Umeå University Medical Dissertations, New Series No 1484

Genetic studies in Rheumatoid Arthritis – familial studies and analysis of relationships to atherothrombotic comorbidity Lisbeth Ärlestig

Akademisk avhandling som med vederbörligt tillstånd av Rektor vid Umeå universitet för avläggande av medicine doktorsexamen framläggs till offentligt försvar i Sal D, 9 trappor, byggnad 1D, Norrlands Universitetssjukhus fredagen den 30 mars 2012, kl. 09:00. Avhandlingen kommer att försvaras på svenska. Fakultetsopponent: Docent Åsa Torinsson Naluai, Institution för Biomedicin, Göteborgs universitet.

Department of Public Health and Clinical Medicine, Rheumatology Umeå University Umeå 2012

Genetic studies in Rheumatoid Arthritis - familial studies and analysis of relationships to atherothrombotic comorbidity

Lisbeth Ärlestig

Department of Public Health and Clinical Medicine, Rheumatology, Umeå University Umeå 2012

Responsible publisher under swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7459-383-9 ISSN: 0346-6612 Ev. info om Omslag/sättning/omslagsbild: Elektronisk version tillgänglig på http://umu.diva-portal.org/ Tryck/Printed by: Print & Media, Umeå Universitet Umeå, Sweden, 2012

To my family

Table of Contents Table of Contents

i

Abstract

ii

Abbrevations

iiv

Gene abbrevations

vii

List of papers

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Enkel sammanfattning på svenska

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Introduktion

1

Rheumatoid arthritis

1

Aetiology

1

Diagnosis

1

Pathogenesis

2

Autoantibodies

4

Assessment of disease activity and outcome

4

Treatment

5

Cardiovascular co-morbidity

6

Genetics

8

Genetic markers

9

Genetic methods

10

Population genetics

11

Rheumatoid arthritis and genetics

11

Cardiovascular disease and genetics

14

Cardiovascular disease and genetics in rheumatoid arthritis

14

Aims

16

Study populations and methods

17

Study populations

17

Clinical examinations and surveys

19

Registration of cardiovascular events

19

Genotyping and serological analysis

20

Statistics

21

Results and discussion

23

Paper I

23

Paper II

28

Paper III

34

Paper IV

36

Conclusions

41

Future perspective

43

Acknowledgements

44

References

45

i

Abstract Background. Rheumatoid arthritis (RA) is an autoimmune disease mainly affecting the joints but has also extra articular manifestations and an increased cardiovascular (CV) co-morbidity. Rheumatoid factor (RF) and antibodies against citrullinated proteins/peptides (ACPA) are diagnostically important and are related to a more severe disease. The aetiology is unknown but RA is considered a complex disease caused by both genetic and environmental factors. The heritability is estimated to be 60% with the main contribution from the HLA region. The relative homogeneity of the population in northern Sweden due to low immigration and founder effects has shown to be suitable for genetic studies. Objectives. The aim of this thesis has been to identify genes contributing to the susceptibility of RA and the CV co-morbidity in particular. To achieve this, multi-case families from the four northern most counties of Sweden were collected for linkage studies to identify susceptibility genes. Association studies with genetic polymorphisms in genes, involved in inflammation or being of importance for atherothrombotic manifestations (ATM) in the general population, were performed in RA-patients concerning ATM e.g. myocardial infarction, angina pectoris with intervention, stroke/TIA, deep vein thrombosis/pulmonary embolism (DVT/PE) at follow-up. Methods & Results. 47 families with 134 affected and 216 unaffected relatives were included in a genome-wide linkage study (GWL) performed with microsatellite markers at an average of 10cM resolution analysed using ABI PRISM 3730 DNA sequencer and non-parametric multipoint linkage in the Merlin program. Eight linked loci were identified with HLA as the most significant and a novel region on chromosome 14. In a follow-up analysis on a custom Illumina chip, with 13 additional families, yielding a total of 198 affected and 197 unaffected relatives. The majority of the 1536 single nucleotide polymorphisms (SNPs) used in the Illumina follow-up analyses was focused on chromosome 14. Statistical analyses with linkage and transmission disequilibrium test narrowed the region to 4 cM, a region containing multiple plausible RA candidate genes (Paper I). In Paper II serum samples from 163 affected and 157 first degree relatives were analysed with EliA ACPA assay on ImmunoCAP250 for ACPA (IgA, IgG, IgM) and RF (IgA, IgM) isotypes. Both concentrations and frequencies were increased among the relatives compared with controls but lower compared with RApatients and with a different relative distribution of the isotypes. The genetic contribution to ATM was studied in Paper III and IV using selected SNPs analysed using ABI PRISM 7900HT sequence detector system. In Paper III, RA-patients (n=467) were compared with age and sex

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matched controls (n=696) with respect to SNPs in tumor necrosis factor receptor II (TNFRII) (M196R), ß-fibrinogen -455 (G-455A), plasminogen activator inhibitor type-1 (PAI-1) (4G/5G) and Factor XIIIA (Val34Leu). Hypertension was predicted by TNFRII R allele and to a higher extent in combination with the A-allele in ß-fibrinogen. The 4G allele in PAI-1 was more frequent in patients with ischemic heart disease (IHD) and the FXIIIA Leu34 variant in patients with DVT/PE. In Paper IV, the minor allele of the polymorphism in growth differentiation factor 15 (GDF15) was found to be associated with RA (n=696) per se but also to ATM, a SNP in the 9p21.3 locus was also associated with ATM. A significant association to stroke was found in female patients homozygote for the minor allele of GDF15. Stoke among male patients was significantly associated with carrying the major allele of two SNPs in the CD40 gene. DVT/PE was associated with the minor allele of GDF15. Conclusion. A novel locus on chromosome 14 of importance for RA susceptibility in northern Sweden was found. The minor allele of TNFRII separately and together with the minor allele of ß-fibrinogen -455 was associated with hypertension and the 4G allele in PAI-1 was associated with IHD and the Leu34 variant was associated with DVT/PE in RA patients. The GDF15 minor allele was associated with RA per se, ATM and DVT/PE in RA patients and a genotype in the SNP on 9p21.3 was associated with ATM. Stroke among females was associated with GDF15 and stroke among males with two SNPs in CD40.

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Abbrevations ACPAs ACR CAD CCP CHD CI cM CNV CRP CVD CVE D’ DAS DAS28 DM DMARD DNA DVT/PE ELISA eQTL ESR EULAR GWAS GWL HAQ HLA IFNJ Ig IHD IL LD Mb MHC MI mRNA NSAID OR oxLDL PAI-1 PCR r2 RA RF SD SNP SPSS TDT

Anti-citrullinated protein/peptide antibodies American College of Rheumatology Coronary artery disease Cyclic citrullinated peptide Coronary heart disease Confidence interval centimorgan Copy number variation C-reactive protein Cardiovascular disease Cardiovascular event Pairwise-disequilibrium coefficient Disease Activity Score Disease Activity Score based on 28 joint counts Diabetes mellitus Disease modifying anti-rheumatic drug Deoxyribonucleic acid Deep vein thrombosis/pulmonary embolism Enzyme-linked immunosorbent assay Expression quantitative trait locus Erythrocyte sedimentation rate European league against rheumatism Genome wide association study Genome-wide linkage Health assessment questionnaire Human leukocyte antigen Interferon gamma Immunoglobulin Ischaemic heart disease Interleukin Linkage disequilibrium Mega base Major histocompatibility complex Myocardial infarction Messenger ribonucleic acid Non steroidal anti-inflammatory drugs Odds ratio Oxidized low density lipoproteins Plasminogen activator inhibitor-1 Polymerase chain reaction Correlation coefficient Rheumatoid arthritis Rheumatoid factor Standard deviation Single nucleotide polymorphism Statistical package for the social sciences Transmission disequilibrium tests

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TGF-β Th17 TIA TNF Treg

Transforming growth factor beta Type 17 helper T-cell Transient ischaemic attack Tumour necrosis factor Regulatory T-cell

v

Gene abbreviations AFF3 ANKRD55 BLK C5orf30 CCL2/5/21 CCR6 CD2 CD244 CD28 CD40 CD58 CTLA4 CX3CL1 CXCL10 FCGR2A FCLR3 FOXN3/CHES1 FXIIIA GDF15/MIC1 HLA IGSF2 IL1/2/6/10/17/21 IL2RA/B IL6ST IRF5 KCNK13/THIK1 KIF5A PADI2/4 PAI-1 PIP4K2C PRDM1 PRKCQ PTPN22 PTPRC PXK RBPJ REL

AF4/FMR2 family, member 3 ankyrin repeat domain 55 protein Tyrosine kinase, B-lymphocyte specific Chromosome 5, open reading frame 30 Chemokine (c-c motif) ligand 2/5/21 Chemokine, CC motif, receptor 6 T-lymphocyte surface CD2 antigen CD244 antigen, Natural killer cell receptor 2B4 Antigen CD28 CD40 molecule, TNFR superfamily member 5 CD58 molecule Cytotoxic T-lymphocyte associated protein 4 Chemokine (CX3C motif) ligand 1 Chemokine (CXC motif) ligand 10 Fc fragment of IgG, low affinity IIa, receptor Fc receptor-like 3 Forkhead box N3/Checkpoint suppressor 1 Factor XIII, A1 subunit Growth/differentiation factor 15/Macrophageinhibiting cytokine 1 Human leukocyte antigen Immunoglobulin superfamily, member 2 Interleukin 1/2/6/10/17/21 Interleukin 2 receptor alpha/beta Interleukin 6 signal transducer Interferon regulatory factor 5 Potassium channel, subfamily K, member 13/Tandem pore domain halothane-inhibited potassium channel 1 Kinesin family member 5A Peptidylarginine deiminase, type II/IV Plasminogen activator inhibitor-1 Phosphatidylinositol-5-phosphate 4 kinase, type II, γ PR domain-containing protein 1 Protein kinase C, theta Protein tyrosine phosphatase, non-receptor type 22 Protein-tyrosine phosphatase, receptor-type, C PX domain containing serine/threonine kinase Recombination signal-binding protein for immuneglobulin kappa J region v-rel reticuloendotheliosis viral oncogene homolog

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SPRED2 STAT4 TAGAP TNFAIP3 TNFRII TNFRSF14 TRAF1-C5 TRAF6 TTC7B VCAM-1/CD106

Sprouty-related EVH1 domain-containing protein Signal transducer and activator of transcription 4 T-cell activation GTPase-activating protein TNF alpha interacting protein 2/3 Tumor necrosis factor receptor superfamily, member 1A TNFR superfamily, member 14 TNFR-associated factor 1 – Complement component 5 TNF receptor-associated factor 6 Tetratricopeptide repeat domain 7B Vascular cell adhesion molecule-1

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List of original papers This thesis is based on the following papers that will be referred to by their roman numerals:

I. Lisbeth Ärlestig, Elisabet Einarsdottir, Dan Holmberg and Solbritt Rantapää Dahlqvist. Novel risk locus on chromosome 14 in multi-case families with rheumatoid arthritis from northern Sweden. Manuscript in preparation II. Lisbeth Ärlestig, Mohammed Mullazehi, Heidi Kokkonen, Joacim Rocklöv, Johan Rönnelid and Solbritt Rantapää Dahlqvist. Antibodies against cyclic citrullinated peptides of IgG, IgA and IgM isotype and rheumatoid factor of IgM and IgA isotype are increased in unaffected members of multicase rheumatoid arthritis families from Northern Sweden. Ann Rheum Dis 2011; Nov 29. [Epub ahead of print]

III. L. Ärlestig, S. Wållberg Jonsson, B. Stegmayr, S. Rantapää-Dahlqvist. Polymorphism of genes related to cardiovascular disease in patients with rheumatoid arthritis. Clin Exp Rheumatol 2007;25:866-71

IV. Lisbeth Ärlestig and Solbritt Rantapää Dahlqvist. Polymorphisms of the genes encoding CD40 and Growth differentiation factor 15 and 9p21.3 region in patients with rheumatoid arthritis and cardiovascular disease. Accepted for publication by J Rheumatol 2012 Jan.

Papers II, III and IV are re-printed with the kind permission of the publishers.

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Enkel sammanfattning på svenska Reumatoid artrit/kronisk ledgångsreumatism (RA) är en autoimmun sjukdom som framför allt drabbar leder, främst i händer och fötter, symmetriskt i båda kroppshalvorna. Sjukdomen medför inflammation i lederna och obehandlad så sker nedbrytning av brosk och ben. Prevalensen för RA är 0,5-1 % och två tredjedelar av dessa är kvinnor, medianåldern för insjuknandet är 55-60 år. Sjukdomen kan yttra sig på olika sätt, mild och självläkande eller aggressiv med snabb påverkan på brosk och ben i leden. Extra-artikulära manifestationer som reumatiska knutor, noduli och vasculit kan uppkomma under sjukdomen. Auto-antikroppar som reumatoid faktor (RF), riktade mot kroppens egna IgG antikroppar, och anti-kroppar riktade mot citrullinerade proteiner/peptider (ACPA) finns hos 70-80 % av patienter med RA. Orsaken till RA är i det närmaste okänd men sjukdomen anses som en komplex sjukdom dvs. med många faktorer som i olika utsträckning bidrar till insjuknandet, framför allt genetiska, hormonella och miljö faktorer tros bidra till utveckling av RA. Ärftligheten för RA har uppskattats till 60 % i tvillingstudier men då varje ärftlig variation ger en liten ökning av risken för RA och många faktorer måste samverka är de enskilda faktorerna svåra att identifiera. I befolkningar som har varit isolerade, geografiskt eller socialt, kan den genetiska variationen vara homogenare. Befolkningen i Norr- och Västerbotten har i genetiska studier visats vara relativt genetiskt homogen population och skiljer sig från övriga Sverige. Denna homogenitet kan underlätta identifieringen av regioner/gener som medför ökad risk för sjukdomar med ärftligt inslag. I de fyra nordligaste länen har jag identifierat familjer med flera familjemedlemmar som har RA och insamlat blodprover från familjemedlemmarna, både med och utan RA, som velat delta i mina studier. 51 familjer med mellan 2 och 11 sjuka medlemmar i varje familj omfattande flera generationer har analyserats. Jag har genomfört en helgenom analys för att identifiera DNA regioner som är kopplade till RA hos 134 individer med RA och 216 friska familjemedlemmar och identifierade flera genetiska områden. En av dessa regioner på kromosom 14 kartlades med högre upplösning och några potentiella riskgener för RA i norra Sverige har identifierats. Förekomst av RF och ACPA har också studerats hos både RA patienter och första grads släktingar. Analyserna visade att antikropparna var vanligare hos släktingarna jämfört med friska kontroller. Patienterna med RA hade ökad förekomst av ACPA och RF än släktingarna men med en annan fördelning av isotyperna. Patienter med RA har en ökad sjuklighet av kardiovaskulär sjukdom (KVS) i jämförelse med övriga befolkningen. I två av projekten har jag analyserat variationer i kandidatgener för KVS hos patienter med RA. Valet av kandidatgener, som har studerats baserar sig dels på andra KVS studier i

x

normalbefolkningen där dessa gener har visat på samband dels har valet av kandidatgener varit gener som är relaterade till koagulation, inflammation och RA. Vi har insamlat blodprover från 681 RA patienter och patienternas sjukdom har noggrant följts dels via journalstudier från sjukdomsdebuten och fram till inklusionsdatum. Information om KVS manifestationer, som inträffat efter insjuknandet i RA samt under en 6-års uppföljningsperiod har inhämtats från journalstudier och från Socialstyrelsens register över Slutenoch Öppenvård samt Dödsorsaksregistret. Jag har analyserat relationen mellan DNA-variationerna i kandidatgenerna och de olika KVS manifestationerna; ischemisk hjärtsjukdom (IHS), stoke/transient iscemisk attack (TIA) och djup ventrombos/lungemboli (DVT/LE) samt hypertension. Jag fann en association mellan hypertension och tumor nekrosis faktor receptor 2 (TNFRII) som är en av receptorerna för TNFα vilken har en viktig pro-inflammatorisk roll vid RA. KVS var associerad med variationer i både growth differentiation factor (GDF15) och i 9p21.3 lokuset, den senare var även associerad med KVS i 6-års uppföljning. GDF15 reglerar aktiveringen av makrofager, och uppregleras vid inflammation och finns i högre plasma nivåer i samband med KVS. Varianten i 9p21.3 lokuset har en stark association med KVS i den allmänna populationen men den direkta funktionen är inte klarlagd. IHS var associerad med en variant i plasminogen aktivator inhibitor 1 (PAI1) genen vilken är inblandad i fibrinolysen som en inhibitor för upplösning av koagulerat blod. Stroke/TIA bland kvinnor var associerat med en variant i GDF15 och en i faktor 13 A (FXIIIA), den senare också inblandad i blodkoagulationen men detta protein förstärker koaglet. Bland män var det två varianter i CD40 antigen (CD40) som var associerat med stroke/TIA. CD40 är en receptor som uttrycks på vissa B-celler och förmedlar ett flertal immun och inflammations svar. DVT/LE var associerat med varianterna i FXIIIA och TNFRII. Sammantaget finns samband mellan flera olika kandidatgener och KV manifestationer delvis i analogi med beskrivning för populationen i allmänhet och delvis unikt vid RA, vilket bör konfirmeras vid uppföljande studier. De ovanligare varianterna av GDF15 och TNFRII var associerade med RA per se, TNFRII dock bara hos män.

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Introduction Rheumatoid arthritis Rheumatoid Arthritis (RA) (MIM 180300) is a systemic autoimmune disease affecting about 0.5-1% of the population worldwide and of these two-thirds are female (1-3). The difference in incidence between men and women is larger in pre-menopausal women whilst in post-menopausal women the incidence declines to approximately the same as that for men, which indicates a hormonal influence on development of RA. The disease mainly affects the synovial lining of the joints, which is infiltrated by T- and Blymphocytes and macrophages followed by neo-angiogenesis and fibrosis. The inflamed, expanded synovial tissue forms a tumour-like pannus which invades and destroys the underlying tissue, cartilage and bone. Extraarticular manifestations, such as nodules, vasculitis, pleuritis and pericarditis can occur although later during the disease. Cardiovascular disease, the most common co-morbidity in patients with RA (4), occurs to the same extent as in diabetes mellitus patients (5). Patients with RA have an increased risk of developing lymphomas, particularly those patients with severe disease and high inflammatory activity (6). Aetiology The aetiology of RA is, as yet, unknown and the disease is considered to be a complex disease with genetic, hormonal and environmental influences (7). As an environmental factor smoking has been shown to contribute to the development of RA (8-9) whilst alcohol intake has been suggested to have a protective role (10). The genetic contribution to an individual’s susceptibility to develop RA will be discussed later. Diagnosis The disease is heterogeneous and is diagnosed when a patient fulfils four out of seven criteria proposed by the American College of Rheumatology, see Table 1 (11). These disease criteria are more a classification of the disease than a help to a clinician in order to make an early diagnosis in clinical practice. The disease varies from a mild, self-limiting form to an aggressive one with erosions and disability. It is important that an early diagnosis is made since early aggressive treatment prevents many of the complications of the disease. To identify those patients with an early form of the disease new criteria has been developed (12). These criteria include the presence of antibodies against citrullinated proteins/peptides (ACPAs) and rheumatoid

1

factor (RF), and acute-phase reactants, such as C-reactive protein (CRP) and an increased erythrocyte sedimentation rate (ESR). Table 1. The American College of Rheumatology (1987) revised criteria for RA. Criteria 1-4 must have been present for at least 6 weeks Criterion

Definition

1. Morning stiffness

Morning stiffness in and around the joints, lasting for at least 1 hour before maximal improvement

2. Arthritis of 3 or more joint areas

At least 3 joint areas simultaneously have had soft tissue swelling or fluid

3. Arthritis of hand joints

At least 1 area is swollen in a wrist, metacarpophalangeal joints (MCP), or proximal interphalangeal joints (PIP) joint

4. Symmetric arthritis

Simultaneous involvement of the same joint areas on both sides of the body

5. Rheumatoid nodules

Subcutaneous nodules

6. Serum rheumatoid factor

Abnormal amounts of serum rheumatoid factor by any method in which 4,000 samples from North America and Sweden: association of susceptibility with PTPN22, CTLA4, and PADI4. Am J Hum Genet. 2005;77:1044-60. 123. Zhernakova A, Alizadeh BZ, Bevova M, van Leeuwen MA, Coenen MJ, Franke B, et al. Novel association in chromosome 4q27 region with rheumatoid arthritis and confirmation of type 1 diabetes point to a

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