Genotype-structure-phenotype relationships diverge in paralogs ATP1A1, ATP1A2, and ATP1A3

Variant distributions in ATP1A2 and ATP1A3

The premise that high sequence conservation predicts variant pathogenicity implies that paralogs with high sequence identity should have similar variant distributions in the protein structure. ATP1A2 and ATP1A3 have the most disease-associated variants for comparison (figure 1A): 77 in ATP1A2 and 130 in ATP1A3 at this writing, including alternative substitutions (e.g., p.D801 to p.D801N and p.D801Y). There are striking differences, however, in the proportion of disease variants found in different protein domains of ATP1A2 and ATP1A3 (figure 1B). Such differences would not be predicted by shared enzyme mechanisms and high sequence identity. Disease-causing variants in ATP1A2 and ATP1A3 should frequently appear at the corresponding amino acids. Instead, of 154 mutated amino acid positions in the 2 genes (68 in ATP1A2 and 86 in ATP1A3), only 18 mutated amino acids overlapped.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1 Dissimilar mutation distributions in 88% identical ATP1A2 and ATP1A3 proteins

(A) Ribbon diagrams of Na,K-ATPase in the K⁺-bound conformation. Bound K⁺ is shown as pink spheres. Extracellular portions include the β-subunit (green; transmembrane portion removed for clarity) and extracellular loops of the α-subunit (magenta). The 10 transmembrane α-helices are white. The cytoplasmic domains are the stalk (S, lavender), phosphorylation domain (P, red), nucleotide binding domain (N, gold), and actuator domain (A, yellow). Spacefill residues are the backbones of each amino acid mutated in ATP1A2 or ATP1A3. Color shading is varied to help distinguish different amino acids. For ATP1A2, 76 different DNA variants occur in 66 amino acids, i.e., 10 amino acids have 2 alternative codon changes. Three of the mutations are single amino acid deletions. For ATP1A3, 130 different DNA variants occur in 86 amino acids; 5 are single amino acid deletions, and there are 25 amino acids with 2–6 alternative codon changes. All 18 overlapping residue pairs (including 3 where one was found in ClinVar) were identical before the mutation. In 6 pairs, the amino acid change was different; in 8, it was the same; and in 4, both identical and different substitutions were found (i.e., alternative codon changes). (B) Linear diagram of the domain substructure. Both the A domain (yellow) and the P domain (red) are composed of 2 parts that are separated in the linear structure. As in (A), the extracellular loops are magenta. The stalk domain comprises 5 separate parts: the cytoplasmic extensions of the M4 and M5 transmembrane spans S1 and S2; the short intracellular loops L6-7 and L8-9; and the C-terminus (lavender). For ATP1A2 and ATP1A3, domain lengths are proportional to the number of distinct variants found (including alternative codon changes). Although variants are found in most domains, ATP1A2 has an excess in the P domain, and ATP1A3 has an excess in the membrane domain.

Although more such overlap should emerge with time, the low level of overlap is not simply that there are not enough cases yet: it contrasts sharply with the number of recurring variants. First, there were many cases in which alternative codons produced different substitutions of an amino acid: 25 amino acids in ATP1A3 had alternative substitutions (up to 6), and 13 amino acids in ATP1A2 had 2 alternative substitutions. Second, for ∼600 independent occurrences of variants in ATP1A3 patients (a family is 1 occurrence), 62% have recurred twice or more, and of those, more than half have recurred 5 or more times. ATP1A2 has many fewer reported occurrences (∼160), but 20% of their mutations recurred. Of the 18 pairs of variants overlapping in ATP1A2 and ATP1A3, none involved highly recurrent mutations. These data suggest that there is little bias toward crucial residues shared by ATP1A2 and ATP1A3 but strong bias for other residues.

Singletons and families in the literature were ascertained by symptoms suspicious of a genetic origin. Separate clinical diagnoses such as RDP and AHC were independently linked to ATP1A3.^10,16 The divergence in structure distribution suggests the existence of genotype-structure-phenotype relationships. Additional phenotypes for each gene may yet be discovered.

Structure-phenotype relationship for ATP1A3

Human disease variants in ATP1A2 and ATP1A3 produce neurologic syndromes with onset at birth, in childhood, or in adulthood, including familial hemiplegic migraine (FHM2 [MIM:602481]),² RDP (MIM:128235),^1,29 AHC (MIM:614820),^1,2 and cerebellar ataxia, areflexia, pes cavus, optic nerve atrophy, and sensorineural deafness (CAPOS [MIM:601338]).¹¹ Disease variants in ATP1A2, expressed in astrocytes, produce FHM2 phenotypes associated with spreading depression or spreading excitation: migraine, hemiplegia, and seizures.² Symptom severity has not been systematically studied for ATP1A2 mutations,^2,17 but ATP1A3 disease variants can be divided by severity.^{10,11,18,–,20} The most severe are defined by onset in infancy and include AHC,¹ severe infantile epilepsy,^18,21 and severe hypotonia.²² Milder phenotypes have onset in children and adults and include RDP, CAPOS, relapsing encephalopathy and cerebellar ataxia¹⁹ also called fever-induced weakness and encephalopathy,²³ and rapid-onset ataxia.^20,24 Ages at symptom onset were taken from original reports.

Figure 2 highlights a structure-phenotype relationship in the membrane domain. ATP1A2 and the milder phenotypes in ATP1A3 have a scattered distribution, but the ion binding site is almost devoid of variants. In contrast, the variants with severe phenotypes in ATP1A3 are clustered around the ion binding sites, as noted previously for AHC.^25,–,27 Three residues in ATP1A3 with either mild or severe manifestations are pale yellow and not close to the ions. The absence of known ATP1A2 mutations near the ions suggests that their effects are too severe to manifest as hemiplegic migraine.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2 Structure-phenotype relationship in the membrane domain

This figure shows the transmembrane α-helices, omitting the portions that extend into the extracellular and intracellular spaces. All the ATP1A2 membrane mutations are shown (left), whereas ATP1A3's are divided into 2 groups: milder ATP1A3 phenotypes (middle) with onset from childhood to adult and severe ATP1A3 mutations (right) with onset in infancy. ATP1A2 and the milder ATP1A3 phenotypes have similar distributions that appear to exclude the residues right around the ions (pink = potassium ions). In contrast, the severe ATP1A3 mutations are usually close to the ions. Almost 70% of the severe mutations are in contiguous stretches in M4, M5, and M6 near the ions. In contrast, 100% of the mild mutations are not adjacent to any other mutations, and the few in M4, M5, and M6 are on either side of the contiguous stretches. The distributions highlight 2 unique features: that mild and severe ATP1A3 mutations have different distributions and that ATP1A2 mutations all look like “mild”. In fact, hemiplegic migraine seldom has onset in infancy.¹⁷ Equivalent “severe” mutations of ATP1A2 have not been found. Variants of 3 ATP1A3 residues in pale yellow can produce either mild or severe phenotypes. Fourteen ATP1A3 residues altogether have produced both RDP and mild AHC or an intermediate phenotype. Eleven of those were recurrent (with the same or different substitution) or appeared also in ATP1A1 or ATP1A2 patients. Here, 2 had different substitutions in patients with differing phenotypes (S137Y or S137F severe,¹⁰ S137del mild⁵³; Q140L severe,¹⁰ Q140H mild⁵⁴), and the third, D923N, reduces affinity for Na⁺⁵⁵ and presents as a continuum between AHC and RDP,^56,57 severe or mild in the same family.⁵⁸

The P domain, the site of ATP hydrolysis, is highly evolutionarily conserved. Figure 3A dissects this domain because its structure is otherwise difficult to appreciate. Two distant stretches of amino acids (P1 and P2) interdigitate in a twisted sheet of β-sheet strands encircled by α-helices. In figure 3A, the top row shows an end view of the β-sheet, the α-helices that surround it, and the 2 parts combined. The active site has an aqua magnesium ion. The second row shows a side view of the same structures. Figure 3B shows the distributions of mutated amino acids. In the β-sheet, all but 3 are clustered at the face of the active site. In the ring of α-helices, all mutations except 1, in magenta, are away from the active site. The helix mutations are confined to the central 5 of the 9 helices. The more distal helices and strands so far have no mutations.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3 Similar but restricted distributions of pathogenic variants in the P domain

The P domain is the most conserved in the gene family. It is covalently phosphorylated during transport, and the Mg²⁺ ion (aqua) is at the active site. (A) The P domain consists of 2 halves that are separated in the linear structure but that interdigitate in a complex way when the protein folds. The first half, P1 in figure 1B, is shown in pale yellow, and the second half, P2 in figure 1B, in pink. There is a twisted β-sheet (left views) surrounded by α-helices (middle views). Strands and helices are numbered in the order found in the sequence. (B) The known mutations are displayed with color coding as indicated. Stick view is used for mutations in the β-sheet to avoid overcrowding. Note how most of the side chains of the mutated residues extend down into the active site surface. Spacefill view of the mutations, without side chains, is used for the α -helices. Note how the mutations are confined to just 5 helices: helix 1, which is associated with the stalk domain, and 4 helices that form a belt around the middle of the P domain: 2, 3, 7, and 8. There are no mutations in the end strands (1 and 4) or the end helices (4, 5, 6, and 9), which may be less important to function. The magenta mutation in an α-helix is the only helix mutation near (but not in) the active site. It is ATP1A3 T613M, a recurrent RDP mutation. The data indicate that the ATP1A2 and ATP1A3 distributions of mutations are very similar in the P domain, but there are 2 types of mutations. Most of the mutations in the β-sheet are at the active site. Most of the ones in the α-helices are distant from the active site. There was no correlation with phenotype severity.

In Figure 3, color distinguishes the variants found in ATP1A2, ATP1A3, or both. All have similar patterns of distribution in the P domain. Figure e-4 (links.lww.com/NXG/A134) compares the mild and severe phenotype variants of ATP1A3. Any suggestion of differences in location would need to be supported by further evidence. Figure e-5 compares the ATP1A2 and ATP1A3 variants in the S domain. On the whole, P domain and S domain mutation distributions do not differ systematically between ATP1A2 and ATP1A3.

Two types of variants in ATP1A1

Presumably because ATP1A1 is essential in early embryonic life, not many disease variants have been discovered. Those fall into 2 groups with different characteristics. Seven ATP1A1 putatively pathogenic variants found in Charcot-Marie-Tooth (CMT2) axonal sensorimotor neuropathy are in the cytoplasmic A, P, and N domains, and 1 is at an ion binding residue frequently mutated in ATP1A3 but with a less disruptive substitution (figure 4A). Adrenal adenomas, like many cancers, have a high somatic mutation rate, and APAs have distinctly different ATP1A1 variants (short deletions and missense), all in the membrane domain (figure 4B).^28,–,31 Those tested for function showed reduced Na,K-ATPase activity, cell depolarization, or a modest inward leak current.^{28,29,32,–,34} The amino acids of the most recurrent adenoma variants, p.Gly99Arg, p.Leu104Arg, and p.Val332Gly, had previously been shown to line the ion pathway or function as gates: their chemical modification blocked palytoxin-induced currents caused by the toxin's ability to bind to Na,K-ATPase and wedge it open.³⁵ In sum, the CMT2 ATP1A1 disease variants are likely to cause a mild loss of function, and the adenoma variants may cause a gain of function with limited currents leading to aberrant aldosterone production.

• Download figure
• Open in new tab
• Download powerpoint

Figure 4 Distinct groups of ATP1A1 pathogenic variants

There are only a handful of known mutations for ATP1A1, but they fall in 2 clearly different groups. (A) Seven germline mutations were found in CMT2 patients.⁴ One is at the interface between the 2 halves of the A domain (A1 dark pink, A2 yellow), where it may interfere with A domain stability. One is at the site equivalent to D801N, the most common AHC pathogenic variant, but the change is from aspartate to alanine, presumably a milder substitution. The other 5 variants are close to the boundary of P and N domains. They do not appear fundamentally different from mutations in ATP1A2 or ATP1A3. I592T in ATP1A1 (orange) is the residue equivalent to I589T in ATP1A2.⁵⁹ (B) The other group is somatic mutations found in aldosterone-producing adenomas.³ (Left) Six of the missense mutations all lie along the ion path through the protein, and some alter amino acids that are believed to be gates. The seventh is at the top of the extracellular mouth (green). Two of them, V332G and L337M, are less disruptive versions of mild pathogenic variants in ATP1A3: V322D and L327del. (Right) Multiple different short deletions (of 2–6 amino acids) appear only in the 3 stretches of amino acids highlighted in membrane spans 1, 4, and 9. Such deletions have not been reported for ATP1A2 or ATP1A3. These somatic mutations are in a class by themselves. They are believed to generate slight leaks, large enough to lead to aldosterone production but not large enough to kill the cells.

Pathogenic variants without evolutionary conservation

To determine whether mutations are in amino acids with high evolutionary conservation, we used ConSurf to estimate conservation based on 94 nonredundant homologous sequences from the P-type ATPase superfamily, from vertebrates to yeast and bacteria.³⁶ Figure 5A shows the conservation scores of all residues as small blue symbols, and the bar shows the locations of domains as in figure 1B. The 2D presentation highlights that for much of the protein, high conservation and low conservation residues are closely interspersed in the linear sequence. Shaded boxes highlight 2 nonrandom features. First, there are 3 large stretches of conserved residues (tan) with practically no low conservation residues (off-white). In the right off-white box, at around amino acid 800, there is a vertical streak of less-conserved residues that correspond to a loop in the extracellular domain, which is magenta on the domain bar. Second, there are 2 stretches of amino acids that are depleted of high conservation residues (lavender boxes). Because conservation was calculated using sequences that are related but have divergent functions, we infer that these regions, rather than being of lesser importance, evolved for functions not shared by more distant homologs: regions of special interest for ATP1A1, ATP1A2, and ATP1A3.

• Download figure
• Open in new tab
• Download powerpoint

Figure 5 Evolutionary sequence conservation

Human ATP1A1, ATP1A2, and ATP1A3 amino acid sequences were aligned with Sus scrofa ATP1A1 (the species used in protein crystal structures 3KDP and 3WGU), and each residue affected by a disease variant was assigned the conservation score calculated for ATP1A1. (A) The individual conservation scores of all aligning amino acids are shown with score on the Y axis and amino acid sequence (1–1,023) on the X axis. As seen in figure e-1 (links.lww.com/NXG/A134), there is no alignment at the N-terminus, so N-terminal residues do not appear. Each blue spot is one amino acid, and the Na,K-ATPase domains are illustrated in the bar at the bottom. Some patterns of conservation can be discerned, highlighted by transparent boxes: two regions with few or no highly conserved amino acids (lavender) and 3 regions of conserved residues (tan) with practically no low conservation residues (off-white). (B) The ATP1A2 variants (green) and the mild ATP1A3 variants (magenta) have similar distributions. There are 14 cases (∼15%) where homology scores were in the less-conserved half. The C-terminal region has many pathogenic variants in both genes despite lacking the highest conservation scores. Residues E174 and R1008 from figure 6 are marked with arrowheads. (C) The severe ATP1A3 variants had a more restricted distribution. It can be seen that many align with the membrane spans. Although their conservation scores varied widely, none were in the less-conserved half. Of the points that do not lie in one of the 3 clusters of high conservation, 8 were shared by mild and severe ATP1A3 phenotypes (visible as 2 colors per symbol at high magnification). This suggests that intermediate phenotypes tend to have less restricted structure distributions than the most severe phenotypes. (D) All the synonymous (brown; n = 281) and missense (coral pink; n = 145) ATP1A3 variants from gnomAD (138,632 sequences) are plotted to compare to the clustering of mutations.

The average conservation scores for missense variants found in patients were similar for all 3 genes at the 30th percentile (data not shown). Two-thirds of the mutated residues were in the highest 20%, and only 3% were in the lowest 20%. The conservation scores were graphed to look for patterns unique to ATP1A2 and mild-phenotype ATP1A3 (figure 5B) and severe ATP1A3 (figure 5C). A difference in the distribution of variants is clear both in the location and conservation score, with the severe-phenotype ATP1A3 variants being more restricted and all falling below zero on the graph (5C). Another difference that cannot be appreciated on the graphs is that 65% of the severe ATP1A3 variants are recurrent, whereas the proportion is 38% for mild ATP1A3 variants and 20% for ATP1A2. The region at the C-terminus with no very high conservation residues has many mutations (figure 5, B and C).

Normally, one would expect that variants with low conservation scores are benign. In the ATP1A2 and mild ATP1A3 variants in figure 5B, 14 amino acids have positive (poor) conservation scores. Of these, 10 were supported as pathogenic by laboratory testing of function, recurrence as an alternative amino acid, or occurrence in a second gene, and 3 of those were de novo. There was no pattern in their scores. Three others had insufficient data, and a fourth was published as possibly disease causing and is analyzed further.

The missense and synonymous variants in gnomAD gave no obvious pattern (figure 5D). More than half (58%) of the synonymous variants were in the higher half of the conservation score, whereas only 39.3% of missense variants were (p = 10⁻⁵ t test, 2 tailed, equal variance) consistent with genetic constraint.

Structure data assist tricky predictions

ATP1A2 variant E174K has a conservation score of 2.077, putting it at the top of figure 5B. The variant appeared in a family that experiences migraine with side-changing paresthesias, which is on the FHM2 spectrum.³⁷ Pedigree data were supportive but incomplete. When expressed in Xenopus oocytes, no reduction of pump activity was seen,³⁷ but the ion concentrations used were high enough to saturate the enzyme. In later experiments using protein expressed in insect cells, it was learned that the enzyme had altered kinetics: reduced affinity for Na⁺ and increased affinity for K⁺.³⁸ Because Na⁺ concentration is rate limiting for enzyme activity in vivo, this mimics reduction of activity. Figure 6, A and B illustrate how the position of E174 in the crystal structures explains the pathogenicity. E174 is at the surface of the A domain, which rotates. In the K⁺-bound structure, E174 faces the cytoplasm, and the side chain is in contact with nothing (figure 6A). In the Na⁺-bound form, however, it binds 2 amino acids in the N-domain (figure 6B), i.e., it is at a domain-domain interface where the change from negative to positive charge will be damaging. This explains the abnormal kinetics because the sodium conformation would be destabilized.³⁸ In this example, an amino acid with the worst conservation score nonetheless is legitimately pathogenic.

• Download figure
• Open in new tab
• Download powerpoint

Figure 6 Structure-based interpretation of variants of uncertain significance

(A) E174K; N, P, and A domains are visible. In the K⁺-bound conformation, the N (gold) and A (yellow) domains are far apart. (B) In the Na⁺-bound conformation, the N and A domains are in contact. The P domain in the background was hidden for clarity. E174K (magenta) is a variant in ATP1A2 that has the poorest conservation score in figure 5B, and it is predicted to be tolerated by SIFT.³⁸ In the K⁺-bound structure, it is fully exposed to the cytoplasm (A), but in the Na⁺-bound structure, it is the point of closest contact between the A domain (yellow) and N domain (gold). There it interacts with 2 adjacent residues, an asparagine N432 and an alanine A433 (blue and aqua). They are also shown in (A). (C) R1008W; close-up of M10, M7, and M8 where these helices terminate in parts of the S domain (lavender). R1008W (magenta) is a variant in ATP1A2 in a patient with compound heterozygosity with R548C. Because the adjacent variant R1007W (green) is known to be pathogenic, it was suggested that R1008W might contribute to the severity.³⁹ R1007 has a better conservation score, but R1008's score is still in the range of other pathogenic variants (close to zero). In the structure, R1007 points into the protein where it makes close association with 2 domains: two residues in M10 (light blue) and 2 residues in L7-8 (lavender), including the protein kinase A phosphorylation site, S933. In contrast, R1008 points out into the cytoplasm, where a substitution may be tolerated.

The second example is ATP1A2 variant R1008W, which was found together with a known pathogenic variant, R548C, in a compound heterozygous child with unusually early onset of severe epilepsy, hemiplegia, and migraine symptoms.³⁹ The severity of the phenotype, not seen in a family with R548C alone⁴⁰ or in the child's mother who carried the R548C mutation, suggested that both variants contributed to disease severity. An adjacent arginine with the same substitution, R1007W, previously described in a family with hemiplegic migraine and epilepsy, was shown to be pathogenic by functional studies in Xenopus oocytes.⁴¹ Although R1007 has a conservation score in figure 5B of −0.967 while R1008 is +0.069, their identical substitutions suggested that the adjacent variants might both be pathogenic.³⁹ However, their positions in the crystal structure tell a different story (figure 6C). R1007's side chain faces into the protein and makes contacts with 2 amino acids in M10 and 2 in the S domain. One of those contacts is the protein kinase A site, and R1007 had been shown previously by mutagenesis to play a complex role in controlling Na,K-ATPase.⁴² In contrast, the adjacent R1008 side chain points away from the protein, where it may associate with phospholipid headgroups.⁴³ Adjacent amino acids can have very different propensities to be pathogenic. In this example, a variant with some apparently credible features, nonetheless, may be benign.