To describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel ...sequencing failed to yield a genetic diagnosis, and to use remnant normal
splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia.
Exome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for
premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle.
Splice-altering intronic single nucleotide variants or structural rearrangements in
were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced
mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%-5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness.
Whole-genome sequencing relied heavily on RNA studies to identify
splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant
mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.
Spontaneous regression is a recognized phenomenon in chronic lymphocytic leukemia (CLL) but its biological basis remains unknown. We undertook a detailed investigation of the biological and clinical ...features of 20 spontaneous CLL regression cases incorporating phenotypic, functional, transcriptomic, and genomic studies at sequential time points. All spontaneously regressed tumors were IGHV-mutated with no restricted IGHV usage or B-cell receptor (BCR) stereotypy. They exhibited shortened telomeres similar to nonregressing CLL, indicating prior proliferation. They also displayed low Ki-67, CD49d, cell-surface immunoglobulin M (IgM) expression and IgM-signaling response but high CXCR4 expression, indicating low proliferative activity associated with poor migration to proliferation centers, with these features becoming increasingly marked during regression. Spontaneously regressed CLL displayed a transcriptome profile characterized by downregulation of metabolic processes as well as MYC and its downstream targets compared with nonregressing CLL. Moreover, spontaneous regression was associated with reversal of T-cell exhaustion features including reduced programmed cell death 1 expression and increased T-cell proliferation. Interestingly, archetypal CLL genomic aberrations including HIST1H1B and TP53 mutations and del(13q14) were found in some spontaneously regressing tumors, but genetic composition remained stable during regression. Conversely, a single case of CLL relapse following spontaneous regression was associated with increased BCR signaling, CLL proliferation, and clonal evolution. These observations indicate that spontaneously regressing CLL appear to undergo a period of proliferation before entering a more quiescent state, and that a complex interaction between genomic alterations and the microenvironment determines disease course. Together, the findings provide novel insight into the biological processes underpinning spontaneous CLL regression, with implications for CLL treatment.
•Spontaneously regressed tumors are composed of a formerly proliferating CLL clone that has transitioned into a quiescent state.•A microenvironmental stimulation change on an indolent genomic background state underpins clonal attrition in spontaneous CLL regression.
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We present the second Swift Ultra-Violet/Optical Telescope (UVOT) gamma-ray burst (GRB) afterglow catalog, greatly expanding on the first Swift UVOT GRB afterglow catalog. The second catalog is ...constructed from a database containing over 120,000 independent UVOT observations of 538 GRBs first detected by Swift, the High Energy Transient Explorer 2 (HETE2), the INTErnational Gamma-Ray Astrophysics Laboratory (INTEGRAL), the Interplanetary Network (IPN), Fermi, and Astro-rivelatore Gamma a Immagini Leggero (AGILE). The catalog covers GRBs discovered from 2005 Jan 17 to 2010 Dec 25. Using photometric information in three UV bands, three optical bands, and a `white' or open filter, the data are optimally co-added to maximize the number of detections and normalized to one band to provide a detailed light curve. The catalog provides positional, temporal, and photometric information for each burst, as well as Swift Burst Alert Telescope (BAT) and X-Ray Telescope (XRT) GRB parameters. Temporal slopes are provided for each UVOT filter. The temporal slope per filter of almost half the GRBs are fit with a single power-law, but one to three breaks are required in the remaining bursts. Morphological comparisons with the X-ray reveal that approximately 75% of the UVOT light curves are similar to one of the four morphologies identified by Evans et al. (2009). The remaining approximately 25% have a newly identified morphology. For many bursts, redshift and extinction corrected UV/optical spectral slopes are also provided at 2000, 20,000, and 200,000 seconds.
Binary neutron-star mergers (BNSMs) are among the most readily detectable gravitational-wave (GW) sources with LIGO. They are also thought to produce short \(\gamma\)-ray bursts (SGRBs), and ...kilonovae that are powered by r-process nuclei. Detecting these phenomena simultaneously would provide an unprecedented view of the physics during and after the merger of two compact objects. Such a Rosetta Stone event was detected by LIGO/Virgo on 17 August 2017 at a distance of \(\sim 44\) Mpc. We monitored the position of the BNSM with ALMA at 338.5 GHz and GMRT at 1.4 GHz, from 1.4 to 44 days after the merger. Our observations rule out any afterglow more luminous than \(3\times 10^{26}~{\rm erg\,s}^{-1}\,{\rm Hz}^{-1}\) in these bands, probing \(>\)2--4 dex fainter than previous SGRB limits. We match these limits, in conjunction with public data announcing the appearance of X-ray and radio emission in the weeks after the GW event, to templates of off-axis afterglows. Our broadband modeling suggests that GW170817 was accompanied by a SGRB and that the GRB jet, powered by \(E_{\rm AG,\,iso}\sim10^{50}\)~erg, had a half-opening angle of \(\sim20^\circ\), and was misaligned by \(\sim41^\circ\) from our line of sight. The data are also consistent with a more collimated jet: \(E_{\rm AG,\,iso}\sim10^{51}\)~erg, \(\theta_{1/2,\,\rm jet}\sim5^\circ\), \(\theta_{\rm obs}\sim17^\circ\). This is the most conclusive detection of an off-axis GRB afterglow and the first associated with a BNSM-GW event to date. Assuming a uniform top-hat jet, we use the viewing angle estimates to infer the initial bulk Lorentz factor and true energy release of the burst.
Background
Chemo-immunotherapy (CIT) with fludarabine, cyclophosphamide, and rituximab (FCR) is the standard of care in frontline treatment of CLL. With this approach, 25% of patients relapse within ...24 months, whereas approximately one third of patients with hypermutated immunoglobulin heavy chains (IgHV) achieve a functional cure (Hallek et al. Lancet. 2010; Tam et al. Blood, 2014, Fischer et al, Blood 2015; Philip A. Thompson et al. Blood 2016). So far, mutations and/or deletions of TP53 remain the only predictive marker screened for in routine clinical practice, accounting for only one third of patients relapsing early after CIT. Recent next-generation sequencing (NGS) studies have revealed novel candidate predictors of early relapse including somatic mutations in RPS15 (Landau et al. Nature, 2015) and SAMHD1 (Clifford et al., submitted). Taken together with TP53disruption, these only occur in a subset of high-risk patients. Here, we present a comprehensive analysis of high-risk patients using Whole Genome Sequencing (WGS).
Patients and Methods
Using WGS we investigated 149 CLL patients from 5 national UK clinical trials: CLEAR (n=8), RIAltO (n=45), CLL 210 (n=22), ARCTIC (n=32) and AdMIRe (n=42). The two first line FCR-based clinical trials (ARCTIC and AdMIRe) were studied in most detail: 56 patients relapsed within 24 months; this group of patients will be referred to as high risk patients. Leukemia samples (peripheral blood) and germline samples (saliva) were collected for each patient. We performed WGS on the HiSeqX (Illumina). After read alignment, we detected somatic variants using Strelka 2.4.7 for small variants detection (SNV and InDels), Manta 0.28.0 for Structural variant (SV) detection, and Canvas 1.3.1 for Copy number variant (CNV) detection (Illumina). Non-coding regions were annotated with information from primary CLL, CLL cell lines and B-cell ENCODE databases. We interrogated the data at a gene scale and global level in order to identify patterns of early relapsing patients. Operative mutational signatures were analysed according to Alexandrov et al. (Nature, 2013). Putative regions of kataegis were calculated based on Lawrence et al. (Nature, 2013) and Alexandrov et al. (Nature, 2013).
Results
The mean coverage for CLL tumour and germline samples was 105.2X and 33.7X, respectively. The analysis of the whole cohort highlighted 1,723,603 somatic SNVs (mean= 11,570/sample) and 555,179 InDels (mean= 3,726/sample). Somatic SNVs spectrum consisted mainly of C>T/G>A mutations (30% of total SNVs reported) as previously described. The analysis of 13,490 somatic functional SNVs and InDels revealed novel candidate genes as most commonly mutated in the cohort. In high-risk patients, we noticed an enrichment of mutations in known genes such as TP53, genes of the NF-κB pathway and novel candidate genes previously reported in other cancers. A specific analysis of the functional coding mutations of known CLL driver genes revealed ATM, SF3B1 and IGLL5 as most commonly mutated genes in FCR responders compared to TP53, RPS15 and EGR2 in high risk patients. In depth analysis of somatic non-coding regions also identified potential new candidate regions associated with early relapse. Next, we investigated 52,871 CNAs (mean= 380/sample) and 29,080 SVs (mean= 195/sample) and identified as expected del13q, del17p, del11q and tri12 as the most frequent aberrations. In addition, we identified SVs across genes of interest in CLL, for instance TP53, ATM and BIRC3. Finally, we performed global genome analyses with investigation of mutational signatures and kataegis analyses highlighting hypermutated candidate regions, including the previously described IGLL5gene.
Conclusion
Here we present initial analysis of WGS data on 149 CLL patients from 5 UK clinical trials. Different patterns of mutations between low and high risk clinical groups are suggested. More detailed analysis with greater numbers of samples is ongoing and will determine the true clinical significance of these preliminary findings. The possibility of using WGS to aid clinical decision-making is becoming a realistic goal.
Becq:Illumina: Employment. He:Illumina: Employment. Pettitt:Celgene: Speakers Bureau; Gilead: Research Funding, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Infinity: Research Funding. Hillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding. Bentley:Illumina: Employment. Schuh:Gilead: Consultancy, Honoraria, Research Funding; Roche, Janssen, Novartis, Celgene, Abbvie: Consultancy, Honoraria.
Background:Historically, the identification of minimal deleted regions (MDRs) has been a useful approach for pinpointing genes involved in the pathogenesis of human malignancies and constitutional ...disorders. Microarray technology has offered increased capability for newly identifying or refining existing MDRs and minimal overlapping regions (MORs) in cancer. Despite this, in chronic lymphocytic leukemia (CLL), published MORs that pinpoint only a few candidate genes have been limited and with the advent of NGS, the utility of high resolution array work as a discovery tool has become uncertain. Here, we show that profiling copy number abnormalities (CNAs) and cnLOH using arrays in a large patient series can still be a valuable approach for the identification of genes that are disrupted or mutated in CLL and have a role in CLL development and/or progression.
Methods: 250 CLL patient DNAs from individuals enrolled in two UK-based Phase II randomised controlled trials (AdMIRe and ARCTIC trials) were tested using Infinium HumanOmni2.5-8 v1.1 according to manufacturer’s guidelines (Illumina Inc, San Diego, CA). Data were processed using GenomeStudioV2009.2 (Illumina Inc.) and analysed using Nexus Discovery Edition v6.1 (BioDiscovery, Hawthorne, CA). All Nexus plots were inspected visually to verify calls made, identify uncalled events and exclude likely false positives. To exclude common germline CNVs, the Database of Genomic Variants (DGV), a comprehensive catalog of structural variation in control data, was used. Copy number (CN) changes that encompassed fully changes noted in the DGV were excluded from further analysis. Regions of copy neutral loss of heterozygosity (cnLOH) were recorded if >1Mb in size, but were not used to define or refine MORs. Data from 1275 age-appropriate control samples minimised the reporting of common cnLOH events. All genomic coordinates were noted with reference to the GRCh37, hg19 assembly. MORs were investigated using Microsoft Excel filtering functions. A subset of genes (n=91) selected from MORs mainly on the basis of event frequency and/or number of genes within the MOR and/or literature interest were taken forward for targeted sequencing (exons only) of appropriate samples with/without CN Losses or cnLOH (Set 1 n=124; Set 2 n=126). These were tested using custom designed TruSeq Custom Amplicon panels (Illumina Inc) and processed according to manufacturer’s instructions. SAMHD1 was excluded from these panels since it had been studied separately within our laboratory. The data were analysed using an in-house bioinformatics pipeline that uses the sequence aligners MSR and Stampy and the variant callers GATK and Platypus, followed by stringent filtering.
Results: Using our datasets we have identified >50 MORs previously unreported in the literature. Six of these showed copy number (CN) losses in >3% of patients studied. Furthermore, we have refined 14 MORs that overlapped with regions described previously and that had also a CN loss frequency of >3%. Thirteen MORs involved only a single reference gene, often a gene implicated previously in cancer (eg. SAMHD1, MTSS1, DCC and RFC1). Of the 91 genes taken forward for targeted sequencing, stringent data filtering led to a subset of 19 genes of interest harbouring exonic mutations. Genes with mutations identified include DCC, BAP1 and FBXW7, also implicated previously in cancer.
Conclusion: We have generated high resolution CNA and cnLOH profiles for 250 first-line chemo-immunotherapy treated CLL patients and used this information to document newly identified MORs, to refine MORs reported previously and to identify mutation harbouring genes using targeted NGS. Functional knowledge supports our hypothesis that these genes may have a contributory role in CLL. For two genes, SAMHD1 and FBXW7, relevance in CLL has been established already. Taken together, our data validate the utility of high resolution arrays studies for the identification of candidate genes that may be involved in CLL development or progression when disrupted. Further studies are required to confirm a role for these genes in CLL and to elucidate the nature of the underlying biological mechanisms.
No relevant conflicts of interest to declare.
We report on a large stellar flare from the nearby dMe flare star EV Lac observed by the Swift and Konus-Wind satellites and the Liverpool Telescope. It is the first large stellar flare from a dMe ...flare star to result in a Swift trigger based on its hard X-ray intensity. Its peak f_X from 0.3--100 keV of 5.3x10^-8 erg/cm2/s is nearly 7000 times larger than the star's quiescent coronal flux, and the change in magnitude in the white filter is >4.7. This flare also caused a transient increase in EV Lac's bolometric luminosity (L_bol) during the early stages of the flare, with a peak estimated L_X/L_bol ~3.1. We apply flare loop hydrodynamic modeling to the plasma parameter temporal changes to derive a loop semi-length of l/Rstar =0.37 +/-0.07. The soft X-ray spectrum of the flare reveals evidence of iron Kalpha emission at 6.4 keV. We model the Kalpha emission as fluorescence from the hot flare source irradiating the photospheric iron, and derive loop heights of h/Rstar=0.1, consistent within factors of a few with the heights inferred from hydrodynamic modeling. The Kalpha emission feature shows variability on time scales of ~200 s which is difficult to interpret using the pure fluorescence hypothesis. We examine Kalpha emission produced by collisional ionization from accelerated particles, and find parameter values for the spectrum of accelerated particles which can accommodate the increased amount of Kalpha flux and the lack of observed nonthermal emission in the 20-50 keV spectral region.
The long and relatively faint gamma-ray burst GRB 060605 detected by \emph{Swift}/BAT lasted about 20 sec. Its afterglow could be observed with \emph{Swift}/XRT for nearly 1 day, while ...\emph{Swift}/UVOT could detect the afterglow during the first 6 hours after the event. Here, we report on integral field spectroscopy of its afterglow performed with PMAS/PPak mounted at the Calar Alto 3.5 m telescope. In addition, we report on a detailed analysis of XRT and UVOT data and on the results of deep late-time VLT observations that reveal the GRB host galaxy. We find that the burst occurred at a redshift of \(z\)=3.773, possibly associated with a faint, \(R_C=26.4 \pm 0.3\) host. Based on the optical and X-ray data, we deduce information on the SED of the afterglow, the position of the cooling frequency in the SED, the nature of the circumburst environment, its collimation factor, and its energetics. We find that the GRB fireball was expanding into a constant-density medium and that the explosion was collimated with a narrow half-opening angle of about 2.4 degrees. The initial Lorentz factor of the fireball was about 250; however, its beaming-corrected energy release in the gamma-ray band was comparably low. The optical, X-ray afterglow, on the other hand, was rather luminous. Finally, we find that the data are consistent within the error bars with an achromatic evolution of the afterglow during the suspected jet break time at about 0.27 days after the burst.
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