Sara Molatore

A novel germline CDKN1B mutation causing multiple endocrine tumors: clinical, genetic and functional characterization

Multiple endocrine neoplasia (MEN) syndromes are characterized by tumors involving two or more endocrine glands. Two MEN syndromes have long been known: MEN1 and MEN2, caused by germline mutations in MEN1 or RET, respectively. Recently, mutations in CDKN1B, encoding the cyclin‐dependent kinase (Cdk) inhibitor p27, were identified in patients having a MEN1‐like phenotype but no MEN1 gene mutations. Currently, the molecular mechanisms mediating the role of p27 in tumor predisposition are ill defined. We here report a novel germline missense variant in CDKN1B (c.678C>T, p.P69L) found in a patient with multiple endocrine tumors. We previously reported a nonsense p27 mutation (c.692G>A, p.W76X) in two patients with MEN1‐like phenotype. Functional assays were used to characterize p27P69L and p27W76X in vitro. We show that p27P69L is expressed at reduced level and is impaired in both binding to Cdk2 and inhibiting cell growth. p27W76X, which is mislocalized to the cytoplasm, can no longer efficiently bind Cyclins‐Cdks, nor inhibit cell growth or induce apoptosis. In the patients tumor tissues, p27P69L associates with reduced/absent p27 expression and in one tumor with loss‐of‐heterozygosity. Our results extend previous findings of CDKN1B mutations in patients with MEN1‐related states and support the hypothesis of a tumor suppressor role for p27 in neuroendocrine cells.

Role of MUTYH and MSH2 in the Control of Oxidative DNA Damage, Genetic Instability, and Tumorigenesis

Mismatch repair is the major pathway controlling genetic stability by removing mispairs caused by faulty replication and/or mismatches containing oxidized bases. Thus, inactivation of the Msh2 mismatch repair gene is associated with a mutator phenotype and increased cancer susceptibility. The base excision repair gene Mutyh is also involved in the maintenance of genomic integrity by repairing premutagenic lesions induced by oxidative DNA damage. Because evidence in bacteria suggested that Msh2 and Mutyh repair factors might have some overlapping functions, we investigated the biological consequences of their single and double inactivation in vitro and in vivo. Msh2(-/-) mouse embryo fibroblasts (MEF) showed a strong mutator phenotype at the hprt gene, whereas Mutyh inactivation was associated with a milder phenotype (2.9 x 10(-6) and 3.3 x 10(-7) mutation/cell/generation, respectively). The value of 2.7 x 10(-6) mutation/cell/generation in Msh2(-/-)Mutyh(-/-) MEFs did not differ significantly from Msh2(-/-) cells. When steady-state levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG) were measured in MEFs of different genotypes, single gene inactivation resulted in increases similar to those observed in doubly defective cells. In contrast, a synergistic accumulation of 8-oxoG was observed in several organs of Msh2(-/-)Mutyh(-/-) animals, suggesting that in vivo Msh2 and Mutyh provide separate repair functions and contribute independently to the control of oxidative DNA damage. Finally, a strong delay in lymphomagenesis was observed in Msh2(-/-)Mutyh(-/-) when compared with Msh2(-/-) animals. The immunophenotype of these tumors indicate that both genotypes develop B-cell lymphoblastic lymphomas displaying microsatellite instability. This suggests that a large fraction of the cancer-prone phenotype of Msh2(-/-) mice depends on Mutyh activity.

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay†

MUTYH‐associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8‐hydroxyguanine (8‐oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh−/− mouse defective cells. Several parameters, including accumulation of 8‐oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell‐based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild‐type protein. Our cell‐based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1–8, 2009.

Constitutional high expression of an APC mRNA isoform in a subset of attenuated familial adenomatous polyposis patients

Familial adenomatous polyposis is an inherited condition associated with hundreds to thousands of colorectal adenomas conferring a very high risk of cancer at a young age. In addition to “classical” form, there is also an attenuated polyposis, with fewer than 100 polyps and a delayed age of cancer onset. Both classical and attenuated polyposis are characterized by a relevant phenotypic heterogeneity. The disease has been linked to constitutive mutations of either APC tumor suppressor gene, or less frequently, MYH base-excision repair gene. However, the genetic cause remains undetected in up to 70–80% of patients with the attenuated form. This analysis was performed on 26 polyposis patients with the attenuated phenotype. All patients had formerly proven to be negative for APC truncating mutations that typically represent the majority of APC gene alterations. We evaluated the APC mRNA constitutional level by real-time quantitative reverse transcription polymerase chain reaction (PCR). Eleven patients (42%) showed an anomalous APC transcription level. One patient with reduced mRNA was a carrier of a whole APC gene deletion. In seven out of the ten remaining cases, we found the increased expression of an APC mRNA isoform resulting from exon 10/15 connection and giving rise to a stable truncated peptide. Mutations neither in the invariant splice sites nor in the known transcription regulatory signals were found. Our results support the notion that in attenuated polyposis patients, a detailed investigation of APC transcription can allow detection of rare alterations. Although functional data are required, the isoform we observed might have some pathogenic role, accounting for the heterogeneous phenotype that characterizes the polyposis syndrome.

Functional Imaging of Pheochromocytoma with 68Ga-DOTATOC and 68C-HED in a Genetically Defined Rat Model of Multiple Endocrine Neoplasia

Rats affected by the MENX multitumor syndrome develop pheochromocytoma (100%). Pheochromocytomas are uncommon tumors and animal models are scarce, hence the interest in MENX rats to identify and preclinically evaluate novel targeted therapies. A prerequisite for such studies is a sensitive and noninvasive detection of MENXassociated pheochromocytoma. We performed positron emission tomography (PET) to determine whether rat pheochromocytomas are detected by tracers used in clinical practice, such as 68Ga-DOTATOC (somatostatin analogue) or 11C-Hydroxyephedrine (HED), a norepinephrine analogue. We analyzed four affected and three unaffected rats. The PET scan findings were correlated to histopathology and immunophenotype of the tumors, their proliferative index, and the expression of genes coding for somatostatin receptors or the norepinephrine transporter. We observed that mean 68Ga-DOTATOC standard uptake value (SUV) in adrenals of affected animals was 23.3 ± 3.9, significantly higher than in control rats (15.4 ± 7.9; P = .03). The increase in mean tumor-to-liver ratio of 11C-HED in the MENX-affected animals (1.6 ± 0.5) compared to controls (0.7 ± 0.1) was even more significant (P = .0016). In a unique animal model, functional imaging depicting two pathways important in pheochromocytoma biology discriminated affected animals from controls, thus providing the basis for future preclinical work with MENX rats.

Association between the p27 rs2066827 variant and tumor multiplicity in patients harboring MEN1 germline mutations

OBJECTIVE To date, no evidence of robust genotype-phenotype correlation or disease modifiers for multiple endocrine neoplasia type 1 (MEN1) syndrome has been described, leaving the highly variable clinical presentation of patients unaccounted for. DESIGN As the CDKN1B (p27) gene causes MEN4 syndrome and it is transcriptionally regulated by the product of the MEN1 gene (menin), we sought to analyze whether p27 influences the phenotype of MEN1-mutated patients. The cohort consisted of 100 patients carrying germline MEN1 gene mutations and 855 population-matched control individuals. METHODS Genotyping of the coding p27 c.326T>G (V109G) variant was performed by sequencing and restriction site digestion, and the genotypes were associated with clinical parameters by calculating odds ratios (ORs) and their 95% CIs using logistic regression. RESULTS There were significant differences in p27 V109G allele frequencies between controls and MEN1-mutated patients (OR=2.55, P=0.019, CI=1.013-5.76). Among patients who are ≥30 years old carrying truncating MEN1 mutations, the T allele was strongly associated with susceptibility to tumors in multiple glands (three to four glands affected vs one to two glands affected; OR=18.33; P=0.002, CI=2.88-16.41). This finding remained significant after the Bonferronis multiple testing correction, indicating a robust association. No correlations were observed with the development of MEN1-related tumors such as hyperparathyroidism, pituitary adenomas, and enteropancreatic and adrenocortical tumors. CONCLUSIONS Our study suggests that the p27 tumor suppressor gene acts as a disease modifier for the MEN1 syndrome associated with MEN1 germline mutations. If confirmed in independent patient cohorts, this finding could facilitate the management of this clinically complex disease.

Abstract 1997: Pheochromocytoma developing within the MENX rat model shares gene expression patterns with its human counterpart

Pheochromocytoma is a neoplasia that arises from the chromaffin cells of the adrenal medulla. Pheochromocytoma develops in 100% of rats affected by the multiple endocrine neoplasia syndrome MENX, caused by a loss-of-function mutation in p27. Affected rats develop adreno-medullary hyperplasia by 3-4 months of age, which progresses to pheochromocytoma with time, providing an interesting model for pheochromocytoma development. MENX-affected rats also develop extra-adrenal pheochromocytoma (paraganglioma) (frequency 20%). Aim: The aim of the study is to discover novel molecular mechanisms involved in the pathophysiology of rat adrenal tumors and to validate the findings in human pheochromocytoma. Our ultimate goal is to identify novel molecular markers for diagnosis, prognosis and targeted therapy of pheochromocytoma. Methods: Gene expression array analysis of 8 hyperplasias and 10 pheochromocytomas from MENX rats was performed on Rat 2.0 Affymetrix Genechips. Samples were compared with adreno-medullary RNA from wild-type age-matched littermates. Selected genes overexpressed in the lesions were validated by RealTime quantitative RT-PCR or in situ hybridization. Results: The adrenal lesions associated to the MENX syndrome show distinct gene expression profiles enriched in genes belonging to neuronal development pathways. Hyperplasia and pheochromocytoma show significant overlap of the most dysregulated gene categories. Among the genes overexpressed in the adrenal lesions are Mash1, Bmp7, Phox2a, Neurod1, Gal, Cxcr4, Cdkn2a, Cdkn2c, Sctr, Dgkh, L1Cam, Tcte1, which were validated in additional samples. These genes are already overexpressed in normal-looking adrenal glands of 1 month-old affected rats. Thus, their up-regulation might be a more direct consequence of the genetic mutation in p27. Among these genes, the only one overexpressed also in rat paragangliomas is Sctr, encoding the secretin receptor, indicating that pheochromocytoma arising within or outside the adrenal gland associates with different expression signatures. Interestingly, Sctr is highly expressed in PC12 cells, indicating that it might be a feature of rat pheochromocytoma. A series of 30 sporadic and 13 familial human pheochromocytoma was analyzed for the expression of selected genes overexpressed in the rat tumors. The results showed that a subset of these genes is highly expressed in human pheochromocytoma at both the mRNA and the protein level. Interestingly, several of them, including L1CAM, have not been previously associated to human pheochromocytoma and represent novel potential markers of this tumor. Conclusions: Expression profiling of rat pheochromocytoma can identify molecular pathways involved in its human counterpart and can provide novel biomarkers for clinical use. Our studies support the hypothesis that the MENX syndrome is a good model of human neuroendocrine tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1997.