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Identification of candidate genes involved in Mercury Toxicokinetics and Mercury Induced Autoimmunity
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

BACKGROUND: Autoimmune diseases require the involvement and activation of immune cells and occur when the body builds up an immune response against its own tissues. This process takes place due to the inability to distinguish self-antigen from foreign antigen. Systemic autoimmunity represents an important cause of morbidity and mortality in humans. The mechanisms triggering autoimmune responses are complex and involve a network of genetic factors. Genome wide association study (GWAS) is a powerful method, used to identify genetic risk factors in numerous diseases, such as systemic autoimmune diseases. The goal of GWAS is to identify these genetic risk factors in order to make predictions about who is at risk and investigate the biological process of disease susceptibility. There are several valuable mouse models to investigate the underlying mechanisms causing systemic autoimmune diseases in which mercury induced autoimmunity (HgIA) is a well- established and relevant model. HgIA in mice includes development of autoantibodies, immune complex glomerulonephritis, lymphocyte proliferation, hypergammaglobulinemia and polyclonal B cell activation. In humans, mercury exposure accumulates with considerable concentrations in kidney, liver, and brain. Toxicokinetics of Hg has been studied extensively but the key for inter-individual variation in humans are largely unclear. Differences in accumulation of renal Hg between inbred mouse strains suggest a genetic inter-strain variation regulating retention or/and excretion of Hg.

OBJECTIVES: To find loci and candidate genes associated with phenotypes involved in the development of autoimmunity and find candidate genes involved in the regulation of renal Hg excretion.

METHODS: MHC II (H-2s) mice were paired (A.SW x B10.S) to obtain F2 offspring exposed to 2.0 or 4.0 mg Hg in drinking water for 6 weeks. Mercury induced autoimmune phenotypes were studied with immunofluorescence (anti-nucleolar antibodies (ANoA)), ELISA anti-DNP/anti-ssDNA (polyclonal B cell activation), anti-chromatin antibodies (ACA) (4.0 mg Hg), and serum IgG1 concentrations. Mercury accumulation in kidney was performed previously and data was included as phenotype. F2 mice exposed to 2.0 mg Hg were genotyped with microsatellites for genome-wide scan with Ion Pair Reverse Phase High Performance Liquid Chromatography (IP RP HPLC). F2 mice exposed to 4.0 mg Hg were genotyped with single nucleotide polymorphisms for genomewide scan with SNP&SEQ technology platform. Quantitative trait loci (QTL) was established with R/QTL. Denaturing HPLC, next generation sequencing, conserved region analysis and genetic mouse strain comparison were used for haplotyping and fine mapping on QTLs associated with Hg concentration in kidney, development of ANoA and serum IgG1 hypergammaglobulinemia. Candidate genes (Pprc1, Bank1 and Nfkb1) verified by additional QTL were further investigated by real time polymerase chain reaction. Genes involved in the intracellular signaling together with candidate genes were included for gene expression analysis.

RESULTS: F2 mice exposed to 2.0 mg Hg had low or no development of autoantibodies and showed no significant difference in polyclonal B cell activation in the B10.S and F2 strains. F2 mice exposed to 4.0 mg Hg developed autoantibodies and significantly increased IgG1 concentration and polyclonal B cell activation (anti-DNP). QTL analysis showed a logarithm of odds ratio (LOD) score between 2.9 – 4.36 on all serological phenotypes exposed to 4.0 mg Hg, and a LOD score of 5.78 on renal Hg concentration. Haplotyping and fine mapping associated the development of ANoA with Bank1 (B-cell scaffold protein with ankyrin repeats 1) and Nfkb1 (nuclear factor kappa B subunit 1). The serum IgG1 concentration was associated with a locus on chromosome 3, in which Rxfp4 (Relaxin Family Peptide/INSL5 Receptor 4) is a potential candidate gene. The renal Hg concentration was associated with Pprc1 (Peroxisome Proliferator-Activated Receptor Gamma, Co-activator-Related). Gene expression analysis revealed that the more susceptible A.SW strain expresses significantly higher levels of Nfkb1, Il6 and Tnf than the less susceptible B10.S strain. The A.SW strain expresses significantly lower levels of Pprc1 and cascade proteins than the B10.S strain. Development of ACA was associated with chromosomes 3, 6, 7 and 16 (LOD 3.1, 3.2, 3.4 and 6.8 respectively). Polyclonal B cell activation was associated with chromosome 2 with a LOD score of 2.9.

CONCLUSIONS: By implementing a GWAS on HgIA in mice, several QTLs were discovered to be associated with the development of autoantibodies, polyclonal B cell activation and hypergammaglobulinemia. This thesis plausibly supports Bank1 and Nfkb1 as key regulators for ANoA development and HgIA seems to be initiated by B cells rather than T cells. GWAS on renal mercury excretion plausibly supports Pprc1 as key regulator and it seems that this gene has a protective role against Hg.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. , p. 93
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1649
National Category
Medical Genetics Immunology in the medical area Immunology Developmental Biology
Identifiers
URN: urn:nbn:se:liu:diva-152259DOI: 10.3384/diss.diva-152259ISBN: 9789176851920 (print)OAI: oai:DiVA.org:liu-152259DiVA, id: diva2:1258279
Public defence
2018-11-09, Belladonna, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2018-10-24 Created: 2018-10-24 Last updated: 2019-09-30Bibliographically approved
List of papers
1. Genome-Wide Association Study to Identify Genes Related to Renal Mercury Concentrations in Mice
Open this publication in new window or tab >>Genome-Wide Association Study to Identify Genes Related to Renal Mercury Concentrations in Mice
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2016 (English)In: Journal of Environmental Health Perspectives, ISSN 0091-6765, E-ISSN 1552-9924, Vol. 124, no 7, p. 920-926Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Following human mercury (Hg) exposure, the metal accumulates in considerable concentrations in kidney, liver, and brain. Although the toxicokinetics of Hg have been studied extensively, factors responsible for interindividual variation in humans are largely unknown. Differences in accumulation of renal Hg between inbred mouse strains suggest a genetic interstrain variation regulating retention or/and excretion of Hg. A. SW, DBA/2 and BALB/C mouse strains accumulate higher amounts of Hg than B10.S.

OBJECTIVES: We aimed to find candidate genes associated with regulation of renal Hg concentrations.

METHODS: A. SW, B10.S and their F1 and F2 offspring were exposed for 6 weeks to 2.0 mg Hg/L drinking water. Genotyping with microsatellites was conducted on 84 F2 mice for genome-wide scanning with ion pair reverse-phase high-performance liquid chromatography (IP RP HPLC). Quantitative trait loci (QTL) were established. Denaturing HPLC was used to detect single nucleotide polymorphisms for haplotyping and fine mapping in 184 and 32 F2 mice, respectively. Candidate genes (Pprc1, Btrc and Nfkb2) verified by fine mapping and QTL were further investigated by real-time polymerase chain reaction. Genes enhanced by Pprc1 (Nrf1 and Nrf2) were included for gene expression analysis.

RESULTS: Renal Hg concentrations differed significantly between A. SW and B10. S mice and between males and females within each strain. QTL analysis showed a peak logarithm of odds ratio score 5.78 on chromosome 19 (p = 0.002). Haplotype and fine mapping associated the Hg accumulation with Pprc1, which encodes PGC-1-related coactivator (PRC), a coactivator for proteins involved in detoxification. Pprc1 and two genes coactivated by Pprc1 (Nrf1 and Nrf2) had significantly lower gene expression in the A. SW strain than in the B10. S strain.

CONCLUSIONS: This study supports Pprc1 as a key regulator for renal Hg excretion.

Place, publisher, year, edition, pages
U.S. Department of Health and Human Services * National Institute of Environmental Health Sciences, 2016
National Category
Other Biological Topics
Identifiers
urn:nbn:se:liu:diva-131584 (URN)10.1289/ehp.1409284 (DOI)000380749300012 ()26942574 (PubMedID)
Note

Funding Agencies|Swedish Research Council Branch of Medicine; County Council of Ostergotland; Linkoping University

Available from: 2016-09-27 Created: 2016-09-27 Last updated: 2018-10-24Bibliographically approved
2. Bank1 and NF-kappaB as key regulators in anti-nucleolar antibody development
Open this publication in new window or tab >>Bank1 and NF-kappaB as key regulators in anti-nucleolar antibody development
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2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 7, article id e0199979Article in journal (Refereed) Published
Abstract [en]

Systemic autoimmune rheumatic disorders (SARD) represent important causes of morbidity and mortality in humans. The mechanisms triggering autoimmune responses are complex and involve a network of genetic factors. Mercury-induced autoimmunity (HgIA) in mice is an established model to study the mechanisms of the development of antinuclear antibodies (ANA), which is a hallmark in the diagnosis of SARD. A.SW mice with HgIA show a significantly higher titer of antinucleolar antibodies (ANoA) than the B10.S mice, although both share the same MHC class II (H-2). We applied a genome-wide association study (GWAS) to their Hg-exposed F2 offspring to investigate the non-MHC genes involved in the development of ANoA. Quantitative trait locus (QTL) analysis showed a peak logarithm of odds ratio (LOD) score of 3.05 on chromosome 3. Microsatellites were used for haplotyping, and fine mapping was conducted with next generation sequencing. The candidate genes Bank1 (B-cell scaffold protein with ankyrin repeats 1) and Nfkbl (nuclear factor kappa B subunit 1) were identified by additional QTL analysis. Expression of the Bank1 and Nfkb1 genes and their downstream target genes involved in the intracellular pathway (Tlr9,II6, Tnf) was investigated in mercury-exposed A.SW and B10.S mice by real-time PCR. Bank1 showed significantly lower gene expression in the A.SW strain after Hg-exposure, whereas the B10.S strain showed no significant difference. Nfkb1, Tlr9, II6 and Tnf had significantly higher gene expression in the A.SW strain after Hg-exposure, while the B10.S strain showed no difference. This study supports the roles of Bank1 (produced mainly in B-cells) and Nfkbl (produced in most immune cells) as key regulators of ANoA development in HgIA.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2018
National Category
Genetics
Identifiers
urn:nbn:se:liu:diva-150265 (URN)10.1371/journal.pone.0199979 (DOI)000438866600014 ()30016332 (PubMedID)
Note

Funding Agencies|Swedish Research Council Branch of Medicine; County Council of Ostergotland; Linkoping University

Available from: 2018-08-17 Created: 2018-08-17 Last updated: 2019-04-24

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