0021-972X/97/$03.00/0
Journal of Clinical Endocrinology and Metabolism
Copyright © 1997 by The Endocrine Society
Vol. 82, No. 8
Printed in U.S.A.
Sporadic Hypoparathyroidism Caused by de Novo Gainof-Function Mutations of the Ca21-Sensing Receptor*
FRANCESCO DE LUCA, KAUSIK RAY, EDNA E. MANCILLA, GAO-FENG FAN,
KAREN K. WINER, PANKAJ GORE, ALLEN M. SPIEGEL, AND JEFFREY BARON
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
ABSTRACT
Activating mutations of the Ca21-sensing receptor (CaR) gene have
been identified in families with autosomal dominant hypoparathyroidism and in one patient with sporadic hypoparathyroidism. Here,
we describe two additional patients with sporadic hypoparathyroidism. One patient presented with mild symptoms at age 18 yr; the other
was severely symptomatic from infancy. A heterozygous missense
mutation was identified in each patient. One mutation (L773R) involved the fifth transmembrane domain of the CaR, the other (N118K)
affected the amino-terminal, extracellular domain. In both cases, the
probands’ parents lacked the mutation, indicating that the mutations
arose de novo. In expression studies the mutations shifted the concentration-response curve to the left and increased maximal activity.
We conclude that 1) sporadic hypoparathyroidism can be caused by de
novo gain-of-function mutations of the CaR; 2) the phenotype can vary
from mild to life-threatening hypocalcemia; 3) gain-of-function mutations can involve not only extracellular regions, as previously reported, but also transmembrane domains of the CaR; and 4) the
mechanism of activation can involve both increased receptor sensitivity to Ca21 and increased maximal signal transduction. (J Clin
Endocrinol Metab 82: 2710 –2715, 1997)
T
ceptor, 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 Ca21 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.
HE CALCIUM-SENSING receptor (CaR) is a member of
the G protein-coupled receptor superfamily, sharing
the motif of seven membrane-spanning domains (1). Binding
of Ca21 favors receptor activation and G protein coupling.
The activated G protein stimulates phospholipase C activity,
leading to accumulation of inositol trisphosphate, which, in
turn, increases cytoplasmic Ca21 (1).
In parathyroid cells, this cascade decreases PTH secretion
(1). In kidney, CaR activation inhibits reabsorption of Ca21
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 Ca21 sensors are present in
parathyroid gland, kidney, placenta, and osteoclasts (6, 7).
The physiological roles of these putative Ca21 sensors are not
known.
Inactivating mutations of the CaR gene (located on chromosome 3q) (8) cause resistance of parathyroid gland and
kidney to extracellular Ca21 (9, 10). Thus, there is decreased
inhibition of PTH secretion, producing hypercalcemia, and
decreased inhibition of renal Ca21 reabsorption, producing
relative hypocalciuria.
Conversely, activating mutations of the CaR are associated
with the reverse phenotype, familial hypoparathyroidism,
and relative hypercalciuria (11–16). Receptor activation
could result from increased activity of the unliganded reReceived November 25, 1997. Revision received April 18, 1997. Accepted May 9, 1997.
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:
[email protected].
* Presented in part at the 65th Annual Meeting of the Society for
Pediatric Research, Washington, D.C., 1996.
Subjects and Methods
Case descriptions
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.
Patient B, a 23-yr-old woman, experienced a hypocalcemic seizure at
7 months of age. Hyperphosphatemia and low PTH levels were also
2710
DE NOVO ACTIVATING CaR MUTATIONS
TABLE 1. Biochemical and clinical characteristics of two patients
with sporadic hypoparathyroidism
Sex
Age (yr)
Age at diagnosis
Serum calcium (2.05–2.4 mmol/L)a
Serum phosphate (2.3– 4.3 mmol/L)a
Serum PTH (10 – 65 pg/ml)b
Urinary calcium (1.25– 6.25 mmol/24 h)a
Clinical findingsc
Patient A
Patient B
F
25
18 yr
1.6
6.4
,3
9.2
f, d, nc
F
22
7 months
1.75
9
,3
7.05
s, b, nc
Normal values are given in parentheses. All measurements were
obtained during treatment with vitamin D and oral calcium.
a
Serum electrolytes and urinary calcium values represent simultaneous measurements.
b
PTH levels were measured while the patients were hypocalcemic.
c
s, Seizures; b, basal ganglia calcifications; nc, nephrocalcinosis; f,
fatigue; d, depression.
detected. She has taken oral calcium and vitamin D analogs since diagnosis. She was repeatedly hospitalized for hypercalcemia. She has
been treated with anticonvulsants for recurrent seizures. At 14 yr of age,
a computed tomography scan revealed calcifications of frontal cortex
and basal ganglia. When seen at the NIH at age 16 yr, physical examination was normal, except for a large hemangioma on the trunk and left
arm. Height, weight, and sexual development were normal. Computed
tomography scan of the kidneys revealed bilateral nephrocalcinosis.
Renal function tests were normal. The results of biochemical studies on
oral calcium and vitamin D are listed in Table 1.
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 mL Lipofectamine (Life Technologies, Gaithersburg, MD) and 0.5 mg 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 mg/mL aprotinin; 5 mg/mL
2711
leupeptin; and 0.7 mg/mL pepstatin) was added at 4°C. The cells were
lysed by passing repeatedly through 22-gauge needles and sedimented
at 16,000 3 g for 30 min to remove nuclei and mitochondria. The supernatant was sedimented at 50,000 3 g for 30 min to pellet plasma
membranes, which were solubilized with 1% Triton X-100.
Western analysis
Membrane proteins (25 mg/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 mg/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 enzymelinked 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 mg/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 mCi/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 Ca21 (in PI buffer) for 30 min
at 37°C. The reactions were terminated with acid-methanol (167 mL 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 Ca21 concentration for IP measurement.
Statistical analysis
Results were expressed as the mean 6 sem. Significance was assessed
by ANOVA and post-hoc Fisher’s protected least significant difference
test.
Results
DNA sequence analysis
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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DE LUCA ET AL.
ELISA. Expression of the mutant receptors was lower than
that of wild-type receptor (Table 2).
Functional analysis
After transient transfection with wild-type or mutant constructs, HEK-293 cells were exposed to graded Ca21 concentrations, and IP accumulation was measured. At the lowest
Ca21 concentration (0.5 mmol/L), the mutant receptors all
induced a slightly greater IP accumulation than did wildtype receptor (P 5 NS; Table 2). With increasing Ca21, IP
accumulation increased for the wild-type and all mutant
receptors (P , 0.001), reaching a plateau at high Ca21 concentrations (Fig. 5). Two of the mutant receptors (L118R and
N773K) showed a greater maximal response to Ca21 (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).
Discussion
FIG. 1. Basepair substitutions in the Ca21 -sensing receptor gene.
PCR-amplified genomic DNA was sequenced directly using fluorescent dideoxynucleotides in an automated DNA sequencing system.
DNA sequence for patient A, shown below that for a normal control,
revealed a heterozygous T to G substitution at position 2318. Sequence for patient B revealed a heterozygous C to A substitution at
position 354, which is not present in her unaffected parents. Arrows
indicate the sites of the mutations.
In patient B, we found a heterozygous C to A basepair
substitution at position 354 in exon 2 (Fig. 1). 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) (22–24). 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).
Assessment of receptor expression
Cell surface expression of mutant and wild-type CaRs on
the plasma membrane of HEK-293 cells was assessed by
We identified heterozygous missense CaR mutations
(N118K and L773R) in two patients with sporadic isolated
hypoparathyroidism. In both cases, the probands’ parents
lacked the mutation, indicating that the mutations arose de
novo.
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
Ca21 (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 hypocalce-
DE NOVO ACTIVATING CaR MUTATIONS
2713
FIG. 2. Schematic diagram of the human Ca21-sensing receptor. The shaded
area depicts the plasma membrane,
with the extracellular space at the top of
the figure. Amino acids are indicated by
circles. The amino acid substitutions
produced by the point mutations are illustrated by the black circles in the
three insets. N118K and L773R are
newly identified substitutions, whereas
F806S was previously reported (11).
FIG. 3. AvaI restriction enzyme digest of PCR-amplified exon 6 DNA
from patient A (closed symbol) and three unaffected family members
(open symbols). AvaI recognizes a site created by the T2318G mutation in exon 6. All three unaffected family members showed a single
band, which represents the two wild-type alleles. In contrast, patient
A showed the wild-type band and two additional fragments, which
represent the mutant allele cut by AvaI.
mic. 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 Ca21 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 re-
FIG. 4. Western analysis of wild-type and mutant Ca21 -sensing receptors. Cell membrane proteins were obtained from HEK293 cells
transfected with wild-type or mutant constructs. The protein samples
(25 mg/well) were subjected to SDS-PAGE on a linear gradient running gel (5–15%). The CaR was identified with affinity-purified monoclonal antibody anti-ADD (made against residues 214 –235 of human
CaR). WT indicates wild-type construct; N118K, L773R, and F806S
indicate the three mutant constructs; 293 indicates the nontransfected HEK293 cells.
ceptors showed a biphasic concentration-response curve,
with decreasing IP accumulation at Ca21 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 Ca21 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
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DE LUCA ET AL.
TABLE 2. IP accumulation, EC50, and cell surface expression for the mutant and the wild-type receptors
Mutation
ELISA
(OD units)
Minimum IP accumulation
(0.5 mmol/L Ca21; 103 cpm)
Maximum IP accumulation
(8 mmol/L Ca21; 103 cpm)
EC50 for Ca21
(mmol/L)
Wild-type
N118K
L773R
F806S
3.7 6 0.1
2.4 6 0.1
1.6 6 0.01
2.4 6 0.07
3.6 6 0.6
6.1 6 1.0
5.6 6 0.9
5.1 6 0.5
33 6 2
50 6 4
50 6 3
33 6 3
4.3 6 0.2
2.7 6 0.2
2.8 6 0.2
4.0 6 0.3
Values are the mean 6
SEM.
ELISA values reflect cell surface CaR expression.
FIG. 5. Ca21 -evoked accumulation of IP in HEK293 cells transiently
transfected with wild-type or mutant CaR cDNA. Transfected
HEK293 cells were incubated with myo-[3H]inositol and then stimulated with the indicated Ca21 concentration for 30 min. Total inositol
phosphates were isolated and counted by liquid scintillation. Each
data point represents the mean 6 SEM of nine independent transfections. Curve labels indicate amino acid substitutions.
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 Ca21 . However, methods for the direct assessment of
Ca21 binding have not been established.
A similar concomitant increase in receptor affinity and
maximal signal transduction has been observed for activating mutations of the b2-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 Ca21 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 Ca21 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.
Acknowledgments
We thank Dr. Paul K. Goldsmith for providing the monoclonal antibodies and participating in the ELISA analysis, Dr. Regina Collins for
tissue culture assistance, and Dr. Domenico Accili for helpful suggestions. We also thank NPS Pharmaceuticals for providing the wild-type
construct.
References
1. Brown EM, Gamba G, Riccardi D, et al. 1993 Cloning and characterization of
an extracellular Ca21 -sensing receptor from bovine parathyroid. Nature.
366:575–580.
2. Riccardi D, Park J, Lee WS, et al. 1995 Cloning and functional expression of
a rat kidney extracellular calcium-sensing receptor. Proc Natl Acad Sci USA.
92:131–135.
3. Aida K, Koishi S, Tawata M, Onaya T. 1995 Molecular cloning of a putative
Ca21 -sensing receptor cDNA from human kidney. Biochem Biophys Res
Commun. 214:524 –529.
4. Garrett JE, Tamir H, Kifor O, et al. 1995 Calcitonin-secreting cells of the
DE NOVO ACTIVATING CaR MUTATIONS
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
thyroid gland express an extracellular calcium-sensing receptor gene. Endocrinology. 136:5202–5211.
Ruat M, Molliver ME, Snowman AM, Snyder SH. 1995 Calcium sensing
receptor: molecular cloning in rat and localization to nerve terminals. Proc Natl
Acad Sci USA. 92:3161–3165.
Lundgren S, Hjalm G, Hellman P, et al. 1994 A protein involved in calcium
sensing of the human parathyroid and placental cytotrophoblast cells belongs
to the LDL-receptor protein superfamily. Exp Cell Res. 212:344 –350.
Zaidi M, Shankar VS, Tunwell R, et al. 1995 A ryanodine receptor-like
molecule expressed in the osteoclast plasma membrane functions in extracellular Ca21 sensing. J Clin Invest. 96:1582–1590.
Finegold DN, Armitage MM, Galiani M, et al. 1994 Preliminary localization
of a gene for autosomal dominant hypoparathyroidism to chromosome 3q13.
Pediatr Res. 36:414 – 417.
Pollack MR, Brown EM, Chou YW, et al. 1993 Mutations in the human Ca21
-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell. 75:1297–1303.
Ho C, Conner DA, Pollak MR, et al. 1995 A mouse model of human hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat
Genet. 11:389 –394.
Baron J, Winer KK, Yanovski JA, et al. 1996 Mutations in the Ca21 -sensing
receptor gene cause autosomal dominant and sporadic hypoparathyroidism.
Hum Mol Genet. 5:601– 606.
Pollack MR, Brown EM, Estep HL, et al. 1994 Autosomal dominant hypocalcemia caused by a Ca21 -sensing receptor gene mutation. Nat Genet. 8:303–307.
Lovlie R, Eiken HG, Sorheim JI, Boman H. 1996 The Ca21 -sensing receptor
gene (PCAR1) mutation T151 M in isolated autosomal dominant hypoparathyroidism. Hum Genet. 98:129 –133.
Pearce SHS, Williamson C, Kifor O, et al. 1996 A familial syndrome of
hypocalcemia with hypercalciuria due to mutations in the calcium-sensing
receptor. N Engl J Med. 335:1115–11225.
Perry YM, Finegold DN, Armitage MM, Ferrell RE. 1995 A missense mutation
in the Ca21 -sensing receptor gene causes familial autosomal dominant hypoparathyroidism [Abstract]. Am J Hum Genet. 55(Suppl):A17.
Pearce SHS, Bai M, Quinn SJ, Kifor O, Brown EM, Thakker RV. 1996
Functional characterization of calcium-sensing receptor mutations expressed
in human embryonic kidney cells. J Clin Invest. 98:1860 –1866.
Bai M, Quinn S, Trivedi S, et al. 1996 Expression and Characterization of
inactivating and activating mutations in the Human Ca21 -sensing receptor.
J Biol Chem. 271:19537–19545.
2715
18. Fitzpatrick LA, Arnold A. 1995 Hypoparathyroidism. In: DeGroot LJ, Besser
M, Burger HG, et al. Endocrinology, 3rd ed. Philadelphia: Saunders; 1123–1135.
19. Smith LM, Sanders JZ, Kaiser RJ, et al. 1986 Fluorescence detection in automated DNA sequence analysis. Nature. 321:674 – 679.
20. Berridge MJ, Dawson RM, Downes CP, Heslop JP, Irvine RF. 1983 Changes
in the levels of inositol phosphates after agonist-dependent hydrolysis of
membrane phosphoinositides. Biochem J. 212:473– 482.
21. Garrett JE, Capuano IV, Hammerland LG, et al. 1995 Molecular cloning and
functional expression of human parathyroid calcium receptor cDNAs. J Biol
Chem. 270:12919 –12925.
22. Wilkie PJ, Krizman DB, Weber IL. 1992 Linkage map of human chromosome
9 microsatellite polymorphisms. Genomics. 12:607– 609.
23. Udalova IA, Nedospasov SA, Webb GC, Chaplin DD, Turetskaya RL. 1993
Highly informative typing of the human TNF locus using adjacent polymorphic markers. Genomics. 16:180 –186.
24. Hammond HA, Jin L, Zhong Y, Caskey CT, Chakraborty R. 1994 Evaluation
of 13 short tandem repeat loci for use in personal identification applications.
Am J Hum Genet. 55:175–189.
25. Porter RH, Cox BG, Heaney D, Hostetter TH, Stinebaugh BJ, Suki WN. 1978
Treatment of hypoparathyroid patients with chlorthalidone. N Engl J Med.
298:577–581.
26. Santos F, Smith MJ, Chan JC. 1986 Hypercalciuria associated with long-term
administration of calcitriol (1,25-dihydroxyvitamin D3). Action of hydrochlorothiazide. Am J Dis Child. 140:139 –142.
27. Winer KK, Yanovski JA, Cutler Jr GB. 1996 Synthetic human parathyroid
hormone. 1–34 vs calcitriol and calcium in the treatment of hypoparathyroidism. Results of a short-term randomized crossover trial. JAMA. 276:631– 636.
28. Peacock M, Robertson WG, Nordin BEC. 1969 Relation between serum and
urinary calcium with particular reference to parathyroid activity. Lancet.
7591:384 –386.
29. Samama P, Cotecchia S, Costa T. 26 Lefkowitz RJ. 1993 A mutation-induced
activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem. 268:4625– 4636.
30. Parent J, Le Gouill C, de Brum-Fernandes AJ, Rola-Pleszczynski M,
Stankova J. 1996 Mutations of two adjacent amino acids generate inactive and
constitutively active forms of the human platelet-activating factor receptor.
J Biol Chem. 271:7949 –7955.
31. Lefkowitz RJ, Cotecchia S, Samama P, Costa T. 1993 Constitutive activity of
receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci. 14:303–307.