Биологический микрочип для определения структуры химерных транскриптов с участием гена MLL у детей, больных острыми лейкозами

Abstract

MLL is involved in fusion genes with more than 100 partner genes, approximately 80 of which have been characterized at the molecular level. MLL fusion genes are often found in infants (60-80% of acute lymphoblastic leukemia (ALL) cases and 40-50% of acute myeloblastic leukemia (AML) cases) and are appreciably rarer (8-10%) in children older than 1 year of age. MLL rearrangements are important markers in diagnosis and treatment choice. To identify the partner gene is of primary importance for prognosis and minimal residual disease monitoring. The structure of the fusion gene, including localization of the MLL breakpoints, is also informative. A method was developed to examine the fusion transcripts in order to identify the partner gene among the six most common ones and to establish the exon structure of the rearranged MLL. The method includes a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify and to fluorescently label a fusion transcript fragment and subsequent hybridization of the product on a biological microchip with immobilized oligonucleotides complementary to exons of MLL and its partner genes AFF1, MLLT1, MLLT3, MLLT4, MLLT10, and ELL. Hybridization results were verified by sequencing the RT-PCR products and, in some cases, performing long-distance inverse PCR (LDI-PCR). The study involved 38 bone marrow samples from ALL patients (including 33 children younger than 1 year of age) and 15 samples from AML patients (including 10 from children younger than 1 year of age). The main partner genes were AFF1 (49%), MLLT1 (27%), MLLT3 (12%), and MLLT10 (12%) in ALL and MLLT3 (80%), MLLT10 (10%), and MLLT4 (10%) in AML. Fusion gene transcripts most commonly included MLL exon 11 (58% of ALL cases and 50% of AML cases), suggesting a breakpoint in MLL intron 11.

Detection of Chimeric Transcript NUP98-NSD1 in Pediatric Acute Myeloid Leukemia

“…The reverse transcription reaction was performed in a volume of 25 μL using the REVERTA-L kit (AmpliSens, Russia): 10 μL RT-mix (AmpliSens, Russia), 10 μL RNA, 1 μL reverse transcriptase MMlv (Ampli-Sens, Russia), 5 pmol specific primer and 20 pmol primer ABL. For the production of a single-stranded labeled DNA fragment for hybridization on a hydrogel microchip, the nested PCR method was used in two stages, as described earlier [8]. PCR was carried out in a T100 thermal cycler (Bio-Rad, United States) according to the following program: 95°C for 5 min; then a further 35 cycles: 95°C for 20 s, 62°C for 20 s, 72°C for 30 s; 72°C for 3 min.…”

“…The fluorescently labeled product of the second stage of PCR was hybridized on a biochip. Biochips were prepared by photoinduced copolymerization of oligonucleotides and acrylamide gel components, as described earlier [6,8]. The sequences of probes are shown in Table 1.…”

The EIMB Hydrogel Microarray Technology: Thirty Years Later

“…A LK-BIOCHIP kit has been developed and approved by the Russian Federal Service for Surveillance in Healthcare and Social Development for the analysis of the 13 most clinically significant chromosomal breakpoints in leukemia [43]. The LK-BIOCHIP was used to diagnose chromosomal translocations in children in multi-center trials aimed at treating acute lymphoblastic leukemia (ALL MB-2002 and ALL MB-2008) in the Russian Federation in 2005–2015 [44]. The new generation of microarrays for leukemia diagnosis is capable of detecting an extended range of chromosomal translocations, including nine additional clinically significant rearrangements t(1;11) MLL/MLLT11, t(1;11) MLL/EPS15, Del1 SIL/TAL1, t(2;5) NPM1/ALK, t(16;21) FUS/ERG, t(1;22) RBM15-MKL1, t(10;11) CALM/AF10, t(17;19) E2A/HLF, and t(6;9) DEK/CAN ( Fig.…”