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*OMIM
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*CALCIUM COMPOUNDS
*CALCIUM, ELEMENTAL
*CHOLECALCIFEROL
*HYDROCHLOROTHIAZIDE
The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 7 3068-3073
Copyright © 2002 by The Endocrine Society

Special Features

Hydrochlorothiazide Effectively Reduces Urinary Calcium Excretion in Two Japanese Patients with Gain-of-Function Mutations of the Calcium-Sensing Receptor Gene

Kohei Sato, Yukihiro Hasegawa, Jun Nakae, Kenji Nanao, Ikuko Takahashi, Toshihiro Tajima, Nozomi Shinohara and Kenji Fujieda

Department of Pediatrics (K.S., J.N., T.T., N.S., K.F.), Hokkaido University School of Medicine, Sapporo 060-0835, Japan; and Endocrinology, Metabolism and Genetics Unit (Y.H., K.N., I.T.), Tokyo Metropolitan Kiyose Children’s Hospital, Tokyo 204-0024, Japan

Address all correspondence and requests for reprints to: Kenji Fujieda, M.D., Ph.D., Department of Pediatrics, Asahikawa Medical College, 2-1-1-1, Midorigaoka, Higashi, Asahikawa 078-8510, Japan. E-mail: . ken-fuji{at}asahikawa-med.ac.jp

Abstract

Gain-of-function mutations of the calcium-sensing receptor (CaR) gene cause autosomal dominant and/or sporadic hypocalcemia with hypercalciuria. Because treatment of the hypocalcemia with vitamin D and/or calcium in patients with such mutations results in increased hypercalciuria, nephrocalcinosis, and renal impairment, its use should be limited to alleviating the symptoms of symptomatic patients. Because thiazide diuretics have been successfully used to treat patients with hypercalciuria and hypoparathyroidism, they are theoretically useful in reducing urine calcium excretion and maintaining serum calcium levels in patients with gain-of-function mutations of the CaR gene. In this study, we report on the clinical course, molecular analysis, and effects of hydrochlorothiazide therapy in two Japanese patients with gain-of-function mutations of the CaR gene. Within a few weeks after birth, they developed generalized tonic seizures due to hypocalcemia (serum calcium values: 1.1 mmol/liter and 1.3 mmol/liter, respectively). Despite treatment with the standard dose of 1,25-dihydroxyvitamin D3 in one patient and 1{alpha}-hydroxyvitamin D3 in the other, acceptable serum calcium levels near the lower limit of normal were not established, and their urinary calcium excretion inappropriately increased. Addition of hydrochlorothiazide (1 mg/kg) reduced their urinary calcium excretion and maintained their serum calcium concentrations near the lower limit of normal, allowing the 1,25-dihydroxyvitamin D3 and 1{alpha}-hydroxyvitamin D3 doses to be reduced, and it alleviated their symptoms. A heterozygous missense mutation was identified in both patients. In one patient, the mutation was A843E in the seventh transmembrane domain of the CaR, and in the other it was L125P in the N-terminal extracellular domain. In vitro transient transfection of their mutant CaR cDNAs into HEK293 cells shifted the concentration-response curve of Ca2+ to the left.

In conclusion, two sporadic cases of hypercalciuric hypocalcemia were due to de novo gain-of-function mutations of the CaR gene. Hydrochlorothiazide with vitamin D3 successfully reduced the patients’ urinary calcium excretion and controlled their serum calcium concentrations and symptoms. Thiazide diuretics are effective in patients with gain-of function mutations of the CaR gene.

THE CALCIUM-SENSING receptor (CaR) belongs to family C of the seven transmembrane G protein-coupled receptor superfamily (GPCRs) (1, 2, 3, 4, 5, 6, 7, 8, 9). This subfamily of GPCRs is characterized by a very long amino-terminal domain of approximately 600 amino acids that has been shown to contain the agonists binding site (2, 5, 6) and one domain for dimerization of the CaR (10, 11, 12). CaR is highly expressed in the parathyroid gland and kidney (1, 3, 4, 5, 6), and extracellular Ca directly activates CaR, causing inhibition of PTH secretion and decreased renal Ca reabsorption (4, 5, 6).

After cloning of the CaR gene, germline mutations of the CaR gene were found to cause abnormalities in serum Ca levels in humans. Loss of function mutations in the CaR gene cause familial benign hypercalcemia (also known as familial hypocalciuric hypercalcemia) and severe neonatal hypeparathyroidism (2, 5, 6). Activating mutations of the CaR gene, on the other hand, are associated with familial autosomal dominant hypocalcemia (ADH) and/or sporadic hypocalcemia with hypercalciuria (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). CaRs with activating mutations inhibit PTH secretion and decrease renal Ca reabsorption at an inappropriately low serum Ca concentration, leading to inappropriately low serum PTH levels, hypocalcemia, and relative hypercalciuria (5, 6, 14). To date, 25 missense activating mutations and one large in-frame deletion of the C-terminal domain of the CaR gene have been identified in patients with ADH and/or sporadic hypocalcemia (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Fifteen of the 25 missense mutations are localized within the CaR’s extracellular domain, which includes the ligand binding and dimerizing domain, whereas the remaining 10 are in the first and third extracellular loops or the third intracellular loop or the fifth, sixth, and seventh transmembrane domains (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23).

The differential diagnosis of ADH and/or sporadic hypocalcemia with hypercalciuria due to the gain-of-function mutations of the CaR gene from hypoparathyroidism is quite important because patients with gain-of-function mutations of the CaR gene are at risk of hypercalciuria and resultant renal insufficiency as a result of vitamin D and calcium supplementation (6, 14, 24). It is recommended that asymptomatic familial hypocalcemic hypercalciuria patients not be routinely treated with vitamin D and that such treatment be reserved for symptomatic patients (6, 14).

Although there are no published reports describing the effectiveness of thiazide diuretics in patients with gain-of-function mutations of the CaR gene, thiazide diuretics seemed to be worth trying. The hypocalciuric action of thiazide diuretics has been successfully used in the treatment of idiopathic hypercalciuria (25, 26, 27, 28), and thiazides efficaciously maintain the serum calcium levels of hypoparathyroidism patients without the complications of vitamin D therapy (29, 30, 31, 32).

We encountered two sporadic Japanese cases of severe hypocalcemia accompanied by hypercalciuria resulting from different novel de novo gain-of-function mutations of the CaR gene. During vitamin D3 treatment, the urinary calcium excretion of both patients increased even when their serum calcium levels were still low and they were symptomatic. Addition of hydrochlorothiazide, however, was effective in reducing their urinary calcium excretion, increasing their serum calcium levels, and alleviating their symptoms.

Subject and Methods

PTH assay

A highly sensitive RIA for PTH consisting of PTH antiserum, 125I-labeled Tyr42 human PTH (43–68) and synthetic human PTH (1–84) as the standard was used (Yamasa Ltd., Tokyo, Japan) (33). The RIA cross-reacted with the mid-region and carboxy terminals of PTH. The within-assay and between-assay coefficients of variations were less than 4.6% and less than 8.6%, respectively. The limit of detection was 50 pg/ml (33).

DNA amplification and sequence analysis

Informed consent to participate the study was obtained from the patients and their parents. Genomic DNA was extracted from peripheral leukocytes as described elsewhere (34). Each exon of the CaR gene was amplified by the PCR using the primers previously described (2, 14). The PCR conditions consisted of 9 min at 94 C, followed by 30 cycles of 30 sec at 94 C, 30 sec at 60 C, and 30 sec at 72 C in a Perkin-Elmer Corp. Gene Amp PCR System 2400 thermal cycler (PE Applied Biosystems, Foster City, CA). After amplification, the PCR products were purified from low melting agarose gel (34), and the purified products were sequenced directly with an ABI PRISM Dye Terminator Cycle Sequencing Kit and a 373A automated fluorescent sequencer (PE Applied Biosystems), as described previously (34).

Site-directed mutagenesis

Human CaR cDNA inserted into a human expression vector (pcDNA3, Invitrogen, San Diego, CA) was obtained from NPS Pharmaceuticals, Inc. (Salt Lake City, UT). Site-directed mutagenesis to produce mutant CaR genes with the point mutation was performed by the overlapping PCR method using missense primers containing the desired nucleotide change, as described previously (35). The accuracy of the construct was confirmed by direct sequencing. Each PCR product was ligated into the human expression vector of CaR and transformed in DH 5{alpha}-competent cells. The mutant clone was confirmed by sequencing again.

Cell culture and transient transfection

HEK-293 cells were cultured in DMEM (Life Technologies, Inc., Gaithersburg, MD) with 10% heat-inactivated FBS, and the cells were then plated in six-well plates (106 cells per well). Transient transfection was performed by using constructs encoding the wild-type (WT) and mutant receptors, with 5 µl lipofectamine (Life Technologies, Inc.) and 1 µg DNA, as described previously (15).

Measurement of Phosphoinositides (IPs)

Forty-eight hours after transfection, HEK293 cells were labeled with 3 mCi/ml myo-[3H] inositol (Amersham Pharmacia Biotech, Buckinghamshire, UK) for 24 h, then incubated for 30 min in DMEM (free of bicarbonate, calcium, and magnesium), supplemented with 20 mM HEPES buffer, 0.2% fetal calf serum albumin, 10 mM lithium chloride, 0.5 mM magnesium chloride, and then stimulated with various concentrations of calcium chloride (0, 0.5, 1, 2, 3, 5, 7, and 10 mM), as described in a previous report (15). The reactions were terminated with a 0.2 volume of ice-cold 20% perchloric acid, and the mixtures were incubated on ice for 20 min. Proteins were sedimented by centrifugation at 2000 x g for 15 min at 4 C, and the supernatant was decanted into a siliconized glass, and neutralized to pH 7.5 by titrating with ice-cold 10 M KOH. Centrifugation at 2000 x g for 15 min at 4 C was performed again, and inositol triphosphate in the supernatant was measured by liquid scintillation in a myo-[3H]-inositol assay system (Amersham Pharmacia Biotech). Six independent transfections were performed at each Ca2+ concentration for IP measurement. The mean EC50 (median effective concentration) values for the WT and mutant receptors in response to increasing concentrations of Ca2+ were calculated from the EC50 values obtained in the individual experiments and are expressed with the SEM. The EC50 values was compared by ANOVA. P values equal to or less than 0.05 were considered to indicate a statistically significant difference.

Results

Case reports

Patient 1. The patient was an 18-yr-old Japanese male born after an uneventful pregnancy and delivery. He had been irritable since10 d of age and was admitted because of intractable seizures at 24 d of age, at which time his serum calcium and phosphate levels were 1.1 mmol/liter and 3.4 mmol/liter, respectively (Table 1Go). PTH was undetectable on admission (Table 1Go). At 9 months of age, 1,25-dihydroxyvitamin D3, 0.5 µg/kg·d, was required to maintain normocalcemia; however, the urinary calcium/creatinine ratios (mmol/mmol) remained in the normal to high range (0.42–4.13; normal range: 0.14–0.4 mmol/mmol) even when the serum calcium levels were low (less than 1.9 mmol/liter). At 7 yr 3 months of age, the daily dose of 1, 25-dihydroxyvitamin D3 to sustain the appropriate serum calcium levels was 1.2 µg/kg·d, and the patient’s 25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D levels were 46.8 nmol/liter (normal range: 20–100 nmol/liter) and 549.5 pmol/liter (normal range: 36–144 pmol/liter), respectively. The serum creatinine levels at the time were in the 0.03–0.05 mmol/liter range. Computed tomography of the skull did not show calcification, but renal echography revealed nephrocalcinosis. Administration of hydrochlorothiazide, 1 mg/kg·d, was started, and was followed by a marked reduction in the hypercalciuria. At the same time, the dose of 1,25-dihydroxyvitamin D3 was reduced to 0.036 µg/kg·d to maintain low to normal serum calcium levels. The urinary calcium/creatinine ratios before and after the start of hydrochlorothiazide are plotted in Fig. 1Go and clearly show that the addition of hydrochlorothiazide was effective in reducing urinary calcium excretion. At 15 yr of age, the mutation of the CaR gene was identified, and serum creatinine level was 0.11 mmol/liter. Treatment at 15 yr of age consisted of 1{alpha}-hydroxyvitamin D3 (0.044 µg/kg·d), hydrochlorothiazide (0.6 mg/kg·d), and potassium supplementation (40 mg/kg·d).


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  		Table 1. Biochemical findings of two patients of gain-of-function mutations of the CaR gene

 


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  		Figure 1. Effects of hydrochlorothiazide on serum Ca and urinary Ca excretion in two patients with CaR gain-of-function mutations. Solid circles indicate serum Ca levels. Open squares indicate urinary Ca/Cr ratio. Doses of 1{alpha}-hydroxyvitamin D3 and hydrochlorothiazide are shown.

 
Patient 2. The patient was a 16-yr-old Japanese girl born at 38 wk of gestation by normal vaginal delivery. At 5 d of age, she developed generalized tonic seizures. At that time, her serum calcium (1.3 mmol/liter) and magnesium (0.7 mmo/liter) levels were low, and her serum phosphate level was high (2.9 mmol/liter) (Table 1Go). PTH was undetectable. Her urinary calcium/creatinine ratios (mmol/mmol) were abnormally high (0.84–1.68), despite low serum calcium levels around that time (Table 1Go, Fig. 1Go). Despite supplementation with oral calcium (3–4 g/d) and 1{alpha}-hydroxyvitamin D3 (0.2–0.5 µg/kg·d), her serum calcium levels fluctuated a great deal (1.4–2.0 mmol/liter), and intermittent neuromuscular irritability, easy fatigue, and tetanic episodes were noted. Increases in the dose of 1{alpha}-hydroxyvitamin D3 to (0.6–0.8 µg/kg·d) were followed by increased urinary calcium excretion [urine calcium/urine creatinine (mmol/mmol), 2.8 to 4.2] (Fig. 1Go). At 5 yr of age, computed tomography scans of the brain and kidney revealed calcification of the basal ganglia and bilateral nephrocalcinosis, and the patient’s serum creatinine levels were 0.03–0.05 mmol/liter. At 12 yr of age, 1 mg/kg hydrochlorothiazide was added. Hydrochlorothiazide reduced urinary calcium excretion, and it increased serum calcium levels to near the lower limit of normal (Fig. 1Go). It also allowed reduction of the vitamin D dosage (Fig. 1Go) and alleviated the patient’s symptoms. At 16 yr of age, the creatinine clearance and serum creatinine values were 104.3 ml/min·m2 and 0.07 mmol/liter, respectively, and a renogram revealed normal kidney function. Treatment currently consists of 1{alpha}-hydroxyvitamin D3 (0.06 µg/kg·d), hydrochlorothiazide (0.5 mg/kg·d), and potassium supplementation (50 mg/kg·d).

DNA sequence analysis

Patient 1 was found to have a heterozygous C to A transition at nucleotide 2528 in exon 7 (Fig. 2AGo). This mutation substitutes glutamic acid for alanine at codon 843 (A843E) in the seventh transmembrane domain of the CaR gene. Patient 2, on the other hand, had a heterozygous T to C change at nucleotide 374 in exon 3 (Fig. 2BGo) that resulted in a missense mutation of L125P in the N-terminal extracellular domain of the CaR gene. Neither of the mutations was detected in 50 normal Japanese subjects. Family analysis demonstrated that neither of the patients’ parent harbored their children’s mutation, indicating that both mutations had arisen de novo.



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  		Figure 2. Missense mutation in the CaR gene. A, DNA sequence for patient 1 revealed a heterozygous C to A substitution at position 2528 in exon 7. This mutation changes alanine to glutamic acid at codon 843 (A843E). His father or mother does not have this mutation. B, In patient 2, a heterozygous T to C change was identified at position 374 in exon 3. This mutation substitutes proline for leucine at codon 125 (L125P). This mutation is not detected in her father or mother.

 
Functional analysis of CaRs

Figure 3Go shows the responses of the WT and mutated CaRs to various concentrations of Ca2+. IP accumulation in response to increasing Ca2+ concentrations increased in the WT and mutants and plateaued at high concentrations (Fig. 3Go). Both mutant receptors showed a leftward shift in the concentration-response curve. The EC50 for Ca2+ calculated from six independent experiments on cells transfected with the A843E and L125P mutants was 2.7 mmol/liter and 2.3 mmol/liter, respectively (Fig. 3Go). The EC50 values of the mutants were significantly lower (P < 0.05) than those of the WT (4.0 mmol/liter).



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  		Figure 3. Ca2+ evoked accumulation of IP in HEK293 cells transiently transfected with WT or mutant CaR DNA. Transfected HEK293 cells were incubated with myo-[3H]-inositol and then stimulated with the indicated Ca2+concentration for 30 min. Total IPs were isolated and counted by liquid scintillation. All responses are normalized to the maximum response of the WT construct. Each data point represents the mean + SEM of six independent transfections. The SEM is indicated with a vertical bar through each point (no error bar is shown when the SEM is smaller than the symbol.

 
Discussion

Heterozygous gain-of-function mutations of the CaR gene in two sporadic Japanese cases of hypercalciuric hypocalcemia were identified. The clinical course of our patients, including the elevation of the urinary calcium/creatinine ratio at the time of diagnosis and further increase after treatment, was consistent with previous reports (5, 6, 14, 17). To date, eight cases of sporadic hypercalciuric hypocalcemia due to de novo gain-of-function mutations of the CaR gene have been reported (17, 18, 23, 36, 37), and our cases were also caused by de novo mutations of the CaR gene. An in vitro functional expression study of the L125P and A843E mutant CaRs revealed a leftward shift of the Ca2+ concentration-IP response curves, confirming that they are those of a gain-of-function mutants. Several gain-of-function missense mutations of the CaR gene have already been described, and at least 14 of them have been located in the N-terminal extracellular domain (14, 15, 16, 17, 18, 21, 23), suggesting that this domain is particularly sensitive to mutationally induced activation. Furthermore, Jensen et al. (38) demonstrated that the alanine 116 to proline136 region of the N-terminal domain of CaR is important to maintenance of the inactive conformation of CaR. Because L125P is present in this region, a similar mechanism may activate the function of the L125P mutant. The A843E mutation in patient 2 was located in the seventh transmembrane domain. This residue is well conserved in human (3), bovine (1), rat (39), chicken (40), and fugu (41) CaRs, indicating its functional importance. Furthermore, F788C and L733R in the fifth transmembrane domain and F806S in the sixth transmembrane domain of patients have also been found to be activated by in vitro functional analysis (18, 19, 20). All of these findings suggest that the A843E mutation affects CaR function and causes activation of CaR, resulting in the disease.

Recently, both the L125P and A843E mutations have been identified in unrelated hypocalcemic families (23). Our result of the in vitro analysis of the L125P mutant is consistent with their study (23). However, regarding the A843E mutant, Zhao et al. (42) and Lienhardt et al. (23) demonstrated that the mutant was constitutively active and that increasing extracellular calcium led to only slight further activation. This result may suggest that the A843E mutation, without Ca2+ binding, may disrupt the inhibitory constraints that G protein coupling by altering the conformation of the CaR. Our result of the A843E mutant was not consistent with their results, and a reason for this difference remains to be established. A structural model of CaR and precise molecular mechanisms involved in activation of G protein will be necessary to understand the mechanism of CaR gain-of-function mutations.

Hydrochlorothiazide reduced urinary calcium excretion and allowed reduction the dose of vitamin D3, and it maintained the serum calcium levels and alleviated our patients’ symptoms. Thiazide diuretics efficaciously maintain the serum calcium concentration, and they reverse and prevent hypercalciuria induced by vitamin D treatment in hypoparathyroidism patients (29, 30, 31, 32). Worsening of the hypercalciuria, and the resultant renal impairment, is the most important factor affecting outcome, and thus vigorous vitamin D and calcium supplementation should be discouraged in patients with a gain-of-function of CaR. Because of this, thiazide diuretics might be the treatments of first choice, especially for patients with a combination of asymptomatic hypocalcemia and hypercalciuria. However, of note is that thiazide diuretics may cause side effects, such as rash, headache, liver disorders, gastrointestinal problems, and acute renal failure, particularly in elderly people (43, 44, 45). More problematic are the consequences of hypokalemia. Especially, high-risk patients who have symptomatic coronary disease, congestive heart failure, and those who are taking digitalis should be protected from hypokalemia (44, 45). It is possible that patients with CaR gain-of-function mutations may have renal insufficiency as a result of long-term vitamin D and calcium supplementation (14). Therefore, careful monitoring for serum electrolytes levels, dosage adjustment, and replacement of electrolyte losses should be performed when thiazide diuretics are used in patients with CaR gain-of-function mutations.

Finally, Winer et al. (46) recently reported that a twice-daily PTH regimen was effective in overcoming the tendency of patients with gain-of-function mutations of the CaR to develop hypercalciuria with near-normal serum Ca levels. The combination of thiazide diuretics and PTH may be more effective in the patients with CaR hyperfunction, and this must be investigated further.

In conclusion, we have reported two sporadic Japanese cases of severe hypercalciuric hypocalcemia caused by de novo gain-of-function mutations in the CaR gene. Because hydrochlorothiazide was effective in our patients, thiazide diuretics should be considered for the treatment of patients with gain-of-function mutations in the CaR gene.

Footnotes

Present address for K.F.: Department of Pediatrics, Asahikawa Medical College 2-1-1-1, Midorigaoka, Higashi, Asahikawa 078-8510, Japan.

Abbreviations: ADH, Autosomal dominant hypocalcemia; CaR, calcium-sensing receptor; EC50, an effective concentration of an agonist giving one-half of the maximal response; GPCRs, G protein-coupled receptor superfamily; IP, phosphoinositide; WT, wild-type.

Received August 7, 2001.

Accepted March 21, 2002.

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