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CLINICAL CASE SEMINAR |
Dipartimento di Endocrinologia e Metabolismo, Università degli Studi di Pisa, 56124 Pisa, Italy
Address all correspondence and requests for reprints to: Claudio Marcocci, M.D., Dipartimento di Endocrinologia e Metabolismo, Università di Pisa, Via Paradisa 2, 56124 Pisa, Italy. E-mail: c.marcocci{at}endoc.med.unipi.it.
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Abstract |
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Introduction |
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Familial hypocalciuric hypercalcemia (FHH) is a benign, autosomal, dominantly inherited disease characterized by a lifelong persistent hypercalcemia and hypocalciuria, without hypercalcemic-related complications (3). The disease is associated with loss-of-function mutation of the calcium-sensing receptor (CaR) gene (3). The CaR belongs to the superfamily of the G protein-coupled receptors. Calcium binding to the receptor stimulates phospholipase C activity, leading to accumulation of inositol trisphosphate, which in turn, increases cytoplasmic calcium. Activation of the CaR in the parathyroid cells inhibits PTH secretion, whereas in the kidney it increases renal calcium excretion (3).
In this report we describe a 45-yr-old woman with metastatic breast cancer in whom the finding of hypercalcemia was initially attributed to HCM and, accordingly, treated with bisphosphonates. A more detailed evaluation suggested the diagnosis of FHH, which was confirmed by the familial nature of hypercalcemia and the identification of a new missense inactivating mutation of the CaR.
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Case Report |
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), a 45-yr-old woman, was referred to our department in April 2001 for evaluation of hypercalcemia. The patient underwent mastectomy and local lymph node resection for breast cancer in 1987 and was subsequently treated with external irradiation, chemotherapy, and GnRH analogs. In 1996, the patient received a further course of chemotherapy for histologically confirmed pleural metastasis and suspected bone and lung metastases. Thereafter, she was in good health, and imaging studies showed an improvement of the pleural lesion with no progression of the bone and lung lesions. In 1999, at the age of 43 yr, routine laboratory investigations disclosed a mild elevation of total serum calcium (s-Ca) [10.4–10.8 mg/dl (2.60–2.70 mmol/liter)]; serum breast cancer markers were normal, and a thorough examination (abdominal ultrasound, total-body computed tomography scan, and bone scintigraphy) showed no change in the lesions previously detected, and no evidence of further metastases was found. Treatment with iv pamidronate was started (90 mg every 4 wk) and maintained until the patient was seen at our clinic. Repeated measurement of s-Ca at the time of each pamidronate administration did not show any significant change of s-Ca concentration [mean, 11.0 mg/dl (2.75 mmol/liter)]. In three of 17 measurements, the s-Ca was in the upper limit of normal range.
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The finding of long-standing mild hypercalcemia with normal PTH levels and no convincing evidence of bone metastases suggested the possibility that the patient might have FHH. Family history was apparently negative for hypercalcemia. Twenty-four-hour urinary calcium clearance to creatinine clearance ratio was 0.007, a value supporting the diagnosis of FHH. The patient has been followed regularly to date, and she is in good health with no evidence of recurrent breast cancer.
Seven family members were available for further investigations and gave their informed consent for biochemical (n = 5) and genetic studies. s-Ca and ionized calcium, PTH, creatinine, phosphate, magnesium, 24-h urinary calcium, and creatinine were measured. Whole blood in EDTA was also collected from the patient and all family members.
Our internal review board approved the study.
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Materials and Methods |
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Serum PTH and ionized calcium were measured, as previously described (4). s-Ca, phosphorus, creatinine, and urinary calcium and creatinine were measured using standard methods. PTHrp was measured using a commercial kit (Nichols Diagnostics, San Clemente, CA). Normal values are reported in Table 1.
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Genomic DNA was isolated from peripheral blood samples using standard procedures. All coding exons and their flanking exon-intron boundaries of the CaR gene were amplified by PCR from the proband as described previously (5). The region of interest detected in the proband (see Results) was also amplified in the family members. PCR products were purified using the Concert Ready purification kit (Life Technologies, Milan, Italy), according to manufacturer’s instructions, and sequenced by a Dye-Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA). The DNA sequences of both strands were determined on an autosequencer (ABI PRISM 310, Genetic Analyzer, PE Applied Biosystems). To confirm the presence of a CaR mutation, the mutation was subcloned in a plasmid, and sequences were repeated on individual clones. The region of interest was also amplified and sequenced in 50 unrelated healthy Italian subjects (100 chromosomes).
Site-directed mutagenesis, transient expression of the wild-type (WT) and mutated CaR in COS-7
Site-directed mutagenesis to produce the CaR mutant and transient expression of the WT and mutated CaR in COS-7 cells were performed as described previously (4). Briefly, COS-7 cells were transfected with 625 ng of cDNA by the diethylaminoethyl-dextran method followed by a 2-min 10% dimethyl-sulfoxide shock after 3 h. For coexpression experiments, equal amounts (625 ng) of WT and mutant CaR cDNA were mixed and used to transfect COS-7 cells. The primer sequences to carry out site-directed mutagenesis were as follows:
Primers B and C are completely overlapping; bold, underlined letters indicate the mismatched bases that introduce the mutation.
Functional studies
Forty-eight hours after transfection, cells were used for inositol phosphate (IP) determinations. Triplicate dishes were used, and each experiment was repeated at least four times. Cells transfected with pcDNA3 alone were used as controls. Cells were incubated with 20 µCi [3H]inositol per milliliter DMEM for 24 h, as described previously (4). Results are expressed as the mean ± SD from a representative experiment.
Western blotting
Membrane fractions were isolated from confluent cultures of COS-7, transfected with 1.25 µg of WT or mutant CaR. Briefly, cells were dislodged with 0.02% EDTA in PBS, were then pelleted and resuspended in 400 µliter of homogenization buffer [50 mmol/liter Tris-HCl (pH 7.4)]; 320 mmol/liter sucrose; 1 mmol/liter EDTA; 10 µg/ml aprotinin, leupeptin, and calpain inhibitor; 40 µg/ml bestatin; and 100 µg/ml Pefabloc (Boehringer Mannheim, Indianapolis, IN) at 4 C. The cells were then homogenized with 15 strokes of a Teflon pestle, and nuclei and mitochondria were sedimented at 18,000 x g for 20 min, followed by 43,000 x g for 20 min to pellet the membranes. Membrane pellets were solubilized in 1% Triton X-100, and 10 µg protein was separated by SDS-PAGE. Gel electrophoresis, binding with primary antibody, and immunostaining were carried out as described previously (6).
Statistical analysis
The mean EC50 (the effective concentration of an agonist giving one half of the maximal response) for the WT and mutant receptor in response to increasing concentrations of calcium was calculated for each individual experiment and expressed as the mean ± SD.
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Results |
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. Three relatives (IV-2, IV-5, and V-2) had mild hypercalcemia with normal PTH levels.
Genetic studies of CaR in the proband revealed a novel heterozygous base substitution T to C, determining a change of isoleucine to threonine at codon 212 (I212T) of exon 4 (Table 1). This mutation was located in the extracellular domain of the CaR. The remaining sequence was entirely normal. Direct sequencing of the region of interest of exon 4 confirmed the presence of the same heterozygous mutation in the affected members (Table 1). Conversely, none of the unaffected relatives (IV-6 and V-1) and none of 50 unrelated normal controls carried the mutation. Expression of the WT CaR in COS-7 cells conferred a 4.5- to 8-fold increase in total IP at high levels of calcium, with an EC50 value of 3.4 ± 0.4 mM (Fig. 2A). On the other hand, transfection of the mutant I212T CaR resulted in no response of IPs to any calcium concentration. Nontransfected cells showed no effect in IP production at any concentration of calcium tested. As shown in Fig. 2A, the calcium dose-response curve of the coexpressed receptors (WT/I212T) was lower than that of the WT receptor alone, suggesting that the mutant receptor interferes with the function of the WT receptor. Western analysis of membrane from COS-7 cells transiently transfected with the WT or the mutant CaR showed that the quantity of the expressed WT CaR protein was comparable to that of the mutant CaR (Fig. 2B). This finding indicates that the abnormal functional responses of the mutant CaR are due to a dominant-negative effect rather than to a lack of receptor expression on the cell membrane.
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Discussion |
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The present case clearly indicates that, in hypercalcemic breast cancer patients, particularly those without extensive bone metastases, other causes of hypercalcemia should be considered. In this regard, it is worth noting that an increased prevalence of primary hyperparathyroidism has recently been reported in breast cancer patients (8). Primary hyperparathyroidism is a rather common disorder and, therefore, this disease should be taken into account in female breast cancer patients with hypercalcemia and measurable or elevated PTH levels. Other conditions characterized by mild hypercalcemia and unsuppressed serum PTH include lithium treatment and FHH. The former can be suspected by medical history and the latter by the finding of familial hypercalcemia and relative hypocalciuria.
When the patient was originally seen in the outpatient clinic, several clinical findings raised suspicions about the previous diagnosis of HCM: the good, healthy condition of the patient, the rather stable disease, the limited bone involvement, and the lack of changes of serum calcium despite pamidronate treatment. Indeed, the careful evaluation of previous biochemistry and additional laboratory tests allowed us to diagnose FHH. Of course, an earlier recognition that hypercalcemia was due to FHH would have spared the patient unnecessary anxiety of possible tumor recurrence with bone metastases and the unindicated pamidronate infusions.
Genetic analysis confirmed the diagnosis of FHH by demonstrating a novel loss-of-function mutation of the CaR gene in exon 4 (I212T). This codon is located in the extracellular domain of the CaR, and the corresponding amino acid is highly conserved between species. Coexpression experiments in COS-7 cells indicated that this mutation acts through a dominant-negative effect. This effect might occur through several mechanisms, including interference in receptor dimerization, which involves the extracellular domain and is essential for receptor activation (3). Alternatively, it may result from a reduction of the effective concentration of the WT receptor on the cell surface. Recent studies have shown that cysteines at positions 101, 129, 131, and 236 are involved in disulfide-linked dimerization (9). There is also evidence that functional dimers may occur even in the absence of intermolecular disulfide linkages, through noncovalent interactions (10). The I212T mutation does not directly affect the disulfide-linked dimerization, because cysteine residues are not involved. However, the isoleucine to threonine substitution at codon 212 and, consequently, the introduction of an idrophilic rather than idrophobic residue, might induce conformational changes of the protein. Such changes, occurring close to the cysteine-rich region, may still interfere with the dimerization process. It is generally believed that CaR mutations acting through a dominant-negative effect occur in families that have higher serum calcium than those observed in families with other mutations (11). Indeed, two of the best characterized CaR mutations acting through a dominant-negative mechanism, i.e. R227L (12) and R185Q (13), were found in patients with s-Ca levels of 2.94 (14) and 3.2 mmol/liter (13). A dominant-negative mechanism has been also proposed in FHH families in whom, in the absence of cotransfection experiments, Western blot analysis of membranes from transiently transfected cells showed a comparable amount of WT and mutated CaR (12). In these families s-Ca ranged from 2.75–2.85 mmol/liter, values that are comparable to those observed in our family.
In conclusion, we describe a case of FHH in a woman with breast cancer in whom hypercalcemia was initially attributed to HCM, giving more weight to the cancer history than to biochemical data.
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Acknowledgments |
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Footnotes |
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Abbreviations: CaR, Calcium-sensing receptor gene; FHH, familial hypocalciuric hypercalcemia; HCM, hypercalcemia of malignancy; IP, inositol phosphate; s-Ca, serum calcium; WT, wild-type.
Received April 28, 2003.
Accepted July 15, 2003.
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References |
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Ser mutations at Cys101 and Cys236. J Biol Chem 274:11629–116334
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