptor member of the TGF-beta family. J Med Genet
8. Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA 3rd, Loyd JE, Nichols
WC, Trembath RC. Heterozygous germline mutations in BMPR2, encoding a TGF-beta
receptor, cause familial primary pulmonary hypertension. The International PPH
Consortium. Nat Genet 2000;26:81–4.
9. Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis
E, Fischer SG, Barst RJ, Hodge SE, Knowles JA. Familial primary pulmonary
hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein
receptor-II gene. Am J Hum Genet 2000;67:737–44.
10. Machado RD, Aldred MA, James V, Harrison RE, Patel B, Schwalbe EC, Gruenig E,
Janssen B, Koehler R, Seeger W, Eickelberg O, Olschewski H, Elliott CG, Glissmeyer
E, Carlquist J, Kim M, Torbicki A, Fijalkowska A, Szewczyk G, Parma J, Abramowicz
MJ, Galie N, Morisaki H, Kyotani S, Nakanishi N, Morisaki T, Humbert M, Simonneau
G, Sitbon O, Soubrier F, Coulet F, Morrell NW, Trembath RC. Mutations of the TGF-
beta type II receptor BMPR2 in pulmonary arterial hypertension. Hum Mutat
11. Cogan JD, Pauciulo MW, Batchman AP, Prince MA, Robbins IM, Hedges LK, Stanton
KC, Wheeler LA, Phillips JA 3rd, Loyd, JE, Nichols WC. High frequency of BMPR2
exonic deletions/duplications in familial pulmonary arterial hypertension. Am J Respir
Crit Care Med 2006;174:590–8.
12. Harrison RE, Berger R, Haworth SG, Tulloh R, Mache CJ, Morrell NW, Aldred MA,
Trembath RC. Transforming growth factor-beta receptor mutations and pulmonary
arterial hypertension in childhood. Circulation 2005;111:435–41.
13. Fujiwara M, Yagi H, Matsuoka R, Akimoto K, Furutani M, Imamura S, Uehara R,
Nakayama T, Takao A, Nakazawa M, Saji T. Implications of mutations of activin receptor-
like kinase 1 gene (ALK1) in addition to bone morphogenetic protein receptor II gene
(BMPR2) in children with pulmonary arterial hypertension. Circ J 2008;72:127–33.
14. Trembath RC, Thomson JR, Machado RD, Morgan NV, Atkinson C, Winship I,
Simonneau G, Galie N, Loyd JE, Humbert M, Nichols WC, Morrell NW, Berg J, Manes
A, McGaughran J, Pauciulo M, Wheeler L. Clinical and molecular genetic features of
pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia.
N Engl J Med 2001;345:325–34.
15. Harrison RE, Flanagan JA, Sankelo M, Abdalla SA, Rowell J, Machado RD, Elliott
CG, Robbins IM, Olschewski H, McLaughlin V, Gruenig E, Kermeen F, Halme M,
Ra¨isa¨nen-Sokolowski A, Laitinen T, Morrell NW, Trembath RC. Molecular and
functional analysis identifies ALK-1 as the predominant cause of pulmonary
hypertension related to hereditary haemorrhagic telangiectasia. J Med Genet
16. Morrell NW, Yang X, Upton PD, Jourdan KB, Morgan N, Sheares KK, Trembath RC.
Altered growth responses of pulmonary artery smooth muscle cells from patients
with primary pulmonary hypertension to transforming growth factor-beta(1) and bone
morphogenetic proteins. Circulation 2001;104:790–5.
17. Yoshida MC, Satoh H, Sasaki M, Semba K, Yamamoto T, Toyoshima K. Regional
location of a novel yes-related proto-oncogene, syn, on human chromosome 6at
band q21. Jpn J Cancer Res 1986;77:1059–61.
18. Matsushita Y, Furukawa T, Kasanuki H, Nishibatake M, Kurihara Y, Ikeda A,
Kamatani N, Takeshima H, Matsuoka R. Mutation of junctophilin type 2 associated
with hypertrophic cardiomyopathy. J Hum Genet 2007;52:543–8.
19. Ferrie R, Schwarz M, Robertson N, Vaudin S, Super M, Malone G, Little S.
Development, multiplexing, and application of ARMS tests for common mutations in
the CFTR gene. Am J Hum Genet 1992;51:251–62.
20. Ishida W, Hamamoto T, Kusanagi K, Yagi K, Kawabata M, Takehara K, Sampath TK,
Kato M, Miyazono K. Smad6 is a Smad1/5-induced smad inhibitor. Characterization of
bone morphogenetic protein-responsive element in the mouse Smad6 promoter. JBiol
21. Wrana JL, Attisano L, Ca´rcamo J, Zentella A, Doody J, Laiho M, Wang XF,
Massague´ J. TGF beta signals through a heteromeric protein kinase receptor
complex. Cell 1992;71:1003–14.
22. Hoffmann A, Pelled G, Turgeman G, Eberle P, Zilberman Y, Shinar H, Keinan-
Adamsky K, Winkel A, Shahab S, Navon G, Gross G, Gazit D. Neotendon formation
induced by manipulation of the Smad8 signalling pathway in mesenchymal stem
cells. J Clin Invest 2006;116:940–52.
23. Kawai S, Faucheu C, Gallea S, Spinella-Jaegle S, Atfi A, Baron R, Roman SR. Mouse
smad8 phosphorylation downstream of BMP receptors ALK-2, ALK-3, and ALK-6
induces its association with Smad4 and transcriptional activity. Biochem Biophys Res
24. Massague´J,Seoane J, Wotton D. Smad transcription factors. Genes Dev
25. Yang X, Long L, Southwood M, Rudarakanchana N, Upton PD, Jeffery TK, Atkinson
C, Chen H, Trembath RC, Morrell NW. Dysfunctional Smad signaling contributes to
abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension.
Circ Res 2005;96:1053–63.
26. Atkinson C, Stewart S, Upton PD, Machado R, Thomson JR, Trembath RC, Morrell
NW. Primary pulmonary hypertension is associated with reduced pulmonary vascular
expression of type II bone morphogenetic protein receptor. Circulation
336 J Med Genet 2009;46:331–337. doi:10.1136/jmg.2008.062703
27. Nishihara A, Watabe T, Imamura T, Miyazono K. Functional heterogeneity of bone
morphogenetic protein receptor-II mutants found in patients with primary pulmonary
hypertension. Mol Biol Cell 2002;13:3055–63.
28. Rudarakanchana N, Flanagan JA, Chen H, Upton PD, Machado R, Patel D,
Trembath RC, Morrell NW. Functional analysis of bone morphogenetic protein type II
receptor mutations underlying primary pulmonary hypertension. Hum Mol Genet
29. Du L, Sullivan CC, Chu D, Cho AJ, Kido M, Wolf PL, Yuan JX, Deutsch R, Jamieson
SW, Thistlethwaite PA. Signaling molecules in nonfamilial pulmonary hypertension.
N Engl J Med 2003;348:500–9.
30. Eblaghie MC, Reedy M, Oliver T, Mishina Y, Hogan BL. Evidence that autocrine
signaling through Bmpr1a regulates the proliferation, survival and morphogenetic
behavior of distal lung epithelial cells. Dev Biol 2006;291:67–82.
31. Gu Y, Jin P, Zhang L, Zhao X, Gao X, Ning Y, Meng A, Chen YG. Functional analysis of
mutations in the kinase domain of the TGF-beta receptor ALK1 reveals different
mechanisms for induction of hereditary hemorrhagic telangiectasia. Blood
32. Cheng KH, Ponte JF, Thiagalingam S. Elucidation of epigenetic inactivation of SMAD8in
cancer using targeted expressed gene display. Cancer Res 2004;64:1639–46.
33. Horvath LG, Henshall SM, Kench JG, Turner JJ, Golovsky D, Brenner PC, O’Neill GF,
Kooner R, Stricker PD, Grygiel JJ, Sutherland RL. Loss of BMP2, Smad8, and Smad4
expression in prostate cancer progression. Prostate 2004;59:234–42.
34. Machado RD, Pauciulo MW, Thomson JR, Lane KB, Morgan NV, Wheeler L, Phillips
JA 3rd, Newman J, Williams D, Galie` N, Manes A, McNeil K, Yacoub M, Mikhail G,
Rogers P, Corris P, Humbert M, Donnai D, Martensson G, Tranebjaerg L, Loyd JE,
Trembath RC, Nichols WC. BMPR2 haploinsufficiency as the inherited molecular
mechanism for primary pulmonary hypertension. Am J Hum Genet 2001;68:92–102.
35. Beppu H, Ichinose F, Kawai N, Jones RC, Yu PB, Zapol WM, Miyazono K, Li E, Bloch
KD. BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired
pulmonary vascular remodeling response to prolonged hypoxia. Am J Physiol Lung
Cell Mol Physiol 2004;287:L1241–7.
36. Song Y, Jones JE, Beppu H, Keaney JF Jr, Loscalzo J, Zhang YY. Increased
susceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice.
Drug and Therapeutics Bulletin (DTB)
Your key source of unbiased, independent advice
For over 45 years DTB has been an independent, indispensable part of evidence-based clinical practice.
DTB offers healthcare professionals detailed assessment of, and practical advice on, individual
medicines and other treatments, groups of treatment and the overall management of disease.
DTB is now also available online at http://dtb.bmj.com:
c browse or search all DTB content from the latest issue back to 1994
c email alerting, sophisticated searching, RSS feeds and full text links from cited references
c interactive services such as My Folders for quick access to articles that you have viewed previously
and My Searches to save and re-use useful searches
c comment online on any DTB article
To subscribe, or for further information, please visit http://dtb.bmj.com
J Med Genet 2009;46:331–337. doi:10.1136/jmg.2008.062703 337