Browsing by Author "Murphy, Daniel"

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    Author Correction: CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity
    (Nature Publishing Group, 2018-04-13) Walton, Josephine B.; Farquharson, Malcolm; Mason, Susan; Port, Jennifer; Kruspig, Bjorn; Dowson, Suzanne; Stevenson, David; Murphy, Daniel; Matzuk, Martin; Kim, Jaeyeon; Coffelt, Seth; Blyth, Karen; McNeish, Iain A.; Biochemistry and Molecular Biology, School of Medicine
    A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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    CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity
    (Nature Publishing group, 2017-12-04) Walton, Josephine B.; Farquharson, Malcolm; Mason, Susan; Port, Jennifer; Kruspig, Bjorn; Dowson, Suzanne; Stevenson, David; Murphy, Daniel; Matzuk, Martin; Kim, Jaeyeon; Coffelt, Seth; Blyth, Karen; McNeish, Iain A.; Biochemistry and Molecular Biology, School of Medicine
    Transplantable murine models of ovarian high grade serous carcinoma (HGSC) remain an important research tool. We previously showed that ID8, a widely-used syngeneic model of ovarian cancer, lacked any of the frequent mutations in HGSC, and used CRISPR/Cas9 gene editing to generate derivatives with deletions in Trp53 and Brca2. Here we have used one ID8 Trp53 −/− clone to generate further mutants, with additional mutations in Brca1, Pten and Nf1, all of which are frequently mutated or deleted in HGSC. We have also generated clones with triple deletions in Trp53, Brca2 and Pten. We show that ID8 Trp53 −/−;Brca1 −/− and Trp53 −/−;Brca2 −/− cells have defective homologous recombination and increased sensitivity to both platinum and PARP inhibitor chemotherapy compared to Trp53 −/−. By contrast, loss of Pten or Nf1 increases growth rate in vivo, and reduces survival following cisplatin chemotherapy in vivo. Finally, we have also targeted Trp53 in cells isolated from a previous transgenic murine fallopian tube carcinoma model, and confirmed that loss of p53 expression in this second model accelerates intraperitoneal growth. Together, these CRISPR-generated models represent a new and simple tool to investigate the biology of HGSC, and the ID8 cell lines are freely available to researchers.
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    Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA
    (Elsevier, 2023) Xiao, Sihao; Kai, Zhentian; Murphy, Daniel; Li, Dongyang; Patel, Dilip; Bielowka, Adrianna M.; Bernabeu-Herrero, Maria E.; Abdulmogith, Awatif; Mumford, Andrew D.; Westbury, Sarah K.; Aldred, Micheala A.; Vargesson, Neil; Caulfield, Mark J.; Genomics England Research Consortium; Shovlin, Claire L.; Medicine, School of Medicine
    Despite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.
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    Repair of Thoracic and Thoracoabdominal Mycotic Aneurysms and Infected Aortic Grafts Using Allograft
    (Elsevier, 2018) Corvera, Joel S.; Blitzer, David; Copeland, Hannah; Murphy, Daniel; Hess, Philip J.; Pillai, Saila T.; Fehrenbacher, John W.; Surgery, School of Medicine
    Background Mycotic aneurysm of the thoracic or thoracoabdominal aorta and infection of thoracic or thoracoabdominal aortic grafts are challenging problems with high mortality. In-situ reconstruction with cryopreserved allograft(CPA) avoids placement of prosthetic material in an infected field and avoids suppressive antibiotics or autologous tissue coverage. Methods Fifty consecutive patients with infection of a thoracic or thoracoabdominal aortic graft or mycotic aneurysm underwent resection and replacement with CPA from 2006 to 2016. Intravenous antibiotics were continued postoperatively for 6 weeks. Long-term suppressive antibiotics were uncommonly used (8 patients). Follow up imaging occurred at 6, 18 and 42 months postoperatively. Initial follow up was 93% complete. Results Males comprised 64% of the cohort. The mean age was 63±14 years. The procedures performed included reoperations in 37, replacement of the aortic root, ascending aorta or transverse arch in 19, replacement of the descending or thoracoabdominal aorta in 27 and extensive replacement of the ascending, arch and descending or thoracoabdominal aorta in 4. Intraoperative cultures revealed most commonly staphylococcus 24%), enterococcus (12%), candida (6%) and gram negative rods (14%). Operative mortality was 8%, stroke 4%, paralysis 2%, hemodialysis 6%, and respiratory failure requiring tracheostomy 6%. Early reoperation for pseudoaneurysm of the CPA was necessary in 4 patients. One, two and five year survival was 84%, 76% and 64%, respectively. Conclusions Radical resection and in-situ reconstruction with CPA avoids placing prosthetic material in an infected field and provides good early and mid-term outcomes. However, early postoperative imaging is necessary given the risk of pseudoaneurysm formation.