ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2020

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1654

Lupus Nephritis – Genetic Impacton Clinical Phenotypes, DiseaseSeverity and Renal Outcome

KARIN BOLIN

ISSN 1651-6206ISBN 978-91-513-0911-8urn:nbn:se:uu:diva-401809

Dissertation presented at Uppsala University to be publicly examined in H:sonHolmdahlsalen, Akademiska sjukhuset, Entrance 100/101, Dag Hammarskjölds väg 8,Uppsala, Thursday, 14 May 2020 at 09:00 for the degree of Doctor of Philosophy (Faculty ofMedicine). The examination will be conducted in Swedish. Faculty examiner: Docent IngerGjertsson (Avd för reumatologi och inflammationsforskning vid Institutionen för medicin,Sahlgrenska akademin, Göteborg).

AbstractBolin, K. 2020. Lupus Nephritis – Genetic Impact on Clinical Phenotypes, DiseaseSeverity and Renal Outcome. Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1654. 62 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-513-0911-8.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease that can affectevery organ system. Lupus nephritis (LN) is one of the more serious SLE manifestations. Whilstthe genetic background of SLE has been thoroughly investigated, less is known about thebackground of LN. The aim of this thesis was to further elucidate the genetic background ofLN, its subtypes and outcome.

In paper I, we analysed genetic variations for association with LN, its severe formproliferative nephritis and renal outcome, in two SLE cohorts. Patients and controls weregenotyped and association analyses were performed for patients versus controls and for patientswith or without a specific clinical manifestation. In the case-control analysis of cohort I,four highly linked risk alleles in the STAT4 gene were associated with LN with genome-wide significance. In the case-only meta-analysis of the two cohorts, a STAT4 risk allelewas associated with severe renal insufficiency. We conclude that genetic variations in STAT4predispose to LN and a worse outcome with severe renal insufficiency.

In paper II, we describe a case of severe SLE on the basis of C1q deficiency. By sequencing,a mutation in the C1qC gene leading to a premature stop codon was found. The patient was alsofound to carry risk alleles in several SLE-associated variants. Interferon alpha (IFN-α) levelswere analysed over time in patient serum, and were found to correlate with disease activity.The patient’s serum had a strong interferogenic capacity when stimulating peripheral bloodmononuclear cells from healthy individuals. With this study, we further emphasise the roleof IFN-α in C1q deficiency and highlight the need to consider inherited impairments in thecomplement system in SLE with childhood onset.

In paper III, we studied the impact of sex on disease manifestations in SLE. Female SLEpatients more often presented with malar rash, photosensitivity, oral ulcers and arthritis, whilstthe frequency of serositis, renal disorder and immunologic disorder were higher among malepatients. Women were younger at LN onset, whereas men had a higher risk for progression intoend-stage renal disease.

In paper IV, we analysed genetic variations for association with LN and its subtypes in threeSLE cohorts. Patients were genotyped and association analyses were performed for patientswith versus without different phenotypes. We found genetic variations in the BANK1 gene tobe associated with LN.

In conclusion, this thesis provides further insight into the genetic background of renalmanifestations in patients with SLE.

Keywords: Systemic Lupus Erythematosus, Nephritis, Lupus Nephritis, Genetics, STAT4,BANK1, C1q Deficiency, Renal Insufficiency, End-Stage Renal Disease, Sex Differences

Karin Bolin, Department of Medical Sciences, Rheumatology, Akademiska sjukhuset, UppsalaUniversity, SE-75185 Uppsala, Sweden.

© Karin Bolin 2020

ISSN 1651-6206ISBN 978-91-513-0911-8urn:nbn:se:uu:diva-401809 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-401809)

To Kalle, Märta and Silje

List of Papers

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

I Bolin, K., Sandling, J.K., Zickert, A., Jönsen, A., Sjöwall, C.,

Svenungsson, E., Bengtsson, A.A., Eloranta, ML., Rönnblom, L., Syvänen, AC., Gunnarsson, I., Nordmark, G. (2013) Associ-ation of STAT4 polymorphism with severe renal insufficiency in lupus nephritis. PLoS One, 2013 Dec 27;8(12)1(2):3–4.

II Bolin, K., Eloranta, ML, Kozyrev, S.V., Dahlqvist, J., Nilsson, B., Knight, A., Rönnblom, L. (2019) A case of SLE with C1q deficiency, increased serum interferon-α levels and high serum interferogenic activity. Rheumatology (Oxford), 2019 May 1;58(5):918-919.

III Ramírez Sepúlveda, JI, Bolin, K., Mofors, J., Leonard, D., Svenungsson, E., Jönsen, A., Bengtsson, C., the DISSECT con-sortium, Nordmark, G., Rantapää Dahlqvist, S., Bengtsson. A.A., Rönnblom, L., Sjöwall, C., Gunnarsson, I., Wahren-Herlenius, M. (2019) Sex differences in clinical presentation of systemic lu-pus erythematosus. Biology of Sex Differences, 2019 Dec16;10(1):60.

IV Bolin, K., Leonard, D., Sandling, J.K., Imgenberg-Kreuz, J., Alexsson, A., Pucholt, P., Loberg Haarhaus, M., Nititham, J., Jönsen, A., Sjöwall, C., Bengtsson, AA., Rantapää-Dahlqvist, S., Svenungsson, E., Gunnarsson, I., Syvänen, AC., Lerang, K., Troldborg, T., Voss, A., Molberg, Ø., Jacobsen, S., Criswell, L., Rönnblom, L., Nordmark, G. (2020) Variants in BANK1 are as-sociated with lupus nephritis. Manuscript.

Reprints were made with permission from the respective publishers.

Related papers

Jönsen, A., Nilsson, S.C., Ahlqvist, E., Svenungsson, E., Gunnarsson, I., Eriksson, K.G*., Bengtsson, A., Zickert, A., Eloranta, ML., Truedsson, L., Rönnblom, L., Nordmark, G., Sturfelt, G., Blom, A.M. (2011) Mutations in genes encoding complement inhibitors CD46 and CFH affect the age at ne-phritis onset in patients with systemic lupus erythematosus. Arthritis Research and Therapy. 2011;13(6):R206. Reid, S., Alexsson, A., Frodlund, M., Morris, D., Sandling, J.K., Bolin, K., Svenungsson, E., Jönsen, A., Bengtsson, C., Gunnarsson, I., Illescas Rodri-guez, V., Bengtsson, A., Arve, S., Rantapää-Dahlqvist, S., Eloranta, ML., Syvänen, AC., Sjöwall, C., Vyse, T.J., Rönnblom, L., Leonard, D. (2019) High genetic risk score is associated with early disease onset, damage accrual and decreased survival in systemic lupus erythematosus. Annals of Rheumatic Diseases. 2020 Mar;79(3):363-369. *Maiden name

Contents

Introduction ................................................................................................... 11 Systemic Lupus Erythematosus ................................................................ 11 

Background .......................................................................................... 11 Epidemiology ....................................................................................... 11 Classification criteria, clinical phenotypes and assessment of disease activity ................................................................................................. 12 Sex differences .................................................................................... 16 Clinical course and treatment .............................................................. 16 

Lupus nephritis ......................................................................................... 17 Etiopathogenesis .................................................................................. 17 Definitions and histopathological classification .................................. 18 Assessment and staging of renal function ............................................ 22 Treatment ............................................................................................. 22 Prognosis ............................................................................................. 23 

The immune system ................................................................................. 24 The interferon system .......................................................................... 24 The complement system and complement deficiencies ....................... 26 

Genetics and epigenetics .......................................................................... 27 The human genome.............................................................................. 27 SNP genotyping ................................................................................... 28 Genetics in SLE ................................................................................... 28 Genetics in LN ..................................................................................... 30 Epigenetics ........................................................................................... 30 

Present investigations .................................................................................... 32 Aims of the thesis ..................................................................................... 32 Materials and methods ............................................................................. 33 

Patients and controls ............................................................................ 33 LN definitions ...................................................................................... 34 Genotyping and genetic association analyses ...................................... 34 Statistical analysis ................................................................................ 35 In vitro cell studies............................................................................... 36 Methylation quantitative trait loci (meQTL) ....................................... 36 

Results and discussion .............................................................................. 36 Paper I .................................................................................................. 36 Paper II ................................................................................................ 39 Paper III ............................................................................................... 41 Paper IV ............................................................................................... 42 

General discussion .................................................................................... 45 Concluding remarks ................................................................................. 46 

Sammanfattning på svenska .......................................................................... 47 

Acknowledgements ....................................................................................... 49 

References ..................................................................................................... 53 

Abbreviations

ACR American College of Rheumatology ANA Anti-nuclear autoantibodies APOL1 Apolipoprotein L1 BANK1 B cell scaffold protein with ankyrin repeats 1 BLK B lymphoid tyrosine kinase BLyS B lymphocyte stimulator C1q Complement factor 1q CI Confidence interval CKD Chronic kidney disease CYC Cyclophosphamide DNA Deoxyribonucleic acid ESRD End-stage renal disease FCGR Fc gamma receptor GFR Glomerular filtration rate HLA Human leukocyte antigen IFN-α Interferon alpha IRF5 Interferon regulatory factor 5 ISN/RPS International Society of Nephrology/ Renal Pathology Society ITGAM Integrin subunit alpha M IV Intravenous JAK Janus kinase LN Lupus nephritis MDRD Modification of diet in renal disease MMF Mycophenolate mofetil OR Odds ratio PBMC Peripheral blood mononuclear cells pDC Plasmacytoid dendritic cell PMS2 Postmeiotic segregation increased 2 pSS Primary Sjögren’s syndrome SLE Systemic lupus erythematosus SLEDAI SLE disease activity index SLICC Systemic Lupus Erythematosus International Collaborating

Clinics SNP Single nucleotide polymorphism STAT4 Signal transducer and activator of transcription 4 TLR Toll like receptor

TNIP1 TNFAIP3 interacting protein 1 WHO

World Health Organization

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Introduction

Systemic Lupus Erythematosus Background Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, char-acterized by hyperactive B cells, decreased clearance of apoptotic material and production of anti-nuclear autoantibodies (ANA) and immune complex for-mation [1].

SLE can affect all organ systems, leading to a diversity of symptoms. These include organ-specific symptoms such as arthritis, cutaneous manifestations, nephritis, cytopenia and serositis, as well as more general symptoms such as fatigue, fever and musculoskeletal symptoms. Disease severity varies from a mild disease with skin and joint involvement, to severe organ- or life-threat-ening manifestations with renal or central nervous system involvement.

The aetiology of SLE is not completely understood, but a combination of environmental and hormonal factors is believed to trigger disease in genet-ically susceptible subjects. The importance of the genetic component is em-phasised by the familial aggregation of SLE, with a concordance rate in monozygotic twins of ~25-50% and in dizygotic twins of ~2-5% [2-4]. The familial concordance is similar to other autoimmune diseases such as RA and primary Sjögrens syndrome (pSS) [2, 5, 6]. Relatives of SLE patients also have a slightly increased risk for other autoimmune diseases [7].

Epidemiology The prevalence of SLE varies across ethnic populations, ranging from approx-imately 50 cases per 100,000 persons among Northern Europeans to more than 200 per 100,000 persons among Afro-Caribbean females [8, 9]. The preva-lence of SLE in Sweden is estimated to be 65/100 000, and the annual inci-dence is approximately 2.8–5.0/100 000 [10, 11]. Disease severity and prog-nosis differ between populations, with a significantly higher risk for SLE-re-lated death in persons of ethnic minorities (Afro-American, Hispanic, and Asian) than in Caucasians [12, 13].

SLE is one of the inflammatory autoimmune diseases with the strongest female preponderance, with a female to male ratio of 9–10:1 [14, 15]. The female:male ratio is markedly higher during the childbearing ages compared

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to the pre-pubertal and post-menopausal period in which it ranges from 2 to 6:1 and 3–8:1, respectively [16, 17].

Classification criteria, clinical phenotypes and assessment of disease activity In clinical practice, Fries and Holman’s diagnostic principle is often used. It is based on the presence of ANAs on at least one occasion in combination with signs of systemic disease with involvement of at least two defined organ sys-tems (including skin, joints, kidney, serosa, blood, lungs and nervous system), in the absence of other diagnosis that can explain the patient’s symptoms [18]. Formal classification criteria often used in clinical studies include the 1982 American College of Rheumatology (ACR) classification criteria and the Sys-temic Lupus International Collaborating Clinics (SLICC) classification crite-ria from 2012 [19, 20].

For classification of SLE according to the ACR criteria, at least four criteria have to be met (Table 1). A new version of the 1982 ACR criteria was pre-sented in 1997, proposing a revision of the immunologic criterion with re-moval of the LE cell preparation and inclusion of IgG or IgM anticardiolipin antibodies or a positive test result for lupus anticoagulant [21]. There was, however, no breakthrough for these criteria because of the absence of a vali-dation.

To be classified as SLE according to the SLICC classification the patient must fulfil at least 4 criteria, including at least one clinical and one immuno-logic criterion, or the patient must have biopsy-proven lupus nephritis (LN) in the presence of ANA or anti-double-stranded DNA antibodies (Table 2).

In paper I of his thesis, the 1982 ACR criteria were used for SLE classifi-cation. In paper III, patients were diagnosed according to the 1982 ACR cri-teria or Fries’ diagnostic principle for SLE. In paper IV, the basis for inclusion was fulfilment of the 1982 ACR criteria, but patients with a biopsy-proved LN and an immunologic criterion according to the SLICC classification could also be included.

In 2019, new EULAR/ACR classification criteria were presented. Classifi-cation according to these criteria is based on a positive ANA as an entry cri-terion, followed by weighted criteria grouped in 7 clinical and 3 immunologic domains, weighted from 2 to 10. Patients accumulating ≥10 points are classi-fied as having SLE [22].

There are different indices for assessment of disease activity. The SLE dis-ease Activity Index 2000 (SLEDAI-2K) is one of the most commonly used. This index is based on 24 weighted clinical and laboratory variables, slightly modified from the original SLEDAI score to include not only new or recurrent manifestations, but also persistent active disease in the items alopecia, mucous membrane ulcers, rash, and proteinuria [23]. Other indices include the British

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Isles Lupus Assessment Group Index (BILAG) and the Systemic Lupus Ery-thematosus Activity Measure (SLAM). During the last years, several treat-ment response indices have been developed for SLE clinical trial use. These include the SLE Responder Index (SRI) and the BILAG-Based Composite Lupus Assessment (BICLA) [24, 25].

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Table 1. The 1982 revised ACR criteria for classification of SLE

1 Malar rash Fixed erythema, flat or raised, over the malar eminences, tending to spare the nasolabial folds

2 Discoid rash

Erythematous raised patches with adherent keratotic scal-ing and follicular plugging; atrophic scarring may occur in older lesions

3 Photosensitivity Skin rash as a result of unusual reaction to sunlight, by patient history or physician observation

4 Oral ulcers Oral or nasopharyngeal, usually painless, observed by a physician

5 Arthritis Non-erosive arthritis involving two or more peripheral joints

6 Serositis a. Pleurisy

b. Pericarditis

7 Renal a. Persistent proteinuria greater than 0.5 g/day or greater than 3+ if quantification not performed, or

b. Cellular casts: red cell, haemoglobin, granular, tubular, or mixed

8 Neurologic Seizures or psychosis, in the absence of other causes

9 Haematologic a. Haemolytic anaemia: with reticulocytosis, or

b. Leukopenia: <4,000 cells/mm3 on 2 or more occasions, or

c. Lymphopenia: <1,500 cells/mm3 on 2 or more occa-sions, or

d. Thrombocytopenia: <100,000 cells/mm3 in the absence of offending drugs

10 Immunologic a. Positive LE cell preparation, or

b. Anti-DNA: antibody to native DNA in abnormal titre, or

c. Anti-Sm: presence of antibody to Sm nuclear antigen, or

d. False positive serologic test for syphilis, known to be positive for at least 6 months and confirmed by Trepo-nema pallidum immobilisation or fluorescent treponemal antibody absorption

11 ANA Abnormal titre of ANA by immuno-fluorescence or an equivalent assay at any point in time and in the absence of drugs associated with drug-induced lupus syndrome

For the purpose of identifying patients in clinical studies, a person shall be said to have systemic lupus erythematosus if any 4 or more of the 11 criteria are present, serially or simultaneously, during any interval of observation [19].

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Table 2. SLICC classification system for SLE

Clinical criteria

1 Acute or subacute cutaneous lupus

2 Chronic cutaneous lupus

3 Oral or nasal ulcers, in the absence of other causes

4 Non-scarring alopecia, in the absence of other causes

5 Synovitis, involving at least 2 joints

6 Serositis (pleurisy or pericarditis), in the absence of other causes

7 Renal: Urine protein/creatinine ratio (or 24-hour urine protein) representing 500 mg of protein/24 hour, or red blood cell casts

8 Neurologic: seizures, psychosis, mononeuritis multiplex, myelitis, peripheral or cranial neuropathy, or acute confusional state (in the absence of other causes)

9 Haemolytic anaemia

10 Leucopenia (<4,000/mm3) or lymphopenia (<1,000/mm3) in the absence of other causes 11 Thrombocytopenia (<100,000/mm3) in the absence of other causes

Immunologic criteria

1 ANA

2 Anti-dsDNA

3 Anti-Sm

4 Antiphospholipid antibodies: lupus anticoagulant and/or false-positive test re-sult for rapid plasma reagin and/or medium or high anti-cardiolipin antibody level (IgA, IgG or IgM) and/or positive test result for anti-β2-glycoprotein I (IgA, IgG or IgM)

5 Low complement: low C3 and/or low C4 and/or low CH50

6 Direct Coombs’ test, in the absence of haemolytic anaemia

The patient is classified as having SLE if the patient satisfies four of the criteria, including at least one clinical criterion and one immunologic criterion, OR the patient has biopsy-proven nephritis compatible with SLE and with ANA or anti-dsDNA antibodies. Criteria are cumula-tive and need not be present concurrently [20].

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Sex differences The exact mechanisms behind the sex differences observed in SLE are un-clear, however many underlying factors have been proposed. These include sex hormones [26], sex chromosomes [27], sex differences of the immune sys-tem [28], the gut microbiome [29], sex differences in gene regulation [30], and gender-dependent environmental factors. Interestingly, an increased preva-lence of Klinefelter’s syndrome (47,XXY) have been observed among men with SLE and pSS than in the general male population [31, 32].

Sex differences do not only affect the risk for SLE, but also its clinical manifestations and prognosis. Male patients more often present with renal in-volvement, hypocomplementemia and anti-dsDNA autoantibodies [33]. Male sex has also been identified as a risk factor for cardiovascular complications, organ damage and premature death [34, 35]. Regarding LN, there is incon-sistency across studies as to whether male sex confers an increased risk for renal failure [36-41].

Clinical course and treatment SLE is a heterogenous disease ranging in severity from mild skin and joint symptoms to a severe life- or organ-threatening disease. Disease activity often follows one of three patterns: relapsing-remitting, chronic active, and long quiescent, the former two accompanied with an increased risk for complica-tions such as osteoporosis and cardiovascular events [42, 43]. Because of this heterogeneity, the treatment must be individualised, with the common aim to achieve and sustain remission.

The cornerstone in all SLE treatment is antimalarial agents, such as hy-droxychloroquine [44]. Antimalarials can prevent lupus flares, reduce the risk for damage accrual and increase long-term survival of patients with SLE [45, 46]. Specifically, antimalarials have been associated with a cardiovascular risk reduction in SLE [47]. Glucocorticoids are often used, but due to their long-term side effects, with increased risks for cardiovascular complications and osteoporosis, the dose should be reduced to the lowest possible. Doses of more than 7.5 mg/day have been associated with an increased risk for organ damage [46]. Other immunosuppressive drugs can help in reducing the corticosteroid dose. Methotrexate, azathioprine and mycophenolate mofetil (MMF) are widely used in patients where the disease is active despite treatment with an-timalarials and low-dose corticosteroids. Cyclophosphamide (CYC) and high-dose corticosteroids are needed in cases of severe disease manifestations. Rituximab (RTX) is sometimes used as second-line treatment, although clini-cal trials in SLE and LN have failed [48].

Much effort has been made during the last years to find new targets for SLE treatment, but many clinical trials in SLE have failed [49]. Possible reasons

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behind this failure have been proposed to be the heterogeneity of patients in-cluded in clinical trials, use of outcome measures that were not developed for clinical trials and the use of concomitant medication [50]. Despite these diffi-culties, some positive results have come out of SLE trials. In 2011, Benlysta® (belimumab) was the first biologic agent approved for treatment of SLE. Promising results have recently been reported from phase 3 trials with anifro-lumab, a monoclonal antibody binding to the type I interferon (IFN) receptor [51]. Several agents targeting B cells, cytokines or intracellular signalling pathways are currently under investigation [49].

Lupus nephritis Lupus nephritis (LN) is one of the more serious SLE manifestations. It occurs in 14-55 % of SLE patients, with the highest cumulative incidence in patients with African or Asian descent [52]. The morbidity and mortality of LN is con-siderable, with up to 10 % of the patients developing end-stage renal disease (ESRD, defined as dialysis or transplantation) after 10 years [53-56]. LN oc-curs most often within 1 year of SLE diagnosis [57]. As the early phase is often asymptomatic, careful surveillance of SLE patients with regards to oc-currence of hypertension or proteinuria/microscopic haematuria is important.

Etiopathogenesis The pathogenesis of LN and SLE in general is characterized by loss of self-tolererance to nuclear antigens, leading to auto-antibodies and immune com-plex formation [1, 58]. There are several theories on the pathophysiological mechanisms in the kidney. The classical theory is that circulating immune complexes deposit in the renal tissue and promote inflammation. Depending on the exact location of the immune complexes (mesangial, subendothelial or subepithelial), different classes of LN can develop [59, 60].

A more recent theory is that rather than the deposition of circulating im-mune complexes in renal tissue, immune complexes are produced locally in the kidneys through the binding of antibodies directly to intrarenal autoanti-gens such as nucleosomes from renal cells or neutrophil extracellular traps (NETs) [58]. Not only the immune complexes, but also their nucleic acid com-ponent, can promote inflammation through activation of Toll-like receptors (TLRs) in macrophages and dendritic cells, and through stimulation of pro-duction of proinflammatory cytokines such as interferon alpha (IFN-α) and interferon beta (IFN-β) [58].

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Definitions and histopathological classification According to the 1982 revised ACR criteria for classification of SLE (Table 1), renal disorder is defined as

a. persistent proteinuria greater than 0.5 g/day or greater than 3+ accord-ing to dipstick measurement if quantification has not been performed, and/or

b. cellular casts: red cell, haemoglobin, granular, tubular, or mixed [19]. According to the more recent SLICC classification criteria (Table 2), renal disorder is defined as a) a urinary protein to creatinine ratio or a 24-hour urine protein excretion representing 500 mg of urinary protein excretion per day or more, and/or b) red blood cell casts [20].

The cornerstone in the investigation is a renal biopsy with histopathological classification according to the World Health Organization (WHO) or Interna-tional Society of Nephrology/ Renal Pathology Society (ISN/RPS) classifica-tion systems (Table 3, Table 4, Figure 1) [59, 60]. Scoring of activity and chronicity indices has been shown to provide important prognostic infor-mation [36].

Proliferative nephritis (WHO or ISN/RPS class III-IV) is the most severe form of LN, characterized by focal segmental or diffuse glomerulonephritis with mesangial, endocapillary, or mesangio-capillary proliferation and/or ex-tensive subendothelial deposits [59, 60]. Subjects with proliferative nephritis are at high risk of developing ESRD, and intense immunosuppressive and anti-hypertensive treatment is needed to prevent this complication [55].

In the absence of biomarkers able to reflect findings in renal tissue, the benefit of repeated renal biopsies has been discussed [61]. A large study on repeated renal biopsies has shown a discordance between clinical and histo-pathological findings, with persistent histopathological renal activity despite apparent clinical quiescent disease in a substantial proportion of patients [62].

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Table 3. World Health Organization (WHO) morphologic classification of lupus ne-phritis (modified in 1982) [59]

Class I Normal glomeruli

a) Nil (by all techniques).

b) Normal by light microscopy, but deposits by electron or

immunofluorescence microscopy

Class II Pure mesangial alterations (mesangiopathy)

a) Mesangial widening and/or mild hypercellularity (+)

b) Moderate hypercellularity (++)

Class III Focal segmental glomerulonephritis (associated with mild or

moderate mesangial alterations)

a) With “active” necrotizing lesions

b) With “active” and sclerosing lesions

c) With sclerosing lesions

Class IV Diffuse glomerulonephritis (severe mesangial, endocapillary or

mesangio-capillary proliferation and/or extensive subendothelial

deposits)

a) Without segmental lesions

b) With “active” necrotizing lesions

c) With “active” and sclerosing lesions

d) With sclerosing lesions

Class V Diffuse membranous glomerulonephritis

a) Pure membranous glomerulonephritis

b) Associated with lesions of class II

c) Associated with lesions of class III

d) Associated with lesions of class IV

Class VI Advanced sclerosing glomerulonephritis

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Table 4. The 2003 ISN/RPS classification of LN (abbreviated)

Class I Minimal mesangial lupus nephritis Class II Mesangial proliferative lupus nephritis Class III Focal lupus nephritisa Class IV Diffuse segmental (IV-S) or global (IV-G) lupus nephritisb Class V Membranous lupus nephritisc Class VI Advanced sclerosing lupus nephritis

a Indicate the proportion of glomeruli with active and with sclerotic lesions. b Indicate the proportion of glomeruli with fibrinoid necrosis and cellular crescents. c Class V may occur in combination with class III or IV in which case both will be diagnosed [60].

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Figure 1. (1) Lupus nephritis class II. Light micrograph of a glomerulus with mild mesangial hypercellularity [periodic acid-Schiff (PAS)]. (2) Lupus nephritis class III (A). Light micrograph showing a glomerulus with segmental endocapillary hyper-cellularity, mesangial hypercellularity, capillary wall thickening, and early segmen-tal capillary necrosis (methenamine silver). (3) Lupus nephritis class III (A). Light micrograph showing a glomerulus with segmental capillary necrosis with sparing of the remainder of the capillary tuft—a vasculitis-like lesion (methenamine silver). (4) Lupus nephritis class IV-G (A). Light micrograph showing a glomerulus with global involvement of endocapillary and mesangial hypercellularity and matrix expansion, influx of leukocytes, and occasional double contours (methenamine silver). (5) Lu-pus nephritis class IV-S (A). Segment of a glomerulus showing endocapillary hyper-cellularity, capillary wall double contours, wireloop lesions, and hyaline thrombi (PAS). (6) Lupus nephritis class IV-G (A/C). Light micrograph of a glomerulus showing global severe endo- and extracapillary proliferation, wireloop lesions, leu-kocyte influx, apoptotic bodies, capillary necrosis, and mesangial expansion with hypercellularity and matrix expansion; marked interstitial inflammatory infiltration (PAS). Reproduced with permission from Weening et al. Journal of the American Society of Nephrology, Copyright American Society of Nephrology 2004 [60].

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Assessment and staging of renal function Renal function is assessed by measuring or estimating the glomerular filtration rate (GFR). There are several methodologies of measuring the GFR through recording clearance of exogenous substances, for example inulin clearance, clearance of chromium 51−labeled ethylenediaminetetraacetic acid (51Cr-EDTA) and iohexol [63]. These methods are, however, complicated and ex-pensive which limits their use in clinical practise. The most commonly used method to estimate the GFR is measurement of serum creatinine. As serum creatinine levels are affected by several factors other than the GFR, including sex and body composition, its precision as a marker for renal function is lim-ited. For that reason, several formulas for calculating renal function using se-rum creatinine have been developed. The Cockcroft-Gault formula is based on serum creatinine, age, sex and weight, but is known to overestimate the GFR due to the tubular secretion of creatinine [64]. The Modification of Diet in Renal Disease (MDRD) Study Group formula estimates body-surface ad-justed GFR using serum creatinine, age, sex and race [65]. The CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) formula uses the same parameters and has been shown to perform better especially in patients with an estimated GFR >60 mL/min/1.73 m² [66].

The Kidney Disease Outcomes Quality Initiative (K/DOQI) classifica-tion system is used to score the severity of chronic kidney disease (CKD) [67, 68]. It contains 5 stages, ranging from CKD stage 1 with an estimated GFR of >90 ml/min/1.73m² to CKD stage 5 with an estimated GFR of <15 ml/min/1.73m² (Table 5).

Table 5. Classification of chronic kidney disease (CKD)

CKD stage Description eGFR (ml/min/1.73m²)

1 Kidney damage with normal or ↑ GFR ≥90 2 Kidney damage with mild ↓ GFR 60-89 3 Moderate ↓ GFR 30-59 4 Severe ↓ GFR 15-29 5 Kidney failure <15

eGFR: estimated glomerular filtration rate [68]

Treatment The basis for treatment of all SLE is antimalarials. In addition, high dose cor-ticosteroids and immunosuppressive agents are commonly used in LN. Severe forms have traditionally been treated with high doses of cyclophosphamide (CYC), a treatment associated with an increased risk for infections and ovar-ian failure.

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The Euro-Lupus trial in 2002 led to a change in treatment recommendations in LN [69]. In this trial, a low-dose intravenous (IV) CYC regimen (6 fort-nightly pulses at a fixed dose of 500 mg) was shown to be as effective as high-dose IV CYC. Following the Aspreva Lupus Management Study in 2005, in which mycophenolate mofetil (MMF) was shown to be equally effective as IV CYC, MMF or low-dose IV CYC have become first-line treatment in in-duction of remission in LN [70]. MMF may be preferred in Hispanic and Afro-American patients which have shown lower response rates to IV CYC [71].

Rituximab (RTX) is sometimes used as second-line treatment, although no additional benefit of adding rituximab to MMF has been shown in clinical trials [72]. The clinical experience of RTX is rather large, and there is data on more than 400 patients where 67-77% achieved complete or partial renal re-sponse after 6-12 months [73].

Calcineurin inhibitors (cyclosporine and tacrolimus) may also be used as second-line agents for induction or maintenance therapy mainly in membra-nous LN [44, 74]. Following induction of remission, MMF or azathioprine is recommended for maintenance therapy [44, 75, 76].

In refractory LN, there is some evidence supporting extracorporeal treat-ment (plasma exchange or immunoadsorption), calcineurin inhibitors, borte-zomib, intravenous immunoglobulin and stem cell transplantation [77, 78]. Clinical trials of belimumab and anifrolumab in LN are currently ongoing [49, 79, 80]. Inhibition of Janus kinase (JAK) and interleukin 17 (IL-17) pathways has been mentioned as potential future treatment options in LN [81, 82].

Prognosis Despite improved treatment regiments, SLE and LN still confer an increased mortality and risk for ESRD. The 10-year survival is lower in LN patients (88%) than in SLE patients without LN (94%) [83]. The prognosis varies across populations with a more favourable outcome in Caucasian populations than in African, Asian and Hispanic populations [84, 85].

Besides ethnicity, a number of demographic, genetic, clinical and histolog-ical factors have been found to influence prognosis in LN. These include:

Age and sex genetic and immunological factors (genetic polymorphisms, anti-

dsDNA, anti-C1q, anti-phospholipid antibodies) histopathological findings (WHO classes, activity and chronicity

indices, cellular crescents and fibrinoid necrosis, tubular atrophy and interstitial fibrosis)

clinical and laboratory findings (elevated serum creatinine, ne-phrotic syndrome, failure to achieve early remission, hypertension, hypocomplementemia), and

treatment regimens, including compliance to treatment [86].

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The immune system The interferon system Interferons (IFNs) are important in the defence against viral infections [87]. There are three different types of IFNs (I–III). Type I IFNs is the largest fam-ily, consisting of five classes (IFN-α, β, ε, κ and ω) [88, 89]. The type II IFN consists of the sole member IFN-γ, and type III IFNs includes four lambda IFNs: IFNλ1/IL29, IFNλ2/IL28A, IFNλ3/IL28B and IFNλ4 (IFNL4) [88].

The main type I IFN-producing cell is the plasmacytoid dendritic cell (pDC) [90, 91]. SLE patients have a reduced number of pDCs in the circula-tion, but activated pDCs can be found in affected tissues [92, 93]. For exam-ple, increased levels of pDCs have been found in kidneys of patients with LN [93]. These pDCs are thought to be activated by immune complexes formed by autoantibodies and deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)-containing autoantigens [94]. Interferogenic immune complexes can then be internalised via the FC Gamma Receptor (FcγR) IIa expressed on pDCs, and through stimulation of TLR7 or -9 this can lead to gene transcrip-tion and an increased IFN-α synthesis [94] (Figure 2).

All type I IFNs bind to the type I IFN receptor (IFNAR), while type II and type III IFNs have their own receptors [88]. The IFNs act via several signal transduction pathways of which the JAK/signal transducer and activator of transcription (STAT) pathway are the best characterized, leading to an in-creased transcription of IFN-stimulated genes [95].

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Figure 2. Inducers and regulators of IFN-α production by plasmacytoid dendritic cells. APC, antigen-presenting cell; GM-CSF, granulocyte-macrophage colony-stim-ulating factor; IC, immune complex; IFN, interferon; IL-3, interleukin 3; LFA1, lymphocyte function–associated antigen 1; MIP-1β, macrophage inflammatory pro-tein-1β; NET, neutrophil extracellular traps; PECAM-1, platelet and endothelial cell adhesion molecule 1; ROS, reactive oxygen species. Reproduced with permission from Rönnblom and Leonard, Lupus Sci Med [88]. Copyright Authors 2019.

Increased IFN levels in serum of SLE patients have been found [88, 96]. In addition, an increased expression of IFN-regulated genes, a so called IFN sig-nature, has been reported in more than 50% of SLE patients [97]. The IFN levels have been found to correlate with disease activity and severity, and an association between high serum IFN-α levels and specific symptoms such as fever and skin rashes has been shown [98].

The pivotal role of IFN-α in SLE pathogenesis has led to clinical trials us-ing IFN-α as a target. The first positive results from phase 3 trials with anifro-lumab, a monoclonal antibody binding to the type I IFN receptor, were re-cently published [51].

Many of the known SLE susceptibility genes are encoding proteins linked to the type I IFN system, emphasizing the importance of the IFN system in SLE [88, 99].

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The complement system and complement deficiencies The complement system is an important part of the innate immune system, functioning as an immune surveillance system to distinguish between healthy tissue, cellular debris, apoptotic cells, and foreign intruders [100]. When com-plement-mediated clearance mechanisms are not available, production of im-mune complexes composed of nucleic acids/nuclear material and autoantibod-ies can lead to auto-antigen presentation, loss of tolerance and production of both type I and type II IFN [101-103].

Complement factor 1q (C1q) is part of the C1 complex which is the first protein in the classical pathway of the complement system [104] (Figure 3). C1q deficiency is a strong risk factor for SLE, with individuals carrying homozygous mutations in the C1q genes being at a high risk (~90%) of developing the disease [105-107]. SLE in C1q deficient subjects is charac-terized by an early disease onset and a severe disease course [107, 108]. C1q deficiency is rare, and less than 100 cases have been reported [107]. In addi-tion to C1q, mutations in genes encoding complement factors C2 and C4 and mutations in complement inhibitors, for example CD46 and complement fac-tor H, have also been associated with SLE [109].

A far more common cause of decreased C1q levels is the presence of anti-C1q antibodies, which have been shown to strongly correlate with active ne-phritis and thereby have a role as a predictive marker in LN [110, 111]. Anti-bodies against complement proteins are believed to cause activation of the complement system, and may also affect the clearance of apoptotic cells through opsonisation and phagocytosis [111].

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Figure 3. The classical pathway of complement activation is activated following binding of the recognition molecule C1q to ligands such as immune complexes. The lectin pathway is activated following binding of recognition molecules, such as man-nose-binding lectin (MBL), collectins or ficolins, to their ligands, which include car-bohydrate structures. Although the alternative pathway is initiated spontaneously, properdin (not shown) might also serve as a recognition molecule for directing acti-vation of this pathway. Following activation via the initiating molecules a cascade of proteolytic activation steps leads to the formation of C3-convertases that cleave C3 into the anaphylatoxin C3a and the opsonin C3b. Next, C5-convertases generate the potent pro-inflammatory anaphylatoxins C5a and C5b, the latter of which, together with C6–C9, forms the membrane attack complex (MAC). Complement inhibition strategies used in rheumatic disease include C5aR blockade in anti-neutrophil cyto-plasmic antibody (ANCA)-associated vasculitis (CCX168) and rheumatoid arthritis (PMX53); and C5 inhibition (eculizumab) in SLE. Reproduced with permission from Trouw et al. Nature Reviews Rheumatology, Copyright 2017, Springer Nature [112].

Genetics and epigenetics The human genome The human genome consists of 22 pairs of chromosomes and two sex chro-mosomes. The Human Genome Project which started in the 1980s, aimed to determine the nucleotide sequence of the entire human nuclear genome [113, 114]. As a result of this and other projects, the nucleotide sequence of a total of 3 billion base pairs included in the human genome have been identified

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[115]. Each base pair consists of two nucleotides, either A-T (adenine - thy-mine) or C-G (cytosine - guanine). When the genome of two humans are com-pared, they are 99.9% identical [116]. In about 1/1,000 nucleotides, there is an individual variation that can account for personal attributes and disease susceptibility. An allele is one of two or more possible nucleotides at the same location in the DNA sequence. The most common form of such genetic variant is a single nucleotide polymorphism (SNP). A SNP is a variation at a single base of the DNA sequence, occurring with a frequency of 1% or higher in the population. Although most SNPs are located outside of protein-coding re-gions, several have been associated with an increased risk for disease [116].

SNP genotyping There are several methods for finding risk genes or variants. Candidate gene association studies and linkage studies were formerly commonly used, whereas today genome-wide association studies (GWAS) and whole-genome sequencing are dominating the field.

The principle behind association studies is to compare allele frequencies of the SNP of interest between two populations, for example patients versus con-trols (case-control study) or between patients with different disease manifes-tations (case-case study). The association study can range from a few SNPs to SNPs covering the whole genome. There are several commercial genotyping arrays available.

Genetics in SLE The familial aggregation of SLE is a support for the genetic role in SLE. The risk for monozygotic twins to SLE patients is as high as ~25-50% [2-4]. Most genetic variants confer a moderately increased risk for disease. Rare mono-genic variants, the main example being C1q deficiency, are on the other hand associated with a high risk for disease [117].

To date, more than 100 SLE risk loci have been identified, most of which are common genetic variants found through candidate gene studies or GWAS [34]. These include genes in pathways related to apoptosis and clearance of apoptotic material, TLR/type I IFN signalling, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signalling and immune cell sig-nalling [3]. The strongest association can be observed in the human leukocyte antigen (HLA) region, in particular the HLA class II alleles DR3 and DR15 [34, 118-120]. Outside the HLA region, associations have been found between SLE and SNPs in the signal transducer and activator of transcription 4 (STAT4) and interferon regulatory factor 5 (IRF5) genes [121-125] (Figure 4).

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Figure 4. Identification of Five Major Loci Associated with Systemic Lupus Erythe-matosus in a Genome-wide Association Study. Data represent 502,033 variants of single-nucleotide polymorphisms (SNPs) that were genotyped in three series of DNA samples from 1311 case subjects and 3340 control subjects. The figure shows the –log10 P values from the combined analysis, according to chromosome. Repro-duced with permission from Hom et al, N Engl J Med., Copyright Massachusetts Medical Society 2008 [126].

Genetic variants have not only been associated with SLE per se, but also with specific clinical phenotypes in SLE. For example, variants in STAT4 and in-terferon regulatory factor 8 (IRF8) genes have been associated with increased risks for ischemic cerebrovascular events and coronary heart disease, respec-tively [127, 128].

STAT4 STAT4 is a transcription factor primarily expressed in lymphoid and myeloid tissues, functioning as an important mediator of pro-inflammatory immune responses [129]. At its latent stage, it is activated by cytokine stimulation and transferred to the cell nucleus where, through DNA binding, it regulates gene transcription [129]. The main STAT4 activating cytokine is interleukin 12 (IL-12), which is expressed by macrophages and dendritic cells (DCs) [130]. IL-12 binds specifically to receptors expressed on natural killer (NK) cells and activated T- and B-cells. STAT4 can also be activated by IL-23 and IFN-α/β. STAT4 activation leads to Th1 and Th17 differentiation and production of the pro-inflammatory cytokines IFN-γ and IL-17 [129]. The exact role of STAT4 in the pathogenesis of SLE is not clear, but several findings indicate a func-tional role. For example, high baseline IL-17 levels have been associated with a less favourable histopathological response to treatment in LN and IL-17 pro-ducing cells have been detected in renal biopsies from LN patients [131, 132]. Furthermore, activated T cells from STAT4 risk allele carriers with SLE have increased levels of STAT4 protein, resulting in increased phosphorylation of

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STAT4 in response to IL-12 and IFN-α, and an enhanced IL-12-induced IFN-γ production [133].

SLE patients carrying the STAT4 risk allele rs7574865 have an increased expression of IFN-α regulated genes [134]. An increased activation of IFN stimulated genes promotes the autoimmune process by activating a number of cells in the immune system. For example, the TNFSF13B gene encodes the B cell activating factor (BAFF)/B lymphocyte stimulator (BLyS), which pro-motes B cell differentiation and autoantibody production, including antibodies against double stranded DNA (anti-dsDNA) [135]. BLyS is the target for belimumab, the first biological drug approved for treatment of SLE.

Variants in the STAT4 gene have been shown to be associated with more severe manifestations of SLE, such as nephritis and production of anti-dsDNA antibodies [122, 123, 136]. Apart from SLE, it has also been associated with other autoimmune diseases such as rheumatoid arthritis, pSS, anti-phospho-lipid syndrome, systemic sclerosis, inflammatory bowel disease and type 1 diabetes [137-139].

Genetics in LN While the genetic background to SLE has been carefully elucidated, less is known of the genetic background to LN. So far, only one GWAS has been performed in LN. This was a meta-analysis of three SLE GWAS, in which female patients with LN were compared with female SLE patients without LN [140]. Some of the general SLE susceptibility genes which function in the immune system seem to also be associated with LN. In addition, there are more renal-specific genes that predispose specifically to LN [141]. The risk genes described in association with LN are involved in several important parts of the immune system including the HLA, Fc gamma receptor (FCGR), T-cell signalling, B-cell signalling and other inflammatory pathways [142]. HLA subtype DR (HLA-DR), integrin subunit alpha M (ITGAM), FCGR, IRF5, TNFAIP3 interacting protein 1 (TNIP1), STAT4 and TNF superfamily mem-ber 4 (TNFSF4) have been associated with both SLE per se and LN, whereas apolipoprotein L1 (APOL1), platelet-derived growth factor receptor A (PDG-FRA) and hyaluronan synthase 2 (HAS2) seem to correlate with LN specifi-cally [141]. Some genes, for example STAT4 and FcγRIIIa, have been pro-posed to associate not only with SLE and LN per se, but also with more severe subtypes of LN [123, 143].

Epigenetics The role of epigenetic regulation is of emerging importance for understanding the pathogenesis of SLE. Epigenetic modifications are changes occurring on a cellular level that can affect gene expression without altering the underlying DNA sequence, for example through modifications of transcription binding

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sites [144]. Examples of epigenetic modifications are DNA methylation, chro-matin remodelling and histone modifications. The most commonly studied re-gions where epigenetic changes occur are the so called CpG sites, regions of DNA where a cytosine is followed by a guanine. There are almost 30 million CpG sites in the human genome [113].

A common method to study epigenetic variations is by commercial meth-ylation arrays, such as the Infinium HumanMethylation450 (HM450k) Bead-Chip (Illumina), which includes analysis of quantitative DNA methylation levels at ~480,000 CpG sites covering virtually all genes in the human genome including regulatory regions [145].

Changes in DNA methylation have been identified in SLE and pSS by epigenome-wide association studies in which affected cases and controls are compared [146-150]. Hypomethylation of type I IFN induced genes in whole blood, PBMCs and neutrophils of patients with SLE has been reported in sev-eral publications [146, 151-153]. A potential role for epigenetic regulation in the pathogenesis of LN has also been proposed, e.g. the type-I IFN regulator gene IRF7 has been found to be hypomethylated in CD4+ T cells in SLE pa-tients with renal involvement, compared to SLE patients without renal in-volvement [154, 155].

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Present investigations

Aims of the thesis The general aim of this thesis was to identify genetic factors with impact on disease susceptibility, clinical phenotypes, disease severity and outcome in patients with LN.

I. The aim of this study was to investigate the genetic impact on LN in Swedish patients with SLE.

II. This study aimed to characterize a rare case of C1q deficiency with

regards to clinical symptoms, molecular mechanisms, genetic back-ground and the relation between disease activity and IFN-α levels.

III. The aim of this study was to identify sex differences in disease man-

ifestations in SLE, with special focus on renal involvement. IV. The aim of this study was to identify new genetic risk variants asso-

ciated with LN and its subtypes using three large SLE cohorts.

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Materials and methods Several methods have been applied in these investigations, and they are de-scribed in detail in the manuscripts. A brief summary is provided below.

Patients and controls In paper I, 567 Swedish Caucasian patients with SLE and 512 controls were included in cohort I. The patients originated from the rheumatology clinics at the Uppsala (n = 143) and Lund (n = 155) University Hospitals and the Karolin-ska University Hospital, Stockholm (n = 269), Sweden. The controls were in-dividually matched for age, sex and area of residence and consisted of healthy blood donors from Uppsala (n = 132) and Lund (n = 91) whereas the controls from Stockholm (n = 289) were samples from the population-based Epidemi-ological Investigation of Rheumatoid Arthritis (EIRA) control cohort [156]. Cohort II consisted of 145 Swedish SLE patients of Caucasian origin from Linköping University Hospital, and 619 healthy Swedish blood donor con-trols. Of these, 552 cases and 499 controls in cohort I, as well as 144 cases and all controls in cohort II, were previously described in a study by Wang et al [157]. All patients fulfilled the 1982 ACR criteria for SLE [19]. Clinical data was extracted from the patient files, and all participants gave informed consent.

In paper II, the patient originating from the Uppsala SLE cohort was treated at the rheumatology clinic in Uppsala. Informed consent was provided.

In paper III, the study population consisted of 1226 patients (1060 women and 166 men) from the DISSECT study, diagnosed according to the 1982 ACR classification criteria or Fries’ diagnostic principle for SLE [18, 19]. DISSECT - “Dissecting disease mechanisms in three systemic inflammatory autoimmune diseases with an interferon signature” - is a multicentre consor-tium comprising the Scandinavian Sjögren’s syndrome research network, the Swedish SLE network and the Swedish Myositis network linked to the Euro-pean Myositis network [158]. Clinical data was retrieved from the patients’ medical records.

In paper IV, 1091 Swedish patients with SLE were included in a discovery cohort. All patients fulfilled either the 1982 ACR criteria [19] or the SLICC nephritis criterion [20] for SLE diagnosis. A total of 996 patients with SLE from the University of California, San Francisco (UCSF) Lupus Genetics pro-ject were included in a replication cohort [159]. The patients from UCSF com-pleted an extensive questionnaire and the SLE diagnosis was confirmed by medical record review according to the ACR criteria. Finally, 854 patients with SLE from Denmark and Norway, all fulfilling ≥4 ACR criteria for SLE, were included. In 833 of the patients, information was available on the pres-ence or absence of LN. All patients were of Caucasian ethnicity.

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Table 6. SLE patients participating in the studies

Centre Paper I Paper III Paper IV

Sweden 712 1226 1091

USA 962

Norway/Denmark 833

LN definitions In paper I and III, LN was defined according to the ACR criterion [19]. In paper IV, LN was defined as fulfilling the ACR criterion or having a biopsy-confirmed LN in the presence of SLE autoantibodies according to the SLICC classification [20]. Renal biopsies were classified according to the WHO or ISN-RPS systems [59, 60]. Proliferative nephritis was defined as class III or IV nephritis in either classification system. The GFR was calculated at follow up with the MDRD formula [160]. Renal function was classified according to the CKD system where stage 4 and 5 (GFR < 30 mL/min/1.73m²) were de-fined as severe renal insufficiency. ESRD was defined as either dialysis or transplantation in paper I and IV, and in paper III as having a GFR of less than 15 mL/min/1.73m².

Genotyping and genetic association analyses In paper I, the individuals in cohort I were genotyped on a custom designed array with 12,000 SNPs in a previous study [161]. The selected SNPs were previously associated with SLE or other autoimmune diseases. The samples in cohort II were genotyped on a custom 384plex Illumina VeraCode Golden-Gate assay (Illumina Inc, CA, USA) in a previous study [157]. Allele frequen-cies in case-control and case-only analyses were compared using Fisher's ex-act test, conditioning on matched pairs. Furthermore, association analyses were performed using logistic regression with age or disease duration and sex as covariates. Meta-analyses were performed with the Cochran-Mantel-Haenszel test.

In paper II, the three genes coding for the A, B and C chains of the C1q complement component were amplified by PCR as fragments of around 2-3 kb in length with a set of primers covering all exons and introns, as well as the sequences 2 kb upstream of the transcription start point and 1 kb downstream of the last exon. All PCR fragments were directly sequenced by Sanger se-quencing at Uppsala Genome Center. In order to assess if there were any other common susceptibility variants contributing to the disease pathogenesis be-yond the C1q deficiency, 15 loci with previous association to SLE were gen-otyped by Sanger sequencing of the PCR-amplified fragments containing a particular SNP.

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In paper IV, the participants in the discovery and replication cohort I were genotyped on the Illumina© Infinium Immunochip. The Immunochip is a cus-tom array containing 196,524 polymorphisms designed to perform deep rep-lication and fine mapping of established autoimmune and inflammatory dis-ease loci [34, 162]. Replication cohort 2 was genotyped using the iPLEX chemistry on a MassARRAY system (Agena Bioscience).

In the discovery cohort, allele frequencies were compared between SLE cases without LN, SLE cases with LN, proliferative nephritis and ESRD, re-spectively, using logistic regression with sex and disease duration as covari-ates. All SNPs with a nominal p-value of <0.001 in LN versus LN negative, proliferative nephritis versus LN negative and ESRD versus LN negative anal-yses in the discovery cohort were further analysed in replication cohort I. Lo-gistic regression was performed adjusting for sex, disease duration and the first principal component for population stratification. SNPs with association to LN in the discovery cohort (p<0.0002) were genotyped in replication cohort II, and association analysis was performed for patients with versus without LN. Sex and disease duration were used as covariates.

Furthermore, meta-analyses were performed for LN (all cohorts), prolifer-ative nephritis and ESRD (discovery + replication cohort I) versus LN nega-tive patients. In these meta-analyses, a Bonferroni corrected p-value of <1.0×10−6 adjusting for 48,000 independent SNPs on the Immunochip was considered significant.

All association analyses were performed using the PLINK software version 1.07 [163].

Statistical analysis In paper I, patient characteristics were compared between SLE patients with and without LN. Chi square tests were used to compare frequencies and Mann-Whitney U-tests and one-way ANOVA were used to compare continuous var-iables, all using Statistica® software version 10. In paper III, continuous variables were compared with Mann-Whit-ney U tests. Categorical data was analysed with the Chi-square test, and Fisher’s exact test was used when the observed frequency of any given cell was < 5 and/or the total number of analysed individuals was < 40. The data was analysed using GraphPad Prism 6. The hazard ratios (HR) for ESRD and death after nephritis diagnosis, comparing males to females, were estimated with the cox proportional hazard modelling, adjusting for age and SLE dura-tion at the time of nephritis diagnosis. Analyses were performed using STATA MP 13.0 (StataCorp LP, College Station, TX, USA). In all analyses, p values < 0.05 were considered statistically significant.

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In vitro cell studies In paper II, peripheral blood mononuclear cells (PBMCs) were isolated from two healthy blood donor buffy coats (Department of Transfusion Medicine, Uppsala University Hospital) by Ficoll-Paque (GE-Healthcare) density gradi-ent centrifugation. PBMCs and monocyte-depleted PBMCs (CD14-Mi-crobead kit, Miltenyi Biotec) were stimulated in 96-well plates with necrotic cell material [164] and patient serum (0-10%). The cells were cultured for 20 hours [165] and the IFN-α levels in supernatants were measured by an in-house dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) with a lowest detection limit 0.5 U/ml [166]. Furthermore, PBMCs from the patient and a healthy control were isolated, stimulated as described above, and IFN-α levels were measured.

Methylation quantitative trait loci (meQTL) To search for genetic variants that may regulate DNA methylation in LN, we performed a cis-meQTL analysis investigating DNA methylation levels in LN patients from the discovery cohort (n= 180), against the genotypes of SNPs with a nominal p-value <0.001 in LN versus LN negative analysis in the dis-covery cohort (n=110 SNPs) and SNPs in genes with previously demonstrated association with LN (n=592 SNPs in total) [141]. DNA methylation data was generated on the HM450k methylation array, quality controlled and normal-ized as described previously [146]. All CpG sites located within a 100kb flanking region of these SNPs were included, and methylation levels were tested in PLINK for genotype association in LN patients assuming an additive model. A Bonferroni corrected p-value of <2.12x10-6 based on 23,535 tests was considered significant.

Results and discussion Paper I Association of STAT4 polymorphism with severe renal insufficiency in lupus nephritis The aim of this study was to identifiy risk genes for LN, one of the more severe manifestations in patients with SLE. Data on 12,000 SNPs, generated in one of our previous studies on SLE, was examined. Of these, 5,676 SNPs passed quality control and remained after removal of ancestry informal markers [161]. Specifically, we wanted to study the association between susceptibility genes and LN, proliferative nephritis and progression into severe renal insuf-ficiency.

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LN patients were more often men, younger at disease onset and fulfilled a higher number of ACR criteria than SLE patients without nephritis. Male sex as a risk factor of LN and worse renal outcome has been reported in several studies [33]. The most frequent finding in renal biopsies was proliferative ne-phritis (112/178 patients, 62.9%). Severe renal insufficiency occurred in 31/235 patients (13.8%) at follow-up (median 14 years from LN onset, range 0-46). This can be compared to previous studies, where up to 10% of patients have been reported to develop ESRD after 10 years [53-55, 167]. In our study, proliferative nephritis was the most frequent cause of developing severe renal insufficiency. Proliferative nephritis is known to represent the most severe form of LN [168].

In association analysis of LN cases versus healthy controls in cohort I, the strongest signals of association were detected for four highly linked SNPs in STAT4 with p-values reaching genome wide significance (p < 5x10-8, Figure 5). Interestingly, this association was stronger than the association with HLA, which is often reported to include the strongest SLE-associated loci [126, 161]. Strong signals of association with LN were also detected for SNPs in IRF5 and HLA-DR3 (p< 1x10-4), and SNPs in the postmeiotic segregation in-creased 2 (PMS2), TNIP1, caspase recruitment domain family, member 11 (CARD11), ITGAM, BLK proto-oncogene src family tyrosine kinase (BLK) and interleukin-1 receptor-associated kinase 1 (IRAK1) genes were associated with LN with p-values < 0.001.

Figure 5. Results from the association analysis of 5676 SNPs in 195 patients with lupus nephritis and 512 healthy controls in cohort I. The negative logarithm of the p-value is plotted against the chromosomal location. The line represents associations with p<0.001 and the nine genes associated with p<0.001 are indicated. The STAT4 SNPs rs11889341, rs7574865, rs7568275 and rs7582694 have an r2=0.98 calculated from the 512 controls.

In analysis of patients with proliferative nephritis versus healthy controls, the STAT4, IRF5 and HLA-DR3 SNP proxy were all associated with proliferative nephritis. Furthermore, amongst LN patients with severe renal insufficiency

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there were signals of association with STAT4 in the case-control analysis. In the case-only analysis, there was a nominal association with LN for SNPs in the PMS2 and TNIP1 genes whereas no associations were found for prolifer-ative nephritis. There was a tendency towards association between STAT4 and development of severe renal insufficiency.

Similar analyses were performed in a replication cohort of 145 Swedish SLE patients with SLE, where the STAT4, IRF5, TNIP1 and BLK genes had been genotyped. In a case-only meta-analysis of both cohorts a significant as-sociation was found between the SNP rs7582694 in STAT4 and severe renal insufficiency (Table 7).

Table 7. Results for STAT4 SNP rs7582694 in case-only meta-analysis of a total of 712 SLE cases

Lupus nephritis n=230a

Proliferative nephritis n=112b

Severe renal insufficiency n=31c

Gene SNP P OR (95% CI) P

OR (95% CI) P

OR (95% CI)

STAT4 rs7582694 0.11 1.21 (0.96-1.54) 0.11

1.28 (0.95-1.72) 1.6x10-3

2.22 (1.34-3.70)

P: p value. OR: Odds ratio. CI: Confidence interval. aCompared against 482 SLE patients without nephritis. bCompared against 548 SLE patients without proliferative nephritis. cCompared against 676 SLE patients without severe renal insufficiency. P value significant after Bonferroni correction for 4 tested SNPs in bold italic.

STAT4 may be involved in LN pathogenesis in different ways. STAT4 is mainly activated by interleukin 12 (IL-12) and IL-23, which lead to differen-tiation of Th1 and Th17 with production of IFN-γ and IL-17. These cytokines are important in the pro-inflammatory immune response, both for initiation and progression of the inflammatory process in the kidney [129, 169]. STAT4 can also act via the type I IFN receptor. SLE patients with the STAT4 risk allele rs7574865 have an increased expression of IFN-α regulated genes and can thus be more sensitive to IFN-α signalling [170].

In conclusion, this study demonstrated an association of STAT4 with LN, as well as with severe renal insufficiency in LN patients. The exact role of this STAT4 genetic variant in LN remains to be clarified.

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Paper II A case of SLE with C1q deficiency, increased serum interferon-α levels and high serum interferogenic activity C1q deficiency is one of the strongest genetic risk factors for development of SLE, commonly associated with severe disease and childhood onset [107, 108]. When a patient treated at our clinic was diagnosed with C1q deficiency, we started to explore the molecular background of the disease, with the aim to provide further knowledge of the pathogenesis behind C1q deficient SLE.

The patient was diagnosed with SLE at 3 years of age. Clinical manifesta-tions over the years included butterfly rash, LN WHO class II and pleurisy. At the age of 24, a Salmonella abony-infection triggered a disease flare with weight loss, leukopenia and neurological symptoms. ANA was speckled with positive anti-SS-A/Ro52, anti-SS-A/Ro60, anti-SS-B, anti-RNP and anti-Sm antibodies. Anti-dsDNA antibodies were negative, which is common in C1q deficient patients [105]. The classical complement function was found to be very low (8%) with normal or close to normal levels of C3 (1.3 [reference level 0.67-1.29]) and C4 (0.28 [reference level 0.13-0.32]). After addition of exogenous C1q, classical complement function in the sample were restored. A western blot on patient serum revealed complete absence of C1q protein confirming the diagnosis of C1q deficiency.

Sequencing of the genes encoding the subunits C1qA, C1qB and C1qC of the C1q molecule identified a premature termination codon mutation rs377549148, R69X, in exon 3 of the C1qC gene (Figure 6).

Figure 6. Schematic representation of the C1q gene cluster. The exon 3 of the C1qC gene is enlarged and shows the positions of the premature termination stop codon mutation rs377549148 and the missense variant rs1033462692. Chr=chromosome. UTR=untranslated region.

The patient was homozygous for the R69X mutation leading to a non-func-tional C1qC subunit. Both parents and a healthy brother were genotyped and found to be heterozygous. The R69X mutation has previously been found in two families with similar geographic background, with a similar phenotype as our patient with malar rash, discoid rash and oral ulceration, and positivity for ANA, anti-RNP and anti-Sm [106]. This is a commonly reported phenotype in C1q deficiency [105]. Our patient was also found to carry risk alleles in 15

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SLE-associated variants in 13 genes. Amongst these, he was homozygous for the IRF5 risk gene variant known to correlate with high serum IFN-α levels [171]. When serum samples were analysed for IFN-α, high levels were de-tected and seemed to correlate with disease activity as measured by the SLEDAI score (Figure 7).

Figure 7. Correlation between IFN-α levels and disease activity. High IFN-α levels were found in patient’s serum, and correlated well with disease activity measured by SLEDAI score. *Fever, pulmonary infiltrates, rash, arthritis, mucosal ulcers. **Fever, rash, cutaneous vasculitis, arthritis, leukopenia. Arrow: Fresh frozen plasma infusion Dashed arrow: Time point of appearance of C1q antibodies (19 U/ml, reference<14). The C1q antibody titre subsequently increased to >100U/ml.

Monocyte-depleted PBMCs from healthy blood donors stimulated with the patient’s serum generated high levels of IFN-α (mean 1958 U IFN-α/ml). When comparing the IFN-α production from the patient’s own PBMCs and PBMCs from a healthy control, both stimulated with patient serum, markedly lower levels were produced from the patient’s cells. This discrepancy could have several explanations. For instance, the low frequency of pDCs among the patient’s PBMCs compared to the healthy donor’s PBMCs, reduced IFN producing capacity of patients pDCs due to treatment or concurrent infections, or a refractory state because of in vivo activation.

C1q has previously been shown to inhibit the production of IFN-α in pDCs, which may indicate a regulatory function of C1q [101, 102]. The continuously high levels of serum IFN-α and its correlation with disease activity provide further support for the role of IFN-α in the pathogenesis.

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Paper III Sex Differences in Clinical Presentation of Systemic Lupus Erythematosus SLE is one of the autoimmune rheumatic diseases with the strongest female preponderance [14]. Although women are at higher risk for developing SLE, disease in men is often characterised by a more severe phenotype and in-creased risk for organ damage and death [33, 35, 172]. With this study, we wanted to explore the correlation between sex, clinical phenotypes and long-term prognosis in patients with lupus nephritis, using a large Swedish SLE cohort.

Included in this study were 1226 SLE patients. Women were younger at diagnosis than men (mean age 36 years for women and 40 years for men, p=0.006). Male patients were significantly more often affected by serositis (both pleuritis and pericarditis). Furthermore, LN and fulfilment of immuno-logic ACR criteria was more common in men. On the other hand, female pa-tients more frequently fulfilled the malar rash, photosensitivity, oral ulcers and arthritis ACR criteria.

Clinical data for in depth analysis of renal involvement were available in 902 patients, of which a higher frequency of men (75/122, 61%) than women (247/780, 32%) presented with renal involvement (p < 0.0001). The histo-pathological examination revealed that most patients of both sexes had prolif-erative nephritis. Women were diagnosed with renal involvement at an earlier age than men.

Interestingly, we found important sex-specific differences with regard to outcome, where men were at higher relative risk for developing ESRD than women (Table 8). Furthermore, men with SLE and renal involvement dis-played a trend towards increased death rate in comparison with the female group (Table 8).

Table 8. Risk of ESRD and death in men compared with women after diagnosis of renal involvements

Event No. events Incidence rate per 1000 person-years (95% CI) Risk estimate (male sex)

Men Women Men Women Hazard ratio

95% CI

ESRD 11 15 19.3 5.7 5.1 2.1–12.5 Death 10 33 15.2 11.6 1.7 0.8–3.8 CI: Confidence interval. ESRD: end-stage renal disease. Death: All-cause death.

The less favourable prognosis in men may partly be explained by concomitant tobacco smoking, hypertension, hyperlipidaemia, or atherosclerosis, which could accelerate progression of the renal disease. Unfortunately, these data were not available for the current study.

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Our study confirms male sex as a risk factor for severe forms of SLE, with an increased risk for more severe phenotypes like serositis and LN, and a less favourable long-term outcome. These sex differences in SLE manifestations could motivate an increased surveillance in male SLE patients, especially in those with renal involvement.

The differences observed between men and women in SLE susceptibility and disease manifestations could partly be explained by underlying genetic differences, mainly outside the sex chromosome. However, several genes in-volved in the immune system are located on the X chromosome and therefore, the role of the sex chromosomes should not be neglected. Normally, one of the X chromosomes in women is suppressed through X chromosome inacti-vation. Impairments in this inactivation can lead to a double dosage of proteins and thus promote autoimmunity [173]. The importance of the X chromosome is further supported by the higher prevalence of Klinefelter´s syndrome (47, XXY) observed in SLE and pSS patients than in the healthy population [31, 32]. Other mechanisms behind the sex-observed differences may also be pos-sible. For example, there are sex-specific differences in DNA methylation that can lead to differences in gene expression between men and women [174, 175]. Differences in sex hormones is another explanation, where oestrogen can potentiate autoimmunity whilst androgens have a more protective role [176]. Finally, gender-specific differences with regard to exposure to environ-mental factors could also partly explain the differences observed between men and women.

Paper IV Variants in BANK1 are associated with lupus nephritis In this study, including almost 3000 SLE patients from three cohorts, the aim was to further explore the genetic background of LN. In all cohorts, patients with LN were more often men, younger at disease onset, had a longer disease duration and fulfilled more ACR criteria compared to SLE patients without LN.

Because of continuous evolution in genotyping arrays, the genotyping in this paper included more than ten times as many SNPs than in paper I. When allele frequencies were compared between patients with and without LN in the discovery cohort, the strongest signals of association were found for four SNPs located close to the NFKB Inhibitor Alpha (NFKBIA) gene (p=1.6x10-5, OR: 0.5, 95% CI: 0.42-0.72). Associations were also found for SNPs in calcium voltage-gated channel subunit alpha1 S (CACNA1S), integrin subunit alpha 1 (ITGA1), B cell scaffold protein with ankyrin repeats 1 (BANK1), olfactory receptor family 2 subfamily Y member 1 (OR2Y1) and alkaline ceramidase 3 (PHCA) (p<1x10-4, Figure 8).

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Figure 8. Manhattan plot displaying results from the association analysis of 112,815 SNPs in 377 patients with lupus nephritis and 714 LN-negative patients in the dis-covery cohort. The negative logarithm of the p-value is plotted against the chromo-somal location.

All SNPs with a p-value of <0.001 in the analysis of LN, proliferative nephritis and ESRD versus LN-negative patients in the discovery cohort were further analysed in replication cohort 1. In the analysis of LN versus LN-negative patients, an association was found for a SNP in BANK1 (p<1x10-3). Meta-analyses were performed for each phenotype using the results from the dis-covery and replication cohort 1. In the meta-analysis of LN, the strongest as-sociation was found for a cluster of SNPs in the BANK1 gene (p=3.3x10-7).

In addition, the minor allele frequency of BANK1 SNP rs4699259 (r2=0.98 to rs4699261) in the second replication cohort was lower in patients with LN than in patients without LN, similar to what was observed in the discovery and replication cohort 1 (Table 9). This SNP is an eQTL for BANK1 expression in multiple tissues [177-179].

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Table 9. Summary of results for BANK1 SNP rs4699259 in logistic regression of LN patients versus SLE patients without LN

MAFLN+ MAFLN- P OR (95% CI)

Discovery cohort 0.22 0.30 9.8x10-5 0.66 (0.54-0.81)

Replication cohort 1 0.24 0.32 1.2x10-3 0.65 (0.50-0.85) Replication cohort 2 0.23 0.28 0.05 0.80 (0.64-1.0)

Meta-analysis, all cohorts 1.7x10-7 MAF: Minor allele frequency. LN+: lupus nephritis. LN-: SLE without nephritis. P: P value. OR: Odds ratio. CI: Confidence interval. Number in bold italic is significant after Bonferroni correction for 48000 independent SNPs on the ImmunoChip The meQTL analysis identified meQTL effects between CpG site cg01116491 and several SNPs in BANK1 (top SNP rs6856202, p=0.0006, r2=0.52 to rs4699259). Another block of SNPs in the BANK1 locus was also associated with methylation levels (top SNP rs7683892, r2=0.57 to rs4699259). How-ever, no direct effect was observed of the top SNP rs4699259 on the level of DNA methylation at BANK1 CpG sites (p=0.57).

BANK1 is a B cell regulating protein. The association between variations in the BANK1 gene and SLE is well established, and associations with renal, immunologic and haematological manifestations have been described [136, 180, 181]. The role of BANK1 in LN has also been studied, and signals toward an association have been shown in case-case analysis, however not surpassing Bonferroni correction [140, 180]. BANK1 protein expression has been found in renal biopsies from SLE patients, indicating a possible functional role of BANK1 in LN [140]. Nonetheless, the exact role of BANK1 in the pathogen-esis of LN needs to be further elucidated.

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General discussion SLE has a multi-factorial aetiology, wherein environmental factors can trigger disease in genetically predisposed individuals.

Although much effort has been made during the last years to study the ge-netics behind SLE, there is still a gap in the knowledge on the genetics in LN and the impact of genetic risk variants on disease manifestations and outcome. The only GWAS performed to date is a meta-analysis of three SLE GWAS, including only female patients [140]. Most of the LN-associated risk variants identified are not specific for SLE or LN, but rather occur in a higher fre-quency in SLE patients than among healthy individuals on a population-level. Thus, the benefit of genotyping single SNPs in patients on an individual level in clinical practise is very limited. Rather, it could provide further understand-ing of the etiopathogenesis of the disease, which in the prolongation may fa-cilitate the identification of novel therapeutic targets.

Two of the main findings in this thesis are the association between risk alleles in the STAT4 gene and severe renal insufficiency, and the association between BANK1 variants and occurrence of LN in SLE. STAT4 functions in the JAK/STAT pathway and interestingly, JAK inhibitors have been shown to block the IL-12 response in SLE patients carrying the STAT4 risk allele [133]. Thus, JAK inhibitors may be a future target for STAT4 risk allele carriers, subjects at high risk for severe disease and worse outcome. The links between LN, STAT4 and IFN-α/IL-17 cytokines are also of great interest for future therapies, providing hope for anifrolumab and IL-17 inhibitors for the treat-ment of LN.

The association between BANK1 and LN has previously been discussed, and this thesis provides further support for the association through the results from a large case-case analysis of LN patients. Although the exact role of this finding in the pathogenesis of LN needs to be further studied, the finding is highly interesting. As BANK1 is a B cell regulating protein, it emphasises the importance of B cells in LN and brings further hope for the role of B cell depleting agents, such as rituximab or belimumab, as therapeutic targets. Alt-hough clinical trials of rituximab in LN have not yet been successful, promis-ing results have been announced from clinical trials of belimumab in LN.

There is also an emerging interest in the use of genetic risk scores in SLE patients [182, 183]. These risk scores are based on SNPs which have previ-ously been shown to associate with SLE, weighted for the strength of the as-sociation. A high genetic risk score has been shown to confer an increased risk for organ damage, renal dysfunction and mortality, and might be a future tool to predict disease outcome and provide guidance in the surveillance and treat-ment of SLE patients.

Another finding with possible clinical implication is the increasing knowledge of the sex impact on disease manifestations and outcome. It is

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well-known that there is a sex difference in SLE susceptibility and many stud-ies have pointed to the differences in clinical manifestations. With the work presented in this thesis, we further emphasise the impact of sex on clinical phenotypes and, most importantly, that male sex confers a higher risk for pro-gression to end-stage renal disease and possibly also an increased mortality. This new information stresses the importance of a careful surveillance of male SLE patients.

The reasons to identify more rare variants, for example deficiencies in the complement system, are several. First, as discussed above, identification of risk genes can increase the understanding of the pathogenesis behind the dis-ease. To identify complement deficiencies is important also on an individual level. For example, impaired production of complement factors can be treated with exogenously added plasma with complement factors [184]. Furthermore, genotyping can be helpful in the investigation of potential hematopoietic stem cell donors. Allogenic hematopoietic stem cell transplantation is a treatment aiming to cure the patient by permanently restoring the C1q levels through transfer of healthy donor mononuclear cells. Although successful case reports have been published, the risk for treatment-associated morbidity and mortality should not be neglected [185].

Future studies of genetic risk factors and their functional consequences can hopefully provide further insight into the pathogenesis of LN, the identifica-tion of high-risk subjects with need for careful surveillance and, finally, pro-vide guidance in the search for new therapeutic targets in LN.

Concluding remarks Within this work, the genetic background to LN has been further elucidated. The results give further support for the impact of genetic factors, both com-mon variants and rare inherited mutations, on disease susceptibility and dis-ease severity. We have presented evidence for the association between risk alleles in the STAT4 gene and LN with increased risk for progression into se-vere renal insufficiency for patients carrying the risk allele. In addition, we described how C1q deficiency, a rare genetic variant, associates with an in-creased interferogenic serum activity and elevated IFN-α levels. Furthermore, we have defined differences in disease manifestations in women and men with SLE. Finally, we have identified risk variants in the BANK1 gene predisposing to renal manifestations in patients with SLE.

Future studies of genetic risk factors and their functional consequences are needed to provide further insight into the pathogenesis of LN, one of the most serious manifestations in SLE.

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Sammanfattning på svenska

Systemisk Lupus Erythematosus (SLE) är en kronisk autoimmun sjukdom som framför allt drabbar kvinnor i fertil ålder. Orsaken till sjukdomen är inte helt kartlagd, men en kombination av ärftliga faktorer och omgivningsfak-torer, t.ex. solljus och östrogen, anses vara viktiga för uppkomst av sjukdo-men.

SLE är en mycket heterogen sjukdom och kan variera från milda former med hudutslag och ledvärk till allvarligare former men engagemang av inre organ såsom hjärt- och lungsäck, njurar och centrala nervsystemet. Njurenga-gemang, eller nefrit, är ett av de allvarligaste symtomen och förekommer hos ungefär en tredjedel av patienterna. Beroende på nefritens svårighetsgrad ges behandling med kortison, immunreglerande läkemedel och cellgifter, men trots det löper patienterna en betydande risk för framtida njursvikt.

Målet med den här avhandlingen var att studera sambandet mellan gene-tiska varianter och förekomst av njurmanifestationer hos patienter med SLE.

I det första arbetet genotypades 567 svenska SLE-patienter och 512 friska kontroller med avseende på ca 12 000 olika genvarianter. Vi studerade kopp-lingen mellan dessa genvarianter och förekomst av SLE, nefrit, olika svårig-hetsgrader av nefrit och njurfunktion. Vi fann ett starkt samband mellan vari-anter i genen STAT4 och nefrit, och att patienter som bar på dessa genvarianter löpte en ökad risk att utveckla svår njursvikt.

I det andra arbetet presenteras ett patientfall med svår SLE på basen av C1q-brist, en ovanlig defekt i komplementsystemet som i de flesta fall leder till svår SLE redan i barndomen. Patienten hade mycket låga nivåer av C1q-protein i serum och visade dig vara homozygot för en mutation i C1qC-genen som ledde till en defekt C1q-produktion. Vi studerade sambandet mellan sjuk-domsaktivitet och nivåer av cytokinet IFN-α, och hur patientens serum påver-kade perifera mononukleära blodcellers (PBMCs) förmåga att bilda IFN-α. Vi fann höga nivåer av IFN-α i patients serum, och ett tydligt samband mellan IFN-α-nivåer och sjukdomsaktivitet. Vidare fann vi att patients serum hade en stark förmåga att stimulera till bildning av IFN-α i PBMCs från friska försöks-personer. Vi betonar med detta arbete den viktiga kopplingen mellan IFN-α och C1q-brist, och visar på vikten av att överväga ärftliga komplementdefek-ter vid debut av SLE i ung ålder.

I det tredje arbetet studerade vi skillnader avseende sjukdomsmanifestat-ioner och prognos mellan män och kvinnor med SLE. Vi fann att sjukdomen hos kvinnor ofta yttrade sig som hudutslag, solkänslighet, munsår och artrit

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medan män oftare hade symtom i form av serosit, nefrit och immunologiska avvikelser. Bland patienter med nefrit var kvinnor yngre vid diagnosen jäm-fört med män, medan män löpte en större risk att utveckla terminal njursvikt.

I det fjärde arbetet studerade vi sambandet mellan genetiska variationer och förekomst av nefrit, olika subtyper av nefrit och prognos i tre kohorter av SLE-patienter från Sverige, USA och Danmark/Norge. Vi fann ett samband mellan varianter i BANK1-genen och förekomst av nefrit hos patienter med SLE. BANK1 kodar för ett B-cellsreglerande protein och har tidigare visats vara kopplad till SLE. Detta protein uttrycks i flera vävnader, bl.a. i njurvävnad hos patienter med SLE. Exakt vilken roll BANK1 har i patogenesen är ännu inte klarlagd.

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Acknowledgements

This thesis was carried out at the Department of Medical Sciences, Faculty of Medicine, Uppsala University. I would like to thank all of you who have sup-ported me and contributed to this thesis. In particular I would like to acknowledge: My very special gratitude to my main supervisor, Gunnel Nordmark, for making this thesis possible. I can clearly recall us working together with a young girl with severe nephritis, where your enthusiasm was the beginning of my deep fascination for this mysterious disease. Thank you for inviting me to participate in your research, for your encouragement, for sharing all your knowledge, for always being there to help and discuss, for being the most well-structured person I have ever met, for believing in me, for your patience despite several breaks in the PhD studies, and especially for always being so friendly and considerate. I sincerely hope that there will be a chance for me to work with you again. Lars Rönnblom, my co-supervisor, for allowing me to be a part of your re-search group. It has been a true honour to be a part of your brilliant team. Thank you for valuable input on all my work, for inviting me to interesting scientific meetings, for being enthusiastic and encouraging and not least for your patience during a long PhD period. Former and current friends and colleagues in the Rheumatology research group: Maija-Leena Eloranta, for always being friendly and helpful, and for all assistance with the C1q paper and the experiments. Johanna Sandling, for all your knowledge, for always having time for good advice, and especially for being a saviour when a major revision was needed just when I came home with a new-born baby. Juliana Imgenberg-Kreuz, for all support both in gen-eral and for your excellent skills in methylation. Lisbeth Fuxler, for your enormous patience when introducing me to the laboratory world (although you would have done it yourself in half the time). Sarah Reid, for excellent proof-reading. Niklas Hagberg, Olof Berggren, Christian Lundtoft and Pascal Pucholt, for always being so friendly and helpful. Cane Yaka, Sule Yavuz, Alina Johansson and Paulina Turesson for nice company. Andrei Alexsson, for your patience with my never-ending questions on the ImmunoChip.

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All colleagues in the Swedish SLE network, especially Iva Gunnarsson, Christopher Sjöwall, Andreas Jönsen, Anders Bengtsson, Solbritt Rantapää Dahlqvist, Elisabet Svenungsson and Agneta Zickert. All other co-workers and co-authors of the papers, not already mentioned: Marie Wahren-Herlenius, Sergey Kozyrev, Bo Nilsson, Ann-Christine Syvänen, Malena Loberg Haarhaus, Jorge Iván Ramírez Sepúlveda, Jo-hannes Mofors, Christine Bengtsson and Jonas Carlsson Almlöf.

Danish, Norwegian and American colleagues for kindly sharing data from their respective SLE cohorts for replication in paper IV: Anne Troldborg, Anne Voss, Søren Jacobsen, Karoline Lerang, Øyvind Molberg, Joanne Nititham, and Lindsey Criswell. Former and current friends and colleagues at the Rheumatology clinic: Ann Knight, for giving me the opportunity to join this fantastic assembly and for all collaboration with the C1q paper. Johan Back, for being a never-ending source of knowledge. Thank you for everything you have taught me - I have never doubted your advice. Stina Östman Blomberg, my clinical supervisor. Thank you for all your advice, care, and the flowers that are still so beautiful in my garden. Maria Lidén, Ann Olofsson Sahlqvist, Ylva Rydvald, Eva Baecklund, Peter Matt, Lilian Vasaitis, Erik Hellbacher, Karin Hjorton, Dag Leonard, Efthymia Sidiropoulou, Nikolas Malliotis, Ágnes Szentpé-tery, Johanna Dahlqvist, Hanna Lindberg, Cecilia Fugmann, Shamisa Adeli, Ioanna Giannakou, Iris Karkamanis, Bjarni Thorsteinsson, Mar-cus Bergström and finally Elisabeth Skoglund, with special warm thanks from all my family. All nurses at the Rheumatology clinic, especially Ann-Charlotte Hoflin for always being a ray of sunshine and bringing happiness into work, and Rezvan Kiani Dehkordi for collecting all the patient samples and always being help-ful in any matter. All of you who inspired me and introduced me to the fascinating world of rheumatology: Roger Hällgren, for you lectures at medical school that made me eager to learn more on the autoimmune diseases. Birgir Arge and col-leagues at the Rheumatology clinic in Eskilstuna which was my first meeting with rheumatology in clinical practice. Esmail Karim and colleagues at the Rheumatology clinic in Gävle, for providing further inspiration to a fantastic speciality. Without all of you, the topic of this thesis (if any) might have been something completely different. Anders Lind, who initiated the collection of the lupus nephritis cohort in Uppsala.

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All colleagues and team leaders at Läkemedelsverket, for making it possible to proceed with my PhD studies. Special thanks to Mari Thörn, Charlotte Söderberg-Nyhem, Ingrid Landberg, Helena Möllby, Annica Nordin and least but not last Anna Vikerfors, for being my scientific, regulatory and hu-manistic mentor, and in particular for being such a good running partner. You make me look forward to going to work every day. My friends during medical school, for all cups of tea making it so much more fun to study and for all ER evenings: Marie Backman, Nina Bylund and Sofia Palmér. I so much appreciate to still hang out with you, although it’s a bit more complicated being 17 of us. “Mammagruppen”- especially Elisabet Vikeved, Jessica Schubert, Shadi Amid Hägg and Sandra Bjerkesjö - for being a necessary break from work, PhD studies, wake-nights, terrible twos, or simply life´s up and downs. Eve-rything lightens after a cava at Hava. My very oldest and best friends Caroline Engberg, Frida Andersson and Karoleen Eckerbert. Although we don’t meet as often as I wish, things are always the same when we meet and I hope it always will. My very deepest thanks to my family. Mamma och Pappa, Farmor, Lina, Örjan, Ida and Vera. I would not have been me without you. Kerstin and Mia, Rolf, Ylva, Clara and Julia, for always being so encouraging. Kalle, Märta and Silje. No work can ever compete with the importance of having you around. I hope this book in your hands will mean more time to-gether from now on. Ni är bäst! All the patients who participated in this research. I wish to send my very deepest thanks and warm thoughts to the patient in paper II and his family, who despite being in middle of a difficult disease were willing to contribute to this research. Without all of you, this thesis would not exist. The studies in this thesis were supported by grants from Swedish Research Council for Medicine and Health, the Knut and Alice Wallenberg Foundation, the Swedish Rheumatism Foundation, the King Gustav V’s 80-year Founda-tion, the Agnes and Mac Rudberg Foundation, the Ragnar Söderberg Founda-tion, the Ingegerd Johansson donation, COMBINE, DISSECT, the Östergöt-land, Stockholm and Uppsala County Councils (ALF funding), Karolinska In-stitutet, Uppsala University, Swedish Society for Medical Research, the Ingrid Asp Research Foundation, the Swedish Heart-Lung foundation, the Fund for

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Renal Research, the Foundation in memory of Clas Groschinsky and the Swe-dish Society of Medicine.

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A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

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ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2020