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Departments of Medicine, Physiology, and Human Genetics (K.Z., L.C., D.G., G.N.H.), McGill University, and Calcium Research Laboratory, Royal Victoria Hospital, Montréal, Québec, Canada H3A 1A1; Institute of Endocrinology (K.Z., J.V.), Prague 1, 116 94, Czech Republic; and Department of Chemistry and Biochemistry (P.D.P.), Concordia University, Montréal, Québec, Canada H4B 1R6
Address all correspondence and requests for reprints to: Geoffrey N. Hendy, Ph.D., Calcium Research Laboratory, Room H4.67, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Québec, Canada H3A 1A1. E-mail: geoffrey.hendy{at}mcgill.ca.
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Abstract |
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Objective: The objective of the study was to identify and characterize a CASR mutation in a moderately hypercalcemic, hyperparathyroid individual and his family and assess the influence of vitamin D status on the clinical expression of the defect.
Subjects: We studied a kindred with FHH, in which the proband (a 34-yr-old male) was initially diagnosed with primary hyperparathyroidism due to frankly elevated serum PTH levels.
Methods: CASR gene mutation analysis was performed on genomic DNA of the proband and family members. The mutant CASR was functionally characterized by transient transfection studies in kidney cells in vitro.
Results: A novel heterozygous mutation (F180C, TTC>TGC) in exon 4 of the CASR gene was identified. Although the mutant receptor was expressed normally at the cell surface, it was unresponsive with respect to intracellular signaling (MAPK activation) to increases in extracellular calcium concentrations. The baby daughter of the proband presented with neonatal hyperparathyroidism with markedly elevated PTH. Vitamin D supplementation of both the proband and the baby resulted in reduction of serum PTH levels to the normal range. The serum calcium level remained at a constant and moderately elevated level.
Conclusion: The identification of a novel CASR gene mutation established the basis of the hypercalcemia in the kindred. Concomitant vitamin D deficiency modulates the severity of the presentation of FHH.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We report a Czech family with FHH resulting from a novel inactivating CASR mutation. Besides hypercalcemia, the proband of the family demonstrated frankly elevated serum PTH levels, confusing a plausible diagnosis of FHH based on clinical findings. The low calcium to creatinine clearance ratio and low serum 25-hydroxyvitamin D [25(OH)D] levels, indicative of vitamin D deficiency, led eventually to the consideration of FHH. We also studied the perinatal and postpartum course of the baby daughter of the proband. The infant who was heterozygous for the mutation presented with neonatal hyperparathyroidism.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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), a 34-yr-old man, was referred for evaluation of asymptomatic hypercalcemia. His total and ionized calcium concentrations were elevated (2.92 and 1.55 mmol/liter, respectively) (Fig. 1B), whereas serum phosphate, magnesium, and creatinine were normal (Table 1). Although FHH was considered as a possible diagnosis due to frankly elevated serum PTH levels (151.8 ng/liter), the patient was thought to have primary hyperparathyroidism. Bone densitometry (dual-energy x-ray absorptiometry) at lumbar spine and hip showed no reduction of bone mineral density. X-ray of the skull and right hand was without any features associated with hyperparathyroidism. On kidney ultrasound, there were no signs of kidney stones or nephrocalcinosis. Imaging techniques (neck ultrasonography, computed tomography, and Tc-99m sestamibi scanning) did not reveal any enlarged parathyroid gland or ectopic tumor. A low calcium to creatinine clearance ratio (<0.01) led to further consideration of the possibility of FHH.
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) was also hypercalcemic with mildly elevated serum PTH (Fig. 1B). However, her calcium to creatinine clearance ratio was over 1% (0.0141). The proband’s 25-yr-old sister (II-1, Fig. 1A) was normocalcemic (Table 1). The proband’s baby girl was born normally: weight 3650 g and length 52 cm (length to weight ratio 45th percentile, SD score 0.12, length 88th percentile, SD score 1.19) and was breast-fed. The newborn (d 0, cord blood) had a modestly elevated calcium level but markedly elevated PTH and osteocalcin levels (Fig. 1C). In addition, the bone-specific alkaline phosphatase level was elevated (80.36 µg/liter). All subjects (or their guardians) gave informed consent, and the studies were approved by the ethical committees of the Institute of Endocrinology (Prague) and the Royal Victoria Hospital (Montréal).
Direct sequence analysis of CASR gene exons
Leukocyte DNA was isolated using standard methods. Protein-coding exons 2–7 of the CASR gene were amplified as described (10). Gel-purified PCR products were directly sequenced. For all family members, CASR exon 4 was amplified and digested with PdmI to test for the presence of the mutation.
Site-directed mutagenesis
The mutation was introduced into a c-Myc-tagged human CASR cDNA in pcDNA3.1 as described (11). The correctness of the construction was confirmed by sequencing.
Transient transfection of human CASR cDNA
Human embryonic kidney (HEK293) cells (provided by NPS Pharmaceuticals, Inc., Salt Lake City, UT) were transfected with c-Myc-tagged human CASR cDNAs, wild-type or mutant, as described (10). Western blot analysis of total cell extracts was performed with the c-Myc 9E10 mouse monoclonal antibody. Membranes were reprobed with ß-tubulin mouse monoclonal antibody as loading control.
CASR intact cell surface ELISA
Cell surface expression of the CASR was measured by an ELISA method as described (12, 13). HEK293 cells, transiently transfected with wild-type or mutant c-Myc-tagged CASR cDNAs, were fixed and washed with PBS. After incubation with c-Myc 9E10 mouse monoclonal antibody, peroxidase-conjugated goat antimouse antibody was added. To develop the reaction, 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) liquid substrate (Sigma, Oakville, Canada) was added and the OD read at 405 nm. Controls included HEK293 cells stably expressing CASR, cells transiently transfected with empty vector or a c-Myc expression vector, nontransfected cells, or omission of the anti-CASR (c-Myc) antibody.
MAPK assay
The ability of wild-type and mutant CASR to activate the MAPK signaling cascade was assessed by examining phosphorylation of ERK1/2 as described (14). HEK293 cells were transiently transfected with wild-type or mutant CASR cDNAs. Cells were treated with increasing CaCl2 concentrations (0.5–10 mM) for 5 min. Whole-cell extracts were made and aliquots were separated by 10% SDS-PAGE and analyzed for expression of phosphorylated and total ERK1/2 by immunoblotting with PhosphoPlus p44/42 MAPK (Thr202/Tyr204) antibody kit (Cell Signaling Technology, Beverly, MA) according to the manufacturer’s protocol.
The Scion Image-National Institutes of Health image processing and analysis program (http://rsb.info.nih.gov/nih-image/) was used for signal densitometry. The ratios of the phosphorylated to nonphosphorylated ERK1/2 signals at various extracellular calcium concentrations were calculated first and then normalized to the ratio of phosphorylated to nonphosphorylated ERK1/2 at 0.5 mM Ca2+.
Homology modeling of the dimeric extracellular domain of the CASR
An alignment of the CASR (accession no. AAA86503) and metabotropic glutamate receptor 1 (mGluR1) (accession no. NP_058707) extracellular domains was made with the T-Coffee program (15) using the Expresso 3D-Coffee version (16) (Centre National de la Recherche Scientifique, http://igs-server.cnrs-mrs.fr/Tcoffee). Homology modeling of the human CASR (based on the mGluR1 structure; 1EWT, mmdbId: 15526) was performed with the DeepView/SwissModel package (17) (Swiss Institute of Bioinformatics, http://ca.expasy.org/spdbv/). To investigate potential disulfide formation between residues 180 and 482, the computer program Pymol (18) was used to mutate computationally F180 to C180. Side-chain rotamer positions at C180 and C482 were sampled, and interatomic distances between sulfhydryl groups were determined, using the program O (19).
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Results |
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The proband, individual II-2, was found to have a low circulating 25(OH)D level indicative of vitamin D deficiency (8.8 nmol/liter) and was treated with vitamin D. Serum total and ionized calcium, PTH, and 25(OH)D levels are shown at a time when proband II-2 was vitamin D deficient and during the course of vitamin D supplementation (1800–3600 IU/d) (Fig. 1B). With continuing vitamin D treatment, the serum 25(OH)D levels rose, and the serum PTH levels fell into the normal range. Throughout the entire period, the serum and ionized calcium levels remained constant at moderately elevated levels (Fig. 1B). For comparison, it can be noted that the proband’s mother (I-1) had a higher serum 25(OH)D level and a less elevated serum PTH than the proband, at the time of initial screening, and calcium levels just above the upper limit of normal.
Influence of vitamin D status on the postpartum course of infant III-1
The proband’s wife (II-3, Fig. 1A) was vitamin D deficient (Table 1) at the time of her pregnancy. At birth, the baby (III-1, Fig. 1A) presented with a mildly elevated calcium level, a phosphate level at the lower end of the normal range, a markedly elevated PTH concentration (Fig. 1C), and a 25(OH)D level of 26.2 nmol/liter (Table 1). The osteocalcin level was elevated. At birth vitamin D supplementation was initiated (600 IU/d). Serum PTH levels fell over time as the hyperparathyroidism resolved. Serum osteocalcin levels decreased, indicating normalization of bone turnover (Fig. 1C). The serum calcium rose slightly throughout the postpartum period, whereas the serum phosphate level remained at the lower end of the normal range (Fig. 1C).
Identification of CASR mutation
Direct sequence analysis of PCR-amplified CASR exons identified a heterozygous mutation (TTC
TGC, F180C) in the CASR extracellular domain encoded by exon 4 of the gene (Fig. 2). The presence of the mutation abolished a PdmI restriction site, which provided a convenient diagnostic test to confirm the presence of the mutation in the proband (II-2), his mother (I-1), and his infant daughter (III-1).
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Mutant and wild-type CASR constructs were generated and transfected into HEK293 cells. The 180C mutant receptor was expressed at equivalent levels and exhibited the same pattern of molecular species as wild-type CASR: the core glycosylated (immature) 140-kDa species and the mature fully glycosylated 160-kDa species, respectively (Fig. 3A). High-molecular-mass forms (>280 kDa), likely to be dimers, were seen equally for wild-type and mutant receptors. Hence, the data suggest that the mutant receptor achieved maturation and was appropriately trafficked to the plasma membrane. In a CASR-intact cell surface ELISA, the OD signals obtained with both the wild-type and 180C mutant receptors were 7-fold those in HEK293 cells transfected with either empty vector (Fig. 3B) or a c-Myc expression vector or untransfected (data not shown). Therefore, the cell surface expression of the 180C mutant receptor was identical with that of wild type.
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Raising the extracellular calcium concentration from 0.5 to 10 mM stimulated the phosphorylation of ERK1/2 MAPKs (as assessed by immunoblot) in wild-type CASR-expressing cells (Fig. 4A). In contrast, cells transfected with the mutant CASR were virtually without MAPK responsiveness to increasing extracellular calcium (Fig. 4B). Quantification of the signals by densitometric analyses showed a half-maximal response (EC50) of 2.7 ± 0.10 mM (mean ± SE; Fig. 4C) for wild-type CASR. When the mutant was cotransfected with wild-type CASR to mimic the heterozygous state, the curve was significantly right shifted to a position intermediate to that of wild-type alone and the mutant alone, with an EC50 of 7.1 ± 1.9 mM (Fig. 4C).
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The homology model of the human CASR extracellular domain predicts a fold (structure) that is highly similar to that of the mGluR1 on which it was based (see Patients and Methods). This is indicated by the very low C
-C root mean square deviation value of 0.001 Å calculated for the two superimposed structures. (C-C refers to the distance between the -carbon atoms of two particular amino acids.) Thus, the protein backbone atoms of the CASR model are predicted to lie in virtually identical positions to those of the mGluR1. Based on the overall fold of the human CASR model (Fig. 5A), residues F180 and C482 are predicted to reside in loops (Fig. 5B), with a C-C distance of 6.11 Å. Upon mutation of F180 to C180 and sampling of preferred side-chain rotamers, one rotamer pair resulted in an interthiol distance of 3.52 Å between C180 and C482. Although this distance might not favor disulfide formation if the structures were fixed, mobility of the loops containing these cysteine residues would bring the thiols proximal to each other for disulfide bond formation.
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Discussion |
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The VFT of the CASR has a number of conserved cysteines: intermolecular bridging between C129 and C131 contributes to dimer formation (20, 22). Dimerization is an early event that is already evident for newly synthesized receptors in the endoplasmic reticulum (23). There was no evidence from our study that dimer formation of the 180C mutant was impaired. The 180C mutant was expressed at normal levels and underwent maturation and trafficking to the plasma membrane like the wild-type receptor. With respect to the other cysteines, by analogy with the mGluR1 VFT structure, it can be surmised that CASR cysteine pairs, 60–101, 358–395, and 437–449, form intramolecular disulfide bonds. In addition, residues 236 and 482 exist as free cysteines. Mutation of any of these residues, with the exception of C482, led to the loss of normal receptor expression (at the cell surface) and function (12). C482 is not conserved, even among vertebrate CASRs consistent with the lack of functional importance.
Molecular modeling of the dimeric CASR extracellular domain predicts that F180 is in the hinge region between lobes 1 and 2 (LB1 and LB2) of each protomer (Fig. 5A) that close on ligand binding (22). Whereas the phenolic ring side groups of both phenylalanine and tyrosine (see above) at position 180 point away from the surrounding protein structure into open space, modeling of the sulfhydryl group of the mutant cysteine indicates that the side chain is oriented toward the protein core (Fig. 5B). Sampling of preferred side-chain rotamers within the model suggests that the sulfhydryl moieties of C180 and C482 could be in close proximity, potentially leading to disulfide bond formation. Such a constraint would likely interfere with the flexibility of the hinge region between LB1 and LB2. So whereas the mutation does not affect dimerization, exit from the endoplasmic reticulum, and trafficking to the plasma membrane, it likely impairs the ligand-stimulated closure of the VFT that initiates receptor activation.
At the cell surface, on ligand binding, the dimeric CASR can activate multiple intracellular signaling pathways. The 180C mutant was markedly defective in ligand-activated cell signaling (endogenous MAPK activity), suggesting that although being properly processed and expressed at the cell surface, it may not attain an active conformation after ligand binding. Cotransfection with equal amounts of wild-type and mutant CASR, mimicking the heterozygous state of FHH patients, produced a response intermediate to that of wild-type or mutant alone. Because a proportion of the CASR dimer population is likely to exist as wild-type/mutant heterodimers, such a finding has been interpreted as indicative that these particular (inactivating) mutant receptors exert a dominant-negative effect on the wild-type receptor.
FHH-type CASR mutations can be broadly classified into two types. The first are those in which the mutant mature receptor is either not synthesized at all, or if it is, it cannot form dimers with the wild-type receptor. The former are represented by mutations in the signal peptide such that little mature CASR is made (24) and the latter by nonsense mutations leading to significantly truncated receptor proteins, e.g. N583X (23). The second type includes the majority of missense mutations in the CASR that can dimerize with the wild-type CASR and reduce its activity, e.g. R185Q (25), R227Q, R227L (26), and the presently described F180C. Mutations in the second group are potentially more inactivating because they have an impact on the activity of the normal CASR encoded by the wild-type allele. Hence, individuals harboring such mutations may present in a more clinically severe manner. However, as illustrated by the present case, other factors are likely to play an equal or greater role in the clinical presentation (27).
The identification of an inactivating mutation in the CASR that segregated with the hypercalcemic trait in the family confirmed the diagnosis of FHH. The proband, however, did not present in a typical fashion. Besides hypercalcemia, severely elevated PTH levels were found, confusing a plausible clinical diagnosis of FHH. The additional features that then pointed to FHH included the low calcium to creatinine clearance ratio and the presence of hypercalcemia in the proband’s mother. However, a major factor in arriving at this conclusion was the consideration of the concomitant vitamin D deficiency in the proband.
Vitamin D insufficiency is common among patients with primary hyperparathyroidism, and vitamin D nutrition has been suggested to be a major influence on the presentation of the disorder, either geographically and/or as the mild form more commonly encountered in the present day (28, 29, 30). Vitamin D deficiency is associated with more severe and progressive disease. Judicious vitamin D repletion in primary hyperparathyroid patients does not exacerbate the hypercalcemia and may decrease circulating PTH levels and bone turnover (31). The active metabolite of vitamin D, 1,25(OH)2D, regulates several aspects of parathyroid gland function, such as parathyroid cell proliferation (32) and expression of the PTH and CASR genes (33). Reductions in serum 25(OH)D levels are associated with a concomitant decrease in intestinal calcium absorption, relatively reduced serum calcium concentration, deactivation of the CASR, and increased PTH secretion. In addition, in view of our previous studies demonstrating vitamin D responsive elements in the CASR promoters and positive regulation of CASR expression by 1,25(OH)2D (33), reduced 25(OH)D leading to decreased 1,25(OH)2D may also contribute to dysregulated PTH secretion and parathyroid cell growth control. Vitamin D supplementation may therefore reverse these events and reduce (to an extent) PTH secretion via the CASR product of the single normal allele in an FHH individual. Because both megalin, an endocytic receptor required for cellular uptake of 25(OH)D, and the 25-hydroxyvitamin D-1-hydroxylase enzyme responsible for production of 1,25(OH)2D, are expressed in parathyroid chief cells, local production of the active vitamin D metabolite is also possible as a modulator of CASR and PTH production (34). Therefore, consideration of the circulating 25(OH)D levels may be of prime importance in correlating vitamin D status to parathyroid gland function.
We were able to demonstrate that the vitamin D status of the proband markedly influenced the severity of PTH elevation. With vitamin D supplementation, the serum 25(OH)D levels rose into the vitamin D replete range and the serum PTH level fell into the normal range. Throughout the entire period, the serum and ionized calcium levels remained constant at moderately elevated levels. It is likely that low urinary calcium excretion in vitamin D deficiency may also contribute to ambiguity in distinguishing primary hyperparathyroidism from FHH. However, after vitamin D supplementation, urinary calcium levels should become normal or elevated in primary hyperparathyroidism (31) but remain low in FHH due to the mutated CASR allele. Consistent with this, the urinary Ca/Cr clearance ratio of proband II-2, which was 0.0012 while he was vitamin D deficient, only rose to 0.0026 when he was supplemented with vitamin D.
Individuals harboring heterozygous inactivating CASR mutations may or may not come to medical attention during their lifetime because of the mainly benign nature of the FHH disorder. Individuals with such mutations that do manifest hyperparathyroidism in the perinatal period may present with a wide spectrum of severity, ranging from stillbirth, severe to moderate neonatal skeletal demineralization that either does or does not resolve over time, to a benign FHH condition. This may occur with or without intervention of either surgical (parathyroidectomy) or medical (e.g. bisphosphonate) therapy (35, 36). Several factors may be at play in contributing to the presentation of individual cases.
In our study, the baby of the proband presented at birth with a mildly elevated calcium level, a markedly elevated PTH concentration, and a low 25(OH)D level. The discordance between the high PTH and the relatively mild elevation in serum calcium may have reflected the vitamin D status and calcium economy of the mother and therefore the neonate. With vitamin D treatment, PTH levels fell over time to be only slightly elevated at d 48 and within the normal range at d 70 after birth. The serum calcium rose slightly throughout the postpartum period to achieve a moderately elevated steady-state level consistent with the expected phenotype of FHH. The early high osteocalcin levels were likely a reflection of the hyperparathyroidism that had developed in utero.
Other cases have been reported in which the neonatal primary hyperparathyroidism associated with FHH is self-limiting either without parathyroidectomy (37, 38, 39) or with subtotal parathyroidectomy (26, 40). In such cases, the serum PTH levels decrease because serum calcium levels are either maintained at a constant moderately elevated level or increase somewhat, although occasionally vitamin D deficiency may mask the hypercalcemia of neonatal primary hyperparathyroidism (41). Besides maternal and fetal vitamin D deficiency, other factors that would influence both the development of fetal hyperparathyroidism and the later postpartum resolution of the condition have been suggested. In the latter part of pregnancy, active placental calcium transport in a maternal-fetal direction results in a higher fetal than maternal extracellular calcium concentration. The normal fetal role of the CASR is to stimulate placental calcium transport via PTHrP (42) and respond to the elevated calcium level and suppress PTH from the parathyroids. Heterozygous and homozygous Casr gene disruption in mice causes the parathyroid glands to misread a normal calcium concentration as low, leading to increases in serum levels of PTH [and 1,25(OH)2D], stimulating bone resorption leading to a further elevation in the fetal calcium concentration, although placental calcium transport is decreased (43). Whereas a heterozygous fetus and heterozygous mother will be matched with respect to compensation of the placental calcium transport defect, a heterozygous fetus of a normal mother is at a disadvantage, and fetal parathyroid activity would have to increase even further to maintain the elevated serum calcium concentration (44).
Postpartum, the kidney (and intestines) plays a much greater role in calcium homeostasis than in the fetus in which mineral ion and water homeostasis is predominantly regulated by the placenta and parathyroids. Renal CASR expression is very low in the fetus but is markedly up-regulated postpartum (45). Hence, postnatally, as the effect of the defective renal calcium-sensing becomes apparent, provoking enhanced renal calcium reabsorption and declining urinary calcium excretion, the parathyroid gland calcium-sensing mechanism will adjust to the elevated serum calcium concentrations and reduce PTH secretion to levels commensurate with the FHH state. To these considerations regarding the pathophysiology of hyperparathyroidism in neonatal FHH should now be added the vitamin D status of the mother, and therefore the neonate, factors that may be readily reversible.
In conclusion, the identification and functional evaluation of this novel mutation in the CASR gene established the basis of the hypercalcemia in the kindred. Demonstration of this mutation will spare family members undergoing unnecessary parathyroidectomy. Moreover, the present study highlights the vitamin D status of the affected individuals as an important influence on the FHH phenotype and therefore identifies a correctible contributor to the pathophysiology of this genetic disorder.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online May 1, 2007
Abbreviations: CASR, Calcium-sensing receptor; FHH, familial hypocalciuric hypercalcemia; LB, lobe; mGluR1, metabotropic glutamate receptor 1; 1,25(OH)2D, active metabolite of vitamin D; 25(OH)D, 25-hydroxyvitamin D; VFT, venus fly trap.
Received January 18, 2007.
Accepted April 23, 2007.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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-hydroxylase expression in normal and pathological parathyroid glands. J Clin Endocrinol Metab 87:2967–2972This article has been cited by other articles:
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) or during (
) vitamin D supplementation (from January 2005 to November 2005, 1800 IU/d, and from November 2005 to January 2006, 3600 IU/d). For comparison, the results of a one-time measurement in the proband’s mother, I-1, are shown. Stippled bars indicate normal ranges or, for 25(OH)D, sufficient levels. C, Serum concentrations of calcium, phosphate, PTH, and osteocalcin during the first 180 d of life in the infant III-1 with neonatal hyperparathyroidism. Stippled bars indicate normal ranges.
