Določitev sekretorskega statusa na podlagi prisotnosti alelov sekretorskega gena FUT2 in določitev genotipa krvnoskupinskega sistema Lewis (FUT3) iz vzorca periferne krvi

Poplašen, Helena (author), Čurin Šerbec, Vladka (mentor)

Gena FUT2 (SE) in FUT3 (LE) nosita zapis za encima fukoziltransferazi, ki sta ključna za sintezo antigenov krvnih skupin AB0 in Lewis (Le), gen FUT2 pa hkrati določa sekretorski status posameznika. Oba gena sta zelo polimorfna ter etnično specifična. Namen naloge je bil določiti prisotnost alelov genov FUT2 in FUT3 v vzorcih genomske DNA slovenskih krvodajalcev ter na podlagi tega sklepati na sekretorski status in fenotip krvne skupine Le preiskovanca. Z določitvijo sekretorskega statusa z metodo qPCR smo želeli zagotoviti novo diagnostično storitev, z določitvijo genotipa krvne skupine Le pa izboljšati diagnostiko, predvsem pa omogočiti določitev fenotipa Le, kadar tega ni mogoče izvesti s serološkimi metodami. S podatki iz literature smo za pravilno določitev sekretorskega statusa izbrali najpogostejšo mutacijo 428G>A v genu FUT2 ter optimizirali metodo qPCR. Rezultate smo primerjali z rezultati serološke določitve ter rezultati genotipizacije, ki smo jih pridobili iz Transfuzijskega centra Zürich, Schlieren. Z izbranim pristopom smo pravilno določili prisotnost alelov Se in se in v 100 % pravilno napovedali sekretorski status preiskovancev. Metoda, ki smo jo postavili, je primerna za rutinsko določanje sekretorskega statusa. Za določitev prisotnosti alelov FUT3 smo s podatki iz literature in pravilnim izborom delov gena FUT3 pripravili ustrezne reakcije qPCR za določitev polimorfizmov 59T>G, 202T>C, 314C>T, 508G>A in 1067T>A za alel le, ki so najpogosteje prisotni v kavkaški populaciji. Primerjali smo rezultate posameznih testov z že znanim fenotipom Le v kontrolni skupini. Ugotovili smo, da smo za mutacije 59T>G, 314C>T, 508G>A in 1067T>A uspešno pripravili reakcije qPCR, test za določitev mutacije 202T>C pa ni bil dovolj specifičen in dobljeni rezultati niso bili skladni z znanim fenotipom Le v kontrolni skupini. Določanje fenotipa Le je zaradi različnih kombinacij mutacij zahtevnejše, zato lahko sklenemo, da metoda, ki smo jo zasnovali, ni primerna za rutinsko določanje fenotipa Le in jo je treba nadgraditi.

• Language: Slovenian
• Keywords: FUT2, FUT3, Lewis, sekretor, qPCR
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2019
• PID: 20.500.12556/RUL-113128
• COBISS.SI-ID: 1538498755
• Publication date in RUL: 05.12.2019

Secondary language

• Language: English
• Title: Determination of secretor status on the basis of presence of alleles of FUT2 secretor gene and determination of genotype of Lewis (FUT3) blood group system from peripheral blood samples

Abstract

FUT2 (SE) and FUT3 (LE) genes encode enzymes fucosyltransferases, which plays a key role in the synthesis of ABO and Lewis blood group antigens. FUT2 gene also determines the secretor status of an individual. Both genes are highly polymorphic and ethnically specific. In our study, we wanted to determine the presence of alleles of genes FUT2 and FUT3 in Slovenian blood donors genomic DNA samples and on the basis of the results determine their secretor status and Lewis phenotype. By secretor status determination using qPCR method, we wanted to provide a new diagnostic test. By Lewis blood group genotype determination we wanted to improve diagnostics, mostly when serological determination of Lewis blood group is not possible. Based on data from the literature, the most common mutation 428G>A in FUT2 gene was selected and the qPCR reaction was optimized. The serological and genotyping results results were compared. The genotype of two samples from Transfusion center Zürich, Schlieren were compared with our our genotyping results as well. The presence of Se and se alleles were predicted correctly and the concordance with secretor status was 100%. We conclude that the method is reliable for routine secretor status determination. Regarding to data from literature, we selected the appropriate FUT3 gene sequences. We optimized qPCR reactions for determination of most common SNPs in Caucasian population, 59T>G, 202T>C, 314C>T, 508G>A and 1067T>A for le allele. The results of each assay with already known Lewis phenotype in the control group were compared. The results show that the qPCR reactions to determine mutations 59T>G, 314C>T, 508G>A and 1067T>A in FUT3 gene were successfully prepared. Assay for determination of 202T>C mutation wasn`t specific enough, and the genotyping results were not consistent with the previously known Lewis phenotype in the control group. Determination of Lewis phenotype is very complex due to multiple mutation combinations, so we conclude that our approach is not appropriate for routine Lewis phenotype determination and need to be adjusted and upgraded.

• Keywords: FUT2, FUT3, Lewis, secretor, qPCR