Targeted exon sequencing of blast cell DNA samples of children with acute lymphoblastic leukemia in relationship of the expression of subunit A of coagulation factor XIII
témavezető: Kiss Csongor
helyszín (magyar oldal): Department of Pediatrics, Faculty of Medicine, University of Debrecen helyszín rövidítés: UD
A kutatási téma leírása:
Background: Leukemic B-cell precursor (BCP) lymphoblasts were recently identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). FXIII-A expression determined by flow cytometry (FC) exhibited characteristically distinct expression patterns in the subgroups of FXIII-A negative, FXIII-A dim, and FXIII-A bright lymphoblasts. The FXIII-A negative subgroup was significantly associated with the ‘B-other’ genetic category and had an unfavorable disease outcome. RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology (GO) analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed (DE) genes were validated by RT-Q-PCR. We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the expression of FXIII-A protein, as determined by FC. Three well-defined categories of FXIII-A expression: FXIII-A negative, FXIII-A dim, and FXIII-A bright subgroups defined by characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays.
Targeted exon sequencing: 20 patients with de novo FXIIIA-negative/MRD-positive ALL will be included in the "index patient" group. Targeted exon sequencing in 10-10 strong FXIII-A positive and FXIII-A negative samples will be performed. Genes will be selected for targeted capture from three sources:
• All genes from the NimbleGen Comprehensive Cancer Gene List (2015. February version), 497 human genes altogether.
• Genes characteristically mutated in childhood ALL.
• Genes with significantly altered expression between FXIII-A negative and FXIII-A postive childhood ALL patients with the following GEO identifiers: GSE47051, GSE13425, GSE13351, GSE10255 and GSE28497. This resulted in a list of 476 genes.
The non-redundant combination of the lists will be used for targeted exon-capture followed by next generation sequencing. Exon coordinates for these genes will be downloaded from the UCSC Genome Browser database. The custom exon capture probe panel for the Roche NimbleGen SeqCap EZ Library SR kit will be designed by Roche NimbleGen, based on the exon coordinates of the final gene list. Genomic DNA isolated from leukemic blasts of the selected patients will be used for exon-capture, performed by UD-Genomed, Debrecen. Next generation sequencing will be done on the Illumina MiSeq platform, using paired-end 125 bp sequencing, aiming for at least 100X coverage per base. Primary data analysis will be performed on the Galaxy platform (usegalaxy.org), with the GATK mutation analysis pipeline, followed by Variant Effect Predictor-Ensembl (https://www.ensembl.org/vep) annotation and with manual evaluation using the Integrated Genomics Viewer (Broad Institute). Basic quality assessment will be done with the FASTQC program. After adapter removal and preprocessing with the Trimmomatic program, reads will be aligned to the hg19 human genome reference sequence with BWA-MEM. Putative mutations will be selected finally based on a) their difference from the reference genome sequence, b) read support, c) sequence context, d) lack of representation in SNP databases, e) on their predicted effect on gene function, supported by the VEP annotation, and f) FXIIIA-group specific representation.
Validation: Putative high-value mutations will be validated by Sanger sequencing in the original patient cohort, and in BM samples of 30 additional patients with the same clinical profile. Mutant genes with potential impact on therapy (known drug targets) will be analyzed further to confirm their altered biological functions. This work will include the following steps:
1) PCR primers will be designed around the selected mutations, and the PCR reaction conditions will be optimized to allow specific amplification of the selected region from genomic DNA.
2) The PCR fragments will be sequenced by traditional sequencing to confirm the presence of mutations.
3) Validated mutations will also be checked on normal genomic DNA of the affected patients to determine the germ-line or somatic origin of the mutations.
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