Functional assessment of the missense variants identified in
MAP3K7
The differences in clinical features seen in FMD2 versus CSCF patients
are thought to be caused by different underlying molecular mechanisms.MAP3K7 variants giving rise to FMD2 are most often recurrent,
gain-of-function variants
(Wade et al., 2016), whereas CSCF is frequently caused by non-recurrent
missense variants, where for some variants a loss-of-function effect was
shown, but the functional effect of other variants remains unknown. Our
cohort, in combination with already published patients, allowed us to
assess whether there is indeed a molecular fingerprint that can
distinguish between FMD2 and CSCF. Since our cohort has only 2 FMD2
patients, we included the previously published FMD2-causing missense
variants in MAP3K7
(Wade et al., 2016).
In searching for a molecular fingerprint, we first assessed the
expression levels of the different MAP3K7 variants upon
overexpression in HEK-293T cells. Whereas the variants causing FMD2
showed similar or even a significantly higher expression level compared
to MAP3K7WT, all CSCF variants showed significantly
lower expression levels compared to MAP3K7WT (FMD2
variants: one-way ANOVA F[5,25]=6.16, p=0.0008;
MAP3K7WT versus MAP3K7E70Q: p=0.39;
MAP3K7WT versus MAP3K7V100E:
p=0.087; MAP3K7WT versus
MAP3K7Y113D: p=0.42; MAP3K7WT versus
MAP3K7G168R: p=0.40; MAP3K7WT versus
MAP3K7P485L: p=0.0079, Dunnett’s multiple comparison
test; CSCF variants: one-way ANOVA F[7,23]=6.39, p=0.0003;
MAP3K7WT versus MAP3K7G48E: p=0.004;
MAP3K7WT versus MAP3K7R83H:
p=0.0017; MAP3K7WT versus
MAP3K7G110D: p=0.041; MAP3K7WTversus MAP3K7M196V: p=0.0013;
MAP3K7WT versus MAP3K7Y206C: p=0.01;
MAP3K7WT versus MAP3K7Y206D:
p=0.0025; MAP3K7WT versus
MAP3K7W241G: p<0.0001, Dunnett’s multiple
comparison test ; Figure 4A ). With the exception of
MAP3K7W241G, reduced expression levels of the CSCF
variants could be normalized to control levels upon co-transfection with
TAB1 (one-way ANOVA F[7,54]=4.3, p<0.001;
MAP3K7WT versus MAP3K7G48E:
p<0.0001; MAP3K7WT versus
MAP3K7R83H: p=0.86; MAP3K7WT versus
MAP3K7G110D: p=0.9; MAP3K7WT versus
MAP3K7M196V: p=0.25; MAP3K7WT versus
MAP3K7Y206C: p0.9; MAP3K7WT versus
MAP3K7Y206D: p=0.06; MAP3K7WT versus
MAP3K7W241G: p=0.02, Dunnett’s multiple comparison
test; Figure 4B ), indicating that these variants cause
instability of the MAP3K7 protein in the absence of TAB1.
Co-expression of MAP3K7 with TAB1 has been shown to result
in MAP3K7 autophosphorylation at Thr187, as well as a slower
migration of the TAB1 band on Western blot (Sakurai et al., 2000).
Therefore, we used this assay as a readout for the kinase activity ofMAP3K7 . Consistent with literature, co-expression of
MAP3K7WT with TAB1 resulted in slower migration of the
MAP3K7, as well as the TAB1 band on Western blot (Figure 4B ),
which corresponded with MAP3K7 autophosphorylation of MAP3K7 at Thr187
(Figure 4C ). In support of previous findings that FMD2 is
caused mainly by gain-of-function variants, co-expression of the
FMD2-related MAP3K7 variants with TAB1, all resulted in equal or
(a trend towards) increased levels of autophosphorylation ofMAP3K7 at Thr187 compared to MAP3K7WT (one-way
ANOVA F[5,42]=5.43, p=0.0006; MAP3K7WT versus
MAP3K7E70Q: p=0.9; MAP3K7WT versus
MAP3K7V100E: p=0.4; MAP3K7WT versus
MAP3K7Y113D: p=0.03; MAP3K7WT versus
MAP3K7G168R: p=0.0004; MAP3K7WTversus MAP3K7P485L: p=0.9, Dunnett’s multiple
comparison test (Figure 4C )). In contrast, upon co-transfection
with TAB1 the majority of the CSCF-related MAP3K7 variants showed
significantly reduced pThr187 autophosphorylation levels compared to
MAP3K7WT (one-way ANOVA F[7,54]=15.9,
p<0.0001; MAP3K7WT versus
MAP3K7G48E: p<0.0001;
MAP3K7WT versus MAP3K7G110D:
p<0.0001; MAP3K7WT versus
MAP3K7M196V: p<0.0001;
MAP3K7WT versus MAP3K7Y206C:
p=0.0002;MAP3K7WT versus
MAP3K7Y206D: p<0.0001;
MAP3K7WT versus MAP3K7W241G:
p<0.0001, Dunnett’s multiple comparison test (Figure
4C )). Interestingly, one CSCF-related MAP3K7 variant
(MAP3K7R83H), did not show reduced pThr187
autophosphorylation levels compared to MAP3K7WT(MAP3K7WT versus MAP3K7R83H: p=0.48,
Dunnett’s multiple comparison test (Figure 4C )).
MAP3K7 is known to have several downstream substrates, through which it
affects different pathways in the cell (Xu and Lei, 2020; Aashaq et al.,
2019). Finding that the stability and the autophosphorylation levels
seem to distinguish between CSCF and FMD2-related MAP3K7variants, we next sought to understand whether there is a difference in
substrate regulation between the CSCF and FMD2-related MAP3K7variants. Consistent with the reduced autophosphorylation, we found that
the CSCF-related MAP3K7 variants resulted in reduced
phosphorylated NFkB compared to MAP3K7WT, again with
the exception of MAP3K7R83H (one-way ANOVA
F[7,51]=23.73, p<0.0001; MAP3K7WT versus
MAP3K7G48E: p<0.0001;
MAP3K7WT versus MAP3K7R83H: p=0.66;
MAP3K7WT versus MAP3K7G110D:
p<0.0001; MAP3K7WT versus
MAP3K7M196V: p<0.0001;
MAP3K7WT versus MAP3K7Y206C:
p<0.0001; MAP3K7WT versus
MAP3K7Y206D: p<0.0001;
MAP3K7WT versus MAP3K7W241G:
p<0.0001, Dunnett’s multiple comparison test (Figure
5A )).
As for the FMD2-related MAP3K7 variants, the results were less
straightforward. Overall, the variants did not cause increased levels of
phosphorylated NFkB, instead most variants showed a trend or significant
reduction in phosphorylated NFkB (one-way ANOVA F[5,49]=4.11,
p=0.0034; MAP3K7WT versus
MAP3K7E70Q: p=0.04; MAP3K7WT versus
MAP3K7V100E: p=0.4; MAP3K7WT versus
MAP3K7Y113D: p=0.54; MAP3K7WT versus
MAP3K7G168R: p=0.018; MAP3K7WTversus MAP3K7P485L: p=0.74, Dunnett’s multiple
comparison test (Figure 5A )). These results indicate that
whereas stability and autophosphorylation of MAP3K7 at Thr187 can
potentially be used as molecular fingerprint for distinguishing FMD2 and
CSCF variants, not all downstream pathways of MAP3K7 are necessarily
differentially affected.
The majority of the CSCF patients showed clinical features in part
similar to NS, where the RAS-MAPK pathway is hyperactivated (van der
Burgt, 2007; Jorge et al., 2009). We therefore assessed whether the
CSCF-related MAP3K7 variants upregulated the RAS-MAPK pathway.
However, in contrast to what is normally seen in NS, we found that most
of the CSCF-related MAP3K7 variants resulted in reduced
phosho-ERK levels compared to MAP3K7WT (one-way ANOVA
F[7,24]=6.78, p=0.0002; MAP3K7WT versus
MAP3K7G48E: p=0.016; MAP3K7WT versus
MAP3K7G110D: p=0.007; MAP3K7WTversus MAP3K7M196V: p=0.005;
MAP3K7WT versus MAP3K7Y206C:
p=0.008; MAP3K7WT versus
MAP3K7Y206D: p=0.009; MAP3K7WTversus MAP3K7W241G: p=0.021, Dunnett’s multiple
comparison test (Figure 5B )). Consistent with our previous
results, only MAP3K7R83H behaved similar to
MAP3K7WT (MAP3K7WT versus
MAP3K7R83H: p=0.9, Dunnett’s multiple comparison test
(Figure 5B )).