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 )).