Heredity and Schizophrenia: Endophenotypes and their Applicability

2014/2015 Daniel Rodolfo Pereira da Mota Bacelar Braga Heredity and Schizophrenia: Endophenotypes and their Applicability março, 2015 Mestrado Integrado em Medicina Área: Psiquiatria e Saúde Mental Tipologia: Monografia Trabalho efetuado sob a Orientação de: Dra Raquel Correia Trabalho organizado de acordo com as normas da revista: Schizophrenia Bulletin Daniel Rodolfo Pereira da Mota Bacelar Braga Heredity and Schizophrenia: Endophenotypes and their Applicability março, 2015 À minha Mãe e ao meu Pai, aos meus Padrinhos e ao clã Pereira da Mota, e ao meu tio Luís; Ao Francisco e à Francisca; Aos meus amigos Marta, Joana, Mariana, Mário, Nascimento, Camarão, Maia, Vanessa e Ana Paula. 1 Title: Heredity and Schizophrenia: Endophenotypes and their Applicability Running Title: Endophenotypes and their Applicability Authors and Affiliations: Daniel Braga1*; Raquel Correia, MD2 1Masters Degree in Medicine, Faculty of Medicine of the University of Porto, Porto, Portugal;2Psychiatrist, Clinical Neurosciences and Mental Health Department, Faculty of Medicine, Porto, Portugal * To whom correspondence should be addressed; Clinical Neuroscience and Mental Health Department, Faculty of Medicine of the University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal; tel: +351914369436; e-mail: [email protected] Abstract Word Count: 240 Text Body Word Count: 2888 2 Endophenotypes and their Applicability Abstract Endophenotypes have shown great promise in furthering our understanding of schizophrenia genetics by breaking the large spectrum of schizophrenia findings into smaller phenotypes, assuming there are smaller genetic links to each one of these endophenotypes. Neurophysiological and neurocognitive measures are assessed through a number of different tests and, based on these findings, genetic linkage studies are then made to try to map relevant loci and match them to the different processes reflected by corresponding endophenotypes. We conducted a review of the available literature on several endophenotypes that are currently being used in genetic research to assess to which degree they are useful and further understand what information can be retrieved by using them. Methods: Pubmed was used to retrieve recent articles regarding schizophrenia endophenotypes, with further articles being selected after analysis of relevant references from our selected pool of articles. Results: Six neurophysiological and neurocognitive endophenotypes were reviewed to assess their fulfillment of endophenotype criteria, and the ease of usage in schizophrenia research. Available data on their association with schizophrenia genetics was also included. Conclusions: Several endophenotypes are currently being used to conduct genetic research in an attempt to map genetic abnormalities in schizophrenia. Current research is still insufficient and further studies are required to replicate some findings, but overall endophenotypes show great promise in uncovering the neurobiology of schizophrenia in a genetic level. Keywords: Prepulse inhibition; antisaccade performance; P50 suppression; working memory; verbal declarative memory; sustained focus attention Braga D., Correia R. 3 Heredity and Schizophrenia: Endophenotypes and their Applicability Schizophrenia is a psychiatric disease affecting 0,5-1% of the world population, typically associated with paranoid delusions, disorganized speech and thinking and auditory hallucinations, severely impairing any individual’s social behavior and undermining social relations1. Even though a large number of genetic loci have been positively identified as schizophrenia-related, the pursue of genetic information related to psychiatric diseases met with difficulties: schizophrenia has a heritability described as as high as 80% - however, research has failed to identify a strong, single causative mutation so far1. There have been many candidate genes and chromosomal sites implied in the etiology of schizophrenia, mainly because of the large size of genetic sites involved, polygenic inheritance and epigenetic influence. 2 Given these challenges, a different approach to genetic research in the schizophrenia context emerged: instead of trying to link large syndromic findings to genetic alterations, some researchers attempted to break down these findings in smaller phenotypes and then try linking them to smaller genetic variations. The concept of endophenotype was created based on this theory, describing “internal phenotypes discoverable by a biochemical test or microscopical examination”3,4. Generally speaking, an endophenotype would allow for dissection of a complex phenotypical image in several smaller phenotypes, since small genetic alterations would be associated with small functional changes, that would, as a whole, match the overall phenotype in study; it would also imply that the genetical and functional abnormality would be more strongly associated than the association with the illness phenotype as a whole 5. The original definition of an endophenotype was further adapted as research progressed, with the current definition of a robust endophenotype being described as fulfilling the following criteria3,6: 1. Showing at least moderate effect sizes between schizophrenics and controls, 2. Deficits also present between unaffected family members and controls, 3. Being heritable, stable and showing reliability when measured between the different sites of the study, 4. Having at least some degree of evidence to a neurobiological substrate related to schizophrenia, 5. Reports on these measures are significant enough to suggest they can be used to test genetic loci associated with schizophrenia, 4 Endophenotypes and their Applicability Medication was not associated with irreversible alterations between schizophrenics and controls, meaning that the alterations in the results of both groups could not be fully attributed to them. Besides these criteria, endophenotypes should also be easily and quickly measured, allowing for the usage of large samples to provide statistic significance to candidate endophenotypes, while also reducing effort for study participants.7 These criteria were recently used in a large multi-centered family-based study organized by the Consortium on the Genetics of Schizophrenia (COGS), the first study of its kind to try to apply endophenotypes to retrieve information from a large sample of schizophrenia patients and their families. The COGS researchers selected six endophenotypes to be assessed from their population samples, and research is currently ongoing with some preliminary results already available8. Methods With the aim of reviewing current information regarding schizophrenia endophenotypes, a search was conducted on Pubmed in December 2014 with the query “schizophrenia AND (endophenotype OR endophenotypes)”. The search was restricted to the last five years, to studies with humans and to articles published in English or Portuguese. From the 114 search results, articles were selected or discarded based on the relevance of their abstracts and/or full texts, according to our objectives. We also gave preference to articles relating to the COGS study and the endophenotypes it assesses, discarding literature related to other endophenotypes. Reference lists from the selected articles were also reviewed and some relevant articles were retrieved for consultation, including some published earlier than the filter applied to the search. 36 articles were eventually included to produce this work. Neurophysiological Endophenotypes Schizophrenic patients show significant impairment of several neural circuits at a physiological level, which are translated in terms of brain function as a diminishment of sensory gating. This means that schizophrenics are not capable of skimming trivial stimuli from the environment and retain the important stimuli, which means they cannot adjust themselves to multiple or repetitive stimuli from their surroundings3,8. Several measures can Braga D., Correia R. 11 2. Greenwood TA, Swerdlow NR, Gur RE, et al. Genome-Wide Linkage Analyses of 12 Endophenotypes for Schizophrenia from the Consortium on the Genetics of Schizophrenia. The American journal of psychiatry. May 2013;170(5):521-532. 3. Gottesman II, Gould TD. The Endophenotype Concept in Psychiatry: Etymology and Strategic Intentions. The American journal of psychiatry. Apr 2003;160(4):636-645. 4. Gottesman II, Shields J. Genetic Theorizing and Schizophrenia. The British journal of psychiatry : the journal of mental science. Jan 1973;122(566):15-30. 5. Braff DL, Freedman R, Schork NJ, Gottesman, II. Deconstructing Schizophrenia: An Overview of the Use of Endophenotypes in Order to Understand a Complex Disorder. Schizophrenia bulletin. Jan 2007;33(1):21-32. 6. Calkins ME, Dobie DJ, Cadenhead KS, et al. The Consortium on the Genetics of Endophenotypes in Schizophrenia: Model Recruitment, Assessment, and Endophenotyping Methods for a Multisite Collaboration. Schizophrenia bulletin. January 1, 2007 2007;33(1):33-48. 7. Turetsky BI, Calkins ME, Light GA, Olincy A, Radant AD, Swerdlow NR. Neurophysiological Endophenotypes of Schizophrenia: The Viability of Selected Candidate Measures. Schizophrenia bulletin. Jan 2007;33(1):69-94. 8. Braff DL, Greenwood TA, Swerdlow NR, Light GA, Schork NJ, The Investigators of the Consortium on the Genetics of S. Advances in Endophenotyping Schizophrenia. World Psychiatry. 2008;7(1):11-18. 9. Gur RE, Calkins ME, Gur RC, et al. The Consortium on the Genetics of Schizophrenia: Neurocognitive Endophenotypes. Schizophrenia bulletin. Jan 2007;33(1):49-68. 10. Smucny J, Wylie K, Rojas D, et al. Evidence for Gamma and Beta Sensory Gating Deficits as Translational Endophenotypes for Schizophrenia. Psychiatry research. Nov 30 2013;214(2):169-174. 11. Light GA, Swerdlow NR, Rissling AJ, et al. Characterization of Neurophysiologic and Neurocognitive Biomarkers for Use in Genomic and Clinical Outcome Studies of Schizophrenia. PLoS ONE. 2012;7(7):e39434. 12. Swerdlow NR, Geyer MA, Braff DL. Neural Circuit Regulation of Prepulse Inhibition of Startle in the Rat: Current Knowledge and Future Challenges. Psychopharmacology. 2001;156(2-3):194-215. 12 Endophenotypes and their Applicability 13. Cadenhead KS, Carasso BS, Swerdlow NR, Geyer MA, Braff DL. Prepulse Inhibition and Habituation of the Startle Response Are Stable Neurobiological Measures in a Normal Male Population. Biological Psychiatry. 2/1/ 1999;45(3):360-364. 14. Swerdlow NR, Weber M, Qu Y, Light GA, Braff DL. Realistic Expectations of Prepulse Inhibition in Translational Models for Schizophrenia Research. Psychopharmacology. 06/21 2008;199(3):331-388. 15. Radant AD, Dobie DJ, Calkins ME, et al. Antisaccade Performance in Schizophrenia Patients, Their First-Degree Biological Relatives, and Community Comparison Subjects: Data from the Cogs Study. Psychophysiology. Sep 2010;47(5):846-856. 16. Karoumi B, Saoud M, d'Amato T, et al. Poor Performance in Smooth Pursuit and Antisaccadic Eye-Movement Tasks in Healthy Siblings of Patients with Schizophrenia. Psychiatry research. Apr 15 2001;101(3):209-219. 17. Egan MF, Goldberg TE, Kolachana BS, et al. Effect of Comt Val108/158 Met Genotype on Frontal Lobe Function and Risk for Schizophrenia. Proceedings of the National Academy of Sciences. June 5, 2001 2001;98(12):6917-6922. 18. Cornblatt BA, Malhotra AK. Impaired Attention as an Endophenotype for Molecular Genetic Studies of Schizophrenia. American journal of medical genetics. Jan 8 2001;105(1):11-15. 19. Jablensky A. The Diagnostic Concept of Schizophrenia: Its History, Evolution, and Future Prospects. Dialogues in Clinical Neuroscience. 2010;12(3):271-287. 20. Nuechterlein KH, Green MF, Calkins ME, et al. Attention/Vigilance in Schizophrenia: Performance Results from a Large Multi-Site Study of the Consortium on the Genetics of Schizophrenia (Cogs). Schizophrenia research. Mar 3 2015. 21. Cornblatt BA, Keilp JG. Impaired Attention, Genetics, and the Pathophysiology of Schizophrenia. Schizophrenia bulletin. 1994;20(1):31-46. 22. Amelsvoort Tv, Henry J, Morris R, et al. Cognitive Deficits Associated with Schizophrenia in Velo-Cardio-Facial Syndrome. Schizophrenia research. 10/1/ 2004;70(2–3):223-232. 23. Liu SK, Chen WJ, Chang C-J, Lin H-N. Effects of Atypical Neuroleptics on Sustained Attention Deficits in Schizophrenia. Neuropsychopharmacology. 03//print 2000;22(3):311-319. 24. Botero S, Munoz CC, Ocampo MV, et al. Verbal Working Memory in Individuals with Schizophrenia and Their First Degree Relatives: Relationship with Negative and Braga D., Correia R. 13 Disorganized Symptoms. Actas espanolas de psiquiatria. Mar-Apr 2013;41(2):106114. 25. Stone WS, Mesholam-Gately RI, Braff DL, et al. California Verbal Learning Test-Ii Performance in Schizophrenia as a Function of Ascertainment Strategy: Comparing the First and Second Phases of the Consortium on the Genetics of Schizophrenia (Cogs). Schizophrenia research. Dec 11 2014. 26. Kurtz MM. Neurocognitive Impairment across the Lifespan in Schizophrenia: An Update. Schizophrenia research. 4/1/ 2005;74(1):15-26. 27. Cannon TD, Hennah W, van Erp TM, et al. Association of Disc1/Trax Haplotypes with Schizophrenia, Reduced Prefrontal Gray Matter, and Impaired Shortand LongTerm Memory. Archives of General Psychiatry. 2005;62(11):1205-1213. 28. Paunio T, Tuulio-Henriksson A, Hiekkalinna T, et al. Search for Cognitive Trait Components of Schizophrenia Reveals a Locus for Verbal Learning and Memory on 4q and for Visual Working Memory on 2q. Human Molecular Genetics. August 15, 2004 2004;13(16):1693-1702. 29. Lussier I, Stip E. Memory and Attention Deficits in Drug Naive Patients with Schizophrenia. Schizophrenia research. 3/1/ 2001;48(1):45-55. 30. Barch DM, Carter CS, Braver TS, et al. Selective Deficits in Prefrontal Cortex Function in Medication-Naive Patients with Schizophrenia. Arch Gen Psychiatry. Mar 2001;58(3):280-288. 31. Lee J, Park S. Working Memory Impairments in Schizophrenia: A Meta-Analysis. Journal of abnormal psychology. Nov 2005;114(4):599-611. 32. Cannon TD, Glahn DC, Kim J, et al. Dorsolateral Prefrontal Cortex Activity During Maintenance and Manipulation of Information in Working Memory in Patients with Schizophrenia. Archives of General Psychiatry. 2005;62(10):1071-1080. 33. Selemon LD, Mrzljak J, Kleinman JE, Herman MM, Goldman-Rakic PS. Regional Specificity in the Neuropathologic Substrates of Schizophrenia: A Morphometric Analysis of Broca's Area 44 and Area 9. Archives of General Psychiatry. 2003;60(1):69-77. 34. Bruder GE, Keilp JG, Xu H, et al. Catechol-O-Methyltransferase (Comt) Genotypes and Working Memory: Associations with Differing Cognitive Operations. Biological Psychiatry. 12/1/ 2005;58(11):901-907. 35. Stefanis NC, Os JV, Avramopoulos D, et al. Variation in Catechol-OMethyltransferase Val158 Met Genotype Associated with Schizotypy but Not 14 Endophenotypes and their Applicability Cognition: A Population Study in 543 Young Men. Biological Psychiatry. 10/1/ 2004;56(7):510-515. 36. Greenwood TA, Light GA, Swerdlow NR, Radant AD, Braff DL. Association Analysis of 94 Candidate Genes and Schizophrenia-Related Endophenotypes. PLoS One. 2012;7(1):e29630. Agradecimentos Gostaria de agradecer à Dra Raquel Correia pelo seu trabalho e disponibilidade a orientar a elaboração desta monografia, e pela amabilidade com que o fez. INFORMATION FOR AUTHORS Please note that the journal now encourages authors to complete their license to publish form online Schizophrenia Bulletin is an international peer-reviewed journal that publishes unsolicited and invited reports and reviews of clinical and experimental research relating to all aspects of schizophrenia. Each issue is based on one or more themes with articles about recent advances in the clinical and basic scientific aspects of that area. A guest editor will be responsible for planning and organizing the theme content and will typically invite contributions from leaders in the field. Themes for future issues will be published in advance online.Schizophrenia Bulletin will consider unsolicited full-length manuscripts relating to any aspect of a future theme issue provided they have scientific merit and represent an important advance in knowledge. The Bulletin will also periodically publish an At Issue section focusing on theory or controversial topics including issues in ethics. Historical perspectives from patients and their families are also welcome. EDITORIAL POLICIES Manuscripts must be written in English and are accepted for consideration with an explicit understanding that the material has not been previously published in whole or substantial part and is not currently under consideration for publication by any other journal. All matters relating to the editorial policies of Schizophrenia Bulletin should be addressed in writing to Prof. William Carpenter, M.D., Editor-in Chief, Schizophrenia BulletinEditorial Office, Maryland Psychiatric Research Center, PO Box 21247, Baltimore, MD 21228, USA. Manuscripts should be submitted through the journal's web-based manuscript submission system as instructed below. Copyright Schizophrenia Bulletin does not require authors to transfer copyright of their submitted material. Rather, it is a condition of publication in the journal that authors grant an exclusive license to the Maryland Psychiatric Research Center and Oxford University Press. This ensures that requests from third parties to reproduce articles are handled efficiently and consistently and will also allow the article to be as widely disseminated as possible. In assigning the license, authors may use their own material in other publications provided that the Journal is acknowledged as the original place of publication, and that the Maryland Psychiatric Research Center and Oxford University Press are notified in writing and in advance. Informed Consent and Ethics Committee Approval Manuscripts reporting experiments on patients or healthy volunteers must record the fact that the subjects' consent was obtained and include a statement that the research was approved by the responsible ethical committee of the institution (e.g., an institutional review board) and was consistent with the principles outlined in an internationally recognized standard for the ethical conduct of human research. Consent must be also recorded when photographs of patients are shown or other details given that could lead to the identification of the individuals. Authors may be required to provide tangible proof that the necessary permissions and consents have been obtained from study participants. Laboratory Animals Manuscripts reporting the results of experiments involving laboratory animals must be contain a statement indicating that the procedures used were in accordance with the guidelines published in the Institute of Laboratory Animals Resources Commission on Life Sciences' 1996 Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academic Press; http://www.nap.edu/readingroom/books/labrats) or a similar internationally recognized standard. The species, sex, source, and genetic background of the animals as well as a detailed description of the experimental procedures, including any anesthetics and/or analgesics, must be provided in the Methods section of the manuscript. Manuscripts containing data from human or animal experimentation may be rejected if the ethical aspects are open to question. The corresponding author will be held responsible for false statements or for failure to meet the aforementioned requirements. Originality Schizophrenia Bulletin does not publish articles that overlap substantially with articles already published or accepted for publication, whether in print or in the electronic media, even if the new submission contains data not included in the published or accepted work. Schizophrenia Bulletin's policy is governed by international copyright laws, ethical conduct, and the cost-effective use of resources. Readers of primary-source periodicals trust that the material they are reading is original unless there is a statement that the article is being republished with the knowledge of the author and Editor and the permission of the original copyright holder. This policy does not preclude consideration of a report that follows a presentation at a meeting or expands preliminary findings published or presented as an abstract. A published article that the author thinks may overlap substantially with the manuscript submitted for review should be included with the submission. By submitting your manuscript to the journal it is understood that this is an original manuscript and is unpublished work not under consideration elsewhere. Plagiarism, including duplicate publication of the author‟s own work, in whole or in part without proper citation is not tolerated by the journal. Manuscripts submitted to the journal may be checked for originality using anti-plagiarism software. If an attempt at undisclosed duplicate publication is identified, the article will be rejected, the owners of the copyright will be notified, and the violation may be reported to the Conflict of Interest At the point of submission, Schizophrenia Bulletin's policy requires that each author reveal any financial interests or connections, direct or indirect, or other situations that might raise the question of bias in the work reported or the conclusions, implications, or opinions stated - including pertinent commercial or other sources of funding for the individual author(s) or for the associated department(s) or organization(s), personal relationships, or direct academic competition. When considering whether you should declare a conflicting interest or connection please consider the conflict of interest test: Is there any arrangement that would embarrass you or any of your co-authors if it was to emerge after publication and you had not declared it? Examples of potential conflicts include a proprietary interest in a drug or product mentioned in the study, equity interest in the sponsor of the study or any other commercial entity with a potential financial interest in its outcome, or payments with a cumulative monetary value exceeding $ 2,000 made by the sponsor to the investigators or their family members during or within two years of the completion of the study. Institutional support for the study should be included in the Acknowledgments section of the manuscript. All manuscripts submitted for publication will contain a Conflict of Interest statement. The corresponding author will describe each circumstance in sufficient detail to enable the editors and reviewers to assess its scope and to identify the author(s) with whom the conflict(s) exist. If the corresponding author has indicated that no conflict exists, the following statement will be inserted by the publisher and will appear at the end of the published manuscript: “The Authors have declared that there are no conflicts of interest in relation to the subject of this study.” Funding Details of all funding sources for the work in question should be given in a separate section entitled 'Funding'. This should appear before the 'Acknowledgments' section. The following rules should be followed:  The sentence should begin: „This work was supported by …‟  The full official funding agency name should be given, i.e. „the National Cancer Institute at the National Institutes of Health‟ or simply 'National Institutes of Health', not 'NCI' (one of the 27 subinstitutions) or „NCI at NIH‟ (full RINapproved list of UK funding agencies) .  Grant numbers should be complete and accurate and provided in parentheses as follows: „(grant number xxxx)‟  Multiple grant numbers should be separated by a comma as follows: „(grant numbers xxxx, yyyy)‟  Agencies should be separated by a semi-colon (plus „and‟ before the last funding agency)  Where individuals need to be specified for certain sources of funding the following text should be added after the relevant agency or grant number 'to [author initials]'. An example is given here: „This work was supported by the National Institutes of Health (P50 CA098252 and CA118790 to R.B.S.R.) and the Alcohol & Education Research Council (HFY GR667789).' MANUSCRIPT PREPARATION All manuscripts are submitted and reviewed via the journal's web-based manuscript submission system accessible at http://mc.manuscriptcentral.com/szbltn. New authors should create an account prior to submitting a manuscript for consideration. Manuscripts submitted to Schizophrenia Bulletin should be prepared following the American Medical Association Manual of Style, 10th edition. The manuscript text (including tables) should be prepared using a word processing program and saved as an .rtf or .doc file. Other file formats will not be accepted. Figures must be saved as individual .tif files and should be numbered consecutively (i.e., Figure 1.tif, Figure 2.tif, etc.). The text must be double-spaced throughout and should consist of the sections described below. Language Editing Non-native English speakers may wish to have their manuscript professionally edited prior to submission. While language editing does not guarantee that your paper will be accepted for publication, it may help to ensure that its academic content is fully understood by journal editors and reviewers. Authors are liable for all costs associated with the use of these services. Click here for additional information. Schizophrenia Bulletin authors have the option to publish their paper under the Oxford Open initiative; whereby, for a charge, their paper will be made freely available online immediately upon publication. After your manuscript is accepted the corresponding author will be required to accept a mandatory license to publish agreement. As part of the licensing process you will be asked to indicate whether or not you wish to pay for open access. If you do not select the open access option, your paper will be published with standard subscription-based access and you will not be charged. Oxford Open articles are published under Creative Commons licenses. RCUK/Wellcome Trust funded authors publishing in Schizophrenia Bulletin can use the Creative Commons Attribution license (CC-BY) for their articles. All other authors may use the following Creative Commons licenses: • Creative Commons Attribution Non-Commercial license (CC-BY-NC) • Creative Commons Attribution Non-Commercial No Derivatives license (CC-BY-NCND) Please click here for more information about the Creative Commons licenses. You can pay Open Access charges using our Author Services site. This will enable you to pay online with a credit/debit card, or request an invoice by email or post. The applicable open access charges vary according to which Creative Commons license you select. The open access charges are as follows. Charges for CC-BY-NC/CC-BY-NC-ND: • Regular charge: £1875/ $3000 / €2438 • List B Developing country charge*: £938/ $1500 / €1219 • List A Developing country charge*: £0 /$0 / €0 Charges for CC-BY: • Regular charge: £2125/ $3400 / €2763 • List B Developing country charge*: £1063/ $1700 / €1382 • List A Developing country charge*: £0 /$0 / €0 *Visit our developing countries page (click here for a list of qualifying countries). Please note that these charges are in addition to any color charges that may apply. Orders from the UK will be subject to the current UK VAT charge. For orders from the rest of the European Union, OUP will assume that the service is provided for business purposes. Please provide a VAT number for yourself or your institution, and ensure you account for your own local VAT correctly. PERMISSIONS FOR ILLUSTRATIONS AND FIGURES In order to reproduce any third party material, including figures or tables, in an article authors must obtain permission from the copyright holder and be compliant with any requirements the copyright holder may have pertaining to this reuse. When seeking to reproduce any kind of third party material authors should request the following: (i) non-exclusive rights to reproduce the material in the specified article and journal; (ii) electronic rights, preferably for use in any form or medium; (iii) the right to use the material for the life of the work; and (iv) world-wide English-language rights. Further guidelines on clearing permissions can be found at:http://www.oxfordjournals.org/access_purchase/permissions_guidelines.doc. Authors should also include a statement indicating that permission has been obtained in the relevant legend/footnote and provide the Editorial Office with copies of any relevant paperwork. A template permissions request letter can be found at the end of the above document.