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Primary hyperparathyroidism is most often caused by a sporadic single-gland parathyroid adenoma (PTA), a tumor type for which cyclin D1 is the only known and experimentally validated oncoprotein. However, the molecular origins of its frequent overexpression have remained mostly elusive. In this study, we explored a potential tumorigenic mechanism that could increase cyclin D1 stability through a defect in molecules responsible for its degradation.We examined two tumor suppressor genes known to modulate cyclin D1 ubiquitination, PRKNand FBXO4(FBX4), for evidence of classic two-hit tumor suppressor inactivation within a cohort of 82 PTA cases. We examined the cohort for intragenic inactivating and splice site mutations by Sanger sequencing and for locus-associated loss of heterozygosity (LOH) by microsatellite analysis.We identified no evidence of bi-allelic tumor suppressor inactivation of PRKNor FBXO4via inactivating mutation or splice site perturbation, neither in combination with nor independent of LOH. Among the 82 cases, we encountered previously documented benign single nucleotide polymorphisms (SNPs) in 35 tumors at frequencies similar to those reported in the germlines of the general population. Eight cases exhibited intragenic LOH at the PRKNlocus, in some cases extending to cover at least an additional 1.7 Mb of chromosome 6q25-26. FBXO4was not affected by LOH.The absence of evidence for specific bi-allelic inactivation in PRKNand FBXO4in this sizeable cohort suggests that these genes only rarely, if ever, serve as classic driver tumor suppressors responsible for the growth of PTAs.

The molecular pathogenesis of sporadic parathyroid adenomas is incompletely understood. The possible role of cyclin-dependent kinase inhibitor (CDKI) genes was raised by recognition of cyclin D1 as a parathyroid oncogene, identification of rare germline mutations in CDKI genes in patients with multiple endocrine neoplasia type 1; that in rodents, mutation in Cdkn1bcaused parathyroid tumors; and subsequently through identification of rare predisposing germline sequence variants and somatic mutation of CDKN1B, encoding p27kip1, in sporadic human parathyroid adenoma. We therefore sought to determine whether mutations/variants in the other six CDKI genes CDKN1A, CDKN1C, CDKN2A, CDKN2B, CDKN2C, and CDKN2D, encoding p21, p57, p14ARF/p16, p15, p18, and p19, respectively, contribute to the development of typical parathyroid adenomas. In a series of 85 sporadic parathyroid adenomas, direct DNA sequencing identified alterations in five adenomas (6 %): Two contained distinct heterozygous changes in CDKN1A, one germline and one of undetermined germline status; one had a CDKN2Bgermline alteration, accompanied by loss of the normal allele in the tumor (LOH); two had variants of CDKN2C, one somatic and one germline with LOH. Abnormalities of three of the mutant proteins were readily demonstrable in vitro. Thus, germline mutations/rare variants in CDKN1A, CDKN2B, and CDKN2Clikely contribute to the development of a significant subgroup of common sporadic parathyroid adenomas, and somatic mutation in CDKN2Cfurther suggests a direct role for CDKI alteration in conferring a selective growth advantage to parathyroid cells, providing novel support for the concept that multiple CDKIs can play primary roles in human neoplasia.