ACVRL1 gene variant in a patient with vein of Galen aneurysmal malformation

ACVRL1 gene variant in a patient with vein

of Galen aneurysmal malformation

Ayako Chidaa,b, Masaki Shintanib, Hajime Wakamatsua, Yoshiyuki Tsutsumic, Yuo Iizukad,Nanako Kawaguchib, Yoshiyuki Furutanib, Kei Inaib, Shigeaki Nonoyamaa and Toshio Nakanishib,*aDepartment of Pediatrics, National Defense Medical College, Tokorozawa-city, Saitama, JapanbDepartment of Pediatric Cardiology, Tokyo Women’s Medical University, Shinjuku-ku, Tokyo, JapancDepartment of Radiology, National Center for Child Health and Development, Setagaya-ku Tokyo, JapandDepartment of Radiology, Toho University Ohashi Medical Center, Tokyo, Japan

Received 10 November 2013

Revised 28 January 2014

Accepted 3 February 2014

Abstract. Although mutations in the RASA1 gene in vein of Galen aneurysmal malformation (VGAM) and an endoglin gene

mutation in a VGAM patient with a family history of hereditary hemorrhagic telangiectasia (HHT) have been identified, most

VGAM cases have no mutation in these genes. We sought to detect mutations in other genes related to HHT. We screened for

mutations in RASA1 and three genes (endoglin, activin receptor-like kinase 1 (ACVRL1), encoding ALK1, and SMAD4) related

to HHT in four VGAM patients. One variant (c.652 C>T p.R218W) in ACVRL1 was identified. Immunoblotting revealed that

the ALK1-R218W protein could not promote SMAD1/5/8 phosphorylation by BMP9 stimulation. On the other hand, wild-type

ALK1 could enhance the phosphorylation as expected. Furthermore, the transcriptional activation of ALK1-R218W was less

efficient than that of wild-type ALK1. We identified 1 variant in ACVRL1 in a VGAM patient. These findings suggest that

the ACVRL1 variant-R218W may be associated with the pathogenesis of VGAM.

Keywords: ACVRL1, gene variant, vein of Galen aneurysmal malformation

1. Introduction

Vein of Galen aneurysmal malformation (VGAM)

is a rare intracranial arteriovenous malformation with

connections between choroidal arteries and the median

prosencephalic vein, representing less than 1% of all

intracranial arteriovenous malformations [1,2]. In chil-

dren, VGAM represents 30% of all vascular malforma-

tions [2]. The outcome of VGAM used to be very

poor, but current techniques including endovascular

treatment have resulted in longer survival times [1,3].

Lasjaunias et al. [4] investigated clinical outcome of

216 VGAM patients and revealed that 193 patients

(89.35%) survived and 143 of them (74% of survivors)

were neurologically normal on follow-up. In addition,

brain magnetic resonance imaging (MRI) contributes

to early detection and prompt treatment [5]. The VGAM

pathogenesis remains unclear. In 2008, Revencu et al. [6]

reported the presence of a RASA1 [MIM 139150] gene

mutation in two VGAM patients. Furthermore, Xu

et al. [7] published a case report of familial-associated

VGAM. Thus, the pathogenesis of VGAMmay be based

on hereditary genetics.

We previously reported an endoglin (ENG) [MIM

131195] gene mutation in a VGAM patient with a

family history of hereditary hemorrhagic telangiecta-

sia (HHT) [8]. In that case, we hypothesized that the

genes related to HHT may have been associated with

*Corresponding author: Dr. Toshio Nakanishi, Department ofPediatric Cardiology, Tokyo Women’s Medical University, 8-1

Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel.: +81 3

3353 8112 ext. 24067; Fax: +81 3 3352 3088; E-mail: pnakanis@

hij.twmu.ac.jp.

Journal of Pediatric Genetics 2 (2013) 181–189DOI 10.3233/PGE-13067IOS Press

181

2146-4596/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved

VGAM onset. The data suggested that VGAM patients

should be screened for not only RASA1, but also the

HHT-associated genes: ENG, activin-like receptor 1

(ACVRL1) [MIM 601284], that encode ALK1, and

SMAD4 [MIM 600993]. In this paper, we investigated

the ALK1 gene in VGAM patients, and found that this

mutation could affect the downstream TGF-beta sig-

naling, a pathway known to be important for tumori-

genesis and/or normal development.

2. Materials and methods

2.1. Subjects

Using previously described methods, we recruited

four VGAM patients and identified ENG mutations [8].

Table 1 shows the clinical characteristics of all four

patients. A diagnosis of VGAM was made by a trained

neonatologist. This study was approved by the Institu-

tional Review Committee of Tokyo Women’s Medical

University. Written informed consent was obtained from

all patients or their guardians in accordance with the

declaration of Helsinki.

2.2. Genome analysis

Genomic deoxyribonucleic acid (DNA) was iso-

lated from peripheral blood lymphocytes or lympho-

blastoid cell lines transformed by the Epstein-Barr

virus, as described previously [9]. RASA1, ENG,

ACVRL1, and SMAD4 coding regions and exon-intron

boundaries were amplified from genomic DNA using

primers. Amplified products were purified using the

QIAquick polymerase chain reaction (PCR) purification

kit (QIAGEN, Hilden, Germany) and screened with

bi-directional direct sequencing using an ABI 3130xl

DNA analyzer (Applied Biosystems, Foster City, CA,

USA). After direct sequencing of these genes, multiplex

ligation-dependent probe amplification (MLPA) was

used to detect exonic deletions/duplications of ACVRL1

and ENG in patients who had no mutations in RASA1,

ENG, ACVRL1, and SMAD4. MLPA was performed

with 100 ng of genomic DNA according to the manufac-

turer’s instructions using a SALSA MLPA HHT/PPH1

probe set (MRC-Holland, Amsterdam, Netherlands).

Probe amplification products were run on an ABI

3130xl DNA Analyzer using a GS500 size standard

(Applied Biosystems). MLPA peak plots were visua-

lized using GeneMapper software v4.0 (Applied

Biosystems). For each sample, BMPR2 probe peak

heights were normalized against the sum of all control

peaks. Patients’ samples were then normalized to the

mean of three healthy control samples.

All generated sequences were compared with wild-

type RASA1, ENG, ALK1, and SMAD4. When a new

mutation was detected, we confirmed that it was not

present in the 460 healthy control samples via direct

sequencing.

2.3. Plasmid generation

Human pcDNA3.0-hemagglutinin (HA)-ACVRL1

and the bone morphogenic protein (BMP)-responsive

promoter reporter construct, 3GC2-Lux, were provided

by Dr. K Miyazono (Tokyo, Japan). The 3GC2-Lux

construct contains three repeats of a GC-rich sequence

derived from the proximal BMP response element in

the Smad6 promoter [10]. We previously utilized the

3GC2-Lux reporter gene for functional analysis of the

SMAD8 mutant and BMPR1B mutants in pulmonary

arterial hypertension (PAH) patients [11,12]. 3GC2-

Lux has also been used in other studies to assess the

interaction of genes belonging to the BMP signal path-

way [13–15].

Site-directed mutagenesis was carried out using a

QuikChange XL site-directed mutagenesis kit (Strata-

gene, California, USA). Constructed plasmids were

verified by sequencing. The antibodies used were as fol-

lows: anti-HA rat antibody (Roche, Mannheim, Baden-

Württemberg, Germany), anti-phospho-Smad1/Smad5/

Smad8 rabbit antibody (Cell Signaling Technology),

and monoclonal anti beta-actin (Sigma, St. Louis, USA).

Recombinant human BMP9 was from R&D Systems

(Abingdon, Oxon, UK).

2.4. Cells, transfection, and Western blotting

NIH-3T3 cells were grown in Dulbecco’s modified

eagle medium (DMEM) with 4.5 g/L glucose (Invitro-

gen, Carlsbad, California, USA) supplemented with

10% fetal bovine serum (FBS) (Gibco, New York,

USA), and 100 U/mL penicillin-streptomycin. Trans-

fection was performed using Lipofectamine 2000

reagent (Invitrogen) according to the manufacturer’s

instructions. For experiments investigating endogen-

ous Smad1/5/8 phosphorylation and gene expression,

the cells were cultured in DMEM with 4.5 g/L glucose

containing 0.1% FBS for 4 h. Then, human bone

182 A. Chida et al. / ACVRL1 variant in vein of Galen aneurysmal malformation

Table 1

Summary of VGAM patients

Patient

number

Serial

number

Gene mutation

or variant

Sex Age at

diagnosis

History and

presentation

Complication and

family history

Unique imaging

findings

Treatment Outcome Reference

1 P3389 - M Fetal age of

34 wk

Bradycardia,

hypoxia

- - Catheter

embolization

İntact -

2 P3431 RASA1

(c.1678 G>TE560X)

M Fetal age of

35 wk

Retractive

breathing, groaning,cardiomegaly

- - Catheter

embolization

Mental and

motor retardation,epilepsy

-

3 P3858 - F 2 d of age Hypoglycemia,

cardiomegaly

Patient’s twin

sibling : VGAM

suspected

- Catheter

embolization

Severe mental and

motor retardation,

epilepsy

-

4 P3865 ACVRL1

(c.652 C>T

R218W)

F 1 d of age Pale skin,

bradycardia, anemia,

hypothemia

- SAH, hematoma

around VGAM

Craniotomy

for removal

hematoma

İntact This study

VGAM = Vein of Galen aneurysmal malformation; RASA1 = RAS p21 GTPase activating protein 1; ACVRL1 = Activin receptor-like kinase 1; M = Male; F = Female; SAH = Subarachnoid hemorrhage.

morphogenetic protein 9 (BMP9) (1 ng/mL) were

added to the culture and incubated for 1 h. Twenty-nine

hours after transfection, cells were lysed in lysis buffer

(50 mM Tris-HCl [pH8.0], 1 mM EDTA [pH8.0],

120 mMNaCl, NP-40 0.25%). ForWestern blots, lysates

were separated on a 10% resolving SDS-polyacrylamide

gel and proteins were transferred to polyvinylidene

fluoride membranes by semidry transfer. For phos-

phorylated-SMAD1/5/8, membranes were blocked in

TBS-T (50 mM Tris-HCl [pH7.6], 137 mM NaCl, 0.1%

[w/v] Tween 20) containing 5% bovine serum albumin

for 1 h at room temperature.Membranes were rinsedwith

TBS-T and incubated with primary antibody against

phosphorylated-SMAD1/SMAD5/SMAD8 (1:5000), HA-

ALK1 (1:2,000), and beta-actin (1:10,000) for 1 h at room

temperature. Membranes were rinsed with TBS-T and

incubated with HRP-goat anti-rabbit IgG (Invitrogen)

for phosphorylated-SMAD1/5/8 detection, or anti-rat

IgG (Rockland, Pennsylvania,USA) forALK1detection,

or anti-mouse IgG (Invitrogen) for beta-actin detection.

Blots were then washed with TBS-T and bound com-

plexes were detected using enhanced chemiluminescence

(ImageQuant LAS 4000 mini, GE Healthcare).

2.5. Luciferase assay

NIH-3T3 cells were transfected using Lipofecta-

mine 2000 reagent (Invitrogen) with 3GC2-Lux and

wild type or mutant pcDNA3.0-ACVRL1. These cells

were treated with human BMP9 (100 pg/ml) in

DMEM with 4.5 g/L glucose containing 0.1% FBS

for 15 h. Twenty-four hours after transfection, cells

were harvested. Firefly and renilla luciferase activities

were measured with the Dual luciferase reporter assay

(Promega, Madison, Wisconsin, USA) following the

manufacturer’s instructions. Results are expressed as

the ratio of firefly luciferase activity to renilla lucifer-

ase activity. All assays were performed in triplicate.

2.6. Flow cytometric analysis of ALK1 expression

NIH-3T3 cells were transfected as described above

with wild-type ACVRL1 or ACVRL1-R218W or beta-

gal. Twenty-four hours later, cells were detached with

0.25% trypsin- EDTA solution (Sigma) and collected

by centrifugation. Cells were labeled for 1 h at 4 °C

with polyclonal anti-ALK1 antibody (R&D Systems,

Minneapolis, USA) or with isotype-matched control

IgG. Labeling was detected with Alexa488-conjugated

secondary antibodies (Invitrogen). The fluorescence

intensity of the labeled cells were analyzed by flow

cytometry with a EPICS ALTRA flow cytometer

(Beckman coulter, California, USA).

2.7. Statistical analysis

All results are expressed as the mean ± standard

deviation. For statistical comparison of two samples,

a two-tailed Student’s t test was used where applicable.

Values of P < 0.05 were considered to be significant.

Statistical analyses were performed using JMP for Win-

dows (version 9; SAS Institute, North Carolina, USA).

3. Results

3.1. Detection of an ACVRL1 variant

We screened for mutations in the RASA1, ENG,

ACVRL1, and SMAD4 genes in four patients with

VGAM. One RASA1 mutation and one suspected

ACVRL1 mutation were detected by direct sequencing

(Table 1). MLPA analysis revealed no exonic dele-

tions/ duplications.

ACVRL1 mutation suspected was c.652 C>T

p.R218W (Fig. 1A). As shown in Fig. 1B, ALK1 con-

sists of an extracellular ligand-binding domain, a trans-

membrane domain, the GS domain that is involved in

phosphorylation, and a serine-threonine kinase domain.

R218W is located in the serine-threonine kinase

domain. The alignment of the ALK1 protein between

nine distantly related species show that this amino acid

is highly conserved (Fig. 1C). Although the identified

variant was absent from the HHT mutation database,

it is present in the single nucleotide polymorphisms

database as rs199874575. Furthermore, we detected

ALK1-R218W in one of 460 healthy controls (0.002%)

by direct sequencing. However, in polymorphism phe-

notyping v2 (Polyphen-2) and the SIFT algorithm,

changes to this sequence was regarded as “probably

damaging (score 1.000)” and “damaging” [16,17].

3.2. Clinical characteristics of the VGAM patient

with an ACVRL1 variant

Proband (Table 1; Patient No. 4, Fig. 1D; III:2):

The patient was a female who was delivered vaginally

with cephalic presentation at a fetal age of 39 1/7 wk.


Her birth weight was 2962 g, length was 49 cm, and

Apgar scores at 1st and 5th min were 8 and 9, respec-

tively. There were no abnormal events during her

mother’s pregnancy and parturition history. Her pater-

nal elder uncle died of meningitis at 22 yr (Fig. 1D;

II:1). There was no family history of VGAM and

HHT, but the patient’s elder sister (III:1) and mother

(II:4) were identified as having the same mutation

(Fig. 1D and E). The patient’s father (II:2) did not have

the mutation (Fig. 1D and E). Other family members

were not screened for ACVRL1mutations because their

blood samples were not available.

After birth, she exhibited pale skin, hypothermia,

bradycardia, and poor suckling. Laboratory testing revea-

led a hemoglobin level of 11.3 g/dL, white cell count

of 14,200/mm3, and platelet count of 284,000/mm3.

She had no abnormal coagulant function. Subarach-

noid hemorrhage with a hematoma around galenic

cistern were identified by emergency brain computed

tomography. Three-dimensional brain computed

tomography demonstrated bleeding in the parietal

superior sagittal sinus, confluence, right transverse

sinus, and right sigmoid sinus (Fig. 2A). Brain color

Doppler imaging at 1-day-old revealed a large hema-

toma around the median vein of the prosencephalon

and turbulent flow in the malformation and superior

sagittal sinus (Fig. 2B). Brain MRI at 1-day-old

revealed a hematoma around the median vein of pro-

sencephalon (Fig. 2C). Craniotomy for removal of

the hematoma was performed at 1-day-old. After sur-

gery, she frequently exhibited apnea but her general

condition gradually and spontaneously improved.

The brain MRI at 39-day-old showed no abnormal

arteriovenous shunt. Furthermore, there were no scar

findings at brain parenchyma (Fig. 2D). She was dis-

charged from the hospital at 43-day-old. Patient

Fig. 1. ACVRL1 variant in vein of Galen aneurysmal malformation. (A) c.652 C>T p.R218W was identified in 1 proband. (B) Schematic repre-

sentation of ALK1 wild type and the location of the variant. (C) Alignment of the ALK1 protein among a human, rhesus, mouse, dog, elephant,

opossum, X_tropicalis, and zebrafish show conservation of arginine 218 in these species. (D) Pedigrees of the patients’ families. (E) Sequence

analysis of ACVRL1 in the family of the proband. The mother and elder sister had the same variant as the proband.

A. Chida et al. / ACVRL1 variant in vein of Galen aneurysmal malformation 185

development was normal at 4 yr of age. There have been

no cardiac or neurological complications. Developmen-

tally, she is meeting all her milestones. As assessed by

an expert pediatrician, their conclusion was that she

and her family have no symptoms of HHT.

3.3. The ACVRL1 R218W variant reduced SMAD

1/5/8 phosphorylation

The ACVRL1 R218W variant reduced SMAD 1/5/8

phosphorylation. The addition of BMP9 strongly

induced endogenous SMAD1/5/8 phosphorylation in

the presence of wild-type ALK1. BMP9 also slightly

enhanced SMAD1/5/8 phosphorylation in the presence

of ALK1-R218W (Fig. 3A). By densitometry, we found

that the ratio of phosphorylated-Smad1/5/8 to beta-actin

with wild-type ALK1 was significantly higher than that

with ALK1-R218W (Fig. 3B).

3.4. Luciferase assay showed impaired BMP signal

with variant

We investigated the transcriptional activity mediated

by wild-type ALK1 or ALK1-R218W to determine

whether ALK1-R218W could increase BMP-responsive

promoter-reporter activity. The luciferase assay showed

that, after stimulation with human BMP9, ALK1-

R218W induced significantly lower activity than wild-

type ALK1 (Fig. 4).

3.5. Comparison of ALK1 localization by flow

cytometry

We investigated whether the variant affects ALK1

protein expression. A beta-gal expressing plasmid was

used as a control. ALK1-R218Wwas expressed at simi-

lar levels as wild-type ALK1 (Fig. 5).

4. Discussion

In this study, we for the first time, describe one

ACVRL1 variant in a VGAM patient. The patient

and family had no symptoms of HHT as described

above. Although we detected ACVRL1-R218W in one

of 460 healthy controls by direct sequencing, R218W

was located in a functional domain of ACVRL1. Further-

more, estimations with Polyphen-2 and SIFT algorithm

Fig. 2. Analysis of images from proband. (A) Axial (left) non-enhanced reconstruction computed tomography on the day of birth demonstrating

subarachnoid hemorrhage with a hematoma around galenic cistern. Three-dimensional computed tomography (right) demonstrating bleeding in

the parietal superior sagittal sinus, confluence, right transverse sinus, and right sigmoid sinus. (B) Axial (left) and sagittal (right) color Doppler

imaging at 1-day-old demonstrating a large hematoma around the median vein of prosencephalon. There was turbulent flow in the malformation

and superior sagittal sinus. (C) Axial (left) and sagittal (right) brain magnetic resonance imaging at 1-day-old demonstrating a hematoma around

the median vein of prosencephalon. (D) There was no abnormal arteriovenous shunt in axial (left) and sagittal (right) brain magnetic resonance

imaging at 38 d after surgical intervention. There were no scar findings at brain parenchyma.


showed that ACVRL1-R218W may cause damage to

protein function. Although the variant has been regis-

tered in the SNP database, that is not always accurate

[18]; therefore, we further investigated the impact of

the missense variant on ACVRL1.

The mother and elder sister did not have VGAM or

HHT, but they have the same heterozygous variant as

the patient. These results indicate that VGAM with

ACVRL1-R218W may be inherited in an autosomal

dominant fashion with very low penetrance. Xu et al. [7]

found that familial VGAM can be caused by gene muta-

tions. In our study, we included one case of suspected

familial VGAM (Table 1: Patient No. 3). In this case,

the patient’s twin sibling died at 1 d of age. Brain

Doppler imaging after death revealed an abnormal vessel

like VGAM. While it is conceivable that VGAM is a

heritable disorder based on these reports, further studies

are needed to accurately determine this. Moreover, it is

possible that the HHT phenotype and PAH may appear

in future patients since ACVRL1 mutations are known

to cause HHT and PAH (e.g., patients’ mother or elder

sister) [19,20].

ALK1 is a member of the bone morphogenetic pro-

tein family that belongs to the transforming growth fac-

tor-beta (TGF-beta) superfamily. The TGF-beta/BMP

Fig. 3. SMAD1/5/8 show reduced ALK1 R218W-mediated phosphorylation with BMP9. (A) Western blots show that the addition of BMP9

induced SMAD1/5/8 phosphorylation in the presence of wild-type ALK1, but with ALK1 R218W, SMAD1/5/8 are not phosphorylated. Con-

fluent cells were stimulated with 1 ng/mL BMP9 in DMEM/0.1% FBS for 60 min, followed by lysis for total protein. (B) The ratio of phos-

phorylated-Smad1/5/8 densitometry to beta-actin densitometry expressed did not increase in the presence of ALK1 R218W with BMP9.

Values represent the mean ± standard deviation of four independent experiments. Differences between groups were assessed by the Student’s

t test. *P = 0.04.

Fig. 4. ALK1 variant modestly induced luciferase activity. After sti-

mulation with human BMP9 (100 pg/mL) for 15 h, ALK1-R218W

induced significantly lower activity than wild-type ALK1. Values

represent the mean ± standard deviation. Differences between

groups were assessed by the Student’s t test. **P < 0.001.


signal pathway has two types of receptors. There are

seven type I receptors (ALK1, ALK2, ALK3, ALK4,

ALK5, ALK6, and ALK7) and five type II recep-

tors (ActR2A, ActR2B, TGF-beta R2, AMHR2, and

BMPR2) [21]. BMPs bind independently to both type

I and type II receptors. BMP9 and BMP10 are specific

ligands for ALK1 [22]. Furthermore, it was revealed

that BMP9 is present in human plasma and contributes

to adult vascular quiescence [23]. Upon ligand binding,

the type II receptors phosphorylate and activate type I

receptors. Activated type I receptors then propagate the

signal by phosphorylating a family of transcription

factors, called Smads.ALK1 activates SMAD1, SMAD5,

and SMAD8 by phosphorylation. These activated

Smads complex with a common partner Smad, speci-

fically, SMAD4, and accumulate in the nucleus where

they interact with transcriptional regulators of target

genes [21,24].

ACVRL1 mutations are known to be associated with

HHT and PAH [19]. Ricard et al. [25] reported that

functional analysis of 15 missense mutations and

one frameshift mutation in ACVRL1 identified in

HHT patients revealed a loss of function in all except

one GS domain mutation. Importantly, our data was

consistent with these data. It has been hypothesized

that an imbalance between increased TGF-beta levels

and decreased BMP signals leads to PAH [26].

Furthermore, we previously raised the possibility that

not only the inhibition, but also promotion of BMP

signals, may be associated with PAH onset [12]. The

mechanism VGAM onset may be similar to that of

PAH. In this study, the expression level of ALK1-

R218W was almost equal to that of wild-type ALK1

and this is expected based on data from Ricard et al.

[25]. They showed that cell surface expression of

all ALK1 mutants were similar to wild-type ALK1

levels, with the exception of mutations in the extracel-

luar domain. R218W is located in the serine-threonine

kinase domain, not the extracelluar domain.

Our results indicate that the phenotype of VGAM

with the ACVRL1 mutation/variant needs to be inves-

tigated in greater detail. To elucidate the disease

mechanism, it may be necessary to search for the

ACVRL1 mutation/variants in other vascular malfor-

mations regardless of HHT presentation. In addition,

to identify the role of ACVRL1 in VGAM pathogen-

esis, further studies using blood vessels of the human

brain and/or animal models with the ACVRL1 muta-

tion/variant are strongly required.

Acknowledgements

We are grateful to the patients and their family

members. We thank Dr. Kohei Miyazono for provid-

ing the plasmids.

Fig. 5. Cell surface expression of ALK1 protein. Flow cytometry analysis of NIH-3T3 cells transfected with plasmid encoding wild-type

ACVRL1 or ACVRL1-R218W or beta-gal for 24 h. Non-permeabilized transfected cells were stained by anti-ALK1 antibody for cell surface

ALK1 expression.


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ACVRL1 gene variant in a patient with vein of Galen aneurysmal malformation

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