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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 6 1772-1775
Copyright © 1997 by The Endocrine Society

Experimental Studies

Vitamin D Receptor Polymorphisms Correlate to Parathyroid Cell Function in Primary Hyperparathyroidism1

Tobias Carling, Peter Ridefelt, Per Hellman, Jonas Rastad and Göran Åkerström

Department of Surgery, Uppsala University Hospital, 751 85 Uppsala, Sweden

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


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Calcitriol acts via its receptor (VDR) and inhibits PTH secretion and parathyroid cell proliferation. Increased prevalence of the polymorphic VDR alleles b, a, and T has been demonstrated in sporadic primary hyperparathyroidism. Sixty-two patients with primary hyperparathyroidism due to parathyroid adenoma (mean age, 69.5 ± 1.4 yr) were genotyped for these VDR polymorphisms. Dispersed cells of the adenomas were exposed to increasing concentrations of extracellular Ca2+ and analyzed for PTH release and cytoplasmic Ca2+ concentrations. Ca2+-mediated PTH inhibition exhibited higher ED50 and less suppression in the cells of patients who were homozygous for the b, a, and T alleles (P < 0.05–0.10). When analyzing haplotypes, the patients with baT demonstrated a ED50 of 1.81 ± 0.15 vs. 1.29 ± 0.10 for BAt (P < 0.05). As VDR alleles were unrelated to parathyroid intracellular Ca2+, influences of polymorphic VDR alleles on PTH secretion seem to involve mechanisms other than the Ca2+-sensing protein of the parathyroid cell surface.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PRIMARY hyperparathyroidism (HPT) exhibits 1–3% prevalence in postmenopausal Swedish women (1, 2). The abnormal parathyroid tissue characteristically demonstrates enhanced ED50 in the inverse sigmoidal relationship between external Ca2+ and PTH secretion and reduced calcium-mediated suppression of the secretion (3, 4). This action of extracellular Ca2+ involves Ca2+-sensing receptors on the parathyroid cell surface (5), and the cytoplasmic Ca2+ concentration ([Ca2+]i) seems to be an important second messenger in this process (4, 6, 7). However, mutations in the Ca2+ receptor gene have not been found in parathyroid tumors of sporadic primary HPT (8).

Calcitriol is a principal regulator of both PTH secretion and proliferation of parathyroid cells (9, 10, 11). Calcitriol acts via its receptor (VDR), and the ligand-bound complex can inhibit PTH gene transcription by binding to vitamin D-responsive elements (12). VDR dysfunction has been implicated in the pathogenesis of both primary and secondary HPT (13, 14). Some studies, but not all, support the idea that linked polymorphisms in intron 8 (B/b and A/a alleles) and exon 9 (T/t alleles) of the VDR gene may relate to bone mineral density and the risk of developing osteoporosis (15, 16). Increased prevalence of the VDR b, a, and T alleles in patients with sporadic primary HPT suggests that these polymorphisms may comprise a risk factor in the development of this disorder (17, 18, 19). Experimental studies on minigene constructs support this idea by suggesting that the VDR haplotype baT relates to reduced VDR gene transcription and/or stability of its messenger ribonucleic acid (mRNA) (15). The present study indicates relationships between VDR polymorphisms and Ca2+ regulation of PTH release in primary HPT.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Sixty-two consecutive patients (56 women and 6 men) with primary HPT due to surgically verified parathyroid adenoma were included in the study. Twenty-two of the patients were detected at population-based screening, as described previously (17). None of the patients had a history of familial hypercalcemia or signs of multiple endocrine neoplasia syndromes. All individuals failed to demonstrate substantially increased serum creatinine values (>160 µmol/L; reference range, 64–106 µmol/L) or postoperatively persistent hypercalcemia during follow-up for 1.1–4.5 yr (mean, 2.4 yr). Total serum calcium (reference range, 2.20–2.60 mmol/L) and intact serum PTH (reference range, 12–55 ng/L) were determined as previously described (17). All patients gave informed consent to participate in the study, which was approved by the local ethical committee.

DNA analysis

Genomic DNA was prepared from whole blood according to standard methods or from parathyroid adenomas (17). VDR genotypes denoted BB, Bb, and bb were determined after Bsm 1 restriction cleavage of genomic DNA amplified by PCR, as previously reported (15, 17). A 740-bp fragment of the VDR gene, including the ApaI and TaqI restriction sites in intron 8 and exon 9, was amplified using specific primers 5'-cagagcatggacagggagcaa-3' and 5'-gcaactcctcatggctgaggtctc-3' (20). All PCR reactions were run at 95 C for 2 min followed by 35 cycles of 95 C for 30 s, 60 C for 45 s, 72 C for 60 s, and final extension at 72 C for 7 min. The PCR products were digested with ApaI (10 U at 37 C) or TaqI (3 U at 65 C) and electrophoresed in a 1.5% ethidium bromide-agarose gel. ApaI digestion reveals genotypes denoted AA (740 bp), Aa (740, 530, and 210 bp), or aa (530 and 210 bp) and TaqI genotypes denoted TT (495 and 245 bp), Tt (495, 290, 245, and 205 bp), or tt (290, 245, and 205 bp) (20).

Parathyroid cells

Parathyroid adenoma cells were suspended enzymatically as previously described (21). PTH release was evaluated by duplicate incubations of 0.5–1.0 x 106 cells for 60 min at 37 C in 0.5 mL of a 25 mmol/L HEPES buffer (pH 7.4) containing 3 mmol/L glucose and 0.1% human serum albumin (21). PTH was assayed radioimmunologically with a sheep antiserum (Giselle) raised against human PTH with 125I-labeled [Tyr44]human PTH-(44–68) as tracer and human PTH-(1–84) as standard (22). Half-maximal inhibition of PTH release (ED50) of each preparation was determined from PTH measurements in three to six Ca2+ concentrations between 0.5–3.0 mmol/L (35 patients). Maximal PTH suppression was calculated as the proportional reduction between 0.5 and 3.0 mmol/L Ca2+ (62 patients). [Ca2+]i was analyzed microfluorometrically after loading cells in 1.0 µmol/L fura-2/AM (Calbiochem, La Jolla, CA) for 30 min at 37 C in the HEPES buffer containing 0.5 Ca2+ and 0.1% BSA (21). Emitted fluorescence was measured at 510 nm, and the ratio of emission at 340/380 nm excitation was used to calculate [Ca2+]i assuming a Kd of 224 nmol/L. The mean values of three to five cells from each preparation were used to calculate [Ca2+]i at 0.5 and 3.0 mmol/L Ca2+ (60 patients) as well as the half-maximal [Ca2+]i rise (ED50) upon three to six stepwise elevations of Ca2+ from 0.5–3.0 mmol/L (20 patients).

ANOVA and Student’s unpaired two-tailed t test were used for statistical evaluation, with P < 0.05 considered significant. All results are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
VDR genotype distributions coincided with previous studies of HPT patients (Table 1Go) and contrasted with the underlying Swedish population demonstrating prevalences of 33% (bb), 21% (aa), and 34% (TT) (17, 18). Consistent with other reports (15, 20), concordance between the presence of the Bsm 1 and ApaI restriction sites and Bsm 1 and absence of TaqI was 74.2% and 98.4%, respectively. No significant association between genotypes and age, serum calcium or intact PTH, or parathyroid adenoma weight was recorded.


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  		Table 1. Clinical characteristics of the 62 HPT patients in relation to VDR genotypes

 
The characteristic sigmoidal suppression of PTH release in response to external Ca2+ exhibited an expected mean increase in ED50 to 1.59 ± 0.07 mmol/L and a decreased maximal PTH suppression of 45 ± 2.3% (4). ED50 in each genotype was highest for the aa (P < 0.05, by ANOVA), bb (P = 0.09), and TT (P = 0.09) allelic pairs, whereas the heterozygotes demonstrated intermediate values (Fig. 1Go). Moreover, patients homozygous for BAt exhibited an ED50 of 1.29 ± 0.10 mmol/L Ca2+ compared to 1.81 ± 0.18 mmol/L Ca2+ for the baT haplotype (P < 0.05). Comparisons of maximal PTH suppression showed higher values for patients with the AA genotype (P < 0.05 vs. aa; Table 2Go). Ca2+-regulated [Ca2+]i was consistent with previous analyses (21, 23) and apparently unrelated to the VDR genotypes.



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  		Figure 1. ED50 (moles per L) for Ca2+ inhibition of PTH release in relation to VDR alleles. Values (mean ± SEM) are based on duplicate estimates of PTH release in three to six Ca2+ concentrations for each of the indicated number of patients. P values were calculated by ANOVA.

 

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  		Table 2. Ca2+-regulated PTH suppression and [Ca2+]i in relation to VDR alleles

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The recent demonstration of increased prevalence of the polymorphic VDR alleles b, a, and T in sporadic primary HPT supports interaction in the largely unknown tumorigenesis of this disorder (17, 18, 19). In addition to influences on the risk of developing HPT, these polymorphisms currently are linked to Ca2+ regulation of PTH release. Homozygosity for VDR alleles b, a, and T and the baT haplotype were associated with greater increases in ED50 and lesser suppression of PTH release. It is tempting to speculate that this relationship depends on hampered regulatory actions of calcitriol from relatively reduced VDR expression in the parathyroid adenomas. Observations of approximately 30% lesser reporter gene activity upon insertion of the baT vs. the BAt haplotype into a minigene construct supports this idea (15). However, any influence of these polymorphisms on VDR transcription and/or its mRNA stability in human parathyroid glands is purely speculative. Nevertheless, the VDR genotypes were apparently unrelated to parathyroid [Ca2+]i. Any allele-related effect on PTH secretion consequently seems to involve actions other than the Ca2+-sensing proteins on the parathyroid cell surface (5). This hypothesis is consistent with the demonstration that calcitriol interferes with PTH gene transcription (12), but not parathyroid [Ca2+]i or calcium receptor mRNA levels (9, 24).

A variety of circumstances may confound the current observations. The examined VDR polymorphisms may be linked to additional polymorphisms in the VDR gene and/or to other genes of importance to parathyroid cell functions. Furthermore, the B/b alleles have been related to circulating calcitriol levels and PTH responses after the administration of calcitriol in healthy premenopausal females (15, 25). Patients with the baT haplotype indeed demonstrated no apparent discrepancy with respect to serum Ca2+ and PTH levels despite the fact that PTH secretion in vitro was less Ca2+ sensitive. Circulating PTH levels, however, may also reflect the parathyroid tissue weight and perhaps even the rate of cell proliferation (26), and serum calcium depends on a variety of factors, including peripheral actions of PTH and calcitriol. Several components, thus, may balance the consequences of any influence of polymorphic VDR alleles on PTH secretory regulation in vivo.


   Footnotes
 
1 This work was supported by the Swedish Medical Research Council, the Swedish Cancer Society, and the Swedish Society for Medical Research. Back

Received December 30, 1996.

Revised February 21, 1997.

Accepted March 11, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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