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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-2605
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 7 2474-2479
Copyright © 2006 by The Endocrine Society

CLINICAL CASE SEMINAR

A Hypocalcemic Child with a Novel Activating Mutation of the Calcium-Sensing Receptor Gene: Successful Treatment with Recombinant Human Parathyroid Hormone

Steven D. Mittelman, Geoffrey N. Hendy, Richard A. Fefferman, Lucie Canaff, Irina Mosesova, David E. C. Cole, Linda Burkett and Mitchell E. Geffner

Division of Endocrinology, Diabetes, and Metabolism (S.D.M., L.B., M.E.G.), Childrens Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, California 90027; Departments of Medicine, Physiology, and Human Genetics (G.N.H., L.C., I.M.), McGill University, and Calcium Research Laboratory and Hormones and Cancer Research Unit, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1; Department of Pediatrics (R.A.F.), Kaiser Permanente, Los Angeles, California 90027; Departments of Laboratory Medicine and Pathobiology, Medicine, and Pediatrics (Genetics) (D.E.C.C.), University of Toronto, Toronto, Ontario, Canada M5G 1L5; and The Saban Research Institute of Childrens Hospital Los Angeles (M.E.G.), Los Angeles, California 90027

Address all correspondence and requests for reprints to: Steven D. Mittelman, M.D., Ph.D., Division of Endocrinology, Diabetes, and Metabolism, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Mail Stop Number 61, Los Angeles, California 90027. E-mail: smittelman{at}chla.usc.edu.


   Abstract
Top
 Abstract
Introduction
 Patient and Methods
 Results
 Discussion
 References
 
Context: Persistent hypercalciuria, with the attendant risk of nephrocalcinosis and eventual renal failure, is common in hypoparathyroid patients, especially those with activating mutations of the calcium-sensing receptor (CASR) gene, being treated with oral calcium and calcitriol. Treatment with replacement PTH may be warranted, although this has yet to be evaluated in children.

Objectives: The objectives of this study were to identify the cause of the disorder in a young hypocalcemic patient and to assess the efficacy of treatment of the patient with recombinant human PTH(1–34).

Subject: An infant presenting with hypocalcemia at 3 wk of age was studied.

Methods: CASR gene mutation analysis was performed on genomic DNA of the proband and family members. The patient was treated with twice-daily administration of recombinant human PTH(1–34) over a 17-month period.

Results: The proband was heterozygous for a de novo novel missense mutation (L727Q), on the border between transmembrane helix 4 and intracellular loop 2 of the CASR. When transiently expressed in a human embryonic kidney 293 cell line, the mutant receptor demonstrated a significant leftward shift in the extracellular calcium/intracellular signaling dose-response curve vs. that for the wild-type receptor [EC50; mutant, 2.59 ± 0.11 mM (mean ± SE) vs. wild-type, 3.78 ± 0.12 mM, P < 0.001]. During treatment with PTH(1–34), the patient had no further serious hypocalcemic episodes, and his urinary calcium excretion declined remarkably.

Conclusion: PTH should be evaluated further as a treatment of autosomal dominant hypocalcemia in young patients.


   Introduction
Top
 Abstract
 Introduction
Patient and Methods
 Results
 Discussion
 References
 
THE CALCIUM-SENSING receptor (CASR), a member of group 3 of the seven-transmembrane domain G protein-coupled receptor (GPCR) superfamily, regulates blood calcium and magnesium homeostasis (1). In the presence of increased extracellular calcium, the activated cell surface CASR suppresses PTH secretion from parathyroid cells and reduces calcium reabsorption in the thick ascending limb of the kidney tubules (2). Activating mutations of the CASR gene are a cause of familial hypocalcemia with hypercalciuria (3, 4, 5). The gain-of-function mutations cause impaired PTH secretion and renal calcium and magnesium wasting, leading to hypocalcemia with hypercalciuria, hyperphosphatemia, and hypomagnesemia (6, 7).

Treatment of patients with activating CASR gene mutations (like patients with hypoparathyroidism of other etiologies) normally involves oral administration of calcium, magnesium, and calcitriol. Persistent hypercalciuria, nephrocalcinosis, and potential renal failure are common long-term sequelae of this condition and its treatment (6). The use of thiazide diuretics to decrease urinary calcium excretion has been reported in young patients (8). In addition, studies comparing treatment with either synthetic human PTH(1–34) or calcitriol have been performed in groups of adults with hypoparathyroidism, including some with autosomal dominant hypocalcemia (ADH) due to activating CASR mutations (9, 10, 11).

The present report describes a child in whom we identified a novel activating mutation of the CASR gene and documented successful short-term treatment using recombinant human PTH(1–34).


   Patient and Methods
Top
 Abstract
 Introduction
 Patient and Methods
Results
 Discussion
 References
 
Protocol approval

Protocols were approved by the Institutional Review Board of the Childrens Hospital Los Angeles (Los Angeles, CA) and by the Ethics Committee of the Royal Victoria Hospital (Montreal, Quebec), and the parents of the proband provided written informed consent.

Patient

The patient was born after an uncomplicated pregnancy (birth weight, 3.2 kg; length, 56 cm). At 3 wk of age, he was noted to have brief episodes of unilateral arm and leg stiffness, accompanied by head turning and eye rolling, that initially occurred one to two times per day but gradually increased in frequency up to every 15 min. The patient was admitted to the hospital and was found to have 5.2 mg/dl serum total calcium (normal range 9–11), 12.4 mg/dl phosphate (4.5–6.7), and 1.2 mg/dl magnesium (1.6–2.3). The symptoms resolved with iv calcium administration. Serum intact PTH was less than 10 pg/ml (10–72) when the serum calcium level was 7.4 mg/dl. The patient was then treated with oral calcium, magnesium, calcitriol, and hydrochlorothiazide (HCT). During his 1-month hospitalization, he continued to require high doses of calcium every 2–4 h to maintain normocalcemia and had a significantly elevated urine calcium:creatinine ratio (Ca:Cr) (Table 1Go). Because of the difficulty maintaining a near-normal serum Ca and the severe hypercalciuria, even when hypocalcemic (e.g. urine Ca:Cr was 4.0 mg/mg, whereas serum Ca was 7.4 mg/dl), an activating CaSR mutation was suspected. Over the next 7.5 months of outpatient management, the patient continued to require calcitriol, HCT (administered twice daily), and high doses of calcium and magnesium. The patient had weekly laboratory tests and medication dose adjustments but continued to suffer from episodes of symptomatic hypocalcemia. More specifically, between 12 and 14 months of age, the patient suffered from cramping/tetany every 2–4 wk, and serum calcium levels ranged between 6.7 and 7.4 mg/dl.


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  		TABLE 1. Patient characteristics

 
At 14 months of age, treatment with sc recombinant human PTH(1–34) (Teriparatide, Forteo, Eli Lilly and Company, Indianapolis, IN) was initiated at an initial dose of 2.5 µg twice daily (0.5 µg/kg·d). After the patient was found to tolerate the medication and the urine and serum calcium levels were improving, this dose was gradually increased over the next 17 months to his current dose of 5 µg twice daily (0.7 µg/kg·d).

Both parents and the patient’s older sister had normal serum and urine biochemical testing.

Direct sequence analysis of CASR gene exons

Exons 2–7 of the CASR gene were amplified from leukocyte DNA, and gel-purified PCR products were directly sequenced as described (12).

Site-directed mutagenesis

The mutant sequence was introduced into a c-Myc-tagged human CASR cDNA in the expression vector pcDNA3.1 using previously described methods (13).

Transient transfection of human CASR cDNA

Human embryonic kidney (HEK) 293 cells (a human kidney cell line kindly provided by NPS Pharmaceuticals, Inc., Salt Lake City, UT) were cultured and transfected with the human CASR cDNAs as described (13). Forty-eight hours after transfection, cells were harvested, and Western blot analysis of total cell extracts was performed with the c-Myc 9E10 mouse monoclonal antibody. Membranes were stripped and reprobed with ß-tubulin mouse monoclonal antibody as a loading control.

MAPK assay

MAPK assays were done as previously described (14). In brief, a trans-reporting system (Stratagene, La Jolla, CA) was used to measure the activity of Elk-1, an erythroblast transformation-specific domain transcription factor targeted by MAPK pathways. HEK293 cells were transiently cotransfected with vectors expressing wild-type (0.5 µg), mutant (0.5 µg), or wild-type and mutant receptors (0.25 µg of each), along with Elk-1 reporter constructs and a Renilla luciferase construct. The next day, cells were serum-starved in DMEM containing 0.5 mM CaCl2 for 8 h and cultured in various concentrations of CaCl2 ranging from 0.25–15 mM for 16 h. The cells were washed in PBS and lysed on ice. Luciferase activities in 45-µl cell lysate aliquots were measured using a dual reporter assay system (Promega, Madison, WI) in a Fluostar Optima (BMG Labtech GmbH, Offenburg, Germany), and the firefly luciferase activity was normalized to Renilla luciferase activity.

Data were fitted to a sigmoid dose-response curve to determine EC50 using GraphPad Prism (GraphPad Software Inc., San Diego, CA). Values for each curve were normalized to the maximal activation of each receptor, obtained by stimulation with 15 mM calcium. Significance was assessed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Patient and Methods
 Results
Discussion
 References
 
Identification of CASR mutation

Direct sequence analysis of PCR-amplified CASR exons of the proband identified a heterozygous mutation (L727Q, CTG -> CAG) in the CASR transmembrane region encoded by exon 7 of the gene (Fig. 1AGo). The mutation led to the gain of an SbfI site providing a convenient diagnostic test to confirm the presence of the mutation in the proband and demonstrate its absence in other family members. Digestion of the mutant allele with SbfI produced 509- and 175-bp fragments (Fig. 1BGo).


Figure 1
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  		FIG. 1. A, Direct sequence analysis of the exon 7 genomic DNA amplicon of the proband and wild-type CASR gene revealed a CTG -> CAG mutation at L727Q. B, This mutation created a new SbfI site, resulting in two additional SbfI digestion products identifiable with gel electrophoresis. C, Western blot analysis of cell extracts of HEK293 cells transiently transfected with empty vector (pcDNA3.1), wild-type (WT), or mutant (L727Q) c-Myc-tagged CASR cDNAs. Recombinant proteins stained with c-Myc monoclonal antibody 9E10. D, Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either wild-type (WT) or mutant (L727Q) CASR cDNAs, alone or together, and a MAPK trans-reporting system. Values shown are mean ± SE of four replicates.

 
Expression of wild-type and mutant CASRs in HEK293 cells

By site-directed mutagenesis, the c-Myc-tagged L727Q mutant was created and transiently transfected into HEK293 cells. Cells were also transfected with either the c-Myc-tagged wild type (positive control) or empty vector (negative control). Western blot analysis was conducted with an antibody to the c-Myc epitope tag. The mutant receptor was expressed at an equivalent level to that of the wild-type receptor (Fig. 1CGo). The CASR appears on immunoblot analysis as both monomeric and dimeric forms; the monomeric unglycosylated species is 120 kDa; the core glycosylated (immature) species is 140 kDa; and the mature, fully glycosylated species is 160 kDa. The predominant monomeric species observed was the 140-kDa form with the 160-kDa form also present in both wild-type and mutant L727Q-transfected cells. High-molecular mass forms, likely to be dimers, were seen equally for wild-type and mutant receptors. The Western blot analysis is consistent with the mutant CASR having achieved mature glycosylation (akin to wild type), suggesting that it was appropriately trafficked to the plasma membrane.

MAPK responsiveness of the CASR L727Q mutant to extracellular calcium

The ability of the mutant receptor to respond to extracellular calcium relative to the wild-type receptor was assessed using MAPK pathway trans-reporting system. The wild-type CASR cDNA, when transiently expressed in HEK293 cells, showed an EC50 of 3.78 ± 0.12 mM Ca2+ (mean ± SE; n = 4; Fig. 1DGo). The mutant L727Q showed a significant (P < 0.001) leftward shift in its dose-response curve (EC50, 2.59 ± 0.11 mM) relative to that of the wild type. When equal amounts of wild-type and L727Q mutant CASR cDNAs were transiently coexpressed, the dose-response curve was leftward-shifted (EC50, 3.24 ± 0.10 mM) to a position intermediate to, but different from, that of the wild-type alone (P < 0.01) and the mutant alone (P < 0.01).

Clinical effects of recombinant human PTH treatment

Serum calcium levels stabilized on a lower calcium dose (130 vs. 372 mg elemental Ca/kg·d) within 1 month (Fig. 2Go, top panel, and Table 1Go), and HCT and calcitriol were discontinued. Urine Ca:Cr ratios decreased from 2.81 ± 1.73 mg/mg, mean ± SE [normal range for age, 0.03–0.81 (15)] to 0.53 ± 0.22 (normal range for age, 0.02–0.50, Fig. 2Go, bottom panel), although part of this reduction may have been due to the normal physiological decline that occurs over this age range. Renal ultrasounds performed before and 17 months after initiation of recombinant human PTH(1–34) treatment showed no evidence of nephrocalcinosis. Plasma and urinary markers showed increased bone turnover during recombinant human PTH(1–34) treatment (data not shown). The patient has had no apparent side effects from the treatment. No episodes of symptomatic hypo- or hypercalcemia occurred during recombinant human PTH(1–34) treatment, and oral medication dosing frequency (with calcium) was decreased from six to three times daily. The serum calcium levels stabilized such that the frequency of blood draws could be reduced to one time per month.


Figure 2
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  		FIG. 2. Biochemical responses to treatment. Top, Stable serum calcium levels (black circles) were maintained just below the lower limit of the normal range (light-gray stippling) as the oral calcium requirements were decreased (dark-gray stippling) by the initiation of recombinant human PTH(1–34) therapy at 14 months of age (black bar). Bottom, Urinary calcium excretion (Ca:Cr ratio; black bars) was markedly reduced by the initiation of recombinant human PTH therapy. The upper limits of normal for age (Ca:Cr ratio) are indicated by the dashed lines.

 

   Discussion
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
References
 
The CASR is activated by elevations in extracellular calcium concentration and promotes normocalcemia by regulating PTH secretion and renal calcium excretion. About 50 activating mutations in the CASR gene that cause an increased sensitivity to extracellular calcium, impairing the ability to sense and correct hypocalcemia, have been described in patients with ADH. To date, there have been no other activating mutations reported in transmembrane helix 4 or intracellular loop 2, and only one inactivating mutation is known for this region (5) [http://www.casrdb.mcgill.ca (CASRdb)]. A leucine residue at codon 727 is absolutely conserved in CASRs of all species characterized so far. The L727Q mutation likely alters the conformation of the unbound CASR to more closely resemble the conformation of the ligand-bound state (16, 17), hence increasing the sensitivity of the mutant receptor to calcium. Although some topological models of the CASR transmembrane region (based upon other group 3 GPCRs) place amino acid 727 within transmembrane helix 4 (Ref. 16 and GPCR transmembrane domain prediction tool, http://gdds.pharm.kyoto-u.ac.jp), a more recent model based upon rhodopsin (a group 1 GPCR) places amino acid 727 within intracellular loop 2 (18). In the latter case, substitution of glutamine for leucine might allow enhanced access to molecules, such as G proteins, important for signaling. Although amino acids in intracellular loop 2 neighboring transmembrane helix 3 have been shown to be important for efficient coupling to signaling by phospholipase C (19), other residues within this loop (closer to transmembrane helix 4) have yet to be examined in this way.

Our functional analysis of the mutant receptor transfected into HEK293 cells demonstrated that it is expressed at the same level and has the same monomeric and dimeric forms as the wild-type receptor. The presence of mature glycosylated forms of the mutant receptor indicates its appropriate trafficking to the plasma membrane. At the plasma membrane, the dimeric CASR can activate multiple signaling pathways and it is not clear which particular pathway signals which specific downstream event, such as PTH release in the parathyroid cell or calcium reabsorption in the distal nephron. Evidence has been presented that activation of the MAPK pathway is relevant to the control of parathyroid secretory function by the CASR (20, 21). In the present study, we used a MAPK assay (14, 22) to assess the signaling capability of the L727Q mutant relative to the wild-type receptor when transfected into a human kidney cell line. The mutant receptor demonstrated a significant leftward shift in the dose-response curve with a reduced EC50 relative to wild type. The relative reduction of approximately 35% in EC50 is fully consistent with what has been observed previously for several different CASR-activating mutants (7, 23). In the present study, when mutant and wild-type receptors were cotransfected to mimic the heterozygous state, the dose-response curve was intermediate to those of mutant or wild-type alone (17).

Molecular genetic analysis can be especially important in identifying patients with ADH caused by CASR-activating mutations. These patients represent a particularly challenging management problem relative to hypoparathyroid cases of other etiologies. Given that hypoparathyroid patients are normally treated with calcium and calcitriol, it is important to note that the expression of the CASR gene is up-regulated by 1,25 dihydroxyvitamin D in the parathyroids and renal tubules (24). Hence, treatment with calcium and vitamin D metabolites may fail to raise the serum calcium level to the normal range because of increased expression of the activated CASR in the nephrons. Therefore, this hypocalcemic condition is difficult to treat due to the high doses of calcium required, and the severe renal calcium wasting that persists and can worsen with calcium treatment often leads to nephrocalcinosis and renal insufficiency (6). Because of the central role that PTH plays in calcium regulation, PTH replacement represents a logical treatment option in this disorder.

Purified bovine PTH was first used in the 1960s to evaluate hypoparathyroid states (25). Winer et al. (9) reported the successful use of synthetic PTH(1–34) to treat adults with PTH deficiency and, more recently, those with activating CASR gene mutations (10, 11). They demonstrated that twice-daily injections of synthetic PTH(1–34), along with calcium and magnesium supplementation, can maintain serum calcium levels close to the lower end of the normal range while lowering urine calcium excretion, when compared with the usual treatment of calcium, magnesium, and calcitriol. In a preliminary report, Sanda et al. (26) have described the successful short-term treatment of a 1-yr-old child with congenital hypomagnesemia, hypocalcemia, and secondary hypoparathyroidism with recombinant human PTH(1–34).

Since 2002, recombinant human PTH(1–34) has been approved for the treatment of postmenopausal osteoporosis (27, 28). However, there is little reported experience of its use in children, and this is likely due in part to the theoretical risk of bone tumors associated with its use in humans. This caution is based on studies that reported an increased incidence of bone neoplasms in juvenile Fischer 344 rats receiving daily high-dose injections of PTH(1–34) for 2 yr (the average life span of rats) (29, 30). Even the lowest dose that was associated with the appearance of bone neoplasms in the long term was much greater than the dose used in the present study (30, 31). Rats appear to be much more sensitive to the effects of PTH on bone formation than are primates treated at comparable doses that may be related to their enhanced susceptibility to develop bone tumors (32, 33). No association between PTH administration and osteosarcomas has been found in any primate study to date. Therefore, the relevance of the rodent toxicity model to correction of human insufficiency is not clear. Nonetheless, before initiating treatment with teriparatide in the present case, the potential risks were discussed with our oncology team, as well as with the subject’s parents, and weighed against the potential (and since realized) benefits of the treatment.

The goal of the present study was to safely achieve replacement of a deficient hormone to physiological levels. It was noted by Winer et al. (11) that PTH(1–34) treatment of adult hypoparathyroid patients resulted in a modest increase in bone turnover. Similarly, we observed biochemical evidence of increased bone turnover with PTH(1–34) treatment in our patient (data not shown). This is likely to represent a beneficial effect. Rubin et al. (34) have presented preliminary evidence that the low bone turnover of adult hypoparathyroidism can be restored to normal by PTH administration.

PTH also acts directly on the renal tubule to promote Ca reabsorption. Our patient demonstrated a significant decline in urinary calcium excretion after treatment with recombinant human PTH(1–34) was initiated, although part of this decline may have reflected the normal physiological decline in this parameter that occurs over this age range. That nephrocalcinosis has been prevented to date is another testament to use of PTH treatment in this condition because calcium deposition in the kidney is quite common with traditional therapies (6).

In conclusion, we have identified a novel activating mutation in the CASR gene in a child with hypocalcemia and demonstrated that the mutant protein has a higher than normal sensitivity to calcium in vitro. We also present data supporting the successful treatment of this patient with sc injections of recombinant human PTH(1–34), which, in the short term, has improved his quality of life and likely has decreased his risk for future complications. Further studies evaluating the long-term safety and efficacy of recombinant human PTH(1–34) in children with PTH deficiency and CASR-activating mutations are warranted.


   Acknowledgments
 
We thank all family members for participation and Andrew Lerman for technical assistance.


   Footnotes
 
The work was supported in part by Canadian Institutes of Health Research Grant MOP-57730 (to G.N.H.) and by a Biomedical Fellowship from The Kidney Foundation of Canada (to L.C.).

The web sites for data in this article are as follows: Online Mendelian Inheritance in Man, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM [ADH (MIM:60119)]; and calcium-sensing receptor mutation database, http://www.casrdb.mcgill.ca.

First Published Online April 11, 2006

Abbreviations: ADH, Autosomal dominant hypocalcemia; Ca:Cr, calcium:creatinine ratio; CASR, calcium-sensing receptor; GPCR, G protein-coupled receptor; HCT, hydrochlorothiazide; HEK, human embryonic kidney.

Received December 1, 2005.

Accepted March 30, 2006.


   References
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
 References
 

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