Fig 2: Identification of the 12T-ins/del polymorphism in the ATP1A1 promoter region and transcriptional activity of ATP1A1 promoter variants.(A) Nucleotide sequence spanning the poly-T sequence involved in the 12T-ins/del polymorphism from the two ATP1A1 (p12T ins, p12T del) reporter gene constructs utilized in the transcriptional assays. On right detection of 12T-insertion and 12T-deletion alleles by PCR-amplification followed by denaturing polyacrylamide gel (6%) electrophoresis used for genotyping of the Sardinian cohort. (B) Illustration of the ATP1A1 promoter region. Non-coding exon is presented as open box and exon encoding the NH2-terminal region is presented as black box. Sequence and location of the ATP1A1 12T-ins/del polymorphism is shown. The positions of TATAAA box, INR (initiator) and DPE (downstream promoter element) core promoter elements within ATP1A1 promoter are shown. Core promoter elements were identified based on 100% homology with corresponding consensus sequences [39,40]. (C) Schematic of two ATP1A1 (p12T ins, p12T del) reporter gene constructs. (D) Relative transcriptional activity of 12T-insertion (p12T ins) and 12T-deletion (p12T del) gene constructs in Cos1, HEK293 and MDA-MB-468 cells. *, P < 0.05; **, P < 0.01 (two-tailed student t-test).

Fig 3: mRNA and protein expression analysis of the wild type and mutant ATP1A1. A mRNA expression of ATP1A1 exons in the patient peripheral blood cells compared to healthy controls (Patient: 58.04 ± 2.16%, Control 1: 100.25 ± 2.32%, Control 2: 122.32 ± 6.54%, Control 3: 111.76 ± 2.77%. P = 0.00002). Three independent qPCRs were performed and samples were run as triplicates in each experiment. B Western blot analysis results of ATP1A1 expression normalized against ß-ACTIN and GAPDH. C Quantification of protein expression of ATP1A1 in the patient versus healthy control LCLs (Patient: 45.49 ± 10.02%, Control 1: 99.01 ± 14.84%, Control 2: 94.15 ± 9.10%, Control 3: 123.94 ± 17.00%. P = 0.002). WB was replicated eight times for quantification. Statistical analysis was performed by one-way ANOVA. P < 0.05 considered statistically significant. D The expression of mutant ATP1A1 (c.1799 C > G, P600R) transcript in patient blood confirmed by Sanger sequencing

Fig 4: Ouabain survival (luciferase) assay. Viability of ouabain insensitive HEK cells transfected with wild type ATP1A1, p.P600R and, positive controls p.G509D and p.G718S normalized to untreated cells transfected with the same plasmid

Fig 5: ATP1A1 protein and respective locations of the pathogenic variants reported up to date. Variants written in black represent CMT related variants identified in other studies, red represents the ATP1A1 variant identified in this study, blue represent hypomagnesaemia and intellectual disability or spastic paraplegia, green represent developmental delay, orange represent complex neurodevelopmental syndrome