No evidence for point mutations of the calcium-sensing receptor in familial idiopathic hypercalciuria

Nicolas Lerolle1, Florence Coulet2, Brigitte Lantz1, Françoise Paillard3, Pascal Houillier4, Florent Soubrier2, Bernard Gattegno5, Xavier Jeunemaitre6, Pierre Ronco7 and Eric Rondeau1,

1 Service de Néphrologie A, Hôpital Tenon, Paris, 2 Laboratoire de génétique moléculaire, Hôpital Tenon, Paris, 3 Laboratoire d'explorations fonctionnelles, Hôpital Tenon, Paris, 4 Laboratoire d'explorations fonctionnelles, Hôpital Broussais, Paris, 5 Service d'Urologie, Hôpital Tenon, Paris, 6 Laboratoire de génétique moléculaire, Hôpital Broussais, Paris and 7 Service de Néphrologie B, Hôpital Tenon, Paris, France



   Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. Idiopathic hypercalciuria (IH) is frequently associated with nephrolithiasis. As 40% of patients have a positive familial history of IH, an autosomal dominant mode of inheritance has been suggested. Numerous genes have been studied in this regard but none have been found to be linked to IH. Mutation of the calcium-sensing receptor (CaR) has never been studied. Therefore, we conducted a study to detect such mutations.

Methods. Seven families with IH and nephrolithiasis were recruited in a prospective study. Forty-two family members underwent 24-h urine calcium measurement. Twenty-five of them with 24-h hypercalciuria also underwent extensive metabolic evaluation. Blood samples were collected in one or two affected family members in each family and exons 2–7 of the CaR gene were sequenced.

Results. In the seven families, at least one parent and more than half of the children had hypercalciuria (21/30), consistent with autosomal dominant inheritance. Among the nine affected family members whose CaR gene has been studied, all nine had absorptive hypercalciuria, three also had fasting hypercalciuria, and one had renal phosphorous leak. No mutation of the CaR gene was detected in these seven families. Two previously reported polymorphisms were detected, each of them in five families: A986S and C-to-T change at -60 in intron 5.

Conclusion. In these seven families, IH is not related to the CaR gene mutation. Although we cannot exclude that point mutations can be found in other families, familial IH does not seem to be generally associated with CaR mutation.

Keywords: calcium-sensing receptor; DNA mutational analysis; idiopathic hypercalciuria; kidney calculi



   Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Idiopathic hypercalciuria (IH) is one of the most common causes of nephrolithiasis, as it is documented in about 40% of patients with calcium nephrolithiasis [1]. The pathogenesis of IH remains unclear. Several lines of evidence suggest that excessive calcium intestinal absorption is a prominent feature of IH in most patients. It has been attributed to an increase in vitamin D synthesis or to a possible over-expression in the intestinal vitamin D receptor (VDR) [2]. However, fasting hypercalciuria sometimes accompanies IH and low bone density has been reported in patients restricted to a low-calcium diet [3]. It is now agreed that IH is a multifactorial disease involving in each individual variable amounts of excessive calcium absorption, renal loss of calcium and, in some patients, renal phosphorous leak [4,5].

Large epidemiological studies have shown familial clustering of IH: 40–60% of patients have a positive family history of IH [57]. Although debated, an autosomal dominant mode of inheritance among these families has been advocated and several candidate genes have been suggested [8]. Prior studies have failed to demonstrate a positive linkage between IH and gene loci expected to be involved in vitamin D metabolism [911]. A mutation of the chloride channel gene CLCN5 has been reported in Dent's disease, X-linked recessive nephrolithiasis and X-linked recessive hypophosphatemic rickets, three conditions associated with hypercalciuric calcium stones and an X-linked inheritance [12]. However, IH is not an X-linked disease and Scheinman et al. [11] reported that mutations in CLCN5 do not represent a common cause of IH.

The calcium-sensing receptor (CaR), a G-protein-coupled receptor, is widely distributed, being present in all tissues involved in extracellular calcium regulation [13]. A large (~600 amino acids) N-terminal extracellular domain is known to play an important role in sensing extracellular calcium. It is expressed by, and may modulate cell functions of, osteoblast and osteoclast precursors, epithelial cells of the cortical thick ascending limb of the nephron, parathyroid cells, and the epithelial cells of the small and large intestines.

Familial benign hypercalcaemia (FBH) is a form of generalized resistance to calcium related to missense mutations within the CaR [14]. More than 20 inactivating mutations have been reported within the locus of the CaR, on the long arm of chromosome 3 [13]. This condition is associated with mild hypercalcaemia, normal (non-supressed) parathryroid hormone (PTH) levels and normocalciuria. Patients with FBH display normocalciuria, but normal or even decreased gastro-intestinal absorption of calcium and normal levels of 1,25(OH)2 D [15]. On the other hand, autosomal dominant hypocalcaemia is a disorder resulting from over-responsiveness to calcium related to an activating mutation of the CaR. Affected kindreds exhibit hypocalcaemia, low levels of PTH, often hypercalciuria, and seem to have enhanced calcium intestinal absorption [16].

In regard to these considerations, we wondered whether gene mutations inducing mild activation of the CaR could explain the increased intestinal calcium absorption and the calcium renal loss observed in IH with the autosomal dominant mode of inheritance reported in some families. We conducted a study to detect such mutations in nine affected family members in seven different families with familial IH and a history of calcium stones.



   Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Pedigrees and intermediate phenotypes
Among the patients with IH and nephrolithiasis attending the Department of Nephrology of Tenon Hospital, Paris, France and the Department of Physiology of Broussais Hospital, Paris, France, a familial evaluation is commonly proposed. In a prospective study, we identified seven families in which at least two affected family members had IH, in at least two generations. This work was performed in agreement with the bioethic French law and after approval by the ethic committee of Faculté de Médecine Saint Antoine. Forty-five family members and nine unrelated spouses were evaluated. Age, gender, and weight were recorded. Twenty-four-hour calciuria on free diet was obtained for all of them. Metabolic evaluation was obtained for 25 of them, depending on willingness and cooperation of the subjects. Screening for human extracellular CaR gene mutations was subsequently performed in one or two representative affected family members of each family. Informed consents were obtained.

Metabolic evaluation
The affected family members were admitted in an outpatient setting after 2 days of a diet restricted in calcium and sodium. Within 2 weeks of evaluation, no subjects took anti-acids, non-steroidal anti-inflammatory drugs, calcium, phosphorous, vitamin supplements, diuretics, or glucocorticoid. Twenty-four-hour urine was collected during the last day of the diet for calcium, sodium, and creatininuria analysis. Fasting blood samples were obtained on admission and were analysed for serum total and ionized calcium concentration, PTH, 1,25(OH)2 D, 25(OH) D, thyroid stimulating hormone (TSH), phosphorous, and creatinine. A 1-h fasting urinary collection was obtained for measurement of calcium, phosphorous, and creatinine, then a oral 1 g calcium load was given before a 2-h period urinary collection. The calciuric response after the calcium load gave an indirect measurement of intestinal calcium absorption.

Blood and urine chemistry
Total calcium concentration was measured by atomic absorptiometry (Perkin Elmer 3300, Norwalk, CT, USA), the serum ionized calcium concentration by a specific electrode (ICA2, Radiometer, Copenhagen, Denmark) and phosphorous using a colorimetric reaction (RAXT, Bayer, Germany). Creatinine concentration was determined according to Jaffé's method (RAXT). Intact (1–84) PTH blood concentration was measured by immunoradiometric assay with a kit from Nichols Institute (San Juan Capistrano, CA, USA). Sodium concentration was determined by flame photometry (Ilometer 943). TSH blood level was measured by immunoluminometric assay (BYK-Sangtec, Dietzenbach, Germany). 1,25(OH)2 D concentration was determined by radioimmunometry (DiaSorin, Stillwater, MN, USA). The maximal tubular reabsorption capacity for phosphorous (TmPi) was determined with Bivjoët nomogram.

Phenotype assignment
Hypercalciuria was defined by a 24-h calciuria on a normal calcium diet exceeding 0.1 mmol/kg/day. IH was defined by hypercalciuria in the absence of elevated PTH, 25(OH) D, 1,25(OH)2 D, or TSH levels. Absorptive hypercalciuria was defined by evidence of hyperabsorption of calcium:calciuric response to an oral calcium load of 1 g >0.5 mmol/mmol of creatininuria. Fasting hypercalciuria was defined by a ratio of fasting hypercalciuria:creatininuria >0.37 mmol/mmol. Renal phosphorous leak was defined by TmPi<0.7 mmol/ml of glomerular filtrate, together with hypophosphataemia.

DNA isolation
Sample collection and storage were carried out according to standard methods. Ten millilitres of peripheral blood were collected at the time of initial diagnosis. Blood samples were collected in EDTA tubes centrifuged for 10 min at 3000 g to separate buffy coats and plasma. Total genomic DNA was isolated from the buffy coat using a QIAmp Blood Kit (Qiagen, S.A., Courtaboeuf, France) according to the blood and body fluid protocol recommended by the manufacturer. Extracted DNA was quantitated by spectrophotometry absorbance measurements.

Polymerase chain reaction (PCR)
The human extracellular CaR DNA (GenBank accession numbers X81086 and U20760) was amplified by PCR covering the whole exons sequence (exon 2–7) using 12 pairs of primers (Table 1Go). Two additional PCRs (CASR8F-CASR10R and CASR10F-CASR11R) were performed in order to fill gaps of sequence. PCR was carried out on a thermal cycler 9700 (Perkin Elmer Biosystems, Courtaboeuf, France), according a touchdown protocol: 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s (after four cycles with annealing temperature of 64°C and four cycles with 62°C) prior to 5 min of final elongation at 72°C. Amplifications were performed in 30 µl reaction volume with 1 U of Taq DNA polymerase (Boehringer-Mannheim, Mannheim, Germany), 100 mM of dNTP, 5 pmol of each forward and reverse primer, 1.5 mM of MgCl2, 3 µl of 10x buffer and 50 ng of total genomic DNA. The PCR products were analysed by electrophoresis on a 2% agarose gel visualized after ethidium bromide staining.


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Table 1. Primers used for genomic amplification of the CaR sequence

 

Sequencing
The PCR products were purified with QIAquick PCR Purification Kit (Quiagen S.A.). Sequencing of the amplified products was performed, using the primers described in Table 1,Go with Big Dye Terminator sequencing kit (Perkin Elmer Applied Biosystems) according to the manufacturer’s instructions followed by ethanol precipitation to remove non-incorporated dyes. Reactions were performed on 9700 thermocycler and the sequence reactions were read on both strands on an ABI 310 genetic analyser (Perkin Elmer Applied Biosystems). Finally, sequences were analysed by Sequence Analysis 3.0 (Perkin Elmer Applied Biosystems).



   Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Pedigrees and intermediate phenotypes
The pedigrees of these families and the results of the metabolic exploration are shown in Figure 1 andGo affected family members chosen for DNA sequencing are illustrated in bold. In the seven families studied, at least one parent and more than half of the children (27/38) were affected, the ratios of affected:unaffected males was 12:4 and the ratio of affected:unaffected females was 15:7. The high prevalence of hypercalciuria among siblings is consistent with the hypothesis of an autosomal dominant pattern of inheritance as in two families both parents were affected. Different patterns of hypercalciuria (absorptive, renal, renal phosphorous leak) were observed inside each family. The characteristics and results of the metabolic investigations of the nine affected family members whose DNA have been studied are reported in Table 2Go. Six of them had absorptive hypercalciuria, two of them had renal hypercalciuria and absorptive hypercalciuria and one of them had renal phosphorous leak with absorptive hypercalciuria. TSH and ionized calcium concentrations were within normal range (data not shown). 1,25(OH)2 D levels were normal in all affected family members except in two that display elevated levels, one with renal phosphorous leak and the other had no evidence of granulomatosis and belongs to an IH family. The 24-h natriuresis was <150 mmol/day in all subjects. Six of them had lithiasis history. The three other patients without lithiasis belong to families with a history of IH and frequent stone formation. Nephrolithiasis was observed in at least half of the IH affected family members in every family studied. No family members without IH had nephrolithiasis.



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Fig. 1. Family trees and intermediate phenotypes. Family trees of the seven families studied. Presence or absence of hypercalciuria, history of calcium stones and the result of the extensive metabolic evaluation is indicated if performed. Affected family members whose DNA has been sequenced are underlined. Grey shading, sibling not studied; no shading, normocalciuria; black, hypercalciuria; *, stone history. A, absorbtive hypercalciuria; R, renal hypercalciuria and absorptive hypercalciuria; PL, renal phosphorous leak and absorptive hypercalciuria.

 

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Table 2. Results of the metabolic investigations of the nine patients studied

 

DNA sequencing
Genomic DNAs from nine individuals were analysed by CaR gene sequencing. The human receptor is encoded by 7 exons that span >20 kb. Each of the 6 coding exons (2–7) of the human CaR gene was amplified from affected family members of each family using the PCR. Amplified products were sequenced and screened for small deletions, insertions, or missense mutations. Due to the position of the primer used in this study, splice-site mutations were also excluded except in the 3' region of exon 3.

No mutation in the coding sequences was detected in any family. Two previously described polymorphisms were detected: in five families, six affected family members were heterozygote with a change in alanine-to-serine at codon 986 in exon 7. In one of these families, two affected family members having hypercalciuria had a different genotype (one homozygote AA at position 986, and one heterozygote AS). In five families also, five affected family members were homozygote TT at position -60 in intron 5 and four affected family members from three families were heterozygote with a T-to-C change at -60.



   Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
We failed to detect any mutation in the 6 coding exons of the CaR, despite entire sequencing, in nine affected family members with verified familial IH associated with frequent stone formation. We cannot exclude that mutations of the CaR could be found in some other families. Nevertheless, our results indicate that mutations in CaR do not represent a common cause of familial IH. Incidentally, a previously described polymorphism (A986S) has been found in six affected family members (70%) in five families. This polymorphism was not associated with a specific pattern of hypercalciuria in our affected family members. However, regarding the few patients studied, no conclusion can be drawn on the role of this polymorphism in hypercalciuria.

Our initial hypothesis was that some partial activating mutations of the CaR gene in IH would have led to mild forms of autosomal dominant hypocalcaemia with primitive renal hypercalciuria and/or intestinal calcium hyperabsorption. Indeed, in familial hypoparathyroidism related to activating CaR mutations, patients often have hypercalciuria and excrete approximately twice the daily amount of urinary calcium found in other forms of hypoparathyroidism [13]. Furthermore, Pearce et al. [16] have evaluated bone mineral density of lumber spine by dual-energy X-ray absorptiometry and disclosed normal results in four affected subjects and increased density in three others, indicating that the source of calcium responsible for the hypercalciuria was of digestive origin rather than bone. So, it seems likely that activating mutations of CaR are associated with increased intestinal calcium absorption as in IH. Autosomal dominant hypercalciuric hypocalcaemia and FBH provide evidence for the importance of renal CaR in directly regulating calcium renal handling as activating mutations lead to excessive renal calcium excretion and inactivating mutations to normocalciuria despite hypercalcaemia. In contrast, intestinal calcium hyperabsorption is observed in case of activating mutation of CaR and hypoabsorption in case of inactivating mutations, in opposition with the expected features if CaR had been involved in the regulation of calcium absorption according to the calcaemia.

However, absence of mutation in the coding sequence of the gene does not rule out the possibility of regulatory mutations that could induce abnormal levels of expression of the CaR. These mutations may rely in non-coding regions and could not be detected by the method used in the present study. Therefore, we cannot exclude a role for such mutations in IH. In addition, the functional consequence of the polymorphisms A986S and C-to-T changes at -60 in intron 5 were not studied but are worth testing as some authors have advocated that IH could be polygenic rather than monogenic and these polymorphisms should, therefore, be studied on a large IH population to test for the polygenic hypothesis.

While we were completing this study, Petrucci et al. [17] reported in a recent article, a linkage analysis and quantitative trait locus analysis using various intragenic and flanking markers in 64 French-Canadian sibships that failed to support an association between IH and the CaR locus in this population. Their study rules out any mutation or linked polymorphism of the CaR gene and its regulating sequences with calcium stone passage and calciuria. Polymorphism A986S was used as one of the intragenic marker and did not correlate with calciuria. However, the results of the study by Petrucci et al. are restricted to a French-Canadian population. It has been shown in some regions of Quebec that 80% of the individuals’ gene pool come from founders who settled in Nouvelle-France in the 17th century. Relatively high frequencies of some rare inherited disorders can be found in this population [18]. Conversely some genetic disorders can be completely absent due to the result of the founder effect. Our study confirms the absence of CaR gene mutation in IH in a French population with a very likely broader genetic diversity.

Until now, the search for a gene mutation or a linked polymorphism in IH has been discouraging. Despite the evidence for an increased number of VDR in the intestine of genetically hypercalciuric rat, as in activated lymphocytes from hypercalciuric patients in the presence of normal levels of circulating vitamin D, no mutation of the VDR gene nor a particular gene polymorphism have been found in IH patients [9]. Similarly, no evidence for linkage was found for any candidate gene loci, including CLCN5, a chloride channel involved in Dent's disease, 1{alpha}-hydroxylase, PMCA1 and PMCA4, the 28K and 9K calbindins [18]. A study by Scott et al. [19] has suggested a susceptibility gene near the VDR locus in idiopathic calcium stones; however, quantitative trait linkage analysis of urinary calcium excretion yielded linkage to some, but not all, markers. In contrast, Reed et al. [20], using a whole genome search, found evidence for linkage between a single locus on chromosome 1q23.3-q24 and the absorptive hypercalciuira phenotype in three kindreds, but were not able to identify any candidate gene of known function in this region. These results suggest that as yet unreported genes may be implicated in IH, either by altering the action of proteins known to be involved in the regulation of calcium homeostasis such as VDR, PTH/PTHrP receptor or CaR, or in relation with entirely unknown regulation pathways to be discovered.



   Notes
 
Correspondence and offprint requests to: Prof. E. Rondeau, Service de Néphrologie A, Hôpital Tenon, 4 rue de la Chine, F-75020 Paris, France. Email: loeric.rondeau{at}tnn.ap\|[hyphen]\|hop\|[hyphen]\|paris.fr Back



   References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received for publication: 21.12.00
Revision received 10. 7.01.