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PROGRAMA DE PÓS-GRADUAÇÃO STRICTO SENSU

MESTRADO EM CIÊNCIAS DA REABILITAÇÃO

MARCOS TADEU PARRON FERNANDES

RELAÇÃO ENTRE OS POLIMORFISMOS DOS GENES IL1, IL6 E TNF E OSTEOARTRITE DE QUADRIL E JOELHO EM

IDOSOS

Londrina

2014


RELAÇÃO ENTRE OS POLIMORFISMOS DOS GENES IL1, IL6 E TNF E OSTEOARTRITE DE QUADRIL E JOELHO EM

IDOSOS

Dissertação apresentada à UNOPAR, como requisito parcial à obtenção do título de Mestre em Ciências da Reabilitação.

Orientadora: Profa. Dra. Regina Célia Poli Frederico

Londrina

2014

AUTORIZO A REPRODUÇÃO TOTAL OU PARCIAL DESTE TRABALHO, POR

QUALQUER MEIO CONVENCIONAL OU ELETRÔNICO, PARA FINS DE ESTUDO E

PESQUISA, DESDE QUE CITADA A FONTE.

Dados Internacionais de catalogação-na-publicação

Universidade Norte do Paraná

Biblioteca Central

Setor de Tratamento da Informação

Fernandes, Marcos Tadeu Parron

F398r Relação entre os polimorfismos dos genes da IL1, IL6 e do TNF e osteoartrite de

quadril e joelho em idosos / Marcos Tadeu Parron Fernandes . Londrina: [s.n], 2014.

96f.

Dissertação (Mestrado). Ciências da Reabilitação. Universidade Norte do

Paraná/Universidade Estadual de Londrina.

Orientadora: Profª Drª. Regina Célia Poli-Frederico

1- Ciências da reabilitação - dissertação de mestrado – UNOPAR/UEL 2- Osteoartrite 3-

Polimorfismo genético 4- Citocinas 5- Idoso 6- Funcionalidade I- Poli-Frederico, Regina

Célia; orient. II- Universidade Norte do Paraná. III- Universidade Estadual de Londrina.

CDU 615.8


Dissertação apresentada à UNOPAR, no Mestrado em Ciências da Reabilitação, área de concentração: Avaliação e Intervenção em Reabilitação, como requisito parcial para a

obtenção do título de Mestre conferido pela Banca Examinadora formada pelos professores:

___________________________________ Profa. Dra. Regina Célia Poli Frederico

UNOPAR

__________________________________ Prof. Dr. Marcus Vinícius Matos Gomes

UNOPAR

____________________________________

Profa. Dra. Fernanda de Freitas Anibal

Universidade Federal de São Carlos-UFSCar

Londrina, 10 de outubro de 2014.

Agradeço a Deus,

por me fazer compreender e aceitar os Seus caminhos.

Dedico, humildemente, este trabalho

aos meus pais Ricardo e Maria, a quem devo o que sou

e cujos princípios de correção e bondade sempre me inspiraram;

a Karen, minha paixão, pelo companheirismo e apoio incondicionais

e por ser a professora que me ensinou o verdadeiro sentido da palavra amor;

a Bianca e Isadora, duas princesas que Deus a mim confiou o dever de educar,

e o privilégio de infinitamente amar.

Amor eterno.

AGRADECIMENTOS

À minha orientadora, Profa. Dra. Regina Célia Poli-Frederico, pelos

ensinamentos, calma, bondade, paciência, educação e por sempre acreditar em

mim. Uma pessoa muito especial. Meu muito obrigado.

Aos professores da Banca Examinadora, Profa. Dra. Fernanda de Freitas

Anibal e Prof. Dr. Marcus Vinícius Matos Gomes, pela disponibilidade e valiosa

contribuição.

À Profa. Dra. Ilce Mara de Syllos Cólus, pela generosidade em me acolher,

abrindo as portas do seu laboratório.

À Srta. Marilésia, pela dedicação e paciência para me ensinar o que,

certamente, não aprenderia facilmente com outra pessoa.

À Srta. Emily, pela disposição e grande ajuda na execução dos experimentos.

Ao João Paulo Manfré dos Santos, pela amizade e ajuda na coleta dos dados.

Aos professores e colegas do programa de pós-graduação, pela ótima

convivência e amizade construídas.

À Universidade Norte do Paraná e à coordenação do Programa de Pós-

Graduação em Ciências da Reabilitação pelo apoio em todas as etapas de execução

deste projeto.

Aos meus amigos do Serviço de Anestesiologia de Londrina e a outros que,

direta ou indiretamente, me ajudaram a cumprir os meus compromissos como aluno

de mestrado.

Ao meu irmão, Parron, o grande amigo que sempre se fez presente em minha

vida.

À minha família, que me apóia e incentiva de maneira irrestrita. A vocês, o

meu amor mais profundo.

Muito obrigado!

“As paixões ensinaram a razão aos homens”.

William Shakespeare

FERNANDES, Marcos Tadeu Parron. Relação entre os polimorfismos dos genes da

IL1, IL6 e do TNF e osteoartrite de quadril e joelho em idosos. 98 fls. Dissertação (Mestrado em Ciências da Reabilitação) – Universidade Norte do Paraná, Londrina, 2014.

RESUMO

A osteoartrite (OA) é a doença mais comum do sistema músculo-esquelético e a forma mais freqüente de artrite. É mais frequente em mulheres, acomete cerca de 30% das pessoas com mais de 65 anos e sua prevalência aumenta com o avanço da idade. Sabe-se que a etiologia da OA é multifatorial, com um forte componente genético identificado. Os polimorfismos genéticos das citocinas inflamatórias constituem importante objeto de estudo na compreensão dos mecanismos envolvidos na fisiopatogênese e progressão da OA. Esse trabalho analisou a associação entre os polimorfismos dos genes IL6 (-572 G/C), IL1β (+3954 C/T) e TNF (-308 G/A) com a presença e a gravidade da OA de quadril e joelho em idosos. Para tanto, este estudo caso-controle recrutou 257 idosos fisicamente independentes (95 homens, 162 mulheres), sendo 92 portadores de OA [21 homens (22,8%), 71 mulheres (77,2%)] e 165 controles [74 homens (44,8%) e 91 mulheres (55,2%)]. Foram coletadas amostras de sangue periférico e realizadas a extração e amplificação dos fragmentos de DNA por meio da reação em cadeia da polimerase (PCR) em tempo real pelo sistema TaqMan. A gravidade da OA foi analisada por meio dos achados radiográficos utilizando-se os critérios de Kellgren e Lawrence e a funcionalidade foi avaliada através dos questionários de LEQUESNE e WOMAC. A prevalência observada de OA na população de estudo foi de 35,8%, (43,8% entre as mulheres e 22,1% entre os homens). Foi observado que portadores do alelo C do

gene da IL6 têm menor prevalência de OA (2 = 7,9, p = 0,0005) e 49% menor chance de desenvolver a doença (OR = 0,51, 95% IC: 0,32- 0,80, p = 0,004) e menores graus de lesões radiográficas (Teste do Qui Quadrado, p<0,05). Por outro lado, os portadores dos alelos raros dos gene da IL1β (alelo T) e TNF (alelo A) têm maior prevalência de OA (Teste do Qui Quadrado, p<0,05) e apresentam maiores graus de lesões na análise radiológica (Teste do Qui Quadrado, p<0,05). Na análise de regressão logística, as variáveis do modelo final associadas à OA foram: gênero (OR: 2,67; IC: 1,48-4,08), polimorfismo do gene da IL1β (OR: 1,79; IC: 1,50-3,10) e do TNF (OR: 1,87; IC: 1,10-3,20); idade e IMC não ficaram associados neste modelo. Em relação à funcionalidade, foi observado que os indivíduos com alelo C do gene IL6 apresentam melhor condição funcional segundo o WOMAC (Teste de Mann-Whitney, p<0,05), enquanto indivíduos portadores do alelo A do gene TNF apresentam pior condição funcional segundo o LEQUESNE (Teste de Mann-Whitney, p<0,05). Os resultados demonstraram que o alelo C do gene da IL6 está associado a menor prevalência e gravidade da OA, o que poderia ser explicado por uma menor expressão dessa citocina. Por outro lado, os alelos T e A dos genes da IL1β e TNF, respectivamente, estão relacionados a um maior risco de ocorrência e maiores graus de lesão da doença, o que poderia dever-se a um ganho de função da IL-1B e a uma maior expressão do TNFA. Portanto, os polimorfismos dessas citocinas desempenham um papel na etiologia e gravidade a OA de quadril e joelho em idosos.

Descritores: osteoartrite; polimorfismo genético; citocinas; idoso; funcionalidade.

FERNANDES, Marcos Tadeu Parron. Relationship between polymorphisms of genes IL1β, IL6 and TNF in hip and knee osteoarthritis in elderly. 98 p. Dissertação (Mestrado em Ciências da Reabilitação) – Universidade Norte do Paraná, Londrina, 2014.

ABSTRACT

Osteoarthritis (OA) is the most common disease of the musculoskeletal system and it is the most common form of arthritis. The disease is more common in women and nowadays, about 30% of elderly individuals suffer from OA, with progressive rise in prevalence aging-related. It is known that OA has a multifactorial etiology with a strong genetic component identified. Genetic polymorphisms of inflammatory cytokines are an important object of study for understanding the mechanisms involved in the pathophysiology and progression of OA. This study examined the association between the presence of polymorphism in the genes of IL6 (-572 G/C), IL1β (+3954 C/T) and TNF (-308 G/A) with the presence and severity of hip and knee OA in elderly. Therefore, this case-control study recruited 257 physically independent elderly (95 men and 162 women), being 92 individuals with OA [ 21 men (22.8%) and 71 women (77.2%)] and 165 individuals without OA [74 men (44.8%) and 91 women (55.2%)]. Blood samples from patients and DNA extraction and amplification of DNA fragments was performed by real time polymerase chain reaction (PCR) using TaqMan system. The severity of osteoarthritis was analyzed using the radiographic findings, according to the criteria described by Kellgren and Lawrence and the functional status was verified using LEQUESNE and WOMAC questionnaires. The prevalence observed of OA at the population study was 35.8% (43.8% among women and 22.1% among men). Considering the IL-6, individuals harboring the allele

C showed a lower incidence of OA (2 = 7.9, p = 0.0005) and 49% less chance to have the disease (OR = 0.51, 95% CI: 0.32- 0.80, p = 0.004) and minor radiographic lesions (Chi Square test, p<0.05). On the other hand, individuals who carry the rare allele for IL1β (T allele) and TNF (A allele) exhibited a higher prevalence of OA (Chi Square test, p<0.05) and worse severity considering major radiographic lesions presented (Chi Square test, p<0.05). At the logistic regression analysis, the variables associated at the final model with OA were: gender (OR: 2.67; CI: 1.48-4.08) and polymorphism at the IL1β (OR: 1.79; CI: 1.50-3.10) and TNF (OR: 1.87; CI: 1.10-3.20) genes. Age and body mass index were not associated with OA at this population. Regarding the functional status, individuals carrying the allele C of the IL6 gene have better functional condition according to WOMAC (Mann-Whitney test, p<0.05), while individuals carrying the allele A of TNF gene have a worse functional status according to LEQUESNE (Mann-Whitney test, p<0.05). The results of this study show that the allele C of IL6 gene is associated with lower prevalence and severity of OA, which could be explained by a lower expression of this cytokine. On the other hand, the rare alleles of IL1β (Allele T) and TNF (Allele A) are related to a higher prevalence and severity of the disease, which could be observed through a function gain of IL-1B and a higher expression of TNFA. Therefore, the polymorphisms of theses cytokines play an important role in the etiology and severity of hip and knee osteoarthritis. Key-words: osteoarthritis; genetic polymorphism; cytokine; elderly; functionality.

LISTA DE ILUSTRAÇÕES

Figura 1 - Representação esquemática adaptada de Kapoor et al. (2011) da cartilagem

normal (A) e a cartilagem com osteoartrite (B)...............................................................18

Figura 2 - Figura esquemática adaptada de Terry et al. (2000) apresentando os

principais polimorfismos relacionados ao gene IL6........................................................19

Figura 3 - Figura esquemática apresentando os principais polimorfismos relacionados

ao gene IL1...................................................................................................................20

Figura 4 - Figura esquemática adaptada de Bayley et al. (2004) apresentando os

principais polimorfismos relacionados ao gene TNF......................................................21

Artigo 1

Figure 1- Comparison of functional status from individuals with osteoarthritis in relation

to IL6 genotypes analyzed with WOMAC and LEQUESNE questionnaires…...…….…32

Artigo 2

Figure 1 - Comparison of functional status from individuals with osteoarthritis in relation

to IL1β genotypes analyzed with WOMAC and LEQUESNE

questionnaires………………………………………………………………………….....……49

Figure 2 - Comparison of functional status from individuals with osteoarthritis in relation

to TNF genotypes analyzed with WOMAC and LEQUESNE

questionnaires………………………………………………………………………..…..……52

LISTA DE TABELAS

Artigo 1

Table 1 – Distribution of genotypes and allelic frequencies from the IL6 gene and the

presence of ostheoarthritis………………………………………………….........……….30

Table 2 – Relationship between the distribution of the IL6 gene genotype frequencies

and the severity of knee and hip osteoarthritis according to the degree of radiological

impairment..................................................................................................................31

Artigo 2

Table 1 – Genotypes and alleles frequency of IL1β gene and the presence of

osteoarthritis………………………………………………………………………………...47

Table 2 – Relation between genotype frequencies distribution of IL1β and the

severity of knee and hip osteoarthritis according to the degree of radiological

impairment………………………………………………………………………….……….48

Table 3 - Genotypes and alleles frequency of TNF gene and the presence of

osteoarthritis………………………………………………….……………………………..50

Table 4 – Relationship between the distribution of the TNF –G308A and the severity

of knee and hip osteoarthritis according to the degree of radiological

impairment…………………………………………………………………………….…….51

Table 5 – Multivariate analysis (Logistic Regression) considering the following

variables: age, gender, body mass index and polymorphism of IL1β and TNF genes

with the prevalence of osteoarthritis in elderly……………………………………….…52

1. INTRODUÇÃO

O envelhecimento da população, que até recentemente era característico dos

países desenvolvidos, passou a ser um fenômeno observado também nos países

em desenvolvimento, representando um dos maiores desafios para os sistemas de

saúde em âmbito mundial (WHO, 2011). Desse processo, resulta um aumento da

prevalência de doenças, sobretudo as crônico-degenerativas, dentre as quais se

destaca a osteoartrite (OA), patologia crônica do sistema osteoarticular cuja

evolução pode afetar a capacidade funcional e levar a limitações e incapacidades

(LIMA et al., 2003).

A OA é uma doença multifatorial cuja prevalência em adultos jovens varia

entre 7 e 11% (MARX et al., 2006; ALBANO et al., 2010) e que, embora não seja

decorrente da idade, progride com o envelhecimento (HARVEY; HUNTER, 2010;

TOFOLLETO et al., 2005). Neste contexto, há estimativas que a doença acometa

cerca de 30% dos indivíduos com mais de 65 anos e 40% das pessoas acima de 70

anos (MARX et al., 2006). Em relação ao gênero, ocorre mais nas mulheres do que

nos homens, podendo atingir até a proporção de 2:1, dependendo da faixa etária

(FELSON, 2003).

A OA pode comprometer o desempenho das atividades de vida diária (AVDs)

em cerca de 25% dos casos (MARX et al., 2006). Dos benefícios concedidos pelo

Instituto Nacional de Seguridade Social (INSS) do governo brasileiro no ano de

2005, 18% corresponderam a incapacidades pela OA (SILVA JÚNIOR, 2007).

Além de incapacitante, a OA representa uma das principais causas de

morbidade entre os idosos, acarretando alto custo social e econômico. As

modalidades terapêuticas disponíveis atualmente são pouco eficazes em impedir o

início e, principalmente, a progressão da doença, estando muitas vezes limitadas ao

controle sintomático (PELLETIER; MARTEL-PELLETIER, 2007; SILVA JÚNIOR,

2007).

A OA é uma doença que apresenta sinais e sintomas inflamatórios como dor,

edema e rigidez articular, ainda que não exista, de fato, um infiltrado de neutrófilos

no líquido sinovial e manifestações sistêmicas da inflamação, eventos que ocorrem

em uma artropatia inflamatória clássica. Na OA, há uma sinovite persistente que

acarreta um desarranjo na função dos condrócitos, levando a um desequilíbrio entre

o anabolismo e o catabolismo da cartilagem. Essa inflamação da sinóvia é

caracterizada por um infiltrado de linfócitos T e células B ativados, com um aumento

da expressão de mediadores inflamatórios (GOLDRING; GOLDRING, 2007). Entre

os principais mediadores envolvidos, estão a interleucina 1 beta (IL-1B), interleucina

6 (IL-6) e fator de necrose tumoral alfa (TNFA) (KAPOOR et al., 2011). Uma maior

compreensão dos mecanismos da resposta inflamatória e da ação dessas citocinas

é fundamental para uma abordagem clínica com melhores resultados.

O componente genético é um fator fundamental na etiologia das doenças, e

os estudos dos polimorfismos genéticos permitem caracterizar os indivíduos quanto

à predisposição às mais variadas patologias. No caso da OA, a presença dos

polimorfismos genéticos e suas implicações são eventos complexos e não

totalmente esclarecidos (SEZGIN et al., 2008; HAN et al., 2012).

Desta maneira, o estudo dos polimorfismos dos genes IL1β, IL6 e TNF têm o

potencial de identificar as pessoas com maior ou menor predisposição à OA, o que

possibilitaria um ajuste individualizado da abordagem preventiva e terapêutica, com

resultados clínicos mais positivos e conseqüente redução da morbi-mortalidade e

dos impactos sócio-econômicos.

2. REVISÃO DE LITERATURA – CONTEXTUALIZAÇÃO

2.1. Envelhecimento

O processo de envelhecimento populacional é um fenômeno mundial. No Brasil,

o número de idosos (idade > 60 anos) passou de 3 milhões em 1960 para 7 milhões

em 1975, e para 14 milhões em 2002, um aumento de quase 400% em pouco mais

de 40 anos. Estima-se que alcançará os 32 milhões em 2020 (MANOEL et al., 2006).

Segundo a Organização Mundial de Saúde (OMS), havia 524 milhões de

pessoas com idade igual ou acima de 65 anos no ano de 2010, o que correspondia a

8% da população mundial. A previsão para 2050 é que esse número chegue a 1,5

bilhão de pessoas (18% da população), o que representaria um aumento, nessa

faixa etária, da ordem de 71% nos países desenvolvidos e de aproximadamente

250% nos países em desenvolvimento (WHO, 2011).

O envelhecimento da população tem sido muito discutido no que se refere às

suas implicações sociais e em relação à saúde coletiva, com aumento da

necessidade de atenção por parte das autoridades e profissionais de saúde

(LOYOLA FILHO et al., 2005). No Brasil, diferentemente de outros países, esse

processo ocorreu dentro de um contexto sócio-econômico desfavorável e em ritmo

acelerado, o que levou o país a ter dificuldades para ajustar-se a essa expansão,

com um notório déficit de projetos assistenciais relacionados à atenção ao idoso

(LIMA; VERAS, 2003).

O aumento da expectativa de vida está relacionado a um aumento da

morbidade por doenças crônicas não-transmissíveis (LESSA, 1998; HOLMAN, 2004).

Um estudo realizado por DUARTE e REGO (2007), constatou alta prevalência

dessas comorbidades em pacientes idosos, sendo as mais freqüentes a hipertensão

arterial, o diabetes mellitus, as dislipidemias e a osteoartrite.

2.2. Osteoartrite: aspectos clínicos

A osteoartrite (OA) é a doença mais comum do sistema músculo-esquelético e

a maior causa de incapacidade, tanto em países desenvolvidos quanto nos países

em desenvolvimento (SHARMA et al., 2006), apresentando aumento progressivo da

sua prevalência com o avanço da idade. Representa a quarta causa de

incapacidade entre as mulheres e a oitava entre os homens, com grande

repercussão social e de custos para os sistemas de saúde (HARVEY; HUNTER,

2010; SILVA JÚNIOR, 2007; MARX et al., 2006; TOFFOLETO et al., 2005;

BROOKS, 2002).

A OA é uma doença crônica progressiva e degenerativa das articulações e

estruturas periarticulares. Caracteriza-se, basicamente, pela destruição da

cartilagem articular, remodelamento do osso subcondral com a formação de

osteófitos, e redução do espaço articular (VASCONCELOS et al., 2007;

VASCONCELOS et al., 2006; GOLDRING, 2000).

Clinicamente, a OA resulta dor articular, perda de força, incapacidade para a

marcha e redução da atividade física (MARX et al., 2006). Contempla uma sinovite

leve e persistente, fibrose da cápsula articular, frouxidão ligamentar e atrofia da

musculatura periarticular (PELLETIER; MARTEL-PELLETIER, 2007; SILVA JÚNIOR,

2007). Apresenta sintomas como dor, edema e rigidez, levando à limitações e

redução da qualidade de vida (GOLDRING; OTERO, 2011; BENDELE, 2001).

Aproximadamente 80% dos pacientes têm algum grau de limitação, com

comprometimento da realização das atividades da vida diária (AVDs) em cerca de

25% dessas pessoas (BROOKS, 2002).

A OA pode ser classificada, segundo a etiologia, em primária, quando

idiopática, e secundária, quando decorrente de condições prévias como traumas,

doenças inflamatórias sistêmicas, dentre outras condições (PORTH; MATFIN, 2010).

Segundo Vasconcelos et al., (2007), a OA pode ser também classificada com base

na análise radiológica, segundo os critérios de Kellgren-Lawrence (1957), em grau 0:

sem sinais de OA; grau I: provável diminuição do espaço articular, com possível

osteofitose; grau II: osteófitos bem definidos e possível diminuição do espaço

articular; grau III: múltiplos osteófitos, clara diminuição do espaço articular, esclerose

e possíveis deformidades nas extremidades ósseas; grau IV: grandes osteófitos,

intensa diminuição do espaço articular, esclerose grave e extremidades ósseas com

deformidades definidas.

Estresses biomecânicos, alterações bioquímicas na cartilagem articular e na

membrana sinovial, além do componente genético, são importantes fatores

envolvidos na etiologia da OA. São fatores de risco a obesidade, o excesso de força

articular, os traumas e lesões periarticulares, e alguns riscos ocupacionais

(HARVEY; HUNTER, 2010; VASCONCELOS et al., 2007; PELLETIER; MARTEL-

PELLETIER, 2007; MARX et al., 2006).

O tratamento objetiva o controle da dor, a manutenção ou melhora da

amplitude dos movimentos, assim como a redução da incapacidade. Para tanto,

utilizam-se medicamentos e fisioterapia, além de alterações no estilo de vida (MARX

et al., 2006; NAITO, 2012). Tradicionalmente, a terapêutica é pouco eficaz em conter

a evolução da OA, porém, esse panorama está mudando com os estudos

envolvendo terapias modificadoras das lesões e das estruturas (LOZADA, 2007;

PELLETIER; MARTEL-PELLETIER, 2007).

2.3. Osteoartrite: aspectos moleculares e envolvimento de citocinas

A anatomia articular é caracterizada pela presença de cartilagem hialina

recobrindo a extremidade óssea. A cartilagem é formada por um tecido conjuntivo

elástico composto por um componente celular, os condrócitos, e uma matriz

extracelular (MEC), rica em colágeno e proteoglicanos. Possui as funções

mecânicas de permitir um suave deslizamento durante os movimentos e de absorver

cargas, dissipando energia. Os condrócitos são os responsáveis pela síntese dos

principais elementos da MEC. Como a cartilagem não possui terminações nervosas

ou vasos sanguíneos, a oferta dos nutrientes e a troca de substâncias anabólicas e

catabólicas é feita entre os condrócitos e a membrana sinovial (sinóvia) por meio do

líquido sinovial (GARNERO et al., 2000; SIMON, 1999; FELICE et al., 1985;

BALASZ; DENLINGER, 1993; FELICE et al., 2002).

Quando, devido a fatores genéticos, bioquímicos ou mecânicos, ocorre uma

lesão da MEC, dois mecanismos básicos são ativados: os condrócitos proliferam e

aumentam a síntese de substâncias reparadoras, principalmente colágeno tipo II e

proteoglicanos, e ocorre uma produção e liberação de mediadores inflamatórios

pelas células sinoviais (sinoviócitos) e pelos condrócitos estimulados. Dessa

maneira, na OA, apesar de não haver um infiltrado de neutrófilos e da ausência de

manifestações sistêmicas da inflamação, a presença dos mediadores e o processo

de lesão tecidual conferem características inflamatórias à doença (DIEPPE, 1998;

GLANSBEEK et al., 1997; PELLETIER et al., 1991; GOLDRING; GOLDRING, 2007).

As citocinas são hormônios protéicos que são produzidos por células do

sistema imunológico, como linfócitos e macrófagos, e dentre outras, também por

células dos tecidos ostoearticulares, desempenhando um importante papel como

mediadores na fisiopatogênese da OA. Entre as principais citocinas estão a

interleucina-1 beta (IL-1B), interleucina-6 (IL-6) e fator de necrose tumoral alfa

(TNFA). Produzidas pelos condrócitos, osteoblastos e sinoviócitos, elas encontram-

se aumentadas na membrana e líquido sinovial, no osso subcondral e na cartilagem

dos pacientes portadores de OA, induzindo a produção de vários fatores

inflamatórios e catabólicos (KAPPOR, 2011; MEULENBELT et al., 2010; MARTEL-

PELLETIER et al., 2008; KUMAR et al., 2005).

A IL-1B e o TNFA são considerados citocinas proinflamatórias devido à suas

ações de estímulo do catabolismo e supressão do anabolismo articular, sendo

fundamentais na fisiopatologia da OA (BONDENSON et al., 2006). Agindo de

maneira sinérgica, essas citocinas atuam nos condrócitos inibindo a síntese dos

componentes da MEC, como o colágeno tipo II e os proteoglicanos, e estimulando a

produção de enzimas proteolíticas, como as metaloproteases, que destroem a

articulação (KAPOOR, 2011; GOLDRING et al., 2008). A IL-1B parece estar mais

diretamente envolvida com a destruição da cartilagem, enquanto o TNFA

desempenha um papel fundamental no início do processo, desencadeando a

cascata de eventos inflamatórios (GOLDRING, 2000).

A IL-6 é uma importante citocina envolvida na OA, estando aumentada tanto

no plasma quanto no líquido sinovial dos pacientes acometidos (KANEKO, 2000).

Sua produção, pelos condrócitos, é estimulada pela IL-1B e pelo TNFA, e sua ação

é controversa, envolvendo tanto mecanismos catabólicos quanto anabólicos. De

maneira isolada, aparentemente não é capaz de acarretar lesão da cartilagem

articular (GOLDRING, 2000). Dentre os mecanismos em que parece atuar como um

mediador regulatório, a IL-6 modula um aumento da expressão das

metaloproteinases e uma redução da expressão do colágeno tipo II (ROWAN et al,

2001; CAWSTON et al., 1998). Há evidências de que os níveis de IL-6 poderiam ser

preditores das alterações radiográficas observadas em pacientes com OA de joelho

(LIVSHITS et al., 2009).

A figura 1 apresenta um esquema simplificado adaptado de Kapoor et al.

(2011) sobre a fisiopatologia da OA.

Figura 1 - Representação esquemática adaptada de Kapoor et al. (2011) da

cartilagem normal (A) e a cartilagem com osteoartrite (B).

2.4. Osteoartrite: bases genéticas

Embora seja bem conhecida a natureza multifatorial da OA, fatores genéticos

têm sido compreendidos como fortes determinantes da doença (LOUGHLIN et al.,

2002), e vários genes têm sido relacionados em diferentes estudos (IKEGAWA,

2007; MIYAMOTO et al., 2008).

Considerando que a OA é primariamente caracterizada pela degeneração da

cartilagem articular, poderia se admitir que a predisposição genética para a

osteoartrite estivesse relacionada aos genes que codificam componentes estruturais

da cartilagem, tais como o do colágeno e dos proteoglicanos, ou aos genes

codificadores de enzimas proteolíticas, como os genes das metaloproteinases.

Contudo, estas hipóteses não foram confirmadas em diferentes estudos

(LOUGHLIN, 2005; AERSSENS et al., 1998; BALDWIN et al., 2002). Nesse

contexto, genes que codificam as citocinas são possíveis candidatos para

marcadores genéticos da OA, dado o importante papel que esses mediadores

desempenham na fisiopatologia da doença (GOLDRING, 2000; PELLETIER et al.,

2001).

Os polimorfismos genéticos podem ser definidos como variações na sequência

de nucleotídeos do DNA que ocorrem com uma freqüência superior a 1% na

população estudada (ROCHA et al., 2007; BALASUBRAMANIAN et al., 2002). Os

polimorfismos são essenciais para as espécies, inclusive para os humanos, pois são

os principais responsáveis pela diversidade genética e a expressão de diferentes

fenótipos nas populações. Desta forma, é possível identificar as características e os

genes determinantes para o desenvolvimento de doenças com base no seu padrão

de polimorfismo (SYVANEN, 2001). Polimorfismos de um único nucleotídeo (SNP) e

suas relações com OA têm sido extensivamente estudados, embora resultados

conflitantes tenham sido reportados.

O gene IL6 localiza-se nas bandas 21-24 do braço curto do cromossomo 7

(7p21-24), possuindo uma região promotora de 303 pb (AI et al., 2014). SNPs na

região promotora, que desempenha um papel de regulação da atividade do gene,

têm o potencial de influenciar a expressão gênica. Os principais SNPs do IL6

relacionados à OA são os das posições -174, com troca de uma guanina por uma

citosina (-174 G/C, rs1800795), -572 G/C (rs1800796) e -596 G/A (rs 1800797),

todos situados na região promotora. A figura 2 mostra, esquematicamente, o gene

da IL6 e os principais polimorfismos, incluindo o SNP -572 G/C.

Figura 2- Figura esquemática adaptada de Terry et al. (2000) apresentando os

principais polimorfismos relacionados ao gene IL6. A seta mostra o sítio polimórfico

-572 G/C (rs1800796).

O gene que codifica a citocina IL-1B está situado na banda 13, no braço longo

do cromossomo 2 (2q13). Polimorfismos, como o SNP +3954 C/T (rs1143634) que

está localizado no éxon 5, têm relação com a OA, sendo que a maior parte dos

estudos relaciona a presença dos polimorfismos com aumento da prevalência e

gravidade da doença (KAARVATN et al., 2013; JOTANOVICH et al., 2012; KANOH

et al., 2008). Abaixo, a figura 3 apresenta um esquema dos principais SNPs do gene

IL1β envolvidos com a gênese da OA.

Figura 3- Figura esquemática apresentando os principais polimorfismos

relacionados ao gene IL1. A seta mostra o sítio polimórfico +3954 C/T (rs1143634).

Em relação ao gene TNF, que codifica o mediador TNFA, ele localiza-se na

região 6p21, e um dos principais SNPs estudados é o -308 G/A, rs1800629,

localizado na região promotora. Aparentemente, esse polimorfismo aumenta a

expressão gênica e tem relação com piora do quadro clínico de OA (MUÑOZ-VALLE

et al., 2014; JI et al., 2013; HAN et al., 2012). A figura 4 apresenta um esquema com

os principais SNPs do gene TNF associados com a OA.

Figura 4 - Figura esquemática adaptada de Bayley et al. (2004) apresentando os

principais polimorfismos relacionados ao gene TNF. A seta indica o SNP -308 G/A

(rs1800629).

A compreensão dos polimorfismos dos genes das citocinas tem o potencial de

caracterizar os indivíduos quanto à predisposição à OA, o que poderia definir as

ações preventivas e terapêuticas, com melhores resultados clínicos. Dessa maneira,

o objetivo desse trabalho foi estudar os polimorfismos -572 G/C do gene IL6, +3954

C/T do gene IL1β e -308 G/A do gene TNF, relacionando-os à presença e gravidade

da osteoartrite de quadril e joelho em idosos.

3. OBJETIVOS

3.1. Objetivo geral

Avaliar a associação entre os polimorfismos genéticos da interleucina-1 beta

(IL1β), interleucina-6 (IL6) e fator de necrose tumoral alfa (TNF) com a

presença e gravidade da osteoartrite em idosos.

3.2. Objetivos específicos

Determinar a prevalência e gravidade da osteoartrite em idosos

fisicamente independentes;

Verificar a prevalência das frequências alélicas e genotípicas do IL1β, IL6

e do TNF na população de estudo;

Avaliar a associação entre os polimorfismos dos genes IL1β, IL6 e TNF e

o grau de acometimento radiológico nas articulações do quadril e joelho;

Determinar a associação entre os polimorfismos genéticos e o grau de

comprometimento funcional avaliado pelos questionários de LEQUESNE

(LEQUESNE, 1997) e WOMAC (IVANOVITCH, 2002).

4. ARTIGOS

ARTIGO ORIGINAL 1 - submetido ao periódico Cytokine

Association of interleukin-6 gene polymorphism (rs1800796) with severity and

functional status of osteoarthritis in elderly individuals

Marcos T. P. Fernandesa,b, Karen B. P. Fernandesa,c, Audrey S. Marquezc, Ilce M. S.

Cólusd, João Paulo M. Santosa, Regina C. Poli-Fredericoya,c

a Master Program in Rehabilitation Sciences UEL/UNOPAR, Londrina-PR, Brazil.

b Irmandade Santa Casa de Londrina Hospital (ISCAL), Londrina-PR, Brazil.

c Biological and Health Sciences Center, University of Northern Parana (UNOPAR),

Londrina-PR, Brazil.

d Department of Biology, State University of Londrina (UEL), Londrina-PR, Brazil.

Corresponding author: Regina Célia Poli-Frederico

Laboratory of Molecular Biology - Biological and Health Sciences Center - UNOPAR.

Rua Marselha, 491 Jardim Piza. CEP:86041-140 - Londrina, PR – Brazil.

Telefone: 55 (43) 33717990

E-mail: [email protected]

mailto:[email protected]

ABSTRACT

Osteoarthritis (OA) is the most prevalent disease of the musculoskeletal system and it has

an important genetic component. Despite several reports have shown the involvement of

pro-inflammatory cytokine such as interleukin-1β and TNF-α, the role of interleukin-6 (IL-6)

in osteoarthritis is still unclear. Thus, this study aimed to analyze the relationship between

the single nucleotide polymorphism in the portion - 572 of the promoter region of the IL-6

gene (SNP - 572G/C) with hip and knee OA in the elderly. In this case-control study, 257

physically independent elderly were recruited (Case Group: 92 individuals with osteoarthritis

and Control Group: 165 individuals without osteoarthritis). Blood samples were collected

from patients for the DNA fragments extraction and amplification by real-time polymerase

chain reaction (PCR) by TaqMan system for subsequent genotyping of IL-6 gene. The

degree of joint damage was assessed by radiographic classification based on the criteria of

Kellgren and Lawrence. The functional status was evaluated by Lequesne and WOMAC

questionnaires. The results demonstrate that patients with polymorphic C allele have lower

incidence of OA (2 = 7.9, p = 0.0005) compared to non-polymorphic and lower level of

radiological impairment (2= 10.01, p = 0.002) in both cases of hip and knee osteoarthritis.

Regarding functional status, individuals carrying the C allele has a lower degree of

functional impairment assessed by WOMAC (Mann-Whitney test, p = 0.04), although no

difference was observed in the Lequesne questionnaire (p>0.05). In conclusion, the

polymorphism - 572 G/C IL-6 is a protective factor for the presence and severity of hip and

knee osteoarthritis in the elderly. Further prospective studies with large sample size and

methods such as the effect of this polymorphism on gene expression or haplotype

analysis for IL-6 promoter polymorphism are needed to validate these study findings.

Key-words: osteoarthritis; genetic polymorphism; IL-6; elderly; functional status.

1. INTRODUCTION Osteoarthritis (OA) is the most common disease of the musculoskeletal system as

well as the most common form of arthritis, with progressive increase in prevalence with

aging. It is estimated to affect about 40% of people above 70 years, representing a major

cause of morbidity and disability worldwide [1-3].

The OA is a chronic rheumatic disease characterized by destruction of component

from articular cartilage and changes in the synovial membrane and the subchondral

bone, resulting in deformity and joint limitations [4,5]. Generally occurring in the hips and

knees, it is clinically described by pain, swelling, loss of strength and periarticular muscle

atrophy [6,7]. Radiologically, it may be observed a joint space narrowing, presence of

marginal osteophytes, sclerosis and bone deformities, making it possible to classify the

disease in degrees related to the severity of findings presented at the RX images [8].

These changes may have negative influence on gait disorders and reduced physical

activity usually observed in these cases [9].

Although its pathophysiology is not completely understood, inflammation plays a

major role in OA. The chronic inflammatory process involves the production of cytokines

that are present in the synovial fluid which induces hyperalgesia and are related to the

breakdown of cartilage. Among the major mediators can highlight the tumor necrosis

factor alpha (TNF-α) and interleukin-1 beta (IL-1β), called pro-inflammatory cytokines.

Produced by chondrocytes, they stimulate the production of proteolytic enzymes such as

metalloproteinases as well as they induce the production of other mediators such as

interleukin-6 (IL-6) [10-14]. IL-6 has a complex action, playing a role as a modulator of

both anabolic and catabolic actions in OA [15]. Studies have shown that IL-6 production

is up-regulated by IL-1β and matrix metalloproteinases and it reduces the production of

collagen-2, also contributing to the joint injury [16].

It is known that OA has a multifactorial etiology with a strong genetic component

identified. Genetic polymorphisms of cytokines are important objects of study for

understanding the mechanisms involved in the genesis and progression of OA, among

which stand out the polymorphisms in the IL-6 gene located in 7p21-24, having a

promoter region of 303 bp [17]. The polymorphisms in this region can influence the level

of gene expression with consequent reduction in the levels of this interleukin [18]. An

important genetic variation is single nucleotide polymorphism (SNP) at position -174

(G174C, rs1800795) promoter region, with the exchange of guanine to cytosine (IL-6 -

174 G/C), results in suppression of IL-6 transcription [18]. However, several studies have

shown conflicting results regarding the presence of this polymorphism and the

susceptibility to OA [19-22]. It is noteworthy that the activity of the promoter region of this

gene is also affected by the polymorphic sites -597 (G597A; rs1800797) and -572

(G572C; rs1800796), that seem to control the influence of the polymorphism at position -

174 [23].

It has been proposed that plasma concentrations of IL-6 is related to joint damage

observed on radiographs of patients with OA, suggesting that IL-6 could act as a marker

for the presence of these lesions [12]. However, it was neither determined in this study if

this relation can be observed in the presence of the polymorphism in the gene encoding

this cytokine nor if these alterations may have any functional implications.

Therefore, the study of polymorphism in the IL-6 gene may represent an important

tool in the early identification of the risk for OA developing, besides being a possible

therapeutic target in the prevention and treatment of disease [11,18]. Thus, this study

examined the association between the presence of the polymorphism -G572C of the IL-6

gene and the susceptibility and severity of OA in the elderly.

2. MATERIALS AND METHODS

2.1. Study design and population

This case-control study comprised a sample from the project “Study on Aging and

Longevity”, which was approved by the Ethics Committee of the institution (PP/

0026/2013).

The inclusion criteria for the study were: being elderly (aged over 60 years),

have OA of the hip and / or knee, confirmed by clinical diagnosis and radiographic

assessment by medical specialist (case group) or being elderly with no osteoarthritis

(control group).

The exclusion criteria for both groups were: having other rheumatic diseases,

hip replacement or knee, use of corticosteroid or immunosuppressant drugs, sequelae of

stroke or Parkinson's disease.

2.2. Genetic polymorphisms of IL-6 analysis

2.2.1. Blood samples collect and DNA extraction

From each patient, 5 ml of peripheral blood were collected by venule puncture. Blood

samples were stored at - 80°C. DNA extraction was performed by using the kit PureLink -

Invitrogen according to the manufacturer's instructions.

The evaluation of the purity and concentration of DNA was performed by analysis of

absorbance in a spectrophotometer (NanoDrop ND-2000 - Thermo Scientific) at 260nm

and 280nm. Subsequently, the DNA dilution was made in ultrapure Milli-Q® water to a

final concentration of 30 ng / µL.

2.2.2. Analysis of the polymorphism in the IL-6 gene (rs1800796) by polymerase chain

reaction (PCR) in real time

Despite there are several SNP of the IL-6 gene, the main studied observed at the

promoter positions -597, -572 and -174 (G-597A, G-572C and G-174C) corresponding to

SNP reference numbers of rs1800797, rs1800796 and rs180095 respectively [24].

The polymorphism at position -572 of the IL-6 (rs1800796) was chosen because it is

a TAG SNP, with a perfect linkage disequilibrium with the other SNPs of the promoter

region (rs rs1800797 and rs180095) [22], which means that these polymorphism are

inherited simultaneously, which justifies to study only one of them.

For this polymorphism analysis, the amplification technique of DNA fragments by

polymerase chain reaction (PCR) in real time by TaqMan system (Applied Biosystems,

Life Technologies, Foster City, USA) was performed. The standard reaction

contained 20μL final volume of 10μl with Genotyping TaqMan Master Mix (1x), 0,5μL

probe (1x) (Applied Biosystems, Foster City, USA) 7,5μL ultrapure Milli-Q® and 1μL of

DNA (30 ng /µL). StepOnePlus thermocycler™ Real-Time PCR System (Applied

Biosystems, Foster City, USA) with the following cycling used: 50 cycles of 60ºC for 30

seconds, 95°C (pre denaturation), 95°C for 10 minutes for initial denaturation for 15

seconds (denaturation) and 60°C for 1 minute and 30 seconds and a final extension

cycle of 30 seconds at 60ºC. The evaluation of the results was performed by the

StepOne Software v2.3.

2.3. Assessment of the severity of osteoarthritis

Individuals in the case group were asked to perform an X-ray of the affected joint. All

radiographs were analyzed by a radiologist, being useful not just to confirm the disease

diagnosis but also to classify the disease severity using the criteria described by Kellgren

and Lawrence [8] and described in other studies [25], where the severity of osteoarthritis

may be determined concerning radiographic findings as follows: Grade 0 - no

radiographic changes; Grade I - doubtful space narrowing and possible marginal

osteophytes; Grade II - possible joint space narrowing and defined osteophytes

observed; Grade III - defined joint space narrowing, moderate multiple osteophytes,

subchondral sclerosis and any possible deformity of bone contour and grade IV -

remarkable joint space narrowing, severe subchondral sclerosis, definite deformity of

bone contour and presence of large osteophytes.

Based on these criteria, the groups were re-categorized into two groups: with mild to

moderate (grades I and II) radiographic changes and severe changes (grade III and IV).

2.4. Evaluation of functionality related to osteoarthritis

In order to assess the functional status of these patients, both Western Ontario

and McMaster Universities Osteoarthritis Index (WOMAC) and Lequesne Index were

used.

The WOMAC questionnaire was previously translated and validated to the

Portuguese language and nowadays it is considered the gold standard for OA functional

status evaluation according to the American College of Rheumatology. This

questionnaire involves questions regarding pain, stiffness and function, where the highest

score is the worst condition of the individual [26]. Additionally, the Lequesne Index was

also used. This questionnaire was also translated to Portuguese version and is

recommended by the European League of Rheumatology (EULAR) to evaluate

osteoarthritis functional status, comprising eleven questions involving pain, discomfort

and functional limitation, specially related to functional status [27].

2.5. Statistical Analysis

The Statistical Package for Social Sciences 20.0 (SPSS, UK) was used for statistical

data analysis, with a confidence interval of 95% and a significance level of 5% (p <0.05)

established for all tests used.

For quantitative variables, the Shapiro-Wilk test was used and data with normal

distribution were shown by mean and standard deviation while the data without normal

distribution were presented as median and interquartile range.

The Chi Square was used as to verify that the genotype frequencies were in Hardy-

Weinberg equilibrium as well as to assess possible associations between the presence of

polymorphisms in the IL-6 gene with the occurrence and severity of osteoarthritis.

Moreover, the Mann-Whitney test was used to compare the functional status

measured by WOMAC questionnaire and Lequesne index of individual with different

genotypes (GG versus GC/CC).

3. RESULTS

In this case-control study, 257 elderly subjects were recruited, being 162 women

(63.0%) and 95 men (37.0%). Concerning this sample, 92 (35.8%) individuals have

osteoarthritis, while 165 (64.2%) didn’t have this disease.

No difference was observed between the age of the case (median = 69, interquartile

range = 9) compared to the age of the control group (median = 68, interquartile range =

7), according to the Mann-Whitney test (p = 0.26). Similarly, no difference was observed

in body mass index when compared the case (median = 28.4, interquartile range = 5.6)

and control group (median = 27.7, interquartile range = 7), according to the test Mann-

Whitney test (p = 0.60). Data concerning general characteristics of the population study is

shown in table 1.

It was observed that the distribution of genotype frequencies of the IL-6 gene is in

accordance with the Hardy-Weinberg equilibrium (P = 0.55). Table 2 shows the

distribution of genotypes and alleles for -572 G/C polymorphism of the IL-6 gene in

subjects with osteoarthritis and controls. For healthy individuals, it was found a proportion

for the GG genotype of 50.9% and the percentage of G and C alleles were 70.6% and

29.4%, respectively. For patients with osteoarthritis, increased frequency of GG genotype

(66.3%) and G allele (84.3%) was found.

It was observed that individuals carrying the C allele have lower susceptibility to

osteoarthritis (2 = 7.9, p = 0.005, Table 2). The C allele conferred a 49 % protection (OR

= 0.51, 95% CI: 0,32- 0.80, p = 0.004) of individuals harboring this allele compared to

those with allele G.

Table 1 - Characteristics of the population study.

C Variables Total Groups p Age, years (median/

interquartile range) Case Control

69 / 9

* Statistically significant, 2 test, p<0.05.

Furthermore, it was also observed that the presence of the C allele is a protective factor

against radiographic damage in patients with osteoarthritis (2 = 10.01, p = 0.002). These

data can be confirmed even when the subjects were divided into subgroups as in the

affected hip joint (2 = 4.2, p = 0.04, Table 2) and knee (2 = 4.7, p = 0, 03, Table 2).

Table 2 - Distribution of genotypes and allelic frequencies from the IL-6 gene and the

presence of osteoarthritis.

Presence of osteoarthritis

Genotype Frequencies

No Yes

GG 84 (50.9%) 61 (66.3%)

GC 65 (39.4%) 29 (31.5%)

CC 16 (9.7%) 2 (2.2%)

Total 165 (100.0%) 92 (100.0%)

Allele Frequencies

G 233 (70.6%) 155 (84.3%)

C 97 (29.4%) 33 (17.7%)

Total 330 (100.0%) 184 (100.0%)


Table 3 – Relationship between the distribution of the IL-6 gene genotype frequencies


impairment.

Hip


Radiological Impairment

Mild Moderate/Severe GG 14 (48.3%) 10 (83.3%) GC 14 (48.3%) 02 (16.7%) CC 01(3.4%) -----

Total 29 (100.0%) 12 (100.0%) Allele

Frequencies

G 42 (72.4%) 22 (84.6%) C 16 (27.6%) 4 (15.4%)

Total 58 (100.0%) 26 (100.0%) Knee


Mild Moderate/Severe

GG 10 (55.6%) 27 (81.8%) GC 07 (38.9%) 06 (18.2%) CC 01(5.5%) -----

Total 18 (100.0%) 33 (100.0%) Allele

Frequencies

G 27 (75.0%) 60 (90.1%) C 9 (25.0%) 6 (9.9%)

Total 36 (100.0%) 66 (100.0%)


Considering our previous observation that the individuals harboring the C allele have

a protective effect against osteoarthritis, in order to assess the functional status, the

individuals were stratified according the presence of allele C (GG versus GC/CC).

Regarding the functional status, it was observed that the individuals carriers of allele

C (CG and CC genotypes) had better functional condition according to WOMAC

questionnaire (Mann-Whitney test, p=0.04, figure 1). However, no differences were

observed between the genotype groups according to Lequesne index (Mann-Whitney

test, p>0.05).

Figure 1- Comparison of functional status from individuals with osteoarthritis in relation to

IL-6 genotypes analyzed with WOMAC and Lequesne questionnaires.

GG

GC/C

C

0

20

40

60

80

100

*

IL-6 Genotypes

WO

MA

C

GG

GC/C

C

0

10

20

30

40

IL-6 Genotypes

LE

QU

ES

NE

4. DISCUSSION

Osteoarthritis (OA) is a chronic inflammatory process that results in progressive

destruction of cartilage and related structures [28,29]. Taking into account that the

genetic component is an important determinant of OA, the study of interleukin gene

polymorphisms can identify degrees of susceptibility or predisposition to minor illness

with potential implications for clinical management and preventive therapy. Despite

contrasting results, the polymorphism of a single nucleotide at position -174 to the

exchange of a cytosine for a guanine in the IL-6 gene (SNP -174 G/C) has been

previously studied because the important role of this interleukin on the pathophysiology

of OA [30,31].

The results of this study demonstrate a strong and significant association between

the -572 G/C polymorphism of IL-6 as a protective factor for the presence and severity of

OA. Similar results were previously described by Kämäräinen et al. [22] where GG

genotype was more prevalent in severe forms of distal interphalangeal osteoarthritis.

Individuals with the CC genotype have lower incidence and severity of disease in clinical

evaluation obtained by the WOMAC questionnaire (overall score). There are few studies

that relate the polymorphism of the IL-6 gene with some evaluation of physical

performance or functional status. Nicklas et al [32] studied an association between

physical performance and this IL-6 gene polymorphism in patients with knee OA and

demonstrated that there is a tendency of those without the polymorphism (GG

individuals) straddling over a greater distance in this walking test.

At our study, the radiological findings from both hip and knee showed a significant

correlation between the presence of the polymorphism (both CG and CC) and lower level

of joint damage. These results corroborate the findings of Pola et al [33] who

demonstrated that the association of the polymorphism with low risk of hip OA. Moreover,

the study of Honsawek et al. [34] showed that individuals carrying the genotype of the

polymorphic GC had higher risk of knee osteoarthritis.

It is important to state that the case and control groups showed no differences in

body mass index (BMI). This fact avoids the risk of bias due to the presence of obesity,

which is known to be a risk factor for the OA development and progression [35].

Additionally, no patient at this study was using corticosteroids, which could influence the

hormonal and cellular inflammatory response and, thus, had some influence on functional

status.

Our study showed a lower frequency of CC genotype in patients with OA, suggesting

a possible influence of this genotype against the development of the disease. The study

by Chua et al [36] who evaluated the polymorphism -572 G/C also demonstrated that the

presence of the polymorphic allele C is a protective factor to erythematosus systemic

lupus. Additionally, the presence of allele C (specially the CC genotype) should result in

a low expression of the IL-6 gene after an inflammatory stimulus as compared to the GG

genotype. This mechanism could explain why individuals carrying the polymorphic allele

had minor radiographic lesions for both hip and knee ostheoarthritis. However, the role of

this interleukin in the joint destruction is complex and not yet fully understood [14].

Furthermore, it was also observed that individuals with the IL-6 gene polymorphism

(genotypes GC and CC) have a better functional status when compared to individuals

with ancestral genotype (GG) through WOMAC questionnaire. However, no differences

on functional status were observed regarding the Lequesne index. These discrepancies

may be explained by the differences between both questionnaires. While the WOMAC

questionnaire is useful for functional assessment, the Lequesne index reflects the

functional limitation related to pain intensity [26,27]. Considering that IL-6 plays a

regulatory role in the pathogenesis of ostheoarthritis [2], its influence may be more

related to the equilibrium regarding joint destruction and regeneration that with the pain

itself.

Some limitations of this study should be pointed out. The most important is the lack

of measurement of IL-6 in synovial fluid of individual and, therefore, further studies are

necessary to understand the role of this interleukin in the OA disease. Moreover, it

should be important to assess all the polymorphisms found in the promoter region of the

IL-6 (G-597A, G-572C and G-174C) together, which present a complex interaction

determined by haplotype influencing transcription of the IL -6 gene, since G alleles of

these loci have been associated with increased transcription of IL-6 [23]. Thus, future

studies are needed to better understand the genetic basis of the complex mechanisms

and signaling as well as inflammatory modulation observed in osteoarthritis.

5. CONCLUSION

The results of this study confirmed that the -572 G/C polymorphism in the 5 'region of

the IL-6 gene seems to be a protective factor for hip and knee osteoarthritis in elderly. The

individuals harboring the allele C have lower prevalence and severity of osteoarthritis when

compared to individuals without this polymorphism. However, further prospective studies

with large sample size and methods such as the effect of this polymorphism on gene

expression or haplotype analysis for IL-6 promoter polymorphism are needed to validate

these study findings.

6. REFERENCES

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[2] Krasnokutsky S, Attur M, Palmer G, Samuels, J et al. (2008). Current concepts in the

pathogenesis of osteoarthritis. Osteoarthritis Cartilage 2008; 16 (S 3): S1-S3.

[3] Dieppe PA, Lohmander LS. Pathogenesis and manegement of pain in osteoarthritis.

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ARTIGO ORIGINAL 2 - a ser submetido ao periódico Clinical and Experimental

Reumathology

ASSOCIATION BETWEEN THE POLYMORPHISM OF IL1 AND TNF GENES

RELATED TO SEVERITY AND FUNCTIONAL STATUS IN ELDERLY WITH

OSTEOARTHRITIS

Marcos T.P. Fernandes (1,2), Karen B.P. Fernandes (3,4), Ilce M. S. Cólus(5) Regina C.

Poli-Frederico(3,4)

(1) Master Program in Rehabilitation Sciences, Londrina-PR, Brazil.

(2) Associate Doctor, Irmandade Santa Casa de Londrina Hospital (ISCAL), Londrina-PR,

Brazil.

(3) Associate Professor, Doctoral/Master Program in Rehabilitation Sciences

UEL/UNOPAR, Londrina-PR, Brazil.

(4) Biological and Health Sciences Center, University of Northern Parana (UNOPAR),

Londrina-PR, Brazil.

(5) Department of Biology, State University of Londrina (UEL), Londrina-PR, Brazil.

Corresponding author: Regina Célia Poli-Frederico

Laboratory of Molecular Biology - Biological and Health Sciences Center - UNOPAR. Rua

Marselha, 491 Jardim Piza. CEP:86041-140 - Londrina, PR – Brazil.

Telefone: 55 (43) 33717990

E-mail: [email protected]

mailto:[email protected]

ABSTRACT

Osteoarthritis (OA) is a major musculoskeletal disease with high prevalence in the

elderly. The study of genetic polymorphisms of inflammatory mediators involved in OA

may contribute to the elucidation of the complex pathophysiology of this disease. Thus,

this study aimed to evaluate the association between polymorphisms at interleukin-1β

(IL1β) and tumor necrosis factor alpha (TNF) genes (SNP +3954 C/T of gene and SNP -

308 G/A TNF) with presence and severity of osteoarthritis in elderly individuals. In this

case-control study, 257 physically independent elderly (Mean Age: 68.55 ± 5.2; Minimum

age: 60 and Maximum age: 82) were recruited. After this selection, the groups were

divided in: 92 elderly individuals with osteoarthritis (named case group) and 165 without

the disease (named control group). The individuals were genotyped for both

polymorphisms by the TaqMan real-time PCR system. The subjects were classified

based on the degree of radiological impairment according to the criteria of Kellgren-

Laurence and regarding functional impairment using the WOMAC and LEQUESNE

questionnaires. The results of this study showed that individuals carrying the +3954

polymorphism of the IL1β gene (subjects harboring T allele) have a higher incidence of

OA (2 = 8.7, p = 0.002) and major radiological hip injury (2 = 7, 2, p = 0.007) compared

to individuals not carrying this polymorphism. Moreover, for SNP -308 of TNF gene

polymorphic individuals (subjects harboring allele A) are more affected by OA (2= 8.7, p

= 0.003), they have greater radiological lesion in both cases of osteoarthritis of the hip (2

= 3.9, p = 0.04) and knee (2= 4.0, p = 0.04) and worse functional status assessed by the

LEQUESNE questionnaire (Mann-Whitney, p = 0.04). At the multivariate analysis, after

adjustment for age, gender, body mass index, the presence of rare alleles for both IL1β

(allele T) and TNF (allele A) increases the susceptibility to OA development [OR: 1.79

(95%CI: 1.05-3.1) for IL1β and OR: 1.87 (95%CI: 1.1-3.2) for TNF]. We conclude that the

SNP +3954 C/T polymorphism of the IL1β gene and SNP - 308 G/A TNF are related to a

higher prevalence and severity of osteoarthritis in elderly.

Key-words: Osteoarthritis; Elderly; Genetic polymorphism; Interleukin-1beta; Tumor

Necrosis Factor-alpha; Functional status.

1. INTRODUCTION

Osteoarthritis (OA) is the most prevalent osteomuscular disease as well as the most

common form of arthritis. It mainly affects elderly individuals, being a significant cause of

limitations and incapacities at this population (1,2). It is characterized by chronic and

progressive loss of articular cartilage components due to an imbalance between

destruction and repair of the extracellular matrix, accompanied by changes in the

synovium and subchondral bone (3,4). Although OA globally affects all joint tissues,

cartilage destruction is the final pathway of the disease (1). Clinically, it focuses mainly

on the joints of the hips and knees, being followed with pain, sweling, loss of strength and

atrophy of the periarticular muscles, inability to gait and reduced physical activity,

representing major social burden and high costs to the health system (5-7).

While inflammatory mechanisms are not fully understood, they play an

important role in OA. The inflammation usually results from biomechanical stress,

changes in chondrocyte function related to the aging process, and dysregulation of

inflammatory cytokine activity (8,9). Chronic inflammation involves the production of

mediators that are present in the synovial fluid, induce cartilage breakdown and

hyperalgesia. Among the main mediators are tumor necrosis factor alpha (TNF-A) and

interleukin-1 beta (IL-1B), called pro-inflammatory cytokines, which play a central role in

the pathophysiology of OA (10). They are produced by chondrocytes, mononuclear cells,

osteoblasts and synovial tissue, and act synergistically regulate the synthesis of

components of the extracellular matrix by inhibiting the anabolic activity, and stimulation

of production and release of catabolic inflammatory factors (11).

In OA, the chondrocyte is the cellular target of the action of these cytokines,

whose receptors, IL-1RI, IL-1RII, TNFRI and TNFRII are mainly in the cells of the joint

surface (12). These mediators induce the synthesis of inflammatory factors, and

enzymes such as nitric oxide, nitric oxide synthase, prostaglandin E2, cyclooxygenase-2,

among others. Also increase the expression of metalloproteinases, which are proteolytic

enzymes that destroy cartilage matrix as well as stimulate the production of other

mediators such as interleukin-6 (IL-6) (12-16). At the same time, suppress the anabolic

activity of chondrocytes through the inhibition of proteoglycan and collagen type II (17).

Genetic factors are important in the etiology of OA. The single nucleotide

polymorphisms (SNP) are an important object of study in understanding the complex

mechanisms involved in the genesis and progression of OA. The IL1β gene is located on

2q13 and among the various polymorphisms, the one presented in exon 5, in the position

+3954 with replacement of cytosine by thymine (+3954 C/T; rs1143634) is the most

studied. The TNF gene is located on 6p21, and a major polymorphisms involved in OA is

the position - 308 of the promoter region, with exchange of guanine for adenine (-308

G/A; rs1800629). Several studies have shown that these two polymorphisms increase

the genetic expression of this cytokine, related to higher prevalence and intensity of OA

(18-26). However, there is lack of information regarding a possible relationship between

this genetic background and functional status in elderly with OA.

The possibility of identifying individuals with increased susceptibility or lower

predisposition to develop the disease has the potential to define preventive and

therapeutic actions as early as possible with better clinical outcomes. Thus, the aim was

to study the polymorphism +3954 C/T of IL1β and -308 G/A of TNF relating to the

presence and severity of OA in the elderly.

2. MATERIALS AND METHODS

2.1. Study design and population

This case-control study comprised a sample from the project EELO (Study on Aging

and Longevity), which was approved by the Ethics Committee of the institution (PP /

0026/2013).

The inclusion criteria for the study were: being elderly (aged over 60 years),

have OA of the hip and / or knee, confirmed by radiographic examination performed by

medical specialist (case group) or being elderly with no clinical signs of osteoarthritis

(control group). The control group was matched with case group considering gender and

age.

The exclusion criteria for both groups were: having other rheumatic diseases,

advanced osteoporosis, hip replacement or knee, corticosteroid use, sequelae of stroke

or Parkinson's disease.

2.2. Analysis of genetic polymorphism of IL-1β and TNF

2.2.1. Blood samples collect and DNA extraction

From each patient, 5 ml of peripheral blood were collected by venule puncture. Blood

samples were stored at - 80°C. DNA extraction was performed by using the kit PureLink -

Invitrogen according to the manufacturer's instructions.

The evaluation of the purity and concentration of DNA was performed by analysis of

absorbance in a spectrophotometer (NanoDrop ND-2000 - Thermo Scientific) at 260nm

and 280 nm. Subsequently, the DNA dilution was made in ultrapure Milli-Q® water to a

final concentration of 30 ng/ul.

2.2.2. Analysis of the polymorphism in the IL1β and TNF gene by polymerase chain

reaction (PCR) in real time

To analyze the SNP of genes IL1β and TNF the amplification technique of DNA

fragments was performed by polymerase chain reaction (PCR) in real time by TaqMan

system (Life technologies, Applied Biosystems, Foster City, USA). Polymorphisms at

positions +3954 C/T IL-1β (assay ID: C_9546517_10) and -308 G/A of TNF (assay ID:

C_7514879_10) were checked.

The standard reaction contained 20 μL final volume of 10 μl with Genotyping

TaqMan Master Mix (1x), 0,5 μL probe (1x) (Applied Biosystems, Foster City, USA) 7,5

μL ultrapure Milli-Q® and 1 μL of DNA (30 ng/uL). StepOnePlus thermocycler ™ Real-

Time PCR System (Applied Biosystems, Foster City, USA) with the following cycling

used: 50 cycles of 60ºC for 30 seconds, 95°C (pre denaturation), 95°C for 10 minutes for

initial denaturation for 15 seconds (denaturation) and 60°C for 1 minute and 30 seconds

(primer annealing) and a final extension cycle of 30 seconds at 60ºC. The evaluation of

the results was performed by the StepOne Software v2.3.

2.3 Assessment of the severity of osteoarthritis

Individuals in the case group were asked to perform an X-ray of the affected joint. All

radiographs were analyzed by a radiologist, being useful not just to confirm the disease

diagnosis but also to classify the disease severity using the criteria described by Kellgren

and Lawrence [27] where the severity of osteoarthritis may be determined concerning

radiographic findings as follows: Grade 0 - no radiographic changes; Grade I - doubtful

space narrowing and possible marginal osteophytes; Grade II - possible joint space

narrowing and defined osteophytes observed; Grade III - defined joint space narrowing,

moderate multiple osteophytes, subchondral sclerosis and any possible deformity of

bone contour and grade IV - remarkable joint space narrowing, severe subchondral

sclerosis, definite deformity of bone contour and presence of large osteophytes (28).

Based on these criteria, the groups were re-categorized into two groups: with mild

to moderate (grades I and II) radiographic changes and severe changes (grade III and

IV).

2.4. Evaluation of functionality related to osteoarthritis

In order to assess the functional status of these patients, both Western Ontario

and McMaster Universities Osteoarthritis Index (WOMAC) and LEQUESNE Index were

used.

The WOMAC questionnaire was previously translated and validated to the

Portuguese language and nowadays it is considered the gold standard for OA functional

status evaluation according to the American College of Rheumatology. This

questionnaire involves questions regarding pain, stiffness and function, where the highest

score is the worst condition of the individual [29]. Additionally, the LEQUESNE Index was

also used. This questionnaire was also translated to portuguese version and is

recommended by the European League of Rheumatology (EULAR) to evaluate

osteoarthritis functional status, comprising eleven questions involving pain, discomfort

and functional limitation, specially related to functional status [30].

2.5. Statistical Analysis

The Statistical Package for Social Sciences 20.0 (SPSS, UK) was used for statistical

data analysis, with a confidence interval of 95% and a significance level of 5% (p <0.05)

established for all tests used.

For quantitative variables, the Shapiro-Wilk test was used and data with normal

distribution were presented by mean and standard deviation as the data without normal

distribution were presented as median and interquartile range.

The Chi Square was used as to verify that the genotype frequencies were in Hardy-

Weinberg equilibrium as well as to assess possible associations between the presence of

polymorphisms in the IL1β and TNF genes with the occurrence and severity of

osteoarthritis.

Moreover, the Mann-Whitney test was used to compare the functional status

measured by WOMAC questionnaire and LEQUESNE index of individual with different

genotypes.

In order to assess the influence of genetic polymorphisms on osteoarthritis etiology, a

multivariate model (Logistic Regression) was used, considering the following variables:

age, gender, body mass index and polymorphism of IL1β and TNF gene. At this analysis,

the dependent variable was the occurrence of osteoarthritis in elderly.

3. RESULTS

3.1 Sample characterization

In this case-control study, 257 elderly individuals were recruited, being 162 women

(63.0%) and 95 men (37.0%). Concerning this sample, 92 (35.8%) individuals have

osteoarthritis [21 men (22.8%) and 71 women (77.2%)], while 165 (64.2%) didn’t have

this disease [74 men (44.8%) and 91 women (55.2%)].

The mean age of this sample was 68.55 ± 5.2 (Minimum age: 60 and Maximum age:

82). No difference was observed between the age of the case (median = 69, interquartile

range = 9) compared to the age of the control group (median = 68, interquartile range =

7), according to the Mann-Whitney test (p = 0.26). Similarly, no difference was observed

in body mass index when compared the case (median = 28.4, interquartile range = 5.6)

and control group (median = 27.7, interquartile range = 7), according to the test Mann-

Whitney test (p = 0.60). Therefore, it may be assumed that the groups were similar

considering age and body mass index.

3.2. Gene polymorphism of IL1β and the susceptibility and functional status

related to osteoarthritis

It was observed that the distribution of genotype frequencies of the IL-1β gene is in

accordance with the Hardy-Weinberg equilibrium. Table 1 shows the distribution of

genotypes and alleles for the polymorphism +3954 C/T IL1β in subjects with osteoarthritis

and controls.

For healthy individuals, a higher proportion was found for the CC genotype (63.6%)

and the percentages of C and T alleles were 77% and 23%, respectively. For patients

with osteoarthritis, the CT and TT genotypes were more frequent (52.2%) and the

percentages of C and T alleles were 63% and 37% respectively.

It was observed that individuals who carriers the T allele have greater susceptibility to

osteoarthritis (2 = 8.7, p = 0.002, Table 1). Thus, carriers of the T allele had twice the

risk of developing osteoarthritis compared to individuals with the C allele (OR = 1.91,

95% CI: 1.14-3.20, p = 0.01).

In subjects with hip osteoarthritis, it was observed that the presence of the T allele is

a risk factor for hip joint injuries (2 = 7.2, p = 0.007, Table 2). Thus, we can estimate that

individuals carrying the T allele present four times higher risk of severe radiological

changes (OR = 4.82, 95% CI: 1,74- 13.4, p = 0.004) compared to individuals carrying the

C allele. On the other hand, no association was found between the polymorphism of the

IL1β and the degree of joint involvement in individuals with knee osteoarthritis according

to Chi Square Test (p> 0.05, Table 2). However, there was no association between the

polymorphism of IL1β and functional status of individuals with osteoarthritis analyzed by

both the WOMAC as LEQUESNE questionnaires (Mann-Whitney, p> 0.05).

Table 1 – Genotypes and alleles frequencies of IL1β gene and the presence of osteoarthritis.


No Yes

Genotype

Frequency

CC 105 (63.6%) 44 (47.8%)

CT 44 (26.7%) 28 (30.4%)

TT 16 (9.7%) 20 (21.8%)

Total 165 (100.0%) 92 (100.0%)

Allele

Frequency

C 254 (76.9%) 116 (63.0%)

T 76 (23.1%) 68 (37.0%)

Total 330 (100.0%) 184 (100.0%)


Table 2 – Relation between genotype frequencies distribution of IL1β and the severity of

knee and hip osteoarthritis according to the degree of radiological impairment.

Hip*


Genotype Frequency Mild Moderate/Severe

CC 15 (51.7%) 03 (25.0%)

CT 11 (38.0%) 02 (16.7%)

TT 03 (10.3%) 07 (58.3%)

Total 29 (100.0%) 12 (100.0%)

Allele Frequency Mild Moderate/Severe

C 41 (70.7%) 08 (33.3%)

T 17 (29.3%) 16 (66.7%)

Total 58 (100.0%) 24 (100.0%)

Knee

Genotype

Frequency


CC 10 (52.6%) 16 (50.0%)

CT 05 (26.3%) 10 (31.2%)

TT 04 (21.1%) 06 (18.8%)

Total 19 (100.0%) 32 (100.0%)


C 25 (65.8%) 42 (65.6%)

T 13 (34.2%) 22 (34.4%)

Total 38 (100.0%) 64 (100.0%)



IL1β genotypes analyzed with WOMAC and LEQUESNE questionnaires.

3.3. Gene polymorphism of TNF and susceptibility to osteoarthritis and related

functionality

The distribution of genotype frequencies are in accordance with Hardy-Weinberg

equilibrium. Table 3 shows the distribution of genotypes and alleles for the polymorphism

-308 G/A of TNF in patients with osteoarthritis and controls.

In healthy individuals, a greater proportion of GG genotype (67.9%) was found and

the percentage of G and A alleles were 78.2% and 21.8%, respectively. For patients with

osteoarthritis, the GG genotype was more frequent (53.3%) and the percentages of G

and A alleles were 67.9% and 32.1% respectively. It was observed that individuals

carrying the A allele have increased susceptibility to osteoarthritis (Chi square = 8.7, p =

0.003, Table 3). Thus, carriers of the A allele had twice the risk of developing

osteoarthritis compared to individuals with the G allele (OR = 1.7, 95% CI: 1.13-2.54, p =

0.01).

An association between the presence of the polymorphism in the TNF gene and the

severity of osteoarthritis assessed by radiographic lesions for both hip (2 = 3.9, p = 0.04,

Table 4) and knee osteoarthritis (2 = 4.0, p = 0.04, Table 4).

Thus, we can estimate that individuals carrying the A allele have three times greater

risk for serious radiological findings in cases of knee and hip osteoarthritis (OR = 2.92,

CC

CT/T

T

0

10

20

30

40

IL-1 Genotypes

LE

QU

ES

NE

CC

CT/T

T

0

20

40

60

80

100

IL-1 Genotypes

WO

MA

C

95% CI: 1.16-7.34; p = 0.03; OR= 3.2. 95% CI: 1.16-9.03; p= 0.04, respectively) when

compared to individuals carrying the G allele.

Additionally, it was observed at the multivariate analysis that the gender and the

polymorphisms of IL1β and TNF genes were associated with osteoarthritis (Table 5).

Regarding the functional status of elderly patients with osteoarthritis, it was

observed that individuals carrying the A allele have a poorer functionality according to the

LEQUESNE questionnaire (Mann-Whitney, p = 0.04), being this data shown in figure 1.

However, no differences between groups concerning the WOMAC questionnaire were

observed (Mann-Whitney test, p>0.05).

Table 3 - Genotype and allele frequencies of TNF gene and the presence of osteoarthritis.


No Yes

Genotype Frequency

GG 112 (67.9%) 49 (53.3%)

GA 34 (20.6%) 27 (29.3%)

AA 19 (11.5%) 16 (17.4%)

Total 165 (100.0%) 92 (100.0%)

Allele Frequency

G 258 (78.2%) 125 (67.9%)

A 72 (21.8%) 59 (32.1%)

Total 330 (100.0%) 184 (100.0%)


Table 4 – Relationship between the genotype and allele distributions of the TNF –G308A


impairment.

Hip

Genotype

Frequency



GG 19 (65.5%) 05 (41.7%)

GA 08 (27.6%) 03 (25.0%)

AA 02 (6.9%) 04 (33.3%)

Total 29 (100.0%) 12 (100.0%)


G 46 (79.3%) 13 (54.2%)

A 12 (21.7%) 11 (45.8%)

Total 58 (100.0%) 24 (100.0%)

Knee

Genotype

Frequency


GG 14 (73.7%) 11 (34.4%)

GA 02 (10.5%) 14 (43.7%)

AA 03 (15.8%) 07 (21.9%)

Total 19 (100.0%) 32 (100.0%)


G 30 (78.9%) 36 (56.2%)

A 08 (21.1%) 28 (43.8%)

Total 38 (100.0%) 64 (100.0%)


Table 5 – Multivariate analysis (Logistic Regression) considering the following variables:

age, gender, body mass index and polymorphism of IL1β and TNF gene with the

prevalence of osteoarthritis in elderly.

Variables Univariate analysis Multivariate analysis

Unadjusted Odds ratio

CI (95%) p Adjusted Odds ratio

CI (95%) p

Age Range

60-70 years old 1 - - 1 - -

> 70 years old 1.03 - 0.21 - - -

Gender

Male 1 - - 1

Female 2.75 1.55 – 4.89 0.001 2.67 1.48 – 4.8 0.001*

Body Mass Index

Normal 1 - - 1 - -

Overweight/ Obesity

1.28 0.77 – 2.20 0.35 - - -

IL1β Genotypes

CC 1 - - 1 - -

TT+CT 1.91 1.14 – 3.20 0.01 1.79 1.05 – 3.1 0.03*

TNF Genotypes

GG 1 - 1 - 0.02*

AA+GA 1.86 1.1 – 3.1 0.02 1.87 1.1 – 3.2

* Statistically significant at the final model.


TNF genotypes analyzed with WOMAC and LEQUESNE questionnaires.

GG

GA/A

A

0

20

40

60

80

100

TNF- Genotypes

WO

MA

C

GG

GA/A

A

0

10

20

30

40

*

TNF- Genotypes

LE

QU

ES

NE

4. DISCUSSION

OA is a progressive degenerative disease of the cartilage and articular structures,

with important inflammatory component involved. Proinflammatory cytokines, such as

IL1β and TNF, are important mediators of this process by activating the production of

metalloproteinases by chondrocytes, and stimulating the production of other proteins and

cytokines, resulting in catabolic action and suppression of articular anabolism (16, 31).

The polymorphisms of these cytokine genes are studied in understanding the complex

etiology and pathophysiology of OA, representing a potential tool for identifying risk for

the disease, which can be useful in preventive and clinical management (18-26). In this

study we assessmnent the polymorphisms +3954 C/T IL-1β and -308 G/A TNF with the

presence and severity of OA in the elderly.

The results for IL1β demonstrated a significant association between the

polymorphism and the presence of disease. Individuals carrying the T allele have higher

incidence of OA compared with individuals with CC genotype. This result is in agreement

with previous studies that showed correlations between SNP of IL1β and / or rare

haplotypes with hip OA and knee OA (18,32,33). On the other hand, conflicts with the

findings of other studies (19,20), including a large meta-analysis of Kerkhof et al. (34),

who showed no associations of the polymorphisms of IL1β with osteoarthritis of the knee

and hip. According to Meulenbelt et al. (35), the association of hip OA observed in

carriers of allele T of the IL1β +3954 polymorphism appears to represent an innate

proinflamatory and cartilage degradation-prone profile. This observation may be

explained either by a relatively high responde of chondrocytes in the hip cartilage to IL-

1B stimulation or by the relatively large mechanical stresses as this joint leading to

increased IL-1B production by the chondrocytes. This process could explain the fact that

the polymorphism within IL1β is most likely to be observed in patients with OA of the hip.

As well as, we not found an association with this locus in the knee joint. As reported by

Meukebbelt et al. (2004), maybe this lack of association could be that the genetic effect

of this locus on knee OA is relatively small than compared with the effects of other

environmental risk factors.

Regarding the degree of radiation injury, the present study showed no correlation

between polymorphism and radiographs of the hip and knee. Finally, in relation to IL1β,

there was no correlation between the polymorphism and the degree of functional

impairment, assessed by LEQUESNE and WOMAC questionnaires.

In relation to TNF gene, the results showed significant correlation between the

presence of the polymorphism and the presence of OA: individuals with polymorphic

allele (GA and AA genotypes) have a higher incidence of the disease. Likewise, a

significant association between the GA and AA genotypes and worse functional status

(assessed by LEQUESNE questionnaire) when compared to homozygous (GG)

individuals. However, similar results were not demonstrated through WOMAC

questionnaire. One hypothesis that could explain this finding is that the LEQUESNE

questionnaire has a higher profile assessment for the painful component when compared

to the WOMAC questionnaire, which seems to have a closer relationship with the

inflammatory component in question. Regarding the degree of radiological involvement,

the SNP -308 G/A has also meant a direct and significant association between the

presence of the A allele and the severity of the lesions observed both hips and the knee

radiographs. Some studies have found no relationship between the -308 SNP TNF and

OA (26). In contrast, there are several studies with results agreeing with our data, where

the presence of the A allele at position -308 has a direct relation to the prevalence and

severity of knee and hip OA (23,24,25,36). However, it is important to state that these

studies have usual specific findings and none of them had evaluated the genetic

polymorphism with presence and severity of OA as well as the functional status of the

same individuals.

One limitation of this study is the inability to classify patients into ethnic subgroups,

due to the wide variability in the population sample. Another limitation is the lack of serum

as synovial levels of cytokines. On the other hand, the study groups showed no

differences in body mass index, which excludes obesity bias, admittedly a predisposing

factor for this disease (37).

A possible explanation for all the results observed is that, in fact, polymorphism

+3954 C/T IL1β may result in a function gain for the IL-1B and the polymorphism -308

G/A of TNF may be related to a higher expression of TNFA. Due to the proinflammatory

action of these two cytokines, an increased secretion of IL-1B may lead to a shift of the

metabolic balance in cartilage in favor of matrix loss, which may ultimately result in OA.

In addition, TNFA appears to play a pivotal role in the imbalance between anabolic and

catabolic process of OA patients. It can down-regulate the synthesis of major

extracellular matrix components through the inhibition of the anabolic activity of

chondrocytes (24). The TNF also stimulates chondrocytes to release matrix

metalloproteinases (MMPs), which have the ability to degradate cartilage matrix proteins

(38). The SNPs of IL1 and TNF appears to have a causal relationship closer, but the

lack of uniformity of the findings and conflicts observed in the literature may be due to

factors related to ethnicity, gender or other yet unidentified. The multifactorial nature of

OA makes it a condition of great complexity. Polymorphisms are undoubtedly one of the

factors involved in the genesis and progression of OA. Therefore, this study contributed

to elucidate the complex mechanisms involved in genetic background of OA. However,

further studies are needed in order to clarify the mechanisms of this important disease.

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4. CONCLUSÃO GERAL

Com a realização deste estudo, foi possível concluir que:

- A presença do polimorfismo do gene IL6 está relacionada à osteoartrite, sendo que

os portadores do alelo C apresentam menor chance de apresentar a doença;

- Os indivíduos portadores do alelo C do gene IL6 possuem menores graus de lesões

radiológicas nas articulações de joelho e quadril;

- A presença do alelo C do gene IL6 está relacionada a uma melhor funcionalidade

em idosos com osteoartrite;

- A presença do alelo T da IL1β está associada à predisposição para osteoartrite e

evidenciou um risco de 1,79 vezes para a doença; os idosos portadores deste alelo

apresentaram maior prevalência de lesões radiográficas em casos de osteoartrite de

quadril;

- Não foi observada associação entre o polimorfismo no gene IL1β e a funcionalidade

de idosos com osteoartrite;

- O alelo A do gene TNF está associado à predisposição para osteoartrite, com um

risco aumentado de 1,87 vezes; a presença do alelo A relaciona-se com a maior

ocorrência de lesões radiográficas tanto em casos de osteoartrite de quadril quanto de

joelho;

- A presença do alelo A do gene TNF está associado à pior funcionalidade em idosos

portadores de osteoartrite;

- Portanto, nossos resultados demonstram que determinados alelos dos genes

estudados desempenham um papel na etiologia e gravidade da OA de quadril e joelho

em idosos.

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APÊNDICES

APÊNDICE A - QUESTIONÁRIO DE COMORBIDADES

Nome:

...............................................................................................................................

Data: ........................................

1) O Sr./Sra. teve alguma doença grave no passado? Sim Não

Se sim,

qual(is)?……………………………………………………………………............................

.......................................................................................................................

Com qual idade (teve o

diagnóstico?)…………………................................................................................

2) O Sr./Sra. tem:

Doença pulmonar /

respiratória

Sim

Não

asma enfisema bronquite outra

qual?______________

Doença reumática Sim

Não

artrite artrose gota outra

qual?______________

Doença do coração Sim

Não

arritmia infarto cir. revasc. ins. cardíaca

outra qual?____

Pressão alta Sim

Não

Diabetes Sim

Não

Osteoporose Sim

Não

Problema de tireóide Sim hipotireodismo hipertireoidismo outro

qual?______________

Não

Problema vascular Sim

Não

trombose IAPC varizes AVE outro

qual?______________

Alergia Sim

Não

poeira prod. químico animais outra

qual?______________

Doença cardíaca na família Sim

Não

arritmia infarto cir. revasc. outra

qual?______________

Doença gastrointestinal Sim

Não

gastrite úlcera constipação outra

qual?______________

Doença neurológica Sim

Não

Alzheimer Parkinson outro

qual?______________

3) O Sr./Sra já foi hospitalizado(a) por um período maior do que um dia?

Sim Não

Se sim, quando (aproximadamente) e por quanto

tempo?….............................................

Por qual razão?………………………………........................................................

Realizou procedimento cirúrgico? Sim Não

Qual? ....................................................................

4) Consultou o médico pelo menos 1 vez nos últimos 12 meses?

Sim Não

Se sim, quando (aproximadamente) e por quantas

vezes?…................................................................................................................

Por qual razão?……………………………………………........................................

5) O Sr./Sra teve algum problema ortopédico que gerou alguma limitação importante

nas suas atividades da vida diária? (por exemplo, problemas sérios nas costas ou

joelhos) Sim Não

Se sim, qual era o problema e desde quando /até

quando?......................................……………………..............................................

6) O Sr./Sra ainda trabalha (profissionalmente)?

Sim Não

Se sim, por quantas horas e quantos dias por semana?

………………………………………………………...................................................

7) Qual é a atividade mais cansativa que o Sr./Sra realiza toda semana?

…………………………………….............................................................................

ANEXOS

ANEXO A – CARTA DE APROVAÇÃO DO COMITÊ DE ÉTICA EM PESQUISA

ANEXO B - QUESTIONÁRIO DE ALGOFUNCIONAL DE LEQUESNE

Nome: _________________________________________ ID: _____________

Data: _____/_____/______

QUESITOS PONTUAÇÃO

Durante o descanso noturno:

Nenhum ou insignificante

Somente em movimento ou em certas posições

Mesmo sem movimento

Rigidez matinal ou dor que diminui após se levantar

1 minuto ou menos

Mais de 1 minuto porém menos de 15 minutos

Mais 15 minutos

Depois de andar por 30 minutos

Enquanto anda

Nenhuma

Somente depois de andar alguma distância

Logo depois de começar a andar e aumenta se continuar a

andar

Depois de começar a andar, não aumentando

Ao ficar sentado por muito tempo (2 horas) (somente se

quadril)

Enquanto se levanta da cadeira, sem ajuda dos braços

(somente se joelho)

Máxima distância caminhada/andada (pode caminhar com

0

1

2

0

1

2

0-1

0

1

2

1

0-1

0-1

dor):

Sem limite

Mais de 1 km, porém com alguma dificuldade

Aproximadamente 1 km (em + ou - 15 minutos)

De 500 a 900 metros (aproximadamente 8 a 15 minutos)

De 300 a 500 metros

De 100 a 300 metros

Menos de 100 metros

Com uma bengala ou muleta

Com 2 muletas ou 2 bengalas

Atividades do dia-a-dia/vida diária (Aplicar somente para

quadril)*

Colocar as meias inclinando-se para frente

Pegar um objeto no chão

Subir ou descer um andar de escadas

Pode entrar e sair de um carro

Atividades do dia-a-dia/vida diária (aplicar somente para

joelho)*

Consegue subir um andar de escadas

Consegue descer um andar de escadas

Agachar-se ou ajoelhar-se

Consegue andar em chão irregular / esburacado

CLASSIFICAÇÃO

Sem dificuldade: 0

Com pouca dificuldade: 0,5

Com dificuldade: 1

Com muita dificuldade: 1,5

Incapaz: 2

0

1

2

3

4

5

6

1

2

0-2

0-2

0-2

0-2

0-2

0-2

0-2

0-2

0

0,5

1

1,5

2

SOMA DA PONTUAÇÃO

Extremamente grave (igual ou maior que 14 pontos)

Muito grave (11 a 13 pontos)

Grave (8 a 10 pontos)

Moderada (5 a 7 pontos)

Pouco acometimento (1 a 4 pontos)

≥ 14

11-13

8-10

5-7

1-4

ANEXO C - QUESTIONÁRIO WOMAC

Instruções:

1- Se você indicar 0 (zero) corresponde a não ter nenhuma dor. 2- Se você

indicar 10 (dez) corresponde a dor máxima. 3- Quanto mais seu número se

aproxima de 0 (zero), sua dor é menos importante. 4- Quanto mais seu

número se aproxima de 10 (dez), sua dor é mais importante.

1- SESSÃO A – DOR

Como você avalia sua DOR

1.1 - Quando você caminha num terreno plano (escala numérica analógica)

1.2 - Quando você sobe ou desce uma escada (escala numérica analógica)

1.3 - Quando você esta deitado(a) à noite (escala numérica analógica)

1.4 - Quando você esta sentado(a) ou deitado(a) (escala numérica analógica)

1.5 - Quando você se levanta em pé (escala numérica analógica)

2- SESSÃO B – RIGIDEZ

Qual é a severidade de sua RIGIDEZ

2.1 - Quando você se levanta pela manhã (escala numérica analógica)

2.2 - Depois de ficar sentado(a) deitado(a) ou repousando durante o dia(escala

numérica analógica)

SESSÃO C – INCAPACIDADE

Qual é o grau de dificuldade que você apresenta?

3.1 - Quando você desce uma escada (escala numérica analógica)

3.2 - Quando você sobe uma escada (escala numérica analógica)

3.3 - Quando você levanta-se da posição sentado(a) (escala numérica analógica)

3.4 - Quando você esta em pé (escala numérica analógica)

3.5 - Quando você se inclina para a frente (escala numérica analógica)

3.6 - Quando você caminha sobre um terreno plano (escala numérica analógica)

3.7 - Quando você entra e sai da condução (escala numérica analógica)

3.8 - Quando você vai fazer compras (escala numérica analógica)

3.9 - Quando você coloca suas meias (escala numérica analógica)

3.10 - Quando você sai do leito (escala numérica analógica)

3.11 - Quando você retira suas meias (escala numérica analógica)

3.12 - Quando você se estende no leito (escala numérica analógica)

3.13 - Quando você entra ou sai do banho (escala numérica analógica)

3.14 - Quando você está sentado(a) (escala numérica analógica)

3.15 - Quando você faz a sua higiene e levanta-se do toalete (escala numérica

analógica)

3.16 - Quando você faz um trabalho doméstico maior (escala numérica analógica)

3.17 - Quando você faz um trabalho doméstico leve (escala numérica analógica)

ANEXO D – NORMAS PARA FORMATAÇÃO DA REVISTA CYTOKINE

GUIDE FOR AUTHORS

INTRODUCTION

The official journal of the International Cytokine Society Cytokine is devoted

exclusively to the study of the molecular biology, biochemistry, immunology,

diagnostic and clinical applications of all known interleukins, hematopoietic factors,

growth factors, cytotoxins, interferons, and new cytokines, Cytokine provides

comprehensive coverage of cytokines and their receptors, 12 times a year, by

publishing original high quality refereed scientific papers from prominent investigators

in both the academic and industrial sectors.

TYPES OF PAPER

Review Articles are comprehensive appraisals of research and clinical outcomes in a

field of current interest related to cytokine biology. All reviews are subject to the

normal peer review process. Reviews are mostly invited by the Reviews Associate

Editor but interested authors may contact Dr. Sarah Gaffen at [email protected] to

discuss ideas or present an outline.

Research Articles are full-length descriptions of original research. The scope may

include basic science, clinical results, or applications. These manuscripts will

undergo standard review.

Short Communications. These submissions will also undergo standard review.

Manuscripts should not exceed 2000 words plus no more than 15 references. Your

cover letter must include your word count. Results and Discussion sections may be

combined. No more than 2 Figures and/or Tables

should be included.

BEFORE YOU BEGIN

Ethics in publishing

For information on Ethics in publishing and Ethical guidelines for journal publication

see http://www.elsevier.com/publishingethics and http://www.elsevier.com/journal-

authors/ethics.

Human and animal rights

If the work involves the use of animal or human subjects, the author should ensure

that the work described has been carried out in accordance with The Code of Ethics

of the World Medical Association (Declaration of Helsinki) for experiments involving

humans http://www.wma.net/en/30publications/10policies/b3/index.html; EU Directive

2010/63/EU for animal experiments

http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm;

Uniform Requirements for manuscripts submitted to Biomedical journals

http://www.icmje.org.

Authors should include a statement in the manuscript that informed consent was

obtained for experimentation with human subjects. The privacy rights of human

subjects must always be observed.

CONFLICT OF INTEREST

All authors are requested to disclose any actual or potential conflict of interest

including any financial, personal or other relationships with other people or

organizations within three years of beginning the submitted work that could

inappropriately influence, or be perceived to influence, their work. See

also http://www.elsevier.com/conflictsofinterest. Further information and an example

of a Conflict of Interest form can be found at:

http://help.elsevier.com/app/answers/detail/a_id/286/p/7923.

Submission declaration and verification

Submission of an article implies that the work described has not been published

previously (except in the form of an abstract or as part of a published lecture or

academic thesis or as an electronic preprint, see

http://www.elsevier.com/journal-authors/ethics

http://www.elsevier.com/journal-authors/ethics

http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm

http://www.icmje.org/

http://help.elsevier.com/app/answers/detail/a_id/286/p/7923

http://www.elsevier.com/postingpolicy), that it is not under consideration for

publication elsewhere, that its publication is approved by all authors and tacitly or

explicitly by the responsible authorities where the work was carried out, and that, if

accepted, it will not be published elsewhere in the same form, in English or in any

other language, including electronically without the written consent of the copyright-

holder. To verify originality, your article may be checked by the originality detection

service CrossCheck http://www.elsevier.com/editors/plagdetect.

AUTHORSHIP

All authors should have made substantial contributions to all of the following: (1) the

conception and design of the study, or acquisition of data, or analysis and

interpretation of data, (2) drafting the article or revising it critically for important

intellectual content, (3) final approval of the version to be submitted.

CHANGES TO AUTHORSHIP

This policy concerns the addition, deletion, or rearrangement of author names in the

authorship of accepted manuscripts:

Before the accepted manuscript is published in an online issue: Requests to add or

remove an author, or to rearrange the author names, must be sent to the Journal

Manager from the corresponding author of the accepted manuscript and must

include: (a) the reason the name should be added or removed, or the author names

rearranged and (b) written confirmation (e-mail, fax, letter) from all authors that they

agree with the addition, removal or rearrangement. In the case of addition or removal

of authors, this includes confirmation from the author being added or removed.

Requests that are not sent by the corresponding author will be forwarded by the

Journal Manager to the corresponding author, who must follow the procedure as

described above. Note that: (1) Journal Managers will inform the Journal Editors of

any such requests and (2) publication of the accepted manuscript in an online issue

is suspended until authorship has been agreed.

After the accepted manuscript is published in an online issue: Any requests to add,

delete, or rearrange author names in an article published in an online issue will follow

the same policies as noted above and result in a corrigendum.

http://www.elsevier.com/editors/plagdetect

ARTICLE TRANSFER SERVICE

This journal is part of our Article Transfer Service. This means that if the Editor feels

your article is more suitable in one of our other participating journals, then you may

be asked to consider transferring the article to one of those. If you agree, your article

will be transferred automatically on your behalf with no need to reformat. More

information about this can be found here: http://www.elsevier.com/authors/article-

transfer-service.

COPYRIGHT

This journal offers authors a choice in publishing their research: Open access and

Subscription.

FOR SUBSCRIPTION ARTICLES

Upon acceptance of an article, authors will be asked to complete a 'Journal

Publishing Agreement' (for more information on this and copyright, see

http://www.elsevier.com/copyright). An e-mail will be sent to the corresponding author

confirming receipt of the manuscript together with a 'Journal Publishing Agreement'

form or a link to the online version of this agreement.

Subscribers may reproduce tables of contents or prepare lists of articles including

abstracts for internal circulation within their institutions. Permission of the Publisher is

required for resale or distribution outside the institution and for all other derivative

works, including compilations and translations (please consult

http://www.elsevier.com/permissions). If excerpts from other copyrighted works are

included, the author(s) must obtain written permission from the copyright owners and

credit the source(s) in the article. Elsevier has preprinted forms for use by authors in

these cases: please consult http://www.elsevier.com/permissions.

FOR OPEN ACCESS ARTICLES

Upon acceptance of an article, authors will be asked to complete an 'Exclusive

License Agreement' (for more information see

http://www.elsevier.com/OAauthoragreement). Permitted reuse of open access

http://www.elsevier.com/authors/article-transfer-service

http://www.elsevier.com/authors/article-transfer-service

articles is determined by the author's choice of user license (see

http://www.elsevier.com/openaccesslicenses).

RETAINED AUTHOR RIGHTS

As an author you (or your employer or institution) retain certain rights. For more

information on author rights for: Subscription articles please see

http://www.elsevier.com/journal-authors/author-rights-and-responsibilities. Open

access articles please see http://www.elsevier.com/OAauthoragreement.

ROLE OF THE FUNDING SOURCE

You are requested to identify who provided financial support for the conduct of the

research and/or preparation of the article and to briefly describe the role of the

sponsor(s), if any, in study design; in the collection, analysis and interpretation of

data; in the writing of the report; and in the decision to submit the article for

publication. If the funding source(s) had no such involvement then this should be

stated. Role of the funding source You are requested to identify who provided

financial support for the conduct of the research and/or preparation of the article and

to briefly describe the role of the sponsor(s), if any, in study design; in the collection,

analysis and interpretation of data; in the writing of the report; and in the decision to

submit the article for publication. If the funding source(s) had no such involvement

then this should be stated of published literature.

CONCLUSIONS

The main conclusions of the study may be presented in a short Conclusions section,

which may stand alone or form a subsection of a Discussion or Results and

Discussion section.

Essential title page information

• Title. Concise and informative. Titles are often used in information-retrieval

systems. Avoid abbreviations and formulae where possible.

• Author names and affiliations. Where the family name may be ambiguous (e.g., a

double name), please indicate this clearly. Present the authors' affiliation addresses

(where the actual work was done) below the names. Indicate all affiliations with a

http://www.elsevier.com/openaccesslicenses

http://www.elsevier.com/journal-authors/author-rights-and-responsibilities

http://www.elsevier.com/OAauthoragreement

lower-case superscript letter immediately after the author's name and in front of the

appropriate address. Provide the full postal address of each affiliation, including the

country name and, if available, the e-mail address of each author.

• Corresponding author. Clearly indicate who will handle correspondence at all

stages of refereeing and publication, also post-publication. Ensure that phone

numbers (with country and area code) are provided in addition to the e-mail address

and the complete postal address.

Contact details must be kept up to date by the corresponding author.

• Present/permanent address. If an author has moved since the work described in the

article was done, or was visiting at the time, a 'Present address' (or 'Permanent

address') may be indicated as a footnote to that author's name. The address at which

the author actually did the work must be retained as the main, affiliation address.

Superscript Arabic numerals are used for such footnotes.

Abstract

A concise and factual abstract is required. The abstract should state briefly the

purpose of the research, the principal results and major conclusions. An abstract is

often presented separately from the article, so it must be able to stand alone. For this

reason, References should be avoided, but if essential, then cite the author(s) and

year(s). Also, non-standard or uncommon abbreviations should be avoided, but if

essential they must be defined at their first mention in the abstract itself.

Graphical abstract

A Graphical abstract is optional and should summarize the contents of the article in a

concise, pictorial form designed to capture the attention of a wide readership online.

Authors must provide images that clearly represent the work described in the article.

Graphical abstracts should be submitted as a separate file in the online submission

system. Image size: Please provide an image with a minimum of 531 × 1328 pixels (h

× w) or proportionally more. The image should be readable at a size of 5 × 13 cm

using a regular screen resolution of 96 dpi. Preferred file types: TIFF, EPS, PDF or

MS Office files. See http://www.elsevier.com/graphicalabstracts for examples.

Authors can make use of Elsevier's Illustration and Enhancement service to ensure

the best presentation of their images also in accordance with all technical

requirements: Illustration Service.

Highlights

Highlights are mandatory for this journal. They consist of a short collection of bullet

points that convey the core findings of the article and should be submitted in a

separate file in the online submission system. Please use 'Highlights' in the file name

and include 3 to 5 bullet points (maximum 85 characters, including spaces, per bullet

point). See http://www.elsevier.com/highlights for examples.

Keywords

Immediately after the abstract, provide a maximum of 6 keywords, using American

spelling and avoiding general and plural terms and multiple concepts (avoid, for

example, 'and', 'of'). Be sparing with abbreviations: only abbreviations firmly

established in the field may be eligible. These keywords will be used for indexing

purposes.

Abbreviations

Define abbreviations that are not standard in this field in a footnote to be placed on

the first page of the article. Such abbreviations that are unavoidable in the abstract

must be defined at their first mention there, as well as in the footnote. Ensure

consistency of abbreviations throughout the article.

Acknowledgements

Collate acknowledgements in a separate section at the end of the article before the

references and do not, therefore, include them on the title page, as a footnote to the

title or otherwise. List here those individuals who provided help during the research

(e.g., providing language help, writing assistance or proof reading the article, etc.).

SUBMISSION CHECKLIST

The following list will be useful during the final checking of an article prior to sending

it to the journal for review. Please consult this Guide for Authors for further details of

any item.

Ensure that the following items are present:

One author has been designated as the corresponding author with contact details:

• E-mail address

• Full postal address

• Telephone

All necessary files have been uploaded, and contain:

• Keywords

• All figure captions

• All tables (including title, description, footnotes)

Further considerations

• Manuscript has been 'spell-checked' and 'grammar-checked'

• All references mentioned in the Reference list are cited in the text, and vice versa

• Permission has been obtained for use of copyrighted material from other sources

(including the Web)

• Color figures are clearly marked as being intended for color reproduction on the

Web (free of charge) and in print, or to be reproduced in color on the Web (free of

charge) and in black-and-white in print

• If only color on the Web is required, black-and-white versions of the figures are also

supplied for printing purposes

For any further information please visit our customer support site at

http://support.elsevier.com.

AFTER ACCEPTANCE

Use of the Digital Object Identifier

The Digital Object Identifier (DOI) may be used to cite and link to electronic

documents. The DOI consists of a unique alpha-numeric character string which is

assigned to a document by the publisher upon the initial electronic publication. The

assigned DOI never changes. Therefore, it is an ideal medium for citing a document,

particularly 'Articles in press' because they have not yet received their full

bibliographic information. Example of a correctly given DOI (in URL format; here an

article in the journal Physics Letters B):

http://dx.doi.org/10.1016/j.physletb.2010.09.059 When you use a DOI to create links

to documents on the web, the DOIs are guaranteed never to change.

ONLINE PROOF CORRECTION

Corresponding authors will receive an e-mail with a link to our online proofing

system, allowing annotation and correction of proofs online. The environment is

similar to MS Word: in addition to editing text, you can also comment on

figures/tables and answer questions from the Copy Editor.

Web-based proofing provides a faster and less error-prone process by allowing you

to directly type your corrections, eliminating the potential introduction of errors.

If preferred, you can still choose to annotate and upload your edits on the PDF

version. All instructions for proofing will be given in the e-mail we send to authors,

including alternative methods to the online version and PDF.

http://dx.doi.org/10.1016/j.physletb.2010.09.059

ANEXO E – CLINICAL AND EXPERIMENTAL RHEUMATOLOGY

GUIDELINES FOR AUTHORS

Clinical and Experimental Rheumatology is a bi-monthly journal which publishes original papers on clinical or experimental research pertinent to the rheumatic diseases; work on connective tissue diseases and other immune disorders also are within the journal's scope. Contributors may submit Editorials, Original Articles, Rapid Papers, Review Articles, Case Reports and Letters to the Editor. Every issue also contains a section dedicated to the area of Pediatric Rheumatology.

Articles will be considered for publication on the condition that they are submitted solely to Clinical and Experimental Rheumatology. The statements and opinions expressed in the articles and communications are those of the Authors, and the Editors and publisher disclaim any responsibility or liability for these statements.

Manuscripts will be acknowledged on receipt. All submitted articles will be read by the Editors and sent to two or more members of the Editorial Board or outside consultants for formal review. The referees' comments will be forwarded to the Authors.

Starting with manuscripts submitted from September 1, 2014 a page charge will be assessed for articles of over 2 printed pages: for each page exceeding this limit, the Authors will be charged € 100.00 per additional page. Instructions for payment will be sent to authors along with the proofs. There is no charge for solicited articles or for correspondence (Letters to the Editors and letters commenting on published articles).

Table of Contents

Submission of Manuscripts Types of Articles References Tables and Figures Send submissions to:

Submission of Manuscripts

Please click on the "Submission on line" button on top of this page to submit a manuscript, and follow the instructions on how to submit a manuscript. Manuscripts should be accompanied by a cover letter containing:

a. a statement by the Authors that the article has not been published and is not under consideration for publication elsewhere;

b. information on prior publication or the submission elsewhere of any part of the work (note: if any of the material in the article has already been published in other than abstract form, enclose a copy of the previous publication);

c. a statement of financial or other relationships that could lead to a conflict of interest;

http://www.clinexprheumatol.org/guidelines-authors.asp#submission

http://www.clinexprheumatol.org/guidelines-authors.asp#types

http://www.clinexprheumatol.org/guidelines-authors.asp#References

http://www.clinexprheumatol.org/guidelines-authors.asp#Tables_and_Figures

http://www.clinexprheumatol.org/guidelines-authors.asp#send

d. a statement that the manuscript has been read and approved by all of the authors;

e. in cases of excessive authorship (more than 6 authors for an original article, or more than 3 for a case report) an explanation of the contribution of each author must be provided;

f. the letter may also give information regarding the type of article the manuscript represents and why the authors feel it would be interest to the readers of Clinical and Experimental Rheumatology;

g. The manuscript must be accompanied by copies of permissions to reproduce published material, report sensitive personal information, to use illustrations of identifiable persons, or to name persons for their contributions.

In preparing their manuscripts authors should adhere to the norms laid out in the Uniform Requirements for Manuscripts Submitted to Biomedical Journals (JAMA 1993; 269: 2282-6). The style should be clear and concise, and authors not fluent in English should have their paper corrected by a native English speaker, preferably one with a scientific background. Manuscripts must be typed double-spaced with wide margins (at least 2 cm on all sides). Single-spaced material is not acceptable. Each manuscript should contain in one single file: one page with title, authors names and all affiliations; one page with abstract and key words; text; acknowledgements; references; individual tables; and legends. Number the pages consecutively, beginning with the title page. The title page should include:

a. a concise but informative title (do not use acronyms);

b. the first name, middle initial and last name of each author, with their highest academic degree(s) and institutional affiliation;

c. the name of the department(s) and institution(s) to which the work should be attributed;

d. the name, address, telephone and fax numbers, and E-mail address of the author responsible for correspondence;

e. the name and address of the author to whom reprint requests should be addressed, or the statement that reprints will not be available from the author;

f. a short running title (45 characters or less).

The second page should contain a concise abstract, proportional in length to the paper itself, and 3 to 10 indexing terms drawn from the Medical Subject Headings (MeSH) list of Index Medicus. All measurements, including hematologic and clinical chemistry data, should be reported in metric units in terms of the International System of Units (SI). Only standard abbreviations should be used.

Table of Contents

Types of articles

Editorials are brief discussions focusing on the significance and practical implications of topics of current interest.

http://www.clinexprheumatol.org/guidelines-authors.asp#toc

Rapid Papers report new and original scientific work supported by adequate data. The text should not exceed 1500 words. Include a structured abstract and a maximum of 15 references (see below for format). Submissions will undergo the standard review process and a decision will be taken within one month. Accepted papers will be published immediately as they stand.

Original articles are reports of new and original work or descriptions of a consolidated body of experience in a given field. Epidemiological and psychosocial studies are also welcome. The text should not exceed 3500 words (see below for format). Starting with manuscripts submitted from March 1, 2012 there will be a page charge applied to articles over 4 printed pages. For each page exceeding this limit the authors will be charged € 100.00 per additional page. Instructions for payment will be sent to authors along with the proofs.

Brief papers are short communications analogous in content to the original articles (see below for format). The text should not exceed 1500 words, and the bibliography no more than 15 references.

Reviews are welcome; they will undergo the same review process as other papers. The text should not exceed 4000 words, and should be preceded by a brief abstract.

Case reports will be considered for publication only if they describe extremely unusual cases or are of particular interest to the clinician. The presentation should be concise (max. 1000 words), and include a short abstract, a clear exposition of the case, and a maximum of 15 references and three tables or figures.

Original articles in the area of Pediatric Rheumatology are welcome. Letters to the Editor may report original work, address problems of current

interest, or comment on articles that have recently appeared in the journal. In the last case, the letter will be sent to the authors of the article in question and their reply, if any, will be published together with the letter. Letters should not exceed 600 words, may include a maximum of one table or figure and 10 bibliographical references, and must be furnished with a title and a set of key words.

NEW: Letter to Editor Rheumatology: Commentaries and Controversies (LER) is the international free to view and peer-reviewed online journal, now supported jointly by Clinical and Experimental Rheumatology and the Rheumatology Society of Turkey. The main mission of LER is to provide a platform for comments about published clinical or basic science articles in the wide speciality of rheumatology and the related disciplines. The recommendations of the International Committee of Medical Journal Editors (www.icmje.org) can be a guide in the preparation of such letters. Hypothesis generating short manuscripts, brief original observations, case-records, viewpoints and even rebuttals with peer reviewers are also within the scope of LER. If a letter commenting on an article is accepted for publication, authors of the addressed article will be notified, giving them the opportunity to respond. Once a letter is accepted it will immediately appear ahead of print free to view

athttp://www.clinexprheumatol.org; once a year Clinical and Experimental

Rheumatology will collect all LER accepted letters in a periodic annual Supplement

which will appear both online and in printed paper issue. All the published letters

will also appear in PubMed. Letters already accepted by the old LER can still be

viewed at http://www.lettertoeditor.org.

http://www.icmje.org/

http://www.clinexprheumatol.org/

http://www.lettertoeditor.org/

Letters commenting on published papers: These will be considered for

publication with no time limit following the publication of the original article. The

editors encourage the authors to disclose if their letter has been previously

submitted to and not accepted by the journal where the commented article had

been published. Letters commenting on published papers should have a maximum

of 600 words and an unstructured abstract of up to 50 words. They may contain 2

figures or tables and a maximum of 10 references. Letters about original

thoughts and observations: Critical opinions on established knowledge ,

hypotheses, brief and original case reports or brief manuscripts in a letter format

about clinical and/or basic science observations are also welcome. These letters

may have a maximum of 1000 words and an unstructured abstract of up to 100

words. They may contain up to 4 figures or tables and a maximum of 15

references. Manuscript preparation: All manuscripts should be prepared in the

form of a letter. They should be typed double-spaced and have wide margins. A

title page, containing the title of the article, the names, academic affiliations of the

authors, the contact numbers and e-mail address of the corresponding author

should accompany each manuscript. Tables, figures, illustrations: Prepare each

table double spaced on a separate sheet. Supply a title for each table. Number

tables consecutively in the order of their citation in the text. Place explanations in

footnotes. Supply a title for each of them and give explanations at the bottom if

needed. Number figures and illustrations consecutively in the order of their

citations in the text. References: These should be prepared in PubMed/Index

Medicus format and should be listed in numerical order according to the order of

their citation in the article. List all authors if their number is 6 or less. List only the

first three followed by “et al.” if there are more than 6 authors.Declaration of

conflict of interest: All authors must provide a statement describing any relevant

conflict of interests for the manuscript.

NOTE: Excessive authorship should be avoided: case reports with more than 6 authors will not be considered for publication. In letters to the Editors with more than six authors the respective contributions of each must be specified in the accompanying cover letter. Original articles, brief papers, and rapid papers should be divided into the following sections (for case reports and letters see above): Structured abstract: A summary (250 words or less) suitable for use by abstracting journals, and divided into the following sections: Objective, Methods, Results, and Conclusion. Introduction: State the purpose of the article and summarise the rationale for the study, giving only the most pertinent references. Materials and methods: Describe how the study subjects (including controls) were selected. Identify the methods, apparatuses (including manufacturer), and procedures used in sufficient detail to allow other workers to reproduce the results. Give references to established procedures; provide references and brief descriptions for published but not well-known methods; describe new or substantially modified methods. Identify all drugs and chemicals used, including their generic name, dose, and route of administration.

Ethics: For experiments with human subjects, indicate whether the procedures followed were in accordance with the standards of the responsible local committee or with the Helsinki Delaration of 1975/83. For animal experiments, indicate which guidelines or national law on the use of laboratory animals were followed. Statistics: Describe the statistical methods used (citing standard works) in sufficient detail that a knowledgeable reader with access to the original data may verify the reported results. Quantify findings with appropriate indicators of measurement error or uncertainty (such as confidence intervals). Avoid sole reliance on statistical hypothesis testing, such as the use of P values, which fail to convey important quantitative information. Results. Present results succinctly and in logical sequence in the text, tables, and illustrations. In the text emphasize or summarise only the most important observations; avoid repeating the data given in the tables and illustrations. Discussion. Highlight only the new and most significant aspects of the study and discuss the conclusions that follow from them. Do not repeat in detail material already presented in the Results. Include the implications of your findings and their limitations. Relate the observations to other relevant studies. Avoid conclusions not completely supported by your data. Acknowledgements. Specify: (a) inputs that deserve acknowledgement but do not justify authorship (scientific advisers, critical review of the proposal, data collection, or participation in a clinical trial [Note: authors must obtain written permission from persons who are acknowledged by name, as readers may infer their endorsement of the data and conclusions]; (b) technical assistance; (c) financial and material support; and (d) financial relationships that may involve a conflict of interest.

Table of Contents

Page charge

Starting with manuscripts submitted from March 1, 2012 there will be a page

charge applied to articles over 4 printed pages. For each page exceeding this

limit the authors will be charged € 100.00 per additional page. Instructions for

payment will be sent to authors along with the proofs.

References

References should be compiled numerically according to their order of citation in the text [identified by arabic numbers in parentheses], and typed double-spaced. Use the format described in the Uniform Requirements, which is based on the US National Library of Medicine's Index Medicus. Journals titles should be abbreviated in accordance with the List of Journals Indexed in Index Medicus (published each January in Index Medicus). "Unpublished observations" and "personal communications" may not be used as references, and citing abstracts should be avoided. References to written, but not oral, communications may be inserted in the text within parentheses. Manuscripts that have been submitted but not yet accepted should be cited in the text as "unpublished observations". References must be verified by the authors against the original documents. List all authors when 6 or less; when 7 or more list only the first three and et al.

http://www.clinexprheumatol.org/guidelines-authors.asp#toc

Standard Journal Article:

YOU CH, LEE KY, CHEY RY, MENGUY R: Electrogastrographic study of patients

with unexplained nausea, bloating and vomiting. Gastroenterology 1980; 79: 311-4.

Books and monographs

Personal Author:

EISEN HN: Immunology: An Introduction to Molecular and Cellular Principles of the

Immune Response. 5th ed., New York, Harper and Row, 1974: 406.

Editor or Compiler as Author:

DAUSSET J, COLOMBANI J (Eds.): Histocompatibility Testing 1972. Copenhagen,

Munksgaard 1973: 12-8.

Chapter in a Book:

WEINSTEIN L, SWARTZ MN: Pathogenic properties of invading microorganisms. In

SODEMAN WA Jr, SODEMAN WA (Eds.): Pathologic Physiology: Mechanisms of

Disease. Philadelphia, WB Saunders 1974: 457-72.

Published Proceedings Paper:

DEPONT B: Bone marrow transplantation in severe combined immunodeficiency with

an unrelated MLC compatible donor. In WHITE HJ, SMITH R (Eds.): Proceedings of

the third annual meeting of the International Society for Experimental Hematology.

Houston, International Society for Experimental Hematology, 1974: 44-6.

Monograph in a Series:

HUNNINGHAKE GW, GADEK JE, SZAPIEL SV et al.: The human alveolar

macrophage. In HARRIS CC (Ed.): Cultured Human Cells and Tissues in Biomedical

Research. New York, Academic Press 1980: 54-6. (Stoner GD (Ed.): Methods and

Perspectives in Cell Biology, vol. 1).

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Dissertation or Thesis:

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California, University of California, 1965, 156 p.

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