Using Massively Parallel Sequencing to Determine the Genetic Basis of Leigh Syndrome, the Most Common Mitochondrial Disorder Affecting Children

Total Page:16

File Type:pdf, Size:1020Kb

Using Massively Parallel Sequencing to Determine the Genetic Basis of Leigh Syndrome, the Most Common Mitochondrial Disorder Affecting Children Using Massively Parallel Sequencing to determine the genetic basis of Leigh Syndrome, the most common mitochondrial disorder affecting children Nicole Janet Lake ORCID ID 0000-0003-4103-6387 Doctor of Philosophy January 2018 Department of Paediatrics Faculty of Medicine, Dentistry and Health Sciences University of Melbourne Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy Abstract Mitochondrial diseases are debilitating illnesses caused by mutations that impair mitochondrial energy generation. The most common clinical presentation of mitochondrial disease in children is Leigh syndrome. This neurodegenerative disorder can be caused by mutations in more than 85 genes, encoded by both nuclear and mitochondrial DNA (mtDNA). When this PhD commenced, massively parallel sequencing for genetic diagnosis of Leigh syndrome was transitioning into the clinic, however its diagnostic utility in a clinical setting was unknown. Furthermore, a significant number of Leigh syndrome patients remained without a genetic diagnosis, indicating that further research was required to expand our understanding of the genetic basis of disease. To identify the maximum diagnostic yield of massively parallel sequencing in patients with Leigh syndrome, and to provide insight into the genetic basis of disease, unsolved patients from a historical Leigh syndrome cohort were studied. This cohort is comprised of 67 clinically- ascertained patients diagnosed with Leigh or Leigh-like syndrome according to stringent criteria. DNA from all 33 patients lacking a genetic diagnosis underwent whole exome sequencing, with parallel sequencing of the mtDNA. A targeted analysis of 2273 genes was performed, which included known and candidate mitochondrial disease genes, and differential diagnosis genes underlying distinct disorders with phenotypic overlap. This study provided a genetic diagnosis for 11 of the 12 unsolved Leigh syndrome patients and 7 of the 21 unsolved Leigh-like patients investigated. Eight reported and eight novel pathogenic variants were identified in twelve disease genes; ALDH18A1, MT-ATP6, MT-ND3, MT-ND5, MT-ND6, MTFMT, NARS2, SCO2, SERAC1, SLC19A3, PDHA1, and PDHX. A genetic diagnosis has now been established in 78% of the total cohort, including in 34 of 35 Leigh syndrome patients and 18 of 32 Leigh-like patients. Candidate genetic diagnoses, including in differential diagnosis genes, were identified in an additional 7 Leigh-like patients, where investigation of synonymous variants, complex CNVs and heterozygous X-linked variants highlight challenges associated with variant follow-up in a diagnostic context. This thesis also describes the study of an additional patient with a firm diagnosis of Leigh syndrome. Whole exome sequencing of this patient with targeted analysis identified a homozygous splice site mutation in MRPS34, which was shown to cause abnormal splicing and loss of wild-type MRPS34 protein. MRPS34 encodes a mitochondrial ribosomal protein, and variants in this gene had not previously been described to cause disease. The data presented in this thesis demonstrate that mutation of MRPS34 causes a disorder of mitochondrial energy generation by destabilising the small mitochondrial ribosomal subunit, and therefore reducing the synthesis of mtDNA-encoded proteins. The rescue of cellular defects by lentiviral-mediated expression of wild-type MRPS34 establishes it as a bona fide disease gene. In conclusion, this thesis describes the identification of novel mutations and a new disease gene, thereby expanding our understanding of the genetic basis of Leigh syndrome. The characterisation of the aetiological basis of disease in the cohort, as well as evaluation of the outcomes of massively parallel sequencing of cohort patients, provides key insight into the diagnostic utility of this testing approach in a clinical setting. i Declaration This is to certify that: (i) The thesis comprises my original work towards the PhD, except where indicated in the Preface, (ii) Due acknowledgement has been made in the text to all materials used, (iii) The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. ii Preface (i) Several colleagues and collaborators have contributed to the research described in this thesis. Except where indicated below, the thesis is my original work towards this PhD. The following contributions were made to the content of Chapters 3, 4 and 5: Patients were referred to the Mitochondrial Diagnostic Service, part of the Mitochondrial Research Laboratory at the Murdoch Children’s Research Institute and Victorian Clinical Genetics Service in Melbourne, Australia, and diagnostic enzymology assays were performed by staff members. Simone Tregoning assisted with collation of enzymology results. Massively parallel sequencing was performed at the Broad Institute in Boston, USA in collaboration with Prof. Vamsi Mootha and Dr Sarah Calvo. Variant call files were generated from the sequencing data by Dr Sarah Calvo. The following contributions were made to the content of Chapter 3: Sections 3.2.1.4, 3.2.1.8, and 3.2.3: The SNP array was performed on DNA from LS7, LS18 and LS20a by the Victorian Clinical Genetic Service, and genotype data from LS7 and LS18 was analysed by staff member Ralph Oertel. Section 3.2.3: Comparative genomic hybridisation arrays were performed previously on DNA from LS20a and LS20b in the Mitochondrial Research Laboratory by Drs Alison Compton, Hayley Mountford and Elena Tucker. The following contributions were made to the content of Chapter 4: Section 4.2.1.3: Blue-Native (BN)-PAGE and SDS-PAGE immunoblots examining complex I in fibroblasts from LL45, and complementation studies in HEK293T cells, were performed at Monash University in Melbourne by Dr Luke Formosa. Data for Figure 4-6(a-c) were generated solely by Dr Luke Formosa. Section 4.2.2.1: The SNP array on DNA from LL43a and LL43b was performed by the Victorian Clinical Genetic Service, and genotype data was analysed by staff member Ralph Oertel. Section 4.2.2.2: Sanger sequencing of the DOK7 mutations in genomic DNA from DT110 was performed in the Mitochondrial Research Laboratory by Shalini Thirukeswaran. Section 4.2.2.5: The mitochondrial translation assay for LL42 was performed at Monash University with the assistance of Dr David Stroud and Elliot Surgenor. Section 4.2.3: The sequencing data from LL40 was analysed by Shalini Thirukeswaran. The following contributions were made to the content of Chapter 5: Section 5.1.1.1: The clinical summary was provided by Prof. John Christodoulou. Section 5.2.1: Analysis of the sequencing data, and Sanger sequencing of the mutation in genomic DNA, was performed by Dr Hayley Mountford. Sections 5.2.3 and 5.2.4: BN-PAGE immunoblotting and the mitochondrial translation assay was performed with the assistance of Dr David Stroud and Elliot Surgenor. Sections 5.2.3 and 5.2.6: Quantitative proteomics was performed by Dr David Stroud. Figure 5-5a, Figure 5-10a and Figure 5-10c were generated with the assistance of Dr David Stroud. iii Section 5.2.5: Lentiviral-mediated correction of cell lines was performed by Dr Alison Compton. Section 5.2.6: Sucrose gradient centrifugation and SDS-PAGE immunoblots examining the mitoribosome were performed at the Henry Perkins Institute in Perth, Australia by Dr Tara Richman. (ii) Several colleagues and collaborators have contributed to publications included in this thesis. The following contributions were made to the content of the article “Leigh syndrome: One disease, more than 75 monogenic causes” which is included in Chapter 1: Professor David Thorburn, Dr Alison Compton and Professor Shamima Rahman contributed to reviewing publications on the genetic, clinical, and pathological features of Leigh syndrome, and to the drafting and review of the manuscript. (iii) The publication status of all chapters presented (in part) in article format are as follows: Chapter 1 includes the article “Leigh syndrome: One disorder, more than 75 monogenic causes”. This was published by Annals of Neurology on 15th December 2015. (iv) Acknowledgement of all sources of funding, including grant identification numbers where applicable: The following organisations provided funding to support this PhD project: the University of Melbourne (Australian Postgraduate Award, Ackman Travelling Scholarship, Department of Paediatrics Travelling Scholarship), the National Health and Medical Research Council (Project Grant GNT 1068409 to D Thorburn, A Compton, D Bruno and M Ryan), the Australian Mitochondrial Disease Foundation (PhD Top-up and Travel Scholarship), the Australian National University (Gowrie-Patrick Hore-Ruthven Memorial Scholarship), the Murdoch Children’s Research Institute (Student Conference Scheme Scholarship), and the Australasian Society for Inborn Errors of Metabolism (Travel Grant). iv Acknowledgments Firstly, I thank my primary supervisor Prof. David Thorburn. It’s a unique opportunity to learn from someone who is an international leader of their field, and I am grateful for the opportunity to do a PhD in his lab. I have greatly appreciated the various opportunities he provided, research and beyond, supporting presentations at local and international conferences, committee involvement, media engagement and peer-review of articles. These activities helped me to mature as an early-career researcher. Thank you for your support and mentorship throughout this PhD. I am also very thankful to my co-supervisor
Recommended publications
  • Identification of the Binding Partners for Hspb2 and Cryab Reveals
    Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity.
    [Show full text]
  • Parallel Molecular Evolution in Pathways, Genes, and Sites in High-Elevation Hummingbirds Revealed by Comparative Transcriptomics
    GBE Parallel Molecular Evolution in Pathways, Genes, and Sites in High-Elevation Hummingbirds Revealed by Comparative Transcriptomics Marisa C.W. Lim1,*, Christopher C. Witt2, Catherine H. Graham1,3,andLilianaM.Davalos 1,4 1Department of Ecology and Evolution, Stony Brook University 2 Museum of Southwestern Biology and Department of Biology, University of New Mexico Downloaded from https://academic.oup.com/gbe/article-abstract/11/6/1552/5494706 by guest on 08 June 2019 3Swiss Federal Research Institute (WSL), Birmensdorf, Switzerland 4Consortium for Inter-Disciplinary Environmental Research, Stony Brook University *Corresponding author: E-mail: [email protected]. Accepted: May 12, 2019 Data deposition: The raw read data have been deposited in the NCBI Sequence Read Archive under BioProject: PRJNA543673, BioSample: SAMN11774663-SAMN11774674, SRA Study: SRP198856. All scripts used for analyses are available on Dryad: doi:10.5061/dryad.v961mb4. Abstract High-elevation organisms experience shared environmental challenges that include low oxygen availability, cold temperatures, and intense ultraviolet radiation. Consequently, repeated evolution of the same genetic mechanisms may occur across high-elevation taxa. To test this prediction, we investigated the extent to which the same biochemical pathways, genes, or sites were subject to parallel molecular evolution for 12 Andean hummingbird species (family: Trochilidae) representing several independent transitions to high elevation across the phylogeny. Across high-elevation species, we discovered parallel evolution for several pathways and genes with evidence of positive selection. In particular, positively selected genes were frequently part of cellular respiration, metabolism, or cell death pathways. To further examine the role of elevation in our analyses, we compared results for low- and high-elevation species and tested different thresholds for defining elevation categories.
    [Show full text]
  • Genome-Wide Identification and Analysis of Prognostic Features in Human Cancers
    bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446243; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Genome-wide identification and analysis of prognostic features in human cancers Joan C. Smith1,2 and Jason M. Sheltzer1* 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724. 2. Google, Inc., New York, NY 10011. * Lead contact; to whom correspondence may be addressed. E-mail: [email protected]. bioRxiv preprint doi: https://doi.org/10.1101/2021.06.01.446243; this version posted June 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Abstract Clinical decisions in cancer rely on precisely assessing patient risk. To improve our ability to accurately identify the most aggressive malignancies, we constructed genome-wide survival models using gene expression, copy number, methylation, and mutation data from 10,884 patients with known clinical outcomes. We identified more than 100,000 significant prognostic biomarkers and demonstrate that these genomic features can predict patient outcomes in clinically-ambiguous situations. While adverse biomarkers are commonly believed to represent cancer driver genes and promising therapeutic targets, we show that cancer features associated with shorter survival times are not enriched for either oncogenes or for successful drug targets.
    [Show full text]
  • PDF Datasheet
    Product Datasheet Cactin Overexpression Lysate NBP2-06542 Unit Size: 0.1 mg Store at -80C. Avoid freeze-thaw cycles. Protocols, Publications, Related Products, Reviews, Research Tools and Images at: www.novusbio.com/NBP2-06542 Updated 3/17/2020 v.20.1 Earn rewards for product reviews and publications. Submit a publication at www.novusbio.com/publications Submit a review at www.novusbio.com/reviews/destination/NBP2-06542 Page 1 of 2 v.20.1 Updated 3/17/2020 NBP2-06542 Cactin Overexpression Lysate Product Information Unit Size 0.1 mg Concentration The exact concentration of the protein of interest cannot be determined for overexpression lysates. Please contact technical support for more information. Storage Store at -80C. Avoid freeze-thaw cycles. Buffer RIPA buffer Target Molecular Weight 88.5 kDa Product Description Description Transient overexpression lysate of chromosome 19 open reading frame 29 (C19orf29), transcript variant 2 The lysate was created in HEK293T cells, using Plasmid ID RC213573 and based on accession number NM_021231. The protein contains a C-MYC/DDK Tag. Gene ID 58509 Gene Symbol CACTIN Species Human Notes HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.This product is manufactured by and sold under license from OriGene Technologies and its use is limited solely for research purposes.
    [Show full text]
  • 1 UST College of Science Department of Biological Sciences
    UST College of Science Department of Biological Sciences 1 Pharmacogenomics of Myofascial Pain Syndrome An Undergraduate Thesis Submitted to the Department of Biological Sciences College of Science University of Santo Tomas In Partial Fulfillment of the Requirements for the Degree of Bachelor of Science in Biology Jose Marie V. Lazaga Marc Llandro C. Fernandez May 2021 UST College of Science Department of Biological Sciences 2 PANEL APPROVAL SHEET This undergraduate research manuscript entitled: Pharmacogenomics of Myofascial Pain Syndrome prepared and submitted by Jose Marie V. Lazaga and Marc Llandro C. Fernandez, was checked and has complied with the revisions and suggestions requested by panel members after thorough evaluation. This final version of the manuscript is hereby approved and accepted for submission in partial fulfillment of the requirements for the degree of Bachelor of Science in Biology. Noted by: Asst. Prof. Marilyn G. Rimando, PhD Research adviser, Bio/MicroSem 602-603 Approved by: Bio/MicroSem 603 panel member Bio/MicroSem 603 panel member Date: Date: UST College of Science Department of Biological Sciences 3 DECLARATION OF ORIGINALITY We hereby affirm that this submission is our own work and that, to the best of our knowledge and belief, it contains no material previously published or written by another person nor material to which a substantial extent has been accepted for award of any other degree or diploma of a university or other institute of higher learning, except where due acknowledgement is made in the text. We also declare that the intellectual content of this undergraduate research is the product of our work, even though we may have received assistance from others on style, presentation, and language expression.
    [Show full text]
  • Expression of the POTE Gene Family in Human Ovarian Cancer Carter J Barger1,2, Wa Zhang1,2, Ashok Sharma 1,2, Linda Chee1,2, Smitha R
    www.nature.com/scientificreports OPEN Expression of the POTE gene family in human ovarian cancer Carter J Barger1,2, Wa Zhang1,2, Ashok Sharma 1,2, Linda Chee1,2, Smitha R. James3, Christina N. Kufel3, Austin Miller 4, Jane Meza5, Ronny Drapkin 6, Kunle Odunsi7,8,9, 2,10 1,2,3 Received: 5 July 2018 David Klinkebiel & Adam R. Karpf Accepted: 7 November 2018 The POTE family includes 14 genes in three phylogenetic groups. We determined POTE mRNA Published: xx xx xxxx expression in normal tissues, epithelial ovarian and high-grade serous ovarian cancer (EOC, HGSC), and pan-cancer, and determined the relationship of POTE expression to ovarian cancer clinicopathology. Groups 1 & 2 POTEs showed testis-specifc expression in normal tissues, consistent with assignment as cancer-testis antigens (CTAs), while Group 3 POTEs were expressed in several normal tissues, indicating they are not CTAs. Pan-POTE and individual POTEs showed signifcantly elevated expression in EOC and HGSC compared to normal controls. Pan-POTE correlated with increased stage, grade, and the HGSC subtype. Select individual POTEs showed increased expression in recurrent HGSC, and POTEE specifcally associated with reduced HGSC OS. Consistent with tumors, EOC cell lines had signifcantly elevated Pan-POTE compared to OSE and FTE cells. Notably, Group 1 & 2 POTEs (POTEs A/B/B2/C/D), Group 3 POTE-actin genes (POTEs E/F/I/J/KP), and other Group 3 POTEs (POTEs G/H/M) show within-group correlated expression, and pan-cancer analyses of tumors and cell lines confrmed this relationship. Based on their restricted expression in normal tissues and increased expression and association with poor prognosis in ovarian cancer, POTEs are potential oncogenes and therapeutic targets in this malignancy.
    [Show full text]
  • Characterization of a Monoclonal Antibody Panel Shows That the Myotonic Dystrophy Protein Kinase, DMPK, Is Expressed Almost Exclusively in Muscle and Heart
    © 2000 Oxford University Press Human Molecular Genetics, 2000, Vol. 9, No. 14 2167–2173 Characterization of a monoclonal antibody panel shows that the myotonic dystrophy protein kinase, DMPK, is expressed almost exclusively in muscle and heart L.T. Lam, Y.C.N. Pham, Nguyen thi Man and G.E. Morris+ MRIC Biochemistry Group, PP18, North East Wales Institute, Mold Road, Wrexham LL11 2AW, UK Received 11 May 2000; Accepted 28 June 2000 DDBJ/EMBL/GenBank accession no. AF250871 Myotonic dystrophy (DM) is a multisystemic disorder ness), brain (mental retardation), eyes (cataracts), testis caused by an inherited CTG repeat expansion which (atrophy) and heart (conduction defects) (10). It is important, affects three genes encoding the DM protein kinase therefore, to establish the normal tissue distribution of all three (DMPK), a homeobox protein Six5 and a protein proteins produced from the DM locus. containing WD repeats. Using a panel of 16 mono- The human DMPK cDNA has 15 exons and predicts a ∼ clonal antibodies against several different DMPK protein of 70 kDa, although there may be variation in the size epitopes we detected DMPK, as a single protein of and sequence of the first exon (1–3,11,12). DMPK is a serine/ threonine protein kinase in which the catalytic domain ∼80 kDa, only in skeletal muscle, cardiac muscle and, (∼43 kDa) is followed by a coiled-coil domain (∼12 kDa) and to a lesser extent, smooth muscle. Many earlier a hydrophobic C-terminal domain. Alternative splicing has reports of DMPK with different sizes and tissue distri- been observed in a short VSGGG sequence between domains butions appear to be due to antibody cross-reactions and in the C-terminal domain (13).
    [Show full text]
  • Whole Exome Sequencing in Families at High Risk for Hodgkin Lymphoma: Identification of a Predisposing Mutation in the KDR Gene
    Hodgkin Lymphoma SUPPLEMENTARY APPENDIX Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene Melissa Rotunno, 1 Mary L. McMaster, 1 Joseph Boland, 2 Sara Bass, 2 Xijun Zhang, 2 Laurie Burdett, 2 Belynda Hicks, 2 Sarangan Ravichandran, 3 Brian T. Luke, 3 Meredith Yeager, 2 Laura Fontaine, 4 Paula L. Hyland, 1 Alisa M. Goldstein, 1 NCI DCEG Cancer Sequencing Working Group, NCI DCEG Cancer Genomics Research Laboratory, Stephen J. Chanock, 5 Neil E. Caporaso, 1 Margaret A. Tucker, 6 and Lynn R. Goldin 1 1Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; 2Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; 3Ad - vanced Biomedical Computing Center, Leidos Biomedical Research Inc.; Frederick National Laboratory for Cancer Research, Frederick, MD; 4Westat, Inc., Rockville MD; 5Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD; and 6Human Genetics Program, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, MD, USA ©2016 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2015.135475 Received: August 19, 2015. Accepted: January 7, 2016. Pre-published: June 13, 2016. Correspondence: [email protected] Supplemental Author Information: NCI DCEG Cancer Sequencing Working Group: Mark H. Greene, Allan Hildesheim, Nan Hu, Maria Theresa Landi, Jennifer Loud, Phuong Mai, Lisa Mirabello, Lindsay Morton, Dilys Parry, Anand Pathak, Douglas R. Stewart, Philip R. Taylor, Geoffrey S. Tobias, Xiaohong R. Yang, Guoqin Yu NCI DCEG Cancer Genomics Research Laboratory: Salma Chowdhury, Michael Cullen, Casey Dagnall, Herbert Higson, Amy A.
    [Show full text]
  • Brain-Specific Knock-Out of Hypoxia-Inducible Factor-1Α
    The Journal of Neuroscience, April 20, 2005 • 25(16):4099–4107 • 4099 Neurobiology of Disease Brain-Specific Knock-Out of Hypoxia-Inducible Factor-1␣ Reduces Rather Than Increases Hypoxic–Ischemic Damage Rob Helton,1* Jiankun Cui,2* John R. Scheel,1* Julie A. Ellison,1 Chris Ames,1 Claire Gibson,2 Barbara Blouw,3 Ling Ouyang,1 Ioannis Dragatsis,4 Scott Zeitlin,5 Randall S. Johnson,3 Stuart A. Lipton,2 and Carrolee Barlow1 1Laboratory of Genetics, The Salk Institute for Biological Studies, and 2Center for Neuroscience and Aging, The Burnham Institute, La Jolla, California 92037, 3Molecular Biology Section, Division of Biology, University of California, San Diego, La Jolla, California 92093, 4Department of Physiology, The University of Tennessee, Health Science Center, Memphis, Tennessee 38163, and 5Department of Neuroscience, University of Virginia School of Medicine, Charlottesville, Virginia 22908 ␣ ␣ Hypoxia-inducible factor-1 (HIF-1 ) plays an essential role in cellular and systemic O2 homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1␣ in the brain, we exposed adult mice with late-stage brain deletion of HIF-1␣ to hypoxic injuries. Contrary to expectations, the brains from the HIF-1␣-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreas- ing the level of HIF-1␣ can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1␣-deficient mice.
    [Show full text]
  • Mouse Ccnl2 Conditional Knockout Project (CRISPR/Cas9)
    https://www.alphaknockout.com Mouse Ccnl2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ccnl2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ccnl2 gene (NCBI Reference Sequence: NM_207678 ; Ensembl: ENSMUSG00000029068 ) is located on Mouse chromosome 4. 11 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 11 (Transcript: ENSMUST00000030944). Exon 5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ccnl2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-128M14 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 5 starts from about 37.9% of the coding region. The knockout of Exon 5 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2559 bp, and the size of intron 5 for 3'-loxP site insertion: 2341 bp. The size of effective cKO region: ~565 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 5 11 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ccnl2 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats.
    [Show full text]
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts
    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
    [Show full text]
  • WO 2012/174282 A2 20 December 2012 (20.12.2012) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2012/174282 A2 20 December 2012 (20.12.2012) P O P C T (51) International Patent Classification: David [US/US]; 13539 N . 95th Way, Scottsdale, AZ C12Q 1/68 (2006.01) 85260 (US). (21) International Application Number: (74) Agent: AKHAVAN, Ramin; Caris Science, Inc., 6655 N . PCT/US20 12/0425 19 Macarthur Blvd., Irving, TX 75039 (US). (22) International Filing Date: (81) Designated States (unless otherwise indicated, for every 14 June 2012 (14.06.2012) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, English (25) Filing Language: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, Publication Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, (30) Priority Data: KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, 61/497,895 16 June 201 1 (16.06.201 1) US MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 61/499,138 20 June 201 1 (20.06.201 1) US OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, 61/501,680 27 June 201 1 (27.06.201 1) u s SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 61/506,019 8 July 201 1(08.07.201 1) u s TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
    [Show full text]