A New Workflow for Whole-Genome Sequencing of Single Human Cells

Binder V, Bartenhagen C, Okpanyi V, et al. A New Workflow for Whole-Genome Sequencing of Single Human Cells. Human mutation. 2014;35(10):1260-1270.: Unbiased amplification of the whole-genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter-linker PCR-based WGA method with second-generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy-number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR-based WGA approach. Sequencing with paired-end reads allowed genome-wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity

Next-generation-sequencing of recurrent childhood high hyperdiploid acute lymphoblastic leukemia reveals mutations typically associated with high risk patients

Chen C, Bartenhagen C, Gombert M, et al. Next-generation-sequencing of recurrent childhood high hyperdiploid acute lymphoblastic leukemia reveals mutations typically associated with high risk patients. Leukemia research. 2015;39(9):990-1001.: 20% of children suffering from high hyperdiploid acute lymphoblastic leukemia develop recurrent disease. The molecular mechanisms are largely unknown. Here, we analyzed the genetic landscape of five patients at relapse, who developed recurrent disease without prior high-risk indication using whole-exome- and whole-genome-sequencing. Oncogenic mutations of RAS pathway genes (NRAS, KRAS, FLT3, n=4) and deactivating mutations of major epigenetic regulators (CREBBP, EP300, each n=2 and ARID4B, EZH2, MACROD2, MLL2, each n=1) were prominent in these cases and virtually absent in non-recurrent cases (n=6) or other pediatric acute lymphoblastic leukemia cases (n=18). In relapse nucleotide variations were detected in cell fate determining transcription factors (GLIS1, AKNA). Structural genomic alterations affected genes regulating B-cell development (IKZF1, PBX1, RUNX1). Eleven novel translocations involved the genes ART4, C12orf60, MACROD2, TBL1XR1, LRRN4, KIAA1467, and ELMO1/MIR1200. Typically, patients harbored only single structural variations, except for one patient who displayed massive rearrangements in the context of a germline tumor suppressor TP53 mutation and a Li-Fraumeni syndrome-like family history. Another patient harbored a germline mutation in the DNA repair factor ATM. In summary, the relapse patients of our cohort were characterized by somatic mutations affecting the RAS pathway, epigenetic and developmental programs and germline mutations in DNA repair pathways

Infection Exposure Promotes ETV6-RUNX1 Precursor B-cell Leukemia via Impaired H3K4 Demethylases

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/peripheral cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss-of-function resulted in increased levels of H3K4me3, which co-precipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches

Infection exposure promotes ETV6-RUNX1 precursor B-cell leukemia via impaired H3K4 demethylases

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches.J. Hauer has been supported by the German Children's Cancer Foundation, DJCLS 02R/2016, KKS A2016/07 and from the “Forschungskommission” of the medical faculty of the Heinrich Heine University. M. Müschen is an HHMI Faculty Scholar (HHMI-55108547) and supported by NIH/NCI through an Outstanding Investigator Award (R35CA197628) to M. Müschen, a Wellcome Trust Senior Investigator Award, the Leukemia and Lymphoma Society, the California Institute for Regenerative Medicine, the William Lawrence and Blanche Hughes Foundation. A. Borkhardt has been supported by the German Children's Cancer Foundation and the Federal Ministry of Education and Research, Bonn, Germany. Research in I. Sánchez-García's group is partially supported by FEDER and by MINECO (SAF2012-32810, SAF2015-64420-R and Red de Excelencia Consolider OncoBIOSAF2014-57791-REDC), Instituto de Salud Carlos III (PIE14/00066), ISCIII- Plan de Ayudas IBSAL 2015 Proyectos Integrados (IBY15/00003), by Junta de Castilla y León (BIO/SA51/15, CSI001U14, UIC-017, and CSI001U16), Fundacion Inocente Inocente and by the ARIMMORA project (European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 282891). I. Sánchez-García's lab is a member of the EuroSyStem and the DECIDE Network funded by the European Union under the FP7 program. A. Borkhardt and I. Sánchez-García have been supported by the German Carreras Foundation (DJCLS R13/26). Research in C. Vicente-Dueñas' group is partially supported by a “Miguel Servet” grant (CP14/00082-AES 2013-2016-FEDER) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad) and by the Lady Tata International Award for Research in Leukaemia 2016–2017. A. Martín-Lorenzo and G. Rodríguez-Hernández were supported by FSE-Conserjería de Educación de la Junta de Castilla y León (CSI001-13 and CSI001-15, respectively).Peer Reviewe