Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons

10.1371/journal.pone.0046152PLoS ONE79

Recurrent Fusion Genes in Gastric Cancer: CLDN18-ARHGAP26 Induces Loss of Epithelial Integrity.

Genome rearrangements, a hallmark of cancer, can result in gene fusions with oncogenic properties. Using DNA paired-end-tag (DNA-PET) whole-genome sequencing, we analyzed 15 gastric cancers (GCs) from Southeast Asians. Rearrangements were enriched in open chromatin and shaped by chromatin structure. We identified seven rearrangement hot spots and 136 gene fusions. In three out of 100 GC cases, we found recurrent fusions between CLDN18, a tight junction gene, and ARHGAP26, a gene encoding a RHOA inhibitor. Epithelial cell lines expressing CLDN18-ARHGAP26 displayed a dramatic loss of epithelial phenotype and long protrusions indicative of epithelial-mesenchymal transition (EMT). Fusion-positive cell lines showed impaired barrier properties, reduced cell-cell and cell-extracellular matrix adhesion, retarded wound healing, and inhibition of RHOA. Gain of invasion was seen in cancer cell lines expressing the fusion. Thus, CLDN18-ARHGAP26 mediates epithelial disintegration, possibly leading to stomach H(+) leakage, and the fusion might contribute to invasiveness once a cell is transformed. Cell Rep 2015 Jul 14; 12(2):272-285

Observations that suggest a contribution of altered dermal papilla mitochondrial function to androgenetic alopecia

Androgenetic alopecia (AGA) is a prevalent hair loss condition in males that develops due to the influence of androgens and genetic predisposition. With the aim of elucidating genes involved in AGA pathogenesis, we modelled AGA with three-dimensional culture of keratinocyte-surrounded dermal papilla (DP) cells. We co-cultured immortalised balding and non-balding human DP cells (DPCs) derived from male AGA patients with epidermal keratinocyte (NHEK) using multi-interfacial polyelectrolyte complexation technique. We observed up-regulated mitochondria-related gene expression in balding compared with non-balding DP aggregates which indicated altered mitochondria metabolism. Further observation of significantly reduced electron transport chain complex activity (complexes I, IV and V), ATP levels and ability to uptake metabolites for ATP generation demonstrated compromised mitochondria function in balding DPC. Balding DP was also found to be under significantly higher oxidative stress than non-balding DP. Our experiments suggest that application of antioxidants lowers oxidative stress levels and improves metabolite uptake in balding DPC. We postulate that the observed up-regulation of mitochondria-related genes in balding DP aggregates resulted from an over-compensatory effort to rescue decreased mitochondrial function in balding DP through the attempted production of new functional mitochondria. In all, our three-dimensional co-culturing revealed mitochondrial dysfunction in balding DPC, suggesting a metabolic component in the aetiology of AGA

Comprehensive long-span paired-end-tag mapping reveals characteristic patterns of structural variations in epithelial cancer genomes

Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long-span paired-end-tag (PET) sequencing approach using 10-Kb genomic DNA inserts to study human genome structural variations (SVs). The use of a 10-Kb insert size allows the identification of breakpoints within repetitive or homology-containing regions of a few kilobases in size and results in a higher physical coverage compared with small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and two noncancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germ-line SVs, whereas tandem duplications, unpaired inversions, interchromosomal translocations, and complex rearrangements are over-represented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA–PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures that are compatible with breakage-fusion-bridge cycles, and we discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers

SV identification based on the mapping pattern of dPET clusters.

<p>The dark red and pink arrows represent the 5′ and 3′ anchor regions of the dPET cluster, respectively. Black, white and blue horizontal lines represent chromosome segments. The red track represents the coverage of cPETs. The dotted lines indicate the connections between the two dPET clusters. The sub-types of insertions are as follows: (1) Intra-chromosomal direct forward insertion. (2) Intra-chromosomal direct backward insertion. (3) Intra-chromosomal inverted forward insertion. (4) Intra-chromosomal inverted backward insertion. (5) Deletion plus intra-chromosomal direct forward insertion. (6) Deletion plus intra-chromosomal inverted forward insertion. (7) Inter-chromosomal direct insertion. (8) Inter chromosome inverted insertion.</p

DNA-PET library construction, sequencing and mapping.

<p>(A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, <i>EcoP15I</i> digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.</p

Reconstruction of the <i>BCR-ABL1</i> amplicon of K562.

<p>(A) Concordant tag distributions representing copy number are shown for amplified genomic regions (top, green track). Genomic segments between predicted breakpoints are indicated by colored arrows and dPET clusters with cluster sizes greater than 35 of predicted somatic rearrangements are represented by horizontal lines flanked by dark red and pink arrows indicating 5′ and 3′ anchor regions (middle). Small to large dPET clusters are arranged from top to bottom. Cluster sizes are indicated. High dPET cluster size of the CML causing <i>BCR-ABL1</i> translocation suggests that the rearrangement occurred early and that it has subsequently been amplified. Fusion points I–III correspond to panels C–D. (B) Fluorescence <i>in situ</i> hybridization (FISH) of <i>BCR-ABL1</i> rearrangement (fusion point I with cluster size 692). Yellow spots represent fusion signals and illustrate the amplification of <i>BCR-ABL1</i>. (C) FISH analysis of metaphase chromosomes of three high copy fusion points: I) probes used in B show fusion signals on two marker chromosomes and on chromosome 2q and normal localization on both rearranged chromosomes 9 and normal chromosome 22; the fusion on chromosome 2 has not been identified by DNA-PET most likely due to low sequence complexity at the break point or complex rearrangements, II) probes spanning the fusion point II (cluster size 259) show fusion signals on the same marker chromosomes and normal localization on both normal and rearranged chromosomes 9 and 13, III) probes spanning fusion point III (cluster size 218) show fusion signals on the same marker chromosomes and normal localization on both normal chromosome 22 and rearranged chromosomes 9. (D) Contigs (indicated by boxes) which were covered by PET mapping were concatenated by fusion-point-guided-concatenation method. The length of a contig is represented by the length of the box. Because of the size difference between chromosomes 1, 3, 9, 13, and 22, the length of chromosome 22 is represented by the length of contig/10,000 while the lengths of chromosomes 1, 3, 9, and 13 are represented by the length of contig/100,000. Any value less than 0.1 is rounded to 0.1; any value larger than 6 is rounded to 6. The thickness of borders of each contig represents the coverage (copy number). Red dashed edges represent dPET edges, while black bold edges represent cPET edges. The thickness of dPET edges represents the size of the corresponding dPET cluster. cPET edges have uniform thickness. Arrow heads pointing towards a contig indicate connections with the lower coordinates, arrow heads pointing away from a contig indicate connections with the higher coordinates.</p

Statistics of PET sequencing.

1)<p>non-redundant.</p>2)<p>physical coverage.</p>3)<p>concordant PET.</p>4)<p>proportion of cPET to total NR PET.</p>5)<p>discordant PET.</p>6)<p>proportion of dPET to total NR PET.</p>7)<p>number of dPET involved in the dPET clusters with cluster count ≥3.</p

Evaluation of prognostic risk models for postoperative pulmonary complications in adult patients undergoing major abdominal surgery: a systematic review and international external validation cohort study

Background Stratifying risk of postoperative pulmonary complications after major abdominal surgery allows clinicians to modify risk through targeted interventions and enhanced monitoring. In this study, we aimed to identify and validate prognostic models against a new consensus definition of postoperative pulmonary complications. Methods We did a systematic review and international external validation cohort study. The systematic review was done in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We searched MEDLINE and Embase on March 1, 2020, for articles published in English that reported on risk prediction models for postoperative pulmonary complications following abdominal surgery. External validation of existing models was done within a prospective international cohort study of adult patients (≥18 years) undergoing major abdominal surgery. Data were collected between Jan 1, 2019, and April 30, 2019, in the UK, Ireland, and Australia. Discriminative ability and prognostic accuracy summary statistics were compared between models for the 30-day postoperative pulmonary complication rate as defined by the Standardised Endpoints in Perioperative Medicine Core Outcome Measures in Perioperative and Anaesthetic Care (StEP-COMPAC). Model performance was compared using the area under the receiver operating characteristic curve (AUROCC). Findings In total, we identified 2903 records from our literature search; of which, 2514 (86·6%) unique records were screened, 121 (4·8%) of 2514 full texts were assessed for eligibility, and 29 unique prognostic models were identified. Nine (31·0%) of 29 models had score development reported only, 19 (65·5%) had undergone internal validation, and only four (13·8%) had been externally validated. Data to validate six eligible models were collected in the international external validation cohort study. Data from 11 591 patients were available, with an overall postoperative pulmonary complication rate of 7·8% (n=903). None of the six models showed good discrimination (defined as AUROCC ≥0·70) for identifying postoperative pulmonary complications, with the Assess Respiratory Risk in Surgical Patients in Catalonia score showing the best discrimination (AUROCC 0·700 [95% CI 0·683–0·717]). Interpretation In the pre-COVID-19 pandemic data, variability in the risk of pulmonary complications (StEP-COMPAC definition) following major abdominal surgery was poorly described by existing prognostication tools. To improve surgical safety during the COVID-19 pandemic recovery and beyond, novel risk stratification tools are required. Funding British Journal of Surgery Society