Nowadays high throughput “Next Generation Sequencing” technologies are revolutionizing genomics and transcriptomics by providing a cost-efficient and single base resolution tool for a unified deep analysis of tumor complexity. Taking advantage from this tool, we used the Illumina/Solexa platform to define in a single procedure and at high sensitivity the full repertoire of leukemia-related mutations in 3 adult BCRABL1+ ALL cases treated with tyrosine kinase inhibitors. Patient median age was 55.3 years (range, 40-70); no additional chromosome abnormalities were detected except for one case. All the selected cases had previously been profiled by high resolution SNP (Affymetrix SNP6.0) and gene expression (Affymetrix Human Exon 1ST Array) arrays, as well as candidate gene re-sequencing (IKZF1, PAX5, JAK2, CDKN2A, CDKN2B, IDH1, IDH2), lacking missense point mutations. Two cases harbored the deletion of IKZF1, and in one case PAX5 and CDKN2A/B losses were also found. Poly(A) RNA from blast cells was used to prepare Illumina cDNA libraries according to the manufacturer’s recommendations. Sequencing by synthesis was performed on an Illumina Genome Analyzer IIx platform, with standard sequencing kits and nucleotide incorporation cycles, generating 75 base pairs (bp) paired end sequence reads. A total of 57, 51 and 9 million reads were obtained from the 3 samples and high quality sequence reads were mapped to the reference sequence of the human genome (UCSC hg19) using the Maq software, finding out 58,205, 48,913 and 136,937 putative new single nucleotide variants (SNVs) in the CDS/EXON regions not reported in the dbSNP build 130. Of these, 874 distributed on 290 genes, affected both samples. A functional analysis was carried out on common mutated genes using the GeneGo software (www.genego.com) and in the list of the most significant GeneGo Pathway Maps we found the “Development Hedgehog Signaling” (p value = 1.400e-4), including genes such as casein kinase 1, alpha 1-like (CSNK1A1L), catenin (cadherin-associated protein) beta 1 (CTNNB1) and heat shock protein HSP 90-beta (HSP90AB1). In conclusion, this study provided, for the first time, a comprehensive overview of a BCR-ABL1+ ALL transcriptome, identifying novel mutations potentially involved in ALL. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione–Università 2007–2009.
NEXT GENERATION PAIRED END TRANSCRIPTOMIC RE-SEQUENCING TECHNOLOGY REVEALS NOVEL COMMON GENETIC ALTERATIONS IN THE HEDGEHOG PATHWAY IN ADULT BCR-ABL1 POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)
IACOBUCCI, ILARIA;Ferrarini A;SAZZINI, MARCO;LONETTI, ANNALISA;PAPAYANNIDIS, CRISTINA;SOVERINI, SIMONA;TRINO, STEFANIA;CATTINA, FEDERICA;MARTINELLI, GIOVANNI
2011
Abstract
Nowadays high throughput “Next Generation Sequencing” technologies are revolutionizing genomics and transcriptomics by providing a cost-efficient and single base resolution tool for a unified deep analysis of tumor complexity. Taking advantage from this tool, we used the Illumina/Solexa platform to define in a single procedure and at high sensitivity the full repertoire of leukemia-related mutations in 3 adult BCRABL1+ ALL cases treated with tyrosine kinase inhibitors. Patient median age was 55.3 years (range, 40-70); no additional chromosome abnormalities were detected except for one case. All the selected cases had previously been profiled by high resolution SNP (Affymetrix SNP6.0) and gene expression (Affymetrix Human Exon 1ST Array) arrays, as well as candidate gene re-sequencing (IKZF1, PAX5, JAK2, CDKN2A, CDKN2B, IDH1, IDH2), lacking missense point mutations. Two cases harbored the deletion of IKZF1, and in one case PAX5 and CDKN2A/B losses were also found. Poly(A) RNA from blast cells was used to prepare Illumina cDNA libraries according to the manufacturer’s recommendations. Sequencing by synthesis was performed on an Illumina Genome Analyzer IIx platform, with standard sequencing kits and nucleotide incorporation cycles, generating 75 base pairs (bp) paired end sequence reads. A total of 57, 51 and 9 million reads were obtained from the 3 samples and high quality sequence reads were mapped to the reference sequence of the human genome (UCSC hg19) using the Maq software, finding out 58,205, 48,913 and 136,937 putative new single nucleotide variants (SNVs) in the CDS/EXON regions not reported in the dbSNP build 130. Of these, 874 distributed on 290 genes, affected both samples. A functional analysis was carried out on common mutated genes using the GeneGo software (www.genego.com) and in the list of the most significant GeneGo Pathway Maps we found the “Development Hedgehog Signaling” (p value = 1.400e-4), including genes such as casein kinase 1, alpha 1-like (CSNK1A1L), catenin (cadherin-associated protein) beta 1 (CTNNB1) and heat shock protein HSP 90-beta (HSP90AB1). In conclusion, this study provided, for the first time, a comprehensive overview of a BCR-ABL1+ ALL transcriptome, identifying novel mutations potentially involved in ALL. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione–Università 2007–2009.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
