Vpeljava izolacije zarodne DNA iz nohtov za razlikovanje med prirojenimi in pridobljenimi genetskimi različicami v rakavem vzorcu

Jug Brešan, Tina (avtor), Preložnik Zupan, Irena (mentor), Pajič, Tadej (komentor)

Dedne oblike raka lahko zahtevajo posebno obravnavo bolnika, saj vplivajo na potek bolezni, izbiro zdravljenja, spremljanje bolnika in njegove družine. Sum na družinsko nagnjenost k razvoju raka lahko vzbudi pogosto pojavljanje rakavih obolenj med sorodniki, pojavnost pri zgodnji starosti ali znana zarodna patogena različica, odkrita v rakavem tkivu, primarno analiziranem za določitev somatskih sprememb. V rakavem tkivu lahko odkrijemo več pridobljenih sprememb, ki so odgovorne za nastanek bolezni in so prisotne samo v malignih celicah, medtem ko so prirojene genetske različice prisotne v vseh celicah telesa. Za potrditev zarodne patogene genetske različice potrebujemo vir DNA, ki ni malignega izvora. Potrditveno genetsko testiranje se po navadi izvaja iz krvi, ki predstavlja bogat vir kakovostne zarodne DNA. Pri hematoloških bolnikih s sumom na dedno obliko raka kri ni primeren vzorec za tovrstne teste, saj vsebuje tudi rakave celice. Primeren vir zarodne DNA lahko predstavljajo nohti, vendar so zaradi svoje keratinske sestave zahtevnejši material za uspešno izolacijo zadostne količine kakovostne DNA. Namen naloge je bil oceniti primernost optimizirane metode izolacije DNA iz nohtov za ugotavljanje prirojenega izvora patogene genetske različice, povezane z dedno nagnjenostjo za razvoj raka, ki je bila predhodno ugotovljena v molekularno-genetskem diagnostičnem postopku v tumorskem vzorcu pri bolniku z rakom. V nalogi smo najprej optimizirali izolacijo DNA iz nohtov s pomočjo reagenčnega kompleta, ki za pridobitev DNA uporablja kolone s silikagelom. Z izbrano metodo smo izolirali DNA iz nohtov 22 preiskovancev, nato izmerili koncentracije in kakovost DNA, preverili uspešnost pomnoževanja z verižno reakcijo s polimerazo za gena TP53 in CEBPA ter zaporedja določili s sekvenciranjem po Sangerju. Iz nohtov vseh preiskovancev smo uspešno pridobili merljive koncentracije DNA. Količine izolirane DNA, merjene fluorometrično, so bile precej nizke z mediano 65,4 ng, medtem ko smo spektrofotometrično določili dosti višje povprečje 1585,9 ng DNA. Razlika v meritvah nakazuje na poškodovano in fragmentirano DNA. Kljub temu smo dosegli več kot 90-% uspešnost pri pomnoževanju DNA z reakcijo PCR in prav tako več kot 90-% učinkovitost pridobitve berljivih sekvenc s sekvenciranjem po Sangerju. Glede na dobljene rezultate bi metoda izolacije DNA iz nohtov lahko bila uporabna za potrjevanje zarodnih patogenih genetskih različic, predhodno odkritih v rakavem tkivu.

• Jezik: Slovenski jezik
• Ključne besede: Izolacija DNA, nohti, testiranje zarodne DNA, dedne oblike raka, hematološka maligna obolenja.
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-141636
• Datum objave v RUL: 03.10.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Introduction of germline DNA isolation from nails to distinguish between congenital and acquired genetic variants in a cancer sample

Izvleček

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.

• Ključne besede: DNA isolation, nails, germline testing, inherited cancer types, haematological malignancies.