Dataset Information

Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing.

ABSTRACT:

Background

High resolution molecular studies have demonstrated that the clonal acquisition of gene mutations is an important mechanism that may promote rapid disease progression and drug resistance in chronic lymphocytic leukemia (CLL). Therefore, the early and sensitive detection of such mutations is an important prerequisite for future predictive CLL diagnostics in the clinical setting.

Material & methods

Here, we describe a novel, target-specific next generation sequencing (NGS) approach, which combines multiplex PCR-based target enrichment and library generation with ultra-deep high-throughput parallel sequencing using a MiSeq platform. We designed a CLL specific target panel, covering hotspots or complete coding regions of 15 genes known to be recurrently mutated and/or related to B-cell receptor signaling.

Results

High-throughput sequencing was performed using as little as 40 ng of peripheral blood B-cell DNA from 136 CLL patients and a dilution series of two ATM- or TP53-mutated cell lines, the latter of which demonstrated a limit of mutation detection below 5%. Using a stringent functional assessment algorithm, 102 mutations in 8 genes were identified in CLL patients, including hotspot regions of TP53, SF3B1, NOTCH1, ATM, XPO1, MYD88, DDX3X and the B-cell receptor signaling regulator PTPN6. The presence of mutations was significantly associated with an advanced disease status und molecular markers of an inferior prognosis, such as an unmutated IGHV mutation status or positivity for ZAP70 by flow cytometry.

Conclusion

In summary, targeted sequencing using an amplicon based library technology allows a resource-efficient and sensitive mutation analysis for diagnostic or exploratory purposes and facilitates molecular subtyping of patient sets with adverse prognosis.

SUBMITTER: Vollbrecht C

PROVIDER: S-EPMC4459702 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing.

Vollbrecht Claudia C Mairinger Fabian Dominik FD Koitzsch Ulrike U Peifer Martin M Koenig Katharina K Heukamp Lukas Carl LC Crispatzu Giuliano G Wilden Laura L Kreuzer Karl-Anton KA Hallek Michael M Odenthal Margarete M Herling Carmen Diana CD Buettner Reinhard R

PloS one 20150608 6

<h4>Background</h4>High resolution molecular studies have demonstrated that the clonal acquisition of gene mutations is an important mechanism that may promote rapid disease progression and drug resistance in chronic lymphocytic leukemia (CLL). Therefore, the early and sensitive detection of such mutations is an important prerequisite for future predictive CLL diagnostics in the clinical setting.<h4>Material & methods</h4>Here, we describe a novel, target-specific next generation sequencing (NGS ...[more]

PMID: 26053404

Similar Datasets

Project description:BackgroundDuring the last decade, remarkable progress with massive sequencing has been made in the identification of disease-associated genes for AIDs using next-generation sequencing technologies (NGS). An international group of experts described the ideal genetic screening method which should give information about SNVs, InDels, Copy Number Variations (CNVs), GC rich regions. We aimed to develop and validate a molecular diagnostic method in conjunction with the NGS platform as an inexpensive, extended and uniform coverage and fast screening tool which consists of nine genes known to be associated with various AIDs.MethodsFor the validation of basic and expanded panels, long-range multiplex models were setup on healthy samples without any known variations for MEFV, MVK, TNFRSF1A, NLRP3, PSTPIP1, IL1RN, NOD2, NLRP12 and LPIN2 genes. Patients with AIDs who had already known causative variants in these genes were sequenced for analytical validation. As a last step, multiplex models were validated on patients with pre-diagnosis of AIDs. All sequencing steps were performed on the Illumina NGS platform. Validity steps included the selection of related candidate genes, primer design, development of screening methods, validation and verification of the product. The GDPE (Gentera) bioinformatics pipeline was followed.ResultsAlthough there was no nonsynonymous variation in 21 healthy samples, 107 synonymous variant alleles and some intronic and UTR variants were detected. In 10 patients who underwent analytical validation, besides the 11 known nonsynonymous variant alleles, 11 additional nonsynonymous variant alleles and a total of 81 synonymous variants were found. In the clinical validation phase, 46 patients sequenced with multiplex panels, genetic and clinical findings were combined for diagnosis.ConclusionIn this study, we describe the development and validation of an NGS-based multiplex array enabling the "long-amplicon" approach for targeted sequencing of nine genes associated with common AIDs. This screening tool is less expensive and more comprehensive compared to other methods and more informative than traditional sequencing. The proposed panel offers advantages to WES or hybridization probe equivalents in terms of CNV analysis, high sensitivity and uniformity, GC-rich region sequencing, InDel detection and intron covering.

Dataset Information

Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing.

Background

Material & methods

Results

Conclusion

Publications

Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing.

Similar Datasets

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