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A Novel Activating Mutation in Calcium-Sensing Receptor Gene Associated with a Family of Autosomal Dominant Hypocalcemia¹

Third Department of Medicine, Teikyo University School of Medicine (R.O., K.T.), 3426–3 Anesaki, Ichihara, Chiba 299-0111; Second Department of Research (M.N., M.A.), Department of Metabolism and Endocrinology (M.A., J.M., Y.To.), Kanto-Teishin Hospital, 5–9-22 Higashi-Gotanda, Shinagawa, Tokyo 141-0022; and Fourth Department of Internal Medicine, University of Tokyo School of Medicine (N.C., Y.Ta., T.F., S. F), 3–8-6 Mejirodai, Bunkyo, Tokyo 112-0015, Japan

Address all correspondence and requests for reprints to: Ryo Okazaki, M.D., Third Department of Medicine, Teikyo University School of Medicine, 3426–3 Anesaki, Ichihara 299-0111, Japan. E-mail: rokazaki{at}med.teikyo-u.ac.jp

	Abstract

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Autosomal dominant hypocalcemia (ADH), caused by activating mutations of the calcium-sensing receptor (CaSR), is characterized by hypocalcemia with an inappropriately low concentration of PTH. Among 11 missense mutations of CaSR reported to date in patients with ADH or sporadic hypocalcemia, functional properties of 8 mutant CaSRs were characterized. Here, we describe a novel mutation of CaSR and its functional property in a family with ADH. The 41-yr-old male proband had asymptomatic hypocalcemia with a history of recurrent nephrolithiasis. His father had asymptomatic hypocalcemia, but his mother was normocalcemic. PCR-single strand conformation polymorphism and sequencing revealed that both the proband and the father had a novel heterozygous mutation in CaSR gene that causes lysine to asparagine substitution at codon 47 (K47N), which is in the extracellular domain of CaSR, like 6 of 11 known activating mutations. Using HEK293 cells transfected with wild-type or K47N CaSR complementary DNA, the intracellular Ca²⁺ concentration was assessed in response to changes in the extracellular Ca²⁺ concentration. The EC₅₀ of the mutant CaSR for the extracellular Ca²⁺ concentration was 2.2 mmol/L and was significantly lower than that of wild-type (3.7 mmol/L). These results confirm that this mutation is responsible for ADH in this family. The fact that several inactivating mutations in familial hypocalciuric hypercalcemia occur in amino acid around K47 suggests the importance of the N-terminal portion of the receptor in extracellular Ca sensing.

	Introduction

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AUTOSOMAL dominant hypocalcemia (ADH) is a disease characterized by hypocalcemia with an inappropriately low concentration of serum PTH and relative hypercalciuria, which leads to nephrolithiasis and nephrocalcinosis, especially after active vitamin D treatment (1). The cause of ADH is gain of function mutations in calcium-sensing receptor (CaSR) gene that inhibit PTH secretion and renal Ca reabsorption despite hypocalcemia. Eleven distinct activating mutations in the CaSR gene (A116T, N118K, E127A, F128L, T151M, E191K, F612S, Q681H, L773R, F788C, and F806S) have been reported to date in patients with ADH or sporadic hypocalcemia (1, 2, 3, 4, 5). However, the functional properties of these mutated CaSRs have been examined only in limited cases (1, 2, 5, 6, 7). In the present study, we report a family with ADH with a new mutation in CaSR gene (K47N) and analyzed the function of CaSR with this mutation. The results show that CaSR with the K47N mutation has an EC₅₀ of 2.2 mmol/L for Ca and confirm that this mutation is responsible for ADH in this family.

	Subjects and Methods

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Case descriptions

A 41-yr-old Japanese man with a history of recurrent nephrolithiasis was referred to Kanto-Teishin Hospital in Tokyo, Japan for the evaluation of hypocalcemia. His serum Ca level had been 7.4–8.0 mg/dL. He denied preferences to diet containing abnormally high or low Ca. Physical examination revealed no abnormality. His serum uric acid level was 5.5 mg/dL. At least five stones were found with ultrasonography in the left kidney. As shown in Table 1, his serum intact PTH and 1,25-dihydroxyvitamin D [1,25-(OH)₂D] level was normal despite mild hypocalcemia. To rule out ADH, we measured serum Ca concentrations in his parents and found that his 68-yr-old father had asymptomatic hypocalcemia. His mother was normocalcemic. His father also had inappropriately low serum intact PTH but normal 1,25-(OH)₂D levels (Table 1). These findings strongly suggested ADH and led to the present study.

Single strand conformational polymorphism (SSCP)

Genomic DNA was obtained from leukocytes using DNA Extractor WB kit (Nippon Gene, Tokyo, Japan). For initial screening, DNA from the proband and his father as well as that from two normal subjects were analyzed with PCR-SSCP for all the coding exons of CaSR using a DNA fragment analysis kit (Pharmacia Biotech, Tokyo, Japan) according to the manufacturer’s instruction. Exons 2, 3, 5, and 6 of CaSR were amplified by one PCR reaction, and exons 4 and 7 were amplified by two PCR reactions, which produce two overlapping PCR products. The primers used were 5'-atcccttgccctggagagacggc-3' and 5'-agagaagagattggcagattaggcc-3' for exon 2; 5'-agcttcccattttcttccacttctt-3' and 5'-cccgtctgagaaggcttgagtacct-3' for exon 3; 5'-actcattcaccatgttcttggttct and 5'-gctgttgctaaacctgtcgc-3', 5'-cccaggaagtctgtccacaatg-3' and 5'-cccaactctgctttattat-acagca-3' for exon 4; 5'-ggcttgtactcattctttgctcctc-3' and 5'-gacatctggttttctgatggacagc-3' for exon 5; 5'-caaggacctctggacctccctttgc-3' and 5'-gaccaagccctgcacagtgcccaag-3' for exon 6; and 5'-agtctgtgccacacaataactcactc-3' and 5'-cttgttgaagaagatgcacgcca-3', 5'-tgctcatcttcttcatcgtctgg-3' and 5'-ctctctgcattctccctagcccagt-3' for exon 7 (8). PCR was performed using GeneAmp PCR system 9600 (Perkin Elmer, Norwalk, CT) with the following protocol: initial denaturation at 95 C for 105 s, followed by 35 cycles of 95, 60, and 72 C, each for 30 s, with a final elongation at 72 C for 7 min. Each PCR product was electrophoresed on a 1% agarose gel, purified with QIAquick gel extraction kit (Qiagen Gmbh, Hilden, Germany), and subjected to SSCP analysis. Gel-purified PCR products were electrophoresed on a 10% precast polyacrylamide gel (Pharmacia Biotech, Tokyo, Japan) at 5 C for 3 h, followed by silver staining (Dai-ichi Kagaku Co., Tokyo, Japan). After establishing the point mutation in exon 2 in the proband and his father, leukocyte DNA samples from 50 normal control subjects were analyzed by PCR-SSCP for exon 2. For better resolution, exon 2 PCR products were electrophoresed on a 15–25% precast polyacrylamide gel (Dai-ichi Kagaku Co.) for 18 h at 10 C, followed by silver staining.

Sequence analysis

PCR products for exon 2 from the proband, the father, and two normal control subjects were gel purified and subjected to automated DNA sequencing analyses with fluorescence-labeled dideoxyterminators (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elmer) according to the manufacturer’s instructions (ABI PRISM 310 genetic analyzer, PE Applied Biosystems, Foster City, CA). Sequencing was performed for both strands.

In vitro functional study of mutated CaSR

To analyze the function of mutant CaSR, we have cloned wild-type CaSR, created a mutation by in vitro mutagenesis, and examined the function of mutated CaSR by transient transfection into HEK cells (6). Wild-type CaSR complementary DNA (cDNA) was cloned by RT-long PCR using ribonucleic acid extracted from human kidney (9). CaSR cDNA covering the full coding region was obtained in two PCR amplifications that produced overlapping PCR products with SacI restriction enzyme sites in the overlapping region. The 5'-PCR product was amplified using forward primer (5'-aaaaagcttagagacggcagaaccatggcatt-3') and reverse primer (5'-tcaaacaccaggaggacacggttg-3'), and the 3' PCR product was obtained using forward primer (5'-gtgctgggtgtgtttatcaagttccg-3') and reverse primer (5'-aaatctagactctctgcattctccctagcccagt-3'). The condition for long PCR was 94 C for 2 min for 1 cycle, 98 C for 20 s and 68 C for 2 min for 35 cycles, and 72 C for 7 min for 1 cycle. The PCR products were then cloned into TA vector (Invitrogen, Carlsbad, CA). We sequenced entire coding regions of CaSR from several clones and selected clones whose sequences are identical with those reported previously (10). HindIII-SacI fragment containing 5'-PCR product and SacI-XhoI fragment with 3'-PCR product were subcloned into TA vector. Finally, HindIII-XhoI fragment with the full coding region of CaSR was subcloned into pcDNA3 expression vector (Invitrogen). In vitro mutagenesis was conducted using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instruction. The primers used were 5'-cattttggagtagcagctaacgatcaagatctcaaatcaagg-3' and 5'-ccttgatttgagatcttgatcgttagctgctactccaaaatg-3' for K47N mutation. The mutated nucleotide was confirmed by DNA sequencing.

HEK293 cells were cultured in DMEM (Life Technologies, Grand Island, NY) with 10% FBS and were transiently transfected using the calcium phosphate precipitation method. The function of the mutated CaSR was assessed after 48 h by measuring the EC₅₀ for Ca. HEK293 cells that had been transfected with wild-type or mutant CaSR cDNAs were loaded for 1 h at room temperature with fura-2/AM (Molecular Probes, Inc., Eugene, Oregon) in HBBS (118 mmol/L NaCl, 4.6 mmol/L KCl, 10 mmol/L D-glucose, and 20 mmol/L HEPES, pH 7.2). Fura-2-loaded cells were washed, pelleted, and placed on ice. Aliquots of cells were resuspended in 2 mL Ca-free HBBS in a UV grade fluorometer cuvette on fluorescence spectrophotometer (F-2000 Hitachi, Tokyo, Japan). CaCl₂ was added to produce the desired extracellular Ca²⁺ concentration (Ca²⁺o). Excitation monochrometers were centered at 340 and 380 nm, with emission light collected at 490 ± 40 nm through a wide band emission filter. The 340/380 excitation ratio of emitted light was used to calculate the intracellular Ca²⁺ concentration (Ca²⁺i). For measuring EC₅₀, the Ca²⁺i in response to 0.5, 1.0, 1.5, 2.0, 2.5, 3, 3.5, 4.0, 6, 8, 16, and 32 mmol/L Ca²⁺o were recorded. The magnitude of the peak of the transient response after an individual stimulus was expressed as a proportion to the maximal response that corresponds to the response to 32 mmol/L Ca²⁺o. The Ca²⁺i was then plotted against Ca²⁺o, and EC₅₀ was assessed using KaleidaGraph software (Synergy Software, Reading, PA). The mean EC₅₀ values from three individual experiments were compared by Student’s t test. P < 0.05 was considered significant.

Western blot analysis

HEK293 cells were transfected with wild-type or mutant CaSR cDNAs and harvested 48 h later. Membrane fraction from these cells was prepared by ultracentrifugation as previously reported (11). Thirty-five micrograms of membrane fraction were electrophoresed on a 7.5% precast polyacrylamide gel (Dai-ichi Kagaku Co., Tokyo, Japan) and electrotransferred to a polyvinylidene difluoride membrane (Immobilon, Millipore Corp., Bedford, MA). After blocking with Blockace (Dainihon Seiyaku, Osaka, Japan), the membrane was incubated with primary antihuman CaSR antibody (12). This antibody is a mouse monoclonal alanine-aspartic acid-aspartic acid (ADD) antibody directed against residues 214–235 of human CaSR and provided by NPS Pharmaceuticals, Inc. (Salt Lake City, UT). After washing, the blot was incubated with secondary antimouse IgG antibody (Amersham, Tokyo, Japan) and visualized by the enhanced chemiluminescence system (Amersham).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
PCR-SSCP analysis and DNA sequencing of CaSR

The initial SSCP screening showed that PCR products for exon 2 of CaSR from the proband and the father exhibited different patterns from those of normal controls when electrophoresed at 5 C. No difference was observed in the SSCP pattern for PCR products of the remaining exons. The difference in the pattern of PCR-SSCP for exon 2 was better visualized when PCR products were electrophoresed on a 15–25% gradient gel at 10 C for 18 h (Fig. 1). None of the exon 2 PCR products from 50 additional normal controls had the same pattern as those from the proband and the father (data not shown). Figure 2 shows the results from automated DNA sequencing, which showed the heterozygous mutation in both the proband and the father at the same position (codon 47 AAA to AAC).

View larger version (57K): [in this window] [in a new window]   Figure 1. SSCP analyses of PCR products for exon 2 of CaSR. PCR products for exon 2 of CaSR from the proband (lane 4), the father (lane 5), and six normal controls (lanes 1–3 and 6–8) were subjected to SSCP analysis as described in Subjects and Methods.

View larger version (39K): [in this window] [in a new window]   Figure 2. Direct sequencing of PCR products for exon 2 of CaSR. PCR products for exon 2 of CaSR from the proband (upper figure) and the father (lower figure) were directly sequenced as described in Subjects and Methods. The left panel shows the sequence of the sense strand; the right panel shows that of the antisense strand, respectively. The arrows indicate heterozygous mutation at codon 47.

Figure 3 shows the typical responses of wild-type and mutated CaSR to various concentrations of Ca²⁺o. The EC₅₀ for Ca calculated from three independent experiments was 2.2 ± 0.1 mmol/L for mutated CaSR and was significantly different from that of wild-type CaSR (3.7 ± 0.1 mmol/L). We expressed the response of Ca²⁺i as a ratio to the maximal increase in Ca²⁺i in each transfected cell. Therefore, the difference in the responses between cells transfected with wild-type and K47N CaSR cDNA cannot be ascribed to the variability of transfection efficiency or expression level between these two receptors. However, the function of mutant CaSR might be modulated by its expression level in vivo. Therefore, we compared the expression level of wild-type and mutant CaSR in transfected cells by Western blot analysis. The results indicate that the expression level of K47N CaSR is the same as that of wild-type CaSR, confirming that the functional activation by K47N mutation is responsible for derangements of Ca metabolism in this family (Fig. 4).

View larger version (14K): [in this window] [in a new window]   Figure 3. Functional analysis of mutant CaSR. Mutant CaSR cDNA harboring the K47N mutation was created by in vitro mutagenesis. Wild-type and mutant CaSR cDNAs were transiently transfected into HEK cells, and changes in intracellular ionized Ca were plotted against alterations in extracellular ionized Ca as described in Subjects and Methods. The figure shows typical results from one of three independent experiments.

View larger version (25K): [in this window] [in a new window]   Figure 4. Expression levels of wild-type and mutant CaSR. Wild-type and K47N CaSR cDNAs were transiently transfected into HEK cells. The expression level of CaSR was analyzed by Western blot analysis, using membrane fraction from these cells.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The EC₅₀ for Ca of this K47N CaSR is comparable to those of the previously reported activating mutations of CaSR, ranging from 2.2–3.3 mmol/L (1, 2, 5, 6, 7), and confirms that this mutation is responsible for ADH in this family. In contrast to relatively narrow range of EC₅₀ of CaSR with activating mutations, the EC₅₀ values of CaSR with inactivating mutations are extremely variable, from 5.6 mmol/L to more than 50 mmol/L (8, 13, 15, 17). Because clinical features of FHH are almost the same in patients with these mutations of variable function, it is suggested that the loss of the wild-type allele, but not the presence of the inactivating mutant allele, is responsible for the development of FHH in most patients. On the other hand, the narrow range of EC₅₀ of CaSR with activating mutation indicate the possibility that patients with mutant CaSR with much lower EC₅₀ are diagnosed with congenital hypoparathyroidism by deficient secretion of PTH. Although clinical features of ADH are variable, from asymptomatic mild hypocalcemia to symptomatic severe hypocalcemia, it is not known whether these clinical variabilities could be explained by different EC₅₀ values of mutant CaSR. More systematic examinations of the function of mutant CaSR would be necessary to elucidate the relationship between the function of mutant CaSR and the clinical presentation of ADH.

In conclusion, we have described a novel mutation in CaSR that causes ADH. The mutation is in the N-terminal half of the extracellular domain of CaSR and indicates the importance of this region of CaSR for Ca sensing.

	Footnotes

 
¹ This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and a grant from Research Society for Metabolic Bone Diseases of Japan.

² Present address: Department of Nephrology, Kanto-Teishin Hospital, 5–9-22 Higashi-Gotanda, Shinagawa, Tokyo 141-0022, Japan.

Received May 20, 1998.

Revised September 11, 1998.

Accepted September 22, 1998.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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