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Familial Hypercalcemia and Hypercalciuria Caused by a Novel Mutation in the Cytoplasmic Tail of the Calcium Receptor¹

Endocrine Surgery Unit, Department of Surgery (T.C., E.S., G.W., P.H., J.R.), Department of Genetics and Pathology (P.G., N.D.), and Department of Clinical Chemistry (P.R.), Uppsala University Hospital, S-751 85 Uppsala, Sweden; and Endocrine-Hypertension Division (M.B., S.T., E.M.B.), Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115

Address correspondence and requests for reprints to: Tobias Carling, M.D., Ph.D., Endocrine Surgery Unit, Department of Surgery, Uppsala University Hospital, S-751 85 Uppsala, Sweden. E-mail: Tobias.Carling{at}kirurgi.uu.se

	Abstract

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Familial hyperparathyroidism (HPT), characterized by hypercalcemia and hypercalciuria, and familial benign hypocalciuric hypercalcemia (FHH) are the most common causes of hereditary hypercalcemia. The calcium-sensing receptor (CaR) regulates PTH secretion and renal calcium excretion. Heterozygous inactivating mutations of the gene cause FHH, whereas CaR gene mutations have not been demonstrated in HPT. In a kindred with 20 affected individuals, the hypercalcemic disorder segregated with inappropriately higher serum PTH and magnesium levels and urinary calcium levels than in unaffected members. Subtotal parathyroidectomy revealed parathyroid gland hyperplasia/adenoma and corrected the biochemical signs of the disorder in seven of nine individuals. Linkage analysis mapped the condition to markers flanking the CaR gene on chromosome 3q. Sequence analysis revealed a mutation changing phenylalanine to leucine at codon 881 of the CaR gene, representing the first identified point mutation located within the cytoplasmic tail of the CaR. A construct of the mutant receptor (F881L) was expressed in human embryonic kidney cells (HEK 293), and demonstrated a right-shifted dose-response relationship between the extracellular and intracellular calcium concentrations. The hypercalcemic disorder of the present family is caused by an inactivating point mutation in the cytoplasmic tail of the CaR and displays clinical characteristics atypical of FHH and primary HPT.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
CALCIUM regulates PTH release by interacting with cation receptors on the parathyroid cell surface (1). Such a calcium-sensing receptor (CaR) has been cloned and mapped to chromosome 3q (2, 3). The CaR is a member of the G-protein-coupled receptor family and is at the highest levels expressed in parathyroid chief cells, thyroid C-cells, and renal cells (2, 4). Heterozygous mutations causing inactivation of the CaR have been found in patients with familial benign hypocalciuric hypercalcemia (FHH), whereas homozygous- or, in some cases, heterozygous-inactivating mutations produce neonatal severe hyperparathyroidism (NSHPT) (3, 4, 5, 6, 7, 8, 9). Conversely, autosomal dominant hypocalcemia with hypercalciuria has been associated with activating CaR mutations (4, 10, 11). Mutations in the CaR gene have not been found in parathyroid tumors of primary HPT (12), although reduced levels of expression of an otherwise normal CaR gene are present in most cases (1, 4).

FHH is inherited as an autosomal dominant trait with mild to moderate hypercalcemia, accompanied by few if any symptoms (13, 14). The condition does not require treatment, and responds poorly to parathyroidectomy. FHH should be distinguished from other hypercalcemic disorders such as primary HPT, in which the elevated serum and urinary calcium levels are normalized by successful parathyroid surgery. FHH also contrasts to primary HPT in that the affected family members exhibit relative hypermagnesemia, inappropriately normal serum PTH levels, histologically normal parathyroid glands, and a low or normal urinary calcium excretion (i.e. relative hypocalciuria) (14, 15, 16). Primary HPT in families is inherited in autosomal dominant fashion either as the single lesion (familial isolated HPT) (17) or as part of tumor susceptibility syndromes, such as multiple endocrine neoplasia (MEN) type 1 or 2A (18) and the HPT-jaw tumor syndrome (19).

A large kindred exhibited a combination of moderate hypercalcemia, inappropriately high serum PTH levels, elevated urinary calcium excretion, relative hypermagnesemia and hyperphosphaturia, and parathyroid gland hyperplasia/adenoma. Because the biochemical derangements of this disorder can be reversed by radical parathyroid resection, clinical characteristics atypical to FHH were substantiated. This study reports the clinical characteristics of the family, the underlying point mutation of the CaR gene, and functional characterization of the mutation.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects and biochemical analysis

Hypercalcemia with hypercalciuria in this Swedish family was initially noted in individual IV-8 (Fig. 1), who was referred for parathyroid exploration in 1985. He presented with hypercalcemia (2.75–2.80 mM or 11.0–11.2 mg/dL; reference range, 2.20–2.60 mM or 8.8–10.4 mg/dL), hypermagnesemia (0.92 mM; reference range, 0.70–0.91 mM), hypercalciuria (8.4 mmol/24 h; reference range, 0.6–5.0 mmol/24 h), and a history of renal stones. Parathyroid chief cell hyperplasia was noted at subtotal parathyroidectomy, and he became normocalcemic without recurrences of renal stones. His mother (III-5) and other close relatives subsequently were found to demonstrate hypercalcemia in an autosomal dominant pattern of inheritance. Family screening in 1995 showed a total of 20 affected (hypercalcemic) family members, whereas 33 were normocalcemic. No individual had borderline serum calcium values, and screening under the age of 18 was not performed due to ethical considerations. Eight affected family members underwent thorough clinical and biochemical screening for indices of MEN type 1 according to a previously described protocol (20). Nine of the affected individuals have undergone parathyroid surgery. Informed consent and blood samples were obtained from the affected, as well as representative, normocalcemic family members. The study was approved by the Ethical Committee of the Uppsala University.

View larger version (15K): [in this window] [in a new window]   Figure 1. Pedigree of the family with hypercalcemia and hypercalciuria. Individuals are identified by generation number and presented by squares (male), circles (female), and slashes (deceased). Clinical status is indicated by filled symbols (hypercalcemic), gray symbols (normocalcemic), and open symbols (unknown). All 20 affected (hypercalcemic), but not all 33 unaffected (normocalcemic), family members are shown in the pedigree. An asterisk indicate those diagnosed at the family screening conducted in 1995.

DNA analysis

Leukocyte DNA was prepared according to standard procedures. Specific primers were used to amplify highly polymorphic microsatellite markers from the following loci: 1p (D1S243, D1S244, D1S228), where a potential parathyroid tumor suppressor gene is situated (22); 1q (D1S212, D1S191), the location of the HPT-jaw tumor gene (19); 2q (D2S112, D2S72, D2S142), locus of the gp330/LRP2 gene (23); 3q (D3S1291, D3S1278, D3S1303, D3S1269, D3S1301), where the CaR gene is located (24); 11q (PYGM(CA), INT-2), which harbors the MEN1 tumor suppressor gene (25); and 19p (D19S209, D19S216), to which one FHH family has been mapped (26). PCR (10 µl) contained 2 pmol ³²P-end-labeled forward primer and 2 pmol reverse primer, 20 ng genomic DNA, 0.2 U Taq-polymerase and 10x PCR buffer (Roche Molecular Biochemicals, Mannheim, Germany), 1.5 mM MgCl₂ and 100 µM dNTPs (Pharmacia Biotech, Uppsala, Sweden). The PCR conditions used an initial denaturation at 95 C, followed by 27 cycles at 95 C for 30 sec, 55–62 C for 30 sec, and 72 C for 30 sec with a final extension at 72 C for 4 min. PCR products were mixed with formamide gel loading solution, heat denatured, run on a denaturing 4.5% acrylamide sequencing gel, and visualized by autoradiography (24–96 h).

Sequence analysis of the CaR gene

Exons 2, 3, 4, and 7 of the CaR gene were sequenced in two affected (hypercalcemic) and two unaffected (normocalcemic) family members using primers 1F-1R, 2F-2R, 3AF-3AR, 3BF-3BR, 7GF-6AR, 6BF-7ER, 7FF-6BR, 6CF-6CR, 6DF-6DR (3, 5) (KEBOLab, Stockholm, Sweden). Exons 5 and 6 have previously shown neither activating nor inactivating mutations (4). Subsequently, all 20 affected and 33 normocalcemic individuals were sequenced using primers 7TCF (5'-ggatctccttcattccagcctatgc-3') and 7TCR (5'-gggctgctgctgagatcgttgctgc-3'), generated in accordance with the sequence of the human CaR gene (27). Briefly, PCR was performed using 200 ng genomic DNA and 25 pmol of each primer, and approximately 60 ng of the PCR product underwent sequencing of both DNA strands using the ABI PRISM Dye terminator cycle sequencing ready reaction kit (Perkin-Elmer Corp., PE Applied Biosystems, Foster City, CA).

Site-directed mutagenesis and transient receptor expression

Site-directed mutagenesis to produce a receptor containing the point mutation in this family’s CaR gene (F881L) was performed as described (28). The dut-1 ung-1 strain of Escherichia coli CJ236 was transformed with mutagenesis cassette 6 (29). Uracil-containing, single-stranded (ss) DNA was produced by infecting the cells with the helper phage VCSM13. The ssDNA was annealed to a mutagenesis primer that contained the desired nucleotide change encoding a single point mutation (changing phenylalanine to leucine at amino acid position 881) flanked on both sides by the wild type sequence. The primer was then extended around the entire ssDNA and ligated to generate closed circular heteroduplex DNA. Incorporation of the desired mutation was confirmed by sequencing the entire cassette.

The DNA for transfection was prepared using the Midi Plasmid Kit (QIAGEN, Hilden, Germany), and LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) was used as a DNA carrier for transfection. The HEK293 cells used for transient transfection (kindly provided by NPS Pharmaceuticals, Inc., Salt Lake City, UT) were cultured in DMEM (Life Technologies, Inc.) with 10% FBS (HyClone Laboratories, Inc. Logan, UT). The DNA-liposome complex was prepared by mixing DNA and LipofectAMINE in OPTI-MEM 1 reduced serum medium (Life Technologies, Inc.) at room temperature for 30 min. The mixture was then diluted with OPTI-MEM 1 reduced serum medium and added to 90% confluent HEK293 cells plated on 13.5 x 20.1-mm glass cover slips (for measurement of intracellular calcium; [Ca²⁺]_i) using 0.625 µg DNA. After a 5-h incubation at 37 C, equivalent amounts of OPTI-MEM 1 medium with 20% FBS were added and replaced with DMEM with 10% FBS at 24 h after the transfection. The expressed CaR protein was assayed 48 h after the start of transfection.

Measurement of [Ca2+]i

Coverslips coated with HEK293 cells that had been transfected with the wild-type or mutant CaR cDNAs were loaded for 2 h at room temperature with Fura-2/AM (Calbiochem, La Jolla, CA) in 20 mM HEPES (pH 7.4) and washed with a bath solution [20 mM HEPES (pH 7.4), 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl₂, 0.5 mM MgSO₄, 0.1% BSA, and 0.1% dextrose] at 37 C for 20 min. The coverslips were placed diagonally in a thermostatted quartz cuvette containing the bath solution by a modified technique (29, 30). Extracellular calcium (Ca²⁺₀) was increased stepwise to the desired final concentrations.

Parathyroid cells from pathological parathyroid glands (n = 10) of four affected family members and normal parathyroid glands of 15 individuals operated on for atoxic goiter were suspended enzymatically, as described previously (31). The normal parathyroid glands were biopsied during operations for atoxic goiter and examined histopathologically due to macroscopical ambiguity of the diagnosis. [Ca²⁺]_i was analyzed microfluorometrically after loading cells in 1.0 µM fura-2/AM for 30 min at 37 C in the HEPES buffer. Emitted fluorescence was measured at 510 nm, and the ratio of emission at 340/380 nm excitation was used to calculate [Ca²⁺]_i, as described (29, 30, 31, 32).

Linkage and statistical analysis

Two-point linkage analysis was performed using the FASTLINK program (33), assuming various penetrance frequencies. The unpaired t test was used for evaluation of biochemical measurements in the subjects. The mean EC₅₀s (the effective concentration of Ca²⁺₀ giving one half of the maximal [Ca²⁺]_i response) were calculated from all EC₅₀s of the individual experiments and expressed with SEM as the index of dispersion. Comparison of EC₅₀s was performed using ANOVA or Duncan’s multiple comparison test. All values are expressed as mean ± SEM, and P < 0.05 was considered significant.

	Results

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Clinical characteristics

The moderate hypercalcemia (range, 2.69–2.87 mM) of the investigated family showed an autosomal dominant pattern of inheritance (Fig. 1). Twenty affected family members, 22–77-yr-old, presented with higher serum calcium (P < 0.001), serum PTH (P < 0.05), and serum magnesium levels (P < 0.005) compared with 33 unaffected family members (Table 1). Serum PTH levels of the affected members generally were in the upper part of the reference range and inappropriate (i.e. nonsuppressed) given the elevated serum calcium concentration.

Parathyroid surgery in nine affected individuals revealed parathyroid gland enlargement with a total parathyroid tissue weight of 240–965 mg (upper normal limit is 208 mg; Table 2) (34). Chief cell hyperplasia of the diffuse or nodular type was present in seven of the subjects, whereas one had a single parathyroid adenoma. The distinction was equivocal in one individual. All but two of them had the hypercalcemia and hypercalciuria reversed postoperatively, which required radical subtotal parathyroidectomy. Postoperative persistence of hypercalcemia, albeit ameliorated, occurred in the two members subjected to extirpation of two enlarged parathyroid glands. Recurrent hypercalcemia has not been observed during postoperative follow-up for 1.8–12 yr.

Because the clinical and biochemical expression of the disease was atypical for FHH, and since FHH is a genetically heterogeneous disorder (26, 35), linkage analysis was conducted. The disease gene was shown to map to the CaR gene locus at chromosome 3q between markers D3S1303 (maximum logarithm of odds; Z_max = 4.25, = 0.00) and D3S1269 (Z_max = 5.39, = 0.00; Table 3). Calculations assuming 90% and 80% penetrance, as well as those including only affected individuals, all showed Z_max ( = 0.00) values above 4.0 for the D3S1303 and D3S1269 markers (data not shown). Linkage to the other candidate loci was excluded by haplotype analysis. Both DNA strands from exons 2, 3, 4, and 7 of the CaR gene were sequenced from two affected and two unaffected family members. Both affected ones had a heterozygous T to C transition at nucleotide position 2641 in exon 7, resulting in a phenylalanine to leucine substitution at codon 881 (F881L; Fig. 2). The mutation was subsequently identified in all 20 affected family members, but not in any of the 33 normocalcemic subjects. The mutation cosegregated with the affected haplotype in all cases.

View larger version (24K): [in this window] [in a new window]   Figure 2. Identification of the CaR mutation. Automated sequence analysis of DNA from a representative patient and control. A heterozygous T to C transition at nucleotide 2641, changing a phenylalanine (TTC) to leucine (CTC) in codon 881 of exon 7, was observed in all 20 patients.

Measurements of [Ca²⁺]_i responses in HEK293 cells transiently transfected with the wild-type receptor or the F881L-receptor showed an EC₅₀ for Ca²⁺₀ of 4.1 ± 0.1 mM (n = 4) for the wild-type receptor. The F881L mutant receptor exhibited a dose-response curve that was significantly right-shifted (P < 0.05), with an EC₅₀ of 4.9 ± 0.1 mM (n = 4; Fig. 3). The Ca²⁺₀-regulated [Ca²⁺]_i in cells of the pathological parathyroid glands (n = 10) from the affected individuals invariably demonstrated a raised EC₅₀ similar to that seen in cells from parathyroid adenomas of sporadic primary HPT (32). Mean EC₅₀ for Ca²⁺₀ in the cells of the affected family members was 1.82 ± 0.06 mM, whereas it was 1.45 ± 0.05 mM in the normal parathyroid cells (n = 15 glands; P < 0.0001).

View larger version (23K): [in this window] [in a new window]   Figure 3. High Ca2+0-evoked increases in Ca2+i in fura-2-loaded HEK293 cells transiently transfected with either the wild-type CaR or the mutant receptor (F881L). The responses are normalized to the maximum response of the wild-type receptor (rHuPCaR4.0). EC50 differed significantly between the wild-type receptor vs. the mutant receptor (P < 0.05). Each data point is the mean value of three to four measurements, and SEM is indicated with a vertical bar.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Our findings indicate that the hypercalcemia and hypercalciuria of the present family are related to a previously unrecognized point mutation in the cytoplasmic tail of CaR. Sequence analysis demonstrated that the disease cosegregated with a heterozygous T to C transition at nucleotide position 2641, resulting in a phenylalanine to leucine substitution at codon 881 of exon 7. This mutation was not detected in the 33 normocalcemic individuals, and previous studies on CaR polymorphisms in 100 healthy Caucasian individuals have failed to identify this sequence variant (9).

When the mutant CaR (F881L) was transiently expressed in HEK293 cells and [Ca²⁺]_i responses were measured, a significant right-shift in the dose-response curve as compared to the wild-type receptor was noted. This finding is consistent with inactivation of the receptor. It is noteworthy that the F881L CaR mutation only produced a mild shift in the EC₅₀ of the transiently transfected renal cells as compared to several other mutations in FHH cases with a similar degree of hypercalcemia (4, 29). It is possible that the CaR mutation of the present family affects intracellular responses (such as activation of phospholipase C, coupling to G proteins, and so on.) differently in the parathyroid and kidney, thereby producing a more severe derangement in Ca²⁺₀-sensing in the former. Indeed, the measurements of Ca²⁺₀-regulated [Ca²⁺]_i in parathyroid cells of the affected family members showed a rather, pronounced right-shift in the EC₅₀, and the attained value coincided with that of parathyroid adenomas of primary HPT (32).

It is likely that there exists derangements in control of both the calcium/magnesium sensing and proliferation of parathyroid cells in the affected family members, which cause their hypercalcemia-hypermagnesemia and parathyroid gland hyperplasia/adenoma. Indications exist that alterations in the cytoplasmic tail of the CaR can promote parathyroid cell proliferation. In one FHH family exhibiting an inserted Alu repetitive sequence at codon 877, with predicted truncation of the CaR protein, 3 of 36 heterozygous gene carriers developed high serum calcium and PTH levels and operatively verified parathyroid gland enlargement (7, 38). Consistent with primary HPT, the hypercalciuria of the present family presumably is secondary mainly to the hypercalcemia per se. The hypothetically mild inactivation of the CaR in renal cells presumably responds to the hypercalcemia by an increased urinary calcium excretion, similar to the normal renal CaR in HPT (1, 4).

The atypical disorder of the present family demonstrated autosomal dominant inheritance with mild to moderate hypercalcemia, relative hypermagnesemia, and an early age at onset similar to FHH (14). In the described part of the pedigree there existed a tendency to overrepresentation of affected males. However, ongoing analyses of a more complete pedigree substantiate no difference in the gender distribution, similar to findings in FHH and NSHPT. However, the trends to hyperphosphaturia, inappropriately high serum PTH levels, and a history of renal stones in two of the subjects resemble mild primary HPT (39, 40, 41). The consistent absence of hypocalciuria, Ca/CrCl values above 0.010 in 7 of 10 affected family members, and frank hypercalciuria in several individuals also contrast to findings in FHH (14, 15, 39). Some FHH patients, nevertheless, may demonstrate high urinary calcium levels (42, 43, 44). The increased urinary calcium excretion of the subjects was an important cause for the evaluation of the effects of parathyroid surgery. The induction of normocalcemia after parathyroidectomy and the presence of nodular hyperplasia or adenoma in some of the enlarged parathyroid glands also is atypical for FHH (14, 16, 45). The interesting clinical and genetic findings of the present family exemplify the heterogeniety of familial hypercalcemic syndromes.

	Acknowledgments

 
This work could not have been performed without the superb collaboration from the family members.

	Footnotes

 
¹ Supported by the Swedish Medical Research Council, the Swedish Cancer Society, and the Swedish Society of Medical Research. Additional grant support was obtained from NIH Grants DK-46422, DK-48330, and DK-52005 (to E.M.B.) and Grant DK-09436 (to M.B.), and from the St. Giles Foundation (to E.M.B.).

Received June 16, 1999.

Revised December 1, 1999.

Accepted December 7, 1999.

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