Dokazivanje točkastih mutacija u genu za FLT3 metodom lančane reakcije polimerazom u stvarnom vremenu

master's thesis

• Title (english): Evidence of point mutations in the FLT3 gene by method of real-time polymerase chain reaction
• Author: Robert Kolman
• Mentor: Renata Zadro (mentor)
• Committee member: Renata Zadro (predsjednik povjerenstva)
• Committee member: Dunja Rogić (član povjerenstva)
• Committee member: Sandra Šupraha Goreta (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2019-09-18, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Akutna mijeloična leukemija najčešća je vrsta leukemije te predstavlja četvrtinu svih leukemija koje se dijagnosticiraju u
odrasloj dobi. Ukoliko se bolest ne liječi, uzrok smrti su najčešće ozbiljne sekundarne infekcije i krvarenja, a kod čak 70%
bolesnika starijih od 67 godina očekuje se smrtni ishod unutar jedne godine od dijagnoze. S obzirom na to, točna procjena
prognoze bolesti ima ključnu ulogu kada se pristupa liječenju bolesnika. U tom procesu stratifikacije te donošenju kliničke
odluke o načinu, vrsti, trajanju i intenzitetu liječenja od velike su važnosti prognostički pokazatelji. Trenutno važeća
klasifikacija SZO uz morfološke prognostičke pokazatelje uključuje citogenetičke, molekularne i biološke prognostičke
pokazatelje te kliničku prezentaciju same bolesti. Iako kariotip bolesnika predstavlja najsnažniji prognostički parametar
kada je riječ o AML, ipak 45% bolesnika pokazuje uredan kariotip i sukladno tome pripada intermedijarnoj prognozi stoga
je velika pažnja usmjerena na potencijalno prisutne molekularne promjene. Jedne od klinički i prognostički značajnih
molekularnih promjena koje su prisutne u čak trećini bolesnika s AML jesu mutacije u genu za FLT3 koja je konstitutivno
aktivna u preko 90% leukemijskih blasta. Najčešća vrsta mutacija u genu za FLT3 prisutnih u do 30% slučajeva odraslih
pacijenata s de novo dijagnosticiranom AML jesu unutarnje tandemske duplikacije, dok su mutacije unutar tirozin-kinazne
domene druge po učestalosti te se pojavljuju u do 14% odraslih bolesnika. Cilj ovog diplomskog rada bio je ispitati
prisutnost točkastih mutacija u genu za FLT3 uspostavom metode lančane reakcije polimerazom u stvarnom vremenu kod
populacije odraslih bolesnika s urednim kariogramom i negativnom mutacijom FLT3-ITD. Uspostavljenom metodom
lančane reakcije polimerazom u stvarnom vremenu dokazana je mutacija FLT3-TKD u 4 (8,8%) odrasla bolesnika s
dijagnozom AML i urednim kariogramom. Prisutnost mutacija potvrđena je sekvenciranjem prema Sangeru, a metoda
lančane reakcije polimerazom u stvarnom vremenu pokazala se brzom i učinkovitom za rutinski probir FLT3-TKD i odabir
odgovarajuće terapije.

Abstract (english)

Acute myeloid leukemia is the most common type of leukemia and accounts for a quarter of all leukemias diagnosed in
adulthood. If untreated, the cause of death are usually serious secondary infections and bleeding and as many as 70% of
patients over 67 are expected to die within one year of diagnosis. In this regard, accurate assessment of the prognosis of the
disease plays a crucial role when treating patients. Prognostic indicators are of great importance in this stratification process
and in making clinical decisions about the method, type, duration and intensity of treatment. The current WHO classification
along with morphological prognostic indicators also includes cytogenetic, molecular and biological prognostic indicators
and clinical presentation of the disease itself. Although the karyotype of patients represents the strongest prognostic
parameter in AML, however, 45% of patients show a normal karyotype and accordingly belong to the intermediate
prognosis. Therefore great attention is focused on potentially present molecular changes. One of the clinically and
prognostically significant molecular changes present in as many as a third of patients with AML are mutations in the FLT3
gene, which are constitutively active in over 90% of leukemic blasts. The most common type of FLT3 gene mutations
present in up to 30% of adult patients with de novo diagnosed AML are internal tandem duplications, while mutations within
the tyrosine kinase domain are second in frequency and occur in up to 14% of adult patients. The aim of this thesis was to
investigate the presence of point mutations in the FLT3 gene by establishing a real-time polymerase chain reaction method
in a population of adult patients with a normal karyogram and a negative FLT3-ITD mutation. The established real-time
polymerase chain reaction method demonstrated the FLT3-TKD mutation in 4 (8.8%) adult patients diagnosed with AML
and normal karyogram. The presence of mutations was confirmed by Sanger sequencing. The real-time polymerase chain
reaction method proved to be rapid and effective for routine FLT3-TKD screening and selection of appropriate therapy

• Keywords: akutna mijeloična leukemija; AML; mutacija; FLT3; reverzna transkripcija; PCR
• Keywords (english): acute myeloid leukemia; AML; mutation; FLT3; reverse transcription; PCR
• Language: croatian
• URN:NBN: urn:nbn:hr:163:638534
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-02-13 09:31:22