This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nagase, T. Articles by Yamamoto, M. Search for Related Content PubMed PubMed Citation Articles by Nagase, T. Articles by Yamamoto, M.

A Family of Autosomal Dominant Hypocalcemia with a Positive Correlation between Serum Calcium and Magnesium: Identification of a Novel Gain of Function Mutation (Ser⁸²⁰Phe) in the Calcium-Sensing Receptor

Growth Factor Division, National Cancer Center Research Institute (T.N., T.T., K.Y.), Tokyo 104-0045, Japan; Third Department of Internal Medicine (T.M., R.K., Y.T., N.N.) and Department of General Medicine (T.A.), National Defense Medical College, Saitama 359-8513, University of Tokyo Branch Hospital (N.C., S.F.), Tokyo 112-8868, Japan; Department of Internal Medicine, Self-Defense Forces Central Hospital (T.N., H.T., N.T., H.Y.), Tokyo 154-8532, Japan; and Department of Health and Nutrition, School of Health Sciences, Niigata University of Health and Welfare, Niigata 950-3198 (M.Y.), Japan

Address all correspondence and requests for reprints to: Michiko Yamamoto, M.D., Department of Health Sciences, Niigata University of Health and Welfare, 1398 Shimami-cho, Niigata 950-3198, Japan. E-mail: . yamamoto{at}nuhw.ac.jp

Abstract

To date about 20 activating mutations in the calcium-sensing receptor (CaR) gene have been identified to cause autosomal dominant hypocalcemia (ADH) or sporadic hypoparathyroidism. We report a novel activating mutation in the CaR gene in a Japanese family with ADH. The proband, a 15-yr-old boy, and 5 other patients in 3 generations were asymptomatic, except for the proband’s grandmother who had a history of seizures. They showed mild hypocalcemia (1.68–1.98 mmol/liter) with normal urinary calcium excretion and low normal serum PTH levels. Their serum magnesium concentrations were below normal in 3 adults and within the normal range in 3 teenagers. There was a significant positive correlation (r = 0.90; P < 0.05) between the serum calcium and magnesium concentrations of 6 affected members. Nucleotide sequencing revealed that the proband had a known polymorphism (Gly⁹⁹⁰Arg) and a novel heterozygous mutation substituting phenylalanine for serine at codon 820 (Ser⁸²⁰Phe) in the sixth transmembrane helix of the CaR. In other family members, the Ser⁸²⁰Phe mutation cosegregated with hypocalcemia. The mutation was not detected in 50 control subjects. The Gly⁹⁹⁰Arg polymorphism was observed in 8 of 9 family members with or without hypocalcemia and in 36 of 50 controls. The sensitivity of the Ser⁸²⁰Phe mutant CaR to calcium was assessed using transiently transfected HEK293 cells and measuring the increases in intracellular Ca²⁺ concentrations in response to the changes in extracellular Ca²⁺. The concentration-response curve of the mutant receptor was left-shifted, and its EC₅₀ (2.5 mM) was significantly (P < 0.05) lower than that of the wild-type CaR (3.3 mM). We conclude that the Ser⁸²⁰Phe mutation in the CaR caused ADH in this family. The positive correlation between serum calcium and magnesium levels observed in this family may support the concept that renal CaR acts as a magnesium sensor as well as a calcium sensor.

THE CALCIUM-SENSING receptor (CaR) was first identified in bovine parathyroid cells (1) and was later found to be expressed in the kidney and other tissues (2, 3, 4). The human CaR gene encodes a polypeptide of 1078 amino acids consisting of three parts; a very large extracellular domain at the amino-terminus, a transmembrane domain with seven membrane-spanning helixes characteristic of G protein-coupled receptors, and an intracellular carboxyl-terminal tail (1, 5). The activated receptor stimulates PLC activity, leading to accumulation of IP3, which increases cytoplasmic Ca²⁺ concentrations (1). In the parathyroid cells, binding of the calcium ion to the CaR decreases PTH secretion, and thus the CaR determines the set-point of PTH secretion by acting as a calcium sensor (3, 4). In the kidney, CaR activation inhibits the reabsorption of calcium (4). Heterozygous loss of function (inactivating) mutations in the CaR gene attenuate the sensitivity of parathyroid cells and renal tubular cells to the elevation in extracellular calcium, leading to mild to moderate hypercalcemia and relative hypocalciuria, a disorder called familial hypocalciuric hypercalcemia (FHH) (6). Conversely, heterozygous gain of function (activating) mutations in the CaR gene are associated with the reverse phenotype, autosomal dominant hypocalcemia (ADH) and sporadic hypoparathyroidism (7, 8). Earlier studies have shown that patients with activating CaR mutations generally have milder hypocalcemia with less symptoms and higher urinary calcium excretion compared with patients with idiopathic hypoparathyroidism (8, 9, 10).

To date about 40 inactivating and 20 activating mutations in the CaR gene have been identified (11). These are mostly missense mutations and are not evenly distributed throughout the coding region of the CaR gene. The mutations cluster in two regions, within the first 300 amino acids of extracellular domain and from amino acids 520–881 located in or near the transmembrane domain (11). Based on these observations along with experimental findings, it is assumed that the first 300 amino acids of extracellular domain are essential for calcium binding to the CaR and/or dimerization of the receptor (12, 13). The transmembrane domain and the nearby extracellular and intracellular regions are believed to be involved in the signal transduction (14). However, details of the functional domains are not completely understood. Case reports with CaR mutations are limited in number, and some of them did not provide much clinical information about the patients or functional data for the mutated receptors. Thus, to better understand the function of a particular structure in the CaR it seems necessary to accumulate clinical data for more patients with CaR mutations and conduct functional analyses of the naturally occurring mutant CaRs.

Here we report a Japanese family with ADH in whom a novel activating CaR mutation, Ser⁸²⁰Phe, was identified, and the function of the mutated receptor was analyzed by transfection into human embryonic kidney cells (HEK293). In addition, as a polymorphism, Gly⁹⁹⁰Arg, was found in this family, we examined in 50 control subjects the frequencies of the Ser⁸²⁰Phe mutation and 5 known polymorphisms (Ala⁸²⁶Thr, Cys⁸⁵¹Ser, Ala⁹⁸⁶Ser, Gly⁹⁹⁰Arg, and Gln¹⁰¹¹Glu) in the CaR gene (8, 15, 16).

Subjects and Methods

Subjects

Studies were conducted on nine (six hypocalcemic and three normocalcemic) members of a Japanese family with familial isolated hypoparathyroidism of autosomal dominant inheritance (Fig. 1). Their clinical and biochemical findings are described in Results. Fifty unrelated Japanese men served as controls to determine the allele frequencies of a mutation identified in the ADH family and of other polymorphisms in the CaR gene. Informed consent was obtained from all subjects.

View larger version (12K): [in this window] [in a new window]   Figure 1. Pedigree of the family. The arrow indicates the proband. Filled symbols, Individuals with hypocalcemia; stippled symbols, status unknown; slash, deceased.

Genomic DNA was extracted from white blood cells with a QIAamp blood kit (QIAGEN, Hilden, Germany). In the proband, all protein-coding exons (exons 2–7) of the CaR gene were amplified by PCR with standard procedures using primer pairs as described previously (6, 17) except for exon 7, for which the following four primer pairs were used: 7A-1 (5'-AGTCTGTGCCACACAATAACTCACTC-3') and 7A-2 (5'-ATCTTGGCCTCAAACACCAGGAGGA-3'), 7B-1 (5'-CTTCGTGCTCTGCATCTCATGCATC-3') and 7B-2 (5'-ATAGGTGCTGGCATAGGCTGGAATG-3'), 7C-1 (5'-GTTCATCACCTTCAGCATGCTCATC-3'), and 7C-2 (5'-GCTGCCAAAGATGACCTTCTGCTTG-3'), and 7D-1 (5'-TCCCACAGCAGCAACGATCTCAGCA-3') and 7D-2 (5'-CCAAGAAACCTCTCTGCATTCTCCC-3'). PCR products were purified with a QIAquick PCR purification kit (QIAGEN). Nucleotide sequences of both strands of the PCR products were determined by direct sequencing with a DNA sequencing kit and an automated DNA sequencer (Dye Terminator Cycle Sequencing Ready Reaction and ABI PRISM 310, Perkin-Elmer Corp., Foster City, CA).

After finding a novel mutation at nucleotide 2459 and a known polymorphism at nucleotide 2968 causing amino acid substitutions of Ser⁸²⁰Phe and Gly⁹⁹⁰Arg, respectively, in the proband (see Results), the CaR genes of other family members and control subjects were amplified by PCR using a primer pair 7C-1 (nucleotides 2415–2439) and 7D-2 (nucleotides 3285–3261). Thus, the two variant sites as well as four other polymorphisms previously reported (Ala⁸²⁶Thr, Cys⁸⁵¹Ser, Ala⁹⁸⁶Ser, and Gln¹⁰¹¹Glu, corresponding to nucleotides 2476, 2551, 2956, and 3031, respectively) were encompassed. The PCR products were sequenced as stated above for both strands in the family members and for the nontranscribed strand in the controls.

Construction of expression vector for mutant CaR

A plasmid expressing the wild-type CaR was constructed by subcloning cDNA with the full coding region of the CaR into pcDNA3 expression vector (Invitrogen, Carlsbad, CA) as reported previously (18). A plasmid expressing Ser⁸²⁰Phe mutant CaR was constructed by replacing the 889-bp Eam¹¹⁰⁵I-SurfI segment of the wild-type CaR with the corresponding fragment of the PCR product obtained from a proband’s cousin (III-2 in Fig. 1) using a primer pair 7A-1 (in intron 6) and 7C-2 (nucleotides 2886–2910). The Gly⁹⁹⁰Arg polymorphism was not induced into the mutant CaR. The mutation was confirmed by direct sequencing.

In vitro functional study

HEK293 cells were cultured in DMEM (Life Technologies, Inc., Grand Island, NY) with 10% FCS and were transiently transfected using Lipofectamine reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instruction. The function of the CaR was assessed after 48 h by measuring the EC₅₀ for extracellular Ca²⁺ concentration (Ca²⁺_o) as described previously (18). Briefly, HEK293 cells that had been transfected with the wild-type or mutant CaR cDNA were incubated for 45 min with fura-2/AM (Molecular Probes, Inc., Eugene, OR) in HBBS (118 mM NaCl, 4.6 mM KCl, 10 mM D-glucose, and 20 mM HEPES, pH 7.2). Then the fura-2/AM-loaded cells were washed and pelleted. Aliquots of cells were resuspended in HBBS in a UV grade cuvette on a fluorescence spectrophotometer (F-2000, Hitachi, Tokyo, Japan). The changes in intracellular Ca²⁺ concentrations (Ca²⁺_i) in response to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 10, 20, and 40 mM of Ca²⁺_o were recorded for both receptor types. The magnitude of response at each concentration of Ca²⁺_o was measured as the maximal increment from baseline. In most experiments the peak responses were observed about 70 sec after stimulation. The result was expressed as a ratio to the response elicited at 40 mM Ca²⁺_o in the cells transfected with the same type of receptor. The EC₅₀ value was calculated by plotting the Ca²⁺_i response against the Ca²⁺_o using KaleidaGraph software (Synergy Software, Reading, PA). The mean EC₅₀ values determined from three independent experiments were compared between wild-type and mutant CaRs by t test. P < 0.05 was considered significant.

Western blot analysis

HEK293 cells transiently transfected with the wild-type or mutant CaR cDNA were rinsed twice with PBS and solubilized with 500 µl cold solubilization buffer [150 mM NaCl, 1% Triton X-100, 50 mM Tris (pH 7.4), 1 mM phenylmethylsulfonylfluoride, and 100 µg/ml aprotinin]. The insoluble fraction was removed after centrifugation at 5000 rpm for 15 min at 4 C. The resultant supernatants containing crude cellular extracts were collected. Eighty micrograms of cellular extracts were electrophoresed on a 7.5% polyacrylamide gel and electrotransferred to a polyvinylidene difluoride membrane (ATTO Co., Tokyo, Japan). After blocking with Blockace (Dainihon Seiyaku, Osaka, Japan), the membrane was incubated with a mouse monoclonal antibody against amino acid residues 214–235 of the human CaR, provided by NPS Pharmaceuticals, Inc. (Salt Lake City, UT) (19). After washing, the membrane was incubated with horseradish peroxidase-labeled antimouse IgG antibody (Bio-Rad Laboratories, Inc. Hercules, CA) and visualized by the ECL system (Amersham Pharmacia Biotech, Tokyo, Japan).

Results

Clinical and biochemical characteristics of the family

The proband (III-1 in Fig. 1) was found to have calcifications of the basal ganglia at age 15 yr when he underwent a computed tomographic examination of the brain because of headache. Laboratory data showed mild hypocalcemia, hyperphosphatemia, and an intact PTH level in the normal range (Table 1). The level of 1,25-dihydroxyvitamin D [1,25-(OH)₂D] was low (26 pmol/liter; normal reference range, 48–182), 24,25-dihydroxyvitamin D was slightly elevated (13.9 nmol/liter; normal reference range, 3.1–9.1), and 25-hydroxyvitamin D was normal (62 nmol/liter; normal reference range, 25–137). A family survey revealed six hypocalcemic subjects in total, compatible with autosomal dominant inheritance (Fig. 1). The affected members showed no symptoms of hypocalcemia, except for the proband’s maternal grandmother (I-2) who had episodes of convulsion. The proband’s mother (II-1) was asymptomatic, but had calcifications of the basal ganglia.

Sequence analysis of the CaR gene in the family members

Direct sequencing of genomic DNA from the proband revealed three heterozygous base pair substitutions, C to G at nucleotide 78 (C78G), C to T at nucleotide 2459 (C2459T), and G to A at nucleotide 2968 (G2968A; Fig. 2). The C78G substitution does not change the coded amino acid. The C2459T mutation produces an amino acid change from serine (TCC) to phenylalanine (TTC) at codon 820 (Ser⁸²⁰Phe) in the sixth transmembrane helix of the CaR. The G2968A transition substitutes arginine (AGG) for glycine (GGG) at codon 990 (Gly⁹⁹⁰Arg) in the carboxyl-terminal tail. In the other family members, the heterozygous Ser⁸²⁰Phe mutation was identified in all individuals with hypocalcemia, but in none with normocalcemia (Table 1). The Gly⁹⁹⁰Arg, which is known to be a common polymorphism in Asians as well as Caucasians (20), was present in all individuals with hypocalcemia and in 2 of 3 with normocalcemia. The proband’s mother (II-1) had heterozygous Ser⁸²⁰Phe and homozygous Gly⁹⁹⁰Arg, indicating that in the other affected members the Gly⁹⁹⁰Arg polymorphism was on the same allele as the Ser⁸²⁰Phe mutation.

View larger version (30K): [in this window] [in a new window]   Figure 2. Direct sequencing of genomic DNA from the proband for exon 7 of the CaR. Upper panels, Sequences of nontranscribed strands; lower panels, sequences of transcribed strands. A novel heterozygous mutation was identified at codon 820 (Ser820Phe, left panels), and a known polymorphism was identified at codon 990 (Gly990Arg, right panels).

The Ser⁸²⁰Phe mutation was detected in none of 50 normocalcemic controls. As for codon 990, 14 were homozygous for Gly⁹⁹⁰ (wild-type), 28 were heterozygous for the Gly⁹⁹⁰Arg polymorphism, and 8 were homozygous for Arg⁹⁹⁰ (variant). The distribution pattern was in agreement with Hardy-Weinberg equilibrium, and the allele frequency of Arg⁹⁹⁰ was 0.44. Concerning the other polymorphisms, only 1 subject was heterozygous for the Gln¹⁰¹¹Glu polymorphism, and none had Ala⁸²⁶Thr, Cys⁸⁵¹Ser, or Ala⁹⁸⁶Ser polymorphisms.

Functional study of the wild-type and Ser⁸²⁰Phe mutant CaRs

The response curve of Ca²⁺_i evoked by Ca²⁺_o in Ser⁸²⁰Phe mutant was left-shifted compared with that in the wild-type CaR (Fig. 3). The EC₅₀ calculated from three independent experiments for the mutant CaR (2.5 ± 0.2 mM) was significantly lower than that for the wild-type CaR (3.3 ± 0.1 mM). Although comparison was made by normalizing the data to the response at 40 mM Ca²⁺_o in the cells with the same type of receptor, there was no significant difference between the wild-type and mutant receptors in the maximal Ca²⁺_i evoked by Ca²⁺_o at 40 mM (data not shown). These results indicate that the Ser⁸²⁰Phe is a ligand-dependent activating mutation of the CaR.

View larger version (19K): [in this window] [in a new window]   Figure 3. Ca2+i response to Ca2+o in transfected HEK293 cells. Cells were transiently transfected with the wild-type or Ser820Phe mutant CaR cDNA and were loaded with fura-2. The Ca2+i response was expressed as a ratio (percentage) to the maximal response (Ca2+o = 40 mM). Each point is the mean value of three experiments, with the SEM shown by a vertical bar.

Immunoblotting of the transiently expressed wild-type and mutant receptors is shown in Fig. 4. The two bands between 140–160 kDa were glycosylated monomeric receptors, and the higher molecular mass species above 200 kDa were presumably oligomeric forms of the receptor (21, 22). The expression pattern and level of Ser⁸²⁰Phe mutant CaR were similar to those of the wild-type receptor, demonstrating that the Ser⁸²⁰Phe mutation did not alter the synthesis and glycosylation of the receptor. No band was detected in cellular extracts from HEK293 cells transfected with empty vector alone (data not shown).

View larger version (53K): [in this window] [in a new window]   Figure 4. Western blot analysis of the wild-type and Ser820Phe mutant CaRs. Crude cellular extracts (80 µg protein) were prepared from HEK293 cells transfected with the wild-type or mutant CaR cDNA and were subjected to SDS-PAGE on a 7.5% gel. A monoclonal antibody was used to detect the CaR bands. Lane 1, Wild-type CaR; lane 2, Ser820Phe mutant CaR.

The affected members of this family showed mild to moderate hypocalcemia with few symptoms, normal urinary calcium (relative hypercalciuria), low normal levels of serum PTH, and tendencies to hyperphosphatemia and hypomagnesemia. As these findings and the autosomal dominant pattern of inheritance are suggestive of ADH, we conducted molecular genetic studies to establish the diagnosis. The following results indicated that the Ser⁸²⁰Phe mutation caused ADH in this family. First, the heterozygous Ser⁸²⁰Phe mutation was found in all six hypocalcemic members, but none of the normocalcemic members of the family or in 50 control subjects. Second, the mutant CaR expressed in HEK293 cells demonstrated that the mutation significantly increased the sensitivity to Ca²⁺_o. The EC₅₀ value of 2.5 mM for the Ser⁸²⁰Phe mutation was comparable to those (2.2–2.8 mM) for other activating mutations determined using similar methods (18, 21, 23), although the value for wild-type CaR in our study was slightly lower than those in others.

To date, 22 activating mutations in the CaR have been reported. Except for 1 large deletion in the carboxyl-terminal tail, all are missense mutations (11): 13 are localized in the extracellular domain (7, 8, 9, 11, 18, 24, 25, 26), 6 in the transmembrane helixes (8, 11, 21, 25), and 2 in the extracellular loops within the transmembrane domain (8, 11). Of 6 mutations in the transmembrane helixes, 1 is located in the first (TM1), 2 each in the fifth (TM5) and sixth (TM6), and 1 in the seventh (TM7) helix. The Ser⁸²⁰Phe mutation was the third mutation identified in TM6 and the first for which a functional study confirmed gain of function of the mutated CaR. Concerning the structure-function relationships, it is not clarified whether the sites of activating mutations in the CaR have any relevance to clinical findings of ADH. However, it was suggested that mutations localized in the transmembrane helixes might produce more severe hypocalcemia than those present in the extracellular domain (21). The serum calcium levels were 1.60 mmol/liter in a patient with Leu⁷⁷³Arg in TM5 (25), 1.23–1.48 mmol/liter in a family with Phe⁷⁸⁸Cys in TM5 (21), and 1.20 mmol/liter in a patient with Phe⁸⁰⁶Ser in TM6 (8). In contrast, patients with mutations in the extracellular domain showed higher serum calcium levels ranging from 1.48–2.25 mmol/liter (7, 8, 9, 18, 24, 25, 26). Our findings did not support the theory mentioned above. The serum calcium levels of 1.68–1.98 mmol/liter documented in the members with the Ser⁸²⁰Phe mutation in TM6 were similar to the values in patients with mutations in the extracellular domain.

All affected members with ADH described here had the Gly⁹⁹⁰Arg polymorphism along with the Ser⁸²⁰Phe mutation. However, it is unlikely that the Gly⁹⁹⁰Arg polymorphism had some effect on the function of the Ser⁸²⁰Phe mutation. Although it was reported that the Ala⁹⁸⁶Ser polymorphism, which was frequently observed in Caucasians, had a modest effect on the serum calcium levels (27), Kanazawa et al. (20) recently reported that the Gly⁹⁹⁰Arg polymorphism had no significant effect on serum calcium levels in Japanese. As for CaR polymorphisms, the present study confirmed racial differences in the frequencies of various CaR polymorphisms. The Gly⁹⁹⁰Arg polymorphism was very common in Japanese, whereas the Ala⁹⁸⁶Ser polymorphism and others were extremely rare.

Since the first report of ADH (7) and especially after identification of sporadic cases (8, 25), investigators have been interested in the differences between ADH and other types of PTH-deficient hypoparathyroidism in their pathophysiology and clinical features. In the previous study we compared the severity of hypocalcemia and hypercalciuria between the groups with ADH and so-called idiopathic hypoparathyroidism and found significant differences in the mean values of serum and urinary calcium before treatment (10). Concerning the metabolism and action of vitamin D, available information is limited and conflicting. It is well established that serum 1,25-(OH)₂D levels are low in patients with PTH-deficient hypoparathyroidism. On the other hand, serum concentrations of vitamin D metabolites were not reported in most studies on ADH, probably because the patients had already been treated with vitamin D analogs when their mutations in the CaR were identified. In the proband of our kindred, the serum 1,25-(OH)₂D level was low, compatible with his low normal serum PTH level. Similarly, Conley et al. (24) reported low normal 1,25-(OH)₂D levels in three families with ADH. In contrast, two members of an ADH family reported by Okazaki et al. (18) had normal to supranormal 1,25-(OH)₂D concentrations despite low normal PTH levels. We have no good explanation for these discrepant clinical observations and their underlying mechanisms.

Hypomagnesemia is another, yet to be characterized finding in ADH. Although it is considered that hypomagnesemia is one of the biochemical characteristics of ADH (9), it is not known whether and to what extent serum magnesium levels are different between ADH and other types of hypoparathyroidism. In fact, mild hypomagnesemia is occasionally seen in patients with any type of hypoparathyroidism (10). On the other hand, serum magnesium levels are not always subnormal in patients with ADH, and there are individual variations in serum magnesium levels even within a kindred carrying the same CaR mutation (7, 9). It is not unusual, therefore, that mild hypomagnesemia was observed in only half of our ADH members. However, our observation is of interest in that there was a significant positive correlation between serum calcium and magnesium levels in six affected members.

It has been stated that activation of the CaR in the thick ascending limb of Henle leads to reduced reabsorption of calcium and magnesium and hence to hypocalcemia and hypomagnesemia (3, 4). In accordance with this, patients with FHH (inactivating CaR mutations) exhibit hypercalcemia and hypermagnesemia. Furthermore, Marx et al. (28) reported a significant positive correlation between serum calcium and magnesium levels in 22 patients with FHH. Thus, the positively correlating hypocalcemia and hypo- or normomagnesemia in our family members might be another clinical evidence supporting that the renal CaR could function as an important regulator of the serum magnesium as well as calcium levels (4, 29).

No previous studies have demonstrated a correlation between serum calcium and magnesium levels in patients with ADH. Therefore, we cannot completely rule out the possibility that our observation was coincidental or exceptional. To examine the generalizability of our finding, we analyzed the data from 24 patients of 8 families with activating CaR mutations in whom serum calcium (pretreatment) and magnesium (unknown time point) were reported previously (7, 9, 18, 21). Any single family did not show a significant correlation between serum calcium and magnesium levels. However, there was a trend (P = 0.107) for a positive correlation between the two variables when all the data were combined (Fig. 5A). The correlation became significant (P = 0.039; Fig. 5C) when the data from our kindred showing a significant (P = 0.014) positive correlation (Fig. 5B) were added to these data.

View larger version (16K): [in this window] [in a new window]   Figure 5. Correlation between serum calcium and magnesium concentrations in patients with activating CaR mutations. Each point represents data from one patient. A, Data from 24 patients with 8 different CaR mutations from previous reports (7 9 18 21 ). It is not known whether serum calcium and magnesium concentrations were measured using the same samples before treatment. When there were multiple data per person, the mean value was used. B, Data from 6 patients with the Ser820Phe mutation reported in this study. Serum calcium and magnesium concentrations were measured using the same samples before treatment. C, Data in A and B are combined. The linear regression equations are as follows: y (magnesium) = 0.136x (calcium) + 0.425, r = 0.337, P = 0.107 for A; y = 0.828x + 0.747, r = 0.900, P = 0.014 for B; and y = 0.179x + 0.364, r = 0.380, P = 0.039 for C.

Acknowledgments

We thank Drs. Takeki Ogata and Katsuhiro Ono at National Defense Medical College, and Dr. Akira Aoki at Self-Defense Forces Central Hospital for their support of this study.

Footnotes

Abbreviations: ADH, Autosomal dominant hypocalcemia; Ca²⁺_i, intracellular Ca²⁺ concentrations; Ca²⁺_o, extracellular Ca²⁺ concentration; CaR, calcium-sensing receptor; FHH, familial hypocalciuric hypercalcemia; 1,25-(OH)₂D, 1,25-dihydroxyvitamin D; TM1, transmembrane domain 1.

Received March 27, 2001.

Accepted January 22, 2002.

References

1. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC 1993 Cloning and characterization of an extracellular Ca²⁺-sensing receptor from bovine parathyroid. Nature 366:575–580[CrossRef][Medline]
2. Aida K, Koishi S, Tawata M, Onaya T 1995 Molecular cloning of putative Ca²⁺-sensing receptor cDNA from human kidney. Biochem Biophys Res Commun 214:524–529[CrossRef][Medline]
3. Brown EM 1999 Physiology and pathophysiology of the extracellular calcium-sensing receptor. Am J Med 106:238–253[CrossRef][Medline]
4. Brown EM, MacLeod 2001 Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 81:239–297[Abstract/Free Full Text]
5. Garrett JE, Capuano IV, Hammerland LG, Hung BC, Brown EM, Hebert SC, Nemeth EF, Fuller F 1995 Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. J Biol Chem 270:12919–12925[Abstract/Free Full Text]
6. Pollak MR, Brown EM, Chou Y-HW, Hebert SC, Marx SJ, Steinmann B, Levi T, Seidman CE, Seidman JG 1993 Mutations in the human Ca²⁺-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75:1297–1303[CrossRef][Medline]
7. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert SC, Seidman CE, Seidman JG 1994 Autosomal dominant hypocalcemia caused by a Ca²⁺-sensing receptor gene mutation. Nat Genet 8:303–307[CrossRef][Medline]
8. Baron J, Winer KK, Yanovski JA, Cunningham AW, Laue L, Zimmerman D, Cutler Jr GB 1996 Mutations in the Ca²⁺-sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism. Hum Mol Genet 5:601–606[Abstract/Free Full Text]
9. Pearce SHS, Williamson C, Kifor O, Bai M, Coulthard MG, Davies M, Lewis-Barned N, McCredie D, Powell H, Kendall-Taylor P, Brown EM, Thakker RV 1996 A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor. N Engl J Med 335:1115–1122[Abstract/Free Full Text]
10. Yamamoto M, Akatsu T, Nagase T, Ogata E 2000 Comparison of hypocalcemic hypercalciuria between patients with idiopathic hypoparathyroidism and those with gain-of-function mutations in the calcium-sensing receptor: is it possible to differentiate the two disorders? J Clin Endocrinol Metab 85:4583–4591[Abstract/Free Full Text]
11. Hendy GN, D’Souza-Li L, Yang B, Canaff L, Cole DEC 2000 Mutations of the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia. Hum Mutat 16:281–296[CrossRef][Medline]
12. Brauner-Osborne H, Jensen AA, Sheppard PO, OíHara P, Krogsgaard-Larsen P 1999 The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain. J Biol Chem 274:18382–18386[Abstract/Free Full Text]
13. Zhang Z, Sun S, Quinn SJ, Brown EM, Bai M 2001 The extracellular calcium-sensing receptor dimerizes through multiple types of intermolecular interactions. J Biol Chem 276:5316–5322[Abstract/Free Full Text]
14. Chang W, Chen T-H, Patt S, Shoback D 2000 Amino acids in the second and third intracellular loops of the parathyroid Ca²⁺-sensing receptor mediate efficient coupling to phospholipase C. J Biol Chem 275: 19955–19963
15. Heath H III, Odelberg S, Jackson CE, Teh BT, Hayward N, Larsson C, Buist NR, Krapcho KJ, Hung BC, Capuano IV, Garrett JE, Leppert MF 1996 Clustered inactivating mutations and benign polymorphisms of the calcium-sensing receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains. J Clin Endocrinol Metab 81:1312–1317[Abstract]
16. Cetani F, Pinchera A, Pardi E, Cianferotti L, Vignali E, Picone A, Miccoli P, Viacava P, Marcocci C 1999 No evidence for mutations in the calcium-sensing receptor gene in sporadic parathyroid adenomas. J Bone Miner Res 14:878–882[CrossRef][Medline]
17. Pearce SHS, Trump D, Wooding C, Besser GM, Chew SL, Grant DB, Heath DA, Hughes IA, Paterson CR, Whyte MP, Thakker RV 1995 Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. J Clin Invest 96:2683–2692
18. Okazaki R, Chikatsu N, Nakatsu M, Takeuchi Y, Ajima M, Miki J, Fujita T, Arai M, Totsuka Y, Tanaka K, Fukumoto S 1999 A novel activating mutation in calcium-sensing receptor gene associated with a family of autosomal dominant hypocalcemia. J Clin Endocrinol Metab 84:363–366[Abstract/Free Full Text]
19. Goldsmith PK, Fan G, Millere JL, Rogers KV, Spigel AM 1997 Monoclonal antibodies against synthetic peptides corresponding to the extracellular domain of Ca²⁺ receptor: characterization and use in studying concanavalin A inhibition. J Bone Miner Res 12:1780–1788[CrossRef][Medline]
20. Kanazawa H, Tanaka H, Kodama S, Moriwake A, Kobayashi M, Seino Y 2000 The effect of calcium-sensing receptor gene polymorphisms on serum calcium levels: a familial hypocalciuric hypercalcemia family without mutation in the calcium-sensing receptor gene. Endocr J 47:29–35[Medline]
21. Watanabe T, Bai M, Lane CR, Matsumoto S, Minamitani K, Minagawa M, Niimi H, Brown EM, Yasuda T 1998 Familial hypoparathyroidism: identification of a novel gain of function mutation in transmembrane domain 5 of the calcium-sensing receptor. J Clin Endocrinol Metab 83:2497–2502[Abstract/Free Full Text]
22. Bai M, Quinn S, Trivedi S, Kifor O, Pearce SH, Pollak MR, Krapcho K, Hebert SC, Brown EM 1996 Expression and characterization of inactivating and activating mutations in the human Ca²⁺_o-sensing receptor. J Biol Chem 271:19537–19545[Abstract/Free Full Text]
23. Pearce SHS, Bai M, Quinn SJ, Kifor O, Brown EM, Thakker R 1996 Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells. J Clin Invest 98:1860–1866[Medline]
24. Conley YP, Finegold DN, Peters DG, Cook JS, Oppenheim DS, Ferrell RE 2000 Three novel activating mutations in the calcium-sensing receptor responsible for autosomal dominant hypocalcemia. Mol Genet Metab 71:591–598[CrossRef][Medline]
25. De Luca F, Ray K, Mancilla EE, Fan G-F, Winer KK, Gore P, Spiegel AM, Baron J 1997 Sporadic hypoparathyroidism caused by de novo gain-of-function mutations of Ca2+-sensing receptor. J Clin Endocrinol Metab 82:2710–2715[Abstract/Free Full Text]
26. Mancilla EE, De Luca F, Ray K, Winer KK, Fan G-F, Baron J 1997 A Ca²⁺-sensing receptor mutation causes hypoparathyroidism by increasing receptor sensitivity to Ca²⁺ and maximal signal transduction. Pediatr Res 42:443–447[Medline]
27. Cole DEC, Peltekova VD, Rubin LA, Hawker GA, Vieth R, Liew CC, Hwang DM, Evrovski J, Hendy GN 1999 Association of the A986S polymorphism of the calcium-sensing receptor with circulating calcium concentrations. Lancet 353:112–115[CrossRef][Medline]
28. Marx SJ, Spiegel AM, Brown EM, Koehler JO, Gardner DG, Brennan MF, Aurbach GD 1978 Divalent cation metabolism: familial hypocalciuric hypercalcemia versus typical primary hyperparathyroidism. Am J Med 65:235–242[CrossRef][Medline]
29. Bapty BW, Dai L-J, Ritchie G, Canaff L. Hendy GN, Quamme GA 1998 Mg²⁺/Ca²⁺ sensing inhibits hormone-stimulated Mg²⁺ uptake in mouse distal convoluted tubule cells. Am J Physiol 275:F353–F360

This article has been cited by other articles:

				L Baumber, C Tufarelli, S Patel, P King, C A Johnson, E R Maher, and R C Trembath Identification of a novel mutation disrupting the DNA binding activity of GCM2 in autosomal recessive familial isolated hypoparathyroidism J. Med. Genet., May 1, 2005; 42(5): 443 - 448. [Full Text] [PDF]

				J. Hu, S. J. McLarnon, S. Mora, J. Jiang, C. Thomas, K. A. Jacobson, and A. M. Spiegel A Region in the Seven-transmembrane Domain of the Human Ca2+ Receptor Critical for Response to Ca2+ J. Biol. Chem., February 11, 2005; 280(6): 5113 - 5120. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nagase, T. Articles by Yamamoto, M. Search for Related Content PubMed PubMed Citation Articles by Nagase, T. Articles by Yamamoto, M.