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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-1543
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 12 5019-5021
Copyright © 2006 by The Endocrine Society

Mutational Analysis of the Vitamin D Receptor Does Not Support Its Candidacy as a Tumor Suppressor Gene in Parathyroid Adenomas

Elizabeth Hanna Samander and Andrew Arnold

Center for Molecular Medicine and Division of Endocrinology and Metabolism, University of Connecticut School of Medicine, Farmington, Connecticut 06030-3101

Address all correspondence and requests for reprints to: Andrew Arnold, M.D., Center for Molecular Medicine, University of Connecticut School of Medicine, 263 Farmington Avenue, Farmington, Connecticut 06030-3101. E-mail: molecularmedicine{at}uchc.edu.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: The vitamin D receptor gene (VDR) is a compelling candidate tumor suppressor gene for parathyroid adenomas based on existing evidence of the vitamin D system’s antiproliferative actions in parathyroid and other tissues, its reported inhibition of PTH gene transcription, and the decreased expression of VDR mRNA and VDR protein observed in parathyroid adenomas.

Objective: Because demonstration of intragenic mutations is required to establish the authenticity and primary role in pathogenesis for any candidate tumor suppressor gene, we examined the VDR gene in parathyroid adenomas for the presence of such mutations and other loss-of-function abnormalities.

Methods and Results: Genomic DNA samples from 37 sporadic parathyroid adenomas and matched normal control DNA from the same individuals were subjected to direct sequencing of the entire VDR coding region and all intron-exon boundaries. No VDR coding region or junctional mutations were identified. The tumors were also analyzed for loss of heterozygosity, a frequent mechanism of tumor suppressor gene inactivation, by molecular allelotyping at three microsatellite markers located near the VDR gene, D12S85, D12S96, and D12S368, and a polymorphism within VDR itself. In all 37 cases, at least one marker was informative and no tumor-specific loss of heterozygosity was observed.

Conclusion: We found no evidence of allelic loss within or near the VDR locus and no mutations within the splice junctions and coding regions of the VDR gene in 37 typical sporadic parathyroid adenomas. Thus, VDR is most unlikely to commonly serve as a classical tumor suppressor gene in sporadic parathyroid adenomas.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
PARATHYROID ADENOMAS ARE common benign tumors that are the predominant cause of primary hyperparathyroidism. The monoclonality of parathyroid adenomas implies that acquired (somatic) mutation in specific oncogenes and/or tumor suppressor genes confers a selective growth advantage on a parathyroid cell leading to its clinically significant clonal expansion (1). Although defects in two such genes, cyclinD1/PRAD1 and MEN1, are established participants in subsets of typical sporadic parathyroid adenomas, additional but still-unidentified oncogenes or tumor suppressor genes undoubtedly contribute to their pathogenesis (2).

The vitamin D receptor gene (VDR) is an attractive tumor suppressor gene candidate in sporadic primary parathyroid adenomas because of the vitamin D system’s effects on parathyroid cell growth and function. First, the intact 1,25(OH)2D3:VDR complex suppresses parathyroid cell proliferation and down-regulates PTH synthesis and secretion (3, 4, 5, 6, 7, 8, 9, 10). Next, the antiproliferative and differentiation-promoting effects of vitamin D are well documented in a wide variety of cell types, including those of hematopoietic, keratinocyte, prostate, colon, and breast origins (11, 12). Finally, decreased expression of VDR mRNA and VDR protein have been observed in human parathyroid adenomas (13, 14, 15), consistent with possible roles for disturbed VDR action, whether direct or indirect, in the abnormal cell proliferation and/or the abnormal set point of calcium-regulated PTH secretion characteristic of parathyroid adenomas (16, 17). The finding of recurrent acquired defects in the VDR gene would provide crucial evidence that VDR alterations provide a selective advantage and are primary contributors to tumorigenesis. Therefore, we sought to examine the VDR gene for intragenic inactivating mutations, the evidence needed to rigorously establish it as a tumor suppressor gene (18) in the pathogenesis of typical sporadic parathyroid adenomas.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Tumor samples

Parathyroid tumor tissue and paired normal peripheral blood leukocytes were obtained, in accordance with institutional review board-approved protocols for human studies, from 37 patients who underwent surgical treatment for typical sporadic primary hyperparathyroidism in the United States and who proved to have single-gland disease with no atypical histopathological features. Patients had no clinical evidence or history of familial hyperparathyroidism. After parathyroidectomy, clinicopathological findings and follow-up were consistent with the diagnosis of single-gland parathyroid adenoma. Resected parathyroid adenoma tissue samples were immediately frozen in liquid nitrogen and stored at –80 C. Genomic DNA was extracted from the tissue and blood samples by standard proteinase K digestion, followed by phenol-chloroform extraction and ethanol precipitation.

Molecular allelotyping

Loss of heterozygosity (LOH) analysis was performed on genomic DNA from tumor and matched peripheral blood leukocyte controls to detect possible allelic loss of polymorphic DNA markers within and near the VDR gene. For each paired sample of DNA, three microsatellite loci located near VDR (D12S85, D12S96, D12S368; see Table 1Go) were amplified by PCR using fluorescently labeled primer pairs specific for each microsatellite (Applied Biosystems, Foster City, CA); PCR conditions were as recommended by the manufacturer. Loci were selected from the Généthon human linkage map, based on chromosomal locations and frequency of heterozygosity. After amplification, samples were electrophoresed on 5% Long Ranger gels (BioWhittaker Molecular Applications, Rockland, ME) on an ABI Prism 377 sequencer (Applied Biosystems) and data were analyzed using Genescan and Genotyper software (Applied Biosystems). Preferential loss of at least 50% of signal from one allele was scored as LOH (19). To analyze allelic loss directly within the VDR locus, the TaqI intragenic polymorphism in exon 9 (refSNP ID: rs7311236; http://www.ncbi.nlm.nih.gov/SNP/) was genotyped. DNA samples were subjected to PCR amplification using oligonucleotide primers spanning the TaqI polymorphism. Sequences of oligonucleotide primers were previously published (20). After the first denaturation at 94 C for 5 min, 30 cycles followed: 94 C for 1 min, 68 C for 1 min, and 72 C for 1 min 30 sec, followed with final elongation at 72 C for 7 min, which terminated the reaction. The 740-bp PCR product was digested using 1 U of Taq{alpha}I restriction enzyme at 65 C for 120 min (New England Biolabs, Beverly, MA). Fragments were separated electrophoretically on 2% agarose gel containing ethidium bromide and visualized under UV light. Samples homozygous for the presence of the polymorphic site (tt) yield 290, 245, and 205 bp, respectively, whereas samples homozygous for the absence of the polymorphic site (TT) yield bands of 495 and 245 bp (21). Heterozygous (Tt) presence of the TaqI restriction site exhibits a 495-, 290-, 245-, 205-bp banding pattern. Loss or retention of heterozygosity was assessed by comparing the corresponding tumor genotype for patients whose leukocyte control DNA was heterozygous (Tt).


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  		TABLE 1. Microsatellite markers used for assessing LOH

 
VDR sequencing

Genomic DNA from each tumor was used as template for PCR amplification for the entire coding region of VDR. Primers for PCRs were designed using the VDR genomic sequence (GenBank ID:7421) and the Primer 3 program (22) and are listed in Table 2Go. PCR was performed in 50-µl reactions using 50 ng genomic DNA, 20 pmol of each primer, 1 µl of 50x TITANIUM Taq DNA polymerase, 5 µl of 10x TITANIUM Taq PCR buffer (BD Biosciences Clontech, Palo Alto, CA). Touchdown PCR cycling conditions were as follows with various denaturation temperatures for each exon (see Table 2Go): 95 C for 1 min, 10 cycles of 95 C for 30 sec, and 66 C for 2 min, followed by 22 cycles of 94 C for 45 sec, denaturation (Table 2Go), 72 C for 45 sec, with a final elongation at 72 C for 10 min to terminate the reaction.


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  		TABLE 2. Primers for PCR amplification and sequencing of VDR

 
PCR products were purified using 1 U of shrimp alkaline phosphatase and 10 U of exonuclease I (Amersham Pharmacia Biotech, Piscataway, NJ). Purified PCR products were subjected to automated sequencing using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) with the same primers used for PCR amplification. Sequencing products were cleaned through Sephadex-G50 columns (Sigma-Aldrich, St. Louis, MO) and run on 4.75% Long Ranger gels on ABI Prism 377 Sequencer, and data were analyzed using Sequencing Analysis and AutoAssembler software (Applied Biosystems).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Molecular allelotyping for allelic losses performed using three microsatellite markers (Table 1Go) in the area of the genome encompassing VDR revealed no tumor-specific LOH among the 37 genetically informative control/tumor pairs (Table 3Go).


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  		TABLE 3. Frequency of loss of heterozygosity in sporadic parathyroid adenomas

 
Detailed sequence analysis of the VDR gene revealed no mutations in any of the 37 tumors. The sequencing method was robust and successfully detected known polymorphisms; for example, nine of 37 (24%) patients were heterozygous for the T>C start codon (refSNP ID: rs10735810; http://www.ncbi.nlm.nih.gov/SNP/) polymorphism, a reasonable frequency recognizing the limitation that its allele distribution varies according to race (23). Identification of such previously reported polymorphisms confirmed that our methodology was precise and sensitive enough to detect mutations if they had been present in the tumors studied.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The VDR gene has been considered a compelling candidate tumor suppressor gene in the molecular pathogenesis of parathyroid adenomas, because it is quite plausible that a parathyroid cell would attain a selective advantage through acquired mutations that would impair responsiveness to the antiproliferative influence of the calcitriol/VDR system. First, the vitamin D system is known to regulate both normal and abnormal cellular proliferation and differentiation in the parathyroids and numerous other organ systems (3, 4, 5, 6, 7, 8, 9, 10). Next, vitamin D regulates tissue-specific transcription of the PTH gene through VDR (11, 12, 24). Finally, published data demonstrate abnormal PTH response to vitamin D by parathyroid tumor cells, as well as evidence for decreased expression of VDR mRNA and VDR protein in parathyroid adenomas (13, 14, 15). Therefore, to examine VDR for the most definitive possible evidence that it fulfills strict criteria as a tumor suppressor (18), we examined parathyroid adenomas for intragenic somatic mutations that would cause loss of function of VDR. We observed no such mutations and also found no evidence for LOH of VDR, another common, although less specific, mechanism by which tumor suppressor genes can be inactivated (18, 25). Although our methods did not exclude some possible inactivating lesions such as mutation of the VDR promoter or its hypermethylation, prior experience with established tumor suppressor genes makes it implausible that frequent clonal VDR inactivation would occur to the complete exclusion of generally common mechanisms like coding region mutation and deletion. Thus, our results suggest that loss-of-function mutations within the VDR structural gene contribute rarely, if at all, as primary drivers of clonal parathyroid growth in the development of typical parathyroid adenomas.

Although VDR does not appear to be a classical tumor suppressor gene for parathyroid adenomas, it remains possible that diminished VDR expression could still play a functionally significant role in the tumor phenotype, as a downstream consequence of more primary acquired genetic abnormalities. Of interest, parathyroid VDR expression was secondarily decreased in a transgenic mouse model of primary hyperparathyroidism due to parathyroid-targeted overexpression of the cyclin D1 oncogene (17, 26). Also, VDR-null mice, 1{alpha}-hydroxylase-null mice, and double mutant (VDR–/–/1{alpha}-hydroxylase–/–) mouse models exhibited massive enlargement of parathyroid glands, whereas VDR-null and double-mutant mouse models (but not 1{alpha}-hydroxylase null) fed rescue diets were unable to normalize parathyroid glands, supporting the premise that VDR function is necessary for inhibiting parathyroid proliferation (27, 28), although more effective normalization was suggested in VDR-knockout patients receiving iv calcium infusions (29). Further investigation of the influence of the vitamin D system on the development of hyperparathyroidism or its clinical expression is certainly warranted.


   Acknowledgments
 
We thank Ms. Kristin Corrado and Dr. Elizabeth Saria for expert technical assistance, and Dr. H. Irene Wu for helpful discussions and preliminary experiments.


   Footnotes
 
This work was supported in part by The Murray-Heilig Fund in Molecular Medicine.

Disclosure statement: The authors have nothing to disclose

First Published Online September 26, 2006

Abbreviations: LOH, Loss of heterozygosity; VDR, vitamin D receptor.

Received July 17, 2006.

Accepted September 19, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Arnold A 2001 Molecular basis of hyperparathyroidism. In: Bilezikian JP, Marcus R, Levine MA, eds. The parathyroids: basic and clinical concepts. 2nd ed. New York: Academic Press; 331–347
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