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Center for Molecular Medicine (L.J.K., T.M.S., A.A.), Division of Endocrinology and Metabolism (A.A.), University of Connecticut Health Center, Farmington, Connecticut 06030-3101
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.
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Abstract |
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Objective/Design/Patients: To determine the frequency of HRPT2 somatic mutations in a substantial series of typical, sporadic parathyroid adenomas, we directly sequenced coding and flanking splice junctional regions of all HRPT2 exons in solitary adenomas from 60 patients.
Results/Conclusions: No intragenic HRPT2 mutations were detected, strengthening the degree of specificity of HRPT2 mutation as a feature of sporadic parathyroid carcinoma as opposed to sporadic adenomas. Our observations encourage additional study of the diagnostic potential of HRPT2 in parathyroid neoplasia and support the view that HRPT2 inactivation is not an important participant in the pathogenesis of typical parathyroid adenomas.
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Introduction |
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Examination of the HRPT2 tumor suppressor gene may be potentially helpful in making this diagnostic distinction. HRPT2 is the target for germline mutation in the majority of families with the rare, autosomal dominant, hyperparathyroidism-jaw tumor syndrome (11), in which parathyroid adenomas and carcinomas can occur (12). Similar germline mutations occur in a subset of kindreds with familial isolated hyperparathyroidism (11, 13, 14, 15). The HRPT2 gene product, parafibromin, is evolutionarily conserved and ubiquitously expressed (11) and is a putative member of the human Paf complex, which has been proposed to play a role in the regulation of transcription and chromatin modification (16).
Of particular relevance to the present study, Shattuck et al. (17) identified somatic or germline mutations of HRPT2 in the majority of 15 patients with sporadic parathyroid carcinomas. Howell et al. (14) documented somatic HRPT2 mutation in one additional parathyroid cancer and HRPT2 mutations of indeterminate origin in three of three other parathyroid cancers examined (Table 1
); more recently, Cetani et al. (15) found HRPT2 mutations in six of seven carcinomas (Table 1). Even the impressive combined prevalence of 77% in these studies is likely to underestimate the true role of HRPT2 mutation in sporadic parathyroid cancer, because noncoding mutations equally capable of inactivating the gene would have escaped detection. Thus, HRPT2 mutation is central to the pathogenesis of most, and perhaps virtually all, sporadic parathyroid carcinomas.
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80–85%), whereas parathyroid carcinoma accounts for less than 1% of primary hyperparathyroidism. Thus, even a low detection rate of mutation in adenomas could adversely impact the diagnostic specificity and positive predictive value of HRPT2 mutation status to differentiate between adenomas and carcinomas.
The extent of HRPT2’s role in routinely encountered benign parathyroid adenomas is uncertain, primarily because of sample size limitations and/or imposition of special selection criteria in prior studies. Carpten et al. (11) detected mutations in two of 47 parathyroid adenomas (Table 1
). However, these adenomas were selected for their cystic features, a characteristic of many lesions in the HRPT2-associated hyperparathyroidism-jaw tumor syndrome, but not representative of typical sporadic adenomas that account for most cases of primary hyperparathyroidism. In contrast, a study of unselected parathyroid adenomas revealed no HRPT2 mutations, but only 25 tumors were examined (14), whereas another study found a single mutation among 35 sporadic adenomas selected to exclude cases containing the otherwise common finding of 11q13 loss of heterozygosity (15) (Table 1). The objective of this study was to determine the frequency of HRPT2 somatic mutations in a substantial series of unselected typical, sporadic parathyroid adenomas.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Sixty typical sporadic parathyroid adenoma samples were obtained from 60 patients (42 women and 18 men) who had been treated surgically for primary hyperparathyroidism. Apart from limiting the study to solitary parathyroid adenomas, no other selection criteria were applied for inclusion in this series. None of the patients had clinical evidence of a familial hyperparathyroid syndrome, and all had correction of hypercalcemia after resection of the solitary adenoma with no evidence of synchronous or asynchronous multigland disease. None of the adenomas had been documented to have any atypical gross or histological features suggestive of malignancy, such as fibrous bands, microinvasiveness, or a trabecular pattern. Immediately after surgical resection, tumor samples were frozen in liquid nitrogen and stored at –80 C until use. Genomic DNA was extracted by proteinase K digestion, followed by phenol-chloroform extraction and ethanol precipitation. All tumor samples were obtained in accordance with institutional review board-approved protocols for human studies.
Detection of HRPT2 mutations
The 60 DNA samples from unselected typical sporadic parathyroid adenomas were analyzed for mutations in the HRPT2 gene by direct sequencing of both strands. The 17 exons of the gene were amplified as 15 different fragments with primers derived from flanking intronic or 3'- or 5'-untranslated region sequences as described previously (17). After amplification, primers were removed by digestion with 10 U exonuclease I (U.S. Biochemical Corp., Cleveland, OH) and 1 U shrimp alkaline phosphatase (U.S. Biochemical Corp.).
Sequencing reactions were performed using the BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) as described previously (20), and sequencing was performed using an ABI 3100 capillary sequencer or ABI 377 sequencer. Data were analyzed with Sequencing Analysis and AutoAssembler software (Applied Biosystems).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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t (National Center for Biotechnology Information reference single nucleotide polymorphism identification no. rs4466634), were detected in this series, as predicted. Although these polymorphisms are not expected to importantly influence parafibromin function, their detection does serve to confirm the sensitivity of our mutation analysis methodology.
Combining our data from 60 tumors with the previous study by Howell et al. (25 unselected cases) (14) yields a cumulative result of zero HRPT2 mutations in 85 unselected typical sporadic parathyroid adenomas. If one were to include the studies in which selection criteria affected the sample sets (11, 15), the cumulative outcome would be three HRPT2 mutations in 167 adenomas or 1.8%, which is, therefore, almost certainly an overestimate of the true prevalence. A better estimate may be 0.8% (one of 120), derived from our data, plus the unselected tumors in the study by Howell et al. (14) and including the tumors studied by Cetani et al. that, although selected using molecular attributes, were not subjected to selection with histopathological criteria (15). This dearth of HRPT2 mutations indicates that typical sporadic parathyroid adenomas driven by inactivation of this tumor suppressor gene must be exceedingly rare, especially compared with the rate of intragenic HRPT2 mutation in parathyroid carcinomas (Table 1).
Our observations importantly strengthen the level of specificity of HRPT2 mutation as a key feature of parathyroid carcinoma as opposed to typical sporadic adenoma. Furthermore, if the application of a test for HRPT2 mutation status were to be focused on diagnostically difficult cases with some atypical features, rather than on any and all parathyroid tumors, its diagnostic value and positive predictive value could be even greater. For example, parathyroid tumors may be encountered containing features such as fibrous bands, mitotic figures, or microscopic invasion, but without distant metastasis or gross invasion of surrounding structures and thus lacking the diagnostically definitive features of carcinoma. We envision that a test of HRPT2 mutation status or a surrogate, such as immunohistochemical detection of parafibromin, may be most useful if focused on such cases. Preliminary reports of immunostaining for parafibromin in parathyroid tumors appear encouraging in this respect, but remain susceptible to potential technical and interpretive difficulties and have not been cross-validated with HRPT2 mutational status (21, 22). Therefore, the present study provides a solid base of mutational data at the DNA level, the gold standard for involvement of a tumor suppressor gene, against which future parafibromin expression data as a reliable clinical test can be judged.
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Acknowledgments |
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Footnotes |
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First Published Online June 14, 2005
Received April 1, 2005.
Accepted June 8, 2005.
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References |
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This article has been cited by other articles:
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  C C Juhlin, A Villablanca, K Sandelin, F Haglund, J Nordenstrom, L Forsberg, R Branstrom, T Obara, A Arnold, C Larsson, et al. Parafibromin immunoreactivity: its use as an additional diagnostic marker for parathyroid tumor classification Endocr. Relat. Cancer, June 1, 2007; 14(2): 501 - 512. [Abstract] [Full Text] [PDF]
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