Germ-line mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and humans

Abstract

MENX is a recessive multiple endocrine neoplasia-like syndrome in the rat. The tumor spectrum in MENX overlaps those of human multiple endocrine neoplasia (MEN) types 1 and 2. We mapped the MenX locus to the distal part of rat chromosome 4, excluding the homologs of the genes responsible for the MEN syndromes (RET and MEN1) and syndromes with an endocrine tumor component (VHL and NF1). We report the fine mapping of the disease locus and the identification of a homozygous frameshift mutation in Cdkn1b, encoding the cyclin-dependent kinase inhibitor p27(Kip1). As a consequence of the mutation, MENX-affected rats show dramatic reduction in p27(Kip1) protein. We have identified a germ-line nonsense mutation in the human CDKN1B gene in a MEN1 mutation-negative patient presenting with pituitary and parathyroid tumors. Expanded pedigree analysis shows that the mutation is associated with the development of an MEN1-like phenotype in multiple generations. Our findings demonstrate that germ-line mutations in p27(Kip1) can predispose to the development of multiple endocrine tumors in both rats and humans.

Fig. 1.

Mapping and cloning of the MENX mutation. (A) Haplotype analysis of affected and unaffected animals produced from a (SD^we × Wistar–Kyoto) F1 × SD^we/SD^we backcross for nine microsatellite markers located in the linkage-defined MENX candidate region. Filled areas indicate homozygosity for a given microsatellite marker; empty areas indicate heterozygosity. The number of animals with a specific haplotype and their disease status are listed on the right. Mb, megabases. (B) Pedigree of a breeding group segregating the MENX phenotype. Genomic DNA of the animals was amplified with primers spanning the Cdkn1b mutation, and the alleles (WT and mutated) were resolved on polyacrylamide gels. For each Cdkn1b genotype, the corresponding sequence chromatogram is shown. The insertion in the mut/mut rat sequence is indicated by a rectangle. Open square/circle, unaffected male/female; filled square/circle, affected male/female. M, molecular size marker. (C) Alignment of annotated rat wild-type p27 protein sequence (wt) with the predicted mutant p27 protein (mut). The mutant protein is predicted to be 21 amino acids longer than the WT.

Fig. 2.

Analyses of the Cdkn1b mRNA and p27 protein in tissues of MENX-affected and control rats. (A Upper) Schematic representation of the Cdkn1b gene and location of the primers used for RT-PCR. Filled areas represent the coding sequence. The position of the MENX mutation (insertion) in exon 2 is indicated. (A Lower) RT-PCR results obtained by amplifying RNA from three tissues [thymus (T), spleen (S), and adrenals (A)] of normal (+/+), heterozygous (+/m), and homozygous mutant (m/m) rats. Fragments were resolved on polyacrylamide gels and stained with ethidium bromide. The primers used are indicated. (B) Real-time RT-PCR to determine the level of expression of Cdkn1b in rat tissues. The level of Cdkn1b mRNA in tissues of WT animals (+/+) is arbitrarily set at 1. The level of Cdkn1b mRNA in heterozygous (+/m) or mutant (m/m) rat tissues is normalized against the values of the WT tissues. Values are the mean of three experiments performed on three animals per genotype ± standard deviation. In adrenal glands derived from mut/mut rats, there is significantly more Cdkn1b mRNA than in WT animals (∗, P < 0.05; double-sided t test). (C) Expression of p27 protein in rat tissues. The Western blot analysis of p27 in total protein lysates obtained from tissues of 2-month-old rats is shown. The membrane was sequentially probed with antibodies against p27, Cdk2, Cdk4, cyclin E, cyclin D1, and with an anti-β-tubulin antibody to control for equal loading. KMS12 and Granta human lymphoma cell lines were loaded in parallel as positive controls.

Fig. 3.

Identification of a germ-line mutation in CDKN1B in a suspected MEN1 patient and segregation analyses. (A Upper) A PCR fragment corresponding to part of CDKN1B exon 1 was obtained from the proband, cloned, and both alleles were separately sequenced. The germ line of the proband showed a heterozygous nonsense mutation at codon 76 (c. G692A). (A Lower) The position of the mutation in the CDKN1B gene and p27 protein is shown. (B) Proband family pedigree. Generation numbers are represented by Roman numerals, and individual numbers are in Arabic numerals. The proband is II-1, indicated by the arrow. Mut+ indicates mutation-positive individuals. AM, acromegaly; PT, pituitary adenoma; PC, parathyroid cancer; RA, renal angiomyolipoma; HT, hypertension; TC, testicular cancer. The age of onset is indicated in parentheses. (C) Haplotype analysis. The microsatellite markers on chromosome 12 and their positions are indicated on the right. The affected haplotype is shown in black. (D) Analysis of the tumor tissue of individual II-4. Sequencing chromatograms show the G692GA mutation in both normal and tumor tissue DNA and RNA. Immunohistochemical staining with an anti-p27 antibody (Right) shows a lack of p27 protein in the tumor tissue. The arrows indicate infiltrating lymphocytes used as a positive control for p27 staining. (Immunoperoxidase original magnification: ×650.)

Fig. 4.

Expression and subcellular localization of exogenous proteins during transfection. (A) Schematic representation of Myc-tagged p27 proteins used for transfections. The shaded area at the C terminus of p27_1020;G177fs represents the p27-unrelated amino acids present in the MENX mutant protein. (B and C) Immunoblots showing the expression of the transfected proteins. (B) MCF-7 cells were transfected with 1 μg of the indicated constructs. Cell lysates were prepared at the indicated times after transfection; 50 μg of total protein was separated by electrophoresis, blotted, and probed with antibodies to the Myc tag. To control for equal loading, the membrane was probed with the anti-tubulin monoclonal antibody. (C) MCF-7 cells were transfected as in B. Twenty-four hours later, cells were harvested, and proteins were extracted. Different amounts of total protein lysates (indicated) were loaded onto the gel. p27_1020;G177fs and of p27_1020;W76X proteins seem to be ≈6-fold less abundant than the WT p27 protein. (D) Subcellular localization of Myc-tagged p27 proteins. Rat2 cells were transfected with the Myc-WTp27, Myc-p27_1020;G177fs, Myc-p27_1020;G177X, and p27_1020;W76X (indicated on the right) constructs. Forty-eight hours later, the transfected cells were fixed and permeabilized, and then they were stained with an antibody to the Myc tag visualized with Cy3 (red). Nuclei were counterstained with Hoechst (blue). (Left) Cy3. (Center) Hoechst. (Right) Merge. (Scale bars: 10 μm.)