Določanje polimorfizmov in mutacij v genih MLH1 in MSH2 pri nepolipoznem raku debelega črevesa Polymorphisms and MLH1 and MSH2 gene mutations in non-polyposis colorectal cancer

Povzetek

Rak debelega črevesa prizadene v Sloveniji okrog 1200 ljudi na leto. Dedni rak debelega črevesa (dedni nepolipozni rak debelega črevesa - HNPCC in družinska adematozna polipoza - FAP) predstavlja približno 10% vseh rakov debelega črevesa in danke. Pregledali smo 32 vzorcev bolnikov z rakom debelega črevesa. Z metodo HRM (analiza talitvene krivulje z visoko ločljivostjo) smo presejali gena MLH1 in MSH2. Z metodo PCR smo pomnožili vse eksone genov MLH1 in MSH2 (»missmach repair« geni – MMR). Vse PCR produkte smo obarvali z interkalirajočim barvilom, nato pa naredili analizo talitvene krivulje produkta. PCR produkte, katerih talitvena krivulja je odstopala od kontrolne smo sekvenirali. Našli smo 5 različnih mutacij, 2 neopredeljene variacije in 13 polimorfizmov. Med mutacijami smo našli 1 delecijo, 2 mutaciji izrezovalnih mest in 2 mutaciji, ki povzročita spremembo aminokisline.

Abstract (Eng)

Colorectal cancer affects approximately 1200 individuals in Slovenia every year. Hereditary colorectal cancer (hereditary non-polyposis colorectal cancer - HNPCC and familial adenomatous polyposis - FAP) accounts for around 10% of all colorectal cancers. Thirty-two colorectal patients were screened for DNA variations in MLH1 and MSH2 genes (miss-match repair genes- MMR genes). All patients were screened using high resolution melting (HRM). The exons of MLH1 and MSH2 genes were amplified using PCR (polymerase chain reaction). Amplified double stranded products were stained with intercalating dye and dissociated by heating. The melting curves of the tested samples were compared to the melting curve of the control and all PCR products, showing different melting curve than that of the control, were sequenced. We found 5 different mutations, 2 UVs (unclassified variants), 13 polymorphisms. Among mutations, 1 deletion, 2 splice-site mutations, and 2 missense mutations were found.