High-Throughput Sequencing to Investigate Associations Between HLA Genes and Metamizole-Induced Agranulocytosis

Anca Liliana Cismaru; Livia Grimm; Deborah Rudin; Luisa Ibañez; Evangelia Liakoni; Nicolas Bonadies; Reinhold Kreutz; Pär Hallberg; Mia Wadelius; Manuel Haschke; Carlo R. Largiadèr; Ursula Amstutz; Niclas Eriksson; Mariam Molokhia; Alfonso Carvajal; M. Isabel Lucena; Javier Esther Sancho Martin Ponce; Lourdes Vendrell; Ramon Puig Treserra; Jose Luis Caro; Eduard Palou; María José Herrero; Esmeralda de la Banda Ledrado; Eva Montané; Elisa Orna Montero; José Tomás Navarro Ferrando; Consuelo Pedrós Cholvi

doi:10.3389/fgene.2020.00951

High-Throughput Sequencing to Investigate Associations Between HLA Genes and Metamizole-Induced Agranulocytosis

Anca Liliana Cismaru, Livia Grimm, Deborah Rudin, Luisa Ibañez, Evangelia Liakoni, Nicolas Bonadies, Reinhold Kreutz, Pär Hallberg, Mia Wadelius, Manuel Haschke, Carlo R. Largiadèr, Ursula Amstutz^*, Niclas Eriksson, Mariam Molokhia, Alfonso Carvajal, M. Isabel Lucena, Javier Esther Sancho Martin Ponce, Lourdes Vendrell, Ramon Puig Treserra, Jose Luis CaroEduard Palou, María José Herrero, Esmeralda de la Banda Ledrado, Eva Montané, Elisa Orna Montero, José Tomás Navarro Ferrando, Consuelo Pedrós Cholvi

^*Corresponding author for this work

Population Health Sciences

Research output: Contribution to journal › Article › peer-review

4 Citations (Scopus)

Abstract

Background and Objective: Agranulocytosis is a rare and potentially life-threatening complication of metamizole (dipyrone) intake that is characterized by a loss of circulating neutrophil granulocytes. While the mechanism underlying this adverse drug reaction is not well understood, involvement of the immune system has been suggested. In addition, associations between genetic variants in the Human Leukocyte Antigen (HLA) region and agranulocytosis induced by other drugs have been reported. The aim of the present study was to assess whether genetic variants in classical HLA genes are associated with the susceptibility to metamizole-induced agranulocytosis (MIA) in a European population by targeted resequencing of eight HLA genes. Design: A case-control cohort of Swiss patients with a history of neutropenia or agranulocytosis associated with metamizole exposure (n = 53), metamizole-tolerant (n = 39) and unexposed controls (n = 161) was recruited for this study. A high-throughput resequencing (HTS) and high-resolution typing method was used to sequence and analyze eight HLA loci in a discovery subset of this cohort (n = 31 cases, n = 38 controls). Identified candidate alleles were investigated in the full Swiss cohort as well as in two independent cohorts from Germany and Spain using HLA imputation from genome-wide SNP array data. In addition, variant calling based on HTS data was performed in the discovery subset for the class I genes HLA-A, -B, and -C using the HLA-specific mapper hla-mapper. Results: Eight candidate alleles (p < 0.05) were identified in the discovery subset, of which HLA-C^∗04:01 was associated with MIA in the full Swiss cohort (p < 0.01) restricted to agranulocytosis (ANC < 0.5 × 10⁹/L) cases. However, no candidate allele showed a consistent association in the Swiss, German and Spanish cohorts. Analysis of individual sequence variants in class I genes produced consistent results with HLA typing but did not reveal additional small nucleotide variants associated with MIA. Conclusion: Our results do not support an HLA-restricted T cell-mediated immune mechanism for MIA. However, we established an efficient high-resolution (three-field) eight-locus HTS HLA resequencing method to interrogate the HLA region and demonstrated the feasibility of its application to pharmacogenetic studies.

Original language	English
Article number	951
Journal	Frontiers in Genetics
Volume	11
DOIs	https://doi.org/10.3389/fgene.2020.00951
Publication status	Published - 21 Aug 2020

Keywords

drug-induced agranulocytosis
genetic association studies
high-throughput sequencing
HLA
metamizole (dipyrone)
next generation sequencing
pharmacogenetics

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10.3389/fgene.2020.00951

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Cismaru, A. L., Grimm, L., Rudin, D., Ibañez, L., Liakoni, E., Bonadies, N., Kreutz, R., Hallberg, P., Wadelius, M., Haschke, M., Largiadèr, C. R., Amstutz, U., Eriksson, N., Molokhia, M., Carvajal, A., Lucena, M. I., Martin Ponce, J. E. S., Vendrell, L., Treserra, R. P., ... Cholvi, C. P. (2020). High-Throughput Sequencing to Investigate Associations Between HLA Genes and Metamizole-Induced Agranulocytosis. Frontiers in Genetics, 11, Article 951. https://doi.org/10.3389/fgene.2020.00951

@article{e93d847840d947b5afcbf6de542e1171,

title = "High-Throughput Sequencing to Investigate Associations Between HLA Genes and Metamizole-Induced Agranulocytosis",

abstract = "Background and Objective: Agranulocytosis is a rare and potentially life-threatening complication of metamizole (dipyrone) intake that is characterized by a loss of circulating neutrophil granulocytes. While the mechanism underlying this adverse drug reaction is not well understood, involvement of the immune system has been suggested. In addition, associations between genetic variants in the Human Leukocyte Antigen (HLA) region and agranulocytosis induced by other drugs have been reported. The aim of the present study was to assess whether genetic variants in classical HLA genes are associated with the susceptibility to metamizole-induced agranulocytosis (MIA) in a European population by targeted resequencing of eight HLA genes. Design: A case-control cohort of Swiss patients with a history of neutropenia or agranulocytosis associated with metamizole exposure (n = 53), metamizole-tolerant (n = 39) and unexposed controls (n = 161) was recruited for this study. A high-throughput resequencing (HTS) and high-resolution typing method was used to sequence and analyze eight HLA loci in a discovery subset of this cohort (n = 31 cases, n = 38 controls). Identified candidate alleles were investigated in the full Swiss cohort as well as in two independent cohorts from Germany and Spain using HLA imputation from genome-wide SNP array data. In addition, variant calling based on HTS data was performed in the discovery subset for the class I genes HLA-A, -B, and -C using the HLA-specific mapper hla-mapper. Results: Eight candidate alleles (p < 0.05) were identified in the discovery subset, of which HLA-C∗04:01 was associated with MIA in the full Swiss cohort (p < 0.01) restricted to agranulocytosis (ANC < 0.5 × 109/L) cases. However, no candidate allele showed a consistent association in the Swiss, German and Spanish cohorts. Analysis of individual sequence variants in class I genes produced consistent results with HLA typing but did not reveal additional small nucleotide variants associated with MIA. Conclusion: Our results do not support an HLA-restricted T cell-mediated immune mechanism for MIA. However, we established an efficient high-resolution (three-field) eight-locus HTS HLA resequencing method to interrogate the HLA region and demonstrated the feasibility of its application to pharmacogenetic studies.",

keywords = "drug-induced agranulocytosis, genetic association studies, high-throughput sequencing, HLA, metamizole (dipyrone), next generation sequencing, pharmacogenetics",

author = "Cismaru, {Anca Liliana} and Livia Grimm and Deborah Rudin and Luisa Iba{\~n}ez and Evangelia Liakoni and Nicolas Bonadies and Reinhold Kreutz and P{\"a}r Hallberg and Mia Wadelius and Manuel Haschke and Largiad{\`e}r, {Carlo R.} and Ursula Amstutz and Niclas Eriksson and Mariam Molokhia and Alfonso Carvajal and Lucena, {M. Isabel} and {Martin Ponce}, {Javier Esther Sancho} and Lourdes Vendrell and Treserra, {Ramon Puig} and Caro, {Jose Luis} and Eduard Palou and Herrero, {Mar{\'i}a Jos{\'e}} and Ledrado, {Esmeralda de la Banda} and Eva Montan{\'e} and Montero, {Elisa Orna} and Ferrando, {Jos{\'e} Tom{\'a}s Navarro} and Cholvi, {Consuelo Pedr{\'o}s}",

note = "Funding Information: We would like to cordially thank the laboratory technicians of the University Institute of Clinical Chemistry (Inselspital Bern University Hospital), Gisela Andrey, Daniel Sch?rer, and Daniela Sommer, for their support during the DNA extraction and library preparation phases. We would like to thank the Transplantation Immunology Laboratory of the Center for Laboratory Medicine (Inselspital Bern University Hospital) for their support with SSP-PCR based HLA typing. We further thank the Interfaculty Bioinformatics Unit of the University of Bern and its head, R?my Bruggmann, for providing access to their computational infrastructure to run our HTS data analyses. Last but not least, we would like to extend our thanks to Dr. Shuji Kawaguchi for his kind help with troubleshooting for the HLA-HD tool. Finally, we gratefully thank all study participants. Funding. This work was supported by funding from the Swiss National Science Foundation (SNF grant no. 31003A_160206), the Carlos III Spanish Health Institute (FIS10/02632), the European Regional Development Fund FEDER, the Swedish Research Council (Medicine 521-2011-2440, 521-2014-3370, and 2018-03307), the Swedish Heart and Lung Foundation (20120557, 20140291, and 20170711) and a grant from the Federal Institute for Drugs and Medical Devices (Bonn, Germany). The EUDRAGENE collaboration has received support from the EC 5th Framework program (QLRI-CR-2002-02757) and the Serious Adverse Events Consortium, SAEC, a collaboration for academia and industry. Mariam Molokhia is supported by the National Institute for Health Research Biomedical Research Center at Guy?s and St Thomas? National Health Service Foundation Trust and King?s College London. Publisher Copyright: {\textcopyright} Copyright {\textcopyright} 2020 Cismaru, Grimm, Rudin, Iba{\~n}ez, Liakoni, Bonadies, Kreutz, Hallberg, Wadelius, EuDAC Collaborators, Haschke, Largiad{\`e}r and Amstutz. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.",

year = "2020",

month = aug,

day = "21",

doi = "10.3389/fgene.2020.00951",

language = "English",

volume = "11",

journal = "Frontiers in Genetics",

issn = "1664-8021",

publisher = "Frontiers Media S.A.",

}

Cismaru, AL, Grimm, L, Rudin, D, Ibañez, L, Liakoni, E, Bonadies, N, Kreutz, R, Hallberg, P, Wadelius, M, Haschke, M, Largiadèr, CR, Amstutz, U, Eriksson, N, Molokhia, M, Carvajal, A, Lucena, MI, Martin Ponce, JES, Vendrell, L, Treserra, RP, Caro, JL, Palou, E, Herrero, MJ, Ledrado, EDLB, Montané, E, Montero, EO, Ferrando, JTN & Cholvi, CP 2020, 'High-Throughput Sequencing to Investigate Associations Between HLA Genes and Metamizole-Induced Agranulocytosis', Frontiers in Genetics, vol. 11, 951. https://doi.org/10.3389/fgene.2020.00951

TY - JOUR

T1 - High-Throughput Sequencing to Investigate Associations Between HLA Genes and Metamizole-Induced Agranulocytosis

AU - Cismaru, Anca Liliana

AU - Grimm, Livia

AU - Rudin, Deborah

AU - Ibañez, Luisa

AU - Liakoni, Evangelia

AU - Bonadies, Nicolas

AU - Kreutz, Reinhold

AU - Hallberg, Pär

AU - Wadelius, Mia

AU - Haschke, Manuel

AU - Largiadèr, Carlo R.

AU - Amstutz, Ursula

AU - Eriksson, Niclas

AU - Molokhia, Mariam

AU - Carvajal, Alfonso

AU - Lucena, M. Isabel

AU - Martin Ponce, Javier Esther Sancho

AU - Vendrell, Lourdes

AU - Treserra, Ramon Puig

AU - Caro, Jose Luis

AU - Palou, Eduard

AU - Herrero, María José

AU - Ledrado, Esmeralda de la Banda

AU - Montané, Eva

AU - Montero, Elisa Orna

AU - Ferrando, José Tomás Navarro

AU - Cholvi, Consuelo Pedrós

N1 - Funding Information: We would like to cordially thank the laboratory technicians of the University Institute of Clinical Chemistry (Inselspital Bern University Hospital), Gisela Andrey, Daniel Sch?rer, and Daniela Sommer, for their support during the DNA extraction and library preparation phases. We would like to thank the Transplantation Immunology Laboratory of the Center for Laboratory Medicine (Inselspital Bern University Hospital) for their support with SSP-PCR based HLA typing. We further thank the Interfaculty Bioinformatics Unit of the University of Bern and its head, R?my Bruggmann, for providing access to their computational infrastructure to run our HTS data analyses. Last but not least, we would like to extend our thanks to Dr. Shuji Kawaguchi for his kind help with troubleshooting for the HLA-HD tool. Finally, we gratefully thank all study participants. Funding. This work was supported by funding from the Swiss National Science Foundation (SNF grant no. 31003A_160206), the Carlos III Spanish Health Institute (FIS10/02632), the European Regional Development Fund FEDER, the Swedish Research Council (Medicine 521-2011-2440, 521-2014-3370, and 2018-03307), the Swedish Heart and Lung Foundation (20120557, 20140291, and 20170711) and a grant from the Federal Institute for Drugs and Medical Devices (Bonn, Germany). The EUDRAGENE collaboration has received support from the EC 5th Framework program (QLRI-CR-2002-02757) and the Serious Adverse Events Consortium, SAEC, a collaboration for academia and industry. Mariam Molokhia is supported by the National Institute for Health Research Biomedical Research Center at Guy?s and St Thomas? National Health Service Foundation Trust and King?s College London. Publisher Copyright: © Copyright © 2020 Cismaru, Grimm, Rudin, Ibañez, Liakoni, Bonadies, Kreutz, Hallberg, Wadelius, EuDAC Collaborators, Haschke, Largiadèr and Amstutz. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2020/8/21

Y1 - 2020/8/21

N2 - Background and Objective: Agranulocytosis is a rare and potentially life-threatening complication of metamizole (dipyrone) intake that is characterized by a loss of circulating neutrophil granulocytes. While the mechanism underlying this adverse drug reaction is not well understood, involvement of the immune system has been suggested. In addition, associations between genetic variants in the Human Leukocyte Antigen (HLA) region and agranulocytosis induced by other drugs have been reported. The aim of the present study was to assess whether genetic variants in classical HLA genes are associated with the susceptibility to metamizole-induced agranulocytosis (MIA) in a European population by targeted resequencing of eight HLA genes. Design: A case-control cohort of Swiss patients with a history of neutropenia or agranulocytosis associated with metamizole exposure (n = 53), metamizole-tolerant (n = 39) and unexposed controls (n = 161) was recruited for this study. A high-throughput resequencing (HTS) and high-resolution typing method was used to sequence and analyze eight HLA loci in a discovery subset of this cohort (n = 31 cases, n = 38 controls). Identified candidate alleles were investigated in the full Swiss cohort as well as in two independent cohorts from Germany and Spain using HLA imputation from genome-wide SNP array data. In addition, variant calling based on HTS data was performed in the discovery subset for the class I genes HLA-A, -B, and -C using the HLA-specific mapper hla-mapper. Results: Eight candidate alleles (p < 0.05) were identified in the discovery subset, of which HLA-C∗04:01 was associated with MIA in the full Swiss cohort (p < 0.01) restricted to agranulocytosis (ANC < 0.5 × 109/L) cases. However, no candidate allele showed a consistent association in the Swiss, German and Spanish cohorts. Analysis of individual sequence variants in class I genes produced consistent results with HLA typing but did not reveal additional small nucleotide variants associated with MIA. Conclusion: Our results do not support an HLA-restricted T cell-mediated immune mechanism for MIA. However, we established an efficient high-resolution (three-field) eight-locus HTS HLA resequencing method to interrogate the HLA region and demonstrated the feasibility of its application to pharmacogenetic studies.

AB - Background and Objective: Agranulocytosis is a rare and potentially life-threatening complication of metamizole (dipyrone) intake that is characterized by a loss of circulating neutrophil granulocytes. While the mechanism underlying this adverse drug reaction is not well understood, involvement of the immune system has been suggested. In addition, associations between genetic variants in the Human Leukocyte Antigen (HLA) region and agranulocytosis induced by other drugs have been reported. The aim of the present study was to assess whether genetic variants in classical HLA genes are associated with the susceptibility to metamizole-induced agranulocytosis (MIA) in a European population by targeted resequencing of eight HLA genes. Design: A case-control cohort of Swiss patients with a history of neutropenia or agranulocytosis associated with metamizole exposure (n = 53), metamizole-tolerant (n = 39) and unexposed controls (n = 161) was recruited for this study. A high-throughput resequencing (HTS) and high-resolution typing method was used to sequence and analyze eight HLA loci in a discovery subset of this cohort (n = 31 cases, n = 38 controls). Identified candidate alleles were investigated in the full Swiss cohort as well as in two independent cohorts from Germany and Spain using HLA imputation from genome-wide SNP array data. In addition, variant calling based on HTS data was performed in the discovery subset for the class I genes HLA-A, -B, and -C using the HLA-specific mapper hla-mapper. Results: Eight candidate alleles (p < 0.05) were identified in the discovery subset, of which HLA-C∗04:01 was associated with MIA in the full Swiss cohort (p < 0.01) restricted to agranulocytosis (ANC < 0.5 × 109/L) cases. However, no candidate allele showed a consistent association in the Swiss, German and Spanish cohorts. Analysis of individual sequence variants in class I genes produced consistent results with HLA typing but did not reveal additional small nucleotide variants associated with MIA. Conclusion: Our results do not support an HLA-restricted T cell-mediated immune mechanism for MIA. However, we established an efficient high-resolution (three-field) eight-locus HTS HLA resequencing method to interrogate the HLA region and demonstrated the feasibility of its application to pharmacogenetic studies.

KW - drug-induced agranulocytosis

KW - genetic association studies

KW - high-throughput sequencing

KW - HLA

KW - metamizole (dipyrone)

KW - next generation sequencing

KW - pharmacogenetics

UR - http://www.scopus.com/inward/record.url?scp=85090384319&partnerID=8YFLogxK

U2 - 10.3389/fgene.2020.00951

DO - 10.3389/fgene.2020.00951

M3 - Article

AN - SCOPUS:85090384319

SN - 1664-8021

VL - 11

JO - Frontiers in Genetics

JF - Frontiers in Genetics

M1 - 951

ER -

High-Throughput Sequencing to Investigate Associations Between HLA Genes and Metamizole-Induced Agranulocytosis

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