Discussion
In this study, we significantly expand the published cohort of patients
with variants in the MAP3K7 gene, causing either CSCF or FMD2 and
assess for the first time the genotype-phenotype correlation forMAP3K7 variants. We show that missense variants in MAP3K7causing CSCF reduce MAP3K7 protein stability and autophosphorylation at
Thr187, whereas MAP3K7 variants causing FMD2 show normal or
enhanced MAP3K7 protein expression and autophosphorylation levels.
However, when assessing one of the downstream targets, NFkB, no
difference was observed between the variants causing CSCF or FMD2,
indicating that further research is required to assess what downstream
targets are differentially affected, to further elucidate the precise
molecular mechanism underlying CSCF and FMD2.
Using whole exome sequencing (WES) in 5 independent Dutch pediatric
patients (from two separate medical centers) suspected to have NS,
variants of unknown significance were found in MAP3K7 . Further
clinical assessment (detailed physical examination and radiologic
assessment of the skeleton) of these patients confirmed the diagnosis of
CSCF. To further characterize genotype/phenotype correlations in these
patients, we studied 13 children (12 CSCF, 1 FMD2) and 3 adults (1 FMD2)
with variants in MAP3K7. Features of our CSCF patients
overlapping NS included short stature (n=9; 64%), congenital cardiac
abnormalities (n=7, 54%), cardiomyopathy (n=4, 33%) posteriorly
rotated and/or low set ears (n=7; 58%), hypertelorism (n=8; 67%),
ptosis (n=8, 62%), widely spaced nipples (n=7, 78%) and epicanthal
folds (n=6; 50%). Downslanting palpebral fissures (n=3; 23%),
triangular face (n=4; 33%), short or webbed neck (n=3, 27%) and
scoliosis (n=2, 22%) were less often present. Distinguishing features
of the CSCF patients are the wide nose with bulbous tip (n=9; 82%),
peri-orbital fullness (n=7, 64%) and the full cheeks (n=9; 69%).
Information about the neurological phenotype was available for 7
patients, who showed normal intelligence; three had autism spectrum
disorder. Hypotonia was found in 7 patients (70%), and joint laxity in
8 (80%). The resemblance of CSCF to NS made us wonder if CSCF should
also be considered a RASopathy; however, the RAS/MAPK pathway is
downregulated instead of upregulated, suggesting an alternative
explanation for the resemblance of CSCF to NS. Despite these molecular
results, CSCF should be considered in the differential diagnosis of
NS-patients based on the described clinical features.
Both our FMD2 patients showed phenotypes compatible with previously
reported patients (Wade et al., 2017). Interestingly, our adult male
FMD2 patient initially presented with ambiguous genitalia and he has a
mild skeletal phenotype with normal/high intelligence. The pediatric
patient with the recurrent variant for FMD2 has a severe cardiac,
neurological and skeletal phenotype. Distinguishing features of the FMD2
patients in comparison to NS and CSCF were prominent supraorbital
ridges, flexion contractures of the elbows, ulnar deviation of the
hands, interphalangeal joint contractures, camptodactyly and keloid
scarring (all present in both patients). Overlapping features with NS
were short stature, congenital cardiac defects, hypertelorism,
epicanthal folds, ptosis (n=1) and failure to thrive in infancy. Neither
patient had a triangular face, short or webbed neck. FMD2 patients have
more severe dysmorphism than CSCF patients (Figure 3 ).
Regarding the occurrence of left sided heart lesions and the potential
progressive aspect in our patient of aortic dilatation, these features
go along with previous reports that genes which belong to TGFβ cascade
are potentially prone to this subcategory of cardiac involvement (Baban
et al., 2018). It might be wise to establish a specific screening
program through serial electrocardiography and echocardiography detect
progressive aortic dilatation or arrhythmias.
Our cohort in combination with the MAP3K7 mutations causing FMD2
described in literature allowed for the first time to search for
genotype/phenotype correlations, i.e. side-by-side comparison of the
different missense mutations causing either FMD2 or CSCF. Confirming
previous reports, we found that the FMD2-causing mutations inMAP3K7 produce a gain-of-function effect on MAP3K7. Additionally,
we now provide clear evidence that the CSCF-causing variants inMAP3K7 have a loss-of-function effect. This is most clearly seen
when looking at the expression and pThr187 levels of MAP3K7.
Interestingly, when assessing one of the downstream targets ofMAP3K7 , NFkB (Xu and Lei, 2020), this distinction between the
FMD2- or CSCF-causing variants is not evident, suggesting other
molecular pathways are involved in the pathogenesis of these two
disorders. Indeed, we have only assessed the effect on this pathway in
co-expression of TAB1 and MAP3K7. Possibly there is a more distinct
phenotype when assessing the function of MAP3K7 when in complex with
TAB2/3. Additionally, other downstream pathways remain to be studied.
In this paper we significantly expanded the cohort of patients with
either CSCF or FMD2 and performed for the first time a side-by-side
comparison of the different missense variants related to either FMD2 or
CSCF. We have shown that both clinical phenotypes exhibit a clear
overlap with other syndromic connective tissue disorders with respect to
bone deformities, short stature, hypermobile joints, contractures and
cardiac anomalies. This fits what is known about MAP3K7 ’s effect
on the TGFβ pathway
(Le Goff et al., 2016; Yu et al., 2014). Additionally, we have
identified that cardiac anomalies are common in patients with pathogenicMAP3K7 variants, which may be severe. The cardiac phenotype
ranged from left sided congenital heart defects to cardiomyopathies
(both hypertrophic and dilated) and arrhythmias. The latter subgroup is
infrequent in NS (Pierpont and Digilio, 2018; Gelb et al., 2015). In
conclusion, we show that expression and autophosphorylation levels ofMAP3K7 can serve as a molecular fingerprint to distinguish
between FMD2- or CSCF-causing MAP3K7 variants. Additionally, we
show that MAP3K7 variants should be considered in the
differential diagnosis of patients with syndromic congenital cardiac
(valve) anomalies and/or cardiomyopathy, syndromic connective tissue
disorders as well as NS.