Določanje različic v genu TP53 s sekvenciranjem po Sangerju

Čurič, Sara (author), Podgornik, Helena (mentor), Šućurović, Sandra (co-mentor)

Kronična limfocitna levkemija (KLL) je najpogostejša oblika levkemije v zahodnem svetu, pri kateri so okvare gena TP53, ki je ključnega pomena za ohranjanje celovitosti genoma, zaradi delecije v kromosomu 17 in/ali različice znotraj gena TP53, povezane z večjo odpornostjo na kemoimunoterapijo in s slabšo prognozo za bolnika. Na podlagi tega je vključitev analize različic gena TP53 v rutinsko klinično diagnostiko ključna za izboljšanje obravnave bolnikov in optimizacijo terapevtskih odločitev. Evropska raziskovalna pobuda za kronično limfocitno levkemijo (ERIC) od 2012 priporoča naslednje metodološke pristope za analizo različic v genu TP53: denaturacijsko tekočinsko kromatografijo visoke ločljivosti, funkcionalno analizo ločenih alelov v kvasovkah, uporabo mikromrež, sekvenciranje po Sangerju in sekvenciranje naslednje generacije (NGS). Slednje, zaradi izboljšane občutljivosti trenutno velja za glavno metodo odkrivanja različic v genu TP53. Smernice prav tako narekujejo, da je vse odkrite različice z zgoraj navedenimi metodami zmeraj potrebno potrditi še s sekvenciranjem po Sangerju. V okviru magistrske naloge smo postavili in overili metodo sekvenciranja gena TP53 po Sangerju pri bolnikih s KLL. Pri tem smo sledili priporočilom ERIC, ki narekujejo, da je potrebna analiza najmanj eksonov 4-10, priporočeno pa je analiziranje celotne kodirajoče regije (eksoni 2-11). Za postavitev metode smo uporabili pet vzorcev bolnikov, pri katerih različic v genu TP53 predhodno z metodo sekvenciranja po Sangerju nismo zaznali. Nasprotno smo za overjanje metode uporabili 12 vzorcev bolnikov, pri katerih smo odkrili prisotnost različice oziroma različic. Med praktičnim delom smo ustrezno optimizirali celoten postopek analize, od uporabljenih začetnih oligonukleotidov za posamezni ekson do pogojev, ki omogočajo uspešno pomnoževanje in sekvenčno analizo eksonov 2-11 gena TP53. Na osnovi analize podatkov sekvenciranja smo zaključili, da je z metodo sekvenciranja po Sangerju možno potrditi vse različice z alelno frekvenco različice nad 10 %. Priporočila narekujejo, da se lahko sprejema terapevtske odločitve le na podlagi različic, ki jih s sekvenciranje po Sangerju lahko potrdimo. Analizo smo uspešno vpeljali v redno klinično prakso, kjer bo služila za redno diagnostiko in potrjevanje predhodno odkritih različic z metodo NGS.

• Language: Slovenian
• Keywords: Kronična limfocitna levkemija, različice v genu TP53, ERIC, sekvenciranje po Sangerju, NGS
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-141499
• Publication date in RUL: 30.09.2022

Secondary language

• Language: English
• Title: TP53 gene variants analysis by Sanger sequencing

Abstract

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in the Western world. TP53 gene is crucial for maintaining genome integrity. Gene defects due to deletion in chromosome 17 and / or variants within the TP53 gene are associated with increased resistance to chemoimmunotherapy and poor prognosis for the patient. Therefore, the inclusion of TP53 gene defects analysis in routine clinical diagnostics is important to improve patients care and optimize therapeutic decisions. Since 2012, the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) has recommended the following methodological approaches for analyzing TP53 variants: high-resolution denaturation liquid chromatography, functional analysis of separate alleles in yeasts, microarrays, Sanger sequencing and next-generation sequencing (NGS). The latter due to improved sensitivity is currently the main method for detecting TP53 gene variants. The guidelines also dictate that Sanger sequencing should always confirm all detected variants. We set up and verified the method of sequencing the TP53 gene by Sanger in patients with CLL. We followed ERIC recommendations, which dictates that the sequenced region of the TP53 gene must include exons 4–10, yet it is recommended to analyze the entire coding region (exons 2-11). To set up the method, we used five samples of patients in whom variants in the TP53 gene had not been previously detected by Sanger sequencing. In contrast, 12 samples with mutation in gene TP53 were used for the verification of the method. We optimized the entire analysis, from the oligonucleotides used for exons to the conditions of PCR programs for successful amplification and sequence analysis of exons 2-11 of the TP53 gene. Based on the sequencing data analysis, we concluded that all variants with variant allele frequency above 10 % can be confirmed by Sanger sequencing. The recommendations dictate that therapeutic decisions can only be made based on changes that can be confirmed by Sanger sequencing. The analysis was successfully introduced into regular clinical practice, where it will serve for standard diagnostics and to confirm variants detected by next-generation sequencing.

• Keywords: Chronic lymphocytic leukemia, TP53 gene variants, ERIC, Sanger sequencing, NGS