FRET studies of a landscape of Lac repressor-mediated DNA loops

DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-β-d-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility

Mosaic structural variation in children with developmental disorders

Delineating the genetic causes of developmental disorders is an area of active investigation. Mosaic structural abnormalities, defined as copy number or loss of heterozygosity events that are large and present in only a subset of cells, have been detected in 0.2–1.0% of children ascertained for clinical genetic testing. However, the frequency among healthy children in the community is not well characterized, which, if known, could inform better interpretation of the pathogenic burden of this mutational category in children with developmental disorders. In a case–control analysis, we compared the rate of large-scale mosaicism between 1303 children with developmental disorders and 5094 children lacking developmental disorders, using an analytical pipeline we developed, and identified a substantial enrichment in cases (odds ratio = 39.4, P-value 1.073e − 6). A meta-analysis that included frequency estimates among an additional 7000 children with congenital diseases yielded an even stronger statistical enrichment (P-value 1.784e − 11). In addition, to maximize the detection of low-clonality events in probands, we applied a trio-based mosaic detection algorithm, which detected two additional events in probands, including an individual with genome-wide suspected chimerism. In total, we detected 12 structural mosaic abnormalities among 1303 children (0.9%). Given the burden of mosaicism detected in cases, we suspected that many of the events detected in probands were pathogenic. Scrutiny of the genotypic–phenotypic relationship of each detected variant assessed that the majority of events are very likely pathogenic. This work quantifies the burden of structural mosaicism as a cause of developmental disorders

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy

Overall survival in IGP model subgroups.

<p>A. Overall survival by cytogenetics and mutational profiles. Among patients with intermediate cytogenetics, three-year overall survival was 59% for those with favorable mutational profiles (A), 33% for those with intermediate mutational profiles (B), 51% for those who were <i>FLT3-</i>ITD negative with high-risk mutations (C) and 11% for those who were <i>FLT3-</i>ITD positive with high-risk mutations (D). Three-year overall survival was 77% among patients with favorable cytogenetics (favorable) and 21% among patients with unfavorable cytogenetics (unfavorable). B. Overall survival among patients with favorable mutational profiles. The overall survival curve for patients with intermediate cytogenetics and mutant <i>NPM1</i> plus mutant <i>IDH1</i> or <i>IDH2</i> was similar to the survival curve for patients with favorable cytogenetics (adjusted p = 0.697) and different from the survival curve for patients in the intermediate IGP risk group (adjusted p = 0.028). C. Overall survival among patients with <i>FLT3-</i>ITD negative AML and high-risk mutations. The overall survival curve for patients with <i>FLT3</i>-ITD negative (<i>FLT3-</i>ITD-) AML and co-occurring high-risk mutations (<i>TET2</i>, <i>ASXL1</i> and/or <i>PHF6</i>) was not significantly different from the survival curves for patients with unfavorable cytogenetics or patients with intermediate IGP risk (adjusted p = 0.111 and p = 0.919, respectively). D. Overall survival among patients with <i>FLT3</i>-ITD positive AML and high-risk mutations. The overall survival curve for patients with <i>FLT3</i>-ITD positive (<i>FLT3</i>-ITD+) AML and co-occurring high-risk mutations (trisomy 8, <i>TET2</i> and/or <i>DNMT3A</i>) was similar to the survival curve for patients with unfavorable cytogenetics (adjusted p = 0.793) and different from the survival curve for patients in the intermediate IGP risk group (adjusted p = 0.022).</p