Rheumatol Int DOI 10.1007/s00296-015-3308-z

Rheumatology INTERNATIONAL

ORIGINAL ARTICLE - GENES AND DISEASE

CCR5Δ32 (rs333) polymorphism is associated with the susceptibility to systemic lupus erythematosus in female Brazilian patients Thiago Hissnauer Leal Baltus1 · Ana Paula Kallaur1 · Marcell Alysson Batisti Lozovoy2 · Helena Kaminami Morimoto2 · Francieli Delongui1 · Daniela Frizon Alfieri1 · Tatiane Mayumi Veiga Iriyoda3 · Isaias Dichi4 · Andrea Name Colado Simão2 · Edna Maria Vissoci Reiche2  Received: 11 March 2015 / Accepted: 3 June 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  The role of CCR5Δ32 (rs333) polymorphism in the pathogenesis of systemic lupus erythematosus (SLE) has been evaluated worldwide. The aim of this study was to determine the association between CCR5Δ32 polymorphism with the susceptibility to SLE and the activity of disease in female Southern Brazilian patients. The study enrolled 169 female SLE patients and 132 unrelated female healthy individuals. Baseline clinical, laboratorial characteristics, and the SLE activity (determined using the SLEDAI) were evaluated according to the CCR5Δ32 genotypes. The CCR5Δ32 polymorphism was determined from genomic DNA using a polymerase chain reaction. The frequencies of the genotypes CCR5/CCR5, CCR5/CCR5Δ32, and CCR5Δ32/CCR5Δ32 were 87.6, 11.8, and 0.6 %, respectively, among the patients and 96.2, 3.8, and 0.0 %, respectively, among the controls [CCR5/CCR5 vs. CCR5/CCR5Δ32  +  CCR5Δ32/CCR5Δ32: p  = 0.0081, odds ratio 3.604 (95 % confidence interval 1.321–9.4836)]. The frequencies of the CCR5 and the CCR5Δ32 alleles were 93.2 and 6.8 % among the patients, and 98.1 and 1.9 % among the controls, respectively (p  = 0.0047, OR 3.758, 95 % CI 1.409–10.80). Patients carrying the * Edna Maria Vissoci Reiche [email protected] 1

Health Sciences Postgraduate Program, Health Sciences Center, State University of Londrina, Londrina, Paraná, Brazil

2

Department of Pathology, Clinical Analysis, and Toxicology, Health Sciences Center, State University of Londrina, Av. Robert Koch, 60, CEP 86.038‑440 Londrina, Paraná, Brazil

3

Outpatient Clinic for Rheumatology, University Hospital, State University of Londrina, Londrina, Paraná, Brazil

4

Department of Internal Medicine, Health Sciences Center, State University of Londrina, Londrina, Paraná, Brazil







genotypes with the CCR5Δ32 allele presented earlier age of onset of disease (p  = 0.0293) and higher levels of anti-dsDNA (p  = 0.0255) than those carrying the wildtype genotype. When the analysis was adjusted for ethnicity, only the age at onset of disease remained significant (p > 0.05). The results suggest that the CCR5Δ32 polymorphism might be associated with SLE genetic predisposition among female Brazilian patients and the age at onset of the disease; however, this genetic variant was not associated with the activity of SLE in this population. Keywords  Systemic lupus erythematosus · Genetic polymorphism · CCR5 · Chemokine receptor

Introduction Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease, clinically heterogeneous and genetically complex that is characterized by widespread breakdown of immune tolerance to self-antigens [1, 2]. The disease affects people worldwide, with higher incidence and prevalence among women than men. The prevalence rate ranges from 20 to 150/100,000 persons with the highest prevalence reported in Brazil [3]. The SLE etiology is multifactorial with an involvement of genetic and environmental factors and chemokines, and their receptors are implicated in the pathogenesis of the chronic inflammatory processes of the disease [4–6]. The CC chemokines are typically chemoattractant for inflammatory cells and are associated with T lymphocyte polarization. The Th1 cell subset expresses CCR5 and CXCR3, and Th2 cell subset expresses CCR4 and CCR8; monocyte/macrophage lineage expresses CCR1, CCR5, CXCR1, CXCR2, and CXCR3; moreover, macrophage

13



differentiation is associated with downregulation of CCR2 and induction of CCR5 and CX3CR1 [6]. CCR5 is a seven-transmembrane chemokine receptor associated with G-protein and is expressed in a wide range of cells of the immune system [7–9] and has several chemokine agonistic ligands, such as CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES) [6]. The CCR5 gene is located on short arm of human 3p21 chromosome, and the deletion of 32 base pairs (bp) in the intron 3 of the CCR5 (named CCR5Δ32, rs333) results in a frame shift, creating a premature stop codon and leading to a non-functional protein that fails to reach the cell surface. Consequently, in homozygous individuals there is absence of CCR5 expression, while in heterozygous individuals there is a reduced CCR5 expression in the cell surface [10]. Homozygosity for CCR5Δ32 showed a relative resistance against human immunodeficiency infection type 1 (HIV-1) since the CCR5 is a co-receptor for the macrophage-tropic strain of HIV-1 to entry in the host cell [10–12]. However, the role of this mutation status in autoimmune diseases evaluated in different populations was conflicting. While some studies showed a protective effect of the CCR5Δ32 variant on rheumatoid arthritis susceptibility [13, 14]; others demonstrated lack of association of this deletion with rheumatoid arthritis [15], SLE, lupus nephritis, and disease severity [16, 17]. In juvenile idiopathic arthritis subtypes, the CCR5Δ32 does not have a protective effect, but instead it could be a factor associated with more inflammatory forms of the disease [18]. Conflicting results of the association between the CCR5Δ32 allele and multiple sclerosis were also reported [19–21]. The results evaluating the role of CCR5Δ32 in the pathogenesis of SLE vary in different populations worldwide. While some studies showed an association with protective effect for disease susceptibility [22], others did not confirm this effect [16, 23, 24]. Therefore, the aim of this study was to determine the association between the CCR5Δ32 polymorphism with SLE susceptibility and activity in a female cohort of SLE patients from Southern Brazilian population.

Materials and methods Ethics and consent The protocol was approved by the Institutional Research Ethics Committee of the State University of Londrina, and a written consent form was obtained from all of the individuals included in this study.

13

Rheumatol Int

Participants A total of 169 unrelated female SLE patients diagnosed according to the American College of Rheumatology criteria for SLE [25] were consecutively recruited, between 2009 and 2015, from the Rheumatology Outpatient Department of the Outpatient Clinical Hospital, State University of Londrina, Paraná State, Southern Brazil. As control group, 132 unrelated female healthy blood donors, age, ethnicity, and body mass index controlled, from the same region of the SLE patients, were recruited from the Blood Bank of Londrina. Demographic, clinical, and laboratory characteristics The demographic and clinical data were obtained through a standard questionnaire and medical records. The ethnicity was self-reported as Caucasians and non-Caucasians (AfroBrazilians and Asiatics) [26]. The anthropometric measurements evaluated were body weight (measured to the nearest 0.1 kg using an electronic scale, with individuals wearing light clothing, but no shoes) and height (measured to the nearest 0.1 cm using a stadiometer). BMI was calculated as weight (kg) divided by height (m) squared. Disease activity was assessed using the SLE Disease Activity Index (SLEDAI) [27]. The serum levels of C3 and C4 were determined using nephelometer method and expressed as mg/dL. Antinuclear antibodies (ANA) were quantified using indirect immunofluorescence with HEp2 cells as substrate (IFI-ANA-HEp2-IgG, VIRO-IMMUN LaborDiagnostika, GmbH, Oberursel, Germany) and were considered significant when titers ≥1:160; antibodies against double-stranded DNA (anti-dsDNA) were quantified using enzyme-linked immunoassay (ELISA, anti-dsDNA, Orgentec Diagnostika, GmbH, Germany) and were considered significant when titers ≥20 IU/mL. Peripheral white blood cell counts were quantified using an automatic method and expressed as cells/mm3. Clinical manifestations and therapeutic data were obtained from medical records. CCR5Δ32 genotyping Genomic DNA was extracted from peripheral blood cells, using a resin column procedure (Biopur, Biometrix Diagnóstica, Curitiba, Brazil) according to the manufacturer’s protocol. CCR5 gene was amplified using polymerase chain reaction (PCR), and a fragment with 225 base pairs was obtained using the primers sense 5′-ACC AGA TCT CAA AAA GAA-3′ and antisense 5′-CAT GAT GGT GAA GAT AAG CTT CA-3′) (Invitrogen™, Life Technologies, Carlsbad, CA, USA) as previously reported [21]. PCR was performed in a thermocycler (Applied

Rheumatol Int Table 1  Baseline characteristics of female patients with systemic lupus erythematosus (SLE) enrolled between 2009 and 2015 from Southern Brazilian population

photo documentation system L-PIX-HE (Loccus Biotecnologia, Cotia, SP, Brazil).

Characteristics

Total SLE patients (n = 169)

Statistical analysis

Age (years) Ethnicity Caucasian/non-Caucasian Body mass index (kg/m2) Age at onset of disease (years) Disease duration (years) SLEDAI  ≤3  >3 C3 (mg/dL)  Low C4 (mg/dL)  Low ANA  Negative ( 0.05). Independently of the CCR5Δ32 polymorphism, the Caucasian SLE patients showed higher anti-dsDNA serum levels than the non-Caucasian SLE patients [median 29.4 UI/mL (IQR: 10.7–89.6) versus median 14.2 UI/mL (IQR 4.8– 43.3), p  = 0.0100]. When the GLM test was used to verify whether ethnicity was interfering with the variables that presented significant association with the CCR5Δ32 polymorphism, such as the anti-dsDNA levels and the age at onset of the disease, only the age at onset of disease remained associated with the CCR5Δ32 polymorphism (p  0.05) (data not shown).

Discussion The results revealed a significant association between the genotypes carrying the CCR5Δ32 allele (in heterozygosis and homozygosis for the allele) with the susceptibility to SLE in female patients of Southern Brazil; moreover, this polymorphism was associated with the age at onset of the SLE. The association between the genotypes carrying the CCR5Δ32 allele with low age at onset of SLE patients suggests that this variant may exert an effect on disease severity. Previous studies showed that how much later is the age at onset of SLE, and the disease tends to have a more insidious onset, less major organ system involvement, lower degrees of activity, and less renal involvement than early onset [28, 29]. Hypocomplementaemia and positive results for anti-dsDNA antibodies were less common in late-onset SLE patients than in early-onset SLE [29]. However, this study failed to demonstrate the association between CCR5Δ32 polymorphism and the activity of the disease. Although SLE patients carrying the genotypes with the CCR5Δ32 alleles presented one of the laboratory marker of the disease activity, such as higher levels of anti-dsDNA antibodies than those carrying the wild-type homozygous genotype, this result did not remain significant when the analysis was adjusted for the ethnicity of the patients. These results demonstrated that ethnicity is

Rheumatol Int Table 3  Baseline characteristics of female systemic lupus erythematosus (SLE) patients enrolled between 2009 and 2015 from Southern Brazilian population, according to the chemokine 5 receptor delta 32 (CCR5Δ32, rs333) polymorphism Characteristic

SLE patients (n = 169) CCR5/CCR5 (n = 148)

CCR5/CCR5∆32 + CCR5∆32/CCR5∆32 (n = 21)

p value

40.5 (31.0–51.0) 90 (60.8)/58 (39.2) 26.6 (23.7–31.6) 33.0 (23.0–39.3) 10.0 (4.0–15.1) 2.0 (0.0–6.0) 88 (59.5) 60 (40.5)

32.0 (28.0–50.5) 18 (85.7)/3 (14.3) 28.3 (26.1–33.1) 23.5 (18.0–35.5) 9.0 (3.3–17.5) 2.0 (0.0–7.0) 11 (52.4) 10 (47.6)

0.1512 0.0613 0.317 0.0293 0.8314 0.6207 0.5377

18 (12.2) 130 (87.8)

3 (14.3) 18 (85.7)

0.7286

28 (18.9) 120 (81.1)

5 (23.8) 16 (76.2)

0.5968

43 (29.1) 105 (70.9)

6 (28.6) 15 (71.4)

0.9636

 Nuclear Speckled  Nuclear Homogeneous  Othera Anti-dsDNA (UI/mL)  Negative (

CCR5Δ32 (rs333) polymorphism is associated with the susceptibility to systemic lupus erythematosus in female Brazilian patients.

The role of CCR5Δ32 (rs333) polymorphism in the pathogenesis of systemic lupus erythematosus (SLE) has been evaluated worldwide. The aim of this study...
477KB Sizes 0 Downloads 12 Views