Heterozygous inactivating CaSR mutations causing neonatal hyperparathyroidism: function, inheritance and phenotype

in European Journal of Endocrinology
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  • 1 Division of Endocrinology and Diabetes, Department of Medicine I, Universitätsklinikum Erlangen, Erlangen, Germany
  • | 2 Endokrinologie in Charlottenburg, Berlin, Germany
  • | 3 Department of Endocrinology, Diabetes and Nutrition
  • | 4 Center for Chronic Sick Children, Pediatric Endocrinology and Diabetes, Charité – Universitätsmedizin Berlin, Berlin, Germany
  • | 5 Endocrinology and Diabetology, Klinikum Bielefeld, Bielefeld, Germany
  • | 6 Endocrine Practice, Heidelberg, Germany

Correspondence should be addressed to B Mayr; Email: bernhard.mayr@uk-erlangen.de
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Background

Homozygous inactivating mutations of the calcium-sensing receptor (CaSR) lead to neonatal severe hyperparathyroidism (NSHPT), whereas heterozygous inactivating mutations result in familial hypocalciuric hypercalcemia (FHH). It is unknown why in some cases heterozygous CaSR mutations cause neonatal hyperparathyroidism (NHPT) clinically similar to NSHPT but with only moderately elevated serum calcium.

Methods

A literature survey was conducted to identify patients with heterozygous CaSR mutations and NHPT. The common NHPT CaSR mutants R185Q and R227L were compared with 15 mutants causing only FHH in the heterozygous state. We studied in vitro calcium signaling including the functional consequences of co-expression of mutant and wild-type (wt) CaSR, patients’ phenotype, age of disease manifestation and mode of inheritance.

Results

All inactivating CaSR mutants impaired calcium signaling of wt-CaSR regardless of the patients’ clinical phenotype. The absolute intracellular calcium signaling response to physiologic extracellular calcium concentrations in vitro showed a high correlation with patients’ serum calcium concentrations in vivo, which is similar in NHPT and FHH patients with the same genotype. Pedigrees of FHH families revealed that paternal inheritance per se does not necessarily lead to NHPT but may only cause FHH.

Conclusions

There is a significant correlation between in vitro functional impairment of the CaSR at physiologic calcium concentrations and the severity of alterations in calcium homeostasis in patients. Whether a particular genotype leads to NHPT or FHH appears to depend on additional predisposing genetic or environmental factors. An individual therapeutic approach appears to be warranted for NHPT patients.

Abstract

Background

Homozygous inactivating mutations of the calcium-sensing receptor (CaSR) lead to neonatal severe hyperparathyroidism (NSHPT), whereas heterozygous inactivating mutations result in familial hypocalciuric hypercalcemia (FHH). It is unknown why in some cases heterozygous CaSR mutations cause neonatal hyperparathyroidism (NHPT) clinically similar to NSHPT but with only moderately elevated serum calcium.

Methods

A literature survey was conducted to identify patients with heterozygous CaSR mutations and NHPT. The common NHPT CaSR mutants R185Q and R227L were compared with 15 mutants causing only FHH in the heterozygous state. We studied in vitro calcium signaling including the functional consequences of co-expression of mutant and wild-type (wt) CaSR, patients’ phenotype, age of disease manifestation and mode of inheritance.

Results

All inactivating CaSR mutants impaired calcium signaling of wt-CaSR regardless of the patients’ clinical phenotype. The absolute intracellular calcium signaling response to physiologic extracellular calcium concentrations in vitro showed a high correlation with patients’ serum calcium concentrations in vivo, which is similar in NHPT and FHH patients with the same genotype. Pedigrees of FHH families revealed that paternal inheritance per se does not necessarily lead to NHPT but may only cause FHH.

Conclusions

There is a significant correlation between in vitro functional impairment of the CaSR at physiologic calcium concentrations and the severity of alterations in calcium homeostasis in patients. Whether a particular genotype leads to NHPT or FHH appears to depend on additional predisposing genetic or environmental factors. An individual therapeutic approach appears to be warranted for NHPT patients.

Introduction

The calcium-sensing receptor (CaSR) is the key sensor for extracellular calcium (1, 2, 3). It is expressed mainly in parathyroid tissue, kidney and bone and regulates parathyroid hormone (PTH) secretion and calcium handling in the kidney. The CaSR belongs to class C of the G protein-coupled receptor superfamily and activates the phospholipase C – calcium and other signaling pathways (4).

Inactivating mutations of CaSR elevate the set point of receptor activation by extracellular calcium ([Ca2+]o) and give rise to hypercalcemic disorders such as neonatal severe hyperparathyroidism (NSHPT) caused by homozygous inactivating mutations and the rather benign disease familial hypocalciuric hypercalcemia (FHH) in adults caused by heterozygous inactivating mutations (1, 2, 5).

However, this clear-cut genotype–phenotype correlation does not hold in several well-documented cases of neonatal hyperparathyroidism, where only one mutated CaSR allele was found (Table 1). In these cases, hypercalcemia with serum calcium levels of around 3 mM was similar to adult patients with the same mutant but only FHH. In contrast, typical patients with neonatal severe hyperparathyroidism (NSHPT) have serum calcium concentrations between 5 and 8 mM (Table 1 and (5, 6, 7, 8)). The milder form of neonatal hyperparathyroidism is therefore more appropriately called NHPT (neonatal hyperparathyroidism).

Table 1

Clinical data of patients with different phenotypes and CaSR mutations tested in this study. Clinical and biochemical data of NSHPT, NHPT and FHH patients at the age of first signs or symptoms.

Phenotype–GenotypeAge at first signs/symptomsMaternal genotypeSigns, symptoms and clinical dataS-Ca (mM)PTH (pg/mL)S-PO4 (mM)25-OH Vit D (nM)U-CaReferences
NSHPT
R227X/R227X7 daysR227X/wtOsteopenia, dehydration65730.423 (75–200)Ca/Cr 0.2–8.3a(31)
NHPT
R185Q/wt1 dayWtOsteopenia, fractures, SGA3.339–4000.973 (27–105)CaCl/CrCl 0.0005–0.006b(9)
7 daysWtOsteopenia, oligohydramnios and SGA3.1663NR25 (49–246)CaCl/CrCl < 0.002b(32)
2 daysWtOsteopenia, fractures, oligohydramnios, NGA, lethargic, hypotonic and apneic3.211540.911 (32–80)CaCl/CrCl 0.003b(33)
1 dayWtOsteopenia, fractures, bell-shaped hypoplastic chest and respiratory distress3.3–3.65630.6NRCa/Cr 2.8–7.9b(34)
After birthWtOsteopenia, bell-shaped chest and hypotonia3.4c76c1.4c42 (62–200)cCa/Cr 6.5b,c(26)
4 weeksWtOsteopenia, fractures and failure to thrive3.31961.475 (62–200)Ca/Cr < 0.2b(26)
R227L/wtAfter birthWtOsteopenia, fractures and respiratory distress2.91700.9NRU-Ca ex 0.05 mmol/kg/24 hb(22, 35)
FHH
L159F/wt11 yearsWtAsymptomatic2.9–3.4721.215 (50–300)NDThis study
R185Q/wtNR4x wt, 8x R185Q/wt12 asymptomatic individuals3.0–3.4NRNRNRMean U-Ca ex 2.7 mmol/24 hb(36, 37)
34 yearsWtAsymptomatic2.9–3.144–690.6–0.9NRCaCl/CrCl 0.005b(38)
NRNRNRNRNRNRNRNR(39)
R227G/wt38 yearsNRAsymptomatic2.8NRNRNRCaCl/CrCl 0.005b(31)
R227Q/wtNRNR12 asymptomatic individuals2.9NRNRNRNR(40, 41)
60 yearsNRBone pain2.8461.1105CaCl/CrCl 0.007b(15)
3x R227Q/wt3 asymptomatic individuals2.7–2.933–651.0–1.4NRCaCl/CrCl 0.004–0.009b(15)
R227X/wt3 asymptomatic individuals2.4–2.628–951.1–1.58–38 (75–200)Ca/Cr 0.02–0.08b(31)
S272I/wt65 yearsNDAsymptomatic2.8460.864 (50–300)NDThis study
S417C/wt73 yearsNDAsymptomatic2.778NDNDNDThis study
Q459R/wt4x Q459R/wt4 asymptomatic individuals2.5–2.635–661.1–1.618–75 (50–200)CaCl/CrCl 0.002–0.008b(13)
8x Q459R/wt8 asymptomatic individuals2.4–2.98–85ND8–35 (25–125)CaCl/CrCl 0.001–0.01b(42)
C568Y/wt22 yearsNRPancreatitis2.840NRNRCa/Cr < 0.03b(43)
68 yearsNRBone pain3.01981.087 (21–112)U-Ca ex < 1.8 mmol/24 hb(11)
G571V/wt18 yearsG571V/wtAsymptomatic3.01361.0NDCaCl/CrCl 0.008bThis study
C582R/wt44 yearsNDAsymptomatic3.1750.941 (50–250)CaCl/CrCl 0.007bThis study
R680H/wtND2 asymptomatic individuals2.5–2.737–500.7–0.8NDNDThis study
V697M/wt9 yearsV697M/wtSeizures3.1191.4NRCaCl/CrCl 0.001b(44)
V697M/wtNDNDOsteopenia2.81090.9102 (>50)U-Ca ex 6 mmol/24 hbThis study
I777L/wt59 yearsNDAsymptomatic2.7890.944 (75–175)CaCl/CrCl 0.01bThis study
C851F/wt5–39 years1x C851F/wt, 3x wt, 2x ND6 asymptomatic individuals2.9–3.225–257NDNDCaCl/CrCl 0.001–0.006bThis study
G1019R/wt54 yearsNDAsymptomatic2.91860.755 (21–112)U-Ca ex 0.72 mmol/24 hbThis study

The patient was briefly treated with furosemide i.v. during initial diagnosis when the Ca/Cr of 8.3 mM/mM was determined; bU-Ca was determined without reported diuretic therapy; cvalues at 11 months of age; age at first signs/symptoms; age at which specific disease manifestations of NSHPT, NHPT or FHH were documented.

25-OH Vit D, 25-OH vitamin D and the normal range of the assay used is given in brackets where available; Ca/Cr, urinary calcium creatinine ratio (mM/mM), diagnostic cutoff for NSHPT, NHPT or FHH < 0.5 (30), original values were converted to SI units if necessary; CaCl/CrCl, ratio of calcium clearance to creatinine clearance, diagnostic cutoff for NSHPT, NHPT or FHH < 0.01–0.02 (30); FHH, familial hypocalciuric hypercalcemia; ND, not determined; NGA, normal for gestational age; NHPT, neonatal hyperparathyroidism; NR, not reported; NSHPT, neonatal severe hyperparathyroidism; S-Ca, total serum calcium; SGA, small for gestational age; S-PO4, serum phosphate; S-PTH, serum parathyroid hormone; U-Ca ex, urinary calcium excretion; U-Ca, urinary calcium; wt, wild type.

Although the total or near-total loss of CaSR function in homozygous patients readily explains the severe biochemical and clinical phenotype in NSHPT, it is unclear why heterozygous CaSR mutations in some cases lead to serious clinical problems shortly after birth (NHPT), whereas in majority of patients no or only minor symptoms exist in adults (FHH).

One explanation proposed is that in NHPT, the mutated CaSR protein has a negative effect on the wild-type (wt) CaSR transcribed from the remaining normal allele, which results in impaired CaSR signaling in cells expressing both mutant and wt-CaSR (1, 9). An alternative explanation attributed the NHPT phenotype to a paternally inherited or de novo CaSR mutant. This leads to the unique situation of a fetus with an impaired CaSR function developing in a maternal environment with a normal CaSR (9, 10).

Neither hypothesis about the pathophysiology of NHPT has been comprehensively tested by studying functional impairment and negative effects of CaSR mutants on wt-CaSR in vitro, the mode of inheritance and the clinical phenotype in vivo.

Subjects and methods

Patients and mutants

A literature search was performed to identify CaSR mutants that can cause NHPT in the heterozygous state. We chose the mutants R185Q and R227L for further in vitro analyses because there are six documented cases of NHPT with a heterozygous R185Q mutation and the amino acid R227 offered the possibility to study an NHPT mutant (R227L) in comparison with point and truncation mutants at the same position that cause FHH (R227G, R227Q and R227X) (Table 1). For additional comparison, we selected CaSR mutants from published reports and our own FHH patients where a complete history and biochemical workup was available to assess the clinical phenotype and its time of manifestation. Patients’ family history and genetic studies were reviewed to determine the mode of inheritance of the CaSR mutation. This study was performed according to the Bavarian state law (Bayerisches Krankenhausgesetz/Bavarian Hospital Law Art. 27 paragraph 4) that allows the use of patient data for research, provided that any person’s related data are kept anonymous. In addition, this study was also examined by a local ethics committee, and a written statement was provided verifying that no ethics committee review was required for this study and that there were no concerns regarding its publication.

Functional analyses of CaSR function

Expression plasmids for wt-CaSR and 17 mutants from patients with NSHPT, NHPT and FHH (Table 1) were generated by site-directed mutagenesis, confirmed by sequencing and expressed in HEK 293T cells cultured on glass coverslips as described before (11, 12, 13, 14). One microgram CaSR expression vector or, for co-transfection experiments, 0.5 µg mutant CaSR and 0.5 µg YFP-tagged wt-CaSR were used for transient transfection. Transiently transfected HEK 293T cells were loaded with 5 µM Fura-2/AM (Invitrogen), placed in superfusion buffer and used for measurements of cytosolic free calcium ([Ca2+]i) by dual-wavelength excitation microfluorometry. Dose–response curves were carried out as described (11, 12, 13, 14). Protein expression was analyzed by Western blotting (Supplementary Methods and Supplementary Fig. 1, see section on supplementary data given at the end of this article).

Statistical analysis

Nonlinear regression of dose–response curves was performed with GraphPad Prism 6 (GraphPad) using ∆[Ca2+]i values. EC50 values, maximum ∆[Ca2+]i response and 95% confidence intervals were determined from the nonlinear regression curves (12, 14). The regression fits of wt and mutant/wt-CaSR were tested for statistically significant differences with an F-test using GraphPad Prism 6 (GraphPad) by comparing two nested models as described (14). In the first model, the parameters EC50 or maximum ∆[Ca2+]i response were common for both wt and mutant CaSR, and in the second model, these parameters were allowed to be different.

Linear correlation analyses of average serum calcium levels in heterozygous patients without prior parathyroidectomy or cinacalcet or bisphosphonate therapy (taken from Table 1) against in vitro parameters were performed by calculating the Pearson product-moment correlation coefficient (r) using Microsoft Excel 2012 (Microsoft). In vitro parameters used were the EC50 and the maximum ∆[Ca2+]i of the dose–response curves for [Ca2+]o-induced changes in [Ca2+]i, as well as the absolute ∆[Ca2+]i at every [Ca2+]o from 0.5 to 30 mM at 0.1 mM intervals (derived from dose–response curves shown in Fig. 1). Results were confirmed using SigmaPlot version 11.0 (Systat, Erkrath, Germany).

Figure 1
Figure 1

Intracellular free calcium [Ca2+]i signaling function and effects on wt-CaSR of 17 inactivating CaSR mutants causing NSHPT, NHPT or FHH. Dose–response curves (±95% confidence interval) of Δ[Ca2+]i in response to a stepwise increase of [Ca2+]o for wt-CaSR (solid line with gray confidence interval) and wt-CaSR co-expressed with mutant (dashed lines). Results from eight to 50 individual HEK-293T cells from at least three independent experiments are shown.

Citation: European Journal of Endocrinology 175, 5; 10.1530/EJE-16-0223

Results

Mutant CaSR attenuates wt-CaSR-induced calcium signaling in vitro

The NSHPT mutant R227X showed no increase in [Ca2+]i in response to stimulation with [Ca2+]o as expected (Supplementary Fig. 1, solid lines). By contrast, the NHPT mutants R185Q and R227L showed less severe signaling impairment with marked shifts of EC50 for extracellular calcium [Ca2+]o to the right but no decrease in maximum [Ca2+]i response (Supplementary Fig. 1, solid lines, and Table 2). All mutants from FHH patients showed an impaired signaling function with increases in EC50 and/or a reduction of the maximal [Ca2+]i response (Table 2).

Table 2

Effects on wt-CaSR function of 17 inactivating CaSR mutants causing different clinical phenotypes.

PhenotypeMutant or wtMutant and wt co-expressed
GenotypeEC50 (mM)Max response (nM)EC50 (mM)Max response (nM)
Normal
Wt3.3 (3–3.6)178 (166–190)
NSHPT
R227Xa2.5 (1.2–5.4)8.6 (6.15–11.1)2.9 (2.5–3.3) NS133 (120–147)***
NHPT
R185Qb17.5 (14–22)181 (138–224)3.7 (3.4–3.9) NS183 (172–194) NS
R227L8.4 (7.5–9.4)231 (212–250)3.9 (3.6–4.2)**203 (191–216)**
FHH
L159F8.8 (7.9–9.8)174 (158–189)4.6 (4.1–5.1)***156 (143–170) NS
R227G8.1 (6.8–9.7)199 (173–224)3.5 (3.2–3.8) NS177 (164–190) NS
R227Q4.7 (4.0–5.4)151 (136–167)4.3 (3.8–4.8)**162 (149–175) NS
S272I46.3 (6.9–310)356 (–200 to 913)6.3 (5.2–7.7)***167 (146–189) NS
S417C5 (4.5–5.5)109 (100–117)3.0 (2.6–3.3) NS115 (103–126)**
Q459R3.9 (3.4–4.5)125 (111–139)3.8 (3.5–4.2)*133 (124–143)***
C568Y6.7 (4.4–10.1)7 (5.4–8.7)5.8 (4.1–8.3)*120 (93–146)*
G571V4.3 (3.9–4.7)124 (111–136)4.3 (3.8–4.8)**134 (123–145)***
C582R15.2 (0.2–946)14 (–6.5 to 34.5)3.7 (3.4–4.0) NS96 (86–106)**
R680H4.7 (4.3–5.0)109 (102–116)3.8 (3.4–4.3) NS140 (128–152)*
V697M6.1 (5.3–7.1)163 (148–177)4.5 (4.0–5.1)***157 (142–171) NS
I777L5.2 (4.3–6.2)177 (154–199)6.4 (5.2–7.9)***214 (187–242) NS
C851F9 (6.9–11.7)89 (73–106)3.9 (3.4–4.5) NS87 (78–97)*
G1019R3.7 (3.4–4.0)134 (125–144)4.2 (3.9–4.5)***140 (132–148)***

EC50, extracellular calcium concentration giving half maximal response; max response; maximal response defined as the top plateau of the dose–response curve determined in the nonlinear regression analysis. 95% confidence intervals are given in brackets. The P values for the regression fit was obtained by comparing nested models with EC50 and maximum response common between mutant co-expressed with wild type and wild type or allowed to be different. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; aR227X/wt causes FHH (Table 1); bR185Q/wt can also cause FHH (Table 1).

Co-expression of most CaSR mutants with wt-CaSR (Fig. 1, dashed lines) resulted in an increase in the EC50 for [Ca2+]o-induced changes in [Ca2+]i and/or a decrease in the maximum [Ca2+]i, response compared with wt-CaSR alone. This indicates a dominant-negative effect of the CaSR mutants on wt-CaSR (Table 2). Most interestingly, however, the two NHPT mutants R185Q and R227L behaved differently. Co-expression of R185Q with wt-CaSR did neither affect the EC50 nor the maximum [Ca2+]i signaling response, and co-expression of R227L only caused a small right shift of the dose–response curve and actually seemed to enhance the maximum [Ca2+]i response of wt-CaSR (Table 2).

A closer look at the dose–response curves at [Ca2+]o below 3 mM, which is approximately in the physiologic in vivo range, revealed that the [Ca2+]o-induced [Ca2+]i increase was clearly attenuated. There was hardly any overlap of the 95% confidence intervals in cells co-expressing wt-CaSR and the R185Q CaSR mutant compared with cells expressing wt-CaSR only (Fig. 1, dashed lines vs gray, confidence interval of the wild type). A similar impairment in cytosolic calcium signaling at low [Ca2+]o was observed when wt-CaSR was co-expressed with the other NHPT mutant R227L. The FHH mutants R227G and R227Q also attenuated the wt-CaSR cytosolic calcium response at low extracellular calcium. By contrast, the truncated CaSR mutant R227X, which in the heterozygous state barely leads to a rise in serum calcium level in affected patients (Table 1), did not seem to affect the wt-CaSR-induced cytosolic calcium response at [Ca2+]o below 4 mM (Fig. 1, dashed lines). This suggests a relationship between the [Ca2+]o-induced absolute [Ca2+]i increases at lower [Ca2+]o in vitro with in vivo serum calcium levels measured in patients.

[Ca2+]o-induced calcium signaling in vitro correlates with serum calcium in vivo

To explore a possible relationship between the [Ca2+]o-induced cytosolic calcium response in vitro with in vivo serum calcium in heterozygous patients, a linear correlation analyses was performed for all 17 CaSR mutants co-expressed with wt-CaSR. In vitro parameters used were the EC50 and the maximum ∆[Ca2+]i of the dose–response curves for [Ca2+]o-induced changes in [Ca2+]i, as well as the absolute ∆[Ca2+]i at [Ca2+]o ranging from 0.5 to 30 mM. The Pearson product-moment correlation coefficient (r) was 0.228 (P = 0.36) for serum calcium and EC50 and −0.147 (P = 0.56) for serum calcium and maximum ∆[Ca2+]i response. By contrast, the correlation between serum calcium in vivo and the cytosolic calcium response at different [Ca2+]o peaked at 2.0 mM [Ca2+]o with a r value of −0.829 (P = 0.000022) (Fig. 2A). This indicates a highly significant negative correlation of patients’ in vivo serum calcium levels with the in vitro ability to activate cytosolic calcium signaling in response to [Ca2+]o in the physiologic range. Accordingly, patients heterozygous for the R185Q CaSR mutation had high serum calcium levels, whereas patients heterozygous for R227X CaSR had only mildly elevated serum calcium levels (Fig. 2B). Remarkably, there is no apparent difference between NHPT and non-NHPT mutants. This demonstrates a strong link between the functional impairment of the CaSR to activate the calcium signaling pathway in vitro and in vivo calcium homeostasis in patients regardless of clinical phenotype.

Figure 2
Figure 2

Relationship between CaSR-activated cytosolic calcium signaling in vitro and serum calcium levels in vivo. Correlation analysis of average serum calcium levels against absolute cytosolic calcium responses (Δ[Ca2+]i) of all 17 CaSR mutants co-expressed with wt-CaSR. Average serum calcium concentrations were calculated from patients shown in Table 1 and the midpoint of the reference range of serum calcium (2.1–2.6 mM) of 2.35 mM was used for wt-CaSR. Absolute cytosolic calcium responses in vitro (Δ[Ca2+]i) at in vitro [Ca2+]o from 0.5 to 30 mM at 0.1 mM intervals were derived from dose–response curves shown in Fig. 1. (A) Pearson productmoment correlation coefficient (r) at different in vitro [Ca2+]o; r max, highest r value. (B) Individual regression analysis at [Ca2+]o of 2.0 mM with the highest r. NHPT mutants R185Q and R227L are indicated by gray circles, wt-CaSR is shown as a black circle.

Citation: European Journal of Endocrinology 175, 5; 10.1530/EJE-16-0223

Paternal inheritance per se does not cause NHPT

There are reports about 14 patients with a heterozygous R185Q CaSR mutation, who had no neonatal symptoms (Table 1). This clearly demonstrates that additional factors exist, which determine the clinical phenotype and its time of manifestation. We reviewed our patient’s family histories in order to assess the impact of paternal inheritance or de novo mutation (Table 1). In two families, we identified a total of four subjects harboring heterozygous CaSR mutations, who were born to mothers with wt-CaSR (Fig. 3). None of the four individuals (one with a L159F/wt and three with a C851F/wt genotype) had clinical symptoms as neonates. They presented as asymptomatic FHH with rather high serum calcium levels and were diagnosed between the age of 11 and 39 years because of family screening or routine blood tests. Both, the L159F and the C851F CaSR mutant, caused a similar impairment of the cytosolic calcium response at 2.0 mM [Ca2+]o as the R185Q mutant when co-expressed with wt-CaSR (Fig. 2B). This demonstrates that paternal inheritance per se does not necessarily lead to NHPT.

Figure 3
Figure 3

Pedigree of families with the CaSR mutations L159F (A) and C851F (B). Half-filled symbols indicate individuals with heterozygous CaSR mutations, open symbols indicate individuals with homozygous wt-CaSR. Squares indicate male, circles indicate female individuals. No patient with a heterozygous CaSR mutation had NHPT.

Citation: European Journal of Endocrinology 175, 5; 10.1530/EJE-16-0223

Discussion

Genotype, function and phenotype

NHPT patients with symptomatic hyperparathyroidism shortly after birth contradict the paradigm that heterozygous CaSR mutations cause rather mild or asymptomatic FHH phenotypes in adults and that a homozygous CaSR mutation is required for a neonatal phenotype. This apparent exception from the genotype–phenotype relationship has been attributed to a dominant-negative effect of CaSR mutants causing NHPT on the wt-CaSR (1, 9). Consistently, the R185Q and R227L CaSR mutants impaired wt-CaSR-dependent cytosolic calcium signaling as described previously (9, 15).

However, each of the 15 mutants we tested, which cause only a FHH phenotype in adults, leads to an even more severe alteration of wt-CaSR-dependent cytosolic calcium signaling with lower maximum [Ca2+]i responses and higher EC50 values. Furthermore, there was no correlation between the EC50 values or the maximum cytosolic calcium responses with serum calcium concentrations in vivo. Thus, the assumption that parameters of overall impairment of CaSR-dependent cytosolic calcium signaling could be associated with the clinical phenotype appears to be misleading. However, there was a highly significant correlation between the [Ca2+]o-induced cytosolic calcium response in the physiologic range in vitro and the in vivo serum calcium levels. For example, expression of the CaSR mutants R185Q and R227L strongly attenuate wt-CaSR cytosolic calcium signaling at 2 mM [Ca2+]o and leads to higher serum calcium levels in affected patients compared with CaSR mutants such as R227X, S417C or I777L that inhibit wt-CaSR-dependent calcium signaling only at higher [Ca2+]o (Fig. 1). The observed correlation at low [Ca2+]o appears to explain most of the changes in the calcium metabolism in affected patients. Thus, a function–phenotype model appears to predict the clinical disturbances of the calcium homeostasis in NHPT and FHH patients better than a simple genotype–phenotype model.

The finding that the vast majority of inactivating CaSR mutants negatively affects wt-CaSR signaling function fits well into this concept. Loss of one CaSR allele leads to only mild FHH in mice (16), and it has been noticed before that FHH patients with mutants that completely abolish protein expression from one allele tend to have lower serum calcium levels compared with FHH patients that express a functionally impaired CaSR protein (17). This is also nicely illustrated by the in vitro and in vivo differences observed between truncation and point mutations occurring at the same amino acid. Patients with the CaSR truncation mutants R227X and R185X have lower serum calcium levels than those with R227G, Q, L or R185Q mutants (Table 1 and (18)). A dominant-negative impact of mutant CaSR has been previously suspected to underlie elevated serum calcium levels in FHH patients (15, 17), and this notion is clearly supported by our results.

Homeostasis and clinical severity

In FHH and NSHPT, there is a good correlation between alterations in calcium metabolism and the overall clinical severity. FHH patients have moderately elevated serum calcium levels and are mostly asymptomatic, whereas homozygous NSHPT patients have grossly elevated serum calcium concentrations and are critically ill. In contrast, NHPT and FHH patients have very similar serum calcium levels, particularly those with the same heterozygous mutations. The mutant R185Q is by far the most frequent mutation in NHPT patients reported so far with six published cases. There are, however, four reports with a total of 14 patients harboring the same genotype but presenting as FHH (Table 1). R185Q/wt patients with NHPT have serum calcium levels between 3.1 and 3.6 mM and R185Q/wt patients with FHH between 2.9 and 3.4 mM (Table 1). NHPT patients with heterozygous mutations in other amino acids have serum calcium levels between 2.8 and 3.2 mM, whereas typical homozygous NSHPT patients have serum calcium levels between 5 and 8 mM (Table 1 and (5, 6, 7, 8)). In fetuses and neonates with heterozygous CaSR mutations, the alteration of calcium homeostasis and the overall severity of the clinical phenotype can obviously be dissociated.

Inheritance

Paternal inheritance or a de novo CaSR mutation has been suggested as one possible explanation for the development of NHPT (9, 10). In this and in previous studies, however, neonates with heterozygous CaSR mutations born to mothers with wt-CaSR did not develop NHPT (Fig. 3 and Table 1). Nevertheless, it is noteworthy that none of the published cases of heterozygous NHPT was born to a mother with a mutated CaSR (Table 1, (19, 20, 21, 22)). Thus, paternal inheritance or a de novo CaSR mutation does not cause NHPT per se, but there may be genetic factors such as allelic imbalance that predispose or contribute to the risk for NHPT.

Calcium and vitamin D supply

Metabolic bone disease and osteopenia are a clinical hallmark of NHPT (Table 1 and (19, 20, 23)). During fetal development, calcium is exclusively supplied by the mother via active placental transport that is stimulated by fetal PTH-related peptide (PTHrP) but not by maternal or fetal PTH (24). PTHrP is highly expressed in murine fetal parathyroid glands and its secretion seems to be regulated through CaSR-dependent mechanism, as in CaSR knockout fetuses plasma PTHrP levels as well as placental calcium transport are reduced (25). If in humans the same regulatory mechanisms exist, impaired CaSR activity would cause decreased PTHrP expression and reduced calcium supply to the fetus. The resulting fetal calcium deficiency can lead to secondary hyperparathyroidism, which is exaggerated by the elevated calcium set point due to the inactivating CaSR mutant. Both low calcium and high PTH promote clinically significant metabolic bone disease. A similar pathomechanism has been observed in a young FHH patient with rickets due to pronounced calcium and vitamin D deficiency (13). In NHPT patients, vitamin D deficiency is common (Table 1 and (19, 20, 23)), and as in FHH patients, sufficient calcium and vitamin D supply reduces PTH and improves metabolic bone disease (13, 20). By contrast, calcium restriction can lead to a further rise in PTH and a lack of clinical improvement (26). This model of calcium deficiency and an exaggerated secondary hyperparathyroidism explains quite well the observed pathologic changes in NHPT patients. Further support is provided by several cases, in which NHPT was self-limited under supportive care and normal nutrition despite stable (27) or even slightly rising (21, 28, 29) serum calcium levels. According to this concept, NHPT may develop if the combined effects of impairment of wt-CaSR function by the mutant and of additional environmental or genetic factors that reduce maternal calcium supply to the fetus reach a critical threshold. This could also explain the variable phenotype in patients heterozygous for the R185Q CaSR mutant, but direct effects of the mutant CaSR on chondrocytes and osteoblasts may also contribute to the phenotype.

Summary

Taken together, there is a strong and highly significant correlation between functional impairment of CaSR-dependent calcium signaling and patients’ serum calcium levels. Patients with complete or near-complete loss of CaSR signaling, for example, due to severely inactivating homozygous CaSR mutations develop NSHPT with excessive PTH secretion that is not suppressed by even grossly elevated serum calcium levels. In contrast, patients with only mild impairment of CaSR function develop FHH. CaSR mutants that cause a more pronounced inhibition of wt-CaSR at physiological calcium concentrations may either lead to a neonatal phenotype (NHPT) or to FHH. Additional environmental and/or genetic factors such as allelic imbalance that critically reduce maternal–fetal calcium transfer may determine whether a particular genotype leads to NHPT instead of FHH. Calcium deficiency during fetal development combined with an exaggerated secondary hyperparathyroidism due to impaired overall CaSR function may then lead to neonatal manifestation as NHPT with serious metabolic bone disease, possibly also in patients with mutations that until now have only led to FHH. Although homozygous NSHPT and heterozygous NHPT have similarities in clinical phenotype, heterozygous NHPT shares pathophysiologic characteristics with FHH. An individual therapeutic approach to NHPT patients appears warranted.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/EJE-16-0223.

Declaration of interest

We certify that no author has a conflict of interest that is relevant to the subject matter or materials included in this work.

Funding

This work was supported by institutional grants from the Charité-University Medicine Berlin and from the Friedrich-Alexander University Erlangen-Nuremberg.

Author contribution statement

Bernhard Mayr and Christof Schöfl conceived and designed the experiments. Markus Glaudo and Saskia Letz performed the experiments. Markus Glaudo, Saskia Letz and Bernhard Mayr analyzed the data. Bernhard Mayr, Christof Schöfl and Markus Glaudo wrote the paper. Marcus Quinkler, Ulrich Bogner, Ulf Elbelt, Christian J Strasburger, Dirk Schnabel, Erwin Lankes, Sandra Scheel, Joachim Feldkamp, Christine Haag, Egbert Schulze, Karin Frank-Raue and Friedhelm Raue contributed human genetic and clinical data.

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    Intracellular free calcium [Ca2+]i signaling function and effects on wt-CaSR of 17 inactivating CaSR mutants causing NSHPT, NHPT or FHH. Dose–response curves (±95% confidence interval) of Δ[Ca2+]i in response to a stepwise increase of [Ca2+]o for wt-CaSR (solid line with gray confidence interval) and wt-CaSR co-expressed with mutant (dashed lines). Results from eight to 50 individual HEK-293T cells from at least three independent experiments are shown.

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    Relationship between CaSR-activated cytosolic calcium signaling in vitro and serum calcium levels in vivo. Correlation analysis of average serum calcium levels against absolute cytosolic calcium responses (Δ[Ca2+]i) of all 17 CaSR mutants co-expressed with wt-CaSR. Average serum calcium concentrations were calculated from patients shown in Table 1 and the midpoint of the reference range of serum calcium (2.1–2.6 mM) of 2.35 mM was used for wt-CaSR. Absolute cytosolic calcium responses in vitro (Δ[Ca2+]i) at in vitro [Ca2+]o from 0.5 to 30 mM at 0.1 mM intervals were derived from dose–response curves shown in Fig. 1. (A) Pearson productmoment correlation coefficient (r) at different in vitro [Ca2+]o; r max, highest r value. (B) Individual regression analysis at [Ca2+]o of 2.0 mM with the highest r. NHPT mutants R185Q and R227L are indicated by gray circles, wt-CaSR is shown as a black circle.

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    Pedigree of families with the CaSR mutations L159F (A) and C851F (B). Half-filled symbols indicate individuals with heterozygous CaSR mutations, open symbols indicate individuals with homozygous wt-CaSR. Squares indicate male, circles indicate female individuals. No patient with a heterozygous CaSR mutation had NHPT.