Číslo abstraktu: P301
Autoři: Ana Pudja; Jelena D. Milosevic (Feenstra), Ph.D.; Thorsten Klampfl; Ashot Harutyunyan; Tiina Berg; Klaudia Bagienski; Doris Chen; Bettina Gisslinger; Elisa Rumi; MD Luca Malcovati; Daniela Pietra, Ph.D.; Chiara Elena; MD Matteo Giovanni Della Porta; MD Lisa Pieri, PhD; MD Paola Guglielmelli; prof. MUDr. Michael Doubek, Ph.D.; Ing. Dana Dvořáková, CSc.; prof. MUDr. Nada Suvajdzic; Dragica Tomin; Natasa Tosic; Prof. MUDr. Zdeněk Ráčil, Ph.D.; MD Michael Steurer; Sonja Pavlovic; MD Alessandro M. Vannucchi; M.D. Mario Cazzola; MD Heinz Gisslinger; Robert Kralovics, PhD
Chromosomal aberrations, together with somatic mutations, contribute to disease onset and progression in myeloid malignancies. The detection of these aberrations in patients has allowed identification of genes involved in the disease pathogenesis. Chromosome 6 shows high complexity of aberrations detected in patients. We previously showed thatJARID2 is a target for deletions amplified to homozygosity by 6pUPD (uniparental disomy). However, JARID2 is not mutated in the rest of the patients with 6pUPD. This implies that other genes are possibly targets of chromosome 6p (chr 6p) aberrations.
Identification of target genes of chromosome 6p aberrations in patients with myeloid malignancies, using cytogenetic mapping and whole exome sequencing.
Peripheral blood samples (n=913) were collected from patients with myeloproliferative neoplasms (MPN), post-MPN acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) post-MDS AML, de novo AML (dnAML) and chronic myeloid leukemia (CML), along with written informed consent, in compliance with local ethics committees. Genomic DNA was isolated from whole blood, granulocytes or mononuclear cells. Affymetrix Genome-Wide Human SNP 6.0 arrays and Genotyping Console software were used for detecting deletions, gains and UPDs. Whole exome sequencing was performed, using the TruSeq DNA sample prep kit and exome enrichment kit (Illumina), on the Illumina HiSeq2000 instrument. Data was analyzed with the GATK Haplotype Caller code. Variants were filtered for exonic and splice site variants, located in the affected region of chr 6 (individually defined for each sample). An allelic frequency filter of >50% was introduced in samples with UPD and gains of chr 6p. All variants were validated by Sanger sequencing. For 2 patients T lymphocyte DNA was used as control tissue DNA.
We combined all chr 6 aberrations, detected by SNP microarrays, from 913 patient samples. A total of 41 chromosomal aberrations were detected on chr 6p in 25 patients (7 MPN, 2 progression phase MPN, 7 post-MPN AML, 2 post-MDS AML, 6 dnAML, 1 CML). Deletions represented 54% of events (n=22). Six patients harbored 6pUPD, two of which had focal deletions of JARID2 amplified to homozygosity by UPD. An additional small deletion mapped to JARID2. Two independent UPD events were detected in one patient. The aberration map was complemented with exome sequencing data from 4 selected patients: 1 with a deletion (post-MPN AML), 2 with a UPD (post-MPN AML, dnAML) and 1 with a chr6 trisomy (secondary myelofibrosis). One somatic, 8 germline and 9 mutations of unknown origin were validated. None of the mutations were found in multiple patients. The somatic L521R mutation in FAM65B was amplified by the trisomy. Overlap of the mutations with chromosomal aberrations identified a commonly affected region containing genes NCR3 andPSORS1C1. Trisomy amplified a germline PSORS1C1 P38 frameshift mutation, whereas the NCR3 R96Q mutation was amplified by UPD. A focal gain overlapped with a UPD-amplified UHRF1BP1 S506P mutation.
Intersecting microarray data from a large pan-myeloid cohort of patients with exome sequencing data of 3 accelerated phase MPN/post-MPN AML and 1 dnAML patient resulted in identification of 4 novel genes, in addition to JARID2, as putative targets of chr6p lesions. As the position of the genes does not overlap with all 6pUPDs, it remains possible that mutations in regulatory regions or epigenetic events are targets amplified by 6pUPD in patients with myeloid malignancies.
Datum přednesení příspěvku: 12. 6. 2015