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.