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Dissecting the Genetic Basis of Systemic Lupus Erythematosus: The Pursuit of Functional Variants
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics. (Ulf Gyllensten)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Systemic lupus erythematosus (SLE) is a chronic and systemic autoimmune disease that primarily affects women during the childbearing years. SLE is characterized by the production of autoantibodies against nucleic acids and their interacting proteins. The exact molecular mechanisms leading to the breakdown of self-tolerance remain to a large extent unknown, but it is well established that they are influenced by both non-genetic (i.e. environmental and hormonal) and genetic factors. SLE is a complex, polygenic disease. Several susceptibility variants have been identified in SLE. However, the functional role in disease pathogenesis for the majority of them remains largely unknown.

This thesis includes case-control association studies where the role of the genes TNFSF4 (Paper I), STAT4 (Paper II), CD226 (Paper III), and BLK (Papers IV and V) in the susceptibility of developing SLE was investigated. The primary focus was on the identification of the functional variants underlying the association. For each of these genes, fine mapping was performed using single nucleotide polymorphisms (SNPs), the linkage disequilibrium (LD) was characterized, and the association was narrowed down to specific haplotypes by means of several different statistical genetic strategies. Candidate variants were prioritized for further functional analysis on the basis of their potential effect on the gene function, their association, and/or biological plausibility. In Paper I, the association of TNFSF4 with SLE was validated and attributed to a risk haplotype tagged by SNPs rs1234317-T and rs12039904-T. Paper II provides evidence supporting the presence of at least two independent genetic effects within the STAT4 gene represented by rs3821236-A and rs7574865-A, which correlated with increased levels of gene expression. In Paper III, a functional allele in CD226 (rs727088-C) was identified, which was responsible for decreased levels in both mRNA and protein expression. In Paper IV, two independent genetic effects in the BLK gene were demonstrated. The first one comprised multiple regulatory variants in high LD that were enriched for NFκB and IRF4 binding sites and correlated with low BLK mRNA levels. The second was a low-frequency missense substitution (Ala71Thr) that decreased the BLK protein half-life. In Paper V, a genetic epistatic interaction between BANK1 rs10516487 (GG) and BLK rs2736340 (TT+TC) was demonstrated. Additional molecular analyses established that these molecules interact physically.  

These studies have contributed to the dissection of the genetic architecture of SLE. They highlight the allelic heterogeneity of the disease and provide functional links to the associated variants, which has significantly aided in the understanding of SLE disease pathogenesis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 88 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 876
Keyword [en]
Systemic Lupus Erythematosus, SLE, Genetic Mapping, Association Studies, Functional Variants, TNFSF4, STAT4, IRF5, CD226, BLK, BANK1
Keyword [sv]
Systemisk Lupus Erythematosus, SLE, Genetik, Genetisk Association, Funktionella Varianter, TNFSF4, STAT4, IRF5, CD226, BLK, BANK1
Keyword [es]
Lupus Eritematoso Sistémico, LES, Estudios de Asociación Genética, Variantes Funcionales, TNFSF4, STAT4, IRF5, CD226, BLK, BANK1
National Category
Medical Genetics Genetics
Research subject
Medical Genetics; Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-196428ISBN: 978-91-554-8620-4 (print)OAI: oai:DiVA.org:uu-196428DiVA: diva2:610261
Public defence
2013-04-26, Rudbecksalen, The Rudbeck Laboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-04-05 Created: 2013-03-08 Last updated: 2013-08-30Bibliographically approved
List of papers
1. Replication of the TNFSF4 (OX40L) promoter region association with systemic lupus erythematosus
Open this publication in new window or tab >>Replication of the TNFSF4 (OX40L) promoter region association with systemic lupus erythematosus
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2009 (English)In: Genes and Immunity, ISSN 1466-4879, E-ISSN 1476-5470, Vol. 10, no 3, 248-253 p.Article in journal (Refereed) Published
Abstract [en]

The tumor necrosis factor ligand superfamily member 4 gene (TNFSF4) encodes the OX40 ligand (OX40L), a costimulatory molecule involved in T-cell activation. A recent study demonstrated the association of TNFSF4 haplotypes located in the upstream region with risk for or protection from systemic lupus erythematosus (SLE). To replicate this association, five single nucleotide polymorphisms (SNPs) tagging the previously associated haplotypes and passing the proper quality-control filters were tested in 1312 cases and 1801 controls from Germany, Italy, Spain and Argentina. The association of TNFSF4 with SLE was replicated in all the sets except Spain. There was a unique risk haplotype tagged by the minor alleles of the SNPs rs1234317 (pooled odds ratio (OR)=1.39, P=0.0009) and rs12039904 (pooled OR=1.38, P=0.0012). We did not observe association to a single protective marker (rs844644) or haplotype as the first study reported; instead, we observed different protective haplotypes, all carrying the major alleles of both SNPs rs1234317 and rs12039904. Association analysis conditioning on the haplotypic background confirmed that these two SNPs explain the entire haplotype effect. This first replication study confirms the association of genetic variation in the upstream region of TNFSF4 with susceptibility to SLE.

Keyword
systemic lupus erythematosus, TNFSF4, OX40L, genetic association study
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-102306 (URN)10.1038/gene.2008.95 (DOI)000265961300006 ()19092840 (PubMedID)
Available from: 2009-05-06 Created: 2009-05-06 Last updated: 2017-12-13Bibliographically approved
2. STAT4 Associates with SLE through two independent effects that correlate with gene expression and act additively with IRF5 to increase risk
Open this publication in new window or tab >>STAT4 Associates with SLE through two independent effects that correlate with gene expression and act additively with IRF5 to increase risk
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2009 (English)In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 68, no 11, 1746-1753 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES: To confirm and define the genetic association of STAT4 and systemic lupus erythematosus, investigate the possibility of correlations with differential splicing and/or expression levels, and genetic interaction with IRF5. METHODS: 30 tag SNPs were genotyped in an independent set of Spanish cases and controls. SNPs surviving correction for multiple tests were genotyped in 5 new sets of cases and controls for replication. STAT4 cDNA was analyzed by 5'-RACE PCR and sequencing. Expression levels were measured by quantitative PCR. RESULTS: In the fine-mapping, four SNPs were significant after correction for multiple testing, with rs3821236 and rs3024866 as the strongest signals, followed by the previously associated rs7574865, and by rs1467199. Association was replicated in all cohorts. After conditional regression analyses, two major independent signals represented by SNPs rs3821236 and rs7574865, remained significant across the sets. These SNPs belong to separate haplotype blocks. High levels of STAT4 expression correlated with SNPs rs3821236, rs3024866 (both in the same haplotype block) and rs7574865 but not with other SNPs. We also detected transcription of alternative tissue-specific exons 1, indicating presence of tissue-specific promoters of potential importance in the expression of STAT4. No interaction with associated SNPs of IRF5 was observed using regression analysis. CONCLUSIONS: These data confirm STAT4 as a susceptibility gene for SLE and suggest the presence of at least two functional variants affecting levels of STAT4. Our results also indicate that both genes STAT4 and IRF5 act additively to increase risk for SLE.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-102290 (URN)10.1136/ard.2008.097642 (DOI)000270700900016 ()19019891 (PubMedID)
Available from: 2009-05-06 Created: 2009-05-06 Last updated: 2017-12-13Bibliographically approved
3. A 3 '-Untranslated Region Variant Is Associated With Impaired Expression of CD226 in T and Natural Killer T Cells and Is Associated With Susceptibility to Systemic Lupus Erythematosus
Open this publication in new window or tab >>A 3 '-Untranslated Region Variant Is Associated With Impaired Expression of CD226 in T and Natural Killer T Cells and Is Associated With Susceptibility to Systemic Lupus Erythematosus
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2010 (English)In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 62, no 11, 3404-3414 p.Article in journal (Refereed) Published
Abstract [en]

Objective. Costimulatory receptor CD226 plays an important role in T cell activation, differentiation, and cytotoxicity. This study was undertaken to investigate the genetic association of CD226 with susceptibility to systemic lupus erythematosus (SLE) and to assess the functional implications of this association. Methods. Twelve tag single-nucleotide polymorphisms (SNPs) in CD226 were typed in 1,163 SLE patients and 1,482 healthy control subjects from Europe or of European ancestry. Analyses of association were performed by single-marker Cochran-Mantel-Haenszel meta-analysis, followed by haplotype analysis. Gene expression was analyzed by quantitative real-time polymerase chain reaction analyses of RNA from peripheral blood mononuclear cells, and by fluorescence-activated cell sorter analysis. To study the functional impact of the associated variants, luciferase reporter constructs containing different portions of the 3'-untranslated region (3'-UTR) of the gene were prepared and used in transfection experiments. Results. A 3-variant haplotype, rs763361; rs34794968; rs727088 (ATC), in the last exon of CD226 was associated with SLE (P = 1.3 x 10(-4), odds ratio 1.24, 95% confidence interval 1.11-1.38). This risk haplotype correlated with low CD226 transcript expression and low CD226 protein levels on the surface of CD4+ and CD8+ T cells and natural killer T (NKT) cells. NK cells expressed high levels of CD226, but this expression was independent of the haplotype. Reporter assays with deletion constructs indicated that only the presence of rs727088 could account for the differences in the levels of luciferase transcripts. Conclusion. This study identified an association of CD226 with SLE in individuals of European ancestry. These data support the importance of the 3'-UTR SNP rs727088 in the regulation of CD226 transcription both in T cells and in NKT cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-134128 (URN)10.1002/art.27677 (DOI)000283776400032 ()
Available from: 2010-11-22 Created: 2010-11-22 Last updated: 2017-12-12Bibliographically approved
4. Genetic and physical interaction of the B-cell systemic lupus erythematosus-associated genes BANK1 and BLK
Open this publication in new window or tab >>Genetic and physical interaction of the B-cell systemic lupus erythematosus-associated genes BANK1 and BLK
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2012 (English)In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 71, no 1, 136-142 p.Article in journal (Refereed) Published
Abstract [en]

Objectives

Altered signalling in B cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signalling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterise the role of BANK1 and BLK in SLE, a genetic interaction analysis was performed hypothesising that genetic interactions could reveal functional pathways relevant to disease pathogenesis.

Methods

The GPAT16 method was used to analyse the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localisation, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK.

Results

Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK. As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, the possibility that BANK1 and BLK could also show a protein-protein interaction was tested. The co-immunoprecipitation and co-localisation of BLK and BANK1 were demonstrated. In a Daudi cell line and primary naive B cells endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies.

Conclusions

This study shows a genetic interaction between BANK1 and BLK, and demonstrates that these molecules interact physically. The results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signalling pathway.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-167163 (URN)10.1136/annrheumdis-2011-200085 (DOI)000298180100024 ()
Available from: 2012-01-31 Created: 2012-01-23 Last updated: 2017-12-08Bibliographically approved
5. Fine mapping and conditional analysis identify a new mutation in the autoimmunity susceptibility gene BLK that leads to reduced half-life of the BLK protein
Open this publication in new window or tab >>Fine mapping and conditional analysis identify a new mutation in the autoimmunity susceptibility gene BLK that leads to reduced half-life of the BLK protein
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2012 (English)In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 71, no 7, 1219-1226 p.Article in journal (Refereed) Published
Abstract [en]

Objectives

To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE).

Methods

Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 single-nucleotide polymorphisms. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL's test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild type form were analysed for their effect on protein half-life using a protein stability assay, cycloheximide and western blot. CHiP-qPCR for detection of nuclear factor. B (NFkB) binding.

Results

Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NF kappa B-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2 Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR = 2.31 (95% CI 1.38 to 3.86). The 71Thr decreased BLK protein half-life.

Conclusions

These results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently, lead to reduced levels of BLK protein.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-178536 (URN)10.1136/annrheumdis-2011-200987 (DOI)000305293400020 ()
Available from: 2012-08-01 Created: 2012-07-31 Last updated: 2017-12-07Bibliographically approved

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