SMAD6-deficiency in human genetic disorders

Abstract

SMAD6 encodes an intracellular inhibitor of the bone morphogenetic protein (BMP) signalling pathway. Until now, SMAD6-deficiency has been associated with three distinctive human congenital conditions, i.e., congenital heart diseases, including left ventricular obstruction and conotruncal defects, craniosynostosis and radioulnar synostosis. Intriguingly, a similar spectrum of heterozygous loss-of-function variants has been reported to cause these clinically distinct disorders without a genotype–phenotype correlation. Even identical nucleotide changes have been described in patients with either a cardiovascular phenotype, craniosynostosis or radioulnar synostosis. These findings suggest that the primary pathogenic variant alone cannot explain the resultant patient phenotype. In this review, we summarise clinical and (patho)genetic (dis)similarities between these three SMAD6-related conditions, compare published Madh6 mouse models, in which the importance and impact of the genetic background with respect to the observed phenotype is highlighted, and elaborate on the cellular key mechanisms orchestrated by SMAD6 in the development of these three discrete inherited disorders. In addition, we discuss future research needed to elucidate the pathogenetic mechanisms underlying these diseases in order to improve their molecular diagnosis, advance therapeutic strategies and facilitate counselling of patients and their families.

Full text

REVIEW ARTICLE OPEN
SMAD6-deficiency in human genetic disorders
Ilse Luyckx
1,2
, Aline Verstraeten
1
, Marie-José Goumans
3
and Bart Loeys
1,2
✉
SMAD6 encodes an intracellular inhibitor of the bone morphogenetic protein (BMP) signalling pathway. Until now, SMAD6-
deficiency has been associated with three distinctive human congenital conditions, i.e., congenital heart diseases, including left
ventricular obstruction and conotruncal defects, craniosynostosis and radioulnar synostosis. Intriguingly, a similar spectrum of
heterozygous loss-of-function variants has been reported to cause these clinically distinct disorders without a genotype–phenotype
correlation. Even identical nucleotide changes have been described in patients with either a cardiovascular phenotype,
craniosynostosis or radioulnar synostosis. These findings suggest that the primary pathogenic variant alone cannot explain the
resultant patient phenotype. In this review, we summarise clinical and (patho)genetic (dis)similarities between these three SMAD6-
related conditions, compare published Madh6 mouse models, in which the importance and impact of the genetic background with
respect to the observed phenotype is highlighted, and elaborate on the cellular key mechanisms orchestrated by SMAD6 in the
development of these three discrete inherited disorders. In addition, we discuss future research needed to elucidate the
pathogenetic mechanisms underlying these diseases in order to improve their molecular diagnosis, advance therapeutic strategies
and facilitate counselling of patients and their families.
npj Genomic Medicine (2022) 7:68 ; https://doi.org/10.1038/s41525-022-00338-5
The protein SMAD6, encoded by SMAD6 (OMIM: 602931), belongs
to the SMAD family of proteins involved in the bone morphoge-
netic proteins (BMP) signalling cascade. Even though these
molecules were initially discovered for their ability to induce
bone formation, it is now clear that BMPs are important in the
embryogenesis and development of many organ systems, as well
as in maintenance of adult tissue homoeostasis. SMAD6 is an
intracellular inhibitor of, predominantly, the BMP signalling
pathway, yet it cross-talks with the closely related transforming
growth factor-β(TGF-β) signalling pathway
1,2
.
Over the past 10 years, genetic variants in SMAD6 were
demonstrated to impinge on the risk of human genetic
disorders
3–13
such as cardiovascular diseases, including congenital
heart defects (CHD), craniosynostosis (CRS) and radioulnar
synostosis (RUS). CHD is among the most common birth defects,
affecting 6–13:1000 live-born infants
14–16
. In association with
SMAD6-deficiency, it encompasses a range of cardiac and outflow
tract abnormalities. Complex lesions consisting of multiple defects
are often severe, even critical, for which treatment with advanced
surgery for definitive correction of malformations or (palliative)
medication is imperative
17
. In addition, adult patients with a sole
congenital aortic valve defect associate with more late-onset
vascular complications like a pathological widening of the thoracic
aorta (~thoracic aortic aneurysm (TAA))
18
. TAAs are also life-
threatening as they are (1) clinically silent
19
, (2) entail a high risk
for acute dissection and/or rupture (mortality rates ≥70%)
20
, and
(3) no therapy currently exists that can stop TAA development or
progression
21
. CRS, which is a skull defect afflicting 1:2100–2500
live births
22,23
, is a second SMAD6-related disease. Surgical
correction is frequently necessary to prevent complications
24
such as developmental delay, facial abnormality, sensory, respira-
tory and neurological dysfunction, anomalies affecting the eye,
and psychological disturbances
25
. Finally, congenital RUS, also
referred to as fused forearm bones, is a rare condition with ~500
cases reported in literature
9,13,26
. This malformation, usually
diagnosed before the age of 5 years, is not life-threatening, but
corrective surgery and/or medication to control pain might, in
some cases, improve the quality of life
13
.
The therapeutic strategies for SMAD6 mutation-positive patients
mainly focus on disease monitoring in order to define the
appropriate time for intervention, medication to control pain, and
surgical repair
19,24,27–30
. Even though surgery is effective, it is
associated with risks, requires early detection of at-risk patients,
only provides relief late in the disease course, and does not target
the underlying driver(s) of the disease. Hence, there is a need for a
better (molecular) understanding for early diagnosis, and to
empower new therapies to prevent disease progression. With this
review, we provide a comprehensive overview on SMAD6-
deficiency in human genetic disorders by summarising the clinical,
(patho)genetic and cellular (dis)similarities observed in human
and mouse models. We conclude with future directions of
research needed to improve patient management based on the
underlying SMAD6-related molecular disease signature.
CLINICAL PHENOTYPE OF PATIENTS WITH SMAD6-DEFICIENCY
The clinical presentation of heterozygous SMAD6 variant-positive
patients is extremely heterogeneous as illustrated by the different
affected organ systems, the varying degree of severity, and the
range of associated complications. Table 1summarises the clinical
phenotype of probands with disease-causative SMAD6 variants. All
disease-related clinical definitions are summarised in Table 2.
Cardiovascular diseases
The cardiovascular phenotype (cases, N=31) (probands, N=28,
Table 1) include left ventricular outflow tract defects (N=21/28,
75%)
4,5,7,8,10
, conotruncal defects (N=4/28, 14%)
5
, and defects
defined as “others”(N=3/28, 11%)
5
as they cannot be categorised
1
Centre of Medical Genetics, Faculty of Medicine and Health Sciences, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium.
2
Department of Human
Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
3
Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands.
✉email: bart.loeys@uantwerpen.be
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Table 1. Clinical phenotype of probands with disease-causative SMAD6 variants.
Study Patient cohort Major defect (sub-category) Number of
probands
NS/S Age Additional features within the same organ system
Tan et al.
10
Cardiovascular
malformations
Bicuspid aortic valve (LVO) 1 NS 1.5 y
a
Aortic stenosis
Cardiovascular
malformations
Bicuspid aortic valve with
coarctation of the aorta (LVO)
1NS30y
a
Aortic stenosis
Timberlake et al.
11
;
Timberlake et al.
12
Craniosynostosis Craniosynostosis 17 NS Paediatric
b
Metopic synostosis (N=12), sagittal synostosis (N=3),
metopic +sagittal synostosis (N=2)
Jin et al.
5
Congenital heart defect Tetralogy of Fallot (CTD) 3 Unknown
c
Paediatric
b
Pulmonary stenosis (subvalvular (N=2), valvar (N=1)), ventricular
septal defect (malalignment) (N=1), coronary artery anomaly (right)
(N=1), left aortic arch with normal branching pattern (N=1), patent
foramen ovale (N=1)
Congenital heart defect Transposition of the Great
Arteries (CTD)
1 Unknown
c
Paediatric
b
Atypical coronary arteries in D-loop Transposition of the Great
Arteries, transposition D-loop of the Great Arteries with intact
ventricular septum, left aortic arch with a normal branching pattern
Congenital heart defect Hypoplastic left heart
syndrome (LVO)
1 Unknown
c
Paediatric
b
Aortic arch hypoplasia, aortic atresia, hypoplasia ascending aorta,
hypoplastic left ventricle (subnormal cavity volume), mitral atresia,
restrictive patent foramen ovale
Congenital heart defect Coarctation of the aorta (LVO) 1 Unknown
c
Paediatric
b
Atrial septal defect (secundum), left-sided patent ductus arteriosus,
tubular hypoplasia of aorta, ventricular septal defect (malalignment,
muscular outlet)
Congenital heart defect Bicuspid aortic valve with
coarctation of the aorta (LVO)
1 Unknown
c
Paediatric
b
Aortic arch hypoplasia, hypoplastic left ventricle (subnormal cavity
volume), patent foramen ovale
Congenital heart defect Other 1 Unknown
c
Paediatric
b
Left aortic arch with normal branching pattern, partially anomalous
pulmonary veins, sinus venosus septal defect (superior type)
Congenital heart defect Other 1 Unknown
c
Paediatric
b
Vascular ring, aberrant left subclavian artery, abnormal branching
right aortic arch, right aortic arch left ligament
Gillis et al.
4
; Luyckx
et al.
7
; Park et al.
8
Cardiovascular
malformations
Bicuspid aortic valve-related
thoracic aortic aneurysm
15 NS Average 64.1 y Aortic valve calcification (N=1), coarctation of the aorta (N=2),
aortic regurgitation (N=1), aortic stenosis (N=1)
Kloth et al.
6d
Cardiovascular
malformations
Coarctation of the aorta (LVO) 1 S
e
6 y Suspected tricuspid aor tic valve
Other 1 NS 10 y Dysplastic and stenotic pulmonary valve, dilated cardiomyopathy,
stenotic left main coronary artery
Yang et al.
13
; Shen et al.
9
Radioulnar synostosis Radioulnar synostosis 77 NS Average 5 y Lateral left radioulnar synostosis (N=17), lateral right radioulnar
synostosis (N=6), bilateral radioulnar synostosis (N=54)
Calpena et al.
3
Craniosynostosis Craniosynostosis 26 S
f
/NS
g
Not reported Metopic synostosis (N=15), sagittal synostosis (N=6), right coronal
synostosis (N=2), sagittal +left coronal synostosis (N=1),
sagittal +bicoronal synostosis (N=2)
NS non-syndromic, Ssyndromic, LVO left ventricular obstruction, CTD conotruncal defect, yyears, NA not applicable.
This table excludes patients with known disease-related genetic hits at other loci in addition to SMAD6.
a
Unclear if the aorta has been evaluated.
b
Age is not specified.
c
Two patients had extra-cardiac abnormalities (i.e., syndromic cases).
d
Only paper describing bi-allelic variants.
e
Consanguineous family with facial dysmorphism, unilateral hypoplasia, bilateral radioulnar synostosis, bilateral toe 2/3 syndactyly, very dry and scaly skin, dysrhythmic electro-encephalogram without seizure
activity and mild intellectual disability.
f
Seven syndromic probands.
g
Nineteen non-syndromic probands.
I. Luyckx et al.
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into the traditional subgroups that arise from disruption of shared
embryonic processes. Left ventricular outflow tract defect refers to
hypoplastic left heart syndrome (HLHS) (N=1/21, 5%), coarctation
of the aorta (CoA) (N=2/21, 10%), and bicuspid aortic valve (BAV),
which is associated with congenital CoA and/or late-onset TAA
(N =17/21, 81%) in all patients, except for one toddler with
isolated BAV (N=1/21, 5%; 1.5 years old). Conotruncal defects
include Tetralogy of Fallot (N=3/4, 75%) and D-loop transposition
of the Great Arteries (N=1/4, 25%). The remaining three patients
presented with either a vascular ring, partially anomalous
pulmonary veins combined with sinus venosus septal defect, or
stenotic pulmonary valve and stenotic left main coronary artery
accompanied with dilated cardiomyopathy.
Craniosynostosis
The clinical outcome of CRS (cases, N=49) (probands, N=43,
Table 1)
3,11,12
involves syndromic (N=7/43, 16%) and non-
Table 2. Clinical description of the disease-related anomalies.
Anomalies Clinical description
Absent corpus callosum A congenital brain defect with partial or complete absence of the region that connects the two
cerebral hemispheres.
Atrial septal defect A congenital heart defect resulting from incomplete atrial septation.
Atrioventricular septal defect A congenital heart defect resulting from incomplete septation of the atrioventricular canal.
Bicuspid aortic valve A congenital heart defect in which the aortic valve has only two leaflets instead of the normal three.
Caudal vertebrae dysplasia A congenital defect of a total or partial failure of the development of the caudal vertebrae.
Coarctation of the aorta A congenital heart defect in which blood flow is blocked by aortic narrowing usually at the region of
the ductus arteriosus.
Coronal synostosis A congenital skull defect in which the coronal suture close prematurely leading to flattening of the
head (unicoronal), or a short head with wide appearance (bicoronal).
Dilated cardiomyopathy A condition in which the heart becomes enlarged and cannot pump blood effectively.
D-loop Refers to the normal rightward (dextro =D) loop or bend of the embryonic heart tube and indicates
that the inflow portion of the right ventricle is to the right of the morphological left ventricle.
Frontal bossing A condition indicating a protuberance of the frontal bones of the forehead.
Hypoplastic left heart syndrome A congenital heart defect in which the heart’s left side (including the aorta, aortic valve, left ventricle
and mitral valve) is underdeveloped.
Macrocephaly A condition in which circumference of the head is more than two standard deviations above the
mean value for a given age and gender.
Metopic synostosis A congenital skull defect in which the metopic suture close prematurely leading to a forehead with
triangular appearance (trigonocephaly).
Microcephaly A condition in which circumference of the head is more than two standard deviations below the
mean value for a given age and gender.
Mitral/pulmonary/tricuspid/aortic valve
regurgitation
A condition in which the valve does not close properly, allowing blood to flow backwards.
Regurgitation is also called insufficiency or incompetence.
Patent ductus arteriosus A congenital heart defect in which the ductus arteriosus fails to close after birth.
Patent foramen ovale A congenital heart defect in which the foramen ovale did not close properly at birth, with the result of
an existing hole between the left and right atria of the heart.
Plagiocephaly A condition in which the skull flattens on one side.
Polydactyly A congenital skeletal condition in which an individual has more than 5 fingers per hand or 5 toes
per foot.
Premature fusion of the anterior fontanel A congenital skull defect in which the anterior fontanel close prematurely.
Radioulnar synostosis A congenital defect in which the radius and ulna of the forearm is abnormally connected (synostosis).
Sagittal synostosis A congenital skull defect in which the sagittal suture close prematurely leading to a long and narrow
head (scaphocephaly).
Sinus venosus septal defect A congenital heart defect in which a deficiency of the common wall between the superior vena cava
and the right upper pulmonary vein is present thereby allowing shunting of blood from the systemic
to the pulmonary circulation.
Stenotic left main coronary artery A condition in which the left main coronary artery is narrowed.
Stenotic pulmonary valve A condition in which the pulmonary valve is narrowed.
Tetralogy of Fallot A congenital heart defect characterised by right ventricular outflow tract obstruction, right ventricular
hypertrophy, ventricular septal defect and overriding aorta.
Thoracic aortic aneurysm A condition in which the aortic diameter is more than two standard deviations above the mean value
for a given age and gender.
Transposition of the Great Arteries A congenital heart defect referring to ventriculoarterial discordance, i.e., aorta arises from a
morphological right ventricle, and the pulmonary artery arises from a morphological left ventricle.
Vascular ring A congenital heart defect in which the aorta or its branches forms a ring around the trachea and the
oesophagus.
Ventricular septal defect A congenital heart defect resulting from incomplete ventricular septation.
Ventriculomegaly A condition in which the brain ventricles are abnormally enlarged.
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syndromic presentations (N=36/43, 84%) in which single and
multiple fusion events of almost all sutures have been identified.
Most common presentation was metopic synostosis (N=27/43,
63%), followed by sagittal synostosis (N=9/43, 21%), right
unicoronal synostosis (N=2/43, 5%), combined metopic and
sagittal synostosis (N=2/43, 5%), combined sagittal and bicoronal
synostosis (N =2/46, 4%), and combined sagittal and left
unicoronal synostosis (N=1/43, 2%). Remarkably, raised intracra-
nial pressure following cranial reconstruction, which is usually a
rather infrequent complication in simple synostosis of midline
sutures, should be specifically monitored in SMAD6 variant-
positive patients
3
. Other recurrent brain or skull anomalies in
(non-)syndromic subjects comprise ventriculomegaly and absent
corpus callosum, macrocephaly, and mild microcephaly, and mild-
to-moderate neurodevelopmental delay (consisting of speech,
educational and global delay)
3,11,12
. More subtle learning difficul-
ties were observed in 36% of the non-syndromic patients
(N=14)
11
, while gross and fine motor delays were only observed
occasionally
3,11
. In syndromic cases
3
, cardiac defects were
common (N=5/7, 71%), but seem to have another pattern which
is further discussed in the following section on the clinical overlap.
Congenital radioulnar synostosis
Patients with RUS
9,13
(cases, N=93) (probands, N=77, Table 1)
are most frequently characterised by bilateral RUS (69%; isolated
(N=42/61), familial (N=22/32)), followed by unilateral left-sided
RUS in sporadic patients (N=15/42, 36%) versus right-sided RUS
(N=4/42, 10%), while no susceptibility for left- or right-sided RUS
was observed in families (N=5/32, 16%). Affected individuals
within a single pedigree can show both bilateral and unilateral
RUS. No syndromic cases have been reported thus far, yet some
subordinate clinical findings have been described in 14
families
9,13
: three families presented with axial skeletal deformities
(including cervical fusion, rib malformation, caudal vertebrae
dysplasia and vertebral malformations), two families had poly-
dactyly (pre- and pro-axial type), five families exhibited CHD
(encompassing patent ductus arteriosus, mitral/tricuspid/aortic
regurgitation, atrial septal defect, BAV, left ventricular hypertrophy
and mitral/pulmonary valve insufficiency), and four families
showed skull-related defects (containing frontal bossing, plagio-
cephaly and premature closure of anterior fontanel, but no
unequivocal description of CRS). Remarkably, in six out of the 14
families a variant-positive SMAD6 carrier without RUS but with
skeleton-, skull-, or CHD-related features
9,13
was reported: two
affected individuals from two families displayed cervical fusion or
caudal vertebrae dysplasia, two patients from two families
exhibited with premature closure of anterior fontanel, frontal
bossing and plagiocephaly or solely plagiocephaly, one subject
presented with polydactyly and, finally, one affected had patent
ductus arteriosus together with mitral regurgitation.
Clinical overlap?
Studies have, in addition to the clinical indication for study
enrolment, to some extent also assessed the presence of other
SMAD6-related clinical associations. Patients with cardiovascular
disease did not exhibit CRS and/or RUS. A child, from a
consanguineous family harbouring a pathogenic homozygous
SMAD6 variant, was reported to present with CoA, suspected
tricuspid aortic valve, bilateral RUS, renal anomalies, facial
dysmorphism and global development delay
6
. In view of
consanguinity, homozygosity at other loci as an explanation for
these multisystemic features cannot be excluded. CRS cases did
not exhibit RUS, but five syndromic CRS cases presented with a
CHD
3
of which none seem to mimic the more severe conotruncal
and outflow tract defects seen in the cardiovascular disease
cohorts. For example, defects in three patients resolved sponta-
neously. One patient with atrioventricular septal defect required
surgery at the age of three years, yet his variant-positive mother
Fig. 1 Graphical representation of identified heterozygous SMAD6 variants in probands with bicuspid aortic valve-related aortopathy,
congenital heart disease, (non-)syndromic craniosynostosis and non-syndromic radioulnar synostosis. SMAD6 protein has several
domains: MH1-domain (grey; inhibitory effect on signalling), PY motif (orange), PLDLDS motif (yellow), MH2-domain (grey; inhibitory effect on
signalling) and L3-loop (blue; determines specificity for interaction with type I receptors)
88,89
. Of note: (1) all variants were called “pathogenic”
in the original publications but there is different criteria for calling “pathogenicity”(e.g., by applying different allele frequency thresholds, and/
or using specific functional tests); (2) identical protein changes (underlined) are described in patients with distinctive phenotypes.
I. Luyckx et al.
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had a normal echocardiogram. The fifth patient had BAV (N=1/
46, 2%) with right bundle branch block. This observation exactly
matches the epidemiological number of 2% for BAV in the general
population though
31
. None of the seven extra screened asympto-
matic parents of SMAD6 variant-positive children with CRS showed
any evidence for BAV or TAA
3
. Finally, no BAV or TAA has been
identified so far in the non-syndromic CRS cohort (personal
communication with A. Wilkie, Oxford). Hence, no clinical overlap
of a variant-positive SMAD6 carrier with cardiovascular disease or
CRS with any abnormality affecting the other organ systems has
been observed to date.
Finally, the phenotypic picture in 14 families with RUS is more
complicated as both CHD as well as skull and skeletal
abnormalities have been observed occasionally (N=12/93,
13%)
9,13
. Although based on their nature and incidence, we
cannot rule out an alternative cause for some abnormalities (e.g.,
valve insufficiency, left ventricular hypertrophy and rib/vertebral
malformation), the occurrence of skeletal- (N=4/93, 4%), skull-
(N=9/93, 10%), or CHD-related (N=3/93, 3%) abnormalities in
families with RUS does hint to some clinical overlap. For example,
two variant-positive SMAD6 carriers from two families without RUS
presented with axial skeletal deformities, either cervical fusion or
caudal vertebrae dysplasia. Extra skull features were observed in
another five families, including frontal bossing (N=4/93, 4%),
plagiocephaly (N=3/93, 3%), and premature fusion of the anterior
fontanel (N=2/93, 2%). Plagiocephaly and premature fusion of
the anterior fontanel was reported in a variant-positive family
member without RUS. And finally, three families had CHD too,
namely patent ductus arteriosus (N=1/93, 1%), atrial septal defect
(N=1/93, 1%), and BAV (N=1/93, 1%).
GENETIC (DIS)SIMILARITIES BETWEEN SMAD6-RELATED
DISORDERS
Intriguingly, similar, or even identical, heterozygous loss-of-
function variants in SMAD6 cause these three distinct disorders
(Fig. 1and Supplementary Table 1)
3–13
. The variant spectrum
includes rare truncating and missense variants locating in the
functional MH1- and MH2-domain of the protein with no
phenotypic correlation with respect to variant type nor location.
Identical nucleotide changes (N=6) have been described in
patients with cardiovascular disease (N=3), CRS (N=5) or RUS
(N=10). Moreover, the phenotype within these families are,
predominantly, restricted to one affected organ system. For
example, the p.(Gly156Valfs*23) variant causes BAV-related aorto-
pathy (N=1), sagittal synostosis (N=1), and non-syndromic RUS
(N=4), for which no clinical overlap has been documented except
for frontal bossing in one family with left-sided RUS. Hence, the
molecular finding cannot predict the clinical presentation of a
patient, and, as such, it is likely that (a) factor(s) inherited together
with the primary SMAD6 mutation drives the resultant patient
phenotype. The latter seems likely as within one family
concordance of the phenotype is frequently observed.
Cardiovascular disease
The aetiology of CHD is multifactorial, involving genetic and
environmental factors such as smoking, alcohol abuse and
infection transmitted by the mother during pregnancy
32
. Familial
studies have demonstrated that the CHD recurrence risk in family
members of affected individuals depends on the type of lesion
33
.
Pathogenic variants cause autosomal dominant, autosomal
recessive, or X-linked traits with variable penetrance and clinical
expressivity. About 132 definitive and strong candidate genes for
CHD in numerous functional classes like chromatin modification,
transcription factors and signal transduction, amongst others,
have been reported. The predominant disease-causative effect is
through loss-of-function
34
. To date, 50% of the patients remain
molecularly undiagnosed though, and the yield is even lower in
non-syndromic cases
34
. Interestingly, pathogenic SMAD6 variants
have been shown to be enriched in isolated paediatric and adult
CHD patients, in which most patients exhibited left ventricular
outflow tract defects (Table 3). So far, patients with recessive
variants do not seem to present with a more severe cardiovascular
phenotype as compared to subjects harbouring heterozygous
variants. However, this observation is based on only two cases,
and no functional analyses have been performed
6
.ASMAD6
genetic uptake of 4.6% was reached in more severely affected
BAV-related aortopathy patients, i.e., BAV patients who underwent
surgical repair for aneurysmal disease before the age of 50, and
with a positive family history for cardiovascular disease. The
estimated penetrance for the disease was 82.4%. SMAD6 is the
most important BAV/TAA gene identified thus far, as none of the
approximately 30 definitive and candidate genes for BAV and/or
TAA explain more than 1% of these patients
7
. The emerging BAV/
TAA disease-related pathways include impaired cardiac transcrip-
tion factor activity (e.g., GATA5)
35,36
, perturbed extracellular matrix
homoeostasis (e.g., LOX)
37
, aberrant TGF-β(e.g., TGFBR1)
19
and
NOTCH (e.g., NOTCH1)
38
signalling, deficiency of the vascular
smooth muscle cell contractile apparatus (e.g., ACTA2)
39
, and
altered endothelial cell function (e.g., ROBO4)
40
. Taken together,
carrying a pathogenic SMAD6 might be insufficient to definitively
cause cardiovascular disease in all cases, and, as such, more
research is required to identify the missing information, and to
understand how it contributes to disease.
A clinical and genetic association between BAV, HLHS, and CoA
have already been thoroughly discussed in familial studies
41,42
,
and some examples of monozygotic twins with discordant
phenotypes, i.e., one has BAV while the other present with HLHS,
have been described
43,44
. As SMAD6-deficiency results in a
spectrum of, mainly, left ventricular outflow tract defects, one
could hypothesise the existence of additional genetic hits in
families. Particular emphasis might be given to ascertain essential
cardiac transcription complexes, and to investigate the
Table 3. SMAD6 variant-positive patients with congenital heart disease.
Tan et al.
10
Jin et al.
5
Gillis et al.
4
Luyckx et al.
7
Total
Left ventricular outflow tract 2/83 (2.4%) 3/797 (0.4%) 11/441 (2.5%) 3/65 (4.6%) 19/1386 (1.4%)
D-loop transposition of the Great Arteries 0/65 (0%) 1/251 (0.4%) –– 1/316 (0.3%)
Conotruncal defects 0/78 (0%) 3/872 (0.3%) –– 3/950 (0.3%)
Heterotaxy 0/10 (0%) 0/272 (0%) –– 0/282 (0%)
Others 0/200 (0%) 2/679 (0.3%) –– 2/879 (0.2%)
Total 2/436 (0.5%) 9/2871 (0.3%) 11/441 (2.5%) 3/65 (4.6%)
This table excludes (1) patients with known disease-related genetic hits at other loci, in addition to SMAD6, and (2) case reports lacking information on the total
number of screened patients.
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accessibility of these factors onto DNA in patient-derived material
in order to reveal novel crucial clues on the pathogenesis of CHD
disease.
Craniosynostosis
CRS is a heterogeneous disease influenced by mechanical and
extrinsic forces as well as genetic components affecting the
intrinsic properties of the suture
45
. In families, an autosomal
dominant mode of transmission is mostly observed, but in about
half of the cases a de novo variant is found. The genetic uptake is
highest in syndromic cases, while isolated cases (i.e., 75% of all
patients) largely remain molecularly undiagnosed
45,46
. Approxi-
mately one quarter of CRS cases harbour a disease-causative
variant in one of the known genes, mostly in FGFR2,FGFR3,TCF12,
ERF, EFNB1,orTWIST1, causing either a loss- or gain-of-function.
These gene products are involved in signal transduction pathways
like FGF signalling (FGR2, FGFR3), Eph/ephrin signalling (EFNB1)
and ERK-MAPK activity (ERF) or they bind DNA to regulate gene
expression (TWIST1, TCF12). As SMAD6 variants account for 5.8% of
all (non-)syndromic patients with metopic synostosis, it became,
by far, the largest monogenic contributor to metopic synostosis
yet identified. Furthermore, SMAD6 variants seem less commonly
associated with other types of suture fusion
3
, making it in
particular relevant to screen patients with metopic synostosis for
SMAD6 deficiency.
SMAD6-related CRS has been associated with reduced pene-
trance (overall penetrance, 16–24%)
3,11,12
. As such, a two-locus
inheritance model for CRS (i.e., metopic, sagittal and combined
metopic and sagittal) was proposed by Timberlake et al., in which
near complete-penetrance was reached upon co-occurrence with
a common BMP2 SNP risk allele (C) (rs1884302)
11
. Upon merger of
datasets, this association still holds true, yet the initial signal has
weakened due to non-replication in an independent cohort. One
explanation might be the underrepresentation of the risk allele
(frequency ~0.33, gnomAD: European non-Finnish) in non-
penetrant SMAD6 variant harbouring individuals in the discovery
studies, which was not observed in a third study
3,11,12
. Addition-
ally, rs1884302 was found to strongly associate with sagittal
synostosis
47
, and more recent data for metopic synostosis reveal
no equivalent association for this SNP
48
. Extra work is necessary to
explore on such relationship between SMAD6 variant-positive
patients with sagittal synostosis, and a larger sample size is
needed to dissect whether this interaction is truly digenic
inheritance or is merely an additive effect of the GWAS signal,
modifying the penetrance of SMAD6 pathogenic variants. Addi-
tional light was shed onto this digenic inheritance model by
revealing the presence of this common SNP in SMAD6 mutation-
positive patients with either BAV-related aortopathy (N=4)
7
or
radioulnar synostosis (N=7)
13
but in the absence of any sign of
CRS. Altogether, current data suggest that the pathogenic SMAD6
variant alone might be insufficient to definitively cause CRS in all
cases, and it still remains to be further investigated what the extra
hits, and what the underlying mechanisms are.
Table 4. Overview of the published Madh6 knock-out mouse models with the phenotypic characterisation of Madh6
−/−
animals.
Galvin et al.
54
Estrada et al.
55
Wylie et al.
56
Generation model Embryonic stem cells with transgene
interrupting SMAD6 function (i.e., insertion of
LacZ/neomycin resistance cassette into the 5´
terminus of the exon encoding the MH2-
domain)
Stem cells from Galvin et al. Stem cells from Galvin et al.
Biological consequence Madh6-LacZ fused transcript See Galvin et al.
54
See Galvin et al.
54
Parents of breeding Heterozygous Heterozygous Heterozygous
Lethality Partial lethality of madh6
−/−
mice (P21; 3–13%
~genetic background
a
)
Lethality of madh6
−/−
mice
(P0; 5%, but all died <24 h)
Lethality of madh6
−/−
mice
(P0; 8%, but all died within
2–6 days of birth)
Cardiac phenotype Hyperplasia cardiac valves, enlarged mitral
valve, enlarged pulmonary valve, abnormal
truncus arteriosus septation
b
, aortic
ossification
c
, hypertension
Not investigated No hyperplastic valves, or
other major defects explaining
cause of death
Vascular phenotype Decreased vasodilation
c
, abnormal
thrombosis
d
Not investigated Blood vessel haemorrhages in
skin and brown fat pads
Craniofacial phenotype Not observed Domed skull, shortened snout Nothing obvious that could
explain the cause of death
Axial skeletal phenotype Not observed Posterior transformation of cervical
vertebrae, bilateral ossification centres in
lumbar vertebrae, bifid sternebrae
Nothing obvious that could
explain the cause of death
Appendicular skeletal
phenotype
Not observed Smaller size, abnormal growth plate
development
e
Nothing obvious that could
explain the cause of death
Genetic background 129/SvEv × BALB/cBy, 129/SvEv × C57Bl/6,
inbred 129/SvEv
C57BL/6J x BALB/c CD1
Madh6 is the murine orthologue of human SMAD6.
a
Background sensitivity: inbred 129S/SvEv (3% versus expected 25%), mixed 129S6/SvEvTac × BALB/cByJ (9% versus expected 25%), mixed 129S6/
SvEvTac × C57BL6/J (13% versus expected 25%).
b
A subset of homozygotes exhibit abnormal septation of the outflow tract leading to a severely narrowed ascending aorta, and an enlarged pulmonary trunk
or the reverse.
c
Only observed in the surviving animals starting at 6 weeks of age.
d
Surviving homozygotes display occasional thrombotic lesions as well as focal ischaemia in the lung, liver and kidney.
e
Abnormal growth plate development: delayed onset of hypertrophic differentiation and mineralisation at midgestation, but expanded hypertrophic zone at
late gestation.
I. Luyckx et al.
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npj Genomic Medicine (2022) 68 Published in partnership with CEGMR, King Abdulaziz University

Radioulnar synostosis
Since the 70’s, congenital RUS is recognised as an inheritable
disease segregating in an autosomal dominant manner
49,50
.In
total, 10% of the RUS patients were identified with a monogenetic
cause (e.g., NOG) or with aneuploidy syndromes, in which the
syndromic subjects presented with additional abnormalities in the
skeleton, heart, urinary tract, blood and males had extra X and Y
chromosomes
51
. At present, SMAD6 deficiency is, by far, the most
important known disease gene for non-syndromic RUS, as it
explains 42% of familial cases and 16% of sporadic patients
9,13
.
The penetrance of disease is incomplete, and has been reported
around 20–25%
9,13
. Other genetic causes include two pathogenic
variants in NOG, explaining less than 1% of the patients
13
.NOG
encodes noggin, which is a major BMP antagonist. Dysregulation
of BMP signalling due to NOG deficiency in mice showed
interference with hedgehog signalling for BMP-induced inter-
digital cell death
52
, and for axial skeleton development
53
. The
contribution of genetic variability in SMAD6 and NOG to syndromic
RUS is yet unexplored. In sum, literature indicates that radioulnar
synostosis is not exclusively caused by one pathogenic SMAD6
variant in all cases. Again, more investigation is needed to fill our
gap in knowledge about the extra hits and underlying
mechanisms.
Current challenges in SMAD6-related diagnosis and
counselling
Patient management for SMAD6-related disorders is challenging
as rare pathogenic loss-of-function variants associate with (1)
reduced penetrance, (2) extreme variability in phenotypical
expression, and (3) distinctive clinical entities without
genotype–phenotype correlation, as outlined above. Hence, every
single case should be discussed thoroughly in a multidisciplinary
team based on phenotype, family history, inheritance pattern, and
pathogenicity of the variant. Given the possibility of a devastating
cardiovascular outcome, echocardiographic evaluation is currently
indicated in a SMAD6 variant-positive proband, irrespective of the
clinical indication for referral. A genetic test is best offered to
family members of SMAD6 variant-positive patients with cardio-
vascular disease or RUS as some clinical overlap with the
cardiovascular disease might exist. In contrast, variant-positive
SMAD6 carriers in CRS cohorts are frequently unaffected making a
genetic test uninformative. There is currently some preliminary
evidence that phenotypes are quite consistent in a single family.
Nevertheless, more insight is needed before we can abandon
echocardiographic evaluation in relatives of SMAD6 variant-
positive probands with CRS. Another counselling challenge is
caused by the observation that the general population well-
tolerates loss-of-function SMAD6 variants (pLI =0, gnomAD
v2.1.1), despite the overwhelming overrepresentation of such
variants in disease cohorts as compared to this control popula-
tion
3–5,11–13
. This is in particular challenging for CRS given the low
penetrance of CRS in individuals heterozygous for pathogenic
SMAD6 variants
3,11
.
Lastly, diagnostic and research laboratories also encounter
difficulties for variant interpretation, in particular for missense
variants. In this regard, Calpena et al.
3
have provided a filtering
strategy able to discriminate high-penetrant rare pathogenic
missense variants, as proven in functional tests assessing protein
stability and/or impaired BMP signalling activity. Even though very
useful, this approach will not classify all type of variants (e.g., 5′
untranslated region), and current bio-informatic tools are not
sufficient sensitive to assess variants with moderate effects, which
are likely to explain, to some extent, the variability in expressivity
and unpredictable penetrance. Nonetheless, implementation of
flexible, preferably high-throughput, functional assays for variant
interpretation, combined with further refinement of bio-informatic
tools, is necessary to address this challenge.
LESSONS FROM MOUSE MODELS
Genetically modified mouse models have, with success, been used
to interrogate the pathomechanisms underlying rare human
disorders. At present, three mouse models lacking the murine
orthologue of SMAD6, i.e., Madh6, have been studied (Table 4).
The Madh6-mutant mice were produced by a LacZ/neomycin
resistance cassette inserted into the 5´ terminus of the exon
encoding the MH2-domain of Smad6
54
. Each model is unique by
its respective genetic background as all models were generated
using embryonic stem cells created by Galvin et al.
In the model on a mixed 129/SvEv × BALB/cBy background
54
,
homozygous animals exhibited hyperplasia of the cardiac valves,
with the mitral and pulmonary valve being more extremely
affected, septation defects, and lethality. The latter was observed
due to an underrepresentation of homozygotes at the time of
weaning. Surviving animals developed aortic ossification with
notable cartilaginous metaplasia and trabeculation of the aortic
media (from 6 weeks of age), decreased vasodilation and
hypertension. Subsequent in-depth characterisation revealed an
excess of mesenchymal cells in the cardiac valves in all
homozygotes, while the following was only observed in a subset
of the animals: (1) abnormal septation of the outflow tract, i.e., a
severely narrowed ascending aorta and an enlarged pulmonary
trunk or the reverse, (2) thrombotic lesions and ischaemia in lung,
liver, and kidney, (3) subepicardial vascular malformations in the
ventricular wall with loss of multiple smooth muscle cell layers in
large vessels, and (4) thickening of the endocardium. Interestingly,
a background sensitivity for the survival of homozygotes up to
weaning was observed by comparing mouse models on different
genetic backgrounds (i.e., 129/SvEv × BALB/cBy, 129/SvEv × C57Bl/
6, inbred 129/SvEv), which corresponded to the severity of cardiac
defects. Heterozygotes were not further studied, and no gender-
specific analyses were performed. Even though similar anomalies
were described in humans, it is still unanswered whether these
mice also present BAV, aortic valve calcification, hypoplastic left
heart and what the relative position of the aorta and pulmonary
artery is. No gross non-cardiovascular anomalies were described,
yet this has not been investigated into detail.
The next-studied knock-out mouse model
55
, on a C57BL/
6J × BALB/c background, was generated to investigate the
consequences of Smad6 loss during cartilage development.
Homozygotes displayed craniofacial anomalies like a domed skull
and shortened snout, but no defects in cranial sutures were found.
Abnormalities in the skeleton were observed too, such as posterior
transformation of cervical vertebrae (C7), flatter thoracic vertebral
bodies, presence of bilateral ossification centres in lumbar
vertebrae, and bifid sternebrae due to incomplete sternal band
fusion. In addition, homozygotes were smaller in size, as
confirmed by shorter appendicular bones, and stage-specific
defects in endochondral bone formation were found like the
delayed onset of hypertrophy at midgestation and expanded
hypertophic zone at late gestation. Furthermore, significant
embryonic and neonatal lethality was observed, as merely 5% of
the progeny were homozygous and all live-born pups died within
24 h after birth due to an unspecified cause. Heterozygotes were
not examined in this model, alike with other organ systems,
especially no data on the cardiovascular system in the homo-
zygotes were reported.
The last published model
56
was generated on a CD1 back-
ground to elucidate the effects of Smad6 loss on blood vessel
development. Wylie et al. reported on embryonic and postnatal
lethality of homozygotes (all died by P2–6), in addition to regions
of haemorrhages in skin and brown fat pads without any sign of
hyperplastic valve thickening in these animals. The observed
vessel phenotype was a consequence of disrupted endothelial cell
junctions, thereby compromising vessel wall integrity. No in-depth
I. Luyckx et al.
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experiments were performed on heterozygous animals, nor other
organ systems were examined.
Altogether, this mouse knock-out data support a role for unique
genetic background-related clinical presentations. Additional gene
expression or pathway analyses in the different Madh6-deficient
mouse models might provide essential insights into the pathogen-
esis of these phenotypes. With respect to the observed cardiovas-
cular phenotype in 129/SvEv × BALB/cBy, 129/SvEv × C57Bl/6 and
inbred 129/SvEv Madh6
−/−
mice, a major codominant modifier
gene for lethality might be present. Alike to Tgfβ1
−/−
mice created
on different genetic backgrounds to study angiogenesis
57–59
,
independent but epistatically interacting genetic loci might be
found that determine the incidence of lethality depending on the
model. Interesting modifying genes have already been described
to alter the response of lack to TGF-β1 in mice, suggesting that
proper TGF-βsignalling is key for embryonic survival. Whether by
analogy, improper BMP or TGF-βsignalling explains the incidence
of lethality in Madh6
−/−
mice with a cardiovascular phenotype
remains to be determined.
CELLULAR MECHANISMS ORCHESTRATED BY SMAD6
Epithelial-to-mesenchymal transition (EMT) is a reversible funda-
mental biological process for (1) the formation of the body plan,
(2) the differentiation of multiple tissues and organs, and (3) to
repair tissues. EMT is an extremely coordinated multifaceted
process, in which cells disrupt their intercellular adhesion
complexes and lose their apicobasal polarity in order to
migrate
60,61
. Two highly conserved and critical regulators of EMT
are the TGF-βand BMP signalling pathway, which either
stimulates or tempers this process, respectively
62
. Hence, SMAD6
modulates EMT by interfering with, predominantly, BMP
signalling
54,63
.
The mechanosensitive BMP signalling pathway (Fig. 2) regulates
cellular lineage commitment, morphogenesis, differentiation,
proliferation and apoptosis
64,65
. BMPs activate numerous path-
ways, of which the SMAD signalling pathway has best been
studied
66
. BMP signalling interferes with its own signalling as
SMAD6, a direct BMP target, selectively recruits SMURF1 to BMP
type 1 receptors
67
or competes with receptor-regulated Smads for
binding to SMAD4
68
, thereby establishing a negative feedback
Fig. 2 Schematic overview of SMAD-(in)dependent bone morphogenetic protein (BMP) signalling pathway (oversimplification). Upon
BMP ligand binding, specific type I and type II receptors form a heterotetrameric complex. The type II receptor phosphorylates the type I
receptor, which, in turn, phosphorylates Smad1, Smad5, and Smad8 (canonical BMP signalling). Phosphorylated Smads propagate the signal
via complex formation with Smad4 and translocates into the nucleus, where it regulates the expression of BMP-responsive target genes. In
addition to Smad activation, activated BMP receptor complexes initiates several intracellular pathways to modulate BMP-dependent cellular
responses like PI3-kinase, ERK, RhoA, and MAPK/JNK. Canonical BMP signalling is intracellularly inhibited by inhibitory Smads (Smad6, Smad7)
and E3 ubiquitin ligases like Smurf1 and Smurf2. Created with BioRender.com. The figure was exported under a paid subscription.
I. Luyckx et al.
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npj Genomic Medicine (2022) 68 Published in partnership with CEGMR, King Abdulaziz University

loop. A further level of control is achieved by cross-talk with TGF-β,
FGF, MAPK, Hedgehog, PI3K/Akt, Wnt/beta-catenin, retinoic acid
and Notch signalling pathways in order to regulate cellular BMP-
related processes in a very tight spatial and temporal
manner
64,65,69
.
SMAD6 signalling in cardiovascular development
Dysregulation of BMP signalling has extensively been investigated
in numerous cardiovascular diseases
1,70
. Interestingly, SMAD6-
deficient patients mainly exhibit defects related to two discrete
cell lineages, namely second heart field and neural crest cells.
Second heart field cells are multipotent progenitors originating
from cardiac progenitor cells and contribute to distinct regions of
the myocardium, cardiac endothelial cells and smooth muscle
cells
71
, while neural crest cells are derived from the dorsal aorta
and migrate as multipotent cells into the developing outflow tract
to coordinate outflow tract septation
72
. During cardiac cushion
development, SMAD6 is specifically expressed in endothelial cells
where it functions in (1) maintaining endothelial to mesenchymal
transition (EndMT)
54,63
, (2) stimulating cardiac cushions to grow
73
and (3) interacting with cardiac neural crest cells
74
, cells required
for aorticopulmonary septum formation. As such, this might
explain the marked clinical variability of SMAD6-deficient patient
with BAV-related aortopathy as predominant phenotype, and,
emphasises the complexity of CHD, in which gene dosage, timing,
haemodynamic flow, and its interplay with other signalling
pathways like Notch and TGF-βare important too. For example,
endothelial cells can undergo EndMT to become either
myofibroblast-like
75
or chrondrocyte- and osteoblast-like cells
76
,
depending on their cellular context.
SMAD6 signalling in cranial suture development
Gene discovery studies, and their subsequent characterisation in
mice, have determined highly conserved molecular pathways and
specific biological processes at different stages in cranial suture
development
45
. Initially, the strongest implication of BMP signal-
ling involvement was shown by BMP type 1 receptor (BMPR1A)
77
,
and by its convergence at key transcriptional factors downstream
of BMP, i.e., Msx2
78
and Twist1
79,80
, to regulate cell proliferation,
mesenchyme condensation, osteoblast differentiation, and osteo-
genesis. Subsequent work further supported a role of SMAD-
dependent signalling by the identification of causal mutations in
SKI
81
and SMAD3
82
in Shprintzen–Goldberg and Loeys–Dietz
patients, both conditions associated with CRS. Additional evidence
has emerged as SMAD6-deficiency increases the risk for CRS, and
in particular for metopic synostosis. In literature, metopic
synostosis has already been hypothesised to be the consequence
of abnormal maturation of neural crest-derived mesenchymal
stem cells via disturbed dynamics of cell identity or migration as a
common predisposing factor, and this can now be further
investigated
83,84
. Alternatively, processes not involved in cranial
suture development but affecting osteogenesis such as osteoblast
and osteoclast activity could be impaired too, and lead to CRS.
SMAD6 signalling in radioulnar development
Studies on BMP signalling in radioulnar development are very
scarce. So far, published data on RUS is limited to genetic
studies
9,13
and clinical descriptive reports lacking in-depth
functional analyses. Our current knowledge is inferred from
studies in axial skeletal development, with molecular pathways
like Wnt, Hedgehog, Notch, and FGF signalling pathways, to be
highly involved
85
. As RUS is believed to be the result of anomalous
differentiation and/or segmentation of the adjacent radius and
ulna, it could be true that BMPs lead to impaired mesenchymal
stem cell differentiation via Runx2 to promote osteoblast
differentiation from mesenchymal precursor cells
86,87
.
SUMMARY AND FUTURE OUTLOOK
In summary, three distinctive human genetic disorders are
caused by SMAD6 deficiency without domain-specificor
mutation-type genotype–phenotype correlation making proper
patient management difficult. Patients with cardiovascular
disease or craniosynostosis do not show any manifestations in
the other organ system within relatives of a single family,
suggesting that, (an)other factor(s) co-segregating with the
primary SMAD6 variant might explain the resultant phenotype.
To further explore this hypothesis, in-depth investigation into
the identification of the responsible cell type(s) and their
identity, as well as defining the predominant affected signalling
cascade(s) driving these disorders, will be fundamental for our
knowledge. Cell lineage tracing and spatial gene expression
analyses in Madh6-deficient mouse models might unravel
important clues to discriminate the afflicted processes leading
to cardiovascular disease, craniosynostosis and radioulnar
synostosis. Furthermore, a detailed clinical and genetic assess-
ment of additional SMAD6 variant-positive patients will be
needed, and, in particular, ascertain the complete phenotypic
picture of families with RUS, in which some clinical overlap with
CHD-, skull-, and skeletal-related anomalies might exist.
Other (additional) genetic factor(s) might explain incomplete
penetrance and extreme variability in phenotypical expressivity in
a patient with SMAD6 deficiency. For example, rare (or common)
variants located in a regulatory element of the trans-wild-type
SMAD6 allele, or variants in genes (e.g., SMAD7)afflicting
expression and/or activity of the BMP and/or the closely related
TGF-βsignalling activity are interesting avenues for further
exploration. It is worthwhile to consider genome-wide association
approaches that look into rare “second-hit”variants with large
effect size in SMAD6-deficient patients in order to add novel
information to the puzzle. Although this would aid to understand
the molecular basis of disease, the current available number of
SMAD6 mutant patients might not be sufficient to detect (a)
signal(s) even when only extreme phenotypes would be selected.
Nonetheless, in the upcoming years we will confidently identify
the SMAD6-related molecular patterns associated with these three
distinctive genetic disorders. This will allow us to detect early at-
risk individuals and empower new therapies.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
DATA AVAILABILITY
All data generated or analysed during this study are included in this publish ed article
(and its supplementary information file).
Received: 29 June 2022; Accepted: 8 November 2022;
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ACKNOWLEDGEMENTS
This research was supported by funding from the University of Antwerp (Methusalem-
OEC grant “Genomed”FFB190208). B.L. holds a consolidator grant from the European
Research Council (Genomia –ERC-C OG-2017 -77194 5). B.L. a nd A.V. are m embers of the
European Reference Network on rare multisystemic vascular disorders (VASCERN -
project ID: 769036 partly co-funded by the European Union Third Health Programme). I.L.
is supported by the Outreach project (Dutch Heart Foundation).
AUTHOR CONTRIBUTIONS
I.L. and B.L. conceived the idea. I.L. drafted the initial manuscript and revised the
manuscript. A.V., M.J.G. and B.L. contributed to the critical review and editing of the
manuscript. All authors contributed to the review and the final approval of the
completed version.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41525-022-00338-5.
Correspondence and requests for materials should be addressed to Bart Loeys.
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