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Editorial |
Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Edward M. Brown, M.D., Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: embrown{at}rics.bwh.harvard.edu.
The extracellular calcium (Ca2+o)-sensing receptor [variably designated as CaR or CasR] plays a key role in maintaining near constancy of Ca2+o by inhibiting PTH secretion and promoting renal calcium excretion in response to increases in Ca2+o (1). In this manner, the CasR serves as the body’s "thermostat" for Ca2+o. Although expressed in numerous cell types—both those involved in mineral ion homeostasis and those that aren’t—its physiological importance in most cases remains to be elucidated. Inherited diseases caused by CasR mutations have taught us much about the receptor’s roles in health and disease (2). Two disorders result from activating mutations. Autosomal dominant hypocalcemia is due to heterozygous or homozygous activating CasR mutations, which reset Ca2+o downward by reducing the set-points of parathyroid and kidney for their respective responses to Ca2+o. Recently, patients with activating mutations have presented with the phenotype of Bartter’s syndrome (3), perhaps because they had mutations activating the CasR more than most. That is, severe, persistent activation of the receptor may have unmasked the CasR’s known "lasix-like" action on the cortical thick ascending limb, namely wasting of sodium chloride, calcium, and magnesium in the urine (4).
Three disorders in Ca2+o-sensing arise from inactivating CasR mutations—familial (benign) hypocalciuric hypercalcemia (FHH, also known as FBH or FBHH), neonatal hyperparathyroidism (NHPT), and neonatal severe hyperparathyroidism (NSHPT) (5). FHH comprises three separate genetic disorders. By far the most common, type 1 FHH, is caused by inactivating CasR mutations—the disorder manifested by the family described by Wystrychowski et al. (6) in this issue of the JCEM (6). Single families have been described with types 2 and 3, which are caused by defects in as-yet-unidentified genes on the short and long arms, respectively, of chromosome 19 (5). FHH is a generally asymptomatic, PTH-dependent, hypercalcemic disorder. Type 1 FHH results from heterozygous inactivating CasR mutations, whose presence in parathyroid and kidney renders those tissues resistant to the usual suppression of PTH release and stimulation of urinary calcium excretion by increases in Ca2+o. As a consequence, patients exhibit mild to moderate hypercalcemia, generally normal serum PTH levels, high normal-range or mildly elevated serum magnesium concentrations, and reduced renal calcium excretion, best quantified by calculating the calcium to creatinine clearance ratio (7). About 80% of individuals with FHH have a calcium to creatinine clearance ratio of less than 0.01.
Occasional infants present with moderate hypercalcemia (viz., 
12–13 mg/dl), markedly elevated PTH levels, overt hyperparathyroid bone disease, and variable symptoms of hypercalcemia (8). This clinical presentation tends to occur in infants heterozygous for CasR mutations and may best be termed NHPT, the condition exhibited by the infant referred to by Wystrychowski et al. (see Refs. 22 and 35 in the article by Wystrychowski et al.). Such patients can often be managed with intensive medical therapy and may eventually revert to a clinical picture resembling FHH. In contrast, infants with homozygous FHH most commonly present with a much more severe disorder, termed NSHPT (1). This condition manifests much more dramatic, symptomatic hypercalcemia (e.g. >15 mg/dl), very high PTH levels, and severe bone disease, often with multiple fractures. The total absence of normally functioning CasRs in this setting not infrequently necessitates total parathyroidectomy to achieve clinical remission. In FHH, NHPT, and NSHPT, removal of anything less than all parathyroid tissue is nearly invariably followed by recurrent disease, as remnant parathyroid tissue retains the genetic defect causing abnormal parathyroid function.
The capacity to screen for mutations in the CasR in patients with PTH-dependent hypercalcemia has identified clinical and biochemical presentations resulting from inactivating CasR mutations that considerably broaden the range of phenotypes resulting from the genotypes described above. A family has been described with type 1 FHH and hypercalciuria as well as renal stone disease (9). Surprisingly, these abnormalities remitted following subtotal parathyroidectomy. Seemingly, renal calcium handling in this family responded relatively normally to the calciuric action of hypercalcemia despite the clearly abnormal calcium-regulated PTH release. Furthermore, about 15% of families with isolated familial hyperparathyroidism harbor FHH mutations (10), emphasizing that in some cases the clinical presentation of "FHH" can be indistinguishable from that of "garden-variety" primary hyperparathyroidism. Patients have been described with homozygous FHH who were not identified in infancy and were only diagnosed in adulthood (e.g. Ref.11). One such patient was diagnosed at age 35 yr and was asymptomatic with normal renal function despite serum calcium concentrations of 15–17 mg/dl. This case supports the notion that the CasR mediates at least some of the symptoms and renal complications of hypercalcemia and that this patient was "resistant" to these complications. Presumably, a relatively mild homozygous mutation enabled maintenance of stable calcium homeostasis reset to a moderate to severe degree of hypercalcemia. Several patients with FHH have presented with lipohyperplasia of their parathyroid glands (12). Whether concurrent hyperplasia of fat cells and parathyroid chief cells represents a distinct manifestation of some inactivating CasR mutations requires further study.
All of this brings us to the article at hand by Wystrychowski et al.(6). These investigators describe a hypercalcemic family in which three generations exhibited autosomal dominant inheritance of an R227Q mutation, which resides within the first half of the CasR’s extracellular domain (6). Affected family members manifested calcium concentrations averaging 11.1 mg/dl, normal PTH levels, and calcium to creatinine clearance ratios well under 0.01, establishing a diagnosis of FHH. A calcium concentration of 11–12 is intermediate within the range encountered in FHH. In the mildest cases, serum calcium concentration may be in the upper part of the normal range or only mildly elevated. In contrast, kindreds have been described with serum calcium concentrations consistently above 12 or even 13 mg/dl (7). The proband of Wystrychowski’s kindred was initially thought to have primary hyperparathyroidism and underwent removal of one slightly enlarged parathyroid gland. Typical of FHH, however, hypercalcemia recurred 2 wk later, precipitating additional work-up that revealed the diagnosis of FHH. The authors contrast the mild presentation of this family with that of an infant described earlier (8), who presented in the neonatal period with serum calcium values of about 12 mg/dl and marked bony demineralization. The infant underwent total parathyroidectomy, which revealed four-gland hyperplasia. Permanent hypoparathyroidism ensued, which was treated effectively with vitamin D replacement. Because of the persistent defect in renal calcium handling in hypoparathyroid FHH or NSHPT patients, one might anticipate that less vitamin D would be needed to maintain normocalcemia, but this issue has not been examined systematically given the scarcity of these patients. Originally described in 1984 (13), this child was reinvestigated in 1995 by Pearce et al. (8) and was shown to harbor a heterozygous R227L mutation at the same codon as that described above. Of note, this was a de novo mutation, ensuring that the proband was gestated in a normal mother, thereby exposing the fetal parathyroid glands to a calcium concentration that they would view as abnormally low. In the family evaluated by Wystrychowski et al. (6), in contrast, all affected children were born to affected mothers in the two younger generations.
The central thrust of the article by Wystrychowski et al. is to compare the wild-type receptor’s function in vitro with those of the two mutant receptors when transiently expressed in human embryonic kidney (HEK) 293 cells to determine whether there were any genotype-phenotype relationships. The authors performed careful in vitro studies, documenting by Western blot that the two receptors were expressed at levels comparable to that of the wild-type receptor. Western analysis, however, only crudely reflects the amount of the mature, fully glycosylated receptor on the cell surface. The authors addressed this point elegantly by performing fluorescence immunocytochemistry and confocal microscopy to compare the cell surface expression of the wild-type and two mutant receptors. By labeling the cell surface receptors of nonpermeabilized cells with a fluorescent anti-CasR antibody (the authors used c-myc-tagged receptors and an anti-myc antibody), they were able to conclusively show that cell surface expression of the two mutant CasRs was equivalent to the wild-type CasR. By permeabilizing the cells and performing confocal microscopy, the authors also showed substantial intracellular CasR, confirming the work of other investigators. Although this may simply represent nascent receptor, the interesting possibility remains that the receptor could function intracellularly. For example, the extracellular domain of the intracellular receptor would face the lumen of the endoplasmic reticulum (ER), where calcium concentrations are approximately 1 mM. Could the CasR play some role in sensing calcium within the ER and regulating ER filling?
When tested for their ability to activate MAPK, the R227Q and R227L receptors showed EC50 values (the calcium concentration needed for half-maximal activation) of about 2- and 3-fold higher, respectively, than that of the wild-type CasR. Both would be considered to show a moderate degree of inactivation. Of note, the authors tried to mimic the heterozygous state of the mutant receptors in vivo by cotransfecting equal amounts of the cDNA for the wild-type and the respective mutant receptors. Both R227L and R227Q exerted a so-called dominant negative effect (14), shifting the EC50 of the wild-type receptor to the right, although the EC50 of the "heterozygous" state for the R227L receptor was still moderately higher than that of R227Q. What is the impact of a dominant negative effect on a dimeric receptor? With a truly null mutation (e.g. the protein isn’t expressed), the remaining approximately 50% of the normal complement of the receptor provided by the wild-type allele would have to mediate parathyroid and renal calcium sensing. With a mutant receptor exerting a severe dominant negative effect, in contrast, the 25% mutant homodimers and 50% mutant-wild-type heterodimers would not contribute to receptor signaling in vivo, leaving only 25% wild-type homodimers to regulate parathyroid and renal function. Wystrychowski et al. argue that the relatively modest difference in function between the two mutant receptors (and the respective wild-type mutant heterodimers) may account for the more severe phenotype of the infant with the R227L genotype. However, several alternative or additional contributory factors should be kept in mind. First, there was only one affected family member in the family with the R227L mutation, and it is entirely possible that additional affected individuals might show a milder phenotype as has been noted in other FHH kindreds. Second, the mother of the infant with the de novo mutation was unaffected, whereas all affected individuals in the other family had affected mothers, which might have reduced the tendency to develop hyperparathyroidism in the second instance. Third, as noted by the authors, factors such as low calcium or vitamin D intake could put added stress on fetal parathyroid glands already prone to hyperfunction. Thus, whereas certain heterozygous inactivating CasR mutations may produce more severe hypercalcemia in some families than is the norm in FHH (14), a definitive explanation for why some infants with heterozygous FHH develop NHPT remains elusive. The present study by Wystrychowski et al. (6) represents a solid step in that direction, but more work is clearly needed.
Footnotes
Abbreviations: Ca2+o, Extracellular calcium; CasR, Ca2+o-sensing receptor; FHH, familial (benign) hypocalciuric hypercalcemia; ER, endoplasmic reticulum; NHPT, neonatal hyperparathyroidism; NSHPT, neonatal severe hyperparathyroidism.
Received December 16, 2004.
Accepted December 20, 2004.
References
cek A, Hendy GN 2005 Functional characterization of calcium-sensing receptor codon 227 mutations presenting either as familial (benign) hypocalciuric hypercalcemia or neonatal hyperparathyroidism. J Clin Endocrinol Metab 90:864–870This article has been cited by other articles:
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  L. Fox, J. Sadowsky, K. P. Pringle, A. Kidd, J. Murdoch, D. E.C. Cole, and E. Wiltshire Neonatal Hyperparathyroidism and Pamidronate Therapy in an Extremely Premature Infant Pediatrics, November 1, 2007; 120(5): e1350 - e1354. [Abstract] [Full Text] [PDF]
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 K. Zajickova, J. Vrbikova, L. Canaff, P. D. Pawelek, D. Goltzman, and G. N. Hendy Identification and Functional Characterization of a Novel Mutation in the Calcium-Sensing Receptor Gene in Familial Hypocalciuric Hypercalcemia: Modulation of Clinical Severity by Vitamin D Status J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2616 - 2623. [Abstract] [Full Text] [PDF]
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 F. Raue and K. Frank-Raue Primary hyperparathyroidism--what the nephrologist should know--an update Nephrol. Dial. Transplant., March 1, 2007; 22(3): 696 - 699. [Full Text] [PDF]
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