Author/Editor     Pajič, Tadej; Rebernik, Veronika; Mali, Polona; Žontar, Darja; Černelč, Peter
Title     Ugotavljanje specifičnih BCR-ABL mRNA-zaporedij z načinom obratnega prepisovanja in verižne reakcije s polimerazo pri bolnikih z akutno limfoblastno levkemijo
Translated title     Detection of specific BCR-ABL mRNA sequences in acute lymphoblastic leukaemia patients by the reverse trancriptase and polymerase chain reaction
Type     članek
Source     Med Razgl
Vol. and No.     Letnik 39, št. Suppl 5
Publication year     2000
Volume     str. 13-23
Language     slo
Abstract     Background. One of the most frequent chromosomal abnormalities seen in acute lymphoblastic leukaemia is a reciprocal translocation t (9,22)(q34;q11). The molecular consequence of translocation is the formation of the BCR-ABL gene on the Philadelphia (Ph) chromoso- me. Patients with Ph+ ALL have been linked with unfavourable course of the disease. We wanted to introduce molecular genetic technique - reverse transcriptase and polymerase chain reaction as a support in the diagnosis of ALL. Methods. RT-PCR was used to amplify to BCR-ABL cDNA after reverse transcription of the mRNA. PCR for BCR-ABL in the ALL was performed by three separate reactions: one for e1a2, second for b2a2 and/or b3a2 mRNA transcripts and third for a control ABL gene in order to demonstrate adequate cDNA quality. The nested PCR strategy was used to increase the specificity of the method. PCR products were analysed by electrophoresis in agarose gels stained with ethidium bromide and detected under UV light. Results. We analysed samples from 7 patients with acute lymphoblastic leukaemia by RT-PCR. Specific e1a2 transcript was determined in one sample with ALL. We also analysed b2a2 and b3a2 transcripts in 7 ALL and 2 KML patients. We established the specific transcript in two of them. Conclusion. We applied the PCR method on blood and bone marrow samples from patients with ALL. The method is suitable for routine work and represents the additional tool that can be used for diagnosis and improved estimate success in the medical treatment of patients with ALL.
Summary     Izhodišča. Pri akutni limfoblastni levkemiji (ALL) je ena od pogostih kromosomskih nepravilnosti recipročna translokacija t (9.22)(q34;q11), ki vodi do nastanka hibridnega BCR-ABL-gena na Philadelphia (Ph) kromosomu. Pri otrocih in odraslih z ALL je prisotnost Ph-kromosoma povezana z neugodnim potekom bolezni. Zato smo želeli uvesti molekularnogenetičen način obratnega prepisovanja in verižne reakcije s polimerazo kot pomoč pri diagnostiki akutnih limfoblastnih levkemij. Načini. Ugotavljanje temelji na dokazovanju specifičnega cDNA-segmenta za BCR-ABL m- RNA-prepis z načinom obratnega prepisovanja in verižne reakcije s polimerazo (RT-PCR). Reakcije smo izvedli ločeno: eno za prepis e1a2, drugo za b3a2 in/ali b2a2 in tretjo zaradi prikaza ustreznosti cDNA-molekule, za kontrolno zaporedje mRNA, ki nastane iz gena ABL. Za povečanje specifičnosti načina smo del pridelka prvega pomnoževanja BCR-ABL c-DNA-segmenta ponovno pomnožili s pomočjo oligonukleotidov, ki so se prilegali znotraj prve- ga pridelka. Velikost pridelkov reakcij pomnoževanj smo ocenjevali z elektroforezo v agaroznem gelu, obarvanem z etidijevim bromidom pod UV-svetlobo. Rezultati. Z RT-PCR-načinom smo ocenili 7 bolnikov z akutno limfoblastno levkemijo in ugotovili prepis e1 a2 pri enem bolniku z ALL. Ugotavljali smo tudi prepis oz. cDNA-segment s stičiščem eksonov b2a2 ali b3a2 pri 7 bolnikih z akutno limfoblastno levkemijo in 2 bolnikih s kronično mieloično levkemijo. Specifičen prepis smo ugotovili pri dveh bolnikih. Zaključek. Vpeljali smo RT-PCR-način na vzorcih periferne krvi in kostnega mozga bolnikov z ALL in ugotovili, da je primeren za rutinsko delo. Predstavlja dopolnilno preiskavo pri ugotavljanju bolezni in pri oceni uspeha zdravljenja pri bolnikih z ALL.
Descriptors     LEUKEMIA, LYMPHOCYTIC, ACUTE
PHILADELPHIA CHROMOSOME
POLYMERASE CHAIN REACTION
RNA, MESSENGER
TRANSCRIPTION, GENETIC
BASE SEQUENCE