Kalıtsal polipoz olamayan kolorektal kanserli üç Türk ailesinde HMLHI genindeki eşey hücresi mutasyonlarının analizi

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common genetic diseases in Western world. It is a clinical syndrome characterized by an inherited predisposition to early onset colorectal and an increased incidence of other cancers. The disease is caused by a germline defect in one of five human DNA mismatch repair genes, hMLHl, hMSH2, hPMSl, hPMS2, and hMSH6. Defects in hMLHl and hMSH2 account for the majority of mutations found in HNPCC families. In this study, a variety of mutation detection methods were used to identify hMLHl germline mutations in three Turkish HNPCC kindreds. Restriction enzyme analysis of genomic DNA was used to analyze five members of an HNPCC family with a previously described G884C mutation. The genotypes of all five individuals were determined by Ddel digestion and the results were confirmed by DNA sequence analysis. HphI restriction enzyme analysis was used to analyze twenty-nine members of an unrelated HNPCC family for a previously identified A1652C mutation. Genotypes were determined for all individuals and the results were confirmed by DNA sequence analysis. Both of these restriction enzyme analyses are reliable, cost-effective methods that can be used in mutation screening programs for family members who request genetic counseling. Single strand conformation polymorphism analysis (SSCP) was used to screen for unknown germline mutations. Nine DNA samples with defined mutations in the hMLHl gene were analyzed using several gel formulations and electrophoretic conditions to determine the most sensitive protocols. These protocols were then used for routine mutation detection. In a third HNPCC family, for whom no mutation has yet been defined, the complete coding sequence of the hMLHl gene was screened by SSCP. Two exons, 7 and 15, showed an altered mobility compared to control sequences. The nucleotide sequence of these two exons was determined by automated fluorescence DNA sequence analysis. The differential mobility observed for exon 15 appears to be due to an intonic polymorphism in the control sample. Preliminary results for exon 7 show no difference between proband and control nucleotide sequences. Thus, the DNA mismatch repair defect in this kindred appears not to be in hMLHl. Further studies will focus on the analysis of hMSH2. iii