Double minutes (dmin)-circular, extra-chromosomal amplifications of specific acentric DNA fragments-are relatively frequent in malignant disorders, particularly in solid tumors. In acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), dmin are observed in approximately 1% of the cases. Most of them consist of an amplified segment from chromosome band 8q24, always including the MYC gene. Besides this information, little is known about their internal structure. We have characterized in detail the genomic organization of 32 AML and two MDS cases with MYC-containing dmin. The minimally amplified region was shown to be 4.26 Mb in size, harboring five known genes, with the proximal and the distal amplicon breakpoints clustering in two regions of approximately 500 and 600 kb, respectively. Interestingly, in 23 (68%) of the studied cases, the amplified region was deleted in one of the chromosome 8 homologs at 8q24, suggesting excision of a DNA segment from the original chromosomal location according to the 'episome model'. In one case, sequencing of both the dmin and del(8q) junctions was achieved and provided definitive evidence in favor of the episome model for the formation of dmin. Expression status of the TRIB1 and MYC genes, encompassed by the minimally amplified region, was assessed by northern blot analysis. The TRIB1 gene was found over-expressed in only a subset of the AML/MDS cases, whereas MYC, contrary to expectations, was always silent. The present study, therefore, strongly suggests that MYC is not the target gene of the 8q24 amplifications.

BackgroundThe assessment of TP53 mutational status is becoming a routine clinical practice for chronic lymphocytic leukemia patients (CLL). A broad spectrum of molecular techniques has been employed so far, including both direct Sanger sequencing and next generation sequencing. Oxford Nanopore Technologies recently released the MinION an USB-interfaced sequencer. In this paper we report our experience, with the MinION technology for the detection of the TP53 gene mutation in CLL patients.Twelve CLL patients at diagnosis were included in this study. All except one patient showed the TP53 gene deletion in Fluorescence in situ hybridization experiments.Patients were investigated for TP53 mutation by Sanger and by MinION sequencing.Analysis by Sanger was performed according with the IARC protocol.Analysis by MinION was performed adopting a strategy based on long template PCR, read error correction, and post variant calling filtering.ResultsDue to the high error rate of nanopore technology, sequence data were both used directly and before correction with two different in silico methods: ALEC and nanocorrect. A mean error rate of 15 % was detected before correction that was reduced to 4-5 % after correction.Analysis by Sanger sequencing was able to detect four patients mutated for TP53. MinION analysis detected one more mutated patient previously not detected from Sanger.ConclusionIn our hands, the Nanopore technology shows correlation with Sanger sequencing but more sensitive, manageable and less expensive, and therefore has proven to be a useful tool for TP53 gene mutation detection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13000-016-0550-y) contains supplementary material, which is available to authorized users.

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