Monoaminergic System in Depression: Evaluation in an Autoimmune Disease

Monoaminergic system in Depression: Evaluation in an autoimmune disease

RITA ISABEL ALVES FERREIRA DISSERTAÇÃO DE MESTRADO APRESENTADA AO INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR DA UNIVERSIDADE DO PORTO EM MEDICINA LEGAL

Rita Isabel Alves Ferreira

Monoaminergic system in Depression: Evaluation in an autoimmune disease

Dissertação de Candidatura ao grau de Mestre em Medicina Legal submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto

Orientador – Doutora Berta Martins da Silva

Categoria – Professora Associada

Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto

Acknowledgements

Durante estes anos de ensino superior várias foram as pessoas que deixaram a sua marca no entanto, apenas as descritas contribuíram verdadeiramente para o término desta grande etapa e também para os meus melhores anos de vida como estudante:

Aos meus pais e irmão, a quem devo tudo aquilo que sou e que pretendo ser, que me ensinaram a ser uma pessoa de valor em vez de uma pessoa de sucesso, pelo apoio incondicional e por me darem a oportunidade de explorar o mundo da Medicina Legal, o maior dos agradecimentos. Ao Tomás por todo o apoio e carinho demonstrados ao longo destes anos. Aos Bioquímicos Miká, Fernando, Ti, Diogo e Amadeu um especial agradecimento pela verdadeira amizade, pelo apoio e bons momentos proporcionados apesar da distância. Aos Oncologistas Berto, Ana e Lu um imensurável obrigada pelo apoio, companheirismo, amizade e pelo proporcionar de tão bons momentos de descontração. Às insubstituíveis Jack, Tânia, Susana, Maria e Rosânia agradeço a amizade e a tão boa disposição demonstradas desde o primeiro dia deste mestrado.

À Professora Doutora Berta Martins agradeço a oportunidade de realização deste trabalho. Ainda por todo o apoio, compreensão, ensinamentos e tempo disponibilizado. Seria impossível a realização de um bom trabalho sem uma orientação tão eficiente.

À Doutora Raquel Faria agradeço as correções e sugestões e aos restantes médicos da Unidade de Imunologia Clínica do Hospital de Santo António – Centro Hospitalar do Porto. Ainda a todos os doentes incluídos neste projeto.

À Mestre Bárbara um especial agradecimento não só pela ajuda prática e conhecimentos científicos como ainda pela disponibilidade, apoio e paciência durante esta etapa. À Mestre Andreia Bettencourt agradeço a ajuda prática e a partilha de ensinamentos. Às Mestres Daniela Boleixa, Cláudia Carvalho e Oriana Marques um obrigada pela ajuda laboratorial e simpatia. À Mestre Sandra Maia e à D. São um também enorme agradecimento pelo apoio, simpatia e pelo ambiente fantástico proporcionado.

À Comissão Científica do Mestrado em Medicina Legal, especialmente á Professora Doutora Maria José Pinto da Costa pela oportunidade de frequentar o mesmo. Ainda um agradecimento generalizado a todos os Professores que partilharam uma pequena parte do seu vasto conhecimento neste ciclo de estudos.

II Abstract

Systemic Lupus Erythematosus (SLE) is a multifactorial and multisystemic chronic inflammatory autoimmune disease that occurs more frequently in women, particularly of childbearing age. Nearly half of SLE patients (56%) will experience depression and/or neuropsychiatric manifestations (NPSLE). These are major contributors to quality of life impairment, morbidity and mortality. Depression is one of the most common psychiatric complaints in SLE, affecting 17-75% of patients. Accordingly to the World Health Organization depression is a mental disorder characterized by persistent sadness, loss of interest, fatigue, suicidal thoughts and attempts.

The causes of depression remain largely unknown although genetic, biochemical and social factors appear to play an important role in its development. Particular attention has been given to the genes that encode proteins of serotonergic neurotransmission. Several polymorphisms in serotonin’s receptors and transporter have been associated with depressive symptoms in different settings.

This study aims to contribute to a better understanding of depressive states in SLE patients and their genetic determinants. For this purpose 96 SLE patients (91F, 5M; 39.7 ± 14.6 years) from the outpatient clinic of Clinical Immunology Unit of Hospital de Santo António – Centro Hospitalar do Porto were studied. Diagnosis of SLE was based in ACR1999 criteria. The Hospital Anxiety and Depression Scale (HADS) was used to characterize anxiety and depression, and the Short Form 36 (SF-36) test was used to evaluate quality of life. 38 patients were diagnosed with depression (HADS ≥8) and 36 with neuropsychiatric SLE. Polymorphisms rs6314 and rs6313 of the 5-HTR2A (5- hydroxytryptamine receptor 2a) gene and the STin-2 of the 5-HTT (5- hydroxytryptamine transporter) gene were genotyped by a Taqman assay, PCR-RFLP and PCR product sizing respectively. A group of 274 healthy individuals from the north of Portugal was used as control for the genetic studies.

Comparing SLE patients and controls no statistically significant differences were observed in genotypic and allelic frequencies of the polymorphisms studied. The frequencies of the rs6313T allele (57.1 vs. 41.3, p=0.041, OR [95%CI]=1.891 [1.02–3.49]) and rs6313TT genotype (31.4 vs. 9.60, p=0.027, OR [95%CI]=5.391 [1.22–23.82]) were higher in depressed SLE patients when compared to non-depressive SLE patients. No significant associations with the other polymorphisms were observed. These results suggest that SLE patients carrying the rs6313T allele are more prone to develop

III depression. This polymorphism may alter the 5-HTR2A receptor expression or function leading to impaired serotonergic neurotransmission. To the best of our knowledge this is the first study to associate the 5-HTR2A rs6313 polymorphism with depression development on SLE patients, so replication studies are warranted.

Medico-legal concerns such as the prevention of suicide, or the consideration of the mental state of an accused in criminal trials, could also be impacted by these results.

IV Resumo

O Lúpus Eritematoso Sistémico (LES) é uma doença autoimune inflamatória, crónica, multifatorial e multisistémica com uma maior incidência em mulheres, em particular em idade fértil. Quase metade dos doentes com LES (56%) desenvolvem depressão e/ou manifestações neuropsiquiátricas (LESNP). Estas têm um papel importante na redução da qualidade de vida, na morbidade e na mortalidade. A depressão é um dos sintomas psiquiátricos mais comuns no LES e afeta cerca de 45% dos doentes com LES. De acordo com a Organização Mundial de Saúde a depressão é um transtorno mental caracterizado por uma tristeza persistente, perda de interesse, fadiga, pensamentos e tentativas de suicídio.

A causa da depressão permanece desconhecida embora fatores genéticos, bioquímicos e sociais possam desempenhar um papel importante no seu desenvolvimento. Particular atenção tem sido dada aos genes que codificam as proteínas reguladoras da neurotransmissão serotonérgica. Vários polimorfismos nos recetores e transportadores de serotonina têm sido associados a sintomas depressivos.

Este estudo pretende contribuir para uma melhor compreensão dos estados depressivos em doentes com LES e as suas determinantes genéticas. Para este efeito, foram estudados 96 doentes com LES (91F, 5M; 39,7 ± 14,6 anos) da Unidade de Imunologia Clínica do Hospital de Santo António – Centro Hospitalar do Porto. O diagnóstico de LES foi baseado nos critérios do ACR1999. O inquérito de saúde Hospital Anxiety and Depression Scale (HADS) foi utilizado para caracterizar a ansiedade e depressão e o teste Short Form 36 (SF-36) para avaliar a qualidade de vida. 38 doentes foram diagnosticados com depressão (HADS ≥8) e 36 com LES neuropsiquiátrico. Os polimorfismos rs6314 e rs6313 do gene 5-HTR2A (5-hydroxytryptamine receptor 2a ) e o STin2 do gene 5-HTT ( 5-hydroxytryptamine transporter ) foram genotipados com tecnologia Taqman, PCR-RFLP e PCR com análise de fragmento, respetivamente. Um grupo de 274 indivíduos saudáveis do norte de Portugal foi usado como controlo no estudo genético.

Comparando os doentes com LES com os controlos não foi observada nenhuma diferença estatisticamente significativa nas frequências alélicas e genótipicas dos polimorfismos estudados. A frequência do alelo rs6313T (57.1 vs. 41.3, p=0.041, OR [95%IC]=1.891 [1.02–3.49]) e do genótipo rs6313TT (31.4 vs. 9.60, p=0.027, OR [95%IC]=5.391 [1.22–23.82]) eram mais elevadas em doentes com depressão quando

V comparados com doentes sem depressão. Não foi detetada uma associação significativa com os outros polimorfismos estudados.

Os nossos resultados sugerem que doentes com LES portadores do alelo rs6313T estão mais propensos a desenvolver depressão. Este polimorfismo pode alterar a expressão ou função do recetor 5-HTR2A levando a uma transmissão serotonérgica anómala. Estudos de replicação são necessários para aprofundar o papel desse polimorfismo no desenvolvimento da depressão em doenças autoimunes, especialmente no LES.

Algumas implicações médico-legais como a prevenção do suicídio ou a consideração do estado mental de arguidos em processos penais também podem ser afetados por estes resultados.

VI Table of Contents

CHAPTER 1 – INTRODUCTION ...... 1 1.1. Systemic lupus erythematosus ...... 2

1.1.1. Epidemiology ...... 2

1.1.2. Etiology/Pathogenesis...... 3

1.1.2.1. Genetic factors ...... 3 1.1.2.2. Environmental factors ...... 4 1.1.2.3. Hormonal factors ...... 5 1.1.3. Clinical Manifestations ...... 5

1.1.3.1. Dermatological manifestations ...... 6 1.1.3.2. Musculoskeletal manifestations ...... 6 1.1.3.3. Renal manifestations ...... 7 1.1.3.4. Neuropsychiatric manifestations ...... 7 1.2. Depression ...... 9

1.2.1. Etiology ...... 10

1.2.1.1. Biochemical factors ...... 10 1.2.1.1.1. Monoaminergic hypothesis of depression ...... 11

1.2.1.1.2. Cytokine hypothesis of depression ...... 13

1.2.1.1.3. Neurogenic hypothesis ...... 14

1.2.1.2. Genetic factors ...... 15 1.2.1.2.1. Serotonin Receptor 2A ...... 15

1.2.1.2.2. Serotonin Transporter ...... 16

1.2.1.2.3. Monoamine degradation enzymes ...... 18

1.2.1.3. Stress ...... 19 1.2.1.3.1. Dysfunction of the HPA axis ...... 20

1.2.1.4. Illness ...... 20 1.2.2. Types of depression ...... 22

1.2.3. Treatment ...... 23

1.2.3.1. Psychotherapeutic treatment ...... 23 1.2.3.2. Pharmacotherapeutic treatment ...... 24 1.2.3.3. Sports as therapy ...... 25

VII CHAPTER 2 – AIMS ...... 27 Aims ...... 28

CHAPTER 3 – MATERIALS AND METHODS ...... 29 3.1. Materials ...... 30

3.1.1. Study population ...... 30

3.2. Methods ...... 31

3.2.1. Biological samples ...... 31

3.2.1.1. DNA extraction ...... 31 3.2.1.2. DNA quantification ...... 32 3.2.2. Genetic studies ...... 32

3.2.2.1. Genetic association studies ...... 32 3.2.2.2. Genetic variations ...... 33 3.2.3. Applied techniques ...... 33

3.2.3.1. PCR ...... 33 3.2.3.2. Real-Time PCR ...... 36 3.2.3.3. TaqMan technology ...... 36 3.2.3.4. PCR-RFLP ...... 38 3.2.4. Statistical Analysis ...... 41

CHAPTER 4 – RESULTS ...... 43 4.1. Studied polymorphisms in SLE patients...... 44

4.2. Patients characteristics ...... 45

4.3. Studied polymorphisms and depression ...... 45

4.4. Studied polymorphisms and NPSLE ...... 47

CHAPTER 5 – DISCUSSION AND CONCLUSIONS ...... 49 5.1. Discussion ...... 50

5.1.1. Role of studied polymorphisms in SLE patients...... 50

5.1.2. Role of studied polymorphisms in depression ...... 52

5.1.3. Role of studied polymorphisms in NPSLE ...... 54

5.2. Conclusions ...... 55

CHAPTER 6 – REFERENCES ...... 57

VIII Figure Index

Figure 1 – Normal immune response and autoimmune response. (Adapted:http://www.womensinternational.com/connections/autoimmune.html. Accessed: 27.05.2015) ...... 2

Figure 2 – Malar rash. (Adapted: http://www.nucleuscatalog.com/lupus- erythematosus/view-item?ItemID=9084. Accessed: 21-06-2015) ...... 6

Figure 3 – The general structure of a chemical synapse. (Adapted: Pereda, A.E. 2014. Electrical synapses and their functional interactions with chemical synapses. Nat Rev Neurosci 15: 250-63) ...... 10

Figure 4 – Classes of neurotransmitters...... 11

Figure 5 – Serotonergic system. MAO – Monoamine Oxidase. COMT – catecol-o- metiltransferase. (Adapted: http://visual-science.com/projects/ssri/infographics/ Acessed: 27.05.2015) ...... 12

Figure 6 – Serotonin pathway and kynurenine pathway from L-tryptophan metabolism. (Adapted: https://neuroendoimmune.wordpress.com/. Accessed: 03-02-2014) ...... 14

Figure 7 – Human SLC6A4 gene organization. (Adapted: Murphy DL, Moya PR. 2011. Human Serotonin Transporter Gene (SLC6A4) Variants: Their Contributions to Understanding Pharmacogenomic and Other Functional G x G and G x E Differences in Health and Disease. Current opinion in pharmacology 11: 3-10) ...... 17

Figure 8 – The stress response mediated by the hypothalamic–pituitary–adrenal (HPA) axis. CRH: Corticotropin releasing hormone; ACTH: adrenocorticotropic hormone. (Adapted: Stephens MA, Wand G. 2012. Stress and the HPA axis: role of glucocorticoids in alcohol dependence. Alcohol Res 34: 468-83) ...... 19

Figure 9 – Diagram of the etiology of depressive symptoms. (Adapted: Sperner- Unterweger B, Kohl C, Fuchs D. 2014. Immune changes and neurotransmitters: possible interactions in depression? Prog Neuropsychopharmacol Biol Psychiatry 48: 268-76).....22

Figure 10 – The PCR steps. (Adapted: http://microfluidics.stanford.edu/Projects/Current/OnChipPCR.html. Accessed: 23-01- 2015) ...... 34

Figure 11 – Genotypes of STin2 obtained by PCR. STin2.12 allele corresponds to 12 repeat units of 17bp; STin2.10 allele corresponds to 10 repeat units of 17bp; STin2.9 allele corresponds to 9 repeat units of 17bp...... 36

Figure 12 – RT-PCR steps with Taqman® probes for genotyping. (Adapted: http://www.applied-maths.com/applications/taqman-based-snp-genotyping. Accessed: 28- 01-2015) ...... 37

Figure 13 – Procedure of PCR-RFLP technique. (Adapted: http://answermeup.com/questions/perform-rflp-pcr.html. Accessed: 27.05.2015) ...... 39

Figure 14 – Genotypes of rs6313 obtained from PCR-RFLP with the MSPI restriction enzyme...... 41

X Table Index

Table 1 – Sequence of primers used from STin2 analysis...... 34

Table 2 – Components and their respective concentrations for PCR used from STin2 analysis...... 35

Table 3 – Thermic profile used from STin2 analysis...... 35

Table 4 – Components and their respective concentrations for PCR used from rs6314 analysis...... 38

Table 5 – Thermic profile used from rs6314 analysis...... 38

Table 6 – Sequence of primers used from rs6313 analysis...... 39

Table 7 – Components and their respective concentrations for PCR used from rs6313 analysis...... 40

Table 8 – Thermic profile used from rs6313 analysis...... 40

Table 9 – MspI restriction enzyme cleavage site used from rs6313 analysis...... 40

Table 10 – Components and their respective concentrations for digestion protocol used from rs6313 analysis...... 41

Table 11 – Allelic frequencies in the control population (CP) and SLE patients...... 44

Table 12 – Genotype frequencies in the control population (CP) and SLE patients...... 44

Table 13 – Demographic characteristics of SLE patients...... 45

Table 14 – Allele frequencies in SLE patients with depression (HADS ≥8) and without depression (HADS<8)...... 46

Table 15 – Genotype frequencies in SLE patients with depression (HADS ≥8) and without depression (HADS<8)...... 46

Table 16 – Allelic frequencies in SLE patients with and without NPSLE ...... 47

Table 17 – Genotype frequencies in SLE patients with NPSLE and with non-NPSLE...... 48

Table 18 – Allelic frequencies of rs6313 in depressed patients...... 53

XII Abbreviations

µL – microliter NDRI – norepinephrine-dopamine µM – micromolar reuptake inhibitors 5-HT – serotonin NPSLE – neuropsychiatric systemic 5-HTR – serotonin receptor lupus erythematosus 5-HTT – serotonin transporter ºC – Celsius ACR – American College of PCR – polymerase chain reaction Reumathology PCR-RFLP – PCR restriction fragment ACTH – adrenocorticotropic hormone length polymorphism BBB – blood, brain barrier RCLB – red cell lysis buffer CBT – cognitive behavioural therapy rs – polymorphism CNS – central nervous system RT-PCR – real time PCR COMT – catecol-o-metiltransferase SDS – sodium dodecyl sulfate CRH – corticotrophin releasing hormone SF-36 – Short Form 36 ddH 2O – double-distilled water SLE – systemic lupus erythematosus DNA – deoxyribonucleic acid SNP – single nucleotide polymorphism dNTPs – deoxynucleotide triphosphates SNRI – selective serotonin- DSM-IV – diagnostic and statistical norepinephrine inhibitors manual of mental disorders 5 th edition SSRI – selective serotonin reuptake EBV – Epstein-Barr virus inhibitors EDTA – ethylenediamine tetraacetic acid STin2 – serotonin transporter located in HADS – Hospital Anxiety and Depression intron 2 scale Taq – thermus aquaticus HPA – hypothalamic-pituitary-adrenal TCA – tricyclic antidepressant IDO – indolamine-2,3-dioxygenase TE – tris-EDTA

IFN – interferon TE 2 – tris-EDTA 2x IL – interleukins TNF – tumor necrosis factor IPT – interpersonal psychotherapy TRF – tryptophan KYN – kynurenine VDR – vitamin D receptor LPR – linked promoter region VNTR – variable number of tandem MAO – monoamine oxidase repeats

MgCl 2 – magnesium chloride WHO – World Health Organization MHC – major histocompatibility complex mL – milliliter NaCl – sodium chloride

XIII

INTRODUCTION

Chapter 1 - Introduction

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1.1. Systemic lupus erythematosus

The immune system is a complex association of cells, tissues and organs and its main goal is protect the body from infectious organisms such as bacteria and virus. In SLE and other autoimmune diseases, the immune system loses its ability to distinguish between its own cells and foreign cells and tissues (antigens), thus the immune system develops autoantibodies directed against it (figure 1) (1). Autoimmune diseases’ incidence has been increasing around the world. Indeed, they affect about 5% of the European population with two thirds of the patients being female (2).

SLE is a chronic autoimmune disease that involves multiple organ systems such as skin, kidneys, lungs, blood and, in the most severe cases, the central nervous system (3). SLE pathogenesis remains unknown; however genetic factors are strongly correlated with SLE susceptibility. Environmental factors may also act as triggers for disease development (4). Figure 1 – Normal immune response and autoimmune response. (Adapted:http://www.womensinternational.com/connections/autoim mune.html. Accessed: 27.05.2015)

1.1.1. Epidemiology

SLE incidence and prevalence seems to be increasing. This may be due to the availability of better diagnostic tools and the increased identification of milder cases, adding to a proper increase of disease cases. In an USA population the incidence was 5.1 per 100.000 per year and the prevalence was 52.2 per 100.000 and 3.8 and 26.2 in the United Kingdom (5). In the North of Portugal population the annual incidence rates of SLE is 2.3 cases per 100 .000 persons (6). The male-to-female ratio is about 1:9 (4) and the female predominance is higher in women in the childbearing years compared to the

Chapter 1 - Introduction

ICBAS - UP juvenile and elderly populations, probably due to the effect of female sex hormones on immune system (5). About 10%-20% of SLE cases manifest initially in childhood (before the age of 16 years) (7).

Mainly due to the immune system impairment, the body’s ability to prevent and fight infection is reduced, thus SLE patients have a superior risk for developing infection and complications (8). During the last 5 years of the Euro-Lupus project, arthritis and malar rash was the most prevalent manifestations and thrombosis was the most common cause of death (9). However the 10 year survival rate was 92%, a significant improvement when compared to 20th century (5).

1.1.2. Etiology/Pathogenesis

The precise cellular and molecular mechanisms and which factors determine organ involvement is not clear (10). Disturbances in the immune system run with anomalies in cytokines, B-cell immunity and T-cell signaling, especially intense B cell activation (5). Indeed, Moulton et al characterizes the disease as an autoantibody production by dysregulated B cells (11). In addition, SLE is characterized by the production of a group of cytokines: the type 1 interferon (12), which in turn activates B and T cells (13). The disrupted immunological tolerance leads to a group of mechanisms associated with immune complexes deposition that are responsible for the manifestations of SLE (12). The therapy has relied on global immunosuppression, however it causes significant morbidity and mortality and side effects including infections and osteoporosis (14).

Although SLE is a complex disease with multifactoral etiology, it might be triggered by genetic and environmental factors such as microbial antigens, drugs and hormones. (13).

1.1.2.1. Genetic factors

Multiple pathways predispose to the development of SLE. The disease has a strong genetic component and familial aggregation: the concordance of the disease was found to be 5.6% among first-degree relatives. The risk is also increased in siblings of SLE patients and in identical twins the concordance of the disease is approximately 25- 50% (15). In addition, around 10% to 15% of the patients have more than one affected

Chapter 1 - Introduction

ICBAS - UP family members (16). The hereditary tendency strongly suggests that DNA polymorphisms influence the susceptibility to SLE. More than a hundred single genetic defects affecting the immune system cause SLE-like disease in mice (14). The most important genes associated with SLE are within the major histocompatibility complex (MHC). The function of MHC molecules is to bind peptide fragments derived from antigen and present them on the cell surface for recognition by the appropriate T cells. The human nomination of the MHC is the human leukocyte antigen (HLA). The HLA class II gene alleles HLA- DRB1*15:01 and HLA-DRB1*03:01 have been associated with SLE susceptibility with a two to three fold increased frequency in patients compared to controls (17).

1.1.2.2. Environmental factors

Recognized environmental factors trigger the disease in a genetically predisposed individual. Ultraviolet light and infections are implicated in the etiology. Ultraviolet light is the most well described environmental trigger in SLE. Due to sunlight exposure, skin disease can be exacerbated in those patients. On the other hand, the consequent avoidance of sunlight can lead to vitamin D deficiency. In fact, several studies show an inverse relationship between levels of vitamin D and disease activity (18).

Molecular mimicry between an infectious antigen and self-components is implicated as a pivotal mechanism by which SLE can be triggered. This phenomenon has been assumed to be a mechanism by which an invading organism evades the host immune response, as it is identified as “self”. On the other hand, due to the similar structure, these invading organisms can initiate an immune response in which both T-cells and B-cells cross-react with self-antigens (19). Infections caused by Epstein-Barr virus (EBV) have been reported as possible mediator in the development of SLE (20). A significant increase in EBV infection in pediatric lupus patients compared to controls was been described (21). In addition, the natural host cells of EBV are B cells which play a central role in SLE (22).

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1.1.2.3. Hormonal factors

SLE is predominantly a female disease, as previously stated. The female predisposition declines with the ending of the reproductive age (15). Patients with Klinefelter’s syndrome, characterized by hypergonadotrophic hypogonadism, have an increased risk of SLE development (23). In patients with SLE of both genders an abnormal estrogen metabolism has also been described (24). A study with rodents demonstrate that administration of estrogen and prolactin can lead to autoimmune disease development (20). Another study with a cohort of 238.308 women followed up for 27 years, revealed that early menarche, oral contraceptive use, early menopause and postmenopausal use of hormones were associated with an increased risk of SLE (25).

SLE also affects pregnancy: increased SLE activity at the conception time and the presence of damage associated with SLE before conception leads to complications in pregnant women, such as preterm birth and caesarean section. Thus, although the mechanism of action is unknown, the female hormones appear to be strong regulators of disease activity (5).

1.1.3. Clinical Manifestations

SLE is a multisytemic disease and the manifestations vary widely (26). The constitutional symptoms include fatigue, fever and weight loss. Fatigue is the most common symptom in SLE patients probably due to the active disease, medications or lifestyle habits. Fever may also result from many causes such as SLE flare, infection and reactions to medications. The most common infections involve the respiratory tract, urinary tract and skin. In addition other opportunistic infections such as salmonella and herpes zoster may arise (8) (27). The severity of symptoms can range from mild cutaneous involvement to severe organ damage (4).

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1.1.3.1. Dermatological manifestations

SLE was initially recognized as a dermatologic condition. The skin involvement occurs in up to 85% of SLE patients. There are three skin diagnostic criteria including malar rash, photosensitivity and discoid rash. The malar rash is characterized by an erythema over the cheeks and nasal bridge (figure 2). It is a butterfly-shaped that typically spares the nasolabial folds. In a group of SLE patients living in Dubai, malar rash was the commonest mucocutaneous manifestation (60,3%) (8). Photosensitivity is diagnosed when patients presents any unusual rash or symptom exacerbation after sun exposure. Exposure to ultraviolet light causes skin rash in face, arms or hands. Discoid skin rash is the most common form of chronic cutaneous lupus erythematosus. It is characterized by patchy redness that can cause scarring and also occurring in sun- exposed areas. This manifestation can be a separate Figure 2 – Malar rash. (Adapted: diagnostic entity (without organ involvement) or be part http://www.nucleuscatalog.com/lup of SLE (27) (28). us-erythematosus/view- item?ItemID=9084. Accessed: 21 - 06-2015)

1.1.3.2. Musculoskeletal manifestations

Musculoskeletal symptoms are also reported in more than 90% of the patients (29), among which arthralgia and arthritis are present in 95% of patients (30). Indeed, joint pain is one of the most prevalent manifestations at presentation. In AlSaleh et al study, arthritis is even the most common clinical manifestation (8). Arthralgia and arthritis occur in the small joints of the hands, wrists, and knees although any joint is at risk. These symptoms may mislead, leading to the diagnosis of other types of inflammatory arthritis instead of SLE (27).

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1.1.3.3. Renal manifestations

Renal failure and infection are the two major causes of death in SLE patients. The kidney is the most affected visceral organ in SLE although only 50% of patients develop clinically evident renal disease. Additionally, biopsy studies showed that almost all patients with SLE present some degree of renal involvement. Clinical nephritis is mainly caused by deposits of immune complexes in the glomeruli. Acute or chronic renal failure may cause symptoms such as uremia and fluid overload. Acute nephritic disease can present with hypertension and hematuria. In addition, the glomerular disease that develops in the first years of the disease is usually asymptomatic (27).

1.1.3.4. Neuropsychiatric manifestations

SLE can involve the central nervous system (CNS), leading to a wide spectrum of neuropsychiatric symptoms. Neuropsychiatric SLE (NPSLE) is one of the most important manifestations of SLE (31). Indeed, about half of SLE patients (56%) will have at least one NPSLE during the course of disease (32). These manifestations can occur at any time during the disease. Headache and mood disorders are described as the most common neurologic manifestations. The rate of depression is higher in SLE patients, particularly those with NPSLE, than in general population (33) (34). In fact it has been reported that 17-75% of SLE patients have depression (34). On the other hand, the diagnosis of depressive symptoms is usually associated with other clinical manifestation and rarely with SLE. Nevertheless, the association between depressive symptoms and SLE has already been described for more than 100 years (35).

The cognitive functions include general intelligence, verbal learning/memory, visual–spatial skills, psychomotor speed/manual dexterity and attention/mental flexibility. It was reported that the majority of SLE patients present impairment in 3 or more domains (31). In addition, a study reported that NPSLE patients present poor performance on verbal and memory tests (36).

Delirium is one of the more uncommon NPSLE manifestations and its severity varies widely from mild confusion to profound disorganization with agitation and hallucinations (31). Likewise, psychotic events refer to paranoia with visual and auditory hallucinations in NPSLE patients (37). Furthermore SLE may precipitate epileptics status

Chapter 1 - Introduction

ICBAS - UP and demyelinating disorders such as myelopathy and aseptic meningitis (27). Nonetheless, the diagnose is complex due to the difficulty of distinguishing psychological manifestations caused by SLE, by the burden of the disease (38), by concomitant drugs, infection or comorbidities.

The general symptoms of NPSLE are not specific however they are major contributors to impaired quality of life, morbidity and mortality (32). Ideally, patients with SLE should be assessed regularly for neuropsychiatric events and referral to psychology departments should be available (12). SLE is a chronic disease not only with physically impairment but it is also emotionally draining (32).

Chapter 1 - Introduction

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1.2. Depression

Depression is a common mental disorder, affecting approximately 121 million people worldwide. In Western Europe it affects between 10-55% of the population (39). According to the World Health Organization (WHO), depression manifests with depressed mood, loss of interest, low energy levels and fatigue; the person experiences feelings of guilt, thoughts of suicide and suicide attempts. It is still often associated with symptoms of anxiety and irritability. To be diagnosed depression should persist for a period, of at least, two weeks. These depressive symptoms are associated with a significant reduction in quality of life and functional capacity (40). WHO data report that in 2020 depression will be the second leading cause of disability throughout the world. Thereafter in 2030 it should become the main cause (41). In fact, even after successfully treatment, remission is rarely accompanied by a total loss of symptoms. Among them are cognitive and functional impairment, which affects quality of life. The burden of depression also influences the mortality risk (42). One particularly tragic consequence of depression is suicide. Almost 1 million lives are lost each year, which translates into 3.000 deaths every day (43).

Depression can occur at any stage of a person's life (44). The disease affects both women and men, however women are more propense to be affected with the disease (45). In fact, it is the leading cause of disease-related disability in women. Epidemiological studies show that the prevalence of major depression is 21.3% and 12.7% in women and men respectively (46). Biological factors are important in the several explanations for this fact. Hormonal factors, particularly at the onset of puberty, may account for the increased likelihood of women developing anxiety or depression (47). Furthermore, Hammen et al demonstrate that children with depressed mothers, have a higher predisposition to depression and social maladjustment (48). Depression in men is frequently undiagnosed and untreated. This is due to their difficulty to seek help since depression is often associated with powerlessness and the uncontrolled expression of emotion (49). Women had a greater propensity to commit suicide, although men presented a higher mortality rate in these circumstances (50).

Social factors have been recognized as essential for healthy aging and depression can arise due to lack of these factors. It was described that decreased social network and social support were related to depression in older people (51). Indeed, poor

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ICBAS - UP social relationships is probably more injurious that drinking abuse (52). However the risk of developing depression is strongly biologically and genetically determined (53).

1.2.1. Etiology

Psychiatric disorders have a complex etiology and it is crucial to consider that multiple causes contribute to a particular disorder. The etiology of depression differs widely from person to person; nonetheless there are risk factors that influence the development and the persistence of depressive episodes. Defining the factor/factors that trigger depression is quite important for, not only the treatment itself, but also for the patient to learn to avoid or deal with this factor. Biochemical, genetic and social factors will be discussed as well as the influence of illness and stress as factors that can lead to depression (54).

1.2.1.1. Biochemical factors

The nervous system plays a role in the integration, coordination and communication between the different body parts of the human being. Neurons are the most important cells responsible for the transmission of nerve impulses. Through synapses, the neuron can pass an electrical or chemical signal to the next neuron. The chemical synaptic transmission uses chemical signals (neurotransmitters) released from presynaptic nerve terminals. Subsequently, the neurotransmitters bind to specific receptors on postsynaptic neurons completing the synapse (figure 3). There are several types of Figure 3 – The general neurotransmitters but the main focus will be the biogenic structure of a ch emical amines. These include two large groups: cholines where synapse. (Adapted: Pereda , A.E. 2014. Electrical acetylcholine is the main neurotransmitter and monoamines synapses and their functional interaction s with including serotonin, histamine and catecholamines chemical synapses. Nat (dopamine, epinephrine, norepinephrine) (figure 4) (55). Rev Neurosci 15: 250-63)

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Figure 4 – Classes of neurotransmitters.

1.2.1.1.1. Monoaminergic hypothesis of depression

Monoamines are neurotransmitters that contain one amino group connected to an aromatic ring by a two-carbon chain. The main monoaminergic systems in the CNS are histamine, dopamine, norepinephrine, epinephrine, serotonin and melatonin. For the development of major depression, the serotonergic, noradrenergic and dopaminergic systems are the most relevant (56). Disturbance of the metabolism of these monoamines and other neurotransmitters appears to play an important role in the development of depression and other neuropsychiatric disorders. Thus, the monoaminergic hypothesis states that depression is caused by a functional deficiency of monoamines particularly serotonin, dopamine and norepinephrine (57). The monoamine hypothesis was first formulated in the late 1960s. Currently, the monoaminergic system is considered the main system involved in the neurobiology of depression. Indeed, of several antidepressant drugs the mechanism of action increase serotonin levels in the synaptic cleft, supports this hypothesis (58). Another piece of evidence that supports the monoaminergic hypothesis is that patients with low levels of serotonin are more likely to committed suicide (59).

Phylogenetically, the serotonergic system is one of the oldest transmitter systems in the brain. Indeed, serotonin has an extensive system of innervations in most cortical and subcortical structures. Due to this diversity of signaling opportunities and functional roles this neurotransmitter has been associated with many different types of psychopathological conditions (60). Serotonin neurons use tryptophan to synthesized serotonin. The majority of intraneuronal serotonin is retained in storage vesicles and subsequently their release occurs by exocytosis. After release, the serotonin accumulates

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ICBAS - UP in the synaptic cleft. In order to control the amount of serotonin in the synaptic cleft there are specific transporter proteins that remove the neurotransmitter from the synapse (figure 5). The remaining serotonin will connect to the receptors in the postsynaptic neuron (61). Fourteen different serotonin receptors have been identified. In fact they are grouped in 7 distinct classes (5-HT(1) to 5-HT(7)) basis on their structural and operational characteristics (62).

Some authors refer the hypothesis that serotonin receptor 1A is involved in the pathophysiology of depression or depressive syndrome. In fact, in animal models of depression highly selective serotonin receptor 1A agonists possess antidepressant properties (63). Another serotonin receptor (HTR2A) has been implicated with depression. An increased density of this receptor in the brain of depressed individuals was described (64).

Indeed, monoamine depletion studies demonstrate that subjects with a family history of depression have decreased mood compared to healthy humans (65). However, depletion of a specific monoamine may be a too simplistic explanation due to its complex interactions. On the other hand, this probably helps to understand the vulnerability to become depressed (65). Therefore, several studies reported the urgent need for alternative hypotheses (66) (67). Figure 5 – Serotonergic system. MAO – Monoamine Oxidase. COMT – catecol-o- metiltransferase. (Adapted: http://visual - science.com/projects/ssri/infographics/ Acessed: 27.05.2015)

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1.2.1.1.2. Cytokine hypothesis of depression

Formulated by Smith in 1991, this theory explains the involvement of cytokines of the innate immune system in depression. This hypothesis postulated that depression is accompanied by immune deregulation. Activation of the immune system leads to increased secretion of proinflammatory cytokines. This is the only model of depression that can bridge the diagnostic gap between physical and mental disorders (68). Cytokines are chemical messengers used by cells of the immune system to communicate with one another. Once in the blood, cytokines can alter the function of every tissue and organ, including the brain. In addition, they are also responsible for physical signs such as inflammation, headache, depressed mood, hormonal and biochemical abnormalities.

Depressed patients have activated immune systems and secrete more cytokines than normal controls. In addition, the most severely depressed patients have the most activated immune systems (69). Furthermore, cytokines have powerful effects on neurotransmitters including serotonin, dopamine and norepinephrine. Thus, cytokines can be an explanation for the neurotransmitter abnormalities found with psychiatric diseases (69) (70).

There are several types of cytokines, for example interleukins (IL-1, IL-2, IL-6), interferon (IFN-γ) and tumor necrosis factor (TNF-α). Dowlati et al reported significantly higher concentrations of proinflammatory cytokines TNF-α and IL-6 in depressive patients compared to psychiatrically healthy subjects (68). Accordingly, the administration of inflammatory cytokines can induce an increased indoleamine-2,3-dioxygenase (IDO) activity. This enzyme catalyzes the first step of essential aminoacid tryptophan degradation to N-formyl-kynurenine influencing negatively the synthesis of serotonin. Consequently, this leads to the appearance of depressive symptoms due to IDO activity pushing tryptophan away from the serotonin pathway and into the kynurenine pathway, leading to an increase of kynurenic acid and quinolinic acid concentrations that are also correlated with depressive behaviors (figure 6) (71) (72).

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ICBAS - UP A study described increased rates of depression induced by lipopolysaccharide administration in healthy male volunteers, suggesting that cytokines are involved in the etiology and symptomatology of illness-associated depression (73). Thus interaction between pre-existing vulnerabilities and inflammatory stimuli may act as a trigger in depression development (74). The ability of cytokines to modulate Figure 6 – Serotonin pathway and kynurenine pathway hippocampal neurogenesis (the from L-tryptophan metabolism. (Adapted: generation of new neurons) has also https://neuroendoimmune.wordpress.com/. Accessed: 03-02-2014) been reported (75). This subject will be focus in the next topic.

1.2.1.1.3. Neurogenic hypothesis

Newborn neurons are continuously added to the adult hippocampus. Researchers were focused on the hippocampus since it is also an area of the brain important for stress regulation, cognition and mood. First formally proposed in 2000, the neurogenic hypothesis claims that depression is at least partially caused by an impairment of the brain's ability to produce new neurons (58).

The neurogenic hypothesis was formulated based on several observations. For instance, evidences from post-mortem and imaging studies reported a significantly lower hippocampal volume in subjects with major depression (76) (77). Their hippocampi also appear to be smaller during periods of depression when compared to periods of remission. Consequently, decreased hippocampal neurogenesis was claimed. In addition, the authors suggest that this decrease leads to a reduced monoaminergic neurotransmission; which in turn promotes an altered activity of emotion and cognition (77) (78). Corroborating to this hypothesis, the chronic administration of antidepressants that raises the levels of serotonin, leads to an increase adult hippocampal neurogenesis in stressed rodents and primates (58).

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ICBAS - UP According to this hypothesis it may be possible to predict risk factors based on factors that decrease neurogenesis.

1.2.1.2. Genetic factors

Genetic factors play an important role in the development of depression. Twin studies suggest a heritability of 40-50%. Also family studies reported a twofold to threefold increase in lifetime risk of developing depression among first-degree relatives (79). Many genetic association studies have been conducted since the first case–control study on the relationship between polymorphisms and depression (80). Since they are candidate genes, greater attention has been given to the serotonergic receptor and transporter genes. to the susceptibility of mental disorders (81). Additionally, the genes encoding the catecholamines enzymes degradation monoamine oxidase A (MAO-A) and catechol-O- methyl-transferase (COMT) will be analyzed (82).

1.2.1.2.1. Serotonin Receptor 2A

The 5-HTR2A (5-hydroxytryptamine receptor 2A) is responsible for the regulation of anxiety, mood, memory and cognition. It is also the target of several antidepressant drugs (83). This receptor is encoded by the 5-HTR2A gene located on chromosome 13 (13q14-21) (84).

It has been showed that the polymorphism rs6313 (102T>C) may interfere with 5-HTR2A mediated neurotransmission. In a sample of 485 European descendent patients with major depression, the C allele of this single nucleotide polymorphism (SNP) was more frequent in depressed patients. It was also associated with a more severe major depressive episode (85). However, in other populations no evidence of genetic association of rs6313 with depression was found (86). Additionally it was reported that rs6313 is associated with suicide attempts in female alcoholic patients (87). This polymorphism was been predominantly analyzed in association with personality traits. One study described the association in Italian mood disorder patients (88).

Another polymorphism, rs6314 (1345C>T) has been reported to decrease the receptor`s intracellular signaling (89). The SNP has been shown to be associated with depression symptoms severity and response to antidepressants in adults with diagnosed depressive disorder. Nevertheless results are not consistent. The rare TT variant of

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ICBAS - UP rs6314 was reported in two patients with severe melancholic major depression (85). On the other hand, there was no significant association between rs6314 with severity of depression symptoms in children with autism spectrum disorder (90). Moreover, a study with 166 European descendent patients receiving paroxetine for major depression treatment demonstrated that the rs6314 heterozygote’s individuals had a better response to this antidepressant (91).

1.2.1.2.2. Serotonin Transporter

Candidate gene studies also involve the serotonin transporter (5-HTT) gene in a wide range of neuropsychiatric disorders such as depression and bipolar disorder. The 5- HTT is encoded by the serotonin transporter gene SLC6A4, located on chromosome 17 (17q11.1-17q12) (92) (93). A VNTR (STin2) polymorphism located in the intronic region has been implicated in the regulation of transcription levels (94). The STin2 polymorphism consists of three alleles: STin2.9; STin2.10 and STin2.12 repeat units of 17bp. Some reports described that STin2, in particular the 12 repeats allele, is associated with higher transcriptional levels. This leads to a superior number of receptors and consequently to lower serotonin availability (92) (95). Thus, impairment in serotonin metabolism, may in part underpin the development of depressive symptoms.

The STin2.12 allele is positively associated with comorbid tobacco use disorder and mood disorders such as depression or bipolar depression (96). Another study described that patients with STin2 9/12 or 12/12 genotype had fourfold higher odds of post stroke depression when compared to STin2 10/10 genotype carriers (97).

An additional polymorphism in the serotonin transporter was described. A 44 bp repeat polymorphism in the promoter region of the gene (5-HTTLPR ) has been shown to influence expression levels of the serotonin transporter in vitro (79). It consists in a 44 bp insertion (long allele “L”) or deletion (short allele “S”). The S allele has been reported to be associated with lower level of transcriptional efficiency of the 5-HTT in membranes. Therefore, this allele is considered to be less efficient than L allele (98). Likewise, Bonvicini et al referred in their study that, in human post-mortem brains, the L allele is associated with higher transcriptional activity. Consequently the rate of serotonin reuptake is higher (95). Recent studies show that depressed individuals usually have a higher frequency of S alleles compared to non-depressed controls. In addition, S allele has been

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ICBAS - UP associated with suicide (99) and several mood disorders like neuroticism and anxiety symptoms (100).

A rs25531 SNP in the serotonin transporter SLC6A4 gene is the nearest polymorphism of 5-HTTLPR (101). It is located immediately outside of the 5-HTTLPR segment resulting in a strong link between them (figure 7). Due to their proximity, the rs25531 SNP appears to modulate the effect of the 5-HTTLPR on transcriptional efficacy (102). The rs25531 (25531A>G) can be found in the context of L and S alleles of 5- HTTLPR. The G allele occurring frequently with L allele diminished the transcription levels when compared to A allele (103). This fact is relevant to neuropsychiatric disorders due to the association of the SNP with differences in emotional and personality characteristics (104). Additionally, a study with 97 healthy individuals concluded that homozygote individuals for the G allele may Figure 7 – Human SLC6A4 gene organization. have a tendency to be more optimists and (Adapted: Murphy DL, Moya PR. 2011. Human Serotonin Transporter Gene (SLC6A4) more resilient to stress than individuals Variants: Their Contributions to Understanding carrying an rs25531 A allele (105). Pharmac ogenomic and Other Functional G x G and G x E Differences in Health and Disease. Current opinion in pharmacology 11: 3-10)

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1.2.1.2.3. Monoamine degradation enzymes

One of the most frequently investigated genes, in major depression studies, is the COMT gene located on chromosome 22q11. The COMT gene codes for one of the principal enzymes involved in catecholamine degradation and a key modulator of dopamine levels in the human brain (106). Functionally, this enzyme is responsible for removing monoamines from the synaptic cleft in the prefrontal cortex due to the absence of a specific transporter in this region (107). Genetic variation in the COMT gene has been associated with a 40% increase in enzyme activity . Analyses of human post-mortem dorsolateral prefrontal cortex support the hypothesis that higher enzyme activity results in lower dopamine bioavailability (107). Consequently, it leads to an impaired dopamine neurotransmission and an increased risk for major depression development (108).

Another important enzyme is MAOA. The MAOA gene maps to the short arm of the X chromosome (Xp11) and codes for a mitochondrial outer membrane enzyme involved in the degradation of neurotransmitters such as serotonin, norepinephrine and dopamine. Human and animal research has supported the involvement of MAOA gene in mood disorders and in aggressive behavior. Genetic variations in MAOA gene cause different transcriptional activities that result in different expression levels of this gene (109). The variation that has been most studied consists of a 30 bp VNTR with 2, 3, 3.5, 4 and 5 repeats. Functional studies revealed that this alleles displayed differential expression. The 3 repeat allele presents lower transcription efficiency than the alleles with 3.5 or 4 repeat that transcribed 2–10 times more efficiently. Indeed, it has been shown an association between the 3 repeated allele and neuropsychiatric conditions, antisocial personality and poor reaction to stress. Higher gene expression has been associated with suicide caused by depression (110). In addition, in a study with 42 post-mortem human brain, a higher level of MAOA was found in the hypothalamic region (111).

Another association study indicated that low activity of both enzymes MAOA and COMT, appears to influence mood during stressful events, in particular the peripartum period (112).

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1.2.1.3. Stress

Stress is a factor implicated in psychiatric disorders. It is well established that stressful life events are associated with risk for depression. When compared to those without vulnerability, depression-prone individuals are active agents in the creation of prior stressful life events, particularly major undesirable events (113). Continuing negative environmental circumstances (chronic stress) such as poor working conditions, financial difficulties, conflicting intimate relationships with romantic partners, parents, children, or friends; continuing health problems and other ongoing burdens are included (114). Their responses are inappropriate and may result in adverse mental and psychological effects such as anxiety, panic disorders and depression. Indeed, when the stress situation occurs over a period of time, the depression may arise insidiously covered by continued symptoms of anxiety, the so called “masked depression” (115).

Glucocorticoids like cortisol, that is released in response to stress, appears to be increased in depression. This is due to the changes in the endocrine and immune systems that are associated with the disorder but also with changes in central neurotransmitter function. Stress is regularly associated with activation of a specific neuroendocrine pathway, the hypothalamic–pituitary–adrenal (HPA) axis. Initially, stress stimulates the hypothalamus to release the corticotrophin-releasing hormone (CRH). This hormone influences the pituitary to stimulate the secretion of adrenocorticotropic hormone (ACTH) that in turn culminates in the release of glucocorticoids from the adrenal gland (mainly cortisol in humans). Glucocorticoids in turn act back on Figure 8 – The stress response mediated by the the hypothalamus and pituitary to hypothalamic–pituitary–adrenal (HPA) axis. CRH: suppress CRH and ACTH production Corticotropi n releasing hormone; ACTH: adrenocorticotropic hormone. (Adapted: Stephens MA, in a negative feedback cycle (figure Wand G. 2012. Stress and the HPA axis: role of glucocorticoids in alcohol dependence. Alcohol Res 8) (116). 34: 468-83)

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1.2.1.3.1. Dysfunction of the HPA axis

Dysfunction of the HPA axis presents a challenging hypothesis in the neurobiology of depression. It has been shown that the inhibitory feedback mechanism, that normally reduces the activity of the HPA axis, is largely insensitive to the effect of raised glucocorticoids in depressed patients. The chronic raised glucocorticoid levels in major depression are associated with activation of macrophage activity. It is already established that both, infections and active autoimmune inflammatory processes are associated with activation of the HPA axis. Indeed, cytokines particularly those produced by macrophages are mediators of inflammation. Of these mediators, the pro-inflammatory cytokines TNF-α, IL-1 and IL-6 appears to play an importance role in the activation of the HPA axis. The activation mediated by these cytokines is greater than the one observed with maximal stimulatory doses of CRH. This suggests that the hypothalamus and other stimulators of ACTH release are also activated by these cytokines (117).

The continuing hyper secretion of glucocorticoids has implications in the normal functioning of the brain and the immune system. The long-term consequence of these endocrine and immune changes leads to increased morbidity in depressed patients.

1.2.1.4. Illness

People affected with some illness, can present mood changes and reduced physical capacity in a way that possibly leads to depression. Depression must occur after the onset of the disease to be considered a direct result of medical condition. In addition, a plausible mechanism connecting depression and the medical condition should exist (118).

Certain illnesses like Parkinson's disease strikingly increase the chance of developing depression however, little is known about its pathological mechanisms (119). Cancer patients have psychiatric comorbidities and the stage of cancer is associated with depression or anxiety. In fact, a study in a Tertiary General Hospital in Singapore refers a higher prevalence of depression in those with terminal cancer. That could be related to pain and physical disability (120) although some studies have reveal a possible association between cancer stage and depression (121) (122). Yoshida et al explain that blood glucose is a strong regulator for mood states in patients with diabetes. These

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ICBAS - UP patients have a poor glycemic control that can induce negative feelings (123). A Portuguese study of multiple sclerosis patients also supports a close linkage between depression and physical disability (124). In addition, it has been estimated that people with type 2 diabetes mellitus are twice as likely compared to the general population to suffer from depression (125).

The high prevalence of depression in patients with inflammatory diseases is a strong evidence of the effect of cytokines in depression. Patients receiving chronic therapy with IFN-α have high prevalence of depressive symptoms (20-50%) (126) (127). Likewise, neuropsychiatric events such as mood disorders and major depressive episodes have a frequent occurrence in patients with SLE (128). Increased SLE disease activity appears to be associated with an increased vulnerability for depressive disorders (35). Accordingly, a Chinese study with a total of 285 SLE patients, reported that 34.4% of SLE patients had current suicidal ideation (129).

It was described that SLE patients with brain lesions in areas of the limbic system, presented an impairment in emotional and behavioral functions (34). Neuroanatomical changes may also play an important role in depression symptoms. Indeed, depressed individuals have a lower cerebral blood flow compared with age-matched healthy control subjects (130). This was also observed in depressive SLE patients (118). Moreover, the loss of cerebral and cerebellum volume is a clinical feature of NPSLE in children and adolescents (131).

Depressive symptoms can be associated with physical limitations or with patient’s response to the burden of disease (34). Some authors consider depression an example of the silent burden of the disease (32). The multiple factors that contribute to increase the overall vulnerability for depression is summarized in figure 9.

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Figure 9 – Diagram of the etiology of depressive symptoms. (Adapted: Sperner-Unterweger B, Kohl C, Fuchs D. 2014. Immune changes and neurotransmitters: possible interactions in depression? Prog Neuropsychopharmacol Biol Psychiatry 48: 268-76)

1.2.2. Types of depression

It is important to taken into account the division of depressive disorders in their subtypes in order to the most appropriate treatment. There are different forms of depressive disorders; major depressive and dysthymic disorder are among the most common. The following division is based on the Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM–V):

• Mood changes are characterized by serious explosions with an abnormal intensity or duration patterns, manifested both verbally and behaviorally;

• Major depression when depressive symptoms persist for at least two weeks;

• Dysthymia is diagnosed when manifestation are prevail for two or more years;

• In premenstrual dysphoric disorder, the symptoms occur in the week before the onset of menses and improves days after the beginning of the same;

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• Substance/medication induced depression is a prominent and persistent disturbance of mood that prevails in the clinical perspective. Alcohol, cocaine, anxiolytics or amphetamine stimulants are examples of substances implicated in this disorder;

• Depressive disorder associated with another medical condition requires confirmation from a physical examination or laboratory findings, to prove the disturbance is a direct result of another illness;

• Specific depressive disorder is a diagnosis applied to cases where the symptoms do not meet the criteria mentioned in the above disorders;

• Non-specific depressive disorder is diagnosed in situations where the physician chooses not to specify any cause (45) (132).

1.2.3. Treatment

There are several well documented treatments for depression. These include many drugs, psychotherapeutic intervention and electroconvulsive therapy; the first two are the most particularly used (133).

1.2.3.1. Psychotherapeutic treatment

The reasons regarding the choice of psychotherapy instead of pharmacotherapy for the treatment of depression are not well understood. Nevertheless, the choice of a psychotherapy reduces the probability of treatment failure, regardless of the number of sessions. There are some types of psychotherapeutic interventions, classified according to their theoretical basis:

• Cognitive behavioural therapy (CBT);

• Interpersonal psychotherapy (IPT);

• Counselling.

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CBT is the most studied therapy for the treatment of depression. The goal is to correct the behavioral functions, showing to patients that their thinking affects their mood. It is based on the understanding that thinking negatively is a habit that keeps depressive symptoms (134). Chen H. et al demonstrated that CBT effectively reduce the symptoms of depression in a group of women with an initial episode of moderate depression. Furthermore, this therapy has also shown efficacy in the treatment of sleep disorders (135).

The IPT is based in social roles and life events. This treatment is based on the fact that depression and interpersonal problems are interrelated. Actually, both CBT and IPT have been found to be efficacious for treating depression (136).

Such interventions are particularly useful in slightly adverse situations. Indeed, the role of psychotherapy in the treatment of depression has been subject to some doubts. With the emergence of new drugs, the proportion of people subject to psychotherapy has been declining (137).

Another study indicates that less intensive interventions improve clinical outcomes of antidepressant treatment. Counseling, in the form of telephone contact by nurses, also increased patient satisfaction. This study included patients who simultaneously started a drug treatment and the referred emotional support. The latter has been focused on the realization of about twelve to fourteen calls, approximately ten minutes each for four months. In these phone calls questions about taking the antidepressant were emphasized, as well as the identification of activities that could make a person more active and that allowed them to find pleasure in their execution (138).

1.2.3.2. Pharmacotherapeutic treatment

A large number of studies show that antidepressants are effective and are the mainstay of treatment for serious and chronic depression. Treatment goal should be the complete remission of symptoms and not just partial improvement. Thus, for about one third of patients the first choice treatment is indifferent; that is due to the response and/or toleration to medication. Indeed, recent studies indicate that only 30-40% of patients

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ICBAS - UP respond optimally to one pharmacotherapeutic treatment. The remaining 60-70% of patients do not have the same favorable response (139).

With scientific development these drugs are increasingly effective in control and treatment of depression. Particularly, drugs interfere with the action of neurotransmitters such as serotonin and norepinephrine. Indeed, the older drugs affect these neurotransmitters: tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), selective serotonin-norepinephrine reuptake inhibitors (SNRIs) and also monoamine oxidase inhibitors (MAOIs). All of these are effective by increasing the availability of monoamines (140).

The first generation of antidepressants, where TCAs and MAOIs are included, showed effectiveness, despite some side effects. The second generation of antidepressants, include SSRIs, are selective noradrenaline reuptake inhibitors (NRIs), selective inhibitors of the reuptake of norepinephrine-dopamine (NDRIs) and SNRIs with improved side effects and improved tolerance. However there are no advances regarding the most important target. In fact, one of the oldest TCA (clomipramine) remains among the most effective antidepressants (141) (142). This is mainly due to the existence of a significant period of time between the beginning of the treatment and the manifestation of the beneficial effects (143) (144).

After remission, treatment should be continued for at least six months since the risk of relapse is high if the duration of treatment is shorter. However the level of effectiveness and tolerance of antidepressant varies from person to person (145).

1.2.3.3. Sports as therapy

As an adjunct to conventional therapies, the role of physical exercise has been the subject of several studies. The therapy has demonstrated its effectiveness in reducing the symptoms of depression (146) (147) (148). Actually, some of these studies have reported that exercise can have equivalent efficacy to the use of pharmacological treatments (149) (150) (151) or psychological interventions (152). Stanton et al described that aerobic exercise performed three or four times per week showed a positive effect in the treatment of depression. It was execute in moderate programs of 30-40 minutes, carried for a minimum period of nine weeks. Mainly, the exercise program that is effective

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ICBAS - UP for the treatment of depression is not substantially different the one recommended for the general population (153).

AIMS

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Aims

SLE is not only a physically impairing chronic disease, but also emotionally draining. SLE is also associated with central nervous system involvement, leading to a wide spectrum of neurological symptoms and varied psychiatric manifestations. The disease can have a severe impact on individual quality of life and present serious obstacles to life goals achievement in young individuals. Recognition of depression in SLE patients is of extreme importance since it is treatable, and can lead to fatal consequences if unrecognised. Further exploration of genetic susceptibility factors for depression in SLE will help to improve patient management and treatment, as well as in prevention strategies.

This study was conducted to:

• Determine if serotonergic system molecules are implicated in the development of depressive symptoms in SLE patients. To this end the polymorphisms rs6313 and rs6314 of the 5-HTR2A gene, and the 5’ VNTR of the 5HHT gene were studied;

• To evaluate the putative association of the polymorphisms studied with SLE susceptibility.

MATERIALS AND METHODS

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3.1. Materials

3.1.1. Study population

This study included 96 patients (91 females and 5 males, age= 39.7±14.6 years) with SLE from the outpatient clinic of the Clinical Immunology Unit of Hospital de Santo António – Centro Hospitalar do Porto. The control population was composed of 274 healthy individuals without physical and psychological diseases, from the same geografical region.

The diagnosis of SLE was made according to the criteria established by the American College of Reumathology (ACR). Patients were referred to neurology clinic where were diagnosed with or without involvement of the central nervous system (NPSLE and without NPSLE) using the ACR criteria (1999) and the doctor's clinical experience.

Surveys such as the Short Form 36 (SF-36) and the Hospital Anxiety and Depression Scale (HADS) were also applied. The first is a general health-based survey of quality of life. The SF-36 includes one multi-item scale that explores eight health concepts: 1) limitations in physical activities because of health problems; 2) limitations in social activities because of physical or emotional problems; 3) limitations in usual role activities because of physical health problems; 4) bodily pain; 5) general mental health (psychological distress and well-being); 6) limitations in usual role activities because of emotional problems; 7) vitality (energy and fatigue); and 8) general health perceptions. SF-36 was composed by 36 items with several responses choices. Each item is scored on a 0 to 100 range and a high score defines a more favorable health state (154). The HADS is used as a screening the existence of elevated levels of anxiety and depressed mood, allowing an evaluation of the emotional state of the patient. This inquiry is made up of a set of seven questions with four possible answers, scored from 0 to 3. The final scores are between 0 and 21 points. With score of 0 to 7 could be considered in the normal range, a score of 8 to 10 being just suggestive of the presence of depressive symptoms and 11 or higher indicating probable presence of the respective state (155).

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After diagnosis, patients were divided into two groups: • SLE patients with depression (HADS ≥ 8) and without depression (HADS < 8); • SLE patients with CNS involvement (Neurolupus) and no CNS involvement (without Neurolupus).

3.2. Methods

3.2.1. Biological samples

Blood, particularly human leukocytes, is usually used from DNA extraction, as provides fresh material for analysis (156). DNA was extracted from peripheral blood, collected in 4.5 mL in 5 mL collection tubes with 5% EDTA.

3.2.1.1. DNA extraction

For DNA extraction the salting out procedure was used. This method involves salting out of the cellular proteins by dehydration and precipitation with saturated NaCl solution. The centrifugation of each EDTA blood collection tubes occurred at 490 g, for 10 minutes, in the centrifuge Rotofix 32A (Hettich Zentifugen). After centrifugation three layers were obtained: plasma, leukocytes ring and erythrocytes. The leukocytes ring was removed with a sterile and disposable Pasteur pipette to a 50 mL Falcon tube. Lymphocytes were separated from whole blood by the lysis of red blood cells through addition up to 50 mL of RCLB (Red Cell Lysis Buffer) to Falcon tube. The RCLB (1 mM tris- HCl pH 7.2, 5 M NaCl and 1 M MgCl2.6H2O) was mixed by inverting thoroughly for 5 minutes and centrifuged (Heraeus Megafuge 16R –Thermo Scientific) at 828 g, at 4°C for 10 minutes. The supernatant was discarded, the pellet was resuspended and RCLB was added and centrifuged at the same conditions. Addition of RCLB and centrifugation steps was repeated two to three times until a clean white pellet of leucocytes was obtained. After the final wash, the supernatant was discarded completely and 3.5 mL of TE2 (10 mM Tris- HCl pH 7.2, 0.4 M NaCl and 2 mM EDTA) were added; 200 µL of 10% SDS (Sodium Dodecyl Sulfate) and 10 µL of proteinase K (50 mg/mL) were added to promote the lysis of leukocyte membranes and digest the proteins bound to DNA, respectively. The sample was incubated for 12 hours at 42ºC in a water bath. Thereafter the solution was

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3.2.1.2. DNA quantification

DNA was quantified by measuring the absorbance at 260 nm in a NanoDrop ND -1000 spectrophotometer v3.6.0 (Thermo Scientific). To evaluate the presence of potential contaminants measurement at wavelength 280 nm (proteins) and 230 nm (organic salts) was undertaken. This measurement is important since the absorbance ratios 260/280 and 260/230, which should be between 1.8 to 2.0 and 1.5 to 2.0 respectively provide information about the purity of DNA.

3.2.2. Genetic studies

3.2.2.1. Genetic association studies

In order to identify candidate genes, these studies test for an association between disease status and genetic variations. The test makes a comparison between the patients with the disease and the control population without the disease. A higher frequency of an allele or genotype in individuals affected with a disease suggests that the tested variant increases the risk of the specific disease. Likewise, a lower frequency in patients indicates a possible protective role in the disease.

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3.2.2.2. Genetic variations

Polymorphisms are variations in the DNA sequence. The SNP represents a difference in a single DNA nucleotide and is the most common type of genetic variation among people. While not as common as SNPs, VNTRs and insertion/deletion polymorphism are widely spread across the genome. VNTRs are linear arrangement of multiple copies of short repeated DNA sequences that vary in length and are highly polymorphic. An insertion/deletion polymorphism is a type of genetic variation in which a specific nucleotide sequence is present (insertion) or absent (deletion).

This project focused in polymorphisms that may affect the function and/or expression of proteins relevant to the serotonergic system.

3.2.3. Applied techniques

3.2.3.1. PCR

For the genotyping of different polymorphisms, techniques based on the polymerase chain reaction (PCR) were used.

In 1993 the Nobel Prize for Chemistry Kary Mullis developed the PCR, a methodology that consists in the exponential amplification of a specific DNA sequence from a reduced amount. Based on the replication of cells in vivo, the principle of PCR involves the enzymatic amplification of a template DNA fragment delimited by two primers. This technique needs several components:

• DNA template containing the sequence to be amplified; • Primers, oligonucleotides that define the region to be amplified; • dNTP 's (deoxynucleotide triphosphates) for the construction of the new chain of DNA; • DNA polymerase, the enzyme that catalyzes the reaction addition of dNTP 's; • The Mg 2 + ion, the enzyme cofactor; • Buffer solution that maintains the appropriate conditions for the activity of the enzyme.

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The PCR technique comprises three steps: denaturation of DNA, binding (annealing) of the primers and extension of the DNA chain. These steps together are referred to as a cycle and are repeated a number (30-40 usually) of times (figure 10).

Figure 10 – The PCR steps. (Adapted: http://microfluidics.stanford.edu/Projects/Current/OnChipPCR.html. Accessed: 23-01 - 2015)

In denaturation, the higher temperature (92-95°C) separates the double- stranded template DNA. Binding of the primers to the complementary DNA sequences then occur. The required temperature is between 50°C and 65°C depending on the primer’s sequence and length. Finally, the temperature is rise to 72°C and the DNA polymerase enzyme synthesizes the new chain. The DNA polymerase used is Taq polymerase, isolated from the bacterium Thermus aquaticus (Taq), whose main feature is stability at higher temperatures. PCR is a highly versatile technique and has been modified for various applications (157).

This method was used to genotype the STin2 polymorphism. PCR was used to amplify a specific sequences and its size was determined by gel electrophoresis.

The sequence of primers is described in table 1. A Mastermix was prepared containing the components described in table 2. This mixture was homogenized and 9 µL was added to reaction tubes containing 50 ng of DNA (for a final volume of 10 µL). The PCR cycles occurred in a TProfessional Thermocycler (Biometra) (table 3).

Table 1 – Sequence of primers used from STin2 analysis. Gene Polymorphism Primer sequence Forward 5’-GGT CAG TAT CAC AGG CTG CGA CTA G-3’ 5-HTT STin2 Reverse 5’-TGT TCC TAG TCT TAC GCC AGT GAA G-3’

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Table 2 – Components and their respective concentrations for PCR used from STin2 analysis.

Stock Reaction concentration concentration

MgCl 2 (Bioline) 50 mM 1,50 mM Reaction Buffer (Bioline) 10 x 1 x dNTPs mix (Bioron) 25 mM 0,5 mM Forward primer 10 µM 0,50 µM Reverse primer 10 µM 0,50 µM GoTaq G2 Flexi DNA Polymerase 5 U/µL 0,04 U/µL (Promega, Madison, WI USA)

ddH2O (Braun) -

Table 3 – Thermic profile used from STin2 analysis. T (ºC) Time Cycles numbers 95 3 minutes 1 94 10 seconds 63 1 minute 35 72 1 minute 72 7 minutes 1 4

DNA fragments were separated in an agarose gel 4% (w/v) electrophoresis gel. Previously, 1 µL of Loading Buffer was added to samples. The DNA is negatively charged due to the presence of phosphate groups when an electrical field is applied, migration occurs from the negative to the positive pole. Smaller molecules migrate faster than the higher weight molecules, thus the fragments are separated according to their fragment size. The fragments are visible in ultraviolet light after staining with ethidium bromide (figure 11).

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Figure 11 – Genotypes of STin2 obtained by PCR. STin2.12 allele corresponds to 12 repeat units of 17bp; STin2.10 allele corresponds to 10 repeat units of 17bp ; STin2.9 allele corresponds to 9 repeat units of 17bp.

3.2.3.2. Real-Time PCR

Introduced in 1992 by Higuchi and coworkers the real-time PCR is a modification of conventional PCR [166]. Real-time PCR allows precise quantification of specific nucleic acids even when the starting amount of material has a very low concentration. This is accomplished by monitoring the amplification of a short DNA fragment containing the polymorphism site and detection, in real time, using fluorescent technology. Fluorescence can be detected after amplification using an intercalating dye (ethidium bromide) or during amplification using sequence specific fluorescent probes such as Taqman probes.

3.2.3.3. TaqMan technology

The exonuclease activity of Taq polymerase enzyme is employed to increase the specificity of PCR detection. The technique is specific, quantifiable and helpful for assaying large numbers of samples (158). A TaqMan probe is a oligonucleotide sequence that binds to a specific sequence where a polymorphism is included, allowing detection and quantification of fluorescence. For genotyping two TaqMan probes that differ only in sequence at the SNP site are used. One of the probes is complementary to the wild-type allele and the other for the alternative allele. Also, they exhibit different fluorescence colors to enable distinction. These probes have attached two types of fluorophores the reporter (at the 5’ end) and the quencher (at the 3’ end). The reporter emits fluorescence and the quencher reduces the fluorescence of the first when nearly.

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ICBAS – UP In the PCR annealing step the TaqMan probes hybridize to the targeted SNP site. During the extension the DNA polymerase removes nucleotides from the probe, from 5’ to 3’, and release the reporter and quencher dyes. When the probes are intact, fluorescence is suppressed because the quencher dyes are in the proximity of the reporter dyes. Separating the reporter from the quencher allows the first to emit fluorescence, making possible its detection. This detection is only possible when the probes are perfectly hybridized, since DNA polymerase will not recognize a probe containing a mismatched base. The fluorescence is detected by PCR equipment with an integrated fluorometer. During the experiment, the fluorescent signal for the two reporter dyes is measured (figure 12).

Figure 12 – RT-PCR steps with Taqman® probes for genotyping. ( Adapted: http://www.applied-maths.com/applications/taqman-based-snp-genotyping. Accessed: 28- 01-2015)

This method was applied to study the SNP rs6314 (1354 C>T) in the gene encoding the 2A receptor of serotonin. Each assay consisted of a total of 8 µL containing 12.5 ng of DNA, sterile double-distilled water Braun (Braun), 1x from TaqMan® Gene SNP Assay kit (C_11696920_20 TaqMan® Gene SNP Assay kit from Applied Biosystems, Foster City, CA, USA) and 1x from TaqMan® Universal PCR MasterMix (Applied Biosystems, Foster City, CA, USA). The TaqMan® Gene SNP Assay contain the probes and primers and the TaqMan® Universal PCR MasterMix contain DNA polymerase, dNTPs, magnesium and buffer solution (table 4). In each protocol three samples with known genotype as a comparative term and a negative control (sterile double-distilled

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Table 4 – Components and their respective concentrations for PCR used from rs6314 analysis.

Stock Reaction concentration concentration

TaqMan® Universal PCR MasterMix 2 x 1 x

TaqMan® Gene SNP Assay 40 x 1 x DNA 12.5 ng/µL 12.5 ng/8µL

ddH2O (Braun) -

Table 5 – Thermic profile used from rs6314 analysis. T (ºC) Time Cycle numbers 95 10 minutes 1 93 15 seconds 58 40 seconds 40 72 20 seconds

3.2.3.4. PCR-RFLP

RFLP (Restriction Fragment Length Polymorphism) is a technique based on detection of fragments of different lengths after digestion of the DNA samples with specific restriction endonucleases. The enzymes require 4-6 base pair recognition sequences and when total DNA is digested a large number of fragments generally result (159). In order to study the restriction pattern of DNA, the fragments need to be separated according to size and the fragments of interest distinguished from all other similarly sized fragments.

In PCR-RFLP, digested PCR product is separated by gel electrophoresis (agarose or polyacrylamide), and individual fragments are identified (figure 13).

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Figure 13 – Procedure of PCR-RFLP technique. (Adapted: http://answermeup.com/questions/perform-rflp-pcr.html. Accessed: 27.05.2015 )

This method was applied to study the SNP rs6313 (102 T>C) in the gene encoding the 2A receptor of serotonin. PCR amplified the specific sequences and their size determined by gel electrophoresis, after MSPI digestion.

The sequence of primers was described in table 6. The Mastermix was prepared for a total reaction volume of 20 µL containing the components described in table 7. The mixture was homogenized and 19 µL were added to reaction tubes with 50 ng of DNA. The PCR program was carried out in GeneAmp® PCR System 2700 (Applied Biosystems) (table 8).

Table 6 – Sequence of primers used from rs6313 analysis.

Gene Polymorphism Primer sequence Forward 5’-TGA TGA CAC CAG GCT CTA CAG T-3’ 5-HTT rs6313 Reverse 5’-CAG GAA AGG TTG GTT CGA TT-3’

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Table 7 – Components and their respective concentrations for PCR used from rs6313 analysis.

Stock Reaction concentration concentration

MgCl 2 (Bioline) 50 mM 2,00 mM

Reaction Buffer (Bioline) 10 x 1 x dNTPs mix (Bioron) 25 mM 0,2 µM Forward primer 10 µM 1,00 µM Reverse primer 10 µM 1,00 µM GoTaq G2 Flexi DNA Polymerase 5 U/µL 1 U/20µL (Promega, Madison, WI USA)

ddH2O (Braun) -

Table 8 – Thermic profile used from rs6313 analysis.

T (ºC) Time Cycles numbers 94 3 minutes 1 94 30 seconds 58 30 seconds 35 72 1 minute 72 3 minutes 1

After amplification of the sequence containing the polymorphism, the samples were subjected to digestion with the Thermo Scientific FastDigest MspI restriction enzyme (table 9). The Mix was prepared for a total of 21 µL (table 10) and was added to reaction tubes with 10 µL of PCR product.

Table 9 – MspI restriction enzyme cleavage site used from rs6313 analysis.

5’…C CGG…3’ MspI restriction enzyme 3’…GGC C…5’

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Table 10 – Components and their respective concentrations for digestion protocol used from rs6313 analysis.

Reaction Stock concentration concentration Tango Buffer (Thermo Scientific) 10x 1x MSP I enzyme (Thermo Scientific) 10 U/µL 10 U/µL

H2O -

After overnight incubation at 37ºC the DNA fragments were separated by an electrophoresis gel. Previously was added 1 µL of Loading Buffer to samples and the migration occurred in 3% (p/v) NuSieve® agarose gel. After separation, the DNA molecules were visualized under ultraviolet light (figure 14).

Figure 14 – Genotypes of rs6313 obtained from

PCR-RFLP with the MSPI restriction enzyme.

3.2.4. Statistical Analysis

Allele frequencies were compared using the chi-square test or the Fishers exact test, as appropriate. For genotype frequencies p values and Odds Ratio (OR) were calculated using binary logistic regression. A student’s t-test was used when comparing continuous data.

A multivariable-logistic regression was performed (with the most frequent homozygote as the reference category), in order to evaluate the association between the

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The level of significance for comparisons was p<0.05. All analyses were carried out with PASW Statistics 20 software.

RESULTS

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4.1. Studied polymorphisms in SLE patients

Allele and genotype frequencies from the polymorphisms of the studied genes were compared between SLE patients (n= 96) and the control population (n= 274). No statistically significant differences were observed in the frequencies of the studied polymorphisms between these two groups (tables 11 and 12).

Table 11 – Allelic frequencies in the control population (CP) and SLE patients.

Allele CP (n=548) SLE (n=192) OR (CI 95%) P % (n) % (n)

rs6313 C 52.0 (285/548) 52.3 (91/174) 1.01 (0.72-1.42) 0.947 T 48.0 (263/548) 47.7 (83/174)

rs6314 C 91.9 (489/532) 94.8 (182/192) 1.60 (0.79-3.25) 0.190 T 8.1 (43/532) 5.2 (10/192)

Stin2 12-rpt 68.2 (304/446) 62.2 (117/188) 0.77 (0.54-1.10) 0.149 10-rpt 31.8 (142/446) 37.8 (71/188)

Table 12 – Genotype frequencies in the control population (CP) and SLE patients.

Genotype CP (n=274) SLE (n=96) OR (CI 95%) p % (n) % (n)

CC 29.9 (82/274) 23.0 (20/87) 1 0.064 rs6313 CT 44.2 (121/274) 58.6 (51/87) 1.73 (0.96-3.11) 0.068 TT 25.9 (71/274) 18.4 (16/87) 0.92 (0.44-1.92) 0.832

CC 85.0 (226/266) 89.6 (86/96) 1 0.664 rs6314 CT 13.9 (37/266) 10.4 (10/96) 0.71 (0.34-1.49) 0.366 TT 1.1 (3/266) 0 (0/96) - 0.999

12-rpt/10-rpt 42.1 (94/223) 54.3 (51/94) 1 0.119 Stin2 12-rpt/12-rpt 47.1 (105/223) 35.1 (33/94) 0.58 (0.34-0.97) 0.039* 10-rpt/10-rpt 10.8 (24/223) 10.6 (10/94) 0.77 (0.34-1.73) 0.524 * loses significance with correction for the number of alleles.

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4.2. Patients characteristics

Patient clinical and demographic characteristics are given in table 13. The patient population has a mean age of 42.5 years and is predominantly composed of female individuals (95.8%). Age and education level were significantly associated with depression (p=0.002 and p=0.006, respectively). The diagnosis of depression (HADS ≥8) was more prevalent among older adults (mean age of 47.5 years). Likewise, depressive symptoms are more prevalent in patients with lower education levels (55.3%). The majority of SLE patients have education levels equal or greater than 9 years.

Table 13 – Demographic characteristics of SLE patients.

Total SLE p HADS ≥8 (n=38) HADS<8 (n=58) (n=96) Gender

Female, n (%) 92 (95.8) 37 (97.4) 55 (94.8) 1.000 a Male, n (%) 4 (4.2) 1 (2.6) 3 (5.2)

Age (mean ± standard deviation) 42.5 ± 13.3 47.5 ± 13.6 39.2 ± 12.2 0.002 b

Education level

< 9 years 37 (38.5) 21 (55.3) 16 (27.6) 0.006 c ≥ 9 years 59 (61.5) 17 (44.7) 42 (72.4) NPSLE

Yes 36 (37.5) 15 (39.5) 21 (36.2) 0.746 c No 63 (62.5) 23 (60.5) 37 (63.8) a Fisher's Exact Test b T student Test c Pearson Chi-Square Test

It is worthy of note that the prevalence of depression is similar in patients with and without NPSLE, 39.5% and 36.2% respectively.

4.3. Studied polymorphisms and depression

In order to understand if the rs6313, rs6314 and Stin2 polymorphisms are involved in the development of depression in SLE patients, the allelic and genotypic frequencies were analyzed in a cohort of 96 patients subdivided in two categories using the depression scale (table 14 and 15).

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The rs6313T allele was more frequent among depressive SLE patients compared to non-depressive SLE patients (57.1 vs. 41.3, p=0.041, OR [95%CI]=1.891 [1.02–3.49]). The frequency of the rs6313TT genotype was significantly higher in depressive SLE patients when compared to SLE patients without diagnosed depression (31.4 vs. 9.60, p=0.027, OR [95%CI]=5.391 [1.22–23.82]).

Table 14 – Allele frequencies in SLE patients with depression (HADS ≥8) and without depression (HADS<8).

HADS≥8 (n=76) HADS<8 (n=116) Allele OR (CI 95%) P % (n) % (n)

C 42.9 (30/70) 58.7 (61/104) rs6313 1.891 (1.02-3.49) 0.041 T 57.1 (40/70) 41.3 (43/104)

C 94.7 (72/76) 94.8 (110/116) rs6314 0.982 (0.27-3.60) 1.000 T 5.3 (4/76) 5.2 (6/116)

10-rpt 34.7 (25/72) 39.7 (46/116) Stin2 0.809 (0.44-1.49) 0.809 12-rpt 65.3 (47/72) 60.3 (70/116)

Table 15 – Genotype frequencies in SLE patients with depression (HADS ≥8) and without depression (HADS<8).

HADS≥8 (n=38) HADS<8 (n=58) OR (CI 95%) P % (n) % (n) Genotype

CC 17.1 (6/35) 26.9 (14/52) 1 0.045 rs6313 CT 51.4 (18/35) 63.5 (33/52) 1.27 (0.42-3.88) 0.672 TT 31.4 (11/35) 9.60 (5/52) 5.13 (1.23-21.3) 0.025

CC 89.5 (34/38) 89.7 (52/58) rs6314 1.02 (0.27-3.88) 1.000 CT 10.5 (4/38) 10.3 (6/58)

12-rpt/10-rpt 47.2 (17/36) 58.6 (34/58) 1 0.535 Stin2 12-rpt/12-rpt 41.7 (15/36) 31.0 (18/58) 1.67 (0.68-4.10) 0.265 10-rpt/10-rpt 11.1 (4/36) 10.3 (6/58) 1.33 (0.33-5.37) 0.686

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As the age and education level presented significant differences in depressed patients (table 13) it was important to take into account these demographic characteristics. Logistic regression modelling confirmed the reported association independently from age and education level (p = 0.027, OR [95%CI] = 5.391 [1.22 - 23.82]).

4.4. Studied polymorphisms and NPSLE

The allelic and genotypic frequencies of the studied polymorphisms were also compared between patients with (n=36) and without (n=60) neuropsychiatric symptoms (NPSLE). The frequencies of the studied polymorphisms were similar in the two groups (table 16 and 17).

Table 16 – Allelic frequencies in SLE patients with and without NPSLE

NPSLE (n=72) Non-NPSLE (n=120) Allele OR (CI 95%) P % (n) % (n)

C 57.8 (37/64) 49.1 (54/110) rs6313 0.704 (0.38-1.31) 0.267 T 42.2 (27/64) 50.9 (56/110)

C 95.8 (69/72) 94.2 (113/120) rs6314 1.42 (0.36-5.69) 0.746 T 4.2 (3/72) 5.8 (7/120)

10-rpt 34.3 (24/70) 39.8 (47/118) Stin2 0.788 (0.43-1.46) 0.448 12-rpt 65.7 (46/70) 60.2 (71/118)

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Table 17 – Genotype frequencies in SLE patients with NPSLE and with non-NPSLE.

NPSLE (n=36) Non-NPSLE (n=60) OR (CI 95%) P % (n) % (n) Genotype

CC 31.3 (10/32) 18.2 (10/55) 1 0.380 rs6313 CT 53.1 (17/32) 61.8 (34/55) 0.50 (0.17-1.43) 0.197 TT 15.6 (5/32) 20.0 (11/55) 0.45 (0.11-1.79) 0.260

CC 91.7 (33/36) 88.3 (53/60) rs6314 0.69 (0.17-2.85) 0.739 CT 8.3 (3/36) 11.7 (7/60)

12-rpt/10-rpt 62.9 (22/35) 49.2 (29/59) 1 0.200 Stin2 12-rpt/12-rpt 34.3 (12/35) 35.6 (21/59) 0.75 (0.31-1.85) 0.537 10-rpt/10-rpt 2.8 (1/35) 15.3 (9/59) 0.15 (0.02-1.24) 0.078

DISCUSSION AND CONCLUSIONS

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5.1. Discussion

5.1.1. Role of studied polymorphisms in SLE patients

A possible relationship between serotonergic pathways and SLE has been suggested. Autoimmune diseases, including SLE, may result from disruption of homeostasis in neurological, immune and endocrine systems. Hereupon some authors have claimed that the serotonergic system plays an important role in this network of neurotransmitters, cytokines and endocrine glands hormones (160). Meyerhoff et al have observed significantly lower serotonin levels in 41 SLE patients, and the lowest levels occurred in patients with active disease (161). In addition, another study found overexpression of serotonin receptor type 3A (5-HTR3A) in peripheral blood cells of SLE patients compared to healthy individuals (160). Several polymorphisms such as rs6313, rs6314 and STin2 may influence the function of the serotonin receptor and transporter. In fact, there are some studies that report association between polymorphisms in serotonergic system and SLE: Birmingham et al reported that the 1019C>G polymorphism in the 5-HTR1A gene is related to fluctuation in self-perceived stress, which in turn leads to the onset of flare in SLE patients (162). A previous small-scale study has reported an association between the 5-HTR2A rs6311A allele and SLE development in a Portuguese population (163). To the best of our knowledge, no previous documented studies exist assessing the rs6313, rs6314 and STin2 serotonergic system polymorphisms in SLE.

Our results do not show an influence of these polymorphisms in SLE susceptibility, however it is possible that the homeostasis of the serotonergic system could be influenced by other polymorphisms in the same pathway. Additional research is needed, including, for instance, of the 5-HTTLPR polymorphism. Studies in larger populations are also needed.

It is known that SLE patients have a greater vulnerability or predisposition to depressive symptoms (164). In fact, it has been reported that depression affects around 17-75% of SLE patients (34). The notion that depression represents a manifestation of SLE is controversial. Nevertheless, it has been concluded that depression is more common among SLE patients that in a similar group of rheumatoid arthritis patients (165).

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Tissue inflammation in SLE might be the link to serotonergic abnormalities in depression. Also, in addition to the general effects of chronic disease, it has been reported that depressed SLE patients have greater cognitive deficits (learning and attentional skills) when compared to depressed controls and healthy control subjects (164). However, the etiology of depression in SLE remains controversial. There is some discrepancy in the literature regarding the association between psychological characteristics and SLE. Some studies have shown a relationship between cognitive and psychological status and depression in non-NPSLE. Nevertheless no significant differences were found in global cognitive impairment between non-NPSLE patients and healthy controls (164) (166). Thus, it is still not known if depression is associated with the effects of a chronic illness or if it represents a manifestation of CNS involvement in these patients.

Depression in our SLE cohort is more prevalent in women (n=37; 40%)) and older adults (47.5 ± 13.6), in agreement with previous reports. Patients with lower education levels (n=21; 57%) are also more prone to depression. A recent systematic review, reported that depression was the most common psychiatric comorbidity (18-75%) in SLE patients, particularly in females (87 –100%) (167). It is known that depression is also more prevalent in women with no underlying disease (45) (46) (168). Some authors suggest that the higher prevalence of depression in women can be due to biological reasons such as hormonal factors at the onset of puberty (47), and/or social factors such as poor social relationships (168). An Iranian study found that 45.5% of depressive SLE patients had more than 35 years of age (169). This suggests a shift in depression patterns with aging. It is likely that older adults may suffer from aggravating psychosocial conditions such as isolation, marital separation, grief and economic impoverishment. Suicide in older adults is also more likely to be preceded by depression compared to suicide at any other age (170). Education levels, as well as socioeconomic factors, have a strong linkage to depression. This may be due to the relation between lower education level and low socioeconomic status. This means that living conditions, environmental stressors and perhaps even impaired cognitive functioning are important in the onset of depression (168).

It is important to note that factors leading to depression in SLE patients include the social consequences and patients’ responses to the burden of the disease. An American study compared the quality of life in SLE patients with the quality of life in other chronic illnesses (hypertension, congestive heart failure, adult onset diabetes mellitus, myocardial

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5.1.2. Role of studied polymorphisms in depression

Depression is an important public health problem due to its devastating consequences. It is associated with an increased risk of suicide and decreased physical, cognitive and social functioning, with greater self-neglect, leading to an increased risk of mortality. Therefore recognition of depression, a treatable condition, is of utmost importance.

Published research provides examples of polymorphisms that may contribute to the risk of depression. Our results showed that the rs6313T allele of the 5-HTR2A gene was associated with depression susceptibility in SLE patients. Likewise, the TT genotype also shows association with depression in our patients. This association was already reported in a smaller SLE Portuguese population (172). Located on exon 1 of the 5- HTR2A gene, the rs6313 polymorphism probably interferes with receptor activity. Khait et al reported that the TT genotype is associated with a higher 5-HTR2A density in platelet in both mood disorders and in the healthy population (173). Platelets are a source of 5- HTR2A with similar characteristics to the brain receptors. This greater 5-HTR2A platelet binding was proposed to be associated with impairment of serotonin neurotransmission, and a likely cause of depressive symptoms (173). The rs6313 polymorphism is in complete linkage disequilibrium with rs6311, located in the promoter region of the 5- HTR2A gene. There is evidence that the rs6311 polymorphism results in inadequate promoter activity, with lower transcription of the gene (174). Thus, the rs6311 may also play an important role in this setting.

Our results are not in agreement with those reported for different populations. In general, the rs6313C allele has been associated with depression (85) (87) (175) (176). This allele has also been associated with suicide in depressed patients (175) and a more frequent CC genotype was found in alcohol-dependent females (87).

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Contradictory results are a very common phenomena in studies of complex diseases, that arise from the interaction of several genes with additional environmental factors. One reason is the differences observed in allele prevalence, characteristic of the studied population. Allele frequencies in our study are very different from the frequencies previously reported for depressed patients in other populations (table 18).

Table 18 – Allelic frequencies of rs6313 in depressed patients. C allele frequency T allele frequency Study Year Patients Nationality (%) (%) This study 2015 Portuguese 43 57 Petit A. -C. et al 2014 French 56 44 Wrzosek M. et al 2011 Polish 68 32 Arias B. et al 2001 Spanish 53 47 Zhang H. -Y. et al 1997 Japanese 59 41

Discrepancies between studies may also be due to differences in phenotype definition (study heterogeneity). Unlike in our study, the other studies have been conducted in subjects with depression and psychological disorders with no other underlying medical conditions. In SLE patient’s depression may develop by different mechanisms. An interesting study, reported that the rs6313T allele and TT genotype were associated with increased depressive symptoms in subjects who live in remote rural areas. Surprisingly, the CC genotype was associated with increased depressive symptoms in urban residents. These results suggest that different mechanisms may be involved in the development of depression depending on the nature of the environmental influences (177). Following this line of reasoning, SLE may function as a life experience that leads to a higher vulnerability to depression in the rs6313T allele carriers.

Other studies, as well as a recent meta-analysis, found no significant involvement of the rs6313 polymorphism in depression (178) (179) (180). Both original studies had a large number of patients, 325 and 420, and a total of 21 studies were included in the meta-analysis. Although our results are based in a lower number of patients, they can be considered an interesting exploratory finding that needs replication in other SLE populations.

No significant association between rs6314 alleles and depression in SLE patients was found. These results are in agreement with those of a study with 684 elderly Danish twins (181). Another study with 485 European descent individuals identified two that

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The 5-HTT STin2.12 (12 repeat) allele has been associated with increased gene transcription, that may decrease serotonin availability when compared to the STin2.10 allele (91). Some studies have found no association between the STin2 polymorphism and depression in an elderly population (181), or with suicidal ideation in depressive patients (179). However, Pizzo de Castro et al showed that patients with both tobacco use disorder and mood disorders (depression or bipolar disorder) have a significant increased frequency of the STin2.12 allele and a lowered frequency of the STin2.10/10 genotype (96). Another study suggested that the presence of STin2.10 allele and the absence of STin2.12 allele might be related to the characteristic cognitive dysfunction of depressive patients (92). In the present study no association was found between serotonin transporter polymorphisms and depressive symptoms. Our sample size may have had insufficient power to detect the contribution of this gene variation to a complex disease such as depression. Also, it is possible that this putative contribution depends on epistatic interaction between 5-HTTLPR and STin2 defining the transcription level of the 5-HTT gene. Furthermore, some authors have suggested that the role of serotonin transporter polymorphisms in gene function could be more complex than generally assumed (181).

5.1.3. Role of studied polymorphisms in NPSLE

The expression patterns of the 5-HTR2A and 5-HTT in the brain, have been implicated not only in various central physiological functions including memory, sleep, eating and reward behaviors, but also in many neuropsychiatric disorders.

We found no statistically significant associations between the studied polymorphisms and NPSLE. A previous study found no differences between depression frequencies in patients with and without NPSLE (182). We had too few patients to replicate this observation.

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5.2. Conclusions

The neurobiology of depression has been widely studied over the years. The monoamine hypothesis was the first theory to explain the etiology of the disease. Nowadays others theories have emerged and it is useful to further explore the theme. A substantial proportion of genetic risk factors for depression appear to be shared in men and women. Several studies have tried to associate polymorphisms in serotonergic system genes with psychiatric disorders. Also other works have referred the higher prevalence of depressive symptoms in SLE patients. So the identification of polymorphisms in the serotonergic system may facilitate a further step in the comprehension of complex topic that is the etiology of depression. Since these genetic variations appear to be associated with the different responses to drugs, these studies may also have an important role in the development of new therapeutic strategies.

In this study, some results are controversial compared to other findings reported in the literature. This may be due to the complex traits involved, which link depressive patients with an underlying autoimmune disease. No relationship was observed between the studied polymorphisms and SLE. Lupus is a multifaceted polygenic disease what means that it is caused by the combined effect of more than one gene. Consequently, the contribution of a single allele or SNP may be greatly reduced making it difficult to detect their effect. The rs6313T allele and rs6313TT genotype may be associated with depression in SLE patients in the studied population. Thus, our results reinforce the existence of a relationship between serotonergic system and depression.

The exposure to stressful events of life might have a possible epigenetic influence in genes responsible for depression, suggesting an area of interest to be taken into account in future researches. Other areas of study may include the evaluation of the quality of life and the presence of stressful life events and the SLE activity.

Medico-legal aspects are also interested due to the strong physical, psychological and social impairment in subjects with SLE. The increased knowledge of brain functions opens the opportunity to produce evidence in cases in which disorders of the brain such as depression are implicated. Indeed, the mental condition of an accused person is frequently a matter for special consideration in a criminal trial. As well as others depressed individuals, depressed SLE patients are also predisposed to commit suicide.

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Concerns arise related to the prevention of suicide in depressed patients, including hospitalization and the appropriated treatment, are of medico-legal importance.

The present study has some limitations since it is a preliminary study. Accordingly, a larger number of patients should be study to validate the association of the studied polymorphisms with depression development in SLE patients. There are some studies in the literature, reporting the involvement of serotonergic polymorphisms in depressive patients. However, to the best of our knowledge, this is the first SLE population where these polymorphisms were studied.

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