RESSALVA Atendendo solicitação do(a) autor(a), o texto completo desta dissertação será disponibilizado somente a partir de 30/05/2024. UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” FACULDADE DE MEDICINA Raphaela Neto Pereira DIVERSIDADE GENÉTICA DO GENE HLA-DRA EM POPULAÇÕES MUNDIAIS E BUSCA DE POLIMORFISMOS FUNCIONAIS Dissertação apresentada à Faculdade de Medicina, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de Botucatu, como requisito para obtenção do título de Mestre no Programa de Patologia. Orientador: Prof. Dr. Erick da Cruz Castelli Botucatu 2022 Raphaela Neto Pereira DIVERSIDADE GENÉTICA DO GENE HLA-DRA EM POPULAÇÕES MUNDIAIS E BUSCA DE POLIMORFISMOS FUNCIONAIS Dissertação apresentada à Faculdade de Medicina, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de Botucatu, como requisito para obtenção do título de Mestre no Programa de Patologia. Orientador: Prof. Dr. Erick da Cruz Castelli Botucatu 2022 Palavras-chave: HLA-DRA; NGS; Polimorfismos; Variabilidade. Pereira, Raphaela Neto. Diversidade genética do gene HLA-DRA em populações mundiais e busca de polimorfismos funcionais. / Raphaela Neto Pereira. - Botucatu, 2022 Dissertação (mestrado) - Universidade Estadual Paulista "Júlio de Mesquita Filho", Faculdade de Medicina de Botucatu Orientador: Erick da Cruz Castelli Capes: 40105008 1. Diversidade genética. 2. Polimorfismo (Genética). 3. Cadeias alfa de HLA-DR. 4. Expressão gênica. DIVISÃO TÉCNICA DE BIBLIOTECA E DOCUMENTAÇÃO - CÂMPUS DE BOTUCATU - UNESP BIBLIOTECÁRIA RESPONSÁVEL: ROSEMEIRE APARECIDA VICENTE-CRB 8/5651 FICHA CATALOGRÁFICA ELABORADA PELA SEÇÃO TÉC. AQUIS. TRATAMENTO DA INFORM. Dedicatória Dedico este trabalho à minha mãe, que esteve sempre ao meu lado, lutando para que eu pudesse ser o que sou e estar onde estou, em busca do que sonho e acredito. Meu coração, minhas palavras e meus pensamentos estarão contigo. “Did you ever know that you're my hero, and everything I would like to be? I can fly higher than an eagle, for you are the wind beneath my wings.” - Bette Midler Agradecimentos “A felicidade pode ser encontrada mesmo nas horas mais difíceis, se você se lembrar de acender a luz” - Alvo Dumbledore Agradeço primeiramente à minha mãe e minha avó, que infelizmente não puderam participar desse momento, pelo menos não da forma como gostaria. Mãe, como foi difícil continuar sem você. Como você dizia, “te amo do tamanho do céu, porquê o céu não acaba”. Ao meu pai, o homem mais carinhoso que conheci na vida. Obrigada por acreditar em mim até quando eu mesma não acreditei. Sua parceirinha te ama. Aos meus irmãos, Mirella e Gustavo, pela paciência em me ouvir ensaiar todas as apresentações e o amor em me acolher sempre com abraços e palavras carinhosas. Amo vocês. Agradeço à minha “mãedrinha”, que esteve sempre presente durante toda minha vida e hoje, mais do que nunca, com muito amor e conselhos. Obrigada por sempre saber o que dizer. Amo você. Aos meus avós, Odete e José Prudêncio: sou extremamente feliz em tê-los como avós. Ao meu avô, um dos meus maiores exemplos nos estudos: Obrigada por me mostrar que nunca é tarde para aprender algo novo. Agradeço a todos os muitos amigos que estiveram ao meu lado, em especial, minhas amigas de escola Maria Julia Esteves, Rafaela Lanças Gomes e Camila Martins Cardoso e família, e meus amigos de faculdade, Thales, Jéssica e Marcos. Obrigada por estarem presente nos momentos difíceis. Hoje sou feliz em poder compartilhar essa conquista com vocês. À minha professora de matemática da graduação, Luciane Rodrigues de Souza, obrigada pelo carinho e por enxergar potencial em mim, me apresentando para meu futuro orientador de iniciação científica pelos próximos 4 anos de faculdade. Serei eternamente grata. Ao meu professor e orientador de IC, Newton Tamassia Pegolo, obrigada pela paciência em explicar tudo quantas vezes fossem necessárias. Obrigada por confiar em mim e me acompanhar em todos os eventos e apresentações; por ensaiar comigo e se preocupar; por dar conselhos relacionados à carreira científica e me incentivar a seguir nela, comemorando minha entrada no mestrado. Obrigada por me apresentar o “Admirável mundo novo” da genética. Às minhas parceiras de laboratório: Nayane S. B. Silva, Heloísa S. Andrade, Isabelle Mira Silva, Amanda Muniz, Lívia Ramos e Joyce Machado. Agradeço a paciência em me ensinar e ajudar com todo o conhecimento que construí até agora e o carinho e ombro amigo em todos os momentos. Vocês conquistaram em mim uma amiga para a vida toda. Contem comigo. Às minhas “roommates”, Viviane Oliveira e Gabriela Sato Paes, obrigada por aceitarem dividir essa jornada de aprendizado e crescimento pessoal e profissional comigo. Foram muitas lágrimas secadas com amor e parceria, e muitas risadas e conversas filosóficas. Ainda tem mais por vir. À professora Dra. Camila Ferreira Bannwart Castro, sempre carinhosa, protetora e amiga. Obrigada por todo amor e incentivo que deposita em todos nós. É uma honra poder aprender e conviver com você. Ao meu orientador Prof. Dr. Erick Castelli, agradeço a oportunidade de viver esse momento tão importante com pessoas especiais. Obrigada pela paciência em ensinar e pelo interesse em ouvir nossas opiniões, sempre incentivando o crescimento profissional de todos. Ao programa de Pós-Graduação em Patologia da Faculdade de Medicina de Botucatu (FMB-UNESP), pela oportunidade de construir minha formação científica. Aos membros da banca avaliadora, por aceitarem o convite e contribuírem com a minha formação. À CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), que possibilitou dedicação exclusiva ao trabalho desenvolvido através da bolsa de mestrado. Espero não ter esquecido ninguém. Felizmente fui agraciada com muitas pessoas boas ao meu redor. A todos: muito obrigada por não me deixarem esquecer de acender a luz. Sumário Lista de Figuras ....................................................................................................................................... 4 Lista de Tabelas ...................................................................................................................................... 5 Lista de abreviaturas e siglas .................................................................................................................. 6 RESUMO ................................................................................................................................................. 8 Capítulo I – Revisão de Literatura........................................................................................................... 9 INTRODUÇÃO ....................................................................................................................................... 10 ESTRUTURA E FUNÇÃO DAS MOLÉCULAS HLA DE CLASSE II ...................................... 11 Restrição do MHC ......................................................................................................................... 15 Apresentação cruzada .................................................................................................................. 15 O gene HLA-DRA .......................................................................................................................... 16 Genotipagem de genes HLA ........................................................................................................ 18 Bancos de dados públicos: 1000Genomes, Human Genome Diversity e SABE ................. 20 1000 Genomes ............................................................................................................................................................20 HGDP – Human Genome Diversity Project ................................................................................................21 SABE – Saúde, Bem-Estar e Envelhecimento .........................................................................................21 Características da população brasileira ..................................................................................... 22 JUSTIFICATIVA ...................................................................................................................................... 24 OBJETIVOS ............................................................................................................................................ 25 Objetivo Geral ................................................................................................................................ 25 Objetivos Específicos .................................................................................................................... 25 REFERÊNCIAS ........................................................................................................................................ 26 Capítulo II – Artigo................................................................................................................................ 31 HLA-DRA genetic diversity across different populations ...................................................................... 32 Abstract ................................................................................................................................................ 33 Introduction ......................................................................................................................................... 34 Methods and samples .......................................................................................................................... 36 Samples included in this survey ....................................................................................................... 36 The HLA-DRA region we have studied .............................................................................................. 37 Read alignment and variant calling .................................................................................................. 37 HLA allele calling .............................................................................................................................. 38 Statistical analysis ............................................................................................................................ 38 Results .................................................................................................................................................. 39 HLA-DRA variants and nucleotide diversity ...................................................................................... 39 HLA-DRA genomic alleles (four-field resolution) .............................................................................. 42 HLA-DRA exonic sequences (three-field resolution) ......................................................................... 43 HLA-DRA protein sequences (two-field resolution) .......................................................................... 43 HLA-DRA 3'UTR region ..................................................................................................................... 44 HLA-DRA expression levels ............................................................................................................... 46 Discussion ............................................................................................................................................ 46 References ........................................................................................................................................... 51 Material Suplementar .......................................................................................................................... 54 Lista de Figuras Figura 1. Localização e organização do complexo HLA no cromossomo 6..........................................................................................................................................11 Figura 2. Estrutura da molécula de HLA classe II...............................................................12 Figura 3. Formação da molécula de HLA de classe II e sua via de apresentação de antígeno..............................................................................................................................14 Figura 4. Apresentação cruzada de células dendríticas específicas..................................16 Figura 5. Estrutura do gene HLA-DRA................................................................................18 Lista de Figuras do artigo Figure 1: Plot illustrating the variants (SNPs, indels, multi-allelic variants) across the HLA- DRA locus, frequency, and effect………………………………………………………………..40 Figure 2: Ternary plot indicating the relative frequency of HLA-DRA variants (SNPs and indels) in different parental populations…………………………………………………………41 Figure 3: Linkage disequilibrium encompassing the HLA-DRA locus, considering 5,347 individuals from worldwide population samples and SNPs with minimum allele frequency higher than 2%....................................................................................................................42 Figure 4: Frequency distribution of the DRA*01:03 allele in worldwide populations……….44 Figure 5: The variable sites in the HLA-DRA 3' untranslated region, and the haplotypes formed by them…………………………………………………………………………………….46 Lista de Figuras do artigo – Material Suplementar Figure S1: Frequency distribution of the DRA*01:01 allele in worldwide populations………………………………………………………………………...…….……...55 Figure S2: Frequency distribution of the DRA*01:02 allele in worldwide populations……………………………………………………………………………………...55 Figure S3: List of microRNAs that are influenced by polymorphisms in the HLA-DRA 3'UTR region and the strength of the bindings measured by the Minimum Free Energy(MFE)…………………………………………………………………….……………...56 Figure S4: The alignment between the target (HLA-DRA 3'UTR sequences) and miR- 106b-3p, illustrating how rs1131541 influences the mRNA/miRNA binding affinity………………………………………………………………………………….………...56 4 5 Lista de Tabelas Lista de Tabelas – Material suplementar Table S1. SNP frequency of the HLA-DRA locus among all biogeographic regions…………………………………………………………………………………ANEXO Table S2. Frequency of HLA-DRA genomic sequences among all populations, countries and biogeographic regions…….…………………………………………ANEXO Table S3. Frequency of HLA-DRA exonic sequences among all populations, countries and biogeographic regions…….………………………………………….…………ANEXO Table S4. Frequency of HLA-DRA allotypes among all populations, countries and biogeographic regions…….………………………………………….…………...…ANEXO 6 Lista de abreviaturas e siglas APC Célula Apresentadora de Antígeno (do inglês, Antigen Presenting Cell) AR Artrite Reumatóide CLIP Cadeia Invariante associada a classe II CTL Linfócito T Citotóxico DC Célula dendrítica (do inglês, Dendritic Cell) EM Esclerose Múltipla GWAS Estudos de associação genômica ampla (do inglês, Genome-wide Association Studies) HLA Antígeno Leucocitário Humano (do inglês, Human Leukocyte Antigens) IFN-α Interferon alfa Ig Imunoglobulina IMGT International Immunogenetics Database LD Desequilíbrio de ligação (do inglês, Linkage Disequilibrium) MHC Complexo Principal de Histocompatibilidade (do inglês, Major Histocompatibility Complex) MIIC Compartimento do MHC de classe II miRNA microRNA mRNA Ácido Ribonucleico mensageiro ou RNA mensageiro NGS Sequenciamento de Nova Geração (do inglês, Next-Generation Sequencing) NK Célula Assassina Natural (do inglês, Natural Killer) PCR Reação em Cadeia da Polimerase (do inglês, Polymerase Chain Reaction) pMHC peptídeo+MHC RE Retículo Endoplasmático SNP Polimorfismo de base única (do inglês, Single Nucleotide Polymorphism) TCR Receptor de célula T (do inglês, T Cell Receptor) UTR Região não traduzida (do inglês, Untranslated region) 8 RESUMO A molécula de HLA-DR, alvo do presente estudo, é um heterodímero de superfície celular responsável pela apresentação de peptídeos de origem exógena aos linfócitos T CD4. HLA-DR é composta por duas cadeias: α, codificada pelo gene HLA-DRA, e β, codificada principalmente pelo gene HLA-DRB1. Enquanto HLA-DRA é pouco polimórfico, HLA-DRB1 é bastante variável e influencia diretamente o perfil de peptídeos apresentados. Os genes HLA se originaram por duplicação gênica e possuem elevada similaridade entre si. Por conta disso, a análise de sequências geradas por estratégias de sequenciamento massivo paralelo se torna um grande desafio, necessitando de ferramentas especializadas que corrigem alinhamentos e detectam adequadamente as variantes genéticas e haplótipos. Este estudo utilizou uma metodologia computacional desenhada exclusivamente para os genes HLA de classe II, avaliando a diversidade do gene HLA-DRA em cerca de 5000 amostras de diferentes populações, de todos os continentes, incluindo o Brasil. O gene HLA-DRA demonstrou-se pouco variável em suas regiões exônicas, com mutações não- sinônimas apresentando apenas 3 proteínas distintas. Dessas, apenas duas são frequentes: DRA*01:01 (67%) e DRA*01:02 (29%). No entanto, há uma grande quantidade de polimorfismos em íntrons (168 SNPs), muitos deles bastante frequentes, com Minor Allele Frequence (MAF) maior do que 1%. Palavras-chave: HLA-DRA, variabilidade, polimorfismos, NGS. 26 REFERÊNCIAS 1. Matzaraki V, Kumar V, Wijmenga C, Zhernakova A. The MHC locus and genetic susceptibility to autoimmune and infectious diseases. Genome Biol. 2017 Dec;18(1):76. 2. Rock KL, Reits E, Neefjes J. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends in Immunology. 2016 Nov;37(11):724–37. 3. Abbas A, Lichtman A. Imunologia Celular e Molecular 8a Edição. :1262. 4. Charles A Janeway J, Travers P, Walport M, Shlomchik MJ. The major histocompatibility complex and its functions. Immunobiology: The Immune System in Health and Disease 5th edition [Internet]. 2001 [cited 2021 Nov 8]; Available from: https://www.ncbi.nlm.nih.gov/books/NBK27156/ 5. Monos DS, Winchester RJ. The Major Histocompatibility Complex. In: Clinical Immunology [Internet]. Elsevier; 2019 [cited 2021 Sep 19]. p. 79-92.e1. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780702068966000053 6. Castelli EC, de Castro MV, Naslavsky MS, Scliar MO, Silva NSB, Andrade HS, et al. MHC Variants Associated With Symptomatic Versus Asymptomatic SARS-CoV-2 Infection in Highly Exposed Individuals. Front Immunol. 2021 Sep 28;12:742881. 7. Meyer D, C. Aguiar VR, Bitarello BD, C. Brandt DY, Nunes K. A genomic perspective on HLA evolution. Immunogenetics. 2018 Jan;70(1):5–27. 8. Goldberg AC, Rizzo LV. MHC structure and function – antigen presentation. Part 1. Einstein (São Paulo). 2015 Mar 24;13(1):153–6. 9. Donadi EA, Castelli EC, Arnaiz-Villena A, Roger M, Rey D, Moreau P. Implications of the polymorphism of HLA-G on its function, regulation, evolution and disease association. Cell Mol Life Sci. 2011 Feb;68(3):369–95. 10. Klein J, Sato A. The HLA system. First of two parts. N Engl J Med. 2000 Sep 7;343(10):702–9. 11. Alicia SM. A review of HLA allele and SNP associations with highly prevalent infectious diseases in human populations. Swiss Medical Weekly. :14. 12. Blum JS, Wearsch PA, Cresswell P. Pathways of Antigen Processing. Annu Rev Immunol. 2013;31:443–73. 13. Unanue ER, Turk V, Neefjes J. Variations in MHC Class II Antigen Processing and Presentation in Health and Disease. Annu Rev Immunol. 2016 May 20;34(1):265–97. 14. The CLIP region of invariant chain plays a critical role in regulating major histocompatibility complex class II folding, transport, and peptide occupancy. J Exp Med. 1994 Sep 1;180(3):1107– 13. 15. Peters PJ, Neefjes JJ, Oorschot V, Ploegh HL, Geuze HJ. Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature. 1991 Feb;349(6311):669–76. 16. Goldberg AC, Rizzo LV. MHC structure and function − antigen presentation. Part 2. Einstein (São 27 Paulo). 2015 Mar 24;13(1):157–62. 17. Pos W, Sethi DK, Call MJ, Schulze MSED, Anders AK, Pyrdol J, et al. Crystal Structure of the HLA- DM - HLA-DR1 Complex Defines Mechanisms for Rapid Peptide Selection. Cell. 2012 Dec 21;151(7):1557–68. 18. Anders AK, Call MJ, Schulze MSED, Fowler KD, Schubert DA, Seth NP, et al. HLA-DM Captures Partially Empty HLA-DR Molecules for Catalyzed Peptide Removal. Nat Immunol. 2011 Jan;12(1):54–61. 19. Mosyak L, Zaller DM, Wiley DC. The Structure of HLA-DM, the Peptide Exchange Catalyst that Loads Antigen onto Class II MHC Molecules during Antigen Presentation. Immunity. 1998 Sep;9(3):377–83. 20. Morris P, Shaman J, Attaya M, Amaya M, Goodman S, Bergman C, et al. An essential role for HLA–DM in antigen presentation by class II major histocompatibility molecules. Nature. 1994 Apr;368(6471):551–4. 21. Denzin LK, Sant’Angelo DB, Hammond C, Surman MJ, Cresswell P. Negative Regulation by HLA- DO of MHC Class II-Restricted Antigen Processing. Science. 1997 Oct 3;278(5335):106–9. 22. Fallas JL, Tobin HM, Lou O, Guo D, Sant’Angelo DB, Denzin LK. Ectopic Expression of HLA-DO in Mouse Dendritic Cells Diminishes MHC Class II Antigen Presentation. J Immunol. 2004 Aug 1;173(3):1549–60. 23. Yi W, Seth NP, Martillotti T, Wucherpfennig KW, Sant’Angelo DB, Denzin LK. Targeted regulation of self-peptide presentation prevents type I diabetes in mice without disrupting general immunocompetence. J Clin Invest. 2010 Apr 1;120(4):1324–36. 24. Kourilsky P, Claverie JM. MHC restriction, alloreactivity, and thymic education: a common link? Cell. 1989 Feb 10;56(3):327–9. 25. La Gruta NL, Gras S, Daley SR, Thomas PG, Rossjohn J. Understanding the drivers of MHC restriction of T cell receptors. Nat Rev Immunol. 2018 Jul;18(7):467–78. 26. Adams EJ, Luoma AM. The Adaptable Major Histocompatibility Complex (MHC) Fold: Structure and Function of Nonclassical and MHC Class I–Like Molecules. Annu Rev Immunol. 2013 Mar 21;31(1):529–61. 27. Kurts C, Robinson BWS, Knolle PA. Cross-priming in health and disease. Nat Rev Immunol. 2010 Jun;10(6):403–14. 28. Dudziak D, Kamphorst AO, Heidkamp GF, Buchholz VR, Trumpfheller C, Yamazaki S, et al. Differential antigen processing by dendritic cell subsets in vivo. Science. 2007 Jan 5;315(5808):107–11. 29. Kumar S, Jeong Y, Ashraf MU, Bae YS. Dendritic Cell-Mediated Th2 Immunity and Immune Disorders. IJMS. 2019 May 1;20(9):2159. 30. Lima THA, Souza AS, Porto IOP, Paz MA, Veiga-Castelli LC, Oliveira MLG, et al. HLA-A promoter, coding, and 3’UTR sequences in a Brazilian cohort, and their evolutionary aspects. HLA. 2019 Feb;93(2–3):65–79. 28 31. Matern BM, Olieslagers TI, Voorter CEM, Groeneweg M, Tilanus MGJ. Insights into the polymorphism in HLA‐DRA and its evolutionary relationship with HLA haplotypes. HLA. 2020 Feb;95(2):117–27. 32. Andersson G. Evolution of the human HLA-DR region. Front Biosci. 1998;3(4):d739-745. 33. Guéant JL, Romano A, Cornejo-Garcia JA, Oussalah A, Chery C, Blanca-López N, et al. HLA-DRA variants predict penicillin allergy in genome-wide fine-mapping genotyping. Journal of Allergy and Clinical Immunology. 2015 Jan;135(1):253-259.e10. 34. Boegel S, Löwer M, Bukur T, Sorn P, Castle JC, Sahin U. HLA and proteasome expression body map. BMC Med Genomics. 2018 Dec;11(1):36. 35. Bontrop RE, Otting N, de Groot NG, Doxiadis GG. Major histocompatibility complex class II polymorphisms in primates. Immunol Rev. 1999 Feb;167:339–50. 36. Klein J, Sato A. The HLA system. Second of two parts. N Engl J Med. 2000 Sep 14;343(11):782–6. 37. Storti-Melo LM, Costa DR da, Souza-Neiras WC, Cassiano GC, Bonini-Domingos CR, Rossit ARB, et al. The frequency of HLA-DRB1 polymorphisms in Brazilian Plasmodium vivax malaria patients and in blood donors from the Amazon Region. Rev Pan-Amaz Saude. 2010 Dec;1(4):51–5. 38. SciELO - Brasil - Alopecia areata: revisão e atualização Alopecia areata: revisão e atualização [Internet]. [cited 2021 Nov 7]. Available from: https://www.scielo.br/j/abd/a/vXCLdmVdz8ct6qzkmjBCSyd/?lang=pt 39. A review of the MHC genetics of rheumatoid arthritis | Genes & Immunity [Internet]. [cited 2021 Nov 7]. Available from: https://www.nature.com/articles/6364045 40. Kochi Y, Suzuki A, Yamada R, Yamamoto K. Genetics of rheumatoid arthritis: underlying evidence of ethnic differences. J Autoimmun. 2009 Jun;32(3–4):158–62. 41. de Vries R. Genetics of rheumatoid arthritis: time for a change! Curr Opin Rheumatol. 2011 May;23(3):227–32. 42. Usnayo MJG, Andrade LEC, Alarcon RT, Oliveira JC, Silva GMF, Bendet I, et al. Estudo da frequência dos alelos de HLA-DRB1 em pacientes brasileiros com artrite reumatoide. Rev Bras Reumatol. 2011 Nov;51(5):474–83. 43. Mourad J, Monem F. Associação do alelo HLA-DRB1 com suscetibilidade a artrite reumatoide e gravidade da doença na Síria. Rev Bras Reumatol. 2013 Feb;53:51–6. 44. Gonzalez-Juanatey C, Testa A, Garcia-Castelo A, Garcia-Porrua C, Llorca J, Vidan J, et al. HLA- DRB1 status affects endothelial function in treated patients with rheumatoid arthritis. Am J Med. 2003 Jun 1;114(8):647–52. 45. Barcellos LF, Sawcer S, Ramsay PP, Baranzini SE, Thomson G, Briggs F, et al. Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis. Hum Mol Genet. 2006 Sep 15;15(18):2813–24. 46. Do MD, Le LGH, Nguyen VT, Dang TN, Nguyen NH, Vu HA, et al. High-Resolution HLA Typing of HLA-A, -B, -C, -DRB1, and -DQB1 in Kinh Vietnamese by Using Next-Generation Sequencing. Frontiers in Genetics. 2020;11:383. 29 47. Anasetti C, Amos D, Beatty PG, Appelbaum FR, Bensinger W, Buckner CD, et al. Effect of HLA Compatibility on Engraftment of Bone Marrow Transplants in Patients with Leukemia or Lymphoma. N Engl J Med. 1989 Jan 26;320(4):197–204. 48. Mahdi BM. A glow of HLA typing in organ transplantation. Clin Transl Med. 2013 Feb 23;2(1):6. 49. Opelz G, Mytilineos J, Scherer S, Dunckley H, Trejaut J, Chapman J, et al. Survival of DNA HLA-DR typed and matched cadaver kidney transplants. The Lancet. 1991 Aug;338(8765):461–3. 50. Park I, Terasaki P. Origins of the first HLA specificities. Human Immunology. 2000 Mar 1;61(3):185–9. 51. Hurley CK. Naming HLA diversity: A review of HLA nomenclature. Human Immunology. 2021 Jul;82(7):457–65. 52. Erlich HA, Opelz G, Hansen J. HLA DNA Typing and Transplantation. Immunity. 2001 Apr;14(4):347–56. 53. Franceschi DAS, Viel DO, Sell AM, Tsuneto LT, Visentainer JEL. Otimização de metodologia PCR- SSP para identificação de polimorfismos genéticos de TNF e IL2. Rev Bras Hematol Hemoter. 2009 Aug;31:241–6. 54. Douillard V, Castelli EC, Mack SJ, Hollenbach JA, Gourraud PA, Vince N, et al. Approaching Genetics Through the MHC Lens: Tools and Methods for HLA Research. Frontiers in Genetics. 2021;12:2403. 55. Castelli EC, Paz MA, Souza AS, Ramalho J, Mendes-Junior CT. Hla-mapper: An application to optimize the mapping of HLA sequences produced by massively parallel sequencing procedures. Human Immunology. 2018 Sep;79(9):678–84. 56. Belsare S, Levy-Sakin M, Mostovoy Y, Durinck S, Chaudhuri S, Xiao M, et al. Evaluating the quality of the 1000 genomes project data. BMC Genomics. 2019 Dec;20(1):620. 57. Byrska-Bishop M, Evani US, Zhao X, Basile AO, Regier AA, Corvelo A, et al. High coverage whole genome sequencing of the expanded 1000 Genomes Project cohort including 602 trios. :51. 58. The 1000 Genomes Project Consortium. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012 Nov;491(7422):56–65. 59. Brandt DYC, Aguiar VRC, Bitarello BD, Nunes K, Goudet J, Meyer D. Mapping Bias Overestimates Reference Allele Frequencies at the HLA Genes in the 1000 Genomes Project Phase I Data. :11. 60. The 1000 Genomes Project Consortium. A map of human genome variation from population- scale sequencing. Nature. 2010 Oct 28;467(7319):1061–73. 61. The 1000 Genomes Project Consortium, Corresponding authors, Auton A, Abecasis GR, Steering committee, Altshuler DM, et al. A global reference for human genetic variation. Nature. 2015 Oct 1;526(7571):68–74. 62. Sudmant PH, Rausch T, Gardner EJ, Handsaker RE, Abyzov A, Huddleston J, et al. An integrated map of structural variation in 2,504 human genomes. Nature. 2015 Oct 1;526(7571):75–81. 63. Cavalli-Sforza LL. The Human Genome Diversity Project: past, present and future. 2005;8. 30 64. Greely HT. Human genome diversity: What about the other human genome project? Nat Rev Genet. 2001 Mar;2(3):222–7. 65. Lebrão ML, Duarte YA de O, Santos JLF, Silva NN da. 10 Anos do Estudo SABE: antecedentes, metodologia e organização do estudo. Rev bras epidemiol. 2018;21(suppl 2):e180002. 66. Naslavsky MS, Scliar MO, Yamamoto GL, Wang JYT, Zverinova S, Karp T, et al. Whole-genome sequencing of 1,171 elderly admixed individuals from the largest Latin American metropolis (São Paulo, Brazil). :29. 67. Fabreti-Oliveira RA, Nascimento E, Fonseca CG, Santos MA. The heterogeneous HLA genetic composition of the Brazilian population and its relevance to the optimization of hematopoietic stem cell donor recruitment: The heterogeneous HLA genetic composition of the Brazilian population. Tissue Antigens. 2014 Aug;84(2):187–97. 68. Pena SDJ, Pietro GD, Fuchshuber-Moraes M, Genro JP, Hutz MH, Kehdy F de SG, et al. The Genomic Ancestry of Individuals from Different Geographical Regions of Brazil Is More Uniform Than Expected. PLOS ONE. 2011 Feb 16;6(2):e17063. 69. IBGE | Brasil: 500 anos de povoamento [Internet]. [cited 2022 Jan 7]. Available from: https://brasil500anos.ibge.gov.br/# 70. Reflexões sobre os Deslocamentos Populacionais no Brasil | IBGE [Internet]. [cited 2022 Jan 7]. Available from: https://www.ibge.gov.br/estatisticas/sociais/populacao/9159-reflexoes-sobre- os-deslocamentos-populacionais-no-brasil.html 71. Ferreira LB, Mendes -Junior CT, Wiezel CEV, Luizon MR, Simões AL. Genomic ancestry of a sample population from the state of São Paulo, Brazil. American Journal of Human Biology. 2006;18(5):702–5. 72. Salzano FM, Hutz MH. Genética, genômica e populações nativas brasileiras – história e biomedicina. :23. 73. Secolin R, Mas-Sandoval A, Arauna LR, Torres FR, de Araujo TK, Santos ML, et al. Distribution of local ancestry and evidence of adaptation in admixed populations. Sci Rep. 2019 Dec;9(1):13900. 74. Gouveia MH, Borda V, Leal TP, Moreira RG, Bergen AW, Kehdy FSG, et al. Origins, Admixture Dynamics, and Homogenization of the African Gene Pool in the Americas. Nielsen R, editor. Molecular Biology and Evolution. 2020 Jun 1;37(6):1647–56. 75. Gogarten SM, Sofer T, Chen H, Yu C, Brody JA, Thornton TA, et al. Genetic association testing using the GENESIS R/Bioconductor package. Valencia A, editor. Bioinformatics. 2019 Dec 15;35(24):5346–8. 76. Zhang F, Flickinger M, Taliun SAG, InPSYght Psychiatric Genetics Consortium, Abecasis GR, Scott LJ, et al. Ancestry-agnostic estimation of DNA sample contamination from sequence reads. Genome Res. 2020 Feb;30(2):185–94. 77. Alexander DH, Novembre J, Lange K. Fast model-based estimation of ancestry in unrelated individuals. Genome Res. 2009 Sep;19(9):1655–64. 78. Popejoy AB, Fullerton SM. Genomics is failing on diversity. Nature. 2016 Oct;538(7624):161–4. 33 Abstract The focus of this study is the HLA-DRA gene, which encodes the α chain of the HLA class II molecule HLA-DR. HLA-DR is a heterodimer composed of two chains, the α (from HLA-DRA) and β (encoded by HLA-DRB genes). HLA-DR present exogenous peptides to T CD4 lymphocytes, and is expressed on antigen-presenting cells such as monocytes, dendritic cells, macrophages, and B lymphocytes. While HLA-DRB1 is one of the most polymorphic HLA genes, HLA-DRA is conserved considering its exonic sequences and the molecule it encodes. Although we have public databases with full genomes from different countries, such as the 1000 Genomes, these databases do not include or present very few samples from Latin America, and Brazil is not represented in any of these genomic initiatives. Characterizing admixed samples may add new alleles and haplotypes to our databases and give a broad knowledge of human genetic diversity. Moreover, HLA- DRA typing is usually not performed before transplantation, and therefore its genetic diversity might be highly underestimated. Here, we applied a pipeline to analyze HLA genes from whole-genome data, reporting the HLA-DRA genetic diversity in worldwide population samples, including Brazilians. HLA-DRA is far more polymorphic than we currently acknowledge, particularly in introns and regulatory sequences, but its low protein diversity is sustained. We also detected that polymorphisms in the HLA-DRA 3'UTR might be related to differential expression profiles by influencing the binding of microRNAs. In addition, we provide datasets that can be used in future evolutionary disease-association studies. Keywords: HLA-DRA, variability, NGS, populations worldwide. 51 References 1. Zajonc, D. M. Unconventional Peptide Presentation by Classical MHC Class I and Implications for T and NK Cell Activation. Int. J. Mol. Sci. 21, (2020). 2. Klein, J. & Sato, A. The HLA system. First of two parts. N. Engl. J. Med. 343, 702–709 (2000). 3. Rock, K. L., Reits, E. & Neefjes, J. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends Immunol. 37, 724–737 (2016). 4. Matern, B. M., Olieslagers, T. I., Voorter, C. E. M., Groeneweg, M. & Tilanus, M. G. J. Insights into the polymorphism in HLA-DRA and its evolutionary relationship with HLA haplotypes. Hladnikia 95, 117–127 (2020). 5. Sarri, C. A. et al. Amino acid signatures in the HLA class II peptide-binding region associated with protection/susceptibility to the severe West Nile Virus disease. PLoS One 13, e0205557 (2018). 6. Stern, L. J. et al. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368, 215–221 (1994). 7. Flajnik, M. F. & Kasahara, M. Origin and evolution of the adaptive immune system: genetic events and selective pressures. Nat. Rev. Genet. 11, 47–59 (2010). 8. Kotsch, K. & Blasczyk, R. The noncoding regions of HLA-DRB uncover interlineage recombinations as a mechanism of HLA diversification. J. Immunol. 165, 5664–5670 (2000). 9. Bontrop, R. E., Otting, N., de Groot, N. G. & Doxiadis, G. G. Major histocompatibility complex class II polymorphisms in primates. Immunol. Rev. 167, 339–350 (1999). 10. Robinson, J. et al. The IPD and IMGT/HLA database: allele variant databases. Nucleic Acids Res. 43, D423–31 (2015). 11. Scally, S. W. et al. A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis. J. Exp. Med. 210, 2569–2582 (2013). 12. Andersson, G. Evolution of the human HLA-DR region. Frontiers in Bioscience vol. 3 d739–745 (1998). http://paperpile.com/b/07uDCz/A3u3 http://paperpile.com/b/07uDCz/A3u3 http://paperpile.com/b/07uDCz/A3u3 http://paperpile.com/b/07uDCz/A3u3 http://paperpile.com/b/07uDCz/A3u3 http://paperpile.com/b/07uDCz/A3u3 http://paperpile.com/b/07uDCz/U4wB http://paperpile.com/b/07uDCz/U4wB http://paperpile.com/b/07uDCz/U4wB http://paperpile.com/b/07uDCz/U4wB http://paperpile.com/b/07uDCz/U4wB http://paperpile.com/b/07uDCz/TJh2 http://paperpile.com/b/07uDCz/TJh2 http://paperpile.com/b/07uDCz/TJh2 http://paperpile.com/b/07uDCz/TJh2 http://paperpile.com/b/07uDCz/TJh2 http://paperpile.com/b/07uDCz/TJh2 http://paperpile.com/b/07uDCz/caTm http://paperpile.com/b/07uDCz/caTm http://paperpile.com/b/07uDCz/caTm http://paperpile.com/b/07uDCz/caTm http://paperpile.com/b/07uDCz/caTm http://paperpile.com/b/07uDCz/caTm http://paperpile.com/b/07uDCz/4qH9 http://paperpile.com/b/07uDCz/4qH9 http://paperpile.com/b/07uDCz/4qH9 http://paperpile.com/b/07uDCz/4qH9 http://paperpile.com/b/07uDCz/4qH9 http://paperpile.com/b/07uDCz/4qH9 http://paperpile.com/b/07uDCz/4qH9 http://paperpile.com/b/07uDCz/4qH9 http://paperpile.com/b/07uDCz/dQtz http://paperpile.com/b/07uDCz/dQtz http://paperpile.com/b/07uDCz/dQtz http://paperpile.com/b/07uDCz/dQtz http://paperpile.com/b/07uDCz/dQtz http://paperpile.com/b/07uDCz/dQtz http://paperpile.com/b/07uDCz/dQtz http://paperpile.com/b/07uDCz/dQtz http://paperpile.com/b/07uDCz/gaUI http://paperpile.com/b/07uDCz/gaUI http://paperpile.com/b/07uDCz/gaUI http://paperpile.com/b/07uDCz/gaUI http://paperpile.com/b/07uDCz/gaUI http://paperpile.com/b/07uDCz/gaUI http://paperpile.com/b/07uDCz/Vtcw http://paperpile.com/b/07uDCz/Vtcw http://paperpile.com/b/07uDCz/Vtcw http://paperpile.com/b/07uDCz/Vtcw http://paperpile.com/b/07uDCz/Vtcw http://paperpile.com/b/07uDCz/Vtcw http://paperpile.com/b/07uDCz/WL5c http://paperpile.com/b/07uDCz/WL5c http://paperpile.com/b/07uDCz/WL5c http://paperpile.com/b/07uDCz/WL5c http://paperpile.com/b/07uDCz/WL5c http://paperpile.com/b/07uDCz/WL5c http://paperpile.com/b/07uDCz/34DF http://paperpile.com/b/07uDCz/34DF http://paperpile.com/b/07uDCz/34DF http://paperpile.com/b/07uDCz/34DF http://paperpile.com/b/07uDCz/34DF http://paperpile.com/b/07uDCz/34DF http://paperpile.com/b/07uDCz/34DF http://paperpile.com/b/07uDCz/34DF http://paperpile.com/b/07uDCz/sXcL http://paperpile.com/b/07uDCz/sXcL http://paperpile.com/b/07uDCz/sXcL http://paperpile.com/b/07uDCz/sXcL http://paperpile.com/b/07uDCz/sXcL http://paperpile.com/b/07uDCz/sXcL http://paperpile.com/b/07uDCz/sXcL http://paperpile.com/b/07uDCz/sXcL http://paperpile.com/b/07uDCz/n2np http://paperpile.com/b/07uDCz/n2np http://paperpile.com/b/07uDCz/n2np http://paperpile.com/b/07uDCz/n2np 52 13. Guéant, J.-L. et al. HLA-DRA variants predict penicillin allergy in genome-wide fine-mapping genotyping. J. Allergy Clin. Immunol. 135, 253–259 (2015). 14. Castelli, E. C., Paz, M. A., Souza, A. S., Ramalho, J. & Mendes-Junior, C. T. Hla-mapper: An application to optimize the mapping of HLA sequences produced by massively parallel sequencing procedures. Hum. Immunol. 79, 678–684 (2018). 15. Fairley, S., Lowy-Gallego, E., Perry, E. & Flicek, P. The International Genome Sample Resource (IGSR) collection of open human genomic variation resources. Nucleic Acids Res. 48, D941–D947 (2020). 16. Bergström, A. et al. Insights into human genetic variation and population history from 929 diverse genomes. Science 367, (2020). 17. Parra, F. C. et al. Color and genomic ancestry in Brazilians. Proc. Natl. Acad. Sci. U. S. A. 100, 177–182 (2003). 18. Ferreira, L. B., Mendes-Junior, C. T., Wiezel, C. E. V., Luizon, M. R. & Simões, A. L. Genomic ancestry of a sample population from the state of São Paulo, Brazil. Am. J. Hum. Biol. 18, 702– 705 (2006). 19. Naslavsky, M. S. et al. Whole-genome sequencing of 1,171 elderly admixed individuals from the largest Latin American metropolis (São Paulo, Brazil). Cold Spring Harbor Laboratory 2020.09.15.298026 (2020) doi:10.1101/2020.09.15.298026. 20. Popejoy, A. B. & Fullerton, S. M. Genomics is failing on diversity. Nature vol. 538 161–164 (2016). 21. Martin, A. R. et al. Human Demographic History Impacts Genetic Risk Prediction across Diverse Populations. Am. J. Hum. Genet. 107, 788–789 (2020). 22. Goetz, L. H., Uribe-Bruce, L., Quarless, D., Libiger, O. & Schork, N. J. Admixture and Clinical Phenotypic Variation. Human Heredity vol. 77 73–86 (2014). 23. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009). http://paperpile.com/b/07uDCz/zSeY http://paperpile.com/b/07uDCz/zSeY http://paperpile.com/b/07uDCz/zSeY http://paperpile.com/b/07uDCz/zSeY http://paperpile.com/b/07uDCz/zSeY http://paperpile.com/b/07uDCz/zSeY http://paperpile.com/b/07uDCz/zSeY http://paperpile.com/b/07uDCz/zSeY http://paperpile.com/b/07uDCz/ufY1 http://paperpile.com/b/07uDCz/ufY1 http://paperpile.com/b/07uDCz/ufY1 http://paperpile.com/b/07uDCz/ufY1 http://paperpile.com/b/07uDCz/ufY1 http://paperpile.com/b/07uDCz/ufY1 http://paperpile.com/b/07uDCz/ufY1 http://paperpile.com/b/07uDCz/Jn4R http://paperpile.com/b/07uDCz/Jn4R http://paperpile.com/b/07uDCz/Jn4R http://paperpile.com/b/07uDCz/Jn4R http://paperpile.com/b/07uDCz/Jn4R http://paperpile.com/b/07uDCz/Jn4R http://paperpile.com/b/07uDCz/Jn4R http://paperpile.com/b/07uDCz/Jn9e http://paperpile.com/b/07uDCz/Jn9e http://paperpile.com/b/07uDCz/Jn9e http://paperpile.com/b/07uDCz/Jn9e http://paperpile.com/b/07uDCz/Jn9e http://paperpile.com/b/07uDCz/Jn9e http://paperpile.com/b/07uDCz/Jn9e http://paperpile.com/b/07uDCz/Jn9e http://paperpile.com/b/07uDCz/IPNS http://paperpile.com/b/07uDCz/IPNS http://paperpile.com/b/07uDCz/IPNS http://paperpile.com/b/07uDCz/IPNS http://paperpile.com/b/07uDCz/IPNS http://paperpile.com/b/07uDCz/IPNS http://paperpile.com/b/07uDCz/IPNS http://paperpile.com/b/07uDCz/IPNS http://paperpile.com/b/07uDCz/dNMK http://paperpile.com/b/07uDCz/dNMK http://paperpile.com/b/07uDCz/dNMK http://paperpile.com/b/07uDCz/dNMK http://paperpile.com/b/07uDCz/dNMK http://paperpile.com/b/07uDCz/dNMK http://paperpile.com/b/07uDCz/dNMK http://paperpile.com/b/07uDCz/Htne http://paperpile.com/b/07uDCz/Htne http://paperpile.com/b/07uDCz/Htne http://paperpile.com/b/07uDCz/Htne http://paperpile.com/b/07uDCz/Htne http://paperpile.com/b/07uDCz/Htne http://paperpile.com/b/07uDCz/Htne http://dx.doi.org/10.1101/2020.09.15.298026 http://paperpile.com/b/07uDCz/Htne http://paperpile.com/b/07uDCz/yTtT http://paperpile.com/b/07uDCz/yTtT http://paperpile.com/b/07uDCz/yTtT http://paperpile.com/b/07uDCz/yTtT http://paperpile.com/b/07uDCz/DA9b http://paperpile.com/b/07uDCz/DA9b http://paperpile.com/b/07uDCz/DA9b http://paperpile.com/b/07uDCz/DA9b http://paperpile.com/b/07uDCz/DA9b http://paperpile.com/b/07uDCz/DA9b http://paperpile.com/b/07uDCz/DA9b http://paperpile.com/b/07uDCz/DA9b http://paperpile.com/b/07uDCz/5MrY http://paperpile.com/b/07uDCz/5MrY http://paperpile.com/b/07uDCz/5MrY http://paperpile.com/b/07uDCz/5MrY http://paperpile.com/b/07uDCz/Mmh2 http://paperpile.com/b/07uDCz/Mmh2 http://paperpile.com/b/07uDCz/Mmh2 http://paperpile.com/b/07uDCz/Mmh2 http://paperpile.com/b/07uDCz/Mmh2 http://paperpile.com/b/07uDCz/Mmh2 53 24. Van der Auwera, G. A. & O’Connor, B. D. Genomics in the Cloud: Using Docker, GATK, and WDL in Terra. (O’Reilly Media, 2020). 25. DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011). 26. Martin, M. et al. WhatsHap: fast and accurate read-based phasing. doi:10.1101/085050. 27. Delaneau, O., Zagury, J.-F., Robinson, M. R., Marchini, J. L. & Dermitzakis, E. T. Accurate, scalable and integrative haplotype estimation. Nat. Commun. 10, 5436 (2019). 28. Nielsen, H., Tsirigos, K. D., Brunak, S. & von Heijne, G. A Brief History of Protein Sorting Prediction. Protein J. 38, 200–216 (2019). 29. Castelli, E. C. et al. HLA-G genetic diversity and evolutive aspects in worldwide populations. Sci. Rep. 11, 23070 (2021). 30. Lee, Y. H., Choi, S. J., Ji, J. D. & Song, G. G. Genome-wide pathway analysis of a genome-wide association study on psoriasis and Behcet’s disease. Mol. Biol. Rep. 39, 5953–5959 (2012). 31. Song, G. G., Choi, S. J., Ji, J. D. & Lee, Y. H. Genome-wide pathway analysis of a genome-wide association study on multiple sclerosis. Mol. Biol. Rep. 40, 2557–2564 (2013). 32. Didonna, A. et al. A splice acceptor variant in HLA-DRA affects the conformation and cellular localization of the class II DR alpha-chain. Immunology 162, 194–207 (2021). 33. Secolin, R. et al. Distribution of local ancestry and evidence of adaptation in admixed populations. Sci. Rep. 9, 13900 (2019). http://paperpile.com/b/07uDCz/6bub http://paperpile.com/b/07uDCz/6bub http://paperpile.com/b/07uDCz/6bub http://paperpile.com/b/07uDCz/6bub http://paperpile.com/b/07uDCz/5sEp http://paperpile.com/b/07uDCz/5sEp http://paperpile.com/b/07uDCz/5sEp http://paperpile.com/b/07uDCz/5sEp http://paperpile.com/b/07uDCz/5sEp http://paperpile.com/b/07uDCz/5sEp http://paperpile.com/b/07uDCz/5sEp http://paperpile.com/b/07uDCz/5sEp http://paperpile.com/b/07uDCz/wnee http://paperpile.com/b/07uDCz/wnee http://paperpile.com/b/07uDCz/wnee http://dx.doi.org/10.1101/085050 http://paperpile.com/b/07uDCz/wnee http://paperpile.com/b/07uDCz/0kuC http://paperpile.com/b/07uDCz/0kuC http://paperpile.com/b/07uDCz/0kuC http://paperpile.com/b/07uDCz/0kuC http://paperpile.com/b/07uDCz/0kuC http://paperpile.com/b/07uDCz/0kuC http://paperpile.com/b/07uDCz/Lx2o http://paperpile.com/b/07uDCz/Lx2o http://paperpile.com/b/07uDCz/Lx2o http://paperpile.com/b/07uDCz/Lx2o http://paperpile.com/b/07uDCz/Lx2o http://paperpile.com/b/07uDCz/Lx2o http://paperpile.com/b/07uDCz/IzNL http://paperpile.com/b/07uDCz/IzNL http://paperpile.com/b/07uDCz/IzNL http://paperpile.com/b/07uDCz/IzNL http://paperpile.com/b/07uDCz/IzNL http://paperpile.com/b/07uDCz/IzNL http://paperpile.com/b/07uDCz/IzNL http://paperpile.com/b/07uDCz/IzNL http://paperpile.com/b/07uDCz/hhUM http://paperpile.com/b/07uDCz/hhUM http://paperpile.com/b/07uDCz/hhUM http://paperpile.com/b/07uDCz/hhUM http://paperpile.com/b/07uDCz/hhUM http://paperpile.com/b/07uDCz/hhUM http://paperpile.com/b/07uDCz/m73a http://paperpile.com/b/07uDCz/m73a http://paperpile.com/b/07uDCz/m73a http://paperpile.com/b/07uDCz/m73a http://paperpile.com/b/07uDCz/m73a http://paperpile.com/b/07uDCz/m73a http://paperpile.com/b/07uDCz/btdz http://paperpile.com/b/07uDCz/btdz http://paperpile.com/b/07uDCz/btdz http://paperpile.com/b/07uDCz/btdz http://paperpile.com/b/07uDCz/btdz http://paperpile.com/b/07uDCz/btdz http://paperpile.com/b/07uDCz/btdz http://paperpile.com/b/07uDCz/btdz http://paperpile.com/b/07uDCz/gKpR http://paperpile.com/b/07uDCz/gKpR http://paperpile.com/b/07uDCz/gKpR http://paperpile.com/b/07uDCz/gKpR http://paperpile.com/b/07uDCz/gKpR http://paperpile.com/b/07uDCz/gKpR http://paperpile.com/b/07uDCz/gKpR http://paperpile.com/b/07uDCz/gKpR