Characterization of neuroendocrine tumors in heterozygous mutant MENX rats: a novel model of invasive medullary thyroid carcinoma.

Rats affected by the MENX syndrome spontaneously develop multiple neuroendocrine tumors (NETs) including adrenal, pituitary and thyroid gland neoplasms. MENX was initially reported to be inherited as a recessive trait and affected rats were found to be homozygous for the predisposing Cdkn1b mutation encoding p27. We here report that heterozygous MENX-mutant rats (p27+/mut) develop the same spectrum of NETs seen in the homozygous (p27mut/mut) animals but with slower progression. Consequently, p27+/mut rats have a significantly shorter lifespan compared with their wild-type (p27+/+) littermates. In the tumors of p27+/mut rats, the wild-type Cdkn1b allele is neither lost nor silenced, implying that p27 is haploinsufficient for tumor suppression in this model. Transcriptome profiling of rat adrenal (pheochromocytoma) and pituitary tumors having different p27 dosages revealed a tissue-specific, dose-dependent effect of p27 on gene expression. In p27+/mut rats, thyroid neoplasms progress to invasive and metastatic medullary thyroid carcinomas (MTCs) accompanied by increased calcitonin levels, as in humans. Comparison of expression signatures of late-stage vs early-stage MTCs from p27+/mut rats identified genes potentially involved in tumor aggressiveness. The expression of a subset of these genes was evaluated in human MTCs and found to be associated with aggressive RET-M918T-positive tumors. Altogether, p27 haploinsufficiency in MENX rats uncovered a novel, representative model of invasive and metastatic MTC exploitable for translational studies of this often aggressive and incurable cancer.

uPAR enhances malignant potential of triple-negative breast cancer by directly interacting with uPA and IGF1R.

BACKGROUND: Due to lack of a targeted therapy for the triple-negative breast cancer (TNBC) patients, it is important to explore this aggressive breast cancer type in more detail and to establish novel therapeutic approaches. TNBC is defined negative for the protein expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). One prominent feature of this cancer type is the frequent overexpression of major components of the urokinase-type plasminogen activator system (uPAS) including uPA, its receptor uPAR and the inhibitor PAI-1, which may be valuable as therapeutic targets. METHODS: Direct interactions of uPAR with interactors were demonstrated by immunoprecipitations and proximity ligation assays. For stable knockdowns of target proteins, lentiviral vectors were used and the effects were analysed by immunoblottings and using in vitro cell viability, migration and invasion assays. Immunohistochemical and statistical analyses of biomarkers and clinical parameters were conducted in a TNBC cohort (n = 174). RESULTS: Direct tumour-promoting interactions of uPAR with uPA and the insulin-like growth factor receptor 1 (IGF1R) were shown in TNBC cells and these interactions were significantly reduced (p = 0.001) when uPAR was downregulated. The combined knockdown of uPAR and uPA or IGF1R additively and significantly reduced cell viability, migration and invasion of the model cell lines. In TNBC tissue, the complexes formed by uPAR with uPA or with IGF1R significantly correlated with the histological grade (p = 0.0019) as well as with cathepsin B and D (p ≤ 0.0001) that are implicated in cell invasion and metastasis. CONCLUSIONS: Our outcomes show that not only overexpressed biomarkers promote tumourigenesis, but rather their interactions further potentiate tumour progression. This study emphasises the potential of combined approaches targeting uPAR and its interactors with regard to an improved therapy of TNBC.

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 Bonferroni's 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.

p27 variant and corticotropinoma susceptibility: a genetic and in vitro study.

Germline mutations in p27(kip1) are associated with increased susceptibility to multiple endocrine neoplasias (MEN) both in rats and humans; however, the potential role of common polymorphisms of this gene in endocrine tumor susceptibility and tumorigenesis remains mostly unrecognized. To assess the risk associated with polymorphism rs2066827 (p27-V109G), we genotyped a large cohort of Brazilian patients with sporadic endocrine tumors (pituitary adenomas, n=252; pheochromocytomas, n=125; medullary thyroid carcinoma, n=51; and parathyroid adenomas, n=19) and 885 population-matched healthy controls and determined the odds ratios and 95% CIs. Significant associations were found for the group of patients with pituitary adenomas (P=0.01), particularly for those with ACTH-secreting pituitary adenomas (P=0.005). In contrast, no association was found with GH-secreting pituitary tumors alone or with the sporadic counterpart of MEN2-component neoplasias. Our in vitro analyses revealed increased colony formation and cell growth rate for an AtT20 corticotropin mouse cell line overexpressing the p27-V109G variant compared with cells transfected with the WT p27. However, the genotypic effects in genetic and in vitro approaches were divergent. In accordance with our genetic data showing specificity for ACTH-secreting pituitary tissues, the overexpression of p27-V109G in a GH3 somatotropin rat cell line resulted in no difference compared with the WT. Pituitary tumors are one of the major clinical components of syndromes associated with the p27 pathogenic mutations MENX and MEN4. Our genetic and in vitro data indicate that the common polymorphism rs2066827 may play a role in corticotropinoma susceptibility and tumorigenesis through a molecular mechanism not fully understood thus far.

Understanding the role of the Q338H MUTYH variant in oxidative damage repair.

The MUTYH DNA-glycosylase is indirectly engaged in the repair of the miscoding 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxodG) lesion by removing adenine erroneously incorporated opposite the oxidized purine. Inherited biallelic mutations in the MUTYH gene are responsible for a recessive syndrome, the MUTYH-associated polyposis (MAP), which confers an increased risk of colorectal cancer. In this study, we functionally characterized the Q338H variant using recombinant proteins, as well as cell-based assays. This is a common variant among human colorectal cancer genes, which is generally considered, unrelated to the MAP phenotype but recently indicated as a low-penetrance allele. We demonstrate that the Q338H variant retains a wild-type DNA-glycosylase activity in vitro, but it shows a reduced ability to interact with the replication sensor RAD9:RAD1:HUS1 (9-1-1) complex. In comparison with Mutyh(-)(/)(-) mouse embryo fibroblasts expressing a wild-type MUTYH cDNA, the expression of Q338H variant was associated with increased levels of DNA 8-oxodG, hypersensitivity to oxidant and accumulation of the population in the S phase of the cell cycle. Thus, an inefficient interaction of MUTYH with the 9-1-1 complex leads to a repair-defective phenotype, indicating that a proper communication between MUTYH enzymatic function and the S phase checkpoint is needed for effective repair of oxidative damage.

Functional Imaging of Pheochromocytoma with Ga-DOTATOC and C-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 (11)C-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 (11)C-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.

Somatic mutation and germline sequence abnormalities in CDKN1B, encoding p27Kip1, in sporadic parathyroid adenomas.

CONTEXT: Typical nonfamilial (sporadic) parathyroid adenomas are common endocrine tumors for which no predisposing germline DNA variants and only a few clonally altered genes that drive parathyroid tumorigenesis have been identified. CDKN1B, encoding cyclin-dependent kinase inhibitor p27(kip1), has recently been implicated in a multiple endocrine tumor phenotype in rats and, rarely, in a human familial MEN1 (multiple endocrine neoplasia type 1)-like disorder. OBJECTIVE: We sought to determine whether mutation of CDKN1B might contribute to the development of common sporadic parathyroid adenomas. PATIENTS AND DESIGN: We sequenced the CDKN1B gene in 86 parathyroid adenomas from patients with typical, sporadic presentations of primary hyperparathyroidism. Identified alterations were categorized as somatic or germline, and their functional consequences were examined. RESULTS: CDKN1B sequence abnormalities were identified in four parathyroid adenomas. Acquired biallelic alteration of CDKN1B, resulting from somatic mutation plus loss of heterozygosity, was detected in one tumor. Germline origin was documented in two cases despite nonfamilial presentations. None of the observed alterations were found in 240 CDKN1B alleles from normal individuals, nor among more than 2,000 previously reported alleles. Most identified variants reduced p27(kip1) protein levels or altered in vitro stability. CONCLUSIONS: In typical, sporadic parathyroid adenomas, CDKN1B mutation can be somatic and clonal, indicative of a directly conferred selective advantage in parathyroid tumorigenesis. Additionally, the identification of germline CDKN1B variants in patients with sporadic presentations provides evidence for CDKN1B as a susceptibility gene in the development of typical parathyroid adenomas.

Pheochromocytoma in rats with multiple endocrine neoplasia (MENX) shares gene expression patterns with human pheochromocytoma.

Pheochromocytomas are rare neoplasias of neural crest origin arising from chromaffin cells of the adrenal medulla and sympathetic ganglia (extra-adrenal pheochromocytoma). Pheochromocytoma that develop in rats homozygous for a loss-of-function mutation in p27Kip1 (MENX syndrome) show a clear progression from hyperplasia to tumor, offering the possibility to gain insight into tumor pathobiology. We compared the gene-expression signatures of both adrenomedullary hyperplasia and pheochromocytoma with normal rat adrenal medulla. Hyperplasia and tumor show very similar transcriptome profiles, indicating early determination of the tumorigenic signature. Overrepresentation of developmentally regulated neural genes was a feature of the rat lesions. Quantitative RT-PCR validated the up-regulation of 11 genes, including some involved in neural development: Cdkn2a, Cdkn2c, Neurod1, Gal, Bmp7, and Phox2a. Overexpression of these genes precedes histological changes in affected adrenal glands. Their presence at early stages of tumorigenesis indicates they are not acquired during progression and may be a result of the lack of functional p27Kip1. Adrenal and extra-adrenal pheochromocytoma development clearly follows diverged molecular pathways in MENX rats. To correlate these findings to human pheochromocytoma, we studied nine genes overexpressed in the rat lesions in 46 sporadic and familial human pheochromocytomas. The expression of GAL, DGKH, BMP7, PHOX2A, L1CAM, TCTE1, EBF3, SOX4, and HASH1 was up-regulated, although with different frequencies. Immunohistochemical staining detected high L1CAM expression selectively in 27 human pheochromocytomas but not in 140 nonchromaffin neuroendocrine tumors. These studies reveal clues to the molecular pathways involved in rat and human pheochromocytoma and identify previously unexplored biomarkers for clinical use.

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 toCdk2 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 patient's 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.

The MENX syndrome and p27: relationships with multiple endocrine neoplasia.

In the past 3 years new insight into the etiopathogenesis of hereditary endocrine tumors has emerged from studies conducted on MENX, a rat multiple endocrine neoplasia (MEN) syndrome. MENX spontaneously developed in a rat colony and was discovered by serendipity when these animals underwent complete necropsy, as they were found to consistently develop multiple endocrine tumors with a spectrum similar to both MEN type 1 (MEN1) and MEN2 human syndromes. Genetic studies identified a germline mutation in the Cdkn1b gene, encoding the p27 cell cycle inhibitor, as the causative mutation for the MENX syndrome. Capitalizing on these findings, we and others identified heterozygous germline mutations in the human homologue, CDKN1B, in patients with multiple endocrine tumors. As a consequence of these observations a novel human MEN syndrome, named MEN4, was recognized which is caused by mutations in p27. Altogether these studies identified Cdkn1b/CDKN1B as a novel tumor susceptibility gene for multiple endocrine tumors in both rats and humans. In this chapter we present the MENX syndrome and its phenotype, and we compare it to the human MEN syndromes; we discuss the current state of knowledge regarding the genes associated to inherited MEN, with a particular focus on CDKN1B; we present recent clinical and basic findings about the MEN4 syndrome and the functional characterization of the CDKN1B mutations identified. These findings are placed in the broader context of how p27 dysregulation might affect neuroendocrine cell function and trigger tumorigenesis.

Characterization of a naturally-occurring p27 mutation predisposing to multiple endocrine tumors.

BACKGROUND: p27Kip1 (p27) is an important negative regulator of the cell cycle and a putative tumor suppressor. The finding that a spontaneous germline frameshift mutation in Cdkn1b (encoding p27) causes the MENX multiple endocrine neoplasia syndrome in the rat provided the first evidence that Cdkn1b is a tumor susceptibility gene for endocrine tumors. Noteworthy, germline p27 mutations were also identified in human patients presenting with endocrine tumors. At present, it is not clear which features of p27 are crucial for this tissue-specific tumor predisposition in both rats and humans. It was shown that the MENX-associated Cdkn1b mutation causes reduced expression of the encoded protein, but the molecular mechanisms are unknown. To better understand the role of p27 in tumor predisposition and to characterize the MENX animal model at the molecular level, a prerequisite for future preclinical studies, we set out to assess the functional properties of the MENX-associated p27 mutant protein (named p27fs177) in vitro and in vivo. RESULTS: In vitro, p27fs177 retains some properties of the wild-type p27 (p27wt) protein: it localizes to the nucleus; it interacts with cyclin-dependent kinases and, to lower extent, with cyclins. In contrast to p27wt, p27fs177 is highly unstable and rapidly degraded in every phase of the cell-cycle, including quiescence. It is in part degraded by Skp2-dependent proteasomal proteolysis, similarly to p27wt. Photobleaching studies showed reduced motility of p27fs177 in the nucleus compared to p27wt, suggesting that in this compartment p27fs177 is part of a multi-protein complex, likely together with the degradation machinery. Studies of primary rat newborn fibroblasts (RNF) established from normal and MENX-affected littermates confirmed the rapid degradation of p27fs177 in vivo which can be rescued by Bortezomib (proteasome inhibitor drug). Overexpression of the negative regulators microRNA-221/222 plays no role in regulating the amount of p27fs177 in RNFs and rat tissues. CONCLUSION: Our findings show that reduced p27 levels, not newly acquired properties, trigger tumor formation in rats, similarly to what has been observed in mice. The molecular characteristics of p27fs177 establish MENX as a useful preclinical model to evaluate compounds that inhibit p27 degradation for their efficacy against endocrine tumors.

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.

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.

Heterogeneous molecular mechanisms underlie attenuated familial adenomatous polyposis.

PURPOSE: Familial adenomatous polyposis is a phenotypically heterogeneous disease predisposing to colorectal cancer. It is dominantly transmitted, when associated with the APC gene, and recessively inherited, when associated with MUTYH gene. We searched for APC and MUTYH germline alterations in Italian and Greek patients with attenuated polyposis, a phenotypic variant whose genetic cause remains unknown in many cases. METHODS: We studied 26 unrelated patients (and 16 relatives) with multiple colorectal adenomas (3-100, by endoscopic analysis) that had screened APC mutation-negative by protein truncation test. We searched for APC rearrangements by multiplex ligation-dependent probe amplification and for MUTYH mutations by sequencing. We performed a screening of five MUTYH recurrent pathogenic mutations in 501 Italian and 144 Greek controls. RESULTS: One patient proved to carry an APC whole-gene deletion; 4 of 25 (16%) patients showed biallelic and 3 of 25 (12%) monoallelic MUTYH mutations. In the three heterozygous subjects no pathogenetic variants were found in OGG1, MTH1, APE1, MSH2, and MSH6 genes. Frequency assessment of MUTYH mutations in healthy subjects showed that only Y165C and G382D reach a subpolymorphic frequency. CONCLUSION: Attenuated polyposis patients without "conventional" APC mutations are genetically heterogeneous, and the phenotype is not directly related to the germline defect. Therefore, the families' appropriate management requires an accurate genetic and clinical investigation.

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 analysis and case-control study of -160C/A polymorphism in the E-cadherin gene promoter: association with cancer risk.

BACKGROUND: A C-->A polymorphism within the CDH1 (E-cadherin) promoter seems to be associated with a reduced efficiency of gene transcription in vitro. Due to the crucial role of E-cadherin in epithelia, tissue-specific effect of C-->A change on CDH1 transcription was tested and a case-control study was performed on patients affected with epithelial tumors. PATIENTS AND METHODS: The -178/+93 CDH1 region containing either C or A nucleotide was inserted upstream of the Luciferase reporter gene in the pGL-2 vector, and the construct activity was assessed by transient transfection assay in HeLa and HCT116 cells. RESULTS: A significantly lower activity for pGL-2A was found compared to pGL-2C, both in HeLa (54% decrease) and in HCT116 (67% decrease) cells. Genotyping of 246 controls and 505 patients affected with breast, gastric, colorectal, cervical and endometrial cancers demonstrated an association between the A allele and an increased risk of colorectal, gastric and endometrial tumors (1.66-, 1.81- and 2.35-fold, respectively). CONCLUSION: Our data support the notion that the A allele may act as a low-penetrance cancer susceptibility gene.

High frequency of MYH gene mutations in a subset of patients with familial adenomatous polyposis.

BACKGROUND & AIMS: Inherited colorectal polyposis has been linked to constitutive mutations of the APC tumor suppressor gene. Recently, germline mutations in the base excision repair gene MYH have been associated with a recessively inherited form of the disease. The aim of this study was to evaluate germline mutation frequencies of both MYH and APC susceptibility genes in Italian patients with attenuated familial adenomatous polyposis. METHODS: The analysis was performed in 14 unrelated patients by using the protein truncation test for APC and genomic DNA sequencing for MYH. RESULTS: Overall, we identified 7 of 14 (50%) mutation carriers. Two patients were heterozygotes for an APC truncating mutation (2 of 14 [14%]), whereas 5 proved to be homozygotes or compound heterozygotes for MYH gene alterations (5 of 14 [36%]). Two MYH missense mutations, Y165C and G382D, already found to be frequent among patients from northern Europe, were also preponderant in our survey. Individuals with APC-associated syndrome showed a dominant family history of polyposis, whereas patients with MYH-associated disease were either apparently sporadic cases or had a family history consistent with recessive inheritance. MYH biallelic mutation carriers were up to 60% (5 of 8) among patients showing at least 30 adenomas and a family history with no vertical transmission of polyposis. CONCLUSIONS: On the basis of our data, patients with attenuated familial adenomatous polyposis with >30 adenomas and no obvious vertical transmission of the disease should be considered for MYH gene testing.