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.