Hereditary gastrointestinal polyposis: Diagnosis, genetic test and risk assessment

Colorectal cancer (CRC) is the second cause of cancer deaths, with over 1 million new cases estimated every year. Familial adenomatous polyposis, MUTYH-associated polyposis and hamartomatous polyposis are inherited syndromes that account for 2%-5% of all colon cancer. The mutated genes responsible for the vast majority of these disorders, are now known (MLH1, MSH2, MSH6, PMS2, APC, MYH, LKB1, SMAD4, BMPR1A, and PTEN) and specific mutations have been identified. Molecular caracterization of inherited CRCs allows pre-symptomatic diagnosis identifying at-risk individuals and improving cancer surveillance. Adenomatous polyposis includes familial adenomatous polyposis (FAP), attenuated FAP (AFAP), and MUTYH-associated polyposis (MAP). Hamartomatous polyposis comprises Peutz-Jeghers syndrome (PJS), juvenile polyposis syndrome (JPS) and "PTEN hamartoma tumour syndrome" (PHTS). MAP is an autosomal recessive condition, while all other disorders are inherited in an autosomal dominant manner. Differential dyagnosis could be very difficult between syndromes because of their phenotypic variability. Attenuated FAP, MAP and Lynch syndrome could be all associated with fewer numbers of adenomas (3-10 polyps), nevertheless, each syndrome has distinct cancer risks, characteristic clinical features, and separate genetic etiologies. Thus, differential diagnosis is essential for correct management of the specific disease. In our laboratory we set up a methodology for genetic tests of the colorectal polyposis syndrome. In these reviews we summarize the literature data and our experience about diagnosis, genetic tests and cancer risk assesment associated with colorectal polyposis. According to literature data, in our experience, there is a portion of analyzing patients that remain without identified mutation, after molecular screening of the specific gene involved in the pathogenesis of the disease. Since the sensibility of used techniques, such as DHPLC, MLPA and sequencing, is now very high, we suggest that a different approach to molecular diagnosis of polyposis syndromes is necessary. In our laboratory, we are now planning to set up analysis of a larger pannel of genes that could be involved in colorectal poliposis syndromes, using a next generation sequencing techniques. In our opinion, a better characterization of molecular basis of the polyposis syndromes will allow a more efficient cancer prevention

Beta catenin and cytokine pathway dysregulation in patients with manifestations of the "PTEN hamartoma tumor syndrome"

Background. The "PTEN hamartoma tumor syndrome" (PHTS) includes a group of syndromes caused by germline mutations within the tumor suppressor gene "phosphatase and tensin homolog deleted on chromosome ten" (PTEN), characterized by multiple polyps in the gastrointestinal tract and by a highly increased risk of developing malignant tumours in many tissues. The current work clarifies the molecular basis of PHTS in three unrelated Italian patients, and sheds light on molecular pathway disregulation constitutively associated to PTEN alteration. Methods. We performed a combination of RT-PCR, PCR, sequencing of the amplified fragments, Real Time PCR and western blot techniques. Results. Our data provide the first evidence of β-catenin accumulation in blood cells of patients with hereditary cancer syndrome caused by germ-line PTEN alteration. In addition, for the first time we show, in all PHTS patients analysed, alterations in the expression of TNFα, its receptors and IL-10. Importantly, the isoform of TNFRI that lacks the DEATH domain (TNFRSF1β) was found to be overexpressed. Conclusion. In light of our findings, we suggest that the PTEN pathway disregulation could determine, in non-neoplastic cells of PHTS patients, cell survival and pro-inflammatory stimulation, mediated by the expression of molecules such as β-catenin, TNFα and TNFα receptors, which could predispose these patients to the development of multiple cancers

Analisi mutazionale dei geni del mismatch repair (MMR) mediante tecniche innovative in pazienti affetti da cancro ereditario non poliposico del colon-retto (HNPCC)

Il cancro ereditario non poliposico del colon-retto (HNPCC) è una sindrome autosomica dominante, che include circa il 6% delle forme di cancro colo-rettale (CRC) ereditarie ed è associato alla presenza di una mutazione germinale in uno dei geni del “MisMatch Repair” (MMR), MSH2, MLH1, PMS2, MSH6 e MLH3. I geni MMR più frequentemente mutati sono MLH1 e MSH2, che risultano mutati rispettivamente nel 49% e 38% dei casi HNPCC. L’alterazione di uno di questi geni nella linea germinale si riflette in un’aumentata velocità di accumulo di errori di replicazione, che si traduce nel fenotipo RER (Replication Errors), particolarmente evidente a livello dei microsatelliti. Tuttavia, essendo il tumore sporadico del colon-retto molto frequente, è necessaria un'attenta e corretta anamnesi clinica per l'identificazione di famiglie HNPCC. Nel nostro studio sono stati analizzati 77 soggetti di 54 famiglie con diagnosi clinica di HNPCC nei quali è stata condotta un’indagine genetico-molecolare dei 2 geni MMR maggiormente coinvolti, MLH1 e MSH2. Mediante DHPLC e successiva analisi di sequenza è stato possibile identificare 17 mutazioni responsabili della malattia (8 in MLH1, 2 in MSH2 e 7 in MSH6) in 16 delle 54 famiglie HNPCC. Otto di queste sono nuove mutazioni non ancora descritte in letteratura. Per valutare il grado di instabilità dei microsatelliti è stato analizzato il cosiddetto “pannello di Bethesda” che comprende 3 ripetizioni dinucleotidiche e 2 ripetizioni mononucleotidiche. In più sono state analizzate tre nuove ripetizioni mononucleotidiche quasimonomorfiche NR21, NR22 e NR24, apparse più instabili in linee cellulari del colon con mutazioni in MSH6. I tessuti tumorali dei pazienti portatori di mutazioni in MSH6 hanno presentato un fenotipo MSI-L con maggiore instabilità a livello delle sequenze mononucleotidiche: in particolare, NR21 e NR22. Questo indica che tali ripetizioni potrebbero essere analizzate per individuare i pazienti portatori di mutazioni nel gene MSH6. Allo scopo di verificare se l’alterazione fosse dovuta ad ampie delezioni o duplicazioni è stata messa a punto una nuova metodica la Multiplex Ligation-Probe Dependent Amplification (MLPA). Mediante tale metodica sono state identificate 2 delezioni nel gene MSH2 e una nel gene MLH1 e la duplicazione di 2 esoni nel gene MSH2. Questo studio conferma l’estrema eterogeneità genotipo-fenotipo che caratterizza l’HNPCC e l’estrema variabilità fenotipica tra i familiari affetti. Inoltre, l’MLPA è risultato un metodo sensibile e specifico per l’identificazione di ampi riarrangiamenti nei geni MLH1 e MSH2 e dunque, potrebbe essere considerato una tappa fondamentale nel protocollo da seguire per la diagnosi di cancro ereditario non poliposico del colon. / [ENGLISH] Hereditary non polyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited predisposition for early onset colorectal cancer; it accounts for at least 6% of all colorectal malignancies and it is associated with germline mutations in mismatch repair (MMR) genes: MSH2, MLH1, PMS2, MSH6 and MLH3. Until now 477 point mutations predisposing to HNPCC have been characterized; so far, few large genomic rearrangements within MMR genes have been described. In this work we present 8 novel mutations in MLH1, MSH2 and MSH6 genes and 4 large rearrangements in MLH1 and MSH2 genes. The identification of mutations in MLH1, MSH2 and MSH6 genes have been performed by a combination of denaturing high performance liquid chromatography (DHPLC) and DNA sequencing techniques. Moreover, we set up a novel tecnique, the multipex ligation dependent probe amplification (MLPA), for detection of genomic deletions in MLH1 and MSH2 genes, notoriously undetectable using conventional diagnostic techniques. We have analysed the DNA of seventy-seven patients of 54 families with clinical diagnosis of HNPCC and identified the specific mutation in 27/54 families: eight mutations are located in MLH1 gene, 3 in MSH2 and 7 in MSH6; of these, eight are novel germline mutations. The microsatellite instability (MSI) phenotype is a characteristic of the HNPCC (95%). To evaluate MSI, a reference panel was proposed at an international consensus meeting, comprised of 2 mononucleotide and 3 dinucleotide repeats. Moreover we have analysed 3 new mononucleotide repeats that are quasimonomorphic and that appear unstable in colon cell lines that show mutations in MSH6. The patients carriers of MSH6 mutations presented MSI-Low, with more instability at the mononucleotide markers. This indicates that the mononucleotide quasimonomorphic repeats might also be used for the detection of tumors mutated in MSH6. Finally, using the MLPA technique, we have identified a deletion of exon 19 in MLH1 gene and two deletions in MSH2 gene: a deletion exons 4 and 5 in one case and of exon six in another; moreover we identified a duplication of exon 1-2 in MSH2 gene. This study reinforces the notion of the remarkable heterogeneity of the mutational spectrum in HNPCC that gives rise to an extreme variability of the clinical expression between affected familiars. Moreover, MLPA appears to be a sensitive, specific and simple method for the detection of genomic deletion in MLH1 and MSH2 and might be considered as a part of the mutation detection protocols in the molecular diagnosis of hereditary non polyposis colorectal cancer

Ipermetilazione del promotore di MLH1 ed instabilità micro satellitare: descrizione dell’analisi molecolare

Valutazione dell'analisi di Instabilità dei microsatelliti nella sindrome di Lynch. Implicazioni nella diagnosi molecolare e nella valutazione oncologica sugli eventuali trattamenti chemioterapici da rivolgere al paziente

Same MSH2 Gene Mutation But Variable Phenotypes in 2 Families With Lynch Syndrome: Two Case Reports and Review of Genotype-Phenotype Correlation.

Lynch syndrome is an autosomal dominant syndrome that can be subdivided into Lynch syndrome I, or site-specific colonic cancer, and Lynch syndrome II, or extracolonic cancers, particularly carcinomas of the stomach, endometrium, biliary and pancreatic systems, and urinary tract. Lynch syndrome is associated with point mutations and large rearrangements in DNA MisMatch Repair ( MMR ) genes. This syndrome shows a variable phenotypic expression in people who carry pathogenetic mutations. So far, a correlation in genotype-phenotype has not been definitely established. In this study, we describe 2 Lynch syndrome cases presenting with the same genotype but different phenotypes and discuss possible reasons for this