Germline De Novo Mutations in ATP1A1 Cause Renal Hypomagnesemia, Refractory Seizures, and Intellectual Disability

Over the last decades, a growing spectrum of monogenic disorders of human magnesium homeostasis has been clinically characterized, and genetic studies in affected individuals have identified important molecular components of cellular and epithelial magnesium transport. Here, we describe three infants who are from non-consanguineous families and who presented with a disease phenotype consisting of generalized seizures in infancy, severe hypomagnesemia, and renal magnesium wasting. Seizures persisted despite magnesium supplementation and were associated with significant intellectual disability. Whole-exome sequencing and conventional Sanger sequencing identified heterozygous de novo mutations in the catalytic Na+, K+-ATPase α1 subunit (ATP1A1). Functional characterization of mutant Na+, K+-ATPase α1 subunits in heterologous expression systems revealed not only a loss of Na+, K+-ATPase function but also abnormal cation permeabilities, which led to membrane depolarization and possibly aggravated the effect of the loss of physiological pump activity. These findings underline the indispensable role of the α1 isoform of the Na+, K+-ATPase for renal-tubular magnesium handling and cellular ion homeostasis, as well as maintenance of physiologic neuronal activity. Over the last decades, a growing spectrum of monogenic disorders of human magnesium homeostasis has been clinically characterized, and genetic studies in affected individuals have identified important molecular components of cellular and epithelial magnesium transport. Here, we describe three infants who are from non-consanguineous families and who presented with a disease phenotype consisting of generalized seizures in infancy, severe hypomagnesemia, and renal magnesium wasting. Seizures persisted despite magnesium supplementation and were associated with significant intellectual disability. Whole-exome sequencing and conventional Sanger sequencing identified heterozygous de novo mutations in the catalytic Na+, K+-ATPase α1 subunit (ATP1A1). Functional characterization of mutant Na+, K+-ATPase α1 subunits in heterologous expression systems revealed not only a loss of Na+, K+-ATPase function but also abnormal cation permeabilities, which led to membrane depolarization and possibly aggravated the effect of the loss of physiological pump activity. These findings underline the indispensable role of the α1 isoform of the Na+, K+-ATPase for renal-tubular magnesium handling and cellular ion homeostasis, as well as maintenance of physiologic neuronal activity. Magnesium is essential for numerous cellular processes, including energy metabolism, protein and nucleic acid synthesis, and the maintenance of the electrical potential of nervous tissues and cell membranes. Genetic investigations in children with inherited forms of hypomagnesemia could identify critical components of epithelial magnesium transport at the molecular level.1Viering D.H.H.M. de Baaij J.H.F. Walsh S.B. Kleta R. Bockenhauer D. Genetic causes of hypomagnesemia, a clinical overview.Pediatr. Nephrol. 2017; 32: 1123-1135Crossref PubMed Scopus (84) Google Scholar Affected children commonly present with seizures, muscle spasms, or tetany. In the majority of cases, magnesium supplementation leads to relief of clinical symptoms and allows for a normal motor and cognitive development despite persistence of subnormal serum magnesium levels. In contrast with this favorable clinical course, we noted a small group of nine children who, despite appropriate magnesium supplementation, experienced prolonged and repeated seizure activity associated with severe intellectual disability. Of this cohort, two individuals had previously been diagnosed with hypomagnesemia, seizures, and mental retardation [HOMGSMR, MIM: 616418] due to bi-allelic mutations in CNNM2 [MIM: 607803].2Arjona F.J. de Baaij J.H. Schlingmann K.P. Lameris A.L. van Wijk E. Flik G. Regele S. Korenke G.C. Neophytou B. Rust S. et al.CNNM2 mutations cause impaired brain development and seizures in patients with hypomagnesemia.PLoS Genet. 2014; 10: e1004267Crossref PubMed Scopus (90) Google Scholar Here, we identified heterozygous de novo mutations in ATP1A1 [MIM: 182310] (RefSeqGene: NG_047036, GenBank: NM_000701), encoding the α1 isoform of Na+, K+-ATPase, in three children from this cohort. Data on clinical symptoms and biochemical measures at the time of disease manifestation were collected retrospectively from medical charts. Affected children with ATP1A1 mutations were clinically reevaluated during follow-up, and biochemical data were obtained. The three infants initially presented between 6 days and 6 months of age with generalized convulsions (Table 1, for the full dataset please refer to Table S1 in the Supplemental Data). At the time of manifestation, severe hypomagnesemia (0.30–0.36 mmol/L) was noted. Calculation of urinary fractional excretion rates of magnesium indicated massive renal magnesium wasting. Although urinary calcium excretion was not uniformly elevated, initial renal ultrasound examinations indicated medullary hyperechogenicity compatible with incipient nephrocalcinosis in individuals B-II-1 and C-II-2. All children were treated with antiepileptic drugs and received intravenous magnesium followed by ongoing oral supplementation. However, seizure activity persisted with frequent generalized seizures and repeated status epilepticus despite amelioration of serum magnesium levels. After a status epilepticus with both tonic-clonic seizure activity and hypoxemia for more than one hour, the cerebral magnetic resonance imaging (MRI) of individual A-II-1 showed bilateral parietooccipital cortical and subcortical diffusion restriction compatible with hypoxic ischemic encephalopathy. This resulted in a marked developmental setback and impaired vision. Follow-up MRI showed cerebral volume loss. All three children uniformly had significant global developmental delay, displayed limited motor skills, and spoke only in single words. Two individuals (B-II-1 and C-II-2) showed clinical features compatible with an autism spectrum disorder. MRI examinations revealed cerebral volume loss in individual C-II-2 also. Blood-pressure measurements repeatedly demonstrated normotension in all children, and cardiac examinations performed in individuals A-II-1 and C-II-2 were unremarkable. Individuals B-II-1 and C-II-2 exhibited significant polyuria of 4–8 ml/kg/h, but renal concentrating ability remained at least partially intact (random urine osmolalities of >400 mosmol/L). Although laboratory analyses did not reveal renal salt wasting or a significant activation of the renin-aldosterone system and serum potassium levels were mostly within the reference range, all affected individuals exhibited repeated episodes with significant hypokalemia (S-K+ of 2.1 to 2.6 mmol/L). Calculation of the transtubular potassium gradient during hypokalemic episodes indicated renal potassium wasting. The most recent laboratory examinations in individuals A-II-1 and B-II-1 demonstrated persisting hypomagnesemia despite high doses of oral magnesium supplementation, and fractional urinary excretion rates of magnesium confirmed major renal magnesium wasting. Parents of all three children were clinically unaffected and showed normal serum magnesium levels.Table 1Overview of Clinical Characteristics and GenotypesIndividualA-II-1B-II-1C-II-2DemographicsOriginof European descentof European descentFirst Nations CanadianGenderfemalefemalemaleAge at manifestation6 months2 months6 daysFirst symptomgeneralized seizuresgeneralized seizuresgeneralized seizuresInitial Laboratory FindingsS-Mg (mmol/L) (0.75–1.1)0.360.350.30FE-Mg (%) (3-5%)26.033.8ndMost Recent FindingsAge at last follow-up4 years10 years6 yearsS-Mg (mmol/L) (0.75.-1.1)0.570.280.62FE-Mg (%) (3-5%)15.327.021.3seizure activityrepeated status epilepticusmonthly seizuresfrequent seizures,repeated status epilepticusneurological outcomeglobal developmental delay, hyperactive behaviorglobal developmental delay, suspected autism spectrum disorderglobal developmental delay, speech delay, diagnosis of severe autism, self-biting behaviorATP1A1 Mutationsnucleotide levelc.905T>Cc.907G>Cc.2576T>Gprotein levelp.Leu302Argp.Gly303Argp.Met859Arg Open table in a new tab Extraction of DNA from whole blood was performed according to standard protocols. All genetic studies were approved by the respective ethics committees of the involved centers. The parents provided written informed consent. The clinical phenotype initially suggested the diagnosis of hypomagnesemia with secondary hypocalcemia (HSH) [HOMG1, MIM: 602014]; however, mutations in TRPM6 [MIM: 607009] were excluded.3Schlingmann K.P. Weber S. Peters M. Niemann Nejsum L. Vitzthum H. Klingel K. Kratz M. Haddad E. Ristoff E. Dinour D. et al.Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family.Nat. Genet. 2002; 31: 166-170Crossref PubMed Scopus (647) Google Scholar, 4Walder R.Y. Landau D. Meyer P. Shalev H. Tsolia M. Borochowitz Z. Boettger M.B. Beck G.E. Englehardt R.K. Carmi R. Sheffield V.C. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia.Nat. Genet. 2002; 31: 171-174Crossref PubMed Scopus (470) Google Scholar Under the assumption of an unknown disease phenotype, we performed whole-exome sequencing in individual A-II-1 as well as the family C trio in order to identify the underlying genetic defect. Details on target enrichment, sequencing, and data analysis are provided in the Supplemental Data. We focused on missense, nonsense, splice-site, and frameshift variants upon all modes of inheritance. After performing sequential filtering and keeping variants predicted as pathogenic, we could identify no common gene with homozygous or compound-heterozygous variants in the two affected individuals. In contrast, both were found to carry a single heterozygous mutation, c.905T>G (p.Leu302Arg) and c. 2576T>G (p.Met859Arg) in ATP1A1, respectively. In silico analyses predicted the variants to be pathogenic; they were predicted to affect highly conserved amino acid residues of the Na+, K+-ATPase α1 protein (Figure 1, Table S2). None of the identified mutations is listed in publicly available exome databases, i.e., ExAC and gnomAD browsers. Identified mutations were confirmed by Sanger sequencing (details of primers are available on request); the trio analysis of family C exomes as well as sequencing of the parents of individual A-II-1 demonstrated that both mutations occurred de novo. We could not identify any additional gene with heterozygous variants shared by both affected individuals (A-II-1 and C-II-2). Therefore, we do not have any genetic evidence that the phenotype observed here results from additive effects of variants in a gene other than ATP1A1. Subsequently, ATP1A1 screening by conventional Sanger sequencing revealed that a third variant, c.907G>C (p.Gly303Arg), existed in a heterozygous state in individual B-II-1 but was not found in either parent and also occurred de novo. In families A and B, paternity was confirmed by analysis of seven independent polymorphic microsatellite markers. Paternity in family C was confirmed by segregation analysis of rare variants identified in the trio exome. For biochemical studies, mutations were introduced into full-length cDNA encoding the ouabain-insensitive rat α1 isoform of Na+, K+-ATPase and expressed in COS-1 cells. Ouabain selection was used for obtaining stable viable cell lines.5Vilsen B. Glutamate 329 located in the fourth transmembrane segment of the alpha-subunit of the rat kidney Na+,K+-ATPase is not an essential residue for active transport of sodium and potassium ions.Biochemistry. 1993; 32: 13340-13349Crossref PubMed Scopus (67) Google Scholar However, although COS cells transfected with wild-type rat Atp1a1 grew normally, several attempts to keep COS cells growing after transfection with mutant rat Atp1a1 cDNA and under ouabain selection failed; this indicates that, in contrast to the wild-type enzyme, none of the three mutants (p.Leu302Arg, p.Gly303Arg, or p.Met859Arg) was able to carry out the Na+ and K+ transport required to support cell growth. Therefore, transient expression was performed in the presence of siRNA to knock down endogenous Na+, K+-ATPase.6Beuschlein F. Boulkroun S. Osswald A. Wieland T. Nielsen H.N. Lichtenauer U.D. Penton D. Schack V.R. Amar L. Fischer E. et al.Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension.Nat. Genet. 2013; 45 (e1–e2): 440-444Crossref PubMed Scopus (414) Google Scholar Leaky plasma membranes were assayed functionally by previously described methods7Toustrup-Jensen M. Hauge M. Vilsen B. Mutational effects on conformational changes of the dephospho- and phospho-forms of the Na+,K+-ATPase.Biochemistry. 2001; 40: 5521-5532Crossref PubMed Scopus (28) Google Scholar (details are provided in the Supplemental Data). Na+, K+-ATPase pump function follows the Post-Albers reaction cycle (Figure 2A), starting with after of Na+, which of Na+ to the of after of the enzyme, is into the of under the Na+ were and was with demonstrated that all three mutants that they were although at a for and and that they were able to the Na+ of the Post-Albers cycle (Figure A Na+ however, for the The other two mutants showed of Na+ as from (Figure and The K+ was also for the other two mutants showed effect on K+ but of K+ (Figure of the by of the of the showed no of the of (Figure that the K+ is a effect on K+ with the state than a in the critical conformational from to of Na+, was used for cells as well as cells with full-length cDNA encoding wild-type or mutant rat cells were identified with were performed as P. B. F. R. S. of an mutant of the plasma membrane PubMed Scopus Google Scholar For the of Na+ and K+ was P. B. F. R. S. of an mutant of the plasma membrane PubMed Scopus Google Scholar Na+ and K+ was under and after with endogenous human Na+, analyses of cells mutant ATP1A1 and revealed an abnormal Na+ to that of wild-type as by at (Figure cells mutant ATP1A1 and also showed a membrane potential in the presence of Na+, but upon of Na+, the membrane potential was to the of cells (Figure In cells the mutant were from cells. of endogenous Na+, K+-ATPase by ouabain changes in Na+ and K+ in cells (Figure These changes were after expression of wild-type rat all three mutants to for the of endogenous ATP1A1. of mutant led to more of Na+ and K+ to that in possibly abnormal ion (Figure of levels revealed an abnormal and significant changes of upon of for mutant (Figure The Na+, K+-ATPase is an membrane protein that the transport of three Na+ out of and two K+ into the cell at the of one of the that essential as neuronal muscle and ion transport. The Na+, K+-ATPase is a consisting of and subunits and is by E. R.K. as of the in the PubMed Scopus Google Scholar The catalytic subunit Na+ and K+ as well as the cell membrane to S. by in the of sodium and potassium ion transport PubMed Google Scholar In that are expressed in a and G. subunit as a for ion Nephrol. PubMed Scopus Google Scholar, isoform and Google Scholar The expressed α1 subunit the major isoform in the kidney and is present in all cell and of the nervous system G. subunit as a for ion Nephrol. PubMed Scopus Google Scholar the effects of a of α1 in the was on the cardiac G. A.L. Walsh of a role for the isoform as a of calcium in the PubMed Scopus Google Scholar of ATP1A1 led to that loss of ATP1A1 function is not compatible with In contrast, heterozygous were and however, they exhibited a cardiac phenotype cardiac G. A.L. Walsh of a role for the isoform as a of calcium in the PubMed Scopus Google Scholar The severe phenotype in the affected children from heterozygous The cardiac of the affected children did not reveal any possibly or in the human Seizures and hypomagnesemia have not been in however, they findings in the affected in the affected individuals presented here is caused by massive renal wasting. the the the segment active transport. Here, the expressed Na+, K+-ATPase favorable for cation ion and also the for Na+ The the and activity of the Na+, α1 the K. Nielsen isoform in rat kidney by and ouabain Google Scholar The critical role of Na+, K+-ATPase activity for in the has been by the of genetic in subunit by [MIM: in with hypomagnesemia MIM: van H. de van renal magnesium loss is caused by of the Genet. PubMed Scopus Google Scholar as well as by the hypomagnesemia observed in individuals with MIM: D. S. S. M. E. G. et and PubMed Scopus Google Scholar This is caused by mutations in [MIM: encoding the potassium that is in the and K+ as a for maintenance of Na+, K+-ATPase activity. serum levels in the affected children remained during despite a high oral supplementation (Table Individuals with genetic in TRPM6 or also present with however, in supplementation leads to a of and and mental development are levels K.P. Weber S. K. L. D. M. et TRPM6 mutations in families with hypomagnesemia and secondary Nephrol. PubMed Scopus Google Scholar In contrast, the children with ATP1A1 exhibited seizures and uniformly significant intellectual disability. Therefore, the phenotype has to be as a of ATP1A1 due to a of Na+, K+-ATPase function in the Here, α1 is expressed and to neuronal the isoform as a pump that is only required during of Na+ i.e., after repeated G. subunit as a for ion Nephrol. PubMed Scopus Google Scholar, R. and of the PubMed Google Scholar of the isoform is to and In the Na+, K+-ATPase activity is required for the membrane potential and for and of K+ during neuronal A. brain during neuronal the physiological role of the subunit PubMed Scopus Google Scholar in Na+, K+-ATPase activity have been in of and in forms of human and V.R. seizures are associated with Na+,K+-ATPase activity and subunit state in the cerebral 2013; PubMed Scopus Google Scholar, S. G. G. Schack L. et in the isoform of Na+,K+-ATPase causes in the sodium pump and in the PubMed Scopus Google Scholar, and PubMed Scopus Google Scholar of neuronal Na+, K+-ATPase by cardiac seizures in T. A. Seizures in associated with cation of PubMed Scopus Google Scholar and changes in membrane potential and activity were after the of Na+, K+-ATPase activity by ouabain in of neuronal caused by of in the rat 2002; PubMed Scopus Google Scholar mutations in ATP1A1 have been in individuals with disease MIM: P. G. F. et in ATP1A1 Genet. PubMed Scopus Google Scholar data from exome sequencing the identified heterozygous ATP1A1 mutations in seven analyses were compatible with inheritance. In with the nervous system phenotype, α1 expression was demonstrated in and of and motor The identified mutations affect conserved amino acid residues in of the α1 a is observed within the in this have been to the of to conformational during the Post-Albers reaction but the Functional of to amino located in the of the of Google Scholar Ouabain demonstrated a significant in cell and studies in showed a significant compatible with a effect on Na+, K+-ATPase P. G. F. et in ATP1A1 Genet. PubMed Scopus Google Scholar In contrast to findings in previously the clinical of the affected children with de novo ATP1A1 mutations did not reveal any of This could possibly be to the age of the the age of of clinical symptoms in the previously families between and of P. G. F. et in ATP1A1 Genet. PubMed Scopus Google Scholar data on serum magnesium levels as well as a potential phenotype were not in the the in one family and the of and symptoms and an spectrum of associated with ATP1A1 the renal and symptoms as well as the clinical of the children presented here with intellectual a clinical caused by ATP1A1 a effect with has been described for mutations in the Na+, K+-ATPase and [MIM: and [MIM: The disease spectrum caused by mutations in two familial forms of MIM: of [MIM: and and MIM: as well as MIM: a and M. R. L. L. L. L. A. P. G. of encoding the pump subunit associated with familial Genet. PubMed Scopus Google Scholar, P. L. M. G. M. S.B. et in the gene are associated with PubMed Scopus Google Scholar, A. A. F. B. S. et disorders with 2014; PubMed Scopus Google Scholar, van S. G. M. A. et in causes 2014; PubMed Scopus Google Scholar or mutations are associated with that or a Functional analyses of with wild-type suggested a than a M. R. L. L. L. L. A. P. G. of encoding the pump subunit associated with familial Genet. PubMed Scopus Google Scholar for both critical features of the human S. G. G. Schack L. et in the isoform of Na+,K+-ATPase causes in the sodium pump and in the PubMed Scopus Google Scholar, Genetic effects of in familial and 2013; PubMed Scopus Google Scholar a with the which is to the ATP1A1 identified in individual was and heterozygous the severe human phenotype associated with this P. S. B. A. E. et in a familial PubMed Scopus Google Scholar the in and data for a loss of function of the P. S. B. A. E. et in a familial PubMed Scopus Google Scholar, M. S. F. L. G. P. M. K. A causes familial with PubMed Scopus Google Scholar, L. F. P. R. A. F. M. A new clinical and analysis of the 31: PubMed Scopus Google Scholar cognitive developmental delay, and are of the A. A. F. B. S. et disorders with 2014; PubMed Scopus Google Scholar, L. S. K. T. A. T. A. G. et as of the phenotype associated with PubMed Scopus Google Scholar Therefore, impaired Na+, K+-ATPase function in the cause seizures of the isoform In with this small in Na+, K+-ATPase pump activity were to be able to G. A. M. of the between normal and brain PubMed Scopus Google Scholar The ATP1A1 mutations identified here affect amino acid residues in the of the Na+ and K+ residues of the Na+, K+-ATPase and In with the studies a Na+ and K+ in a loss of Na+ and K+ transport were impaired in all three mutants a expression for p.Gly303Arg, could a role in showed a effect on the for both Na+ and and mutants between the for both Na+ and a function of the and that with Na+ and K+ during the pump and abnormal were observed for mutant upon of These abnormal ion that the Na+, K+-ATPase into a ion to the of the severe phenotype here and could the between the affected children and which one of as well as individuals who ATP1A1 mutations the to conformational during the Post-Albers reaction a effect with by and Na+ has also been for ATP1A1 mutations identified in aldosterone-producing adenomas in individuals with F. Boulkroun S. Osswald A. Wieland T. Nielsen H.N. Lichtenauer U.D. Penton D. Schack V.R. Amar L. Fischer E. et al.Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension.Nat. Genet. 2013; 45 (e1–e2): 440-444Crossref PubMed Scopus (414) Google Scholar, H. P. A. S. et al.Somatic mutations in ATP1A1 and a common of hypertension.Nat. Genet. 2013; PubMed Scopus Google Scholar of genetic and we also the affected children for the presence of we observed episodes of hypokalemia and serum findings as levels or blood were in the children presented In we describe a clinical hypomagnesemia, seizures, and severe intellectual associated with heterozygous de novo mutations in ATP1A1, encoding the α1 subunit of Na+, ATP1A1 be suspected in individuals with seizures and developmental delay, the not to an amelioration of genetic renal magnesium wasting disorders and underline the role of Na+, K+-ATPase for magnesium findings the critical role of the α1 subunit of Na+, K+-ATPase for the maintenance of the of membrane and the of neuronal activity in the nervous they also the effects associated with this the molecular a for studies on the and potential of hypomagnesemia, and intellectual disability. The no We are to the children and families for in this We to the and laboratory involved in the of the children and molecular and C-II-2 was of the with support from the as of the of and by and is a of the for Scholar The was by to from the and the to and R.K. was provided by for and The and and the European for in S.B. and were by the to S.B. and to and received support from the with and S1 and in

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