Rare germline mutations in cyclin-dependent kinase inhibitor genes in multiple endocrine neoplasia type 1 and related states

Abstract

Context: Germline mutation in the MEN1 gene is the usual cause of multiple endocrine neoplasia type 1 (MEN1). However, the prevalence of identifiable germline MEN1 mutations in familial MEN1 cases is only 70%. Some cases may have a germline mutation in another gene such as the p27 cyclin-dependent kinase inhibitor (CDKI).

Objective: The aim of the study was to investigate cases of MEN1 or related states for germline mutations in all CDKI genes.

Methods: A total of 196 consecutive index cases were selected with clear or suspected MEN1 and no identifiable germline MEN1 mutation. Every case was analyzed for germline mutation in each of the seven CDKI genes.

Results: We identified benign polymorphisms of the CDKI genes and also 15 other initially unclassified sequence variants. After detailed gene/protein analysis, seven of these 15 variants were classified as probably pathological mutations. Three of these seven were probable mutations of p27. The remaining four were probable pathological mutations in three of the other CDKI genes, thereby implicating these three genes in the germline of human tumors. The identification rates for probably pathological mutations among the 196 index cases were similarly low for each of four CDKI genes: p15 (1%), p18 (0.5%), p21 (0.5%), and p27 (1.5%). No characteristic clinical subtype related to MEN1 was identified among the seven index cases and their families.

Conclusion: Rare germline mutation in any among four (p15, p18, p21, and p27) of the seven CDKIs is a probable cause of MEN1 or of some related states.

Figure 1

Molecular pathways distal to CDKIs. Mitogenic signals promote entry into the G₁ phase through assembly and activation of cyclinD-CDK4 or cyclinD-CDK6 complexes. Subsequent progression through G₁/S transition and S phase is dependent on active cyclinE-CDK2 complexes and active cyclinA-CDK2 complexes. The complexes of cyclin (green solid) and CDK (green striped) phosphorylate members (only one shown) of the Rb (the retinoblastoma gene product) family. This releases from them the E2F family of transcription factors (only one factor shown). The E2F factors in turn transactivate genes for G₁ to S phase progression. Some other substrates (data not shown) of active cyclin-CDK complexes during G₁ and S phase are: for cyclinE-CDK2, NPAT (nuclear protein mapped to ATM locus), p27, and histone-H1; for cyclinA-CDK2, DNA polymerase α primase. Members of two families of CDKIs, INK4 family (orange striped) and Cip/Kip family (orange solid), negatively regulate the cell cycle. p14 is from an alternative reading frame of the p16 gene (see Supplemental Fig. 4); it does not bind to any cyclin-CDK complex, and it is not a CDKI. p14 inhibits the cell cycle through activating the inhibitory functions of p21 (9). Arrows, Activation; ⊣, inhibition.

Figure 2

Function assays of p27 variants among index cases. A, mRNA. RT-PCR of HEK293 cells untransfected or transfected with wild-type or p27 variant (ATG-7G>C) genomic constructs. Top panel shows RT-PCR products (at 30 cycles) with p27 primers, and lower panel shows products with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. B, Protein. Western blot of whole cell lysates from HEK293 cells untransfected or transfected with wild-type or p27 variant (ATG-7G>C) genomic constructs. Top panel was probed with anti-p27 antibody and lower panel with antitubulin antibody. C, Binding to GRB2. GST pull-down assay for binding with GST-GRB2 of ³⁵S labeled in vitro translated (IVT) wild-type p27 or missense variant P95S or benign polymorphism T142T. The bound IVT protein was detected by SDS-PAGE and autoradiography. Input lane contained 10% of the IVT product incubated with GST or GST-GRB2. D, Protein. Western blot of whole cell lysates from HEK293 cells transfected with wild-type or p27 (stop>Q) expression constructs, untreated or treated with 20 μm MG132 (proteasome inhibitor). Top panel was probed with anti-p27 antibody, middle panel with antitubulin antibody, and lower panel with anti-NPTII antibody (index of transfection efficiency). E, mRNA. RT-PCR of HEK293 cells from panel D. Top panel shows RT-PCR products (at 25 cycles) with p27 primers, and lower panel shows RT-PCR products with GAPDH primers. untrf, Untransfected; wt, wild-type.

Figure 3

Expression and interaction assays of several CDKI missense variants. A and B, Protein. Western blot of whole cell lysates from HEK293 cells transfected with wild-type or mutagenized cDNA expression constructs. Top panels were probed with the indicated specific anti-CDKI antibody, middle panels with antitubulin antibody, and lower panels with anti-NPTII antibody. C and D, Protein. Binding of ³⁵S-labeled IVT wild-type or variants of p15 or p18 to CDK6 in GST pull-down assay. The bound IVT protein was detected by SDS-PAGE and autoradiography. Input lane contained 10% of the IVT product incubated with GST or GST-CDK6.

Figure 4

Pedigrees and tumors of index cases with probably pathological CDKI mutations, their affected relatives, and all of their other first-degree relatives (see Table 2 for details). Germline heterozygous CDKI mutation is shown above each pedigree. Roman numerals indicate generation number. Arrows indicate index case. Circle, Female; square, male; diamond, sex not known; small diamond, miscarriage of unknown gender; double line below descent line, no offspring; number in symbol, number of unaffected offspring of that gender; empty symbol, unaffected family member; slashed symbol, deceased; stippled circle, obligate carrier by family position; question mark in symbol, offspring is adopted; question mark near symbol, offspring information not known; filled quadrant in symbol, tumor; dot in quadrant, likely tumor; upper right quadrant, parathyroid tumor; upper left quadrant, gastrinoma; lower right quadrant, pituitary tumor; lower left quadrant, other MEN1-associated tumor [e.g. adrenal cortex (n = 2), meningioma (n = 1), uterine fibroids (n = 1)] (see also Table 2); (+), CDKI mutation positive; (−), CDKI mutation negative; numbers on top of symbols, year of birth for all carriers. First-degree relatives without CDKI mutation information were shown as affected syndrome carrier if they expressed at least one of the three main tumors of MEN1.