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A Novel Mutation in the Calcium-Sensing Receptor Gene in a Chinese Subject with Persistent Hypercalcemia and Hypocalciuria¹

Section of Biochemistry, Department of Pathology and Laboratory Medicine, Division of Endocrinology and Metabolism, Veterans General Hospital–Taipei, School of Medicine, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, ROC.112

Address correspondence and requests for reprints to: Tjin-Shing Jap, M.D., Section of Biochemistry, Department of Pathology and Laboratory Medicine, Veterans General Hospital–Taipei, Taiwan 112. E-mail: tsjap{at}vghtpe.gov.tw

	Abstract

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Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant disorder characterized by high penetrance of relatively benign, lifelong persistent hypercalcemia and hypocalciuria. By contrast, neonatal severe hyperparathyroidism represents a life-threatening form of hypercalcemia that can cause the early newborn mortality if immediate intervention is not undertaken. Both disorders are due to inactivation mutation of the human calcium-sensing receptor (CaSR) gene on chromosome 3q21-24. Up to now, more than 30 mutations in the CaSR gene associated with FHH have been described. In this study, we analyzed one 79-yr-old male with hypocalciuric hypercalcemia without siblings or children to compare with an additional group of 50 normal Chinese subjects in Taiwan. DNA sequence analysis of the CaSR gene was performed. The result showed that the proband had a heterozygous nonsense mutation in exon 7 of the CaSR gene at codon 648 (CGATGA/ArgTer). This mutation, located in the COOH-terminal of the first intracellular loop of the CaSR, predicts a markedly truncated protein.

We have identified a novel R648X mutation in the CaSR gene in one patient with FHH in Taiwan

	Introduction

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FAMILIAL HYPOCALCIURIC hypercalcemia (FHH) is an autosomal dominant trait with high penetrance, clinically manifesting a relatively benign, lifelong persistent hypercalcemia and hypocalciuria without hypercalcemicrelated complications (1, 2). The calcium-sensing receptor (CaSR) plays an important role in the regulation of PTH secretion and calcium metabolism. It is a cell surface protein that consists of 1078 amino acids and belongs to the superfamily of the seven-transmembrane domain, G proteincoupled receptors. The CaSR is expressed abundantly in the parathyroid gland, thyroid C cells, and kidney (3, 4). The vast majority of FHH kindreds have their genetic abnormality link to chromosome 3q21-24, the locus for the CaSR gene (5, 6). The identification of mutations in the CaSR in most patients with FHH and the development of hypocalciuric hypercalcemia or neonatal severe hyperparathyroidism in mice in which one or both CaSR genes have been inactivated (7) have clearly established the mutation of the CaSR gene as the basis of this syndrome (8, 9, 10, 11, 12). Most mutations in the CaSR associated with FHH are single point mutations localized either the region encoding the extracellular calcium-sensing domain, particularly exons 3 and 4, or to exon 7 (8, 9, 10), the region encoding the signal-transducing domain. These regions in the CaSR are highly conserved across species, and any variations in these residues may cause clinical outcomes. However, only two thirds of FHH kindreds have mutations in the CaSR gene. Mapping of another FHH locus to 19p13.3 suggests the possible involvement of additional related CaSRs or its signaling components (13). We report here a Han Chinese with FHH carrying a mutation in the CaSR gene.

	Research Design and Methods

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Subjects

One patient with hypocalciuric hypercalcemia was studied. An additional group of 50 normal Chinese subjects was recruited as controls. The study protocol was approved by the institutional review board of the hospital, and informed consent was obtained from each patient and normal subjects.

PCR amplification

The six coding exons (exons 2 through 7) of the CaSR gene were amplified by PCR using previously described primers and conditions (14).

Sequence analysis of the CaSR gene

All coding exons of the CaSR gene were first amplified by PCR. Automated DNA sequencing analysis was performed according to the manufacturer’s instructions (ABI 377-36 Autosequencer; Perkin-Elmer Corp., Foster City, CA).

Amplification-created restriction site (ACRS) analysis of mutation

To confirm the presence of the R648X mutation in the CaSR gene, we used modified PCR primers to introduce base substitutions adjacent to a codon of interest and thereby to create an artificial restriction site on only one allelic form (wild or mutant). We amplified exon 7 of the CaSR with a modified primer to create a MspI recognition site only if codon 648 contained the wild-type sequence (Fig. 1). After digestion, aliquots of the samples were electrophoresed on a 3% agarose gel.

View larger version (20K): [in this window] [in a new window]   Figure 1. A, Strategy for insertion of diagnostic MspI restriction site to detect the CaSR R648X mutation. The arrows indicate the nucleotide mismatch, whereas an asterisk indicates the position of the CaSR mutation. B, Restriction analysis of the ACRS amplication products of the CaSR gene exon 7. Each ACRS amplication product was untreated or treated with MspI restriction enzyme. mk, 25 bp DNA ladder; N, normal subjects; P, proband; -, PCR product without MspI digestion; +, PCR product after MSPI digestion. The size of the PCR product is 282 bp. MspI treatment would yield in two restriction products of 257 bp and 25 bp in the wild type and an undigested 282 bp fragment in R648X mutant. The 25 bp has run off the gel.

A 79-yr-old male, single, born in China and emigrated to Taiwan in 1949, visited the hospital because of polyuria. About 19 yr ago, he underwent a laboratory evaluation that showed hypercalcemia of 2.85 mmol/L (normal, 2.10–2.65) and phosphate of 0.94 mmol/L (normal, 0.68–1.52). Sonography of the neck showed no evidence of parathyroid enlargement. A neck exploration done later in 1986 failed to disclose any parathyroid pathology.

Recently, he again underwent a comprehensive evaluation of hypercalcemia. The plasma calcium showed 2.83 mmol/L (normal, 2.10–2.65), alkaline phosphatase activity of 87 U/L (normal, 10–100), intact PTH level of 14.8 ng/L (normal, <35.3), PTH-related protein less than 1.5 pmol/L (normal, <9.2), osteocalcin of 1.48 µg/L (normal, 0.11–0.33), testosterone of 8.09 nmol/L (normal, 8.18–34.53), and 24-h calcium was 32.4 mg with a ratio of calcium clearance to creatinine clearance ranging from 0.0045–0.0054. The N-Telopeptide was 35.7 mmol/mmol creatinine (normal, 20–80). Thyroid function and adrenal function were normal with serum free T₄ of 11.7 pmol/L (normal, 10.3–21.9), TSH of 2.57 mIU/L (normal, 0.4–4.0), and cortisol of 502 nmol/L (normal, 119–618). The bone mineral density of the femoral neck and lumbar spines performed with a Hologic QDR-4500A densitometer (Hologic, Inc., Waltham, MA) showed 0.828 g/cm² (T score, -0.1; Z score, 1.3) and 1.166 g/cm² (T score, 1.2; Z score, 1.5), respectively. He took no medications known to affect calcium metabolism, such as hydrochlorothiazide.

On physical examination he was 151 cm in height, 51 kg in weight, and had normotension. A previous operation scar over the neck was seen. Chest x-ray showed no evidence of tuberculosis or sarcoidosis. The biochemical and clinical evaluation was most consistent with FHH, and, thus, we analyzed his CaSR gene.

	Result

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The result showed that the proband had a heterozygous mutation at codon 648 of the CaSR gene (CGATGA/ArgTer) in exon 7.

ACRS

The R648X missense mutation did not alter a restriction cleavage site, so we used PCR to create a MspI restriction cleavage site (i.e. ACRS) to confirm this site of mutation. The wild-type PCR product (282 bp) is digested by MspI enzyme to yield two fragments of 257 and 25 bp, whereas the PCR product amplified from the mutant allele is not cleaved (Fig. 1). Restriction analysis of PCR products from 50 normal subjects all showed a wild-type pattern.

	Discussion

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This hypercalcemic patient underwent a neck exploration about 14 yr ago without definite diagnosis. In addition, the duration of hypercalcemia was more than 14 yr without development of hypercalcemic-related complications. Analysis of urinary excretion revealed hypocalciuria, and the calcium clearance to creatinine clearance ratio was less than 0.01. Other causes of hypercalcemia, such as hyperthyroidism or adrenal insufficiency, were excluded, and serum concentration of PTH and PTH-related protein were normal. These observations suggested the diagnosis of hypocalciuric hypercalcemia.

More than 30 inactivation mutations in the CaSR gene have been identified in patients with FHH or neonatal severe hyperparathyroidism, and activating mutations have been described in patients with autosomal dominant hypocalcemia. The prevalence of CaSR gene mutation associated with FHH is largely unknown; to our knowledge, the R648X mutation is the first one described in Chinese.

Several lines of evidence indicate that the R648X mutation is related causally to hypocalciuric hypercalcemia in this patient: 1) the mutation observed was not present in genomic DNA from 50 unrelated subjects, suggesting that the R648X is not a polymorphism; 2) the R648X residue in the exon 7 is evolutionally conserved in human, rat, mouse, pig, and cattle CaSR genes; and 3) the R648X mutation occurs in the first intracellular loop and predicts a markedly truncated protein. Other missense or truncated mutations in the carboxyl end of the CaSR, such as Arg796Trp, can cause FHH (8); thus, a truncated protein that lacks six of the seven membranes spanning helices and the intracellular loops and termini would certainly be expected to be inactive. As with other inactivating mutation in the patients with FHH, the R648X produces hypercalcemia and hypocalciuria reducing by half the number of normally functional CaSRs on the surface of parathyroid and kidney cells, respectively. A mutant CaSR with an Alu-repetitive element insertion at codon 876 has been reported in affected members of families with FHH (15). This insertion resulted in truncated protein that had molecular masses some 30 kD less than that of the wild-type CaSR and exhibited no Ca2+(I) responses to either Ca2+(o) or Gd3+(o). Thus, the R648X mutation is the most likely cause of hypercalcemia in the patient we described.

The patient we report has no family history of hypercalcemia and no surviving relatives. Thus, we cannot determine whether the mutation was inherited or arose de novo. Of all mutations identified to date, no mutational "hot spot" has been found in any ethnic groups or populations. Thus far, there does not seem to be any significant relationship between the nature of mutations observed (i.e. heterozygous or homozygous, frameshift, nonsense or missense) and the specific clinical features of hypercalcemia (age of onset, severity). The clustering of CaSR mutation at NH₂-terminal extracellular and membrane-spanning regions of the receptor protein suggests that these regions are critical functional domains serving calcium binding and signal transduction (12).

In conclusion, the long-standing benign hypercalcemia in this patient is due to R648X mutation of the CaSR gene. To our knowledge, it is the first case of FHH reported with CaSR gene mutation in China and Taiwan.

	Acknowledgments

 
We gratefully acknowledge the assistance of Prof. Michael A. Levine for critical reading of the manuscript.

	Footnotes

 
¹ Supported by grants from Medical Research—VGH 1999, Taiwan/ROC.

Received June 5, 2000.

Revised August 24, 2000.

Accepted September 15, 2000.

	References

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