Hereditary cancers may require special patient management, as they affect the course of the disease, the choice of treatment, and the follow-up of the patient and his/her family. Suspicion of a familial predisposition to develop cancer may be triggered by the frequent occurrence of cancer among relatives, the occurrence of cancer at an early age, or a known germline pathogenetic variant detected in cancer tissue primarily analysed for somatic mutations. Several acquired mutations responsible for the disease can be detected in cancer tissue and are present only in malignant cells, whereas inherited genetic variants are present in all cells of the body. To confirm germline pathogenic variant, we need a DNA source that is not of malignant origin. Confirmatory genetic testing is usually performed on blood, which is a rich source of good quality germline DNA. In haematological patients with suspected hereditary cancer, blood is not a suitable sample for such tests as it also contains cancer cells. Nails may be a suitable source of germline DNA; however, their keratin composition makes them a more challenging material for the successful isolation of sufficient quantities of good quality DNA.
The aim of this thesis was to assess the suitability of an optimised method for isolating DNA from nails to identify the inherited origin of a pathogenetic variant associated with an inherited predisposition to develop cancer, previously identified in a molecular genetic diagnostic procedure in a tumour specimen from a cancer patient.
In this work, the isolation of DNA from fingernails was first optimised using a reagent kit that uses silica gel columns to obtain DNA. Using the selected method, DNA from the nails of 22 individuals was isolated, DNA concentrations and quality were measured, amplification performance by polymerase chain reaction for the TP53 and CEBPA genes was verified, and the sequences were determined using Sanger sequencing. Measurable DNA concentrations were successfully extracted from the fingernails of all participants. The amounts of isolated DNA measured fluorometrically were quite low, with a median of 65.4 ng, while spectrophotometrically, a much higher average of 1585.9 ng DNA was determined. The difference in measurements is indicative of damaged and fragmented DNA. Nevertheless, a success rate of more than 90 % was achieved in amplifying DNA by PCR, as well as an efficiency rate of more than 90 % in obtaining readable sequences by Sanger sequencing. According to the results obtained, the method of DNA isolation from fingernails could be useful for the validation of germline pathogenic genetic variants previously detected in cancer tissue.
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