Colorectal cancer and endometrial cancer are among the most common forms of cancer globally and in Slovenia. One potential cause for their development is Lynch syndrome (LS), an autosomal dominant hereditary predisposition for the development of cancers. Lynch syndrome is associated with pathogenic variant in the MLH1, MSH2, MSH6, PMS2, and EPCAM genes. The first four of these genes encode proteins that form a complex involved in the DNA mismatch repair mechanism (MMR). However, the development of colorectal and endometrial cancers can also be attributed to sporadic cases, often resulting from hypermethylation of the MLH1 gene promoter region. Distinguishing between hereditary and sporadic causes of cancer is essential for appropriate patient management. Several methods are available for determining the presence of hypermethylation in the MLH1 promoter region, including MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification) and qPCR-HRM (methylation-sensitive quantitative PCR high-resolution melting). The MS-MLPA technique has already been established in the Laboratory of Molecular Genetics at the Clinical Institute of Genomic Medicine (KIGM), University Medical Center Ljubljana (UKC Ljubljana). The aim of this master's thesis was to optimize and validate the qPCR-HRM method. We used DNA isolated from 25 formalin fixed paraffin embedded tissue samples, comparing the results of qPCR-HRM analysis with those obtained from the MS-MLPA method. Validation of the qPCR-HRM method was achieved by confirming that the results were consistent with those from the MS-MLPA analysis. This advancement significantly expands the range of diagnostic tests available for cancer patients. Consequently, reducing the number of individuals unnecessarily referred for genetic counselling due to suspected LS.
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