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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 12 5936-5941
Copyright © 2004 by The Endocrine Society

CLINICAL CASE SEMINAR

Severe Hypercalcemia in a 9-Year-Old Brazilian Girl Due to a Novel Inactivating Mutation of the Calcium-Sensing Receptor

Kozue Miyashiro, Ilda Kunii, Thais Della Manna, Hamilton C. de Menezes Filho, Durval Damiani, Nuvarte Setian and Omar M. Hauache

Laboratório de Endocrinologia Molecular (K.M., I.K., O.M.H.), Disciplina de Endocrinologia, Escola Paulista de Medicina–Universidade Federal de São Paulo 04039-032; and Unidade de Endocrinologia Pediátrica do Instituto da Criança–Faculdade de Medicina da Universidade de São Paulo (T.D.M., H.C.d.M.F., D.D., N.S.), 05403-900 São Paulo, Brazil

Address all correspondence and requests for reprints to: Omar M. Hauache, Laboratorio de Endocrinologia Molecular, Disciplina de Endocrinologia, Escola Paulista de Medicina–Universidade Federal de São Paulo, Rua Pedro de Toledo, 781, 12° andar, 04039-032 São Paulo–SP, Brazil. E-mail: omar.hauache{at}fleury.com.br.


   Abstract
Top
 Abstract
Introduction
 Patient and Methods
 Results
 Discussion
 References
 
Familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) are consequent to inactivating mutations of the calcium-sensing receptor (CaR) gene. FHH is usually associated with heterozygous inactivating mutations of the CaR gene, whereas NSHPT is usually due to homozygous inactivation of the CaR gene. FHH is generally asymptomatic and is characterized by mild to moderate lifelong hypercalcemia, relative hypocalciuria, and normal intact PTH, whereas individuals with NSHPT frequently show life-threatening hypercalcemia. In this study, we report a novel inactivating mutation of the CaR gene, identified in a 9-yr-old Brazilian girl who was found to be severely hypercalcemic during investigation of a 6-month history of headaches and vomits. Direct sequencing of the CaR gene from this patient showed a novel homozygous mutation (L13P) in exon 2. Functional characterization by intracellular calcium measurement by fluorometry showed that the mutant receptor had a dose-response curve shifted to the right relative to that of wild type. The proband’s consanguineous parents, who had mild asymptomatic hypercalcemia, showed the same mutation in the heterozygous form. The mutation described in this study is the inactivating missense mutation present at the most N-terminal end among the known CaR missense mutations. This study reinforces the fact that patients with homozygous inactivation of the CaR gene may present with severe hypercalcemia in different phases of life.


   Introduction
Top
 Abstract
 Introduction
Patient and Methods
 Results
 Discussion
 References
 
THE EXTRACELLULAR CALCIUM-SENSING receptor (CaR) is a G protein-coupled receptor that plays an essential role in the regulation of extracellular calcium homeostasis (1). Cloning of the CaR cDNA (2) was immediately followed by the association of genetic human diseases with inactivating and activating mutations of the CaR gene: familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) are caused by inactivating mutations of the CaR gene, whereas autosomal dominant hypocalcemia is secondary to activating mutations of the CaR gene (3, 4, 5).

In FHH and NSHPT, loss-of-function mutations in the CaR gene lead to generalized resistance to extracellular calcium. FHH is an autosomal dominant disorder characterized by asymptomatic, mild to moderate lifelong hypercalcemia, relative hypocalciuria, and normal intact PTH, usually associated with the inheritance of a single copy of a mutated CaR gene. When two inactive gene copies are inherited, the homozygous individuals may present with NSHPT, displaying life-threatening hypercalcemia in the neonatal period. Most of these inactivating mutations associated with FHH and NSHPT are missense mutations and are mainly found in the first half of the long extracellular domain, which may constitute the calcium-binding site (6, 7, 8).

In this study, we describe a 9-yr-old Brazilian girl who was found to be severely hypercalcemic. Sequencing of the CaR gene from this patient revealed a novel homozygous mutation (L13P), present in the heterozygous form in her consanguineous parents. Functional studies demonstrated that this mutation strongly impaired the function of this mutant CaR, clearly associating this mutation with the severe hypercalcemia observed on this patient.


   Patient and Methods
Top
 Abstract
 Introduction
 Patient and Methods
Results
 Discussion
 References
 
Parents involved in this study gave fully informed consent to participate in the study, which was approved by the Ethical Committee of Escola Paulista de Medicina, Universidade Federal de São Paulo.

Case description

J.G.M. was a 9-yr-old Brazilian girl admitted to the hospital with a history of periorbicular headache and vomiting, which were preceded by nausea and attenuated with sleeping and common analgesics for the last 6 months. After a treatment for sinusitis, she was referred to a neuropediatrician for investigation when a cranial computed tomography revealed calcifications in the falx cerebri and tentorium cerebelli. Her parents were consanguineous, but there was no family history of metabolic disorders. The patient has developed normally since birth. On physical examination she looked thin, frail, and chronically ill. Height was 132 cm (height SD score = –0.42), and weight was 21.5 kg (weight SD score = –1.81). The remainder of the examination was normal.

Laboratorial evaluation revealed hypercalcemia [total calcium 14.1 mg/dl (reference values 8.2–10.2 mg/dl) and ionized calcium 2.1 mmol/liter (reference values, 1.12–1.32 mmol/liter], hypophosphatemia [phosphorus 2.1 mg/dl (reference values, 3.5–6.0 mg/dl)], serum magnesium 1.12 mg/dl (reference values, 1.9–2.5 mg/dl); intact PTH levels ranged from 50 to 110 pg/ml (reference values,11–62 pg/ml) and relative hypocalciuria (urinary calcium to creatinine clearance ratio, 0.0047; reference range, >0.02). There were no signs of osteopenia on radiographic investigation. Parathyroid radioisotope scan with technetium sestamibi demonstrated three enlarged parathyroid glands. Table 1Go illustrates the biochemical evaluation performed in the patient and her family. Both her parents disclosed mild asymptomatic hypercalcemia and relative hypocalciuria, whereas her brother was normocalcemic.


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  		TABLE 1. Biochemical characteristics of the reported patient with L13P homozygously expressed mutation of the CaR gene and her family

 
The patient was treated with hydration with normal saline plus furosemide (2 mg/kg·d), acetazolamide (60 mg/kg·d), and phosphorus (60 mg/kg·d). After 2 wk, serum calcium levels still remained very high. Bisphosphonate treatment was initiated with pamidronate on 3 consecutive days (0.5, 1, and 1 mg/kg) and later on with oral alendronate (10 mg/d). A transient decrease in serum calcium was observed. Surgical cervical exploration and total parathyroidectomy was then indicated. Ten small fragments (1 mm) of parathyroid tissue were implanted in the left forearm at same time. Dimensions of the four parathyroid glands removed were approximately 10 times bigger than commonly seen in children and averaged 11 x 7 x 5 mm. Parathyroid histopathology was compatible with parathyroid hyperplasia.

Soon after surgery, serum calcium levels decreased, followed by a marked clinical improvement. At the time of discharge, laboratory data were: serum total calcium, 10.4 mg/dl, ionized calcium, 1.23 mmol/liter; serum phosphorus, 4.15 mg/dl; magnesium, 1.83 mg/dl; and serum PTH, 33 pg/ml.

One month after surgery, serum total calcium rose to 12.35 mg/dl and serum ionized calcium to 1.38 mmol/liter. Serum intact PTH collected from the right forearm was 66 pg/ml, whereas from the left forearm was 150 pg/ml. All the implanted fragments were then removed, but serum calcium levels remained high, and there was an upward trend in PTH concentrations. No PTH level gradient was observed anymore (right forearm PTH, 126 pg/ml; and left forearm PTH, 118 pg/ml) and a cervical radioisotope scan with technetium sestamibi was negative. She is currently asymptomatic and under no medications. On her last evaluation, serum total calcium was 11.4 mg/dl.

Sequence analysis of the CaR gene

Leukocyte DNA was isolated using standard methods. Twelve pairs of primers (3, 9) were used to amplify exons 2–7 of the CaR gene from the proband, which encode the CaR protein. DNA sequence of the CaR gene (including its splicing sites) of the proband was analyzed by PCR amplification of these coding exons of the CaR gene and direct sequencing of PCR products using the BigDye terminator cycle sequencing kit and automated sequencer ABI PRISM 377 and 3100 (PE Applied Biosystems, Foster City, CA). Sequencing was performed for both strands.

Restriction enzyme analysis

Cosegregation of the DNA sequence abnormality in affected family members was confirmed by digesting PCR fragments with restriction enzyme BanI. The mutation L13P created a specific restriction enzyme site not present in the wild-type sequence. Restriction enzyme analysis was also performed in 50 healthy controls.

Site-directed mutagenesis

The human CaR (hCaR) cDNA construct subcloned in pCR3.1 vector has been described (10). Site-directed mutagenesis was performed using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer’s instructions. A pair of complementary primers with 30–40 bases was designed for the L13P mutagenesis (sequences available on request), and the desired point mutation was placed in the middle of the primers. Parental hCaR cDNA inserted in pCR3.1 was amplified using Pfu Turbo DNA polymerase with these primers for 15 cycles in a DNA thermal cycler (Perkin-Elmer Corp., Norwalk, CT). After digestion of the parental DNA with DpnI for 1 h, the amplified DNA with the nucleotide substitution incorporated was transformed into Escherichia coli (DH-5{alpha} strain). The mutated nucleotide was confirmed by automated DNA sequencing.

Transient transfection of wild-type and mutant receptor cDNAs in human embryonic kidney (HEK)-293 cells

Calcium receptor cDNA in pCR3.1 (kindly provided by Dr. Allen M. Spiegel, National Institutes of Health) was prepared with a maxi Plasmid Maxi DNA preparation kit (QIAGEN Inc., Chatsworth, CA) and was introduced into HEK-293 cells by the Lipofectamine transfection method (Life Technologies, Inc., Gaithersburg, MD). For transfection, 12 µg of the plasmid DNA were diluted in DMEM and mixed with diluted Lipofectamine. The mixture was then incubated at room temperature for 30 min. The DNA-Lipofectamine complex was further diluted in 6 ml of serum-free DMEM and added to 80–90% confluent HEK-293 cells plated in 75-cm2 flasks. After 5 h of incubation, 15 ml DMEM containing 10% fetal bovine serum were added, and the media were replaced 24 h after transfection with complete DMEM containing 10% fetal bovine serum. Membrane protein extraction for immunoblotting and measurement of intracellular calcium by fluorometry were performed 48 h after transfection. Values shown regarding measurement of intracellular calcium and immunoblotting analysis are representative ones from six independent experiments.

Measurement of intracellular calcium (Ca2+i) by fluorometry

Function of the wild-type and mutated CaR were assessed using dual wave-length fluorometry 48 h after transfection by measuring the increases in Ca2+i in response to different concentrations of extracellular calcium. HEK-293 cells transfected with wild-type or mutant CaR cDNAs were loaded for 2 h at room temperature with fura-2/AM (Molecular Probes Inc., Eugene, OR) in Hank’s balanced salt solution (118 mM NaCl, 4.6 mM KCl, 10 mM D-glucose, and 20 mM HEPES adjusted to pH 7.2 by NaOH). Fura-2 loaded cells were washed, pelleted, and placed on ice. Aliquots of cells were resuspended in 2 ml Calcium-free Hank’s balanced salt solution in a UV grade fluorometer cuvette on fluorescence spectrophotometer (Photon Technology International, South Brunswick, NJ). Calcium chloride was added to give indicated extracellular calcium concentrations. 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 Ca2+i.

Immunoblotting analysis

Confluent cells in 75-cm2 flasks were rinsed with ice-cold PBS and scraped on ice in buffer B containing 20 mM Tris-HCl (pH 6.8), 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, and 1% Triton X-100 with freshly added protease inhibitors cocktail (Complete, Roche Molecular Biochemicals, Indianapolis, IN). To prevent nonspecific disulfide bond formation during protein extraction, the intact cells were incubated and washed in PBS containing 50 mM iodoacetamide, and 10 mM iodoacetamide were included in the lysis buffer. Proteins were eluted with gel-loading sample buffer containing ß-mercaptoethanol as reducing agent and run on SDS-PAGE and then analyzed on immunoblots stained with anti-hCaR monoclonal antibody ADD (kindly provided by Dr. Paul K. Goldsmith and Dr. Allen M. Spiegel, NIH) to detect total hCaR immunoreactive species. The protein content of each sample was determined by the modified Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA), and 60 µg of protein per lane were separated on 6% SDS-PAGE. The proteins on the gel were electrotransferred to nitrocellulose membrane and incubated for 90 min with protein A-purified mouse monoclonal anti-hCaR antibody ADD (raised against a synthetic peptide corresponding to residues 214–235 of hCaR protein at a dilution of 1:10,000). After washing three times for 15 min with Tris-buffered saline (TBS) with Tween 20 [containing 0.05 M Tris (pH 8.0) and 0.05 M NaCl with 0.1% Tween 20], the membrane was incubated for 90 min with a secondary goat antimouse antibody conjugated to horseradish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD) at a dilution of 1:5000. After washing three times for 15 min with TBS with Tween 20 and once for 15 min with TBS, the hCaR protein was detected with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington Heights, IL).


   Results
Top
 Abstract
 Introduction
 Patient and Methods
 Results
Discussion
 References
 
DNA sequencing and restriction enzyme analysis of CaR gene

Sequencing of CaR from the proband showed a novel mutation (L13P) in exon 2 of the CaR gene. In this context, nucleotide sequence analysis identified a T->C point mutation at nucleotide 38, resulting in the conversion of CTC (leucine) to CCC (proline) at codon 13 of the CaR gene. This mutation is located in the 19-amino acid signal peptide region of CaR and was homozygously expressed. Both her parents, who had asymptomatic mild hypercalcemia, disclosed the same mutation (L13P) on CaR but heterozygously expressed (Fig. 1Go). This mutation was confirmed by restriction endonuclease analysis using the enzyme BanI. The T->C transition creates a BanI restriction enzyme site (G{downarrow}GCACC). In the mutant sequence, the digestion results in two products of 189 and 61 bp (Fig. 2Go). Restriction analysis of PCR products from 50 healthy subjects all showed a wild-type pattern.



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  		FIG. 1. DNA sequence for CaR gene in the patient with severe hypercalcemia and her parents. Automated sequence analysis of PCR-amplified genomic DNA from the proband shows a homozygous T to C transition at position 38 in exon 2 of the CaR gene, changing leucine (CTC) to proline (CCC). The same mutation was heterozygously expressed in her parents.

 


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  		FIG. 2. BanI restriction enzyme digestion: T->C transition creates a BanI restriction enzyme site (G{downarrow} GCACC), which is not present in the wild-type sequence. In the heterozygous mutant sequence, the digestion results in two products of 189 and 61 bp. M, 100-bp molecular marker. Lane 1 corresponds to the patient’s sample (homozygous mutation L13P); lanes 2 and 3 correspond to the mother’s and father’s samples, respectively (heterozygous mutation). Lanes 4 and 5 correspond to a normal and undigested sample, respectively.

 
Functional study of L13P CaR mutant

We studied the function of the CaR harboring a L13P mutation and compared the result with that verified for the wild-type CaR.

Figure 3Go shows the responses of wild-type and mutated CaR to various concentrations of extracellular calcium. L13P is shown to strongly impair the CaR function, which is compatible with the severe hypercalcemia presented by the reported patient. When this mutation is heterozygously expressed as was found in her parents, mild hypercalcemia was observed. Reduction of maximal activity for the L13P mutant was also observed when compared with the results obtained for the wild-type CaR.



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  		FIG. 3. Extracellular calcium evoked increases in Ca2+i in fura-2-loaded and immunoblot (right) of CaR in transiently transfected HEK-293 cells expressing wild-type CaR L13P and mutant L13P CaR cDNAs. Nontransfected HEK-293 cells are shown as negative controls. Transfection, measurement of Ca2+i by fluorometry, and immunoblotting analysis with monoclonal anti-CaR ADD were performed as described in Patient and Methods. Results of measurement of Ca2+i by fluorometry are expressed as percent of wild-type CaR’s maximal response. The immunoblot was done with cells from the same transfection as the cells used for the functional assay. Values shown are representative ones from six independent experiments.

 
Immunoblotting analysis

To verify whether the impairment of function observed for the L13P mutant was caused by altered expression, we transfected hCaR cDNA into HEK-293 cells and analyzed receptor expression on immunoblots stained with anti-hCaR monoclonal antibody ADD to detect total CaR immunoreactive species. Under reducing conditions, ADD detected two major bands of 130 and 150 kDa for the wild-type and the mutant L13P CaR, but the mutant 150-kDa band was expressed at lower levels when compared with the wild-type 150-kDa band (Fig. 3Go). Previous studies have shown that the monomeric form 150-kDa band represents hCaR forms expressed at the cell surface and modified with N-linked, complex carbohydrates; the 130-kDa band represents high mannose-modified forms, trapped intracellularly and sensitive to Endo-H digestion (10, 11, 12).


   Discussion
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
References
 
The present study describes a new inactivating mutation in the CaR gene that causes leucine to proline substitution at codon 13 of the CaR (L13P). When this mutation was homozygously expressed, severe hypercalcemia was the hallmark of our reported patient. Her consanguineous parents had mild asymptomatic hypercalcemia and were found to have the same mutation, heterozygously expressed.

To date, 112 naturally occurring mutations (64 inactivating and 48 activating) in the CaR gene have been published, including 17 inactivating and 15 activating mutations that are recurrent (13). Most of these mutations are localized in the first half of the long extracellular domain of this receptor, which may constitute the calcium-binding site (7, 8, 14). The mutation described in this study is the inactivating missense mutation which is present at the most N-terminal end among the known CaR missense mutations.

Interestingly, two additional similar cases have been already described (15, 16), in which the probands were born to consanguineous parents and presented with severe hypercalcemia in the adulthood. These probands also had homozygous inactivating mutations [P39A (15) and Q27R (16)] of the CaR and their respective parents were heterozygotes for the same mutation. Curiously, these mutations were very close to the N terminus of the extracellular domain of the CaR, and this observation may indicate that this initial region, close to the N-terminal end, has an important role for calcium binding and/or activation of the receptor. Moreover, this finding may also be indicative that missense mutations occurring at this region are associated with higher tolerance to hypercalcemia in early periods of life, including the neonatal period and early infancy.

FHH is inherited as an autosomal-dominant trait with a high penetrance of more than 90%. FHH is a rare disorder of mineral metabolism characterized by lifelong, mild to moderate but usually asymptomatic hypercalcemia. Another feature of this disease is the presence of inappropriately low rates of urinary calcium excretion (a calcium to creatinine clearance ratio of < 0.01) and nonsuppressed circulating levels of PTH, regardless the presence of hypercalcemia (17, 18, 19).

In short, the diagnosis of FHH can be established by documenting the combination of a low urinary calcium to creatinine clearance ratio; a normal PTH level; and the autosomal-dominant inheritance of mild, asymptomatic hypercalcemia. Due to severe primary hyperparathyroidism with enlargement of all four parathyroid glands, the degree of hypercalcemia in NSHPT is usually more severe than that observed in FHH, and this disorder can be fatal if parathyroidectomy is not carried out within the first weeks of life (20). Some infants with NSHPT represent the homozygous form of FHH (21),or, as seen in one case, a compound heterozygote in which a different inactivating CaR mutation was inherited from each of the parents (22). In some cases, NSHPT may be caused by the presence of heterozygous-inactivating mutations of the CaR, either in a familial setting or as a de novo mutation in the offspring of normal parents, and possibly in these cases the mutant CaR can exert a dominant negative action, impairing the function of the normal receptor (23). In our study, the mutant CaR L13P was homozygously expressed in the described proband. The consequent phenotype observed when she was 9 yr old consisted of severe hypercalcemia in the presence of relative hypocalciuria and mildly elevated levels of PTH. Her consanguineous parents disclosed the same mutation in a heterozygous form and clinically had mild hypercalcemia, inappropriate normal levels of PTH and relative hypocalciuria, suggestive of FHH. Persistence of hypercalcemia in the patient after parathyroidectomy may be related to the fact that fragments of parathyroid tissue were implanted in her left forearm and could not be totally removed later on. However, the fact that no PTH level gradient was observed anymore between both forearms may suggest the additional possibility of the presence of ectopic parathyroid tissue. Even though she is still hypercalcemic (serum total calcium levels ranging from 11.0 to 12.0 mg/dl), she is currently asymptomatic, which may reflect a chronic clinical adaptation to higher calcium levels.

Impairment of extracellular calcium sensing by the CaR in FHH patients has been proven by functional studies in which HEK-293 cells were transfected with CaRs bearing FHH mutations, whereas described natural activating mutations in patients with autosomal dominant hypocalcemia were shown to increase the CaR sensitivity to extracellular calcium (12, 24, 25, 26, 27). The functional study of the mutant CaR L13P showed a severely impaired activity. Cell surface expression for the L13P mutant was lower than the wild-type CaR expression, suggesting that the impaired function of the mutant CaR is partially due to inadequate cell surface expression.

In conclusion, this case reinforces that inactivation of the CaR gene may be an important cause of hypercalcemia in the pediatric patient and should be suspected in the setting of hypercalcemia, nonsuppressible levels of PTH, and relative hypocalciuria. The description of this case also reinforces that the disease may present with a more variable spectrum than previously believed. Patients with homozygous inactivation of the CaR gene may present with severe hypercalcemia in late phases of life. Based on reports of us and others (15, 16), it is interesting to speculate that homozygous mutations found in the very beginning N terminus portion of the CaR may be associated with the phenotype of severe hypercalcemia presenting later in life, which may be highly suggestive of a possible genotype-phenotype association.


   Acknowledgments
 
We are grateful to Maria Adelaide Cunha (Disciplina de Nefrologia, Escola Paulista de Medicina-Universidade Federal de São Paulo) for assistance with fluorometry functional assays.


   Footnotes
 
This work was supported by Grant 00/06480-4 from the Fundação de Amparo à Pesquisa do Estado de São Paulo.

Preliminary results from this work were presented in abstract form at the 24th Annual Meeting of the American Society for Bone and Mineral Research, San Antonio, Texas, September 2002.

Abbreviations: Ca2+i, Intracellular calcium; CaR, calcium-sensing receptor; FHH, familial hypocalciuric hypercalcemia; hCaR, human CaR; HEK, human embryonic kidney; NSHPT, neonatal severe hyperparathyroidism; TBS, Tris-buffered saline.

Received June 2, 2004.

Accepted September 8, 2004.


   References
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 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
 References
 

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S. Pidasheva, L. Canaff, W. F. Simonds, S. J. Marx, and G. N. Hendy
Impaired cotranslational processing of the calcium-sensing receptor due to signal peptide missense mutations in familial hypocalciuric hypercalcemia
Hum. Mol. Genet., June 15, 2005; 14(12): 1679 - 1690.
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