DOCSLIB.ORG

I Design, Optimization and Validation of a Diagnostic Panel Comprised Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
I Design, Optimization and Validation of a Diagnostic Panel Comprised Of Design, optimization and validation of a diagnostic panel comprised of ABCA4 pathogenic variants causing Stargardt disease in the Newfoundland and Labrador population by © Hoda Rajabi A Thesis submitted to the School of Graduate Studies in the partial fulfillment of the requirements for the degree of Master of Science in Medicine (Human Genetics) Faculty of Medicine, Discipline of Genetics Memorial University of Newfoundland October 2016 St. John’s Newfoundland and Labrador I ABSTRACT The objective of this translational research project was to develop, optimize and validate a diagnostic panel of the 20 pathogenic variants in the ABCA4 gene previously identified to cause Stargardt disease in the population of Newfoundland and Labrador (NL). Two different laboratory developed test (LDT) panels (1x20- plex & 2x20-plex) were designed and genotyping was performed on the Sequenom MassARRAY 4 system using genomic DNA from both Stargardt patients and population controls. A set of minimum criteria was established to accurately determine genotyping calls; 1x20-plex LDT panel was selected based on quality metrics. Assessment of the validation cohort including 78 previously tested genomic DNA samples, blind to the investigator, resulted in establishing analytical sensitivity (100%), specificity (100%), accuracy (100%), and reproducibility (100%). A total of 1039 control samples were assessed using the 1x20-plex LDT panel and the minor allele frequencies of 0% - 0.76% for NL, and 0% - 0.60% for non-NL samples were determined. II ACKNOWLEDGEMENTS First and foremost, I would like to thank my lovely family, especially my husband. I spent much time and energy pursuing my goals that took me away from my family. Without my husband’s support, encouragement and patients, I would not have been able to finish this challenging project. I would like to express my deepest appreciation to my supervisor Dr. Darren O’Rielly. He consistently guided me in the right direction and consulted regarding the best solutions. The door to Darren’s office was always open whenever I ran into a trouble or had a question about my research or writing. I would like to thank my co-supervisor Dr. Jane Green and my supervisory committee members Drs. Terry-Lynn Young and Proton Rahman for their warmest support and constructive advice. It was a great honour to work with these respected and well-known Faculty members in the area of human genetics. I would like to thank all my friends and staff in the Drs. Young, Rahman and O’Reilly laboratories. They were always there to train, help and guide me whenever needed. Finally, I would like to acknowledge the funding agencies and all participants who donated samples to finish this project. III IV TABLE OF CONTENTS ABSTRACT .................................................................................................................... II ACKNOWLEDGEMENTS ..............................................................................................III LIST OF TABLES ........................................................................................................ VIII LIST OF FIGURES ......................................................................................................... X ABBREVIATIONS ......................................................................................................... XI 1.0 – INTRODUCTION ................................................................................................... 1 1.1 – Eye, Stargardt disease and Genetics ..................................................................... 1 1.1.1 – Structure of the Retina .................................................................................... 1 1.1.2 – The visual cycle............................................................................................... 3 1.1.3 – Retinal degeneration disorders ........................................................................ 5 1.1.4 – Stargardt disease ............................................................................................ 7 1.1.5 – STGD1 ............................................................................................................ 8 1.1.6 – Fundus Flavimaculatus ................................................................................... 8 1.1.7 – The ABCA4 gene and ABC transporters ......................................................... 9 1.1.8 – Variants in ABCA4 and genotype-phenotype correlations ..............................10 1.1.9 – Clinical examinations and diagnostic tools .....................................................12 1.1.10 – STGD1 pathogenic variants .........................................................................15 1.1.11 – Phenotypic variations in STGD1 ...................................................................15 1.1.12 – Animal models and potential therapy ............................................................16 1.2 – Newfoundland and Labrador population and Stargardt disease ............................19 1.2.1 – History of Newfoundland and Labrador ..........................................................19 1.2.2 – Genetic drift, founder effect and the NL population .........................................19 1.2.3 – Hereditary eye diseases in NL ........................................................................20 1.3 – Molecular detection of STGD1 ..............................................................................21 1.3.1 – Sequencing technology ..................................................................................21 1.3.2 – Genotyping using microarray technology........................................................22 1.3.3 – Previous studies on Stargardt in NL ...............................................................24 1.3.4 – Genotyping using Sequenom MassARRAY technology ..................................26 1.4 – Knowledge translation ..........................................................................................27 1.5 – analytic validity, clinical validity, and clinical utility .................................................28 V 2.0 – MATERIALS & METHODS ...................................................................................30 2.1 – Study cohort and ethics ........................................................................................30 2.2 – In-silico design of multiplex panel .........................................................................32 2.2.1 – In-silico design of 20 pathogenic variants causing Stargardt disease .............34 2.3 – Primer mixtures and adjustments..........................................................................36 2.4 – Sequenom MassArray and iPLEX chemistry ........................................................37 2.4.1 – Preparation of samples ..................................................................................38 2.4.2 – DNA amplification ...........................................................................................38 2.4.3 – Neutralize unincorporated dNTPs (SAP reaction) ...........................................39 2.4.4 – iPLEX extension reaction ...............................................................................40 2.4.5 – Condition the iPLEX reaction products ...........................................................41 2.4.6 – Dispense onto SpectroCHIP arrays and defining assays and plates ..............41 2.5 – Assessment and selection of best performing LDT panel......................................43 2.6 – Confirmation of genotypes by Sanger sequencing ................................................44 2.6.1 – Designing PCR primers ..................................................................................44 2.6.2 – Preparing the DNA samples at the required concentration .............................45 2.6.3 – PCR procedure, examining the amplification on the gel electrophoresis, and purifying the products using Sephacryl .......................................................................45 2.6.4 – Cycle sequencing, ethanol precipitation and sample setup on the ABI 3130 or 3730 XL DNA Analyser ..............................................................................................47 2.6.5 – Analyzing Sanger sequencing data ................................................................48 2.7 – Optimization of the 1x20-plex LDT panel ..............................................................48 2.7.1 – Adjustments to eliminate automated genotype calls in NTC wells ..................48 2.7.2 – Adjustments to increase allele height .............................................................50 2.7.2.1 – Nanodispenser adjustments ....................................................................50 2.7.2.2 – Adjustment of PCR/extension primer pool based on optimization cohort .51 2.7.2.3 – Allele frequency determination .................................................................52 2.7.2.4 – Final optimization of the 1x20-plex LDT panel .........................................52 2.7.3 – Final calling algorithm parameters ..................................................................53 2.8 – Analytical validation ..............................................................................................53 2.9 – Clinical validity of the 1x20-plex LDT panel ...........................................................54 2.10 – Statistical analysis ..............................................................................................54 VI 3.0 – RESULTS .............................................................................................................56
Load more

Recommended publications
  • EXTENDED CARRIER SCREENING Peace of Mind for Planned Pregnancies
    Focusing on Personalised Medicine EXTENDED CARRIER SCREENING Peace of Mind for Planned Pregnancies Extended carrier screening is an important tool for prospective parents to help them determine their risk of having a child affected with a heritable disease. In many cases, parents aren’t aware they are carriers and have no family history due to the rarity of some diseases in the general population. What is covered by the screening? Genomics For Life offers a comprehensive Extended Carrier Screening test, providing prospective parents with the information they require when planning their pregnancy. Extended Carrier Screening has been shown to detect carriers who would not have been considered candidates for traditional risk- based screening. With a simple mouth swab collection, we are able to test for over 419 genes associated with inherited diseases, including Fragile X Syndrome, Cystic Fibrosis and Spinal Muscular Atrophy. The assay has been developed in conjunction with clinical molecular geneticists, and includes genes listed in the NIH Genetic Test Registry. For a list of genes and disorders covered, please see the reverse of this brochure. If your gene of interest is not covered on our Extended Carrier Screening panel, please contact our friendly team to assist you in finding a gene test panel that suits your needs. Why have Extended Carrier Screening? Extended Carrier Screening prior to pregnancy enables couples to learn about their reproductive risk and consider a complete range of reproductive options, including whether or not to become pregnant, whether to use advanced reproductive technologies, such as preimplantation genetic diagnosis, or to use donor gametes.
    [Show full text]
  • ABCB6 Is a Porphyrin Transporter with a Novel Trafficking Signal That Is Conserved in Other ABC Transporters Yu Fukuda University of Tennessee Health Science Center
    University of Tennessee Health Science Center UTHSC Digital Commons Theses and Dissertations (ETD) College of Graduate Health Sciences 12-2008 ABCB6 Is a Porphyrin Transporter with a Novel Trafficking Signal That Is Conserved in Other ABC Transporters Yu Fukuda University of Tennessee Health Science Center Follow this and additional works at: https://dc.uthsc.edu/dissertations Part of the Chemicals and Drugs Commons, and the Medical Sciences Commons Recommended Citation Fukuda, Yu , "ABCB6 Is a Porphyrin Transporter with a Novel Trafficking Signal That Is Conserved in Other ABC Transporters" (2008). Theses and Dissertations (ETD). Paper 345. http://dx.doi.org/10.21007/etd.cghs.2008.0100. This Dissertation is brought to you for free and open access by the College of Graduate Health Sciences at UTHSC Digital Commons. It has been accepted for inclusion in Theses and Dissertations (ETD) by an authorized administrator of UTHSC Digital Commons. For more information, please contact [email protected]. ABCB6 Is a Porphyrin Transporter with a Novel Trafficking Signal That Is Conserved in Other ABC Transporters Document Type Dissertation Degree Name Doctor of Philosophy (PhD) Program Interdisciplinary Program Research Advisor John D. Schuetz, Ph.D. Committee Linda Hendershot, Ph.D. James I. Morgan, Ph.D. Anjaparavanda P. Naren, Ph.D. Jie Zheng, Ph.D. DOI 10.21007/etd.cghs.2008.0100 This dissertation is available at UTHSC Digital Commons: https://dc.uthsc.edu/dissertations/345 ABCB6 IS A PORPHYRIN TRANSPORTER WITH A NOVEL TRAFFICKING SIGNAL THAT
    [Show full text]
  • Transcriptional and Post-Transcriptional Regulation of ATP-Binding Cassette Transporter Expression
    Transcriptional and Post-transcriptional Regulation of ATP-binding Cassette Transporter Expression by Aparna Chhibber DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmaceutical Sciences and Pbarmacogenomies in the Copyright 2014 by Aparna Chhibber ii Acknowledgements First and foremost, I would like to thank my advisor, Dr. Deanna Kroetz. More than just a research advisor, Deanna has clearly made it a priority to guide her students to become better scientists, and I am grateful for the countless hours she has spent editing papers, developing presentations, discussing research, and so much more. I would not have made it this far without her support and guidance. My thesis committee has provided valuable advice through the years. Dr. Nadav Ahituv in particular has been a source of support from my first year in the graduate program as my academic advisor, qualifying exam committee chair, and finally thesis committee member. Dr. Kathy Giacomini graciously stepped in as a member of my thesis committee in my 3rd year, and Dr. Steven Brenner provided valuable input as thesis committee member in my 2nd year. My labmates over the past five years have been incredible colleagues and friends. Dr. Svetlana Markova first welcomed me into the lab and taught me numerous laboratory techniques, and has always been willing to act as a sounding board. Michael Martin has been my partner-in-crime in the lab from the beginning, and has made my days in lab fly by. Dr. Yingmei Lui has made the lab run smoothly, and has always been willing to jump in to help me at a moment’s notice.
    [Show full text]
  • ABC Transporter Subfamily D: Distinct Differences in Behavior Between ABCD1–3 and ABCD4 in Subcellular Localization, Function, and Human Disease
    Hindawi Publishing Corporation BioMed Research International Volume 2016, Article ID 6786245, 11 pages http://dx.doi.org/10.1155/2016/6786245 Review Article ABC Transporter Subfamily D: Distinct Differences in Behavior between ABCD1–3 and ABCD4 in Subcellular Localization, Function, and Human Disease Kosuke Kawaguchi and Masashi Morita Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan Correspondence should be addressed to Kosuke Kawaguchi; [email protected] Received 24 June 2016; Accepted 29 August 2016 Academic Editor: Hiroshi Nakagawa Copyright © 2016 K. Kawaguchi and M. Morita. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ATP-binding cassette (ABC) transporters are one of the largest families of membrane-bound proteins and transport a wide variety of substrates across both extra- and intracellular membranes. They play a critical role in maintaining cellular homeostasis. To date, four ABC transporters belonging to subfamily D have been identified. ABCD1–3 and ABCD4 are localized to peroxisomes and lysosomes, respectively. ABCD1 and ABCD2 are involved in the transport of long and very long chain fatty acids (VLCFA) or their CoA-derivatives into peroxisomes with different substrate specificities, while ABCD3 is involved in the transport of branched chain acyl-CoA into peroxisomes. On the other hand, ABCD4 is deduced to take part in the transport of vitamin B12 from lysosomes into the cytosol. It is well known that the dysfunction of ABCD1 results in X-linked adrenoleukodystrophy, a severe neurodegenerative disease.
    [Show full text]
  • The Role of Mitochondrial ATP-Binding Cassette Transporter
    The Role of Mitochondrial ATP-Binding Cassette Transporter ABCB6 in Metabolism and Energy Balance By © 2019 Robert T Tessman Submitted to the graduate degree program in Toxicology and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Committee Chair: Partha Kasturi, PhD Bruno Hagenbuch, PhD Hao Zhu, PhD Tiangang Li, PhD Luciano DiTacchio, PhD Date Defended: April 19th, 2019Title Page ii The dissertation committee for Robert T Tessman certifies that this is the approved version of the following dissertation: The role of Mitochondrial ATP-Binding Cassette Transporter ABCB6 in Metabolism and Energy Balance Committee Chair: Partha Kasturi, PhD Acceptance Page Date Approved: April 19th, 2019 iii Abstract Obesity and the associated health risks represent a world-wide health and financial crisis. Lack of physical activity combined with excessive caloric intake are the root cause of the problem. Despite the increased advocation for healthy lifestyle choices, the trend has yet to reverse and indeed, seems to be on the rise especially among pre- teens and adolescents, a constituent that had not been previously part of the obesity epidemic. Mitochondria are the “fuel-burners” of the body and like other combustion devices, become inefficient in the context of fuel surplus. Moreover, with chronic over-feeding, the physiological mechanisms that regulate energy balance become permanently dysfunctional leading to the progression of pathologies such as Type II diabetes and cardiovascular disease. Medical and scientific evidence confirms that mitochondria are integral to the responses necessary to adapt to over-nutrition. However, success in mitochondria- based therapies has been extremely limited in the context of metabolic diseases.
    [Show full text]
  • The Putative Mitochondrial Protein ABCB6
    Shifting the Paradigm: The Putative Mitochondrial Protein ABCB6 Resides in the Lysosomes of Cells and in the Plasma Membrane of Erythrocytes Katalin Kiss, Anna Brozik, Nora Kucsma, Alexandra Toth, Melinda Gera, Laurence Berry, Alice Vallentin, Henri Vial, Michel Vidal, Gergely Szakacs To cite this version: Katalin Kiss, Anna Brozik, Nora Kucsma, Alexandra Toth, Melinda Gera, et al.. Shifting the Paradigm: The Putative Mitochondrial Protein ABCB6 Resides in the Lysosomes of Cells and in the Plasma Membrane of Erythrocytes. PLoS ONE, Public Library of Science, 2012, 7 (5), pp.e37378. 10.1371/journal.pone.0037378. hal-02309092 HAL Id: hal-02309092 https://hal.archives-ouvertes.fr/hal-02309092 Submitted on 25 May 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Shifting the Paradigm: The Putative Mitochondrial Protein ABCB6 Resides in the Lysosomes of Cells and in the Plasma Membrane of Erythrocytes Katalin Kiss1, Anna Brozik1, Nora Kucsma1, Alexandra Toth1, Melinda Gera1,
    [Show full text]
  • Whole-Exome Sequencing Identifies Novel Mutations in ABC Transporter
    Liu et al. BMC Pregnancy and Childbirth (2021) 21:110 https://doi.org/10.1186/s12884-021-03595-x RESEARCH ARTICLE Open Access Whole-exome sequencing identifies novel mutations in ABC transporter genes associated with intrahepatic cholestasis of pregnancy disease: a case-control study Xianxian Liu1,2†, Hua Lai1,3†, Siming Xin1,3, Zengming Li1, Xiaoming Zeng1,3, Liju Nie1,3, Zhengyi Liang1,3, Meiling Wu1,3, Jiusheng Zheng1,3* and Yang Zou1,2* Abstract Background: Intrahepatic cholestasis of pregnancy (ICP) can cause premature delivery and stillbirth. Previous studies have reported that mutations in ABC transporter genes strongly influence the transport of bile salts. However, to date, their effects are still largely elusive. Methods: A whole-exome sequencing (WES) approach was used to detect novel variants. Rare novel exonic variants (minor allele frequencies: MAF < 1%) were analyzed. Three web-available tools, namely, SIFT, Mutation Taster and FATHMM, were used to predict protein damage. Protein structure modeling and comparisons between reference and modified protein structures were performed by SWISS-MODEL and Chimera 1.14rc, respectively. Results: We detected a total of 2953 mutations in 44 ABC family transporter genes. When the MAF of loci was controlled in all databases at less than 0.01, 320 mutations were reserved for further analysis. Among these mutations, 42 were novel. We classified these loci into four groups (the damaging, probably damaging, possibly damaging, and neutral groups) according to the prediction results, of which 7 novel possible pathogenic mutations were identified that were located in known functional genes, including ABCB4 (Trp708Ter, Gly527Glu and Lys386Glu), ABCB11 (Gln1194Ter, Gln605Pro and Leu589Met) and ABCC2 (Ser1342Tyr), in the damaging group.
    [Show full text]
  • Analysis of Arabidopsis Abcb Auxin Transporter Mutants Reveals a Primary Role in Membrane Exclusion
    ABSTRACT Title of Dissertation: ANALYSIS OF ARABIDOPSIS ABCB AUXIN TRANSPORTER MUTANTS REVEALS A PRIMARY ROLE IN MEMBRANE EXCLUSION Mark Kubo Jenness, Doctor of Philosophy, 2018 Dissertation directed by: Professor Angus S. Murphy, Department of Plant Science and Landscape Architecture Polar transport of the phytohormone auxin regulates multiple of aspects of plant growth and development. A subset of plant ATP-binding cassette subfamily B (ABCB) transporters mediate cellular auxin export. Loss of these transporters results in reduced polar auxin movement and altered plant architecture but no significant defects in embryogenesis or organ formation. Several of lines of evidence suggest that isotropically-localized ABCB transporters mediate auxin exclusion from the plasma membrane and prevention of reuptake after directional PIN-mediated efflux. Examination of the Arabidopsis auxin transporters ABCB1 and ABCB19 indicates a primary role in exclusion from small auxin producing cells in apical regions and prevention of leakage from polar auxin transport streams. Analysis of abcb mutants identifies a contribution from ABCB21 in restricting auxin to within the root vasculature in seedlings. In mature tissues, ABCB6, ABCB21, and ABCB11 make additional contributions to polar auxin transport in inflorescence stems, leaves, and flowers, respectively. The results presented herein reflect an evolutionarily conserved function for ABCB transporters in maintaining polar transport streams and prevention of cellular reuptake via exclusion. ANALYSIS OF ARABIDOPSIS ABCB AUXIN TRANSPORTER MUTANTS REVEALS A PRIMARY ROLE IN MEMBRANE EXCLUSION by Mark Kubo Jenness Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2018 Advisory Committee: Professor Angus Murphy, Chair Professor David Hawthorne Professor Jianhua Zhu Professor Wendy A.
    [Show full text]
  • Precision Medicine in the Management of Type 2 Diabetes
    Review Precision medicine in the management of type 2 diabetes Anna L Gloyn, Daniel J Drucker The study of type 2 diabetes has been driven by advances in human genetics, epigenetics, biomarkers, mechanistic Lancet Diabetes Endocrinol 2018 studies, and large clinical trials, enabling new insights into disease susceptibility, pathophysiology, progression, and Published Online development of complications. Simultaneously, several new drug classes with different mechanisms of action have April 23, 2018 been introduced over the past two decades, accompanied by data about cardiovascular safety and non-glycaemic http://dx.doi.org/10.1016/ S2213-8587(18)30052-4 outcomes. In this Review, we critically examine the progress and integration of this new science into clinical practice, Oxford Centre for Diabetes, and review opportunities for enabling the use of precision medicine in the diagnosis and treatment of type 2 diabetes. Endocrinology and We contrast the success in delivering personalised medicine for monogenic diabetes with the greater challenge of Metabolism, Radcliffe providing a precision medicine approach for type 2 diabetes, highlighting gaps, limitations, and areas requiring Department of Medicine, and further study. Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Introduction a marked sensitivity to sulfonylureas, a finding that was (Prof A L Gloyn DPhil); NIHR There has been much interest in our increased ability to elegantly substantiated in a randomised controlled trial Oxford Biomedical Research incorporate data from human genetics, along with that provided the first example of personalised medicine Centre, Churchill Hospital, Oxford, UK (Prof A L Gloyn); 3 lifestyle and environmental information, to individualise in diabetes. The precise molecular mechanism for and Department of Medicine, treatment decisions.
    [Show full text]
  • Role of Genetic Variation in ABC Transporters in Breast Cancer Prognosis and Therapy Response
    International Journal of Molecular Sciences Article Role of Genetic Variation in ABC Transporters in Breast Cancer Prognosis and Therapy Response Viktor Hlaváˇc 1,2 , Radka Václavíková 1,2, Veronika Brynychová 1,2, Renata Koževnikovová 3, Katerina Kopeˇcková 4, David Vrána 5 , Jiˇrí Gatˇek 6 and Pavel Souˇcek 1,2,* 1 Toxicogenomics Unit, National Institute of Public Health, 100 42 Prague, Czech Republic; [email protected] (V.H.); [email protected] (R.V.); [email protected] (V.B.) 2 Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic 3 Department of Oncosurgery, Medicon Services, 140 00 Prague, Czech Republic; [email protected] 4 Department of Oncology, Second Faculty of Medicine, Charles University and Motol University Hospital, 150 06 Prague, Czech Republic; [email protected] 5 Department of Oncology, Medical School and Teaching Hospital, Palacky University, 779 00 Olomouc, Czech Republic; [email protected] 6 Department of Surgery, EUC Hospital and University of Tomas Bata in Zlin, 760 01 Zlin, Czech Republic; [email protected] * Correspondence: [email protected]; Tel.: +420-267-082-711 Received: 19 November 2020; Accepted: 11 December 2020; Published: 15 December 2020 Abstract: Breast cancer is the most common cancer in women in the world. The role of germline genetic variability in ATP-binding cassette (ABC) transporters in cancer chemoresistance and prognosis still needs to be elucidated. We used next-generation sequencing to assess associations of germline variants in coding and regulatory sequences of all human ABC genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105).
    [Show full text]
  • Influence of ABCA1 and ABCA7 on the Lipid Microenvironment of the Plasma Membrane
    I Influence of ABCA1 and ABCA7 on the lipid microenvironment of the plasma membrane Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von Dottore Magistrale in Biotecnologie Industriali Anna Pia Plazzo geb. 17.10.1981 in San Giovanni Rotondo, Italien Präsident der Humboldt-Universität zu Berlin Prof. Dr. Dr. h.c. Christoph Markschies Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Lutz-Helmut Schön Gutachter: 1. Prof. Dr. Andreas Herrmann 2. Prof. Dr. Thomas Günther-Pomorski 3. Prof. Dr. Thomas Eitinger Tag der mündlichen Prüfung: 12.06.2009 II Alla mia famiglia III Zusammenfassung Der ABC-Transporter ABCA1 ist unmittelbar in die zelluläre Lipidhomeostasie einbezogen, in dem er die Freisetzung von Cholesterol an plasmatische Rezeptoren, wie ApoA-I, vermittelt. Trotz intensiver Untersuchungen ist dieser molekulare Mechanismus nicht verstanden. Verschiedene Studien deuten daraufhin, dass durch die Aktivität von ABCA1 bedingte Veränderungen in der Lipidphase der äußeren Hälfte der Plasmamembran (PM) wichtig für die Freisetzung des Cholesterols sind. In der vorliegenden Arbeit wird die Lipidumgebung von ABCA1 in der PM lebender Säugetierzellen unter Anwendung der Fluoreszenzlebenszeitmikroskopie von fluoreszierenden Lipidsonden untersucht. Es wurde eine breite Verteilung der Fluoreszenzlebenszeiten der Sonden gefunden, die sensitiv gegenüber Veränderungen der lateralen und transversalen Organisation der Lipide ist. Im Einklang mit Studien an riesengroßen unilamellaren Vesikeln und Plasmamembranvesikeln weisen unsere Ergebnisse die Existenz einer größeren Vielfalt submikroskopischer Lipiddomänen auf. Die FLIM-Untersuchungen an ABCA1 exprimierenden HeLa-Zellen weisen eine die Lipidphase destabilisierende Funktion des Transportes aus. Dieses wurde unterstützt durch die Lipidanalyse von Fraktionen der PM.
    [Show full text]
  • 06-0718 Index: Benefits
    State of California—Health and Human Services Agency Department of Health Care Services JENNIFER KENT EDMUND G. BROWN JR. DIRECTOR GOVERNOR DATE: July 10, 2018 N.L.: 06-0718 Index: Benefits TO: COUNTY CALIFORNIA CHILDREN’S SERVICES (CCS) ADMINISTRATORS, MEDICAL DIRECTORS, AND MEDICAL CONSULTANTS, INTEGRATED SYSTEMS OF CARE DIVISION (ISCD) STAFF, CCS PROGRAM-APPROVED SPECIAL CARE CENTERS (SCC) SUBJECT: AUTHORIZATION OF DIAGNOSTIC AND TREATMENT SERVICE FOR INFANTS REFERRED BY THE CALIFORNIA NEWBORN SCREENING (NBS) PROGRAM FOR X-LINKED ADRENOLEUKODYSTROPHY (ALD) I. PURPOSE The purpose of this Numbered Letter is to establish CCS policy on the authorization of diagnostic and treatment services for X-linked ALD for individuals with a positive NBS test. II. BACKGROUND X-linked ALD is an inherited peroxisomal disorder in which accumulation of very long chain fatty acids (VCLFA) in the brain, spinal cord and adrenal glands leads to demyelination in the brain and spinal cord, and impaired adrenal corticoid function in the adrenal cortex. The condition results from the mutations of the ABCD1 gene on the X chromosome. The ABCD1 gene codes for the ALD protein, which facilitates transport of VLCFA into peroxisomes. Absence of the protein results in an inability to transport and degrade VLCFA. Over 1,000 mutations associated with ALD have been identified. The incidence is between 1 in 20,000 and 1 in 50,000 births. There are multiple presentations of ALD, including childhood cerebral ALD, adolescent cerebral ALD, ‘Addison’s disease only’ and adrenomyeloneuropathy (AMN). Childhood cerebral ALD most commonly presents in boys between the ages of four to eight years old, with intellectual or behavioral impairments.
    [Show full text]