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J Neurol
2023 May 01;2705:2576-2590. doi: 10.1007/s00415-023-11581-w.
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The phenotypic spectrum of pathogenic ATP1A1 variants expands: the novel p.P600R substitution causes demyelinating Charcot-Marie-Tooth disease.
Cinarli Yuksel F
,
Nicolaou P
,
Spontarelli K
,
Dohrn MF
,
Rebelo AP
,
Koutsou P
,
Georghiou A
,
Artigas P
,
Züchner SL
,
Kleopa KA
,
Christodoulou K
.
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BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a genetically and clinically heterogeneous group of inherited neuropathies. Monoallelic pathogenic variants in ATP1A1 were associated with axonal and intermediate CMT. ATP1A1 encodes for the catalytic α1 subunit of the Na+/ K+ ATPase. Besides neuropathy, other associated phenotypes are spastic paraplegia, intellectual disability, and renal hypomagnesemia. We hereby report the first demyelinating CMT case due to a novel ATP1A1 variant.
METHODS: Whole-exome sequencing on the patient's genomic DNA and Sanger sequencing to validate and confirm the segregation of the identified p.P600R ATP1A1 variation were performed. To evaluate functional effects, blood-derived mRNA and protein levels of ATP1A1 and the auxiliary β1 subunit encoded by ATP1B1 were investigated. The ouabain-survival assay was performed in transfected HEK cells to assess cell viability, and two-electrode voltage clamp studies were performed in Xenopus oocytes.
RESULTS: The variant was absent in the local and global control datasets, falls within a highly conserved protein position, and is in a missense-constrained region. The expression levels of ATP1A1 and ATP1B1 were significantly reduced in the patient compared to healthy controls. Electrophysiology indicated that ATP1A1p.P600R injected Xenopus oocytes have reduced Na+/ K+ ATPase function. Moreover, HEK cells transfected with a construct encoding ATP1A1p.P600R harbouring variants that confers ouabain insensitivity displayed a significant decrease in cell viability after ouabain treatment compared to the wild type, further supporting the pathogenicity of this variant.
CONCLUSION: Our results further confirm the causative role of ATP1A1 in peripheral neuropathy and broaden the mutational and phenotypic spectrum of ATP1A1-associated CMT.
Fig. 1
Genetic findings of the Cypriot proband. A Electropherogram of the control ATP1A1 sequence (upper) versus patient ATP1A1: c.1799C > G (p.P600R) sequence (lower). B In silico analysis of the ATP1A1: c.1799C > G (p.P600R). C Conversation analysis of amino acid sequences of ATP1A1 (NM_000701.8) across species compared to p.P600R
Fig. 2
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 β-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. 3
mRNA and protein expression analysis of ATP1B1. A mRNA expression analysis of ATP1B1 cDNA in the patient peripheral blood cells compared to healthy controls (Patient: 44.71 ± 3.63%, Control 1: 100.04 ± 3.40%, Control 2: 94.63 ± 4.31%, Control 3: 103.99 ± 3.21%. P = 0.00001). Four independent qPCRs were performed and samples were analysed as triplicates in each run. B Western blot analysis results of ATP1B1 expression normalized against β-ACTIN and GAPDH. C) ATP1B1 protein expression quantification in patient versus healthy control LCLs (Patient: 30.22 ± 6.43%, Control 1: 118.20 ± 13.21%, Control 2: 110.05 ± 10.23%, Control 3: 103.64 ± 11.04%. P = 0.0002). WB was replicated four times for quantification. Statistical analysis was performed by one-way ANOVA. P < 0.05 considered statistically significant
Fig. 4
Electrophysiological findings. A Representative traces from an oocyte expressing WT (that is the RD ouabain resistant version) and P600R pumps. Oocytes were held at − 50 mV and exposed to the indicated [K+] (in mM) to activate pump current. Vertical deflections indicate application of 100 ms-long pulses to measure voltage-dependent parameters. B K0.5 for K+ activation of pump current obtained from Hill fits (“Methods”) to the [K+] dependencies at each voltage, as a function of the applied voltage. C Current, normalized to the mean from oocytes expressing WT pumps on the same day under the same conditions (results from 3 batches of oocytes, 11 WT and 13 P600R oocytes total. D Ouabain-sensitive transient currents elicited by pulses from − 50 mV to voltages ranging from − 140 mV to + 40 mV (in 20 mV increments). E Charge (integral of current transients) as a function of voltage normalized to the total charge moved in the whole voltage range (Qtot = 21.6 ± 10.6 for WT and 15.3 ± 11.1 for P600R). Line plots are Boltzmann distributions fitted to the data (mean V0.5 = − 47.5 ± 4.3 mV for WT and − 38.1 ± 4.3 mV for P600R, n = 29 each). F Mean total charge (measured from Boltzmann distributions in individual experiments) normalized to the mean from oocytes expressing WT pumps on the same day
Fig. 5
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. 6
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
Figure 2. 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 β-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
Figure 3. mRNA and protein expression analysis of ATP1B1. A mRNA expression analysis of ATP1B1 cDNA in the patient peripheral blood cells compared to healthy controls (Patient: 44.71 ± 3.63%, Control 1: 100.04 ± 3.40%, Control 2: 94.63 ± 4.31%, Control 3: 103.99 ± 3.21%. P = 0.00001). Four independent qPCRs were performed and samples were analysed as triplicates in each run. B Western blot analysis results of ATP1B1 expression normalized against β-ACTIN and GAPDH. C) ATP1B1 protein expression quantification in patient versus healthy control LCLs (Patient: 30.22 ± 6.43%, Control 1: 118.20 ± 13.21%, Control 2: 110.05 ± 10.23%, Control 3: 103.64 ± 11.04%. P = 0.0002). WB was replicated four times for quantification. Statistical analysis was performed by one-way ANOVA. P < 0.05 considered statistically significant
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