NEXT-GENERATION SEQUENCING OF AMPLIFIED DNA FRAGMENTS WITH LENGTH POLYMORPHISMS ASSOCIATED WITH CHRONIC MYELOID LEUKEMIA REVEALED POLYMORPHISMS IN REPETITIVE ELEMENTS AND CANCER RISK LOCI
(Abstract release date: 05/19/16)
EHA Library. Koblihova J. 06/09/16; 132637; E1088
Dr. Jitka Koblihova
Contributions
Contributions
Abstract
Abstract: E1088
Type: Eposter Presentation
Background
A genetic predisposition and molecular mechanisms of chronic myeloid leukemia development (CML) and of resistance to the treatment with tyrosine kinase inhibitors (TKI) is the matter of the research worldwide. High-throughput techniques such as next generation sequencing (NGS) enable whole-genome analysis per each individual, however, often with limitations in a large volume of obtained data requiring extensive bioinformatics analysis.
Aims
We created an approach to scan the individual CML genomes using the combination of highly reproducible amplified fragment length polymorphism (AFLP) analysis and of NGS in order to identify polymorphic loci associated with CML or response to imatinib (IM).
Methods
AFLP was performed on 65 CML patients treated with IM first line (39 pts with optimal response; 26 pts with IM failure) and 30 healthy donors; for confirmatory analysis next 30 controls were added. The median age of patients was 55 years (range 18-84). AFLP analyses were performed using MseI and EcoRI endonucleases and commercially available kits and protocols. Fragmentation was run on ABI PRISM 3130 sequencer. The lengths of DNA fragments were analyzed with GeneMapper. DNA fragment frequency among CML patients and controls were analyzed by the exact binomial test. Nine selected DNA fragments were further characterized by NGS and the obtained data were evaluated using NextGENe. Polymorphisms in identified DNA fragments were confirmed by Sanger sequencing.
Results
AFLP generated 3912 AFLP markers/DNA fragments with diverse distribution among CML patients and healthy controls. Among them 199 were significantly associated with CML and 5 with IM response. Using NGS, we characterized 7 loci associated with CML and 2 with response to IM. In CAC_ACC_25, we confirmed SNP rs113864098 (C/T) resulting in an introduction of EcoRI restriction site. The locus is on chromosome 1 and is a part of L1MC1 and AluSz repetition. The significantly higher frequency of rs113864098-T allele was found in the patient cohort (60 %) in comparison to healthy donors (13 %) (P = 0.016). SNP rs7906704 (G/T) was confirmed in the CAT_ACC_54 fragment localized on chromosome 10 between NRAP and CASP7 genes resulting in the introduction of MseI restriction site in this locus. The frequency of minor rs7906704-T allele was significantly higher in CML patients (38 %; P = 0.04) in comparison to healthy controls. This SNP was found to be involved in apoptosis pathway in pancreatic tumorigenesis (Li et al., 2012). Further, we identified DNA fragment CTT_ACA_57, which was significantly frequently present in patients with optimal response to IM (65 %) in comparison to patients with IM failure (19 %) (P = 0.03). The fragment is a part of satellite sequence ALR/Alpha occurring in centromeric regions with the highest frequency on chromosome 5. Wide evidence of small genomic aberrations of ALR/Alpha was found in chronic lymphocytic leukemia (Kim et al., 2010).
Conclusion
In this work, we applied whole-genome fragmentation analysis to identify DNA polymorphisms associated with CML and response to IM and subsequently confirmed these potential novel DNA markers by genotyping. Up to now, we identified and confirmed 2 SNPs significantly associated with CML and 1 repetitive element polymorphism associated with response to IM. We assume identification of more genomic markers associated with CML and suppose potential use of AFLP/NGS combinational approach as the alternative to whole-genome sequencing.Supported by Ministry of Health of CZ grant IGA NT11555 and project 00023736.
Session topic: E-poster
Keyword(s): Chronic myeloid leukemia, Polymorphism, Resistance, Single nucleotide polymorphism
Type: Eposter Presentation
Background
A genetic predisposition and molecular mechanisms of chronic myeloid leukemia development (CML) and of resistance to the treatment with tyrosine kinase inhibitors (TKI) is the matter of the research worldwide. High-throughput techniques such as next generation sequencing (NGS) enable whole-genome analysis per each individual, however, often with limitations in a large volume of obtained data requiring extensive bioinformatics analysis.
Aims
We created an approach to scan the individual CML genomes using the combination of highly reproducible amplified fragment length polymorphism (AFLP) analysis and of NGS in order to identify polymorphic loci associated with CML or response to imatinib (IM).
Methods
AFLP was performed on 65 CML patients treated with IM first line (39 pts with optimal response; 26 pts with IM failure) and 30 healthy donors; for confirmatory analysis next 30 controls were added. The median age of patients was 55 years (range 18-84). AFLP analyses were performed using MseI and EcoRI endonucleases and commercially available kits and protocols. Fragmentation was run on ABI PRISM 3130 sequencer. The lengths of DNA fragments were analyzed with GeneMapper. DNA fragment frequency among CML patients and controls were analyzed by the exact binomial test. Nine selected DNA fragments were further characterized by NGS and the obtained data were evaluated using NextGENe. Polymorphisms in identified DNA fragments were confirmed by Sanger sequencing.
Results
AFLP generated 3912 AFLP markers/DNA fragments with diverse distribution among CML patients and healthy controls. Among them 199 were significantly associated with CML and 5 with IM response. Using NGS, we characterized 7 loci associated with CML and 2 with response to IM. In CAC_ACC_25, we confirmed SNP rs113864098 (C/T) resulting in an introduction of EcoRI restriction site. The locus is on chromosome 1 and is a part of L1MC1 and AluSz repetition. The significantly higher frequency of rs113864098-T allele was found in the patient cohort (60 %) in comparison to healthy donors (13 %) (P = 0.016). SNP rs7906704 (G/T) was confirmed in the CAT_ACC_54 fragment localized on chromosome 10 between NRAP and CASP7 genes resulting in the introduction of MseI restriction site in this locus. The frequency of minor rs7906704-T allele was significantly higher in CML patients (38 %; P = 0.04) in comparison to healthy controls. This SNP was found to be involved in apoptosis pathway in pancreatic tumorigenesis (Li et al., 2012). Further, we identified DNA fragment CTT_ACA_57, which was significantly frequently present in patients with optimal response to IM (65 %) in comparison to patients with IM failure (19 %) (P = 0.03). The fragment is a part of satellite sequence ALR/Alpha occurring in centromeric regions with the highest frequency on chromosome 5. Wide evidence of small genomic aberrations of ALR/Alpha was found in chronic lymphocytic leukemia (Kim et al., 2010).
Conclusion
In this work, we applied whole-genome fragmentation analysis to identify DNA polymorphisms associated with CML and response to IM and subsequently confirmed these potential novel DNA markers by genotyping. Up to now, we identified and confirmed 2 SNPs significantly associated with CML and 1 repetitive element polymorphism associated with response to IM. We assume identification of more genomic markers associated with CML and suppose potential use of AFLP/NGS combinational approach as the alternative to whole-genome sequencing.Supported by Ministry of Health of CZ grant IGA NT11555 and project 00023736.
Session topic: E-poster
Keyword(s): Chronic myeloid leukemia, Polymorphism, Resistance, Single nucleotide polymorphism
Abstract: E1088
Type: Eposter Presentation
Background
A genetic predisposition and molecular mechanisms of chronic myeloid leukemia development (CML) and of resistance to the treatment with tyrosine kinase inhibitors (TKI) is the matter of the research worldwide. High-throughput techniques such as next generation sequencing (NGS) enable whole-genome analysis per each individual, however, often with limitations in a large volume of obtained data requiring extensive bioinformatics analysis.
Aims
We created an approach to scan the individual CML genomes using the combination of highly reproducible amplified fragment length polymorphism (AFLP) analysis and of NGS in order to identify polymorphic loci associated with CML or response to imatinib (IM).
Methods
AFLP was performed on 65 CML patients treated with IM first line (39 pts with optimal response; 26 pts with IM failure) and 30 healthy donors; for confirmatory analysis next 30 controls were added. The median age of patients was 55 years (range 18-84). AFLP analyses were performed using MseI and EcoRI endonucleases and commercially available kits and protocols. Fragmentation was run on ABI PRISM 3130 sequencer. The lengths of DNA fragments were analyzed with GeneMapper. DNA fragment frequency among CML patients and controls were analyzed by the exact binomial test. Nine selected DNA fragments were further characterized by NGS and the obtained data were evaluated using NextGENe. Polymorphisms in identified DNA fragments were confirmed by Sanger sequencing.
Results
AFLP generated 3912 AFLP markers/DNA fragments with diverse distribution among CML patients and healthy controls. Among them 199 were significantly associated with CML and 5 with IM response. Using NGS, we characterized 7 loci associated with CML and 2 with response to IM. In CAC_ACC_25, we confirmed SNP rs113864098 (C/T) resulting in an introduction of EcoRI restriction site. The locus is on chromosome 1 and is a part of L1MC1 and AluSz repetition. The significantly higher frequency of rs113864098-T allele was found in the patient cohort (60 %) in comparison to healthy donors (13 %) (P = 0.016). SNP rs7906704 (G/T) was confirmed in the CAT_ACC_54 fragment localized on chromosome 10 between NRAP and CASP7 genes resulting in the introduction of MseI restriction site in this locus. The frequency of minor rs7906704-T allele was significantly higher in CML patients (38 %; P = 0.04) in comparison to healthy controls. This SNP was found to be involved in apoptosis pathway in pancreatic tumorigenesis (Li et al., 2012). Further, we identified DNA fragment CTT_ACA_57, which was significantly frequently present in patients with optimal response to IM (65 %) in comparison to patients with IM failure (19 %) (P = 0.03). The fragment is a part of satellite sequence ALR/Alpha occurring in centromeric regions with the highest frequency on chromosome 5. Wide evidence of small genomic aberrations of ALR/Alpha was found in chronic lymphocytic leukemia (Kim et al., 2010).
Conclusion
In this work, we applied whole-genome fragmentation analysis to identify DNA polymorphisms associated with CML and response to IM and subsequently confirmed these potential novel DNA markers by genotyping. Up to now, we identified and confirmed 2 SNPs significantly associated with CML and 1 repetitive element polymorphism associated with response to IM. We assume identification of more genomic markers associated with CML and suppose potential use of AFLP/NGS combinational approach as the alternative to whole-genome sequencing.Supported by Ministry of Health of CZ grant IGA NT11555 and project 00023736.
Session topic: E-poster
Keyword(s): Chronic myeloid leukemia, Polymorphism, Resistance, Single nucleotide polymorphism
Type: Eposter Presentation
Background
A genetic predisposition and molecular mechanisms of chronic myeloid leukemia development (CML) and of resistance to the treatment with tyrosine kinase inhibitors (TKI) is the matter of the research worldwide. High-throughput techniques such as next generation sequencing (NGS) enable whole-genome analysis per each individual, however, often with limitations in a large volume of obtained data requiring extensive bioinformatics analysis.
Aims
We created an approach to scan the individual CML genomes using the combination of highly reproducible amplified fragment length polymorphism (AFLP) analysis and of NGS in order to identify polymorphic loci associated with CML or response to imatinib (IM).
Methods
AFLP was performed on 65 CML patients treated with IM first line (39 pts with optimal response; 26 pts with IM failure) and 30 healthy donors; for confirmatory analysis next 30 controls were added. The median age of patients was 55 years (range 18-84). AFLP analyses were performed using MseI and EcoRI endonucleases and commercially available kits and protocols. Fragmentation was run on ABI PRISM 3130 sequencer. The lengths of DNA fragments were analyzed with GeneMapper. DNA fragment frequency among CML patients and controls were analyzed by the exact binomial test. Nine selected DNA fragments were further characterized by NGS and the obtained data were evaluated using NextGENe. Polymorphisms in identified DNA fragments were confirmed by Sanger sequencing.
Results
AFLP generated 3912 AFLP markers/DNA fragments with diverse distribution among CML patients and healthy controls. Among them 199 were significantly associated with CML and 5 with IM response. Using NGS, we characterized 7 loci associated with CML and 2 with response to IM. In CAC_ACC_25, we confirmed SNP rs113864098 (C/T) resulting in an introduction of EcoRI restriction site. The locus is on chromosome 1 and is a part of L1MC1 and AluSz repetition. The significantly higher frequency of rs113864098-T allele was found in the patient cohort (60 %) in comparison to healthy donors (13 %) (P = 0.016). SNP rs7906704 (G/T) was confirmed in the CAT_ACC_54 fragment localized on chromosome 10 between NRAP and CASP7 genes resulting in the introduction of MseI restriction site in this locus. The frequency of minor rs7906704-T allele was significantly higher in CML patients (38 %; P = 0.04) in comparison to healthy controls. This SNP was found to be involved in apoptosis pathway in pancreatic tumorigenesis (Li et al., 2012). Further, we identified DNA fragment CTT_ACA_57, which was significantly frequently present in patients with optimal response to IM (65 %) in comparison to patients with IM failure (19 %) (P = 0.03). The fragment is a part of satellite sequence ALR/Alpha occurring in centromeric regions with the highest frequency on chromosome 5. Wide evidence of small genomic aberrations of ALR/Alpha was found in chronic lymphocytic leukemia (Kim et al., 2010).
Conclusion
In this work, we applied whole-genome fragmentation analysis to identify DNA polymorphisms associated with CML and response to IM and subsequently confirmed these potential novel DNA markers by genotyping. Up to now, we identified and confirmed 2 SNPs significantly associated with CML and 1 repetitive element polymorphism associated with response to IM. We assume identification of more genomic markers associated with CML and suppose potential use of AFLP/NGS combinational approach as the alternative to whole-genome sequencing.Supported by Ministry of Health of CZ grant IGA NT11555 and project 00023736.
Session topic: E-poster
Keyword(s): Chronic myeloid leukemia, Polymorphism, Resistance, Single nucleotide polymorphism
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