To Gabbe & Emi

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Löfgren SE*, Yin, H*, Delgado-Vega AM*, Sánchez E, Lewen

S, Pons-Estel BA, Witte T, D’Alfonso S, Ortego-Centeno N, Martin J, Alarcón-Riquelme ME, Kozyrev SV. (2009) Promoter insertion/deletion in the IRF5 gene is highly associated with susceptibility to systemic lupus erythematosus in distinct popu-lations, but exerts a modest effect on gene expression in periph-eral blood mononuclear cells. Journal of Rheumatology, 37(3): 574-578.

II Löfgren SE, Silva JA, Alarcon-Riquelme ME, Kozyrev, SV (2012). Estrogen-dependent up-regulation of IRF5 in human immune cells. Manuscript.

III Löfgren SE*, Delgado-Vega AM*, Gallant CJ, Sánchez E, Frostegård J, Truedsson L, de Ramón E, Sabio JM, González-Escribano MF, Pons-Estel BA, D’Alfonso S, Witte T, Lauwerys BR, Endreffy E, Kovács L, Vasconcelos C, da Silva BM, Mar-tin J, Alarcón-Riquelme ME, and Kozyrev SV. (2010) A 3’UTR variant is associated with impaired expression of CD226 in T and NK T cells and susceptibility to systemic lupus ery-thematosus. Arthritis & Rheumatism, 62(11):3404-3414.

IV Löfgren SE, Frostegård J, Truedsson L, Pons-Estel BA,

D’Alfonso S, Witte T, Lauwerys BR, Endreffy E, Kovács L, Vasconcelos C, da Silva BM, Kozyrev SV and Alarcón-Riquelme ME. (2012) Genetic association of miRNA-146a with Systemic Lupus Erythematosus in Europeans through decreased expression of the gene. Genes & Immunity, In press.

Reprints were made with permission from the respective publishers.

Additional publication

Review Delgado-Vega AM, Sánchez E, Löfgren SE, Castillejo-Lopez C, Alarcón-Riquelme ME. (2010) Recent findings on genetics of system-ic autoimmune diseases. Current Opinion in Immunology, 22(6): 698-705.

Contents

Introduction ................................................................................................... 11 Genes, variations & disease...................................................................... 11 Genes & Autoimmunity ........................................................................... 13 Genes & systemic lupus erythematosus ................................................... 14

Systemic lupus erythematosus (SLE) .................................................. 14 Genetics studies of SLE ....................................................................... 16 Identification of causative variants ...................................................... 21

The interferon pathway, IRF5 & SLE ...................................................... 23 The interferon pathway ........................................................................ 23 The interferon regulatory factor 5 (IRF5) ............................................ 24 Estrogen, SLE & IRF5 ......................................................................... 27

CD226 & SLE .......................................................................................... 28 MicroRNAs & SLE .................................................................................. 30

microRNAs .......................................................................................... 30 miR-146a ............................................................................................. 33

Aims of this study ......................................................................................... 34

Results and discussion .................................................................................. 36 Paper I - Promoter insertion/deletion in the IRF5 gene is highly associated with susceptibility to SLE in distinct populations but exerts a modest effect on gene expression in PBMCs ....................................................... 36 Paper II - Estrogen-dependent up-regulation of IRF5 in human immune cells .......................................................................................................... 39 Paper III - A 3’UTR variant is associated with impaired expression of CD226 in T and NK T cells and susceptibility to systemic lupus erythematosus. .......................................................................................... 42 Paper IV - Genetic association of miRNA146a with Systemic Lupus Erythematosus in Europeans through decreased expression of the gene . 46

Concluding remarks and perspectives ........................................................... 52

Acknowledgments......................................................................................... 54

References ..................................................................................................... 56

Abbreviations

AID ATG5 BAFF BANK1 BLK BlyS CD CNV E2 FCR2A GWAS HLA IBD IC IFN IRF5 ITGAM JAZF1 LD LFA-1 MS NF- B OR PBMC pDC PDCD1 PEST PTPN22 PXK RA SLE SNP SS STAT4 TD1 TLR TNF

Autoimmune disease Autophagy related 5 homolog (S. cerevisiae) B-cell activating factor B-cell scaffold protein with ankyrin repeats 1 B lymphoid tyrosine kinase B lymphocyte stimulator Cluster of differentiation Copy number variant 17 -estradiol Fc receptor 2- Genome–wide association study Human leukocyte antigen Inflammatory bowel disease Immune complex Interferon Interferon regulatory factor-5 Integrin, alpha M JAZF zinc finger 1 Linkage disequilibrium Lymphocyte function-associated antigen-1 Multiple sclerosis Nuclear factor- B Odds ratio Peripheral blood mononuclear cells Plasmacytoid dendritic cells Programmed cell death-1 proline, glutamic acid, serine and threonine (domain) Protein tyrosine phosphatase, non-receptor type 22 PX domain containing serine/threonine kinase Rheumatoid arthritis Systemic lupus erythematosus Single nucleotide polymorphisms Sjögren’s syndrome Signal transducer and activator of transcription-4 Type 1 diabetes Toll-like receptor Tumor necrosis factor

TNFSF4 TNIP1 TYK2 UHRF1BP1 UTR XKR6

Tumor necrosis factor (ligand) superfamily, member 4 TNFAIP3 interacting protein 1 Tyrosine kinase 2 Kell blood group complex subunit-related family member 6 Untranslated region UHRF1 binding protein 1

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Introduction

Genes, variations & disease As individuals, we share approximately 99,9% of our genetic composition and most of the genetic difference between us has no effect on the phenotype or is just part of what makes us all unique, not having any direct effects on health. However, a very small proportion of this genetic variability may lead to various degrees of physiological variation that might be either associated with a higher susceptibility to develop different diseases or, alternatively, give an individual an advantage of protection. There are a number of different genetic variants that can occur in the ge-nome, and this variation may be considered as polymorphic or common when the frequency of the minor allele is at least 1%, or rare, when the mi-nor allele has a frequency lower than 1% in a given population. The most frequent genetic variations are represented by single nucleotide polymor-phisms (SNPs), insertion/deletion polymorphisms (indels), repetitive se-quences and copy number variations (CNVs). SNPs represent the most common variation in the genome, which occurs in about every 1:1200 bases in human DNA. It takes place when a single nu-cleotide at a locus can differ between individuals of a given population. To date, with several large-scale genome analyses and the completion of a 1000 genomes project, more than 50 million SNPs have been identified according to the main SNP database. Of those, about 30% have been annotated as in-tronic SNPs, 1% as coding non-synonymous SNPs and 1% as found in 5’/3’ UTRs or splice site regions (dbSNP, www.ncbi.nlm.nih.gov/projects/SNP/, build 135). Among those a very long list of SNPs are associated with a wide range of different disorders, including dozens with SLE 1-3. Indels are the second most common type of variant found in the human ge-nome. They are believed to account for about 20% of all human genetic var-iation and are considered to be responsible for most of the differences among species 4-5. Indels are typically represented by short sequences of a few ba-ses, generally 2-16 bp, that can be either present or absent and can be in linkage disequilibrium (LD) with common SNPs. Indels are believed to have a higher functional impact compared to SNPs because of the potential more

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drastic consequence on gene regulation and/or protein function6. But due to the difficulty of genotyping or sequencing these variants, they have received relatively little attention. However, the advent of new technologies is ena-bling several large-scale indel detection projects with the potential to greatly expand our understanding of the heritability of many diseases 5, 7. In SLE, there is evidence that indels in the human leukocyte antigen-G 8, the interfer-on regulatory factor 5 (IRF5) 9, in the angiotensin converting enzyme 10 and nuclear factor B (NF- B) 11 contribute to disease susceptibility. Repetitive sequences are believed to comprise 46% of the human genome and consist of diverse lengths of varying number of repeat units 12. Those repeats can be in tandem, like microsatellites (1 to 9bp) and minisatellites (around 10-100bp), or interspaced across the genome. Examples that have been associated with SLE are microsatellites in the tumor necrosis factor (TNF) 13 and minisatellites in the interleukin-6 gene 14. CNVs are larger DNA segments in the kb-Mb range present at variable copy numbers and represent a newly characterized source of susceptibility factors for human disorders. Up to 12% of the human genome may have variable copy numbers caused by insertions, deletions or duplications, which may be inherited or caused by de novo mutations 15. As for indels, CNVs are not as easy as SNPs to detect in large-scale, but with new platforms such as next-generation sequencing and comparative genomic hybridization arrays, to-gether with advanced bioinformatic analysis, an enormous amount of data related to CNVs are today being generated. At present, it is known that CNVs comprise far more nucleotide content than SNPs in the genome and a growing number of associations with disease phenotypes are being reported 16-17. CNVs in a few genes (e.g. the complement component C4 18 and the Fc fragment of IgG, low affinity IIIb, receptor -FCGR3B 19) have also been associated with SLE. For the last several years, there has been a very active debate about the im-portance of common genomic variants of low penetrance versus rare genetic variants of high penetrance in conferring disease susceptibility. Today, there seems to be an agreement that complex human disease susceptibility is the result of both. As mentioned, new technologies has made possible large-scale analysis to define nearly comprehensive maps of genetic variation, including several million SNPs, hundreds of thousands of small indels and thousands of struc-tural variants in human genomes. Genome-wide association studies (GWAS) have also significantly contribut-ed to genetic association studies in disease. It allows the use of large sample sizes with higher statistical power to identify associated loci previously un-

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detectable. At the same time, current arrays permit the analysis of millions of variants in a single run. For example, GWAS have already identified asso-ciations between ~1,300 loci and ~200 diseases or traits 20. However, the completion of hundreds of GWASs today has exposed the fact that the associations found often explain only a small proportion of the phe-notypic variation or the extent of the excess familial risk of many diseases, highlighting the so called “missing heritability” of human diseases 20. Thus, the search continues for the missing pieces in the puzzle of this heritability. New research is focusing on gene–environment and gene-gene interactions, copy number variants, epigenetics, and the rare variants missed by former analyses and now more accessible by next generation technologies 21.

Genes & Autoimmunity The immune system is a very complex network designed to identify and combat infectious agents and other foreign antigens, and in this process it is crucial to distinguish between self and non-self. When this normally well regulated self-tolerance is broken and an inappropriate immune response is raised against self-antigens, it leads to autoimmune disease (AID). AIDs comprise more than 100 disorders with a common feature of disturb-ance of self-tolerance that today is estimated to affect approximately 5% of the world population 22. Many AIDs disproportionally affect women and as a group they represent one of the main causes of death among young and middle-age women in the U.S. 23. But in addition to the risk of mortality, these diseases are the second highest cause of chronic illness, bringing poor quality of life, frequently with a lot of pain and physical limitations, both as a consequence of the disease itself and the treatments 24. AIDs are the result of a join contribution from genetic predisposition and environmental factors. This may result in both self-reactive B- and T- lym-phocytes and the effect of the autoimmune process can be either or-gan/tissue-specific (localized), such as type 1 diabetes (TD1) and celiac dis-ease, or against ubiquitously expressed antigens resulting in a systemic effect such as rheumatoid arthritis (RA), Sjögren’s syndrome (SS) and systemic lupus erythematosus (SLE) 25. The reason behind the greater incidence of AIDs among women versus men is not fully understood. However, research suggests that estrogen is correlat-ed to increased autoimmune responses and empirically it has been observed that pregnancy can trigger some AIDs as well as cause other to go into re-mission 26.

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The majority of AIDs have a more or less clear genetic predisposition. In-creased heritability within families and the decrease in risk with the degree of relatedness all argue in favor of genetic factors, although the modest con-cordance rate in monozygotic twins suggests that environmental factors are as well important players in most AIDs 27. The technological improvements in the genome analysis over the past few years have greatly contributed to our knowledge about the genetic back-ground of many AIDs. Some genes and/or pathways have shown to be shared among multiple AIDs, while others are essentially unique to a specif-ic AID or a group of related AIDs 28-29. Indeed, certain AIDs commonly co-occur in a single individual or within families more often than expected sug-gesting the presence of genetic variants that predispose to autoimmunity 30. Also, as more and more genetic associations arise more genes are found to be shared among AIDs demonstrating a probable common etiology underly-ing autoimmunity.

Genes & systemic lupus erythematosus Systemic lupus erythematosus (SLE) SLE is a prototypic systemic autoimmune disorder with a prevalence of 20-69 cases per 100 000 individuals and a very strong sex biased incidence (90% women) reaching a peak in the reproductive age 31. It is a chronic in-flammatory disease characterized by immune dysregulation resulting in pro-duction of autoantibodies, generation of immune complexes (IC), increased apoptosis and defective clearance. Excess of autoantibody-antigen IC cou-pled with its impaired clearance leads to complement activation and finally to persistent activation of inflammatory processes and damage of organs and tissues, e.g. joints, kidney, heart 32. Due to the variety of symptoms and organ systems involved, SLE diagnosis is difficult and a lupus patient usually takes approximately 4 years to be di-agnosed, mainly because of similarities with other diseases and the lack of adequate diagnostic tests.

Because of this difficulty, The American College of Rheumatology estab-lished in 1982 eleven criteria for diagnosis of SLE, which were revised in 1997, as a classificatory instrument to define SLE patients in clinical trials 33. These criteria span the clinical spectrum of SLE, including skin criteria (ma-lar ‘butterfly’ rash, oral ulcers, photosensitivity, and discoid rash), systemic criteria (pleuritis or pericarditis, arthritis, renal disease, or central nervous system involvement), and laboratory criteria (cytopenias, anti-double-stranded DNA or anti-phospholipid / antinuclear antibodies). According to

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these criteria, a patient is classified as having SLE if any 4 out of the 11 symptoms are present simultaneously or serially on two separate occasions. It is important to note that by these criteria, different SLE patients do not necessarily have any shared clinical outcomes, illustrating the great hetero-geneity of patients with this disorder and justifying discussions about their classification as one single disease.

The prevalence of SLE varies considerably between countries, ethnicity, age and gender. Several studies indicate that in average the incidence is higher in African-Caribbean, Asians and Hispanics, while Europeans have the lowest incidence rates of all ethnic groups 34-35. The onset of the disease has also been shown to be later in life in European patients (around 50 years) when compared to Asians and Caribbeans (around 27 years) 35. It is unclear the extent of the influence of the genetic and the environmental factors be-hind this heterogeneity.

With the advances in diagnosis and treatment over the last fifty years, the survival of SLE patients has improved from a 5-year survival rate of only 50%, to a 20-year survival rate of 70% 36-37. But despite improved survival rates the current treatment is not always effective with many medications having major side effects and the mortality among patients with SLE re-mains in average 3-5 times higher than in the general population 37.

In fact, although the disease is known for over a century, today there is still no adequate specific treatment for SLE patients. Standard therapy includes corticosteroids, anti-malarial drugs, immunosuppressive agents, and non-steroidal anti-inflammatory drugs targeting important features of the immune system in a non-specific manner with important side effects 38. Thus, there is a major need to improve therapies towards more selective targets.

This year (2011) Belimumab (Benlysta®), the first drug for treatment of SLE in more than 50 years, was approved by the U.S. FDA. Belimumab is a monoclonal antibody against B lymphocyte stimulator (BlyS)/B-cell activat-ing factor (BAFF), produced mainly by monocytes/macrophages and that promotes the maturation of B cells, including development into plasmablasts and plasma cells that secrete autoantibodies 39-40. However, much is still discussed about the weak efficacy of this drug when compared to placebo, as well as whether specific sub-groups of SLE patients or ethnic groups will benefit most from this treatment.

Certain environmental factors are believed to play an important role in SLE development, exacerbating existing SLE conditions as well as trigger-ing the initial onset of the disease in genetically susceptible individuals. They include certain medications (such as some antidepressants and antibiot-ics), stress, sunlight exposure, infections and hormones 1. There is increasing evidence suggesting that sex hormones such as estrogen might play an im-portant role in immune responses, directly modulating the development and function of different immune cells, although the mechanism by which this might occur is still not well understood 41.

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The familial occurrence of SLE on the other hand was first noted by Leon-hardt in 1954 and later studies by Arnett and Shulman at Johns Hopkins (in 42). It has now been well described by family studies that siblings of SLE patients have 8-20 times higher risk of developing the disease than in the general population. Moreover, twin studies revealed that the concordance rate for monozygotic twins is 10-20 times higher than for dizygotic twins, indicating a clear genetic component contributing to disease susceptibility43.

Genetics studies of SLE SLE demonstrates a complex pattern of genetic inheritance. It is believed to be caused by a number of genetic and environmental factors that together determine the development of the disease and the clinical manifestations, and thus impeding the hunt for the causative genetic factors.

Prior to 2008, two main strategies were applied to identify susceptible ge-netic variants: linkage studies and candidate gene association studies.

Two genetic loci are linked if they are transmitted together from parents more often than expected under independent inheritance, and they are in LD if, across the population as a whole, they are found together on the same haplotype more often than expected 44. Linkage studies of complex diseases as SLE have been applied to identify genomic regions in affected siblings pairs and are based on the hypothesis that the affected siblings will share more often than expected haplotypes at disease loci. But this method only identifies large genomic regions which often contain a high number of genes. For SLE, linkage studies have had some success and contributed to the identification of some susceptibility genes including HLA-DRB1, TNF, PDCD1 and Fc gamma receptors (FCGR) 45.

However, for SLE the most common approach to identify risk variants has been candidate gene association studies which directly test the associa-tion of selected genetic variants with a certain trait or disease. This approach requires an initial hypothesis of a ‘candidate gene’, which is normally cho-sen either based on its biological plausibility in regards to the phenotype or disease, by results of linkage studies or previous associations with related diseases or specific traits.

The model assumes that if a certain allele is associated with the disease, its frequency would be statistically different when compared to a carefully matched group of healthy controls. The magnitude of this association is measured by the significance of this difference as well as the odds-ratio (OR), reflecting the probability of the event (e.g. disease) to occur in the presence or the absence of the risk variant 46. In the case of disease associa-tion studies, it assesses the contribution of the variant to disease susceptibil-ity, and the vast majority of susceptibility variants for complex diseases such as SLE have low or moderate OR (< 1.7) 47.

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In genetic association studies, LD between polymorphisms must be con-sidered, as association of a particular variant may in reality reflect the asso-ciation of a marker in LD. The measurement of LD may be measured by D’ or by the correlation coefficient, r2. Both ranges from 0 to 1 where 0 denotes no LD and 1 means complete LD. r2 is more frequently used in the assess-ment of LD as it can predict how well one SNP can act as a surrogate (proxy) for another, i.e. with the given allele frequencies of one SNP it is possible to predict the allele frequencies for the other SNP. The availability of this information is also of great advantage as it permits the genotyping of only a fraction of polymorphisms in association studies, as many more can be predicted if in high LD with the genotyped markers. This association approach has lead to identification of a modest number of genes reproducibly associated with SLE, such as the HLA region, Fc recep-tor 2- (FCR2A), IRF5, signal transducer and activator of transcription 4 (STAT4) and protein tyrosine phosphatase 22 (PTPN22) 43.

In the last years however, GWAS and the development of commercial 'SNP chips' or arrays without any predetermined hypothesis on the candidate genes, has become a very popular approach also for SLE. It is still an asso-ciation study, but in a large-scale mode which analyze a great number of individuals and tests for thousands to millions of variants. Taking into con-sideration the expansion of the genetic variation by exploiting LD patterns between typed and untyped markers, it is considered to represent most of common variation in the genome. Thus, the assessment of six GWASs, four in Caucasians and two in Asians 48-53, has led to the rapid identification of several new genetic associations in SLE in the last couple of years. Getting back to the missing heritability issue, based on sibling recurrence risk it is estimated that GWAS associations so far can account for about 15% of the SLE heritability 21. However, one im-portant contribution of this ‘candidate-gene free’ approach in understanding complex diseases is illustrated by the fact that many identified genes or loci in these studies were not previously suspected to be involved in the patholo-gy of the disease, such as a role for autophagy in Crohn's disease 54.

But as expected, the great majority of the recently identified genes for SLE are involved in immune responses, both innate and adaptive. Among more than 30 loci convincingly related to SLE today, as summarized in table 1, the majority are implicated in immune-complex (IC) processing, T/B cell signaling and/or development and Toll-like receptor (TLR)/type I interferon signaling. Impaired IC clearance and deposition is an important pathological aspect in SLE and susceptibility genes with important roles in IC processing known from previous studies are the FcGR family of genes, and more re-cently, ITGAM [10] coding for the surface antigen CD11b (or CR3). Signal

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transduction in immune cells, particularly T and B cells, is another pathway that has revealed multiple lupus susceptibility genes, as TNFSF4 (OX40L), PDCD1 and PTPN22, modulating T cell signaling, and BANK1 and BLK, potentially involved in B cell activation and tolerance 28.

Other genes include ATG5, an important component of the autophagy pathway; the transcription factors JAZF1 and UHRF1BP1, TNIP1 which interacts with TNFAIP3, and TNFAIP3 regulating inflammation28. However, in the GWAS era the strongest association in Europeans is still the HLA, the first associated region, which consist of a large and strongly corre-lated region involving more than 400 genes, which pose a challenge for analysis. Other recently identified loci, such as PXK, XKR6 and KIAA1542, with no known function or clear correlation to SLE pathology, have the potential to lead to discovery of novel pathways involved in SLE. Studies in Asian populations have replicated some of the genes found in Europeans such as STAT4, IRF5, BANK1, BLK, TNFAIP3, and TNFSF4. They also identified new susceptibility genes for SLE, in particular ETS1 and WDFY4 50, 55. Those genes might be specific of Chinese population as such association has not yet been found in other ethnicities. Recently a case-control study of 15 known susceptibility loci in Cauca-sian/Asian confirmed several associations including BLK, BANK1, TNFSF4, KIAA1542, ITGAM, STAT4 and FCGR2A also in African-American 56. Further African-American and European-Amerindian admixed populations studies are expected to be published and might contribute to the understand-ing of common and private genetic factors behind SLE in different ethnici-ties. One pathway that has been both biologically and genetically strongly related to SLE pathogenesis, as well as other AIDs, is the Type I interferon (IFN). To date several genes for factors both up and downstream of IFN production, such as IRF5, STAT4, and more recently TNFAIP3, TYK2, TREX1 and IRAK1, have been correlated to susceptibility to SLE 57-59. Among these loci, IRF5 is the most compellingly associated gene, and studies point to at least two independent variants in the gene, each correlated with higher IRF5 tran-script and protein expression in PBMCs 9, 60-61. IRF5 has also been shown to act in an additive way with STAT4, another strongly associated gene in this pathway 62-63.

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Table 1 Susceptible genes in SLE (adapted from 28)

Chromosome Gene SNPs Populationsa References

6p21 HLA region DRB1*0301 and several other alleles

Eu, several As, Af-Am, mixed Eu-Ami 64-65

7q32 IRF5 5bp promoter indel,

rs2004640, rs2070197, rs10954213

Eu, several As, mixed Eu-Ami, Af-Am

49-50, 52-53, 66

2q32 STAT4 rs7574865, rs3821236, rs7601754

Eu, mixed Eu-Ami, several As, Af- Am

49-50, 52-53, 66

6q23 TNFAIP3 rs5029939, rs2230926 Eu, As, Af- Am 48-50, 52-53, 66-67

16p11 ITGAM rs9888739, rs1143679, rs4548893

Eu, mixed Eu-Ami, As, Af- Am

49-50, 52-53, 66, 68

4q24 BANK1 rs10516487, rs17266594, rs3733197 Eu, Eu-Ami, As 50-51, 53, 55

1p13 PTPN22 rs2476601 Eu 49

8p23 BLK rs13277113, rs2736340 Eu, several As 49-50, 52-53, 66

2q37 PDCD1 (CD279) PD1.3A Eu, Eu-Ami, Ch 69

1q25 TNFSF4 Risk haplotype; rs3850641 Eu, As 50, 53, 55, 70-71

18q22.3 CD226 rs763361, rs727088 Eu, Eu-Ami 72-73

1q21-23 FCGR2A ARG131HIS Eu, Eu-Ami, Af-Am 49, 52, 66

19p13.2 TYK2 rs280519 Eu 64

3p21.3 TREX1 rs72556554, R114H and other 11 nonsynonymous

substitutions Eu 74

Xq28 MECP2-IRAK1 rs2269368 Eu, Ch, Korean, Eu-Ami

(Mexican) 64,75

3p14.3 PXK rs6445975, rs2176082 Eu 49, 66

2q24 IFIH1 rs1990760 Eu 64

11p15.5 KIAA1542 (PHRF1) rs4963128 Eu 49

8p23.1 XKR6 rs6985109 Eu 49

6q21 ATG5-PRMD1 rs6568431, rs2245214 Eu, Ch 49, 66

22q11.2 UBE2L3 rs5754217 Eu, Ch 48, 64

5q33.3 PTTG1/miR146a rs2431697 Eu, Ch 48, 64, 76

6p21 UHRF1BP1 rs11755393 Eu 64

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5q32 TNIP1 rs7708392 Eu, Ch, Thai 64, 50

7p15.2 JAZF1 rs849142 Eu 64

7p21.3 ICA1 rs10156091 Eu 49, 66

1q24 IL10 rs3024505 Eu 64

1q25.3 NMNAT2 rs2022013 Eu, Ch 49-50

11q23.3 ETS1 rs6590330 Ch, Thai 50, 53

10q11.23 WDFY4 rs877819 Ch, Thai 50, 53

7p12.2 IKZF1 rs4917014 Ch 50

12q24.32 SLC15A4 rs10847697, rs1385374 Ch 50

2p22.3 RASGRP3 rs13385731 Ch 50

a Population abbreviations: Eu, European; As, Asian; Af, African; Am, Amerindian; Ch, Chinese; Af-Am, African-American; Eu-Ami, European-Amerindian.

One important aspect in genetic studies of SLE is the substantial heterogene-ity of the disease. Since, as mentioned, patients may have very diverse clini-cal manifestations, it might be reasonable to think that although there is like-ly an underlying pathological mechanism, different patients may have differ-ent causal genetic risk factors acting in specific pathways leading to different disease manifestations. Such associations are very difficult to detect when SLE patients are considered as a single phenotype. A detailed and accurate classification and analysis of SLE sub-phenotypes will greatly contribute to the success in genetic association studies and provide greater insight into pathogenic mechanisms in such a heterogeneous disease. For example, studies on STAT4 have revealed a strong correlation of a risk allele with presence of anti-double-stranded DNA (anti-dsDNA) autoanti-body, nephritis, and early disease onset 77. A SNP in ITGAM has also been correlated with nephritis and neurological disorder in Asians 78. Patients’ positive for anti-dsDNA and anti-Ro antibodies were as well each associated with different IRF5 haplotypes by a different combination of functional vari-ants 79. However, taken as a whole, we are still in the early stages of understanding the biological role of the identified genetic susceptibility variants, and in the vast majority of cases, the actual causative variant has not been clearly estab-lished. Moreover, the individual contribution to disease susceptibility by an associated SNP is in general relatively low (odds ratio generally between 1.15 and 2). In addition, the role of environmental factors is still not well understood, as well as the emerging number of rare variants, copy number

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variants, epistasis and epigenetic effects, which importance only recently started to be explored. Indeed, current studies indicate that most of the genes found to be associated with SLE are predicted to be regulated by miRNAs - including genes in the IFN pathway, and hypomethylation in T cells and aberrant methylation of SLE candidate genes has also been shown 80, suggesting an even more elabo-rated layer of gene regulation. CNVs, with the possibility to affect large ex-tent of the genome, are also a promising field to explore. Improved tools to analyze these aspects may soon reveal many more complex aspects in SLE susceptibility.

Identification of causative variants As mentioned throughout the text, we are now witnessing a real explosion of genomic data generation through different approaches, with the ability to generate information quickly and cheaply. The information gathered has been very important to get a comprehensive overview on genomic variation between individuals, and has greatly contributed to evolution understanding, population studies, and for disease association analysis. However, in the case of disease, the ultimate goal is to be able to correlate the variation found with biological pathways to better understand the disor-der, develop better diagnostic tests, and improve prevention and treatment for the patients. In that matter it is important to highlight the significant gap between the identification of a susceptible locus in the genome and the actu-al understanding of the functional importance of this variant in the disease pathogenesis, and also the outcome to a particular patient. The functional characterization of susceptibility variants is not straightfor-ward and often cannot be done in a high-throughput manner. Therefore, fur-ther characterization requires considerable time for experimental confirma-tion and validation. Accordingly, among the more than thousand of disease-associated loci identified today the precise identification of the causal variant has been achieved for only a handful of those 81. Indeed, in the case of SLE, the documentation of genetic associations is only the first step and the identification of polymorphisms with a true genetic role in disease susceptibility, and specially understanding their biological func-tion in the development of the disease has proven to be a challenge. Indeed, the role of the great majority of the disease-associated polymorphisms today are still awaiting to be elucidated. Examples of such studies include IRF5, where some functional variants have been identified and experimentally demonstrated. The variants give rise to

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different isoforms or affect gene transcription and mRNA stability 9, 82, as further discussed in the text. The associated variants in BANK1 influenced splicing efficiency of the transcript and protein properties changed by amino acid substitution, relevant in SLE 51. Studies on PDCD1 also revealed an intronic SNP that changes the strength of the binding site for the RUNX1 transcription factor 83. A non-synonymous SNP in the FCGR2A gene located in the ligand binding domain of the receptor also significantly reduced the binding affinity to circulating IgG2, leading to impaired Fc R-mediated phagocytosis and poor clearance of ICs 84-85. A non-synonymous SNP in the FCGR2B gene on the other hand was correlated to an exclusion of the recep-tor from lipid rafts leading to impaired inhibitory signaling 86. There are also a number of studies mentioning SNPs as causative but without any experi-mental verification. When an associated variant is located in a coding region and directly affects the gene product, the underlying molecular mechanism of action is relatively easy to predict and study. However, in complex diseases, where the relative contribution of each risk factor is rather low or modest, variants are often non-coding and frequently have a regulatory role 87. Indeed, causative vari-ants may occur at splice sites, which may result in exon skipping or intron retention; in untranslated regions affecting transcript translation, stability, secondary structure or sub-cellular localization; in promoter regions, affect-ing gene expression, by altering transcription factor binding sites, or even in other distal regulatory regions of the gene. Several of the genetic variants identified via GWAS are located in intergenic or intronic regions. Obviously, in these cases, it is more difficult to assign the variants a functional mechanism. One could suggest the existence of long-range LD between the associated variant and a yet unknown causative variant. It might be also that we simply do not have enough knowledge of the complex regulatory architecture of the genome to understand the mecha-nism behind the association. Either way, it is very probable that as we acquire a better understanding of the genome structure and gene expression regulation mechanisms, the data generated today will get much more useful and interesting.

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The interferon pathway, IRF5 & SLE The interferon pathway Interferon (IFN) was first described more than 50 years ago and was named by its ability to ‘interfere’ with virus replication 88. Today, there are 3 types of IFN recognized 89. The group of type I IFNs includes mainly IFN- and -

, while only IFN- represent the type II, and IFN- belong to the type III group 89-90. Most cells produce type I interferon during early responses to viral infection, with plasmacytoid dendritic cells (pDCs) being the major producers. pDC are able to secrete IFN- at levels up to 1000 fold higher than any other blood cell following viral infection. IFN- production is induced mainly via pathogen recognition receptors toll-like receptor 7 (TLR7; recognizes single-strand RNA), and TLR9 (recognizes DNA with unmethylated CpGs present in bacterial or viral genomes) 91-92. Its production can also be stimulated by endogenous factors such as the self- derived nucleic acid containing immune complexes found in SLE 93. Because of IFN- ’s clear value as an important clinical drug and a potent immune adjuvant, it was used in several clinical trials against a variety of diseases in the 1980’s. However, it was soon observed in a relatively high portion of the patients an increase of autoantibodies and occurrence of auto-immune diseases, including SLE 94. It was described also that SLE patients have higher serum levels of type I IFN when compared to healthy individu-als 95. After this first indications of the involvement of type I IFN in auto-immune pathology, many studies followed and today the pronounced up-regulation of type I IFN as well as of IFN-inducible genes, the so called ‘IFN-signature’ 96-97 has been well established as a frequent feature in SLE. It has also later been observed in other AIDs, such as RA, SS and multiple sclerosis (MS), suggesting a common underlying feature in autoimmunity 95,

98. Thus, while in healthy individuals the production of this very strong im-mune activator is normally under tight control, in AID patients this homeo-stasis doesn’t seem to be completely achieved. The higher type I IFN levels potentially promote B cell activation, differen-tiation, Immunoglobulin isotype switch, and the generation of antibody-producing plasma cells as well as memory cells 99-100. In addition, type I IFNs prolong the survival of activated T and B lymphocytes and suppress regulatory T cells 101. It also leads to an increased production and exposure of autoantigens, such as Ro52, increased self- derived nucleic acid-containing IC formation that further stimulate pDC to produce more IFN- . This results in a chronic activation of the type I IFN system in a vicious cir-

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cle manner, driving an autoimmune process through inflammation and tissue damage 91, 102. Polymorphisms in genes that are involved in the IFN pathway were evident-ly strong candidates to contribute to this up-regulation. Thus, in 2005 a ge-netic study was performed that screened variants in several genes involved type I IFN production and signaling and led to the identification of IRF5 as a risk factor for SLE 103.

The interferon regulatory factor 5 (IRF5) Interferon regulatory factors (IRFs) constitute a family of 9 transcription factors (IRF1 to IRF9). The members share extensive homology in the DNA-binding domain at their N-termini which recognize a core DNA se-quence normally in a context of IFN-stimulated responsive elements (ISRE). The C-termini on the other hand is rather distinct between IRFs, allowing specific interactions and regulation of both common and distinct target genes 104. IRFs are classically activated through different TLR pathways present in different cell types, including TLR-3, -4, -7/8 and -9. The precise expression of particular TLRs and IRFs in different immune cells allow defined cell-specific effects 105-107. In addition, competition by some IRFs for the same adaptor molecules and/or DNA binding sites contributes to enhance these effects. Besides IRF5, polymorphisms in IRF7 and IRF8 have recently also been genetically associated with SLE 108-109, and nearly all have been in different ways biologically correlated to the disease. However, IRF5 is recognized as one of the strongest and most consistent genetic risk factor outside the MHC region for SLE 106. IRF5 genetic associations has also been replicated in different ethnicities as well as other AIDs, including RA, SSc, SS, primary biliary cirrhosis, inflammatory bowel disease (IBD), and MS, indicating that it may play an important role in the development of autoimmunity 43, 50, 106,

110-112. IRF5 is a key transcription factor in the type I IFN pathway. It is activated by IFN / and regulates the expression of other IFN-dependent genes, in-flammatory cytokines such as TNF , IL-12 and IL-6, genes involved in apoptosis and, importantly, IFN / itself 58, 103. It has been described that SLE patients have higher expression of IRF5 in PBMCs 113 which might partially explain the up-regulation of IFN / and the ‘IFN signature’ in SLE. The up-regulation of IRF5 may also lead to accelerated apoptosis, contrib-uting to the increased burden on the immune clearance system (Figure 1).

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IRF5 is expressed mainly in pDCs, monocytes and B cells 105 and primarily resides in the cytoplasm in the absence of any activating stimuli. Upon liga-tion of the intracellular TLR7/8 or 9 with specific ligands, IRF5 binds to tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1), is phosphorylated by TBK1/IKK kinases 114, forms dimers and translocates to the nucleus, where it binds to ISRE sequences in the promoter regions and activate target genes 105, 107, 114. Upon activation, IRF5 can either form homodimers as well as heterodimers with IRF3 or IRF7, where homodimers or IRF5/IRF3 complexes activates and IRF5/IRF7 heterodimers inhibits expression of type I IFN genes 115.

Figure 1. A model on the role of IRF5 up-regulation and the interferon pathway in pathogenesis of SLE. Viral infection or IC trigger TLR signaling in pDCs, in which IRF5 is highly expressed in genetically predisposed individuals. This promotes the production of inflammatory cytokines including type I IFNs and induces the 'IFN signature' in various immune cells (gray arrows). IFN / induces maturation of DCs, which capture and presents autoantigens and activate autoreactive Th cells and the production of autoantibodies by autoreactive B cells, which results in self-nucleic acid-IC formation. IFN / may also disturb the normal trafficking and chemotaxis of cytotoxic T cells, macrophages and NK cells thus contributing to inflammation in multiple organs. IC and large amount of nucleosomes resultant from tissue damage can be captured by pDCs and mature DCs, further amplifying the autoreactive process (red arrows) 101 .

Many IRF5 variants have been associated with SLE, among them four have plausible functional importance: SNP rs10954213, alters the poly(A) signal; exon 6 insertion/deletion, potentially affects the function of the PEST do-

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main of the protein; rs2004640 may create a splice site; and the most recent-ly identified promoter CGGGG indel may strengthen the binding site for the Sp1 transcription factor (Figure 2) 105, 116. Interestingly, the most robustly associated variant (rs2070197) tags the risk haplotype but has no obvious functional role as judged from its sequence context and location.

Figure 2. Schematic representation of the IRF5 gene showing the relative position and sequence details of four described putative functional variants and rs2070197 which is a marker of the risk haplotype. Coding exons are in black and non-coding exons in white. Adapted from 105.

The risk alleles of these putative functional polymorphisms segregate in a strongly associated haplotype. But while the genetic association is well es-tablished, the functional consequence of these variants and how they predis-pose to autoimmunity is not completely understood and requires additional studies. The variant rs2004640 does not seem to play an important functional role because of the very low expression of the alternative isoforms that arise due to the T allele of this SNP 116.

The indel of two tandem repeats (30bp) in exon 6 lies in a PEST domain (proline (P), glutamate (E), serine (S) and threonine (T)) and could potential-ly affect the interaction of the protein with other factors. But although this 10 amino acid indel is an attractive candidate for having a functional role, no clear biological consequence of this indel alone has been established 105, 116-

117. It is not per se associated with SLE or with altered IRF5 expression, but its presence in the risk haplotype suggests a potential contributing to risk for SLE.

The risk allele of rs10954213 creates an early polyadenylation site leading to a shorter transcript lacking two strong ARE elements (AU-rich elements), associated with rapid RNA turn-over. Thus, the risk allele leads to a shorter and more stable mRNA. This SNP is the most established variant associated with elevated expression of IRF5, described in both PBMCs and LCLs 118.

A recent study described a promoter CGGGG indel located 64 bp up-stream of the first untranslated exon of IRF5 which is highly associated with

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SLE 9. The insertion of one repeat (4x) strengthen the binding of the Sp1 transcription factor in the IRF5 promoter and correlated with higher gene expression. It is in a relatively high LD with rs2004640 and was regarded as a functional variant that could account for the whole association of the other putative causative variants in the gene. This study was however fairly small using only Swedish individuals and no replication of these results were per-formed so far.

Estrogen, SLE & IRF5 In SLE, as well as in other AIDs, it is true that among several environmental and genetic factors the major risk factor for developing the disease is the female gender.

There are many possible explanations for this bias, including the expres-sion of the extra X-chromosome genes - by for example inactivation-skipping, as well as differential genomic imprinting patterns. And, im-portantly, the significant difference between genders concerning the pres-ence of sex- hormones. In that regard, there are some lines of evidence indi-cating that the primary female sex hormone, estrogen, has in general a pro-inflammatory / pro-autoimmune effect, whereas testosterone would have the opposite result 119. In SLE it has been suggested, although there is still some controversy, that estrogen is a key player in the development of the disease. Some empirical lines of evidence include the fact that after passage from pre-puberty to re-productive years (highest production of sex-hormones), the discrepancy in frequency of SLE increases from 3 times to 10 times higher in females than in males 36. In addition, the fact that flares of the disease are aggravated dur-ing pregnancy and that men with Klinefelter syndrome - a XXY genetic syn-drome characterized by an abnormal estrogen/androgen pattern resembling the female model, develop SLE at a rate similar to women, indicate a key role for estrogen in SLE development 120. The effect of hormone replacement therapy and oral contraceptive in the risk of developing and/or exacerbation SLE is still unclear, but administration of 17-beta estradiol (E2) has been observed to aggravate the disease in New Zealand brown/New Zealand white F1 female mice (a model of SLE) 121. Further studies in mice models have shown that E2 administration breaks B cell tolerance and induces a lupus-like phenotype with expansion of anti-dsDNA B cells, elevation of anti-dsDNA antibody titers and glomerular immunoglobulin deposition 122-123.

In humans, E2 treatment has revealed a number of SLE-relevant effects in immune cells, including an increase in anti-DNA antibodies in SLE PBMCs, and altered T cell apoptosis and signaling in patients 124-126. No difference in

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plasma estrogen levels has been observed between women with or without SLE although a higher expression of estrogen receptor- in PBMCs from SLE patients has been reported, which could indicate a higher responsive-ness to estrogen of immune cells in patients 127. Estrogens exert their effects by activating their intracellular receptors, estro-gen receptors alpha (ER- ) and beta (ER- ), encoded by their respective genes ESR1 and ESR2. These receptors contain a N-terminal transcriptional regulation domain, a C-terminal ligand-binding domain, and a DNA-binding domain, which binds to estrogen responsive elements (EREs) in the regulato-ry regions of target genes 26, 120, 128.

The transcription regulation may occur in a direct way when the estrogen-ER complex binds directly to their consensus EREs, or in an indirect way when the complex further binds to a transcription factor such as AP-1, SP-1 or NF B, and modulating its binding to their specific DNA binding sites and thus indirectly regulating their target gene expression. These genes indirectly regulated by estrogen do not require a full ERE sequence, but may present a half-ERE in proximal combination with the transcription factor binding site 129-130.

The first piece of evidence that estrogen could be related to IRF5 came from a microarray analysis of murine NK cells treated with E2, revealing an up-regulation of IRF5 131. It was also shown that splenic B cells from lupus-prone female mice expressed higher levels of IRF5 than strain-matched male mice. In addition, the expression of IRF5 was enhanced when treated with exogenous E2 in a dose-dependent manner 132.

There was no study directly testing the effect of estrogen on IRF5 expres-sion in human immune cells.

CD226 & SLE CD226, or DNAX accessory molecule-1 (DNAM-1), was first identified by Shibuya et al (1996) 133, describing its role in primary adhesion during cyto-toxic T lymphocyte-mediated cytotoxicity and NK–mediated lysing of tumor cells. It is a type I transmembrane receptor of the immunoglobulin superfam-ily with two Ig-like extracellular domains that is today known to be broadly expressed on the surface of hematopoietic cells such as T cells, natural killer (NK) cells, natural killer T cells (NKT), monocytes/macrophages, dendritic cells, cells of megakaryocyte/platelet lineage and in a small subset of B cells 30. CD226 acts as an adhesion molecule and is important for both intercellu-lar binding and intracellular signal transduction 134. Recent studies indicate that CD226 is involved in a wide range of immunological functions, includ-ing T cell activation and differentiation, NK cell cytotoxicity, NKT cells apoptosis, platelet activation and aggregation, monocyte extravasation, den-

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dritic cell maturation, and platelet and megakaryocytic cell adhesion to vas-cular endothelial cells 135-136.

The poliovirus receptor (CD155) and its family member nectin-2 (CD112) have been identified as ligands for CD226. CD155 and CD112 are broadly distributed on hematopoietic, epithelial, and endothelial cells, and highly expressed in many types of tumor cells. Engagement of its ligands induces lipid rafts recruitment and the physical association of CD226 with leukocyte function-associated antigen-1 (LFA-1), inducing serine and tyro-sine phosphorylation of the cytoplasmatic domain of CD226 by the protein kinase C and tyrosine kinase Fyn, mediating co-stimulatory signals and cell activation 133, 137-138. In the last few years, CD226 has been associated with a number of disorders, principally different types of cancer and most recently also with acute graft-versus-host disease 139, HIV 140 as well as several AIDs 141-144 . In 2007, a GWA study for type 1 diabetes (T1D) identified the association of a non-synonymous risk variant (rs763361) in the CD226 gene 145, and since then the association of this SNP has been studied in MS, celiac disease, auto-immune thyroid disease, RA, SSc and Wegener’s granulomatosis 145-148. In the course of this study, a weak association of rs763361 with SLE in Chi-nese was also published, but only the genetic association for the previously known SNP was analyzed. No association was found for Japanese or Co-lombians, the other two populations analyzed in this study 73.

The SNP rs763361 changes the amino acid glycine to serine at the C-terminal region (position 307) of the protein (figure 3) and has been specu-lated to alter RNA splicing by disrupting a splice site enhancer/silencer or alternatively to affect the two nearby phosphorylation sites at positions 322 and 329 thus affecting CD226-mediated signaling 145. But up to today no experimental study has been performed to verify this hypothesis.

Figure 3. Gene structure and corresponding protein domains of CD226. The non-synonymous variant rs763361, is located in exon 7, which in part codes for the cyto-plasmic tail 30.

Concerning SLE, it has been found that CD226 levels are altered on the sur-face of T cells in patients 149. Moreover, CD226 expression is down regulat-

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ed specifically in NKT cells in patients with active SLE, which correlates with increased susceptibility of these cells to apoptosis and could potentially explain the observed lower frequency of these cells in SLE patients 144.

Also, the numbers of NK cells seem to be reduced in active patients. Re-cently, it was reported that this reduction is mainly composed by CD226+ NK cells 150. Interestingly, the observed reduction of circulating CD226+ NK cells in MRL/lpr mice is accompanied by an increase in activated CD226+ NK cells migration to kidneys during disease progression, with a possible role in disease pathology. In the same study, a link between the IFN system and CD226 was also proposed, suggesting that IFN- stimulation down-regulates the proportion of specifically CD226+ NK cells 150.

However, whether or not CD226 is a susceptibility gene for SLE in Euro-peans was not known, as well as any comprehensive study of the gene or functional analysis of the SNP had been conducted so far.

MicroRNAs & SLE microRNAs The first miRNA, lin-4, was discovered in 1993 when a locus that controlled developmental timing in Caenorhabditis elegans was surprisingly found to encode not a protein, but only a 22 nt transcript 151. Only 7 years later the second miRNA, let-7, was described, again in C. elegans, 152 later also found in diverse metazoan species, and soon this class of small non-coding regula-tory RNA was named microRNAs.

The number of miRNA-related publications in Pubmed has grown from 5 in 2001 to more than 14,000 in 2011, evidencing an explosion in the study of miRNAs and their biological roles in the last 10 years.

miRNAs have revealed a new and exciting mechanism of gene expression regulation, playing a crucial role in ‘fine-tuning’ negative regulation in many physiological and pathophysiological processes 153-156. Currently, it has been well established that these miRNAs are highly conserved in animals and plants and have the capacity of post-transcriptionally regulate certain subsets of messenger RNAs (mRNAs) by binding to their 3 untranslated region (UTR), leading to translational repression, mRNA destabilization or target-ing them for degradation 157-158.

Whereas some miRNA are widely expressed, others have a more restrict-ed developmental stage -, tissue- or time-dependent pattern of expression. They can be coded by their own genes whose expression can be regulated by transcription factors just as regular genes, or very commonly may be derived from intronic regions of other genes and in this case its regulation is proba-bly dependent on the ‘host’ gene 159.

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miRNAs/mRNA binding show a pattern of imperfect complementary base pairing with the target mRNA allowing for several mismatches. However, the so called ‘seed region’ comprising typically a short segment of the miR-NA comprising nucleotides 2–8 from 5’ usually matches perfectly, and per-mit the interaction of a miRNA with several different sites in a gene or a number of different genes 160. However, the full process has not been eluci-dated yet and there might be further interesting features of the biology of miRNAs regulatory mechanisms.

As it is known today, the processing of a mature (active) form of miRNAs includes (figure 4): 1. miRNA encoded genes are transcribed by RNA polymerase II into a long primary miRNA transcript. 2. This primary miRNA is processed into a precursor miRNA, usually with an average 100 nt size, by the nuclear RNase III enzyme Drosha. 3. After being actively transported to the cytoplasm by exportin 5, the pre-cursor miRNA is further processed by the RNase III enzyme Dicer to an approximately 20-25 nt long sequence. 4. The miRNA-induced silencing complex (miRISC) recognizes the miRNA duplex and loads the functional strand of the duplex (mature miRNA) onto the target mRNA, typically on the 3’ UTR region (the role, if any, of the free antisense or ‘passenger’ strand is still not clear). 5. Once bound, the complex causes either inhibition of translation, with or without destabilization, or degradation of the mRNA.

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Figure 4. Biogenesis of microRNAs, details are given in the text 157.

To date, the importance of miRNAs in regulation of many immune processes has been widely shown and includes examples of regulation of granulopoie-sis, T- and B-cell development and maturation, antigen presentation, TLR signaling and cytokine production, immunoglobulin class-switch recombina-tion in B-cells, and T-cell receptor (TCR) signaling 161.

The central role of miRNAs in regulating in a ‘fine-tuning’ manner the immune response can be explained by the crucial need of control of the im-mune system to avoid uncontrolled and overreacted responses, with great potential to lead to substantial self-damage. In fact, the importance of this regulation is demonstrated by the severe impairment of the development and/or function of immune cells when miRNA expression is experimentally altered or by the fact that miRNA dysregulation can be correlated with many different immune-related diseases 156, 162-163.

An increasing number of miRNAs like miR-125b, miR-150, miR-155, miR-181a/b, miR-223, miR-101, miR-16 and miR-146a have been associat-ed with regulation of diverse immune functions including T-cell selection, B cell maturation and selection, and development of regulatory T cells (Tregs), also suggesting that the microRNA regulatory mechanism may be central in immunological tolerance (reviewed in 156, 164).

1.

2.

3.

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4.

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miR-146a The microRNA miR-146a was first described in 2006 by Taganov et al 165 in human acute monocytic leukemia cell line. The authors also described its up-regulation in response to stimulation with lipopolysaccharides and its role as a negative feedback regulator of the TLR signaling by targeting TRAF6 and IRAK-1, suggesting for the first time its important role as a negative regula-tor of inflammation.

More recently, the ablation of miR-146a in Treg cells in a knockout mouse model led to the development of autoimmune disease with severe break-down of immunological tolerance, leading to fatal IFN- -dependent immune-mediated injury in many different organs 166. Changes in miR-146a expression have been observed in a number of human disorders, such as inflammatory and autoimmune diseases, viral infections, sepsis, multiorgan failure, and cancer (reviewed in 167). In the case of AIDs, numerous studies have shown that miR-146a is upregulated in synovial tis-sue and PBMCs in RA 162, 168-169 as well as in skin lesions of psoriasis pa-tients 170. On the other hand, miR-146a expression levels were significantly decreased in PBMCs from patients with Type 2 diabetes 171. The controver-sial observations on the expression levels of miR-146a in various autoim-mune diseases may be attributed to the differences in the pathogenic path-ways important for different diseases as well as the stage at which the sam-ples have been taken during disease development.

In SLE patients, Tang et al 172 showed a 3-fold down regulation of miR-146a, which negatively correlated with disease activity and with IFN scores. The authors also reported that beside TRAF6 and IRAK1, miR-146 also regulates the expression of the signal transducer and activator of transcrip-tion 1 (STAT1), and IRF5, all key players in the type I IFN pathway. The overexpression of miR-146a in normal PBMC greatly reduced the induction of type I IFN, while on the other hand the inhibition of endogenous miR-146a increased its production in response to TLR-7 activation. Thus, de-creased expression of miR-146a in PBMC may contribute to the enhanced type I IFN production observed in SLE.

In addition, the analysis of serum miR-146a also revealed reduced amounts in SLE patients, and that the levels could be correlated with renal function, proteinuria and disease activity 173, further indicating that there might be an alteration in miR-146a regulation involved in SLE, and poten-tially other AIDs, pathogenesis.

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Aims of this study

The aim of this study was to perform genetic and functional analysis of IRF5, CD226 and miR-146a genes, as well as the effect of estrogen on IRF5 expression, and contribute to the knowledge of the pathways involved in pathogenesis of SLE.

IRF5 At the time, a study in a Swedish population highlighted the involvement of a novel 5 bp indel polymorphism in the promoter region of the IRF5 gene with SLE. This variant was described by statistical conditional analysis as being able to account for the association of the other putative functional pol-ymorphisms in the gene, and as the main functional variant responsible for IRF5 expression deviation in SLE patients.

The aim of the current study was to replicate the involvement of this pol-ymorphism in a larger cohort study including five distinct populations. We also further investigated the relative contribution of the putative functional polymorphisms on the IRF5 expression in peripheral blood mononuclear cells.

IRF5 and estrogen In humans, although controversial, the effect of estrogen has been implicated with SLE in several ways. However, the mechanism how estrogen contrib-utes to the development of SLE is still largely unknown. Evidences from animal models have recently indicated a relationship between estrogen and IRF5 where E2 treatment could enhance IRF5 expression in lupus-prone mice. This effect has to date not been investigated in humans.

To contribute to the knowledge of the mechanisms behind the strong sex-biased prevalence of SLE, we thus investigated the role of estrogen in the expression of IRF5, one of the strongest associated gene in SLE, in human immune cells.

CD226 A candidate functional variant (rs763361) in the CD226 gene had recently been genetically associated with several AIDs in a number of replication studies, with speculations about the functionality of this variant. However, there were not any functional studies demonstrating that this SNP is causa-

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tive or through which mechanism. Importantly, it was also not known if the gene was also associated with SLE.

Here, we aimed to analyze if CD226 was genetically associated with SLE in a large European cohort and investigate by statistical and functional anal-ysis if indeed this was the only variant associated, and its potential functional effect on the gene.

miR-146a GWAS data indicated an association of a variant, rs2431697 with SLE. This SNP is located approximately in the middle between two genes, PTTG1 and the microRNA miR-146a. PTTG1 codes for a protein called securin, primari-ly important in the regulation of sister chromatid separation during cell divi-sion, and that has been consistently related to several types of cancer. miR-146a is a miRNA recently described as an important player in the regulation of innate and adaptive immune system. To date, neither the functional role of the SNP, nor the gene to which this association could be related to, have been investigated.

Here we aimed to perform a meta-analysis of candidate variants in the re-gion in a large cohort of individuals from different European populations. We also aimed to access if this or other SNPs could be related to expression levels of any of the genes in PBMCs in order to get better insight of the role of these polymorphisms and these genes in SLE.

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Results and discussion

Paper I - Promoter insertion/deletion in the IRF5 gene is highly associated with susceptibility to SLE in distinct populations but exerts a modest effect on gene expression in PBMCs To first replicate the association of the recently characterized 5 bp indel in the promoter region of IRF5 9, we performed genetic association analysis using a large cohort of individuals from five distinct populations from south (Italy and Spain) and north Europe (Germany) and Latin America (Mexico and Argentina). This analysis included also 2 known putative functional variants in the gene (rs2004640 and rs10954213) and a tag SNP of the risk haplotype in this gene (rs2070197) to test the independency of this variant by conditional statistical analysis.

The genetic analysis replicated the association for the insertion allele of the 5 bp indel with susceptibility to SLE in all populations tested, with very strong association signal in the meta-analysis (OR = 1.60, P = 1.58 x 10-19). However, an even stronger association was observed for rs2070197 (OR = 2.11, P = 2.24x10-23) (table 2).

The LD between those variants varied across populations but in the meta-analysis rs2004640 and rs10954213 showed some linkage with the promoter indel (r2 = 47 and 31, respectively), whereas rs2070197 was rather independ-ent (r2 = 9). To confirm the independent contribution of these variants to the genetic association, we performed conditioning analysis on the promoter indel and rs2070197. As shown, the promoter indel and rs2070197 had inde-pendent genetic association with SLE, which seems to account for the asso-ciation of rs2004640 and rs10954213. Interestingly, the effect of those SNPs were independent of the promoter indel in all populations except Argentina and Spain, which could be explained by the higher LD between these two SNPs and the indel compared with the other populations analyzed. The hap-lotype formed by the risk alleles of the four polymorphisms (iTCA) dis-played a very high association with SLE [OR = 2.72, P = 3.17 x 10-25].

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Table 2. Genetic association and conditional analysis of IRF5 polymorphisms with systemic lupus erythematosus

Marker Risk allele frequencies1

Odds-R (95% c.i.)

Single marker P-values P-values for conditional analysis

LD (r2) with promoter

indel

cases controls promoter indel

rs2070197 promoter indel + rs2070197

Promoter indel 0.551 0.434 1.60 (1.44-1.80) 1.58 x 10-19 - 5.00 x 10-7 - -

rs2004640 0.604 0.497 1.54 (1.39-1.71) 2.38 x 10-16 5.83 x 10-5 1.46 x 10-7 0.045 47

rs2070197 0.194 0.104 2.11 (1.81-2.45) 2.24 x 10-23 3.39 x 10-15 - - 9

rs10954213 0.664 0.592 1.35 (1.21-1.51) 3.49 x 10-7 0.001 0.003 0.610 31

1Risk alleles are: insertion for promoter indel, T for rs2004640, C for 2070197, A for rs10954213.

We next sought to investigate the relative contribution of these variants on expression levels of the gene.

Among the analyzed polymorphisms, promoter indel and rs10954213 have previously been associated to IRF5 expression levels 9. We then ana-lyzed IRF5 expression in healthy individuals with different genotypes for those two variants to perform a conditional analysis also for expression anal-ysis.

Although both associated variants exerted an additive effect on gene ex-pression, indel risk allele showed a non-significant trend of upregulation when stratifying by rs10954213, while the latter altered gene expression significantly, independently of the indel (figure 5).

Neither rs2004640 nor rs2070197 had any effect on IRF5 gene expression (data not shown), in concordance with previous studies. We also tested the 30 bp exon 6 indel that gives rise to different isoforms but is not by itself associated with SLE, and it also did not have any effect on expression levels.

The lower impact of the promoter indel on gene expression here as com-pared to the previous study 9 could be explained by the considerable differ-ence in methodologies applied. A more complex architecture of the promoter in the real scenario in immune cells compared to the promoter in the context of the plasmid DNA transfected into unrelated cells (HEK293T), as it was used in the minigene approach 9 could explain this difference.

Our study replicated the association of the promoter indel with SLE in five distinct populations, and revealed that the poly(A) SNP rs10954213 is the major contributor for altered IRF5 gene expression in PBMCs.

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Figure 5. Relative contribution of polymorphisms promoter indel and rs10954213 in mRNA expression levels of IRF5 in PBMCs.

Using a different approach, a recent study analyzed expression data and gen-otypes available in different LCLs databases from Caucasian Europeans 118. Despite some differences between data sets, their statistical results strongly pointed to rs10954213, the promoter indel, and variants in high LD with those as responsible for a large fraction of variability in IRF5 expression. Only rs10954213, however, showed a significant independent contribution in the combined models analyzed.

The combined data supports the idea that those two polymorphisms play the major role in the regulation of IRF5 gene in SLE. However, none of the best models for any of the IRF5 expression data sets was strongly correlated with the defined genetic risk haplotypes for SLE 118. Thus, cis-regulation does not fully account for the genetic association of IRF5 with SLE. This could indicate that there are other variants in the gene potentially important for the disease which does not correlate with IRF5 expression.

Thus, the complex expression pattern of IRF5 with multiple splice vari-ants and several isoforms may reveal further mechanisms that plays a role in SLE pathogenesis and deserves further investigation.

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Paper II - Estrogen-dependent up-regulation of IRF5 in human immune cells Since a gender-differential expression of IRF5 was reported in mice, we first attempted to analyze if this also was true in human immune cells. We thus investigated the IRF5 mRNA levels in PBMCs from healthy donors from both females and males immediately after PBMC purification. We observed a trend of higher expression in women, although it was not statistically sig-nificant (Figure 6a). The standard variation between individuals was also greater in women than in men, which might in part be explained by different menstrual cycle, and hence the different concentrations of estrogen in the blood of the women analyzed. While, when PBMCs from both women and men were left in the stripped medium for 12 h, the expression of IRF5 be-came equal for both genders.

We then investigated if the treatment with exogenous E2 could possibly regulate IRF5 expression in PBMCs in vitro. And in fact, after 24 h, E2-stimulated PBMCs showed a more than 2-fold increase in IRF5 expression in comparison to untreated PBMCs from the same individuals (Figure 6b). This effect was seen in both genders, but was more pronounced in females (P = 0.022) than in males, which was barely significant (P = 0.057).

Figure 6. IRF5 expression analysis in PBMCs from women and men at the time of collection and after 12 h in culture (a) and upon treatment with 100 nM E2 for 24 h (b).

There have been speculations that the effect of estrogen on the development of SLE could be due to a higher number and affinity of estrogen receptor in SLE patients, but results have been rather contradictory between studies 174-

176. Here, we could see a roughly 1.4-fold higher level of ER in women PBMCs, but could not detect any statistical difference in ER or ER ex-pression between women and men or in response to exogenous E2 (Figure 7a, b).

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Figure 7. Estrogen receptors ER (a) and ER (b) expression in PBMCs at the time of collection and upon treatment with 100 nM E2 for 24 h.

Since PBMCs comprise a quite diverse group of immune cells, we attempted to further analyze IRF5 expression in sub-populations of leukocytes to better understand which cell types are predominantly affected by estrogen.

IRF5 is mainly expressed in pDCs and monocytes, and to a lesser extend in B-, T- and NK-cells 114. Since the frequency of pDCs in PBMCs is very low (around 0.4 %) and monocytes constitute one of the most representative cell type within PBMCs, we hypothesized that the latter could potentially be responsible for the increased expression seen even when diluted in PBMCs. In addition, the monocytes/macrophage system plays a key role in initiating the immune response; and higher levels of activated IRF5 specifically in monocytes have been demonstrated in SLE patients 177.

We analyzed IRF5 expression in monocytes and monocyte-derived mac-rophages treated in vitro with different concentrations of E2.

In monocytes we found a statistically relevant up-regulation of IRF5 ex-pression with 10 and 100 nM E2 (P = 0.015 and P = 0.05) (Figure 8a). Alt-hough there was a tendency of upregulation, IRF5 expression was not statisti-cally affected by E2 treatment in differentiated macrophages (Figure 8b). We could not detect any difference in E2-mediated IRF5 regulation and the gen-der of the donors. We further analyzed IRF5 expression in LCLs from healthy male and female individuals with known genotypes for IRF5 and found that in those cells IRF5 expression was not affected by E2 treatment (Figure 8c). Importantly, we could not detect any expression of ER and relatively small amounts of ER (data not shown) in LCLs. Although LCLs are derived from (EBV-transformed) B-cells, many lines of evidence indicate that those cells may have considerably different pattern of gene expression when compared to primary B-cells 178. These differences seem to be related both to the infec-tion itself and to the number of passages that the LCLs have been subjected to 179. It has in particular been shown that both ER and ER are among the nuclear receptors that are down-regulated upon B-cell EBV-transfection 180. It could also be that cells cultured in vitro conditions and in the absence of the hormone may have down regulated the expression of their estrogen receptors.

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Figure 8. IRF5 expression analysis in monocytes (a) , monocyte-derived macro-phages (b) and lymphoblastoid cell lines (LCLs) (c) from healthy individuals upon treatment with increasing concentrations of E2 for 24 h.

To investigate if the observed estrogen effect was restricted to IRF5 or was common to the IRF family of genes we analyzed the expression of a few other members that have been correlated with SLE: IRF3, IRF4, IRF7 and IRF9 in PBMCs, monocytes and macrophages in relation to E2 treatment. We could not detect any differential expression on any of those genes, re-gardless of E2 concentration or gender (data not shown). Thus it does not seem to be a shared characteristic of the group; however, we cannot discard the possibility of another of the remaining 4 members (IRF1, IRF2, IRF6 and IRF8) not analyzed here to be regulated by estrogen.

Of note, the observed IRF5 regulation by estrogen in PBMCs was not cor-related with any specific IRF5 genotypes (data not shown). As mentioned, E2 may modulate expression of target genes by direct or indi-rect way. We first investigated the genomic sequence of IRF5 gene for EREs but could not find any full sites. We then looked for sequences which could potentially enable an E2 indirect effect through an ERE half-site in combina-tion with other transcription factor binding sites known to participate in such interaction Indeed, there is a 1/2 palindromic ERE site in proximity of an SP1 site in the promoter region of IRF5: GGTCA (N)24 GGGCGG site, very similar to functional element demonstrated to be responsive to E2 in heat shock protein 27 gene: SP1 (N)23 ERE 181, thus making IRF5 a plausible target gene for E2 regulation.

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Interestingly, the SP1 binding site is the first repeat of the region where the discussed 5 bp promoter indel is located (that thus create an additional SP1 binding site). However, as mentioned, by genotyping all individuals we found that the presence of 3 or 4 repeats did not affect the E2 responsive-ness. It is possible that only one SP1 binding site would be enough in this context to enable the E2 / SP1 effect, if the site would be functional at all.

A recent study also proposes a sex-specific genetic effect in the associa-tion of IRF5 with SLE. A sex-specific case-control analysis of 18 known susceptibility loci in men and women with SLE pointed to a sex-gene inter-action of the variant tagging the risk haplotype in IRF5 (rs2070197), with men having a higher prevalence of the risk allele compared to women 182. One could thus speculate that in the case of IRF5 men require a higher ge-netic contribution to develop SLE in comparison with women, who already have estrogen potentially contributing to higher IRF5 expression. In conclusion, our results suggest that IRF5 gene expression can be up-regulated in vitro by estrogen in total PBMCs and monocytes. The effect in PBMCs was seen in both genders but it was more pronounced in women. This data could to some extent explain the sex bias in the development of SLE.

The mechanism behind this regulation and the cell-specific effect of es-trogen on IRF5 expression is still unknown and deserves further studies.

Paper III - A 3’UTR variant is associated with impaired expression of CD226 in T and NK T cells and susceptibility to systemic lupus erythematosus. The first question we wanted to answer was if CD226 was genetically asso-ciated with susceptibility to SLE. We performed a fine mapping of the gene locus (±20kb) in a European cohort (BIOLUPUS) which included 1163 SLE patients and 1481 ethnicity-matched healthy controls from 8 different coun-tries. Twelve SNPs, including the previously reported rs763361 variant, were tested for association. Association analysis revealed the strongest sig-nificant signal in exon 7 of CD226 with the correlated SNPs: rs763361 (P = 0.01, OR = 1.20), rs34794968 (P = 0.03, OR = 1.17), and rs727088 (P = 0.01, OR = 1.21). The LD structure of the gene revealed that these SNPs were part of a haplo-type block of five SNPs covering the last exon and 5 kb downstream of the gene, and which was associated with the disease (Omnibus test P = 0.02) (figure 9).

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Figure 9. Linkage disequilibrium structure of the SNPs analyzed in the CD226 gene.

These SNPs were in very tight LD, making it thus rather difficult to dissect the haplotype to find the exact susceptibility variant. Thus, sliding windows of two and three SNPs were tested, and the best model was given by the window rs763361-rs34794968-rs727088 (Omnibus test, P = 8.63 x 10-4). By testing each haplotype against all others, only one (ATC) was found reliably associated with SLE (P = 1.30 x 10-4, OR 1.24) (figure 10 and table 3).

Figure 10. Haplotype analysis by sliding windows showing the three-SNPs haplo-type in the 3’UTR of CD226 associated with SLE.

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Table 3. Association of the CD226 haplotype block rs763361-rs727088 in exon 7 with SLE.

Since it was not possible to identify the causative variant by statistical meth-ods, we moved on to functional analysis. We first investigated if this 3’UTR risk haplotype was correlated with CD226 mRNA levels in PBMCs of healthy donors. We found that individuals homozygous for the risk haplo-type ATC expressed the lowest transcript levels of all haplotypes found (P = 0.02) (figure 11a).

Figure 11. CD226 expression analysis in PBMCs of healthy donors by CD226 mRNA (a) and by FACS analysis of surface CD226 (b).

Since CD226 is a surface protein, we further explored if the low mRNA levels led to the reduction of the protein on the surface of immune cells. We measured CD226 protein expression by FACS analysis on the surface of T helper cells, cytotoxic T cells, NK cells, B cells and NKT cells from healthy individuals carrying different haplotypes. We observed that the highest ex-pression of CD226 was on NK and NKT cells, whereas only a very small fraction of B cells were CD226 positive. Importantly, we found that individ-uals with the risk haplotype had significantly lower surface expression of CD226 in T helper cells (P = 0.002), cytotoxic T cells (P = 0.03) and NKT cells (CD3+CD56+) (P = 0.03) (figure 11b).

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NKT cells represent a unique T cell lineage that has been implicated in the regulation of several different autoimmune diseases in mice and humans, including Type 1 diabetes, scleroderma and SLE. They comprise a subset of regulatory cells characterized by their ability to rapidly produce large amounts of immune-modulating cytokines. It is known that SLE patients have decreased NKT cell number and impaired function, and this is partially believed to be due to an increased sensitivity of those cells to apoptosis in patients 183.

It has been shown that CD226 is down regulated on NKT cells of patients with active SLE. Furthermore, the fact that CD226 up-regulates the expres-sion of the anti-apoptotic protein survinin led to the suggestion that the re-duced numbers of NKT cells in patients is due to increased apoptosis caused by decreased expression of CD226 (and thus of survinin) in those cells 144. This reduced numbers of NKT cells could consequently lead to sustained activation of T cells and promote autoimmunity. Here we found that the expression of CD226 on those cells is also modulated by genetic polymor-phisms. In order to identify the real functional variant responsible for the downregu-lation of CD226 expression, we prepared several reporter constructs with firefly luciferase, each containing different deletions of the CD226 3’UTR sequence. The analysis of these reporter constructs transfected in HEK293 cells indicated that rs727088 was still significantly associated with differen-tial expression of the luciferase reporter after removal of all other variations (P = 0.01). In contrast, removal of rs763361, previously suggested to have a role in transcription regulation, had no impact on luciferase activity. Alt-hough we cannot exclude the possibility that rs763361 may have an effect on protein function, our results suggest that rs727088 is the main causative var-iant responsible for the altered CD226 gene expression (figure 12).

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Figure 12. Reporter constructs expression analysis of the 3’UTR region of CD226.Deletion ( ) constructs of the variants are shown with the relative positions for rs763361 (A/G), rs34794968 (T/G), and rs727088 (C/T), along with the inser-tion/deletion (ins/del). SNP rs727088 is located 245 bp upstream of the polyadenylation signal and thus may influence the efficiency of transcription termination. We tested the hypothesis that it could affect the stability of mRNA by measuring the deg-radation rate of the different transcripts but no such effect could be detected. Thus, the molecular mechanism whereby this SNP affects the transcription of CD226 is an interesting question that still needs to be elucidated.

Our study provides strong evidence in support of the association of CD226 with SLE. Moreover, we demonstrated that the risk allele of rs727088 correlates with a reduced gene expression in T and NK T cells.

Paper IV - Genetic association of miRNA146a with Systemic Lupus Erythematosus in Europeans through decreased expression of the gene Following an association signal detected in GWAS for SLE and located in an intergenic region between PTTG1 and miR146a, we first investigated the genetic association of three candidate SNPs in the region in a case-control study in a European multicenter collection. The SNPs rs2910164, rs2277920 and rs2431697 were tested in 1 324 SLE patients and 1 453 ethnicity-matched healthy controls from 7 European countries.

The SNP rs2910164 is an interesting functional variant located in the pas-senger strand of the mature miR-146a itself and has been shown to affect the efficiency of the pre-miR-146a processing leading to differential expression levels of the mature miRNA184. In the last years several studies have de-scribed discordant association of this SNP with different types of cancer, including a recent large meta-analysis of 19 case-control studies with cancer

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showing that this variant is only associated with cancer in Asians and not in Caucasians 185.

During the course of this work a lack of association of this variant with SLE and RA was reported in Han-Chinese 186-187. Its genetic association with SLE or any other autoimmune diseases in Europeans had not been investi-gated yet.

In our cohort we could not detect any correlation of rs2910164 with SLE susceptibility (table 4), indicating that although the SNP has a functional consequence it is not genetically correlated with SLE in any of these popula-tions.

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Table 4. Genetic association of the variants in PTTG1-miRNA-146a region

Allelic association

Risk allele counts and frequencies

Case Control Ca-Freq Co-Freq P-value OR (95% CI) rs2431697 1370 1586 0.620 0.569 0.00028 1.23 (1.10-1.38) rs2277920 66 72 0.029 0.026 0.360 1.17 (0.84-1.64) rs2910164 550 687 0.248 0.240 0.541 1.04 (0.92-1.18)

Genotypic association

rs2431697 P = 0.0006

C/C 157 240 0.142 0.172 0.040 1 C/T 524 718 0.475 0.516 0.041 1.12 (0.89-1.41) T/T 423 434 0.383 0.312 0.00019 1.49 (1.17-1.90)

rs2277920 P = 0.166

A/A 1044 1338 0.942 0.949 0.461 1 G/A 62 72 0.056 0.051 0.587 1.10 (0.78-1.57) G/G 2 0 0.002 0 0.111 8.2x109 a

rs2910164 P = 0.553

G/G 623 819 0.562 0.574 0.553 1 C/G 422 531 0.381 0.372 0.655 1.05 (0.89-1.23) C/C 64 78 0.058 0.055 0.737 1.08 (0.76-1.53)

Risk alleles: rs2431697-T, rs2277920-G, rs2910164-C

a Since the frequency of this genotype is very rare and present only in the case group the OR for this genotype was disproportionally high.

Also, during the course of this work another interesting variant, rs57095329, in the promoter region of miR-146a, was found to be associated with SLE in Asians 76. The risk allele leads to a weaker binding affinity of the transcrip-tion factor Ets-1, leading to reduced expression of the microRNA.

The variant rs2277920 we analyzed here is located 257 bp downstream of exon 1 donor splice site of the miR-146a primary transcript, and could pos-sibly affect splicing of the full-length transcript. It is also in complete LD and can be considered as a proxy of rs57095329. However, in our study rs2277920 was not associated with SLE, and in fact the frequency of the risk allele of this SNP in Europeans is very low (about 10 times less frequent) when compared with Asians (table 4). Our results highlight the ethnical difference in the genetic background of SLE susceptibility and the im-portance of genetic studies in distinct populations. The polymorphism rs2431697 on the other hand, has been identified as a risk variant for SLE in a European GWAS. It has also been associated with pso-riasis in a Chinese GWAS188 but failed to show any association with RA in a European case-control study 189. Considering the opposite expression pattern

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shift of miR-146a found in SLE and RA patients 168, it is not surprising that this SNP may not be genetically associated with RA.

In our meta-analysis we replicated a robust association of rs2431697 with SLE in Europeans (P = 0.00028, OR = 1.23) (table 4).

This variant was also found associated with SLE in the same Asian study that described rs57095329, suggesting that this polymorphism is a common risk factor among Asians and Europeans.

The LD between those three variants is extremely low, and thus these var-iants can be considered as completely independent markers in this region (figure 13). Also, considering the whole PTTG1-miR146a region in Hap-Map, rs2431697 was found to be rather independent of any major LD block in this region.

Figure 13. Schematic representation of the PTTG1-miR146a genomic region and the position of the SNPs analyzed.

We thus attempted to explore the functional role of rs2431697. We started by examining if it could be correlated with PTTG1 expression levels in PBMC from healthy individuals. PTTG1 expression proved to be very low in these cells; however no difference was seen between the genotypes.

We next analyzed if the SNP could be associated with the expression of the mature form of miR-146a, and we found a decrease of about 1.6-fold in the individuals homozygotes for the risk allele (P = 0.008) (figure 14a).

To further clarify at which stage this effect occurred, we analyzed the ex-pression of the full-length primary miR-146a transcript consisting of two exons. We found a correlation of the risk allele with a down regulation of about 2-fold (P = 0.009) in the primary miR-146a (figure 14b), indicating that the SNP altered its expression at a transcription level and not in the pos-terior stages of microRNA processing. We could also detect this effect when analyzing LCLs obtained from healthy individuals (figure 14d).

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Figure 14. miR-146a gene expression analysis in PBMCs (a and b) and LCLs (c and d) from healthy donors, stratified by genotypes

The molecular mechanism of how the T allele of rs2431697 leads to the downregulation of miR-146a is not easy to predict and thus not clear at the moment. The SNP is located in a region with high regulatory potential – it overlaps with the DNase I hypersensitivity cluster and H3K4me3 trimethyla-tion marks as well as ChIP-seq signals for Pol II and transcription factors NF-kB and PAX5. So even rather far upstream from the gene, it might thus have a regulatory role on miR-146a expression.

However, we cannot exclude the possibility that this association is due to a further unknown functional variant to which it is in LD with. Future fine mapping and targeted re-sequencing of the region in European SLE patients could potentially help to elucidate this question. Interestingly, the previous Asian study did not find a correlation of rs2431697 with gene expression levels as seen here, only with rs57095329. However, given the extremely low frequency of the risk allele of rs57095329 in Europeans and lack of LD between rs2431697 and rs57095329, we find it difficult to explain the differential expression of mir-146a observed in Euro-peans by rs57095329. Because of the very low frequency, in our samples we did not have any individual homozygous for the risk allele of this SNP. However we did test if the presence of the risk allele (heterozygosis) could have an effect on gene expression, but no correlation could be detected (data not shown).

a b

c d

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In summary, we replicated the association of rs2431697 with SLE in Euro-peans and found that the T allele of this SNP correlates with lower expres-sion of the miR-146a gene.

As mentioned, miR-146a has been described as a negative regulator of key factors in the type I IFN pathway, which is normally under rigorous regulation, suggesting that miR146a might be an important element in such control. Reduced levels of this miRNA could lead to enhanced function of the target genes involved in type I IFN and thus contribute to the enhanced IFN production observed in SLE patients. It is interesting to point that the expression level of mRNA within a cell is the result from contributions of both RNA synthesis and degradation. While advances in transcriptome profiling has helped to reveal enormous transcrip-tional complexity, much remains to be learned concerning the role of mRNA degradation (or ‘degradome’) in contributing to gene expression levels, in-cluding the role of miRNAs in transcript instability. Here we could illustrate this implication by showing the genetic association of a variant in a miRNA which potentially regulates expression of important genes in SLE pathology.

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Concluding remarks and perspectives

Although the disease has been described more than 100 years ago, and many years of research has followed, we are still far from having a complete un-derstanding of the complex etiology of SLE. In this study we were able to report a novel association and the identification of putative functional vari-ants in two genes with SLE in Europeans: CD226 and miR-146a. We also further characterized the contribution of functional variants in IRF5 on its expression levels and found that expression of IRF5 might also be modulated by the sex-hormone estrogen, which may play an important role in the gen-der-bias of SLE. For IRF5 we were able to shed some light on the functional contribution of polymorphisms in the IRF5 gene, showing that among the main putative functional polymorphisms known today, the poly(A) SNP rs10954213 is the main responsible for altered gene expression. However, there is still a long way towards the full understanding of the contribution of several polymor-phisms in the function of the gene and the protein itself. Also, the down-stream consequences of alterations in this key gene in the IFN pathway and SLE are still interesting issues to be explored. It was very interesting to find that the primary female sex hormone, estro-gen, can modulate the expression of IRF5 in human immune cells. The search for explanations for the sex bias in SLE has been an issue of discus-sion for many years. And since the female bias exists for several AIDs, this issue is very important to better understand the etiology of autoimmunity in general. If estrogen plays a role on expression levels of genes/proteins that are important for SLE or autoimmunity pathogenesis, it could partially ex-plain the greater susceptibility of women to develop the disease. However, to confirm our results, further analysis of the effect of estrogen on IRF5 expression is needed. The analysis of a larger group of samples could improve the statistic power of the study. Also, the analysis of other cell pop-ulations, particularly B cells or pDCs, could shed some more light on the effect of E2 on different immune cells. Due to the high variability in re-sponse to E2 stimuli observed between individuals, it would be also im-portant to investigate the mechanism behind those differences, what makes

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immune cells from some individuals be more or less responsive to E2. And importantly, it would be interesting to investigate the effect of this hormone on the expression of other relevant genes for SLE. Here, we could also describe a novel functional variant in CD226 that leads to altered gene expression in specific subsets of immune cells. We demon-strated that beyond genetic analysis, further wet-bench experiments are cru-cial to identify causative variants and their functional consequences. As a follow up of this work, the investigation of the mechanism behind the down-regulation effect of rs727088 is warranted and deserves further effort. Since a cell-specific outcome of this variant was observed, it would be important also to further explore this effect in more detail. The analysis of more precise cell-populations, for example sub-groups of Th cells, could contribute to more defined understanding of the role of CD226 in immune responses and its relationship to SLE. microRNAs are a new and exciting field in transcription regulation that is just starting to be explored. Considering the crucial importance of having immune responses under controlled levels, the role of miRNAs is believed to be decisive in the context of autoimmunity. Here, we could describe, to our knowledge for the first time, the genetic association of a microRNA with SLE in Europeans. We found that an upstream SNP is correlated with lower expression of miRNA-146a in immune cells. Considering the relevance of this miRNA in the negative feedback control of IFN pathway and other im-mune processes, it is possible that this variant contributes to the altered im-mune responses observed in lupus patients. It would thus be of great interest to further investigate the role of this SNP, to understand the mechanism be-hind the altered expression and the consequences of this effect in the context of the disease. It is interesting to highlight that in this study the three major causative vari-ants found are located in non-coding and non-promoter regions of the genes. This illustrates the importance of searching for functional polymorphisms beyond non-synonymous variants, and to also focus on regulatory variants with the potential to affect gene function. The major challenge today is gradually switching from the question of how to generate genetic information towards the question of how to biologically interpret it. Much effort will be needed to further understand the role of ge-netic variation in disease. A knowledge that hopefully soon will be able to be put into practice and contribute to the improvement in the diagnosis, preven-tion and treatment of many diseases, including SLE.

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Acknowledgments

This work is a result of a joint effort of the Institution, supervisors, col-leagues, friends and family, all with their piece of indispensable contribution to this journey of mine and to which I am immensely grateful. I would spe-cially like to thank: My supervisors: Ulf Gyllensten, for accepting to ‘take over’ me under this unusual circum-stance and by that allowing me to finish my work here. I also thank you for the tranquilizing words and wise advices when I needed it the most.

Marta Alarcón-Riquelme first for accepting me here and giving me this great opportunity to join the ‘SLE group’ and do exciting research, for all the support during this time and finally for generously making it possibly to me to stay and finish my PhD. And I have to say that your passion and dedica-tion to the research on lupus is really admirable.

Sergey Kozyrev, for your daily and patient supervision, all the sharing of knowledge and for always being there when I needed to discuss ideas and projects. And, importantly, for all the encouragement and trust through the years as well as the understanding I sometimes really needed. All the administrative personnel at IGP – Ulla, Birgitta, Pirkko, Gunilla, Christina, Johanna, and also Britt-Marie.. for always being so very kind whenever I needed help and for making this department such a great place to work. The former ‘SLE group’; thank you for the great times both in the lab and outside, and for all the support in different circumstances.

Special thanks to: Caroline for the fun fikas and nights out, and super big thanks for so nicely reviewing this thesis even when sleepless with a bohe-mian baby at home, I just can’t thank you enough! Angelica for always standing up when I needed all sorts of statistical assistance and specially for being such a good friend and giving a decisive help in one of the most im-portant moments of my life ; ) . Casimiro, for many fun, interesting and sometimes bizarre discussions, your admirable passion for research and for demonstrating that it is definitely possible to speak English, Swedish and Spanish all at the same time. And Ammar for being the best bench-side col-

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league these years, for the good laugh to break the stress of the day - and our daily 2-3min (now) fun complaining sessions while waiting by the centrifuge.

All my PhD colleagues at IGP, for the always so friendly environment, all help in different situations through the years, pleasant corridor small talks, lunches together,.. All my friends outside Rudbeck, for all the fun times, all the music, our nice Brazilian dinners/luch/fikas and to make me forget about science and my experiments for a while. Nelson, I want to thank above all for always encouraging me to follow my dreams (at least this one it seems I may soon achieve!) and for at the same time giving me all the support possible for this. Believe me or not, I am and will always be enormously grateful for that! My loving family that has helped me in so many different ways that I don’t even know where to start, so just a huge OBRIGADA POR TUDO! Special thanks to João for drawing the beautiful picture in the cover. Although not having much of a clue on what I work on he put some thoughts and pictured the butterfly (symbol of lupus) facing ahead to represent our understanding moving forward and looking into the future. I believe it reflects well our wishes in regards to our work and SLE. To my sunshines Gabriel and Emily, for your unconditional love and support no matter how tired, stressed and inattentive mother I sometimes have been during this journey of ours. I hope you know how enormously blessed I feel to have you in my life.

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