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Clinical Studies |
Developmental Endocrinology Branch, National Institute of Child Health and Human Development (F.D.L., E.E.M., K.K.W., J.B.), and Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Disease (K.R., G.-F.F., A.M.S.), National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Francesco De Luca, M.D., Building 10, Room 10N262, National Institutes of Health, 10 Center Drive, MSC 1862, Bethesda, Maryland 20892-1862. E-mail: delucaF{at}cc1.nichd.nih.gov
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In parathyroid cells, this cascade decreases PTH secretion (1). In kidney, CaR activation inhibits reabsorption of Ca2+ in the thick ascending limb and water reabsorption in the collecting duct (2, 3). CaR is also expressed in thyroidal C cells (4) and in brain (5). Other Ca2+ sensors are present in parathyroid gland, kidney, placenta, and osteoclasts (6, 7). The physiological roles of these putative Ca2+ sensors are not known.
Inactivating mutations of the CaR gene (located on chromosome 3q) (8) cause resistance of parathyroid gland and kidney to extracellular Ca2+ (9, 10). Thus, there is decreased inhibition of PTH secretion, producing hypercalcemia, and decreased inhibition of renal Ca2+ reabsorption, producing relative hypocalciuria.
Conversely, activating mutations of the CaR are associated with the reverse phenotype, familial hypoparathyroidism, and relative hypercalciuria (11, 12, 13, 14, 15, 16). Receptor activation could result from increased activity of the unliganded receptor, increased activity of the liganded receptor, or increased affinity of the receptor for ligand. Four mutant CaRs associated with familial hypocalcemia increase the sensitivity of the receptor to Ca2+ in vitro (14, 16, 17).
Although idiopathic isolated hypoparathyroidism can be familial, most cases are sporadic (18). Because familial hypoparathyroidism can be caused by inherited CaR mutations, we hypothesized that sporadic hypoparathyroidism can be caused by de novo CaR mutations. Our hypothesis was confirmed by finding the first de novo CaR mutation in a child with sporadic hypoparathyroidism (11).
In this study, we examined two additional patients with sporadic hypoparathyroidism. In each patient, a de novo missense mutation of the CaR was identified. To elucidate the mechanism of activation, we studied the functional properties of all three de novo CaR mutations associated with sporadic hypoparathyroidism.
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Subjects and Methods |
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Patient A, a 25-yr-old woman, had one generalized seizure of
unknown etiology at 2 weeks of age. She presented with fatigue and
depression at 18 yr of age and was found to be hypocalcemic and
hyperphosphatemic, with an inappropriately low normal serum PTH level
(39 pmol/L; normal range, 0–100). She was treated with oral calcium
and vitamin D, which she discontinued 1 yr later. When evaluated at the
NIH at 20 yr of age, physical examination was normal, except for short
stature (145 cm). No signs of neuromuscular irritability were noted.
Computed tomography scans showed minimal basal ganglia calcifications
and right-sided nephrocalcinosis. Renal function was normal. Serum and
urinary calcium values (off-treatment) are shown in Table 1
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. This study was approved by the NICHD institutional review board. Informed consent was obtained from all subjects.
DNA amplification and sequence analysis
Most of the reported CaR mutations associated with familial hypoparathyroidism have been found in exons 2 and 6. Therefore, we PCR amplified these exons using genomic DNA from white blood cells, as previously described (9). Both strands of the PCR products were sequenced in a fluorescence-based DNA sequencing system (19).
Restriction analysis
PCR-amplified genomic DNA was digested with restriction enzyme AvaI (Promega, Madison, WI), subjected to electrophoresis through a 6% polyacrylamide gel, and stained with ethidium bromide.
Site-directed mutagenesis
The human CaR complementary DNA (cDNA) inserted into the mutagenesis vector pAlterI (Promega) was obtained from NPS Pharmaceuticals (Salt Lake City, UT). The two detected mutations (L773R and N118K) and one previously reported (F806S) were introduced into this construct by site-directed mutagenesis using the Altered Sites II system (Promega). The mutated cDNAs were then isolated with restriction enzymes XbaI and HindIII and inserted into the expression vector pcDNA I/Amp (Invitrogen, San Diego, CA). Mutations were confirmed by DNA sequencing.
Cell culture and transfection
HEK-293 cells were cultured in DMEM. The cells were plated in 24-well plates (105 cells/well) and transiently transfected with constructs encoding the wild-type and mutant receptors, using 5 µL Lipofectamine (Life Technologies, Gaithersburg, MD) and 0.5 µg DNA.
Preparation of cell membranes
Confluent transfected cells from 75-mm plates were washed with phosphate-buffered saline. Extraction buffer (50 mmol/L Tris-HCl, pH 6.8; 0.32 mol/L sucrose; 1 mmol/L ethylenediamine tetraacetate; 1 mmol/L phenylmethylsulfonylfluoride; 10 µg/mL aprotinin; 5 µg/mL leupeptin; and 0.7 µg/mL pepstatin) was added at 4°C. The cells were lysed by passing repeatedly through 22-gauge needles and sedimented at 16,000 x g for 30 min to remove nuclei and mitochondria. The supernatant was sedimented at 50,000 x g for 30 min to pellet plasma membranes, which were solubilized with 1% Triton X-100.
Western analysis
Membrane proteins (25 µg/well; determined by BCA protein assay, Pierce Chemical Co., Rockford, IL) were subjected to SDS-PAGE using a linear gradient of polyacrylamide (5–15%). After transfer to nitrocellulose membrane, the blot was incubated with 2 µg/mL affinity-purified monoclonal antibody (anti-ADD, made against residues 214–235 of human CaR, and provided by Dr. P. K. Goldsmith) and then with a goat antimouse secondary antibody conjugated to alkaline phosphatase (Kierkegaarde & Perry Laboratories, Gaithersburg, MD; 1:1000 dilution). CaR bands were detected with 4-choloronapthol.
Assessment of cell surface receptor expression by enzyme-linked immunosorbent assay (ELISA)
Transfected cells were suspended in 1% BSA-DMEM for 30 min at 4°C, and then incubated with monoclonal antibody 7F8 (20 µg/mL) for 1 h at 4°C. This antibody was made by immunization with the purified extracellular domain of the human CaR (Goldsmith, P. K., manuscript in preparation). After washing, cells were further incubated with peroxidase-conjugated goat antimouse secondary antibody (Kierkegaarde & Perry Laboratories; 1:1000 dilution). After washing, peroxidase substrate was added. Absorbance was measured at 405 nm. Three independent transfections were performed for ELISA.
Measurement of phosphoinositides (IPs)
Forty-eight hours after transfection, HEK-293 cells were labeled with 3 µCi/mL myo-[3H]inositol (New England Biolabs, Beverly, MA) in DMEM for 16–24 h. Cells were then incubated in PI buffer (99 mmol/L NaCl, 5 mmol/L KCl, 5.6 mmol/L glucose, 0.4 mmol/L MgCl2, and 0.5 mmol/L CaCl2) containing 20 mmol/L LiCl for 1 h. Cells were stimulated with the indicated concentrations of Ca2+ (in PI buffer) for 30 min at 37°C. The reactions were terminated with acid-methanol (167 µL HCl in 120 mL methanol). Total inositol phosphates were extracted, separated on Dowex AG1-X8 columns as previously described (20), and counted by liquid scintillation. Nine independent transfections were performed at each Ca2+ concentration for IP measurement.
Statistical analysis
Results were expressed as the mean ± SEM. Significance was assessed by ANOVA and post-hoc Fisher’s protected least significant difference test.
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Results |
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Direct sequencing of PCR-amplified genomic DNA from patient A
revealed a heterozygous T to G basepair substitution at position 2318
in exon 6 (21) (Fig. 1). This mutation produces a
leucine to arginine substitution at position 773 in the fifth
transmembrane domain of the receptor (Fig. 2). It also
creates a new recognition site for restriction enzyme AvaI.
Therefore, genomic DNA from the proband, her brother, and both parents
was screened for the mutation by PCR amplification, AvaI
digestion, and gel electrophoresis (Fig. 3). The brother
and both parents lacked the additional AvaI site and, thus,
the mutation. Approximately half of the proband’s PCR product showed
the additional site, confirming that she was heterozygous for the
mutation. We screened 50 normal controls using AvaI; none
had the mutation (data not shown).
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). This mutation causes an asparagine to
lysine substitution at position 118 in the amino-terminal extracellular
domain of the receptor (Fig. 2). As this mutation did not alter any
known restriction site, we analyzed the parents’ genotype by
sequencing (Fig. 1). Neither had the mutation. We confirmed maternity and paternity for both patients by typing seven polymorphic red cell antigens (ABO, Rh, MN, S, Kell, Kidd, and Duffy) and analyzing four PCR-amplified highly polymorphic DNA loci (D9S52, D9S287, TNF, and F13A1) (22, 23, 24). All were confirmatory.
Western analysis
Plasma membrane preparations from cells transfected with wild-type
construct produced two bands between 100–200 kDa and a band of high
molecular mass. A similar pattern was observed for each of the three
mutant constructs (Fig. 4).
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Cell surface expression of mutant and wild-type CaRs on the plasma
membrane of HEK-293 cells was assessed by ELISA. Expression of the
mutant receptors was lower than that of wild-type receptor (Table 2).
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After transient transfection with wild-type or mutant constructs,
HEK-293 cells were exposed to graded Ca2+ concentrations,
and IP accumulation was measured. At the lowest Ca2+
concentration (0.5 mmol/L), the mutant receptors all induced a slightly
greater IP accumulation than did wild-type receptor
(P = NS; Table 2). With increasing
Ca2+, IP accumulation increased for the wild-type and all
mutant receptors (P < 0.001), reaching a
plateau at high Ca2+ concentrations (Fig. 5). Two of the mutant receptors (L118R and N773K) showed
a greater maximal response to Ca2+ (at 8 mmol/L) than
wild-type receptor (P < 0.001; Table 2 and Fig. 5). These two mutant receptors also showed a leftward shift in the
concentration-response curve (Fig. 5) and, thus, a decrease in
EC50 compared to wild-type receptor (Table 2;
P < 0.001). The concentration-response curve
for mutation F806S also appeared slightly left-shifted (Fig. 5), but
the change in EC50 did not reach statistical significance
(Table 2).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We previously reported the first de novo CaR mutation (F806S) in a child with sporadic hypoparathyroidism (11). The two cases in the present report confirm this association. They demonstrate that these mutations can cause severe disease, presenting in infancy, or mild disease, occurring in adulthood. Therefore, even patients presenting in adulthood without affected relatives should not be assumed to have an autoimmune etiology.
Both patients in the current report were hypercalciuric even while hypocalcemic. We and others (11, 14) reported this disproportionate hypercalciuria in patients with autosomal dominant hypoparathyroidism due to CaR mutations. In the kidney, CaR activation inhibits tubular reabsorption of Ca2+ (2, 3). CaR mutations may increase this inhibition, thus producing hypercalciuria (11, 14). The disproportionate hypercalciuria may increase the risk of nephrocalcinosis, nephrolithiasis, and renal insufficiency. Thus, patients with CaR hyperfunction need careful monitoring of urine calcium and may require more conservative use of vitamin D analogs, addition of thiazide diuretics (25, 26), or PTH administration (27).
The firm diagnosis of CaR hyperfunction requires molecular genetic studies that may not be available outside of the research setting. However, the diagnosis should be suspected when hypercalciuria is present in a patient taking vitamin D analogs despite serum calcium concentrations below or near the lower limit of normal. Patients with acquired hypoparathyroidism also tend to have increased urinary calcium because they lack the calcium-retaining effect of PTH (28), but generally are not hypercalciuric while hypocalcemic. Further studies are needed to define the diagnostic accuracy of this approach.
Mutation N118K lies in the amino-terminal, extracellular domain of the receptor. The same mutation was recently found in a family with autosomal dominant hypoparathyroidism, but its expression was not studied (14). The other mutations described in familial hypoparathyroidism also occurred within extracellular regions of the CaR. In contrast, the other two mutations that we identified in sporadic cases, L773R and F806S, lie in the fifth and sixth transmembrane domains.
Mutant and wild-type CaRs were expressed in HEK-293 cells. For all four receptors, IP production increased with increasing Ca2+ concentrations and then reached a plateau. This finding, typical of ligand-receptor interactions in general, contrasts with a previous report of three different CaR mutations in familial hypocalcemia (14). These mutant receptors showed a biphasic concentration-response curve, with decreasing IP accumulation at Ca2+ concentrations above 2 mmol/L. It is not clear whether this discrepancy reflects methodological differences or intrinsic differences in the mutations studied.
In the current study, mutations N118K and L773R caused a significant leftward shift in the concentration-response curve and a significant increase in the maximal IP response to high Ca2+ concentrations. The increase in maximal activity probably reflects increased activity per receptor, since cell surface expression of the mutant receptors was decreased compared to that of the wild-type receptor. Similarly, the mutations did not affect glycosylation patterns as assessed by Western analysis. Thus, the amino acid substitutions may affect receptor function directly. The observed leftward shifts are probably due to enhanced affinity of the mutant receptors for Ca2+ . However, methods for the direct assessment of Ca2+ binding have not been established.
A similar concomitant increase in receptor affinity and maximal signal transduction has been observed for activating mutations of the ß2-adrenergic receptor (29) and the platelet-activating factor receptor (30). Conversely, concomitant decreases in sensitivity and maximal activity have been reported for inactivating mutations in the CaR (17). The observed dual effect may be explained by the allosteric ternary complex model for G protein-coupled receptors (31). According to this model, the receptor is in equilibrium between an inactive and an active conformation. Ligand binding increases signal transduction by shifting the equilibrium toward the active state. According to this model, the active conformation has a greater affinity for ligand. We speculate that mutations N118R and L773K shift the equilibrium toward the active conformation. Because the active conformation is proposed to have a greater affinity for ligand, the observed decrease in their EC50 values would thus be explained. At saturating Ca2+ concentrations, such mutations would increase the proportion of receptors in the active conformation, thus explaining the observed increase in maximal signal transduction.
Mutation F806S did not produce a significant activating effect. However, in these experiments, cell surface receptor expression was decreased for the mutant receptors compared to that for the wild type. Thus, the actual increase in activity per receptor may be underestimated.
Mutations N118K and L773R produced similar effects on signal transduction in vitro, yet resulted in a different phenotype in vivo. This discrepancy could reflect differences between HEK-293 and parathyroid cells or differences in the two patients’ genetic background.
We conclude that sporadic hypoparathyroidism can be caused by de novo gain-of-function mutations in the CaR. The phenotypic severity can vary greatly, ranging from mild hypocalcemia presenting in adulthood to hypocalcemic seizures presenting in infancy. The characteristic disproportionate hypercalciuria may help identify these patients and cause renal complications. The activating mutations can occur not only in the extracellular regions of the receptor, as previously described, but also in the transmembrane domains. The mechanism of activation can involve increases both in sensitivity to Ca2+ and maximal signal transduction. These findings have implications not only for the molecular pathogenesis of sporadic hypoparathyroidism but also for genetic counselling and treatment of affected patients.
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Acknowledgments |
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Footnotes |
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Received November 25, 1997.
Revised April 18, 1997.
Accepted May 9, 1997.
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