Figure Legends
Figure 1. Identification of a de novo mutation in CHRM1
in a patient with developmental and epileptic encephalopathy. A . Brain
MRI at age 1y (top) and 4y showed right occipital plagiocephaly, mild
enlargement of subarachnoidal spaces, a relatively thick corpus callosum
and marked cerebellar atrophy with vermian predominance (arrows).B . The patient at age 8y, exhibiting poor eye contact,
hypotonia and quadriparesis. C. Left panel: Pedigree of the
affected patient, showing the p.Pro380Leu heterozygous carrier (filled
symbol), and her unaffected parents and half-sister. Right panel: Sanger
sequencing validation. Chromatograms of patient (bottom) and the
wild-type sequence as found in parents and half-sister sequencing (top),
confirmed the de novo occurrence of the heterozygous CHRM1c.1139C>T (NM_000738.2) variant. D . 3D CHRM1
protein structure model shows the 7 transmembrane helixes (in red) and
the position of Proline 380 (in pink), which determines a kink in the
distal part of helix 6.
Figure 2. The mutation p.P380L causes a trafficking and a
functional defect to the CHRM1 protein. A . Western-Blot of transfected
cells with flag-tagged CHRM1 WT or carrying p.P380L mutation was
performed with an antibody detecting the flag epitope. Different protein
band pattern was obtained corresponding to (1) mature glycosylated
protein form, (2) and (3) immature forms. Cells transfected with the
mutated vector showed an increase in forms 2 and 3 and a decrease in
mature glycosylated CHRM1 compared to cells transfected with wild-type
vector. β-actin was used to normalize CHRM1 protein levels. Statistics
were performed as described in Materials and Methods from 5 independent
experiments. B. HeLa cells co-transfected with
pcDNA3-hCHRM1 -3Flag or pcDNA3-hCHRM1 (P380L)-3Flag (P380L)
and PH-GFP, were studied by immunofluorescence and colocalization
between CHRM1 (red label) and PH-GFP (green label) was analyzed (Merge)
by Pearson´s correlation analysis. Statistics were performed from 3
independent experiments. (C, D) Signaling pathways associated
to CHRM1 were studied by a luciferase signal assay in HEK293T cells
transfected with pcDNA3-hCHRM1(wt)-3Flag] or
pcDNA3-hCHRM1(P380L)-3Flag- when treated with Carbachol, an agonist.C. cAMP signaling pathway activation was studied through CRE
gene reporter. Negative controls and forskolin treated cells (positive
control) were used to normalize results. D.IP3-Ca2+ signaling pathway activation
was analyzed through NFAT transcription factor. Negative controls and
CA+PMA treated cells (positive control) were used to normalize results.
Statistics were performed from 3 independent experiments.