Figure 3. Clinical description and light photography/radiological pictures of the patient with the recurrent P485L variant causing FMD2 (patient 16).
Cranio-facial dysmorphisms at 2 months (panels 1-3): bi-temporal narrowing, hypotelorism, down-slanting palpebral fissures, short nose with flat nasal bridge and depressed root, long and deeply grooved filter, microretrognathia. Radiological findings (panels 4-7) at 7 months: a partial fusion of cervical vertebrae (C2-C3) and reduced bone length in relation to age. Magnetic resonance imaging (MRI) at 7 months: T2-weighted hyperintensity of presumed vascular lesion in the coronal and transverse planes (panels 8-9); T1 weighed scans in the sagittal plane: cerebral atrophy, reduced corpus callosum, and Chiari 1 malformation (panel 10). Cranial time-of-flight (TOF) MR angiogram (3T) at 7 months: flow reduction of the M2 branch of left middle cerebral artery (MCA) (panel 11). Facial appearance at 3 y.o. (panel 12).
Figure 4. Differential expression and autophosphorylation of variants in MAP3K7 causing CSCF or FMD2. A) Western blot analysis showing reduced expression levels of CSCF-causing MAP3K7variants (black circles), compared to MAP3K7WT, but normal or even increased expression levels of FMD2-causing MAP3K7variants (blue circles), compared to MAP3K7WT, when overexpressed in HEK-293T cells. N=3 for all CSCF-causing variants, except MAP3K7W241G (N=6). N=6 for all FMD2-causing variants, except MAP3K7Y113D and MAP3K7G168R (N=3). WT N=7. B) Western blot analysis showing that expression levels of CSCF-causing MAP3K7variants is normalized upon TAB1 co-expression, except for MAP3K7W241G. N=7 for all conditions, except for WT (N=14) and MAP3K7W241G (N=6). C) Western blot analysis of the autophosphorylation of MAP3K7 at Thr187 showing that except for MAP3K7R83H, all CSCF-causingMAP3K7 variants show reduced pThr187 compared to MAP3K7WT, whereas FMD2-causing variants show normal or increased levels of pThr187 compared to MAP3K7WT. N=7 for all conditions, except for WT (N=14) and MAP3K7Y206C and MAP3K7Y113D (N=6). Error bars represent standard error of the mean (SEM). * p<0.05; ** p<0.01; *** p<0.0001.
Figure 5. Western blot analysis of downstream targets ofMAP3K7. A) Western blot analysis reveals reduced phosphorylation of NFkB upon overexpression of most CSCF-causing (black circles) and some FMD2-causing (blue circles) MAP3K7 variants in HEK-293T cells, compared to MAP3K7WT. N=7 for all conditions except for WT (N=10). B) Western blot analysis of phosphoERK1/2 reveals reduced phosphorylation of ERK1/2 upon overexpression of most CSCF-causing MAP3K7 variants compared to MAP3K7WT, except for MAP3K7R83H. N=4 for all conditions, except for WT (N=8). Error bars represent standard error of the mean (SEM). * p<0.05; *** p<0.0001.