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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 8 2716-2719
Copyright © 1997 by The Endocrine Society

Experimental Studies

Two Novel Missense Mutations in Calcium-Sensing Receptor Gene Associated with Neonatal Severe Hyperparathyroidism1

Megumi Kobayashi, Hiroyuki Tanaka, Kazuo Tsuzuki, Masumi Tsuyuki, Hiroshi Igaki, Youjirou Ichinose, Kunihiko Aya, Naho Nishioka and Yoshiki Seino

Department of Pediatrics, Okayama University Medical School (M.K., H.T., Y.I., K.A., Y.S.), Shikata-cho Okayama 700; the Department of Pediatrics, Social Insurance Chukyo Hospital (K.T., M.T.), 1–1-10 Sanjo-cho Minami-ku, Nagoya 457; and the Department of Surgery, Social Insurance Chukyo Hospital (H.I.), 1–1-10 Sanjo-cho Minami-ku, Nagoya 457, Japan

Address all correspondence and requests for reprints to: Hiroyuki Tanaka, M.D., Department of Pediatrics, Okayama University Medical School, Shikata-cho, Okayama 700, Japan. E-mail: hrtanaka{at}hospital.okayama-u.ac.jp


   Abstract
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 Abstract
Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 
Familial hypocalciuric hypercalcemia (FHH) is characterized by lifelong asymptomatic hypercalcemia without PTH hypersecretion and is inherited as an autosomal dominant trait with near 100% penetrance. In contrast, neonatal severe hyperparathyroidism (NSHPT) is a life-threatening disorder characterized by marked hypercalcemia and PTH hypersecretion. FHH/NSHPT results from inactivating mutations of the human calcium-sensing receptor (Casr) gene on chromosome 3q13.3–24. Nearly 30 different mutations of the Casr gene associated with FHH/NSHPT have been reported previously. In this report, genetic analysis of 1 Japanese NSHPT family revealed 2 novel mutations at codon 185 (CGA->TGA/Arg->Ter) in exon 4 of the Casr gene and at codon 670 (GGG->GAG/Gly->Glu) in exon 7. The Arg185Ter change was shown to occur in the proband’s unaffected father and paternal grandmother as well as in the proband. The other mutation in exon 7 was shown in the proband’s unaffected mother of Philippine origin as well as in the proband. This family is the first case of manifestation of more than 1 mutation in a proband’s chromosomes; 1 mutation was obtained from the unaffected father, and the other was from the unaffected mother. Our observations have given us important keys to help elucidate the structure-function relationships of the Casr.


   Introduction
Top
 Abstract
 Introduction
Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN calcium-sensing receptor (Casr), consisting of 1078 amino acids, is a cell surface protein belonging to the superfamily of the 7-membrane-spanning, G protein-coupled receptor, which is expressed in the parathyroids, thyroid C cells, and kidney (1, 2). Inactivating mutations on the Casr gene mapped to chromosome 3q21-q24 have been reported in familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) (3, 4, 5, 6, 7, 8). Recently, Heath et al. summarized mutations and benign polymorphisms of the Casr gene found in FHH. They indicated 2 distinct regions containing the mutations causing this disease. One is in the N-terminal extracellular domain, and the other is in or near the transmembrane domain (6). Interestingly, an activating mutation located in the N-terminal extracellular domain of the Casr gene has also been described in the hypocalcemic disorder inherited as an autosomal dominant trait (ADH) (9, 10). Thus, these regions of the Casr gene probably play an important role in the tight regulation of the extracellular calcium concentration.

The relationship between FHH and NSHPT has been discussed in previous reports (3, 4, 7, 8, 11, 12, 13). Pearce et al. performed a mutation search of the Casr gene in 9 unrelated kindreds with a total of 39 affected members with FHH and in 3 unrelated children with sporadic NSHPT (7). In 6 of 9 FHH kindreds, heterozygosity for a novel mutation (1 nonsense and 5 missense) was found. On the other hand, in the 3 children with NSHPT, 2 de novo heterozygous missense mutations and 1 homozygous frameshift mutation were identified. In this report, we describe 1 Japanese NSHPT family associated with 2 novel missense point mutations in the Casr gene.


   Experimental Subjects
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 Abstract
 Introduction
 Experimental Subjects
Materials and Methods
 Results
 Discussion
 References
 
The family is of Japanese origin, including the Philippina mother of the proband. The proband presented at 5 days of age with a history of poor feeding since birth. Initial examination displayed generalized muscle hypotonia and a serum calcium concentration of 26.1 mg/dL. At the age of 21 days, 4 hyperplastic parathyroid glands were excised. In all family members except the proband there was no evidence of hypercalcemia. Diagnosis was based on the clinical observation of the proband case, which indicated hypercalcemia with hypersecretion of PTH (MIM 239200) (14). The serum concentrations of total calcium, magnesium, inorganic phosphorus, and albumin were determined using an automated clinical chemistry analyzer. Plasma intact PTH (Allegro, Nichols Institute, San Juan Capistrano, CA) was quantified by immunoradiometric assay. The clinical data of the family are summarized in Table 1Go. We also examined 25 unrelated normal control subjects to detect the mutations responsible for the disorder.


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  		Table 1. Laboratory data and pedigree

 
Informed consent was obtained from all participants or their guardians in accordance with standards established by local institutions.


   Materials and Methods
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 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
Results
 Discussion
 References
 
PCR and sequence analysis

Leukocyte DNA was obtained from each of the family members and 25 normal subjects using the QIAamp Blood Kit (Qiagen, Chatsworth, CA). To detect the regions causing the disorder, 100 ng genomic DNA were amplified using a previously described protocol (7, 8). Each primer pair (10 pmol of each) was used for PCR of Casr gene exons 2 and 7 in a 100-µL reaction mixture containing 10 mmol/L Tris-HCl (pH 8.4), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.2 mmol/L deoxy-NTPs, and 1 U AmpliTaq DNA polymerase (Perkin-Elmer, Foster City, CA). For exon 4, the concentration of the primer pair was changed to 50 pmol for 100 ng genomic DNA. The primer sequences used to amplify exons 2, 4, and 7 were previously described (7, 8). After an initial denaturing at 95 C for 5 min, 40 cycles of PCR amplification were carried out in a Perkin-Elmer GeneAmp PCR System 2400 thermal cycler with the following protocol: 94 C for 30 s, 63 C for 30 s, and 72 C for 30 s. For exon 7, the annealing temperature was increased to 65 C. After amplification, the products were purified by spin dialysis using the Wizard DNA Clean-Up System (Promega, Madison, WI) for direct double strand DNA sequencing. Automated DNA sequencing analysis with fluorescent-labeled dideoxy-terminators (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin-Elmer) was performed according to the manufacturer’s instructions (373A sequencer, Applied Biosystems, Foster City, CA). To confirm heterozygosity at codon 185, some PCR products from exon 4 were subcloned into a pCR II vector using the Original TA Cloning Kit (Invitrogen Corp., San Diego, CA), and sequencing analysis was performed for each chromosome.

Restriction enzyme digestion

To confirm the suspected heterozygosity of the single base change at codon 670 found by direct sequencing analysis, the 424-bp PCR products from exon 7 of the 25 normal controls and the family members were digested with restriction endonuclease TaqI (Boehringer Mannheim, Indianapolis, IN) at 65 C for 1 h.


   Results
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 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
Discussion
 References
 
Automated direct sequence analysis after amplification of genomic DNA from exons 2, 4, and 7 of the Casr gene suggested that the proband had two novel mutations, showing heterozygosity at the first position of codon 185 and at the second site of codon 670, respectively (Fig. 1AGo). To confirm the heterozygous allele codon 185, the 335-bp of PCR products from exon 4 were subcloned into a pCR II vector. Direct sequence analysis using PCR products from subcloned vectors confirmed both sequences; one was normal, and the other was a mutant (data not shown). The wild-type DNA sequence at codon 185 was CGA encoding for an arginine residue, whereas the mutant sequence indicating TGA predicted a stop codon that introduced a truncated protein. The same heterozygous base change at codon 185 was identified in the proband’s father and paternal grandmother.



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  		Figure 1. Analysis of Casr gene exons 4 and 7. A: 1) Automated DNA sequence analysis of the 335-bp PCR products from exon 4 of the proband, the unaffected father and paternal grandmother, showing a novel heterozygous point mutation at codon 185 ({downarrow}) that produced a truncated protein (CGA->TGA/Arg->Ter); 2) sequence analysis of the amplified exon 7 indicating a novel heterozygous missense mutation at codon 670 ({downarrow}; GGG->GAG/Gly->Glu) for the proband and the unaffected mother. B: PCR/restriction enzyme digestion for detection of the heterozygous state at codon 670 in exon 7. The 424-bp PCR products from normal control subjects and the present NSHPT family members are shown in the even lanes. The TaqI digestion pattern in the wild-type and the mutant indicating 326- and 98-bp fragments are shown in lanes 3, 5, 7, 9, and 11. PCR products were sized relative to a marker generated from a HaeIII digest of {phi}X174 Rf DNA (lane 1).

 
The second novel mutation in our case was the GGG to GAG transition, predicting a Gly670Glu substitution in the receptor protein. As this base change created a TaqI restriction enzyme recognition site, restriction enzyme analysis was performed. TaqI digestion showed clearly that the proband was heterozygous for the mutation, as was his mother, who demonstrated a normal phenotype (Fig. 1BGo). In addition, analysis of 25 unrelated normal subjects showed an absence of this mutation (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
References
 
A large number of mutations in the Casr gene associated with FHH, NSHPT, or ADH have previously been described. The mutation at codon 185 has been described in two FHH families. However, both reported mutations were G to A at the second position of this codon, resulting in the substitution of an Arg for a Glu (6, 8). A missense mutation at codon 670 (GGG to AGG transition) was also reported in one FHH family by Pearce et al., resulting in the alteration from a Gly to an Arg (7).

Previous functional studies of Casr have been focused on the first half of the N-terminal extracellular domain because this region was hypothesized to be a primary ligand-binding domain. Ten known inactivating mutations and 1 activating Casr mutation were expressed in Xenopus and/or HEK293 cells previously to elucidate which receptor functions were affected by the mutations (6, 8, 15). According to those results, 10 inactivating mutations causing FHH or NSHPT reduced functional receptor activity, whereas an activating mutation in ADH increased the affinity of the receptor for its agonist (6, 15). With 1 exception, these inactivating mutations were all reported to be in the first half of the N-terminal extracellular domain. Moreover, according to a comparison of the response curves of intracellular Ca ion to extracellular Ca ion concentration in the Arg185Glu mutation and Arg795Trp, Bai et al. suggested that the primary abnormality in the receptor function was in ligand-binding activity rather than in signal transduction (15). In experiments using Casr-deficient mice (Casr+/+, Casr±), phenotypic similarities between Casr± mice and FHH and between Casr-/- mice and NSHPT were confirmed, and the results suggested that reduction of the number of functional receptor molecules on the cell surface may cause these disorders (16). However, despite the presence of the truncated Casr protein resulting from the Arg to Ter amino acid substitution at codon 185, both the father and paternal grandmother showed a normal phenotype and the proband with both mutations (Agr185Ter and Gly670Glu) demonstrated severe hypercalcemia. Thus, our NSHPT case indicates that previously reported mutations may not simply result in loss of function, but may, in fact, exert some dominant negative effects.

In many cases reported previously, FHH is considered to be an autosomal dominant disorder with a high penetrance (17, 18, 19). The two novel mutations in this report appear to act as a recessive form, because the heterozygotes did not show any clinical manifestation. The serum calcium level depends on calcium intake and intestinal absorption; therefore, low calcium intake in the Japanese population (542 ± 8 mg/day) may have masked hypercalcemia in the father and paternal grandmother. Dawson-Hughes et al. described the link between polymorphism at the vitamin D receptor gene and rates of calcium absorption during low calcium intake (20). Their findings suggest that an environmental factor such as calcium intake also play an important role in determining serum calcium homeostasis.

Identification of the compound novel missense mutations, including one truncated change in the Casr gene, helps to clarify the relationship between ligand binding and G protein coupling.


   Acknowledgments
 
We thank R. Abe for expert secretarial assistance.


   Footnotes
 
1 This work was supported by grants from the Ministry of Health and Welfare of Japan and the Ministry of Education of Japan. Back

Received December 6, 1996.

Revised April 4, 1997.

Accepted April 21, 1997.


   References
Top
 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brown EM, Gamba G, Riccardi D, et al. 1993 Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature. 366:575–580.[CrossRef][Medline]
  2. Garrett JE, Capuano IV, Hammerland LG, et al. 1995 Molecular cloning and functional expression of human parathyroid calcium receptor cDNA. J Biol Chem. 270:12919–12925.[Abstract/Free Full Text]
  3. Janicic N, Pausova Z, Cole DEC, Hendy GN. 1995 Insertion of an Alu sequence in the Ca2+-sensing receptor gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Am J Hum Genet. 56:880–886.[Medline]
  4. Chou YHW, Pollak MR, Brandi ML, et al. 1995 Mutations in the human Ca2+-sensing receptor gene that cause familial hypocalciuric hypercalcemia. Am J Hum Genet. 56:1075–1079.[Medline]
  5. Aida K, Koishi S, Inoue M, Nakazato M, Tawata M, Onaya T, et al. 1995 Familial hypocalciuric hypercalcemia associated with mutation in the human Ca2+-sensing receptor gene. J Clin Endocrinol Metab. 80:2594–2598.[Abstract]
  6. Heath H III, Odelberg S, Jackson CE, et al. 1996 Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains. J Clin Endocrinol Metab. 81:1312–1317.[Abstract]
  7. Pearce SHS, Trump D, Wooding C, et al. 1995 Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. J Clin Invest. 96:2683–2692.
  8. Pollak MR, Brown EM, Chou YHW, et al. 1993 Mutations in the human Ca2+-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell. 75:1297–1303.[CrossRef][Medline]
  9. Pollak MR, Brown EM, Estep HL, et al. 1994 Autosomal dominant hypocalcaemia caused by a Ca2+-sensing receptor gene mutation. Nat Genet. 8:303–307.[CrossRef][Medline]
  10. Pearce SHS, Williamson C, Kifor O, et al. 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]
  11. Marx SJ, Attie MF, Spiegel AM, Levine MA, Lasker RD, Fox M. 1982 An association between neonatal severe primary hyperparathyroidism and familial hypocalciuric hypercalcemia in three kindreds. N Engl J Med. 306:257–264.[Abstract]
  12. Marx SJ, Fraser D, Rapoport A. 1985 Familial hypocalciuric hypercalcemia: mild expression of the gene in heterozygotes and severe expression in homozygotes. Am J Med. 78:15–22.[Medline]
  13. Pollak MR, Chou YHW, Marx SJ, et al. 1994 Familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism: effect of mutant gene dosage on phenotype. J Clin Invest. 93:1108–1112.
  14. Mckusick V. 1994 Mendelian inheritance in man: #239200. In: McKusick V, ed. Hyperparathyroidism, neonatal severe primary, 11th ed. Baltimore and London: Johns Hopkins University Press; vol 2:1916.
  15. Bai M, Quinn S, Trivedi S, et al. 1996 Expression and characterization of inactivating and activating mutations in the human Ca2+0-sensing receptor. J Biol Chem. 271:19537–19545.[Abstract/Free Full Text]
  16. Ho C, Conner DA, Pollak MR, et al. 1995 A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat Genet. 11:389–394.[CrossRef][Medline]
  17. Foley Jr TP, Harrison HC, Arnaud CD, Harrison HE. 1972 Familial benign hypercalcemia. J Pediatr. 81:1060–1067.[CrossRef][Medline]
  18. Marx SJ, Attie MF, Levine MA, Spiegel AM, Downs Jr RW, Lasker RD. 1981 The hypocalciuric or benign variant of familial hypercalcemia: clinical and biochemical features in fifteen kindreds. Medicine. 60:397–412.[Medline]
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  20. Dawson-Hughes B, Harris SS, Finneran S. 1995 Calcium absorption on high and low calcium intakes in relation to vitamin D receptor genotype. J Clin Endocrinol Metab. 80:3657–3661.[Abstract]



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