Mutations affecting G-protein subunit α11 in hypercalcemia and hypocalcemia

Abstract

Background: Familial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide-binding protein (G-protein)-coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia.

Methods: We performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells.

Results: The kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein structures, and assessment on the basis of in vitro expression showed that familial hypocalciuric hypercalcemia type 2-associated mutations decreased the sensitivity of cells expressing calcium-sensing receptors to changes in extracellular calcium concentrations, whereas autosomal dominant hypocalcemia type 2-associated mutations increased cell sensitivity.

Conclusions: Gα11 mutants with loss of function cause familial hypocalciuric hypercalcemia type 2, and Gα11 mutants with gain of function cause a clinical disorder designated as autosomal dominant hypocalcemia type 2. (Funded by the United Kingdom Medical Research Council and others.).

Figure 1. GNA11 Mutation in a Patient with Familial Hypocalciuric Hypercalcemia Type 2

Panel A shows the predicted outcomes of DNA sequence analysis (Fig. S1 in the Supplementary Appendix) in the proband of the kindred with familial hypocalciuric hypercalcemia type 2 (identified as Kindred 11675 in Table S1 in the Supplementary Appendix), who had a heterozygous 3-bp (ATC) deletion, as compared with a normal unrelated person. The 3-bp deletion leads to an in-frame deletion of the Ile200 residue and gain of an XmnI restriction-endonuclease site (GAACA/TCTTC). Panel B shows the resulting XmnI restriction maps of nonmutant and mutant polymerase-chain-reaction (PCR) products. Panel C shows the use of XmnI to confirm the mutation, which was not present in 110 alleles from 55 unrelated persons with normocalcemia (the findings in 3 of the 55 persons [N1, N2, and N3] are shown), and cosegregation of the Ile200del mutation with disease in the kindred with familial hypocalciuric hypercalcemia type 2 (LOD score, +3.60 at 0% recombination fraction). Each member of the kindred with familial hypocalciuric hypercalcemia type 2 is represented above the corresponding XmnI-digested PCR product and identified with the use of numbers previously reported^,; a sample from Person II.6 was not available. All unaffected persons with normocalcemia were homozygous for nonmutant alleles, whereas affected persons were heterozygous for nonmutant and mutant alleles; these findings are consistent with an autosomal dominant inheritance of familial hypocalciuric hypercalcemia type 2. Squares represent male family members, circles female family members, and black symbols affected family members. S denotes size marker. The proband is indicated by an arrow.

Figure 2. Three-Dimensional Modeling and Functional Characterization of Familial Hypocalciuric Hypercalcemia Type 2–Associated Mutant Gα₁₁ Residues

Panel A shows a three-dimensional model of the Gα₁₁ helical and GTPase domains, which are the locations of the Leu135 (L135) and Ile200 (I200) residues (red), respectively. Gα₁₁ has 90% identity to Gα_q at the amino acid level, and the model is therefore based on the reported three-dimensional structure of Gα_q (Fig. S2 in the Supplementary Appendix).^, I200 is located in the β3 sheet and adjacent to the β2–β3 loop, which is formed by the tetrapeptide comprising Ile(I)199-Asn(N)198-Glu(E)197-Leu(L)196. Panel B shows the β2–β3 loop region of nonmutant and mutant Gα₁₁ and the structural effects of the I200del Gα₁₁ mutant on hydrogen bonds (broken lines). Residues (red) mutagenized in this study (Fig. S3 and S7 in the Supplementary Appendix). Panels C and D show the responses of intracellular calcium concentrations to changes in extracellular calcium concentrations, in HEK293 cells stably expressing calcium-sensing receptors that were transiently transfected with nonmutant, familial hypocalciuric hypercalcemia type 2– associated mutant (I200del, Gln[Q]135), or empty GNA11–pBI-CMV2–green fluorescent protein (GFP) expression vectors (Fig. S3 in the Supplementary Appendix). The intracellular calcium responses to changes in extracellular calcium concentrations were expressed as a percentage of the maximum normalized response and are shown as the mean (±SE) of 8 to 45 assays from 3 to 12 independent transfections. P<0.001 for the comparisons of the mutant vector with the nonmutant and empty vectors. The familial hypocalciuric hypercalcemia type 2–associated mutants (I200del and Q135) led to a rightward shift in the concentration–response curves, with significantly higher half-maximal effective concentration (EC₅₀) values (i.e., the extracellular calcium concentration required to produce a half-maximal increase in intracellular calcium concentration values) (Table S4 in the Supplementary Appendix), as compared with cells expressing nonmutated GNA11–pBI-CMV2-GFP. The familial hypocalciuric hypercalcemia type 2–associated Leu135Gln mutation conferred on the mutant protein a significantly increased EC₅₀ as compared with nonmutant Gα₁₁ or empty vector alone, suggesting a possible dominant-negative effect.

Figure 3. GNA11 Mutation in a Patient with Autosomal Dominant Hypocalcemia

Panel A shows the predicted outcomes of DNA sequence analysis (Fig. S8 in the Supplementary Appendix) in Patient 3, who is the proband of a family with autosomal dominant hypocalcemia type 2 (Table S2 in the Supplementary Appendix) who had a heterozygous G→A transition at c.542, as compared with a normal unrelated person. The effect of the G→A transition is an alteration at codon 181 from CGG (encoding the nonmutant Arg [R] residue) to CAG (encoding a mutant Gln [Q] residue) and gain of a PstI restriction-endonuclease site (CTGCA/G). Panel B shows the resulting PstI restriction maps of nonmutant and mutant PCR products. Panel C shows the use of PstI to confirm the mutation, which was not present in 110 alleles from 55 unrelated persons with normocalcemia (the results in 1 of the 55 persons [normal control 1, or N1] are shown). Each person is rep resented above the corresponding restriction enzyme–digested PCR products. The unaffected persons with normocalcemia are homozygous for the nonmutant alleles, whereas the affected proband is heterozygous for the nonmutant and mutant alleles; this finding is consistent with an autosomal dominant inheritance of autosomal dominant hypocalcemia type 2. S denotes the size marker.

Figure 4. Location of Autosomal Dominant Hypocalcemia Type 2–Associated Gα₁₁ Mutants in a Three-Dimensional Model and Effects on the EC₅₀ of Calcium-Sensing Receptor–Expressing Cells

Panel A shows a three-dimensional model of the helical and GTPase domains of Gα₁₁. The Arg181 (R181) and Phe341 (F341) residues (red) are located in the helical and GTPase domains, respectively. Panel B shows a three-dimensional model of the guanosine diphosphate (GDP) (blue)–aluminum fluoride (AlF₄) (gray) binding pocket. Shown are the locations of R181 (red); the hydrophobic phenylalanine (F) cluster in which F341 (red), located in the α5 helix, interacts with F194 (magenta) and F201 (magenta), which are on the β2 and β3 sheets, respectively (Fig. S9 in the Supplementary Appendix); and R183 (black) and Q209 (orange), which are involved in somatic activating mutations in uveal melanomas. R181 and R183 form hydrogen bonds with GDP-AlF₄, thereby stabilizing the GTP hydrolysis transition state. Panels C and D show responses of intracellular calcium concentrations to changes in extracellular calcium concentrations, in HEK293 cells stably expressing calcium-sensing receptors that were transiently transfected with nonmutant, autosomal dominant hypocalcemia–associated mutant (Q181 or L341), or empty GNA11–pBI-CMV2-GFP expression vectors (Fig. S10 in the Supplementary Appendix). The intracellular calcium responses to changes in extracellular calcium concentrations were expressed as a percentage of the maximum normalized response and are shown as the mean (±SE) value of 8 to 45 assays from 3 to 12 independent transfections. P<0.001 for the comparisons of the mutant vectors with the nonmutant and empty vectors. The autosomal dominant hypocalcemia type 2–associated mutants (Q181 and L341) led to a leftward shift in the concentration–response curve, with significantly lower EC₅₀ values (Table S4 in the Supplementary Appendix).