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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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This corrects the article DOI: 10.1038/leu.2017.177.
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Chronic lymphocytic leukaemia (CLL) consists of two biologically and clinically distinct subtypes defined by the abundance of somatic hypermutation (SHM) affecting the Ig variable heavy-chain locus (IgHV). The molecular mechanisms underlying these subtypes are incompletely understood. Here, we present a comprehensive whole-genome sequencing analysis of somatically acquired genetic events from 46 CLL patients, including a systematic comparison of coding and non-coding single-nucleotide variants, copy number variants and structural variants, regions of kataegis and mutation signatures between IgHVmut and IgHVunmut subtypes. We demonstrate that one-quarter of non-coding mutations in regions of kataegis outside the Ig loci are located in genes relevant to CLL. We show that non-coding mutations in ATM may negatively impact on ATM expression and find non-coding and regulatory region mutations in TCL1A, and in IgHVunmut CLL in IKZF3, SAMHD1,PAX5 and BIRC3. Finally, we show that IgHVunmut CLL is dominated by coding mutations in driver genes and an aging signature, whereas IgHVmut CLL has a high incidence of promoter and enhancer mutations caused by aberrant activation-induced cytidine deaminase activity. Taken together, our data support the hypothesis that differences in clinical outcome and biological characteristics between the two subgroups might reflect differences in mutation distribution, incidence and distinct underlying mutagenic mechanisms.
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Genes de Cadeia Pesada de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação/genética , Idoso , Idoso de 80 Anos ou mais , Citidina Desaminase/genética , Elementos Facilitadores Genéticos/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteína 1 com Domínio SAM e Domínio HD/genética , Sequenciamento Completo do Genoma/métodosRESUMO
The immunoglobulin heavy-chain variable region gene (IgHV) mutational status is considered the gold standard of prognostication in chronic lymphocytic leukemia (CLL) and is currently determined by Sanger sequencing that allows the analysis of the major clone. Using next-generation sequencing (NGS), we sequenced the IgHV gene from two independent cohorts: (A) 270 consecutive patient samples obtained at diagnosis and (B) 227 patients from the UK ARCTIC-AdMIRe clinical trials. Using complementary DNA from purified CD19+CD5+ cells, we demonstrate the presence of multiple rearrangements in independent experiments and showed that 24.4% of CLL patients express multiple productive clonally unrelated IgHV rearrangements. On the basis of IgHV-NGS subclonal profiles, we defined five different categories: patients with (a) multiple hypermutated (M) clones, (b) 1 M clone, (c) a mix of M-unmutated (UM) clones, (d) 1 UM clone and (e) multiple UM clones. In population A, IgHV-NGS classification stratified patients into five different subgroups with median treatment-free survival (TFS) of >280(a), 131(b), 94(c), 29(d), 15(e) months (P<0.0001) and a median OS of >397(a), 292(b), 196(c), 137(d) and 100(e) months (P<0.0001). In population B, the poor prognosis of multiple UM patients was confirmed with a median TFS of 2 months (P=0.0038). In conclusion, IgHV-NGS highlighted one quarter of CLL patients with multiple productive IgHV subclones and improves disease stratification and raises important questions concerning the pre-leukemic cellular origin of CLL.
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Genes de Cadeia Pesada de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Linfócitos B/metabolismo , Biomarcadores , Evolução Clonal/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Mutação , Fenótipo , Prognóstico , Modelos de Riscos ProporcionaisRESUMO
Although the tandem pore potassium channel TASK-3 is thought to open and shut at its selectivity filter in response to changes of extracellular pH, it is currently unknown whether the channel also shows gating at its inner, cytoplasmic mouth through movements of membrane helices M2 and M4. We used two electrode voltage clamp and single channel recording to show that TASK-3 responds to voltage in a way that reveals such gating. In wild-type channels, P(open) was very low at negative voltages, but increased with depolarisation. The effect of voltage was relatively weak and the gating charge small, 0.17. Mutants A237T (in M4) and N133A (in M2) increased P(open) at a given voltage, increasing mean open time and the number of openings per burst. In addition, the relationship between P(open) and voltage was shifted to less positive voltages. Mutation of putative hinge glycines (G117A, G231A), residues that are conserved throughout the tandem pore channel family, reduced P(open) at a given voltage, shifting the relationship with voltage to a more positive potential range. None of these mutants substantially affected the response of the channel to extracellular acidification. We have used the results from single channel recording to develop a simple kinetic model to show how gating occurs through two classes of conformation change, with two routes out of the open state, as expected if gating occurs both at the selectivity filter and at its cytoplasmic mouth.
