Publication Date:
2016-12-02
Description:
Background: Recurrent and clonal genetic alterations are characteristic of different subtypes of pediatric acute lymphoblastic leukemia (ALL) and several of them are strong independent predictors of patient outcome. Multiplex Ligation-dependent Probe Amplification (MLPA) has become one of the standard methods for detection of common copy number alterations (CNAs), including IKZF1 deletions, which are associated with a poor outcome. However, standard MLPA assays only accommodate a maximum of 60 MLPA probes. In order to increase the number of target sequences in one assay, a next generation sequencing (NGS)-based MLPA variant has been developed. This digitalMLPA assay is based on the well-known MLPA procedure but can include up to 1000 probes in a single reaction, uses minute amounts of DNA (≥20ng) and can be analysed on all Illumina NGS platforms. Methods: A digitalMLPA assay was designed and optimised to detect CNAs of 55 key target genes and regions in childhood ALL, including deletions of B-cell differentiation and cell cycle control genes (PAX5, IKZF1/2/3, EBF1, RB1, CDKN2A/B, ETV6, and BTG1), iAMP21 and rearrangements of the PAR1 region, T-cell ALL associated aberrations (STIL-TAL1, LEF1, CASP8AP2, MYB, EZH2, MLLT3, NUP214-ABL1, PTEN, LMO1/2, NF1, SUZ12, PTPN2, PHF6), ERG and TP53 deletions. Several genetic alterations of potential prognostic and/or therapeutic relevance in ALL have also been included (NOTCH1, CD200/BTLA, VPREB1, TBL1XR1, PDGFRB-EBF1, IGHM, NR3C1/2, CREBBP, CTCF, ADD3, EPHA1, FHIT, SPRED1 and TOX). All target genes are covered by at least 3 digitalMLPA probes (~450 probes in total). Moreover, a set of 200 probes was designed for genome-wide detection of gross ploidy changes (high hyper- or hypodiploidy) and to determine the extent of CNAs, while also acting as reference probes for data normalization. Performance of all probes has been extensively tested on genomic DNA from healthy individuals and positive cell lines. Only probes with a standard deviation
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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