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Original Studies |
Department of Pediatrics, Chiba University School of Medicine (T.W., K.M., M.M., H.N., T.Y.), Chiba 260-8670, Japan; Endocrine-Hypertension Division, Department of Medicine (M.B., C.R.L., E.M.B.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Saitama Yorii Children’s Hospital (S.M.), Yorii, Saitama 369–1200, Japan
Address all correspondence and requests for reprints to: Toshiyuki Yasuda, M.D., Department of Pediatrics, Chiba University School of Medicine, 1–8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: toshi{at}med.m.chiba-u.ac.jp
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Abstract |
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Introduction |
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Inactivating mutations of the CaR gene cause resistance of the parathyroid and kidney to Ca2+e. Heterozygous inactivating mutations cause a form of familial hypercalcemia with inappropriately low urinary calcium excretion, familial hypocalciuric hypercalcemia (FHH), whereas neonatal severe hyperparathyroidism can be caused by inactivating CaR mutations in either the homozygous or the heterozygous state (5, 6, 7). Heterozygous CaR mutations causing neonatal severe hyperparathyroidism can, in some cases, exert a dominant negative effect on the normal CaR allele, thereby producing more severe hypercalcemia and hyperparathyroidism than are generally present in the heterozygous state (e.g. in FHH) (8).
Pollak et al. first reported that an activating mutation in the extracellular domain of the CaR can be a cause of familial hypocalcemia, with levels of PTH insufficient to maintain normocalcemia, thereby resembling hypoparathyroidism in its pathophysiology (9). Receptor activation lowers the set-point for maintenance of serum Ca levels, in part by suppressing PTH secretion at normal or even frankly low levels of Ca. For this reason we refer to this condition in the present report as familial hypoparathyroidism. Subsequent studies have shown higher levels of urinary Ca excretion in this disorder than in patients with hypoparathyroidism alone (10, 11), probably because activated CaRs in the kidney promote a greater degree of calciuria than would otherwise be present at any given level of serum Ca. Recent studies have identified 11 missense mutations, mainly localized in the extracellular domain of the CaR, that cause hypoparathyroidism (9, 10, 11, 12, 13, 14, 15, 16), of which 7 have been functionally characterized by expression in heterologous mammalian cell systems (10, 12, 13, 16, 17). Although there is considerable heterogeneity in the severity of the hypocalcemia caused by activating CaR mutations, most of them are associated with mild hypocalcemia.
We report here a Japanese family with severe, autosomal dominant hypocalcemia due to the presence of a novel gain of function mutation in the fifth transmembrane domain of the CaR gene. This is the third reported case of an activating mutation of the transmembrane domains of the CaR causing hypoparathyroidism and the first in which such an activating mutation in a transmembrane domain caused familial hypoparathyroidism.
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Subjects and Methods |
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and Table 1)Propositus (patient II-4). A female infant, aged 6 days, developed seizures. She was born at 38 weeks gestation without asphyxia. She had none of the clinical features of the loss of 22q11.2 syndrome (cardiac anomaly, peculiar face, thymic hypoplasia, or hypocalcemia) except for hypocalcemia (1). Her initial laboratory data revealed a low level of serum Ca and a high level of serum phosphorus (P): 4.9 (normal range, 8.5–10.5) and 11.5 mg/dL (normal range, 4–7), respectively. She showed a sharp increase in plasma cAMP in response to infusion of human PTH-(1–34) (before, 12 nmol/L; after, 140 nmol/L). She had relative hypercalciuria (urinary Ca/creatinine ratio of 0.4–0.8 when serum Ca levels were 7–8 mg/dL) during infancy. A computed tomographic scan showed severe calcifications of the basal ganglia at 12 yr of age.
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Patient I-2. The mother of the proband had a history of
tetany. Biochemical findings confirmed the presence of
hypoparathyroidism (Fig. 1
and Table 1). She showed a sharp increase in
urinary excretion of phosphate and cAMP in response to infusion of
human PTH-(1–34) (data not shown).
The other family members shown in Fig. 1 had normal serum Ca and P
levels. All three of the affected patients were initially treated with
1
-hydroxyvitamin D3, initially alone and subsequently,
since 1989, in combination with thiazide diuretics. The biochemical
data for these three subjects while they were being treated are shown
in Table 1. They have sometimes complained of polyuria, polydipsia, and
weakness when their serum Ca levels were raised to levels approaching
the lower limit of normal, and their serum Ca concentrations were,
therefore, usually maintained at 7–8 mg/dL. These clinical and
biochemical features led us to suspect an abnormality in the regulation
of the PTH gene or in the Ca2+e-sensing receptor gene.
Informed consent was obtained from all subjects for the studies described below.
DNA amplification and sequence analysis
Genomic DNA was prepared from white blood cells with an Easy-DNA extraction kit (Invitrogen, San Diego, CA). Exons 2–7 of the CaR gene, comprising the entire coding sequence, were amplified by PCR (6, 7). Both strands of the PCR products of the propositus were sequenced using an Applied Biosystems mode 373A automated sequencer with the Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). A heterozygous nucleotide substitution, T to G, at nucleotide 2363 that changed the phenylalanine normally present at codon 788 to a cysteine (e.g. F788C using the single letter amino acid code) was identified in the propositus (see Results).
PCR-restriction fragment length polymorphism (RFLP) and PCR-single strand conformation polymorphism (SSCP) analysis
Primers MBF and MBR in exon 7 of the CaR were prepared to screen for the presence of the missense mutation (F788C or T2363G) identified in the propositus in other family members by the use of PCR-RFLP and to carry out a similar analysis of DNA from normal controls: MBF, 5'-ATCACGTGCCACGAGGGCTC-3' (nucleotides 2287–2306); and MBR, 5'-GATGAGCATGCTGAAGGTGATG-3' (nucleotides 2439–2418) (18). PCR-amplified genomic DNAs were digested with MboII and then subjected to electrophoresis through a 3% agarose gel to determine whether the DNA contained a MboII restriction site destroyed by the point mutation present in the propositus (see Results). The linkage in the family was also examined by PCR-SSCP analysis. Two microliters of PCR products amplified with [32P]deoxy-CTP were denatured by heating with formamide and electrophoresed on a 6% polyacrylamide gel (acrylamide-N,N'-methylenebisacrylamide, 49:1) containing 5% glycerol, in 0.5 x TBE (Tris-borate/EDTA) buffer with 950 V, for 4 h. After electrophoresis, the gels were dried and autoradiographed.
Site-directed mutagenesis
Site-directed mutagenesis to produce a CaR with the identified
point mutation, F788C, was performed as described previously (17) using
the approach described by Kunkel (19). The dut-1 ung-1 strain of
Escherichia coli, CJ236, was transformed with mutagenesis
cassette 6, as described in Bai et al. (17).
Uracil-containing, single stranded DNA was produced by infecting the
cells with the helper phage, VCSM13. The DNA was then annealed to a
mutagenesis primer containing the desired nucleotide change,
e.g. substitution of a G for the T normally present at
position 2363 flanked on both sides by wild-type sequence. The primer
was then extended around the entire single stranded DNA and ligated to
generate closed circular heteroduplex DNA. DH5-competent cells were
transformed with the heteroduplex DNA, and incorporation of the desired
mutation was confirmed by sequencing the entire cassette.
Transient expression of the wild-type and mutated CaRs harboring the F788C mutation in HEK293 cells
The DNA for transfection was prepared using the Midi Plasmid kit (Qiagen, Chatsworth, CA). Lipofectamine (Life Technologies, Gaithersburg, MD) was employed as the DNA carrier for transfection (17). HEK293 cells (provided by NPS Pharmaceuticals, Salt Lake City, UT) were cultured in DMEM (Life Technologies). Transient transfection was performed as described previously (17) by adding a DNA-Lipofectamine mixture diluted with OPTI-MEM 1 Reduced Serum Medium (Life Technologies) to 90% confluent HEK293 cells plated in 13.5 x 20.1 mm glass coverslips [for measurement of the cytosolic Ca2+ concentration (Ca2+i)] or in six-well plates (for obtaining membrane protein for Western analysis) using 0.625 µg DNA. After 5-h incubation at 37, an amount of OPTI-MEM 1 Reduced Serum Medium with 20% FBS equal to that present in the wells was added to the transfected cells, which was then replaced with fresh DMEM and 10% FBS at 24 h after the start of transfection. The expressed CaR protein was assayed at 48 h after transfection. To perform coexpression of wild-type and mutant receptors, 0.625 µg of each complementary DNA (cDNA) were mixed and used to transfect HEK293 cells as described above.
Preparation of cellular lysates and performance of Western analysis
HEK293 cells transiently transfected with the wild-type or mutant CaRs were rinsed twice with phosphate-buffered saline and solubilized with 1 mL cold solubilization buffer [1% Triton X-100, 0.5% Nonidet P-40, 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.4), 2 mmol/L ethylenediamine tetraacetate, 1 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and protease inhibitors (83 µg/mL aprotinin, 30 µg/mL leupeptin, 1 mg/mL Pefabloc (Boehringer-Mannheim, Indianapolis, IN), 50 µg/mL calpain inhibitor, 50 µg/mL bestatin, and 5 µg/mL pepstatin)] at room temperature (17). Insoluble material was removed by sedimentation at 5000 rpm for 15 min at 4 C. The resultant supernatants containing the crude cellular lysates were collected and employed for Western analysis. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL). An appropriate amount of protein (15 µg) from each of the cell lysates was subjected to SDS-PAGE (17) using a linear gradient of polyacrylamide (4–12%). The proteins on the gel were subsequently electrotransferred to a nitrocellulose membrane. After blocking with 5% milk, the blot was incubated with primary anti-CaR antiserum (rabbit polyclonal antiserum 4641, directed against amino acids 215–237 of the bovine CaR; provided by Drs. Forrest Fuller and Rachel Simin at NPS), which is specific for the CaR, as detailed previously (17). After washing, the blots were incubated with the secondary goat antirabbit antiserum conjugated to horseradish peroxidase (Sigma Chemical Co., St. Louis, MO; diluted 1:500). The CaR protein was detected with an enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
Measurement of Ca2+e-evoked changes in Ca2+i
Coverslips with attached HEK293 cells transiently transfected with the wild-type CaR, the mutant CaR, or both were loaded for 2 h at room temperature with fura-2/AM in 20 mmol/L HEPES, pH 7.4, containing 125 mmol/L NaCl, 4 mmol/L KCl, 1.25 mmol/L CaCl2, 1 mmol/L MgSO4, 1 mmol/L NaH2PO4, 0.1 (wt/vol) dextrose, and 0.1% (wt/vol) BSA (16, 17). After washing without the fura-2/AM (16, 17), the coverslips were placed diagonally in a thermostatted quartz cuvette containing the bath solution, using a modification of the technique employed previously in this laboratory (20). Extracellular calcium was increased in a stepwise fashion to give the desired concentrations, using 1-mmol/L increments up to 5.5 mmol/L Ca2+e followed by 5-mmol/L increments (16, 17) as shown in Results. Excitation monochrometers were centered at 340 and 380 nm, with emission light collected at 510 ± 40 nm through a wide band emission filter. The emission of light after excitation at both 340 and 380 nm was used to calculate Ca2+i, as described previously (17).
Statistical analysis
The mean EC50 values (the effective concentrations of Ca2+e eliciting half of the maximal Ca2+i responses) for the wild-type CaR, mutant CaR, or both in response to increasing concentrations of Ca2+e were calculated from the EC50 values for all of the individual experiments and are expressed with the SEM as the index of dispersion. Comparisons of the EC50 values was performed using ANOVA or Duncan’s multiple comparison test (16, 17).
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Results |
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A heterozygous T to G base pair substitution was identified in the
genomic DNA of the propositus at position 2363 in exon 7 (Fig. 2A). This mutation produces an amino acid
change from phenylalanine to cysteine at position 788 (F788C) within
the predicted fifth transmembrane domain of the CaR. No other mutations
were identified.
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The missense mutation identified in the propositus destroys a
recognition site for the restriction enzyme MboII. The
cleavage of the PCR products (153 bp) by MboII normally
generates two fragments of 69 and 84 bp. Therefore, genomic DNA from
this family as well as from 50 normal control subjects were screened
for the mutation by PCR-RFLP using MboII after PCR
amplification with primers MBF and MBR. The missense mutation was found
in the DNA of the affected individuals in the family and was absent in
the DNA from unaffected family members (Fig. 2B) as well as in that
from 50 normal controls. The mutation was also demonstrated by PCR-SSCP
(see Fig. 2C).
Western analysis of the transiently expressed CaR containing the F788C mutation
Figure 3 shows the results of
Western analysis performed on crude cellular lysates prepared from
HEK293 cells transiently transfected with the cDNAs encoding the
wild-type CaR, the mutant CaR harboring the F788C mutation, or the
vector used for the transfections without any cDNA insert. The bands
that were not present in the vector-transfected HEK293 cells were
specific for the CaR (Fig. 3). The lower molecular mass species between
140–200 kDa were monomeric, N-glycosylated forms of the
receptor, containing high mannose and complex carbohydrates,
respectively (17). The high molecular mass species above 200 kDa were
presumably dimeric and higher oligomeric forms of the CaR. The
expression patterns and levels of the wild-type and mutant receptors
were similar.
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Although the wild-type receptor, when transiently expressed in
HEK293 cells, showed an EC50 for Ca2+e-elicited
increases in Ca2+i of 4.1 ± 0.1 mmol/L (n = 4),
the mutant receptor exhibited a left-shifted dose-response curve, with
an EC50 of 2.7 ± 0.1 mmol/L (n = 4; Fig. 4). Coexpression of the wild-type and
mutant receptors, to mimic the heterozygous state present in affected
family members in vivo, revealed an EC50 of
3.0 ± 0.1 mmol/L, close to that observed in cells transfected
with the mutant receptor alone (Fig. 4).
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Discussion |
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We have identified a novel heterozygous missense mutation (F788C) in the fifth transmembrane helix of the CaR. There was complete concordance of the presence of the mutation with disease status in the family, and the mutation was absent in the genomic DNA from 50 controls as assessed by the PCR-RFLP (and PCR-SSCP) analysis, strongly supporting the causal role of the mutation in this family’s hypoparathyroidism. Moreover, the receptor, when expressed in HEK293 cells, showed a substantial leftward shift in its activation by the major physiological agonist of the CaR in vivo, Ca2+e, with a reduction in EC50 from 4.1 mmol/L Ca2+e for the wild-type receptor to 2.7 mmol/L for the mutant receptor. Expressions of the wild-type and mutant receptors were similar as assessed by Western blot analysis. Although the latter does not directly document cell surface expression of the biologically active form(s) of the receptors, we have shown (17) that the upper band of the doublet present at 140–160 kDa on Western analysis corresponds to the position of the mature, cell surface form of the CaR as assessed by direct cell surface labeling of the receptor, and the levels of this band were comparable for the wild-type and mutant receptors. Coexpression of the wild-type and mutant CaRs, to mimic the heterozygous state present in vivo, gave an EC50 of 3 mmol/L. The observed abnormality in the mutant CaR’s function, therefore, does not result from increased activity of the unliganded receptor, but, rather, from either enhanced activity of the liganded receptor (e.g. more efficient signal transduction for any given degree of receptor occupancy by ligand) or greater affinity of the receptor for ligand. We did not directly compare the maximal responses of the mutant and wild-type CaRs, because the greater sensitivity of the former to the ambient level of Ca2+e present before the measurement of Ca2+e-evoked increases in Ca2+i could have altered intracellular Ca2+ stores in ways that would artifactually perturb apparent maximal responses. Recent studies have compared the function of the wild-type CaR with those of mutant receptors containing activating mutations by measuring high Ca2+e-elicited accumulation of inositol phosphates (IPs) in HEK293 cells preincubated with [3H]inositol (12, 13). In several cases, the mutant CaRs showed not only lower EC50 values but also greater maximal levels of [3H]inositol-labeled IPs at high Ca2+e than those observed with the wild-type receptor. Although these results may indicate that the mutant CaRs cause a greater degree of activation of phospholipase C than the wild-type receptor, it is also possible that the activated CaRs produce more complete labeling of the phosphoinositide precursor pool than the wild-type receptor during the preincubation with [3H]inositol. Direct measurement of high Ca2+e-evoked increases in the mass of inositol 1,4,5-trisphosphate (9) in HEK293 or other cells transiently transfected with wild-type or mutant CaRs might resolve this uncertainty regarding the maximal biological responses produced by CaRs harboring activating mutations.
Most gain of function mutations in the CaR localize in its
extracellular regions: A116T (11), N118K (10, 12), E127A (9), F128L
(10, 16), T151M (10, 14, 16), E191K (10, 16), Q245R (15), F612S (10, 13), and E681H (11) (Table 2). Functional
analysis has revealed that all of these mutations induce leftward
shifts in EC50. The phenotype produced by activating
mutations of the CaR was thought to include relatively mild
hypocalcemia. Mutation L773R, which also resides within transmembrane
helix 5, has been reported very recently as a cause of sporadic
hypoparathyroidism (12). L773R caused a significant leftward shift in
the concentration-response curve as well as an augmentation of maximal
IP production as assessed in HEK293 cells prelabeled with
[3H]inositol (12). The Ca level in this case was 6.4
mg/dL at age 18 yr, with a history of convulsions at age 2 yr. The
F788C mutation in the CaR’s fifth transmembrane helix, which we report
here, produced the most severe familial hypocalcemia reported to date
with activating mutations of this receptor, with levels of serum Ca
ranging from 4.9 to 5.9 mg/dL, high urinary Ca excretion, and prominent
calcifications of the basal ganglia. Therefore, it is possible that
activating mutations present in the CaR’s transmembrane domains may
produce more severe hypocalcemia than those present in the receptor’s
extracellular domain. Moreover, additional studies of the effects of
mutations in various regions of the receptor in the experiments of
nature afforded by both activating and inactivating CaR mutations may
provide valuable information on its structure-function
relationships.
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In conclusion, severe familial hypoparathyroidism can be caused by a gain of function mutation in the fifth transmembrane domain of the CaR and further emphasizes the role of such mutations as a cause of familial hypoparathyroidism of widely varying severity.
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Footnotes |
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Received January 29, 1998.
Revised March 18, 1998.
Accepted March 25, 1998.
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