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Absence of Stabilizing Mutations of ß-Catenin Encoded by CTNNB1 Exon 3 in a Large Series of Sporadic Parathyroid Adenomas

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

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

	Abstract

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Context: The molecular mechanisms underlying the pathogenesis of sporadic parathyroid adenomas are incompletely understood. Dysfunction of the Wnt signaling pathway is an established pathogenetic contributor to human tumorigenesis and, recently, the role of stabilizing mutations in ß-catenin, a cause of abnormal Wnt signaling, has been examined in parathyroid tumors with conflicting results.

Objective: The objective of the present study was to determine the frequency of stabilizing mutations in exon 3 of CTNNB1, encoding ß-catenin, in a large series of parathyroid adenomas.

Patients and Design: Ninety-seven sporadic parathyroid adenomas were examined for mutations in exon 3 of CTNNB1 by direct DNA sequencing.

Results: No mutations were identified in any of the adenomas.

Conclusions: The absence of stabilizing mutations of ß-catenin, including the previously reported S37A, encoded in CTNNB1 exon 3 among 97 tumors suggests that such mutations contribute rarely if at all to the development of sporadic parathyroid adenomas. A primary role for abnormal Wnt signaling in parathyroid tumor formation remains to be established.

	Introduction

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PARATHYROID ADENOMAS, the most common cause of primary hyperparathyroidism, are well-differentiated, benign, clonal tumors, which produce hypercalcemia through excessive secretion of parathyroid hormone (1). Activation of the cyclin D1 (CCND1) oncogene and inactivation of the MEN1 tumor suppressor gene are established pathogenetic contributors, but understanding of the molecular pathogenesis in these tumors remains incomplete (2).

A role for dysfunctional Wnt pathway signaling has been hypothesized for parathyroid tumors, given the established importance of Wnt pathway abnormalities in various types of human tumors (reviewed in Ref. 3), plus evidence that expression of cyclin D1, the only known parathyroid oncogene, is regulated in part by Wnt signaling (4, 5). Germline mutations of the APC tumor suppressor gene are seen in patients with the hereditary cancer syndrome familial adenomatous polyposis (6), and APC is biallelically inactivated in the majority of sporadic colorectal cancers (7). Inactivation of APC leads to inappropriate stabilization of ß-catenin, which translocates to the nucleus and results in excessive transcriptional activation of lymphoid enhancer factor/T-cell factor family responsive target genes. In that phosphorylation of ß-catenin by GSK3ß normally leads to its proteosomal degradation (8), stabilization and accumulation of nonphosphorylated ß-catenin in human tumors can also be accomplished by mutation of the ß-catenin gene CTNNB1 (9). Virtually all CTNNB1 mutations identified in human tumors are located in exon 3, encoding the GSK3ß recognition motif, and most affect serine-threonine phosphorylation sites or adjacent residues, making this a "hotspot" for mutational activation of CTNNB1 (10). In addition, parafibromin, the protein product of the CDC73 (HRPT2) tumor suppressor gene that is central to the pathogenesis of parathyroid carcinoma, was recently implicated in Wnt signaling (11).

The status of ß-catenin in sporadic parathyroid adenomas is controversial. Ikeda et al. (12) found no mutation of exon 3 of CTNNB1 and no accumulation of ß-catenin in a series of 24 sporadic parathyroid adenomas, consistent with the results of an earlier study of 12 parathyroid adenomas (13). More recently, and in sharp contrast, Bjorklund et al. (14) identified a specific, homozygous, stabilizing mutation (S37A) in exon 3 of CTNNB1 in 15% (3 of 20) of parathyroid adenomas studied; they also found accumulation of nonphosphorylated ß-catenin in all 37 parathyroid adenomas studied. If confirmed, this substantial prevalence of a clonally selected ß-catenin mutation would constitute compelling evidence for a primary, driving role of abnormal Wnt signaling in parathyroid tumor development (whereas ß-catenin expression abnormalities may be primary or secondary and are much less definitively interpretable than are somatic mutations). Therefore, we sought to rigorously and directly determine the frequency of stabilizing mutations in exon 3 of CTNNB1 in a large series of parathyroid adenomas.

	Patients and Methods

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Patients and samples

Ninety-seven sporadic parathyroid adenomas were collected from 97 patients who had undergone parathyroidectomy in the United States for the management of primary hyperparathyroidism. Samples were obtained in accordance with institutional review board-approved protocols for human studies. Patients were surgically and pathologically proven to have typical single gland disease with no malignant features. Patients had no history of multiple endocrine neoplasia syndromes or familial hyperparathyroidism. Surgical samples were grossly dissected and quickly frozen in liquid nitrogen. Genomic DNA was extracted using proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation.

CTNNB1 sequencing

Exon 3 of CTNNB1 was amplified using PCR with the following primers: ß-catenin 3F, 5'-GCTGATTTGATGGAGTTGGA-3'; ß-catenin 3R, 5'-GCTACTTGTTCTTGAGTGAA-3', yielding a 227-bp PCR fragment. The location of primer sequences is shown in Fig. 1. PCRs were performed in 20-µl reaction volumes containing 25 ng genomic DNA, 20 pmol of each primer, 200 µM of each dNTP, 1.25 U of Amplitaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA), and 1.5 mM magnesium chloride. Thermocycling consisted of an initial denaturation step of 95 C for 10 min; 35 cycles of 95 C for 30 sec, 55 C for 30 sec, 72 C for 30 sec; and a final extension step at 72 C for 10 min. PCR products were purified using ExoSapIT (Amersham Pharmacia Biotech, Piscataway, NJ). Purified PCR fragments were then sequenced in both forward and reverse directions using the Dye Terminator Cycle Sequencing Quick Start kit (Beckman-Coulter, Fullerton, CA) and the same primers used for PCR, under conditions recommended by the manufacturer. Resulting sequence fragments were purified through Sephadex G-50 columns (Sigma Aldrich, St. Louis, MO) and electrophoresed on a CEQ 8800 Genetic Analysis System (Beckman). Reliable sequence results were obtained from the beginning of the coding region through c.185. Resulting sequence data were analyzed and compared with the published sequence (RefSeq ID:NM_001904) using Sequencher software (GeneCodes Corporation, Ann Arbor, MI).

View larger version (3K): [in this window] [in a new window]   FIG. 1. Schematic diagram of CTNNB1 exon 3. Exon 3 is a "hotspot" for mutational activation of CTNNB1, with virtually all ß-catenin mutations identified in human tumors being located here, primarily in the region encoding the GSK3ß binding domain. The organization of exon 3 is shown, flanked by intronic sequences, with coding sequence depicted in white and noncoding sequence in light gray. The forward PCR primer, indicated by the arrow above the box, spans nucleotides –22 to –2 (upstream of the start codon), and the reverse primer, indicated by the arrow below the box, spans c.186 to c.206 (c.186 denotes nucleotide 186 of the coding region), resulting in PCR amplification of the entire coding region of exon 3. The sequence corresponding to the GSK3ß consensus binding domain spans nucleotides c.87 to c.141 (codons 29 to 47). The location of the mutation reported by Bjorklund et al. (14 ) (c.109 T>G; S37A) is marked with an asterisk.

	Results

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	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
The molecular mechanisms underlying benign sporadic parathyroid tumorigenesis are incompletely understood, with inactivation of MEN1 and activation of cyclin D1 as the only established contributors. The recent finding that the Wnt signaling pathway, an upstream activator of cyclin D1 expression, may contribute to parathyroid tumorigenesis (14) prompted us to investigate the frequency of exon 3 CTNNB1 mutations in a large series of parathyroid adenomas. Our finding of the lack of exon 3 CTNNB1 mutations in 97 parathyroid adenomas suggests that involvement of ß-catenin mutation in benign parathyroid tumorigenesis is, at best, exceedingly rare.

Our observations are consistent with the results of two smaller studies of 12 and 24 parathyroid adenomas by Semba et al. (13) and Ikeda et al. (12), respectively, and fail to confirm the recent finding of CTNNB1 exon 3 mutation in 15% of parathyroid adenomas by Bjorklund et al. (14). The latter report was especially remarkable in that of the 3 of 20 adenomas with mutations, the precisely identical mutation (c.109 T>G; S37A) was present in each and was documented to be an acquired somatic event in two of three. This pattern strongly suggested a "hotspot" for parathyroid-specific CTNNB1 mutation, so its complete absence among 97 adenomas in our study (and among 133 adenomas across three studies) is all the more striking. The reasons for this discrepancy are unclear but might relate to chance clustering with an extremely low mutation frequency of CTNNB1, or to differences in the sample populations in Sweden vs. the United States and Japan. Additional studies are required to further elucidate the molecular mechanisms underlying benign parathyroid tumorigenesis and which, if any, members of the Wnt signaling pathway may contribute.

	Acknowledgments

 
The authors thank Kristin Corrado for expert technical assistance.

	Footnotes

 
This work was supported in part by Grant DHHS/NIDCR 5T32-DE07302 from the National Institutes of Health and by the Murray-Heilig Fund in Molecular Medicine.

The authors have nothing to disclose.

First Published Online February 6, 2007

Received November 21, 2006.

Accepted January 25, 2007.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: 92/4/1564    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Costa-Guda, J. Articles by Arnold, A. Search for Related Content PubMed PubMed Citation Articles by Costa-Guda, J. Articles by Arnold, A. Related Collections Calcium and Bone Metabolism Endocrine Oncology