Recurrent ATP1A1 variant Gly903Arg causes developmental delay, intellectual disability, and autism

Abstract

ATP1A1 encodes a sodium‐potassium ATPase that has been linked to several neurological diseases. Using exome and genome sequencing, we identified the heterozygous ATP1A1 variant NM_000701.8: c.2707G>A;p.(Gly903Arg) in two unrelated children presenting with delayed motor and speech development and autism. While absent in controls, the variant occurred de novo in one proband and co‐segregated in two affected half‐siblings, with mosaicism in the healthy mother. Using a specific ouabain resistance assay in mutant transfected HEK cells, we found significantly reduced cell viability. Demonstrating loss of ATPase function, we conclude that this novel variant is pathogenic, expanding the phenotype spectrum of ATP1A1.

Introduction

Sodium‐potassium pumps play a vital role in maintaining the electrochemical gradients across cell membranes through ATP‐dependent processes. ATP1A1 encodes the α1 subunit of Na⁺/K⁺‐ATPase, which is highly expressed in the nervous system. Heterozygous missense mutations can lead to reduced ATPase function, which has previously been associated with an axonal to intermediate neuropathy phenotype ¹ , with hereditary spastic paraplegia, ² and a complex severe intellectual disability syndrome, partially accompanied by hypomagnesemia, ³ , ⁴ all being inherited in an autosomal dominant manner.

Patients and Methods

Patients were examined by experienced neurologists. We obtained written informed consent from both patients' legal representatives prior to study enrollment. Exome and genome sequencing were conducted following site‐specific protocols. Sanger sequencing was used for variant confirmation and co‐segregation studies. To assess reduced ATPase function, we transfected HEK cells with a mutant ATP1A1 plasmid that otherwise confers resistance to ouabain in vitro. We measured cell viability after 72 h of treatment with the unselective ATPase inhibitor ouabain. ⁴

Results

We identified the variant c.2707G>A; p.(Gly903Arg) in ATP1A1 (ENST00000295598.10/NM_000701.8) (Fig. 1A) in a heterozygous state in two unrelated probands. The two individuals were male, one 10, and the other 7 years old at the time of examination. Patient 1 was born in the United States of America with mixed German/Irish and Colombian descent; patient 2 originated from France. Both patients presented with intellectual disability as the main symptom, accompanied by delayed motor and speech development. Patient 1 was diagnosed with autism; patient 2 had mild dysmorphic facial features. Variants were validated by Sanger sequencing, and co‐segregation analyses revealed de novo status in patient 2. Patient 1's unaffected mother was found to be mosaic for the variant (Fig. 1B), and his 23‐year‐old maternal half‐sister, who also has a history of developmental delay and intellectual disability, was likewise found to be heterozygous for the same ATP1A1 variant.

Figure 1.

Genetic and experimental evidence for p.Gly903Arg pathogenicity. (A) Chromosomal position of the ATP1A1 variant c.2707G>A;(p.Gly903Arg) with its high regional constraint (CRR), high conservation (GERP), and absence from control databases (gnomAD). The red arrow indicates the variant's nucleotide position on chromosome 1. (B) Sanger sequencing shows c.2707G>A mosaicism in saliva and hair follicle in patient 1's unaffected mother. (C) HEK cells were transfected with ouabain‐insensitive ATP1A1 plasmids for mutant and wildtype (oua‐wt), as well as wt‐ATP1A1 (not ouabain‐insensitive) and treated with ouabain (0.5 μmol/L) for 72 h. Cell viability was measured in replicates of eight, using the luminescence‐based CellTiterGlo assay. Ratios are shown in comparison with untreated cells (mean) transfected with the same plasmid. Statistics: one‐way ANOVA, α‐error correction: Tukey's post‐test. ***P < 0.0001

The variant has not been published in association with any disease phenotype, nor has it been listed in control databases (gnomAD v2). Located in exon 19 (of 23), it affects the longest extracellular loop (52 residues) between transmembrane regions 7 and 8, which both contain other pathogenic missense variants (c.2590G>C; p.(Gly864Arg) and c.2768T>A; p.(Phe923Tyr)) that have previously been linked to intellectual disability. ⁴ , ⁵ In the same extracellular domain, another reported pathogenic variant c.2629G>A; p.(Gly877Ser) has been associated with intermediate sensorimotor neuropathy. ⁶ The novel p.(Gly903Arg) variant affects a highly conserved amino acid position and a highly constrained coding region (CCR > 95^th percentile). ⁷ In silico predictions are pathogenic (CADD score: 32), and the transformer‐based neuronal network MAVERICK ⁸ assigned a pathogenicity probability of 91.9% for autosomal dominant inheritance.

To functionally assess the variant's pathogenicity, we transfected HEK cells with ouabain‐insensitive ATP1A1 plasmids encoding either the wildtype (wt) or the mutant, in replicates of eight. As a negative control, we used ouabain‐insensitive wt‐ATP1A1 plasmids, and as a positive control, we chose ouabain‐insensitive plasmids containing the previously reported p.Phe923Tyr variant that is located in close vicinity, as well as the ouabain‐sensitive wt, and untransfected cells. 24 h after transfection, we treated the cells with the ATPase inhibitor ouabain at a concentration of 0.5 μmol/L. We measured cell survival after 72 h, using the previously established luciferase (CellTiter‐Glo) assay. To demonstrate that cell death was not an effect of transfection, we normalized each treated mutant to untreated cells transfected with the same plasmid. Compared to the ouabain‐insensitive wt, cell viability was significantly reduced for the p.(Gly903Arg) variant (Fig. 1C), and cell survival ratios were even lower than compared to the previously reported variant p.(Phe923Tyr). This result indicates reduced ATPase function, rendering otherwise ouabain‐insensitive cells more vulnerable for cell death.

Discussion

We herein describe a novel, pathogenic ATP1A1 variant p.(Gly903Arg) causing developmental delay and intellectual disability in two unrelated families. Based on its absence from control populations and its affecting of a highly conserved position and constrained protein region, it is very likely to be pathogenic. Our established bioassay revealed a significant loss of ATPase function in cells transfected with the mutant, consistent with other pathogenic mutations reported in the literature, supporting haploinsufficiency as the pathomechanism for this variant. Considering that other missense mutations in the same gene cause late‐onset hereditary neuropathy as an allelic disorder, ¹ , ⁶ the herein described clinical phenotype is relatively severe. Interestingly, no signs of neuropathy or spastic paraplegia have been reported in these two children, which may be attributed to the patients' young age. The specific phenotypic outcome of one point mutation over another, as well as potential modifications by other inherited or environmental factors contributing to the disease spectrum requires further investigation.

Autism is known for its broad underlying genetic heterogeneity. As commonly observed in neurodevelopmental syndromes, effects on fecundity may complicate the recognition of heritability, and large Medelian ancestries are rare to find. Like in the herein described families, de novo mutations and mosaicism are relevant mechanisms to take under consideration when searching for the underlying genetic cause. ⁹ To validate potentially pathogenic mutations, we therefore recommend to perform genetic sequencing on parent samples, including DNA derived from tissue other than blood.

We conclude that variant p.(Gly903Arg) is pathogenic, associated with a childhood‐onset neurological phenotype mainly manifesting with developmental delay and intellectual disability. The gene ATP1A1 should therefore be considered for genetic testing in patients with a comparable neurodevelopmental phenotype, including spectrum disorders such as autism.

Members of the Undiagnosed Diseases Network

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Conflict of interest

The authors declare that there are no conflicts of interest.

Written informed consent for publication of case

The patients' parents provided written informed consent for the publication of these cases.

Funding Statement

This work was funded by Deutsche Forschungsgemeinschaft, DFG grant DO 2386/1‐1; National Institutes of Health grant R01NS105755; Office of Strategic Coordination/Office of the NIH Director grant U01HG010230.

Data availability statement

Original data are available upon reasonable request to the corresponding author.