Germline and somatic mutations in cyclin-dependent kinase inhibitor genes CDKN1A, CDKN2B, and CDKN2C in sporadic parathyroid adenomas

Abstract

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 Cdkn1b caused parathyroid tumors; and subsequently through identification of rare predisposing germline sequence variants and somatic mutation of CDKN1B, encoding p27(kip1), 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, p14(ARF)/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 CDKN2B germline 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 CDKN2C likely contribute to the development of a significant subgroup of common sporadic parathyroid adenomas, and somatic mutation in CDKN2C further 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.

Fig. 1

CDKI sequence variants. a Mutation in CDKN1A, encoding p21. Germline and tumor sequences show heterozygosity [cytosine/thymine] at position 350 of the coding sequence. b Mutation in CDKN1A, encoding p21. Germline sequence shows guanine at position 26. The tumor shows acquired heterozygosity [guanine/adenine] at this position. c Mutation and LOH in CDKN2B encoding p15. Germline sequence (N) shows heterozygosity at position 256 of the coding sequence. In the tumor (T), the normal guanine is lost and the abnormal adenine remains. d Mutation in CDKN2C, encoding p18. Germline sequence shows a thymine at position 62 of the coding sequence. The tumor shows acquired heterozygosity [thymine/adenine] at this position. e Mutation and LOH in CDKN2C, encoding p18. Germline sequence shows heterozygosity at position 454 of the coding sequence. In the tumor, the normal cytosine is lost, and the abnormal thymine remains

Fig. 2

Functional characterization of CDKI variants. a Protein expression in whole cell lysates of HEK293 cells transfected with wild type or mutagenized cDNA expression constructs. Top panel was probed with the indicated specific anti-CDKI antibody, middle panel with anti-NPTII antibody (to assess transfection efficiency), and lower panel with anti-tubulin antibody (to assess protein loading). Relative level of wild-type (wt) and mutant protein expression is shown (n = 3), with wt protein level = 1. b Protein level in nuclear and cytoplasmic extracts of HEK293 cells transfected with wild-type or mutagenized cDNA expression constructs. Top panels of cytoplasmic extract (CE), and bottom panels of nuclear extract (NE) were probed with the indicated specific anti-CDKI antibody, anti-tubulin (to assess protein loading in CE, and to assess the efficiency of sub-cellular fractionation in NE), or with anti-p84 (to assess protein loading in the NE, and to assess the efficiency of sub-cellular fractionation in the CE). Relative level of wt and mutant protein expression in CE and NE is shown (n = 3), with wt protein level = 1, *p = 0.04. c In vitro translated (IVT) proteins used for GST pull-down assay in 'D'. ³⁵S-Methionine labeled wild-type or mutagenized proteins were generated by in vitro translation from cDNA expression constructs. IVT proteins were detected by SDS-PAGE and autoradiography. d Protein-binding to CDK2 or CDK6. GST pull-down assay for binding to GST, GST-CDK2, or GST-CDK6 of ³⁵S-methionine labeled IVT wt or mutagenized proteins. The bound IVT protein was detected by SDS-PAGE and autoradiography. Input lane contained 10 % of the IVT product incubated with GST, GST-CDK2, or GST-CDK6. Relative binding of wt and mutant protein to GST-fusion proteins is shown with wt protein binding = 1 (n = 3 for p15 and p18; n = 2 for p21), **p < 0.001