Molecular Biology Reports

, Volume 39, Issue 11, pp 9965–9970

Influence of BLK polymorphisms on the risk of rheumatoid arthritis

Authors

  • Hui Zhang
    • Department of OrthopedicsChangzhou Second People’s Hospital, Affiliated Hospital of Nanjing Medical University
  • Liqun Wang
    • School of Pharmaceutical Engineering & Life ScienceChangzhou University
  • Yong Huang
    • Department of OrthopedicsChangzhou Second People’s Hospital, Affiliated Hospital of Nanjing Medical University
  • Chao Zhuang
    • Department of OrthopedicsChangzhou Second People’s Hospital, Affiliated Hospital of Nanjing Medical University
  • Gongyin Zhao
    • Department of OrthopedicsChangzhou Second People’s Hospital, Affiliated Hospital of Nanjing Medical University
    • Department of OrthopedicsChangzhou Second People’s Hospital, Affiliated Hospital of Nanjing Medical University
    • Central LaboratoryChangzhou Second People’s Hospital, Affiliated Hospital of Nanjing Medical University
    • Central LaboratoryChangzhou Second People’s Hospital, Affiliated Hospital of Nanjing Medical University
Article

DOI: 10.1007/s11033-012-1865-8

Cite this article as:
Zhang, H., Wang, L., Huang, Y. et al. Mol Biol Rep (2012) 39: 9965. doi:10.1007/s11033-012-1865-8

Abstract

B cell lymphocyte kinase (BLK) encodes a member of the Src kinase family and thus may influence the proliferation and differentiation of cells. A single nucleotide polymorphism (SNP) located in the first intron of BLK has shown that the risk C allele of rs2248932 is associated with lower levels of messenger RNA expression of BLK. We hypothesized that this polymorphism may contribute to rheumatoid arthritis (RA) susceptibility. We studied BLK rs2248932 T/C gene polymorphisms in 329 patients with RA and 697 controls in a Chinese population. Genotyping was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). When the BLK rs2248932 TT homozygote genotype was used as the reference group, the CC genotype was associated with a significantly increased risk of RA. In the recessive model, when the BLK rs2248932 TT/TC genotypes were used as the reference group, the CC homozygote genotype was associated with a significantly increased susceptibility to RA. In stratification analyses, a significantly increased risk for RA associated with the BLK rs2248932 CC genotype was evident among younger patients, CRP-negative patients and anti-CCP-positive patients compared with the BLK rs2248932 TT/TC genotype. The risk was also significantly evident among RF-positive patients, patients with lower ESR levels, patients with lower or higher DAS28 score and patients with a lower functional class. These findings suggested that the functional SNP BLK rs2248932 T/C variant allele was associated with RA development. However, our results were obtained from a moderate-sized sample, and therefore this is a preliminary conclusion. Validation in a larger study from a more diverse ethnic population is needed to confirm these findings.

Keywords

BLKPolymorphismsRheumatoid arthritisMolecular epidemiology

Abbreviations

CI

Confidence interval

BLK

B cell lymphocyte kinase

LD

Linkage disequilibrium

OR

Odds ratio

SNP

Single nucleotide polymorphism

Introduction

Rheumatoid arthritis (RA) is a type of systemic autoimmune disease caused by a failure of immune self-tolerance. RA is characterized by synovitis, progressive erosions and cartilage destruction [1, 2]. RA affects ≈1 % of the world population, and has a moderate female predominance [3].

RA and systemic lupus erythematosus (SLE) are autoimmune rheumatological diseases thought to have a substantial genetic contribution [4]. Recent genome-wide association studies (GWAS) in SLE have identified several novel associations, some of which have not been investigated in RA. The human B cell lymphocyte kinase (BLK) gene has been mapped to chromosome 8p23.1, and is expressed only in B lymphocytes [5]. BLK encodes a member of the Src kinase family involved in signal transduction downstream of the B cell receptor, and thus may influence the proliferation and differentiation of B cells [6]. The international consortium on the genetics of systemic lupus erythematosus (SLEGEN) study showed that rs2248932 and rs10903340 single nucleotide polymorphisms (SNPs) were associated with SLE in Caucasians [6]. Furthermore, two recent GWAS revealed BLK to be a novel risk locus for SLE [7, 8].

A study in a USA population confirmed the BLK locus to be associated with RA [9]. Interestingly, the BLK locus had also shown to be associated with RA in a Japanese population [10]. BLK encodes a tyrosine kinase that is involved in the regulation of B cell activation. B cells have a key role in the pathogenesis of RA and SLE through the production of auto-antibodies, antigen presentation to T cells, and cytokine production, and B cell depletion has proved successful in the treatment of these diseases [11].

The risk C allele of the rs2248932 SNP has been shown to be associated with lower levels of messenger RNA expression of BLK [7]. Homozygotes for the C allele had an expression level ≈30 % that of homozygotes for the T allele, and C/T heterozygotes had intermediate levels [7]. BLK protein is involved in the adaptive immune response, and altered protein levels of BLK may influence tolerance mechanisms in B cells, thereby predisposing persons to systemic autoimmunity [8]. However, even though BLK plays an important part in the immune responses, few studies have focused on the influence of BLK rs2248932 T/C polymorphisms in the susceptibility to RA in a Chinese population. Functional variations in BLK genes may contribute to the development of RA. We therefore undertook genotyping in a hospital-based case–control study in a cohort of 329 patients with RA and 697 controls in a Chinese population.

Patients and methods

Study subjects

We obtained approval of the study protocol from the Ethics Committee of Nanjing Medical University (Nanjing, China). All patients provided written informed consent to be included in the study.

Three hundred and twenty-nine RA patients who fulfilled the criteria for RA set by the American College of Rheumatology classification in 1987 [12] were consecutively recruited from the Changzhou Second Hospital-Affiliated Hospital of Nanjing Medical University (Changzhou, China), the Changzhou First Hospital (Changzhou, China), and the Changzhou Traditional Chinese Medical Hospital (Changzhou, China), between September 2010 and October 2011. The controls were patients without RA, matched for age (±5 years) and sex, and recruited from the same institutions during the same time period; most of the controls were admitted to the hospitals for the treatment of trauma.

Each patient was interviewed by trained personnel using a pre-tested questionnaire to obtain information on demographic data and related risk factors for RA. After the interview, 2 mL of peripheral blood was collected from each subject.

Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediamine tetra-acetic acid (EDTA). Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genotyping was done by MALDI-TOF-MS using the MassARRAY system (Sequenom, San Diego, CA, USA) as previously described [13]. Cases and controls at a proportion of ≈1:2 were assayed. Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY Nanodispenser (Sequenom), and analyzed by MALDI-TOF-MS. Genotype calling was done in real time with MassARRAY RT software (version 3.1; Sequenom), and analyzed using MassARRAY Typer software (version 4.0; Sequenom; Fig. 1). For quality control, repeated analyses were undertaken on 10 % of randomly selected samples.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-012-1865-8/MediaObjects/11033_2012_1865_Fig1_HTML.gif
Fig. 1

Genotyping of BLK rs2248932 T/C by MALDI-TOF-MS

Statistical analyses

Differences in demographics, variables, and genotypes of the BLK rs2248932 T/C, polymorphism variants were evaluated using a Chi-squared test. The associations between BLK rs2248932 T/C genotypes and risk of RA were estimated by computing odds ratios (ORs) and 95 % confidence intervals (CIs) using logistic regression analyses, and by using crude ORs. The Hardy–Weinberg equilibrium (HWE) was tested by a goodness-of-fit Chi-squared test to compare the observed genotype frequencies to the expected frequencies among controls. All statistical analyses were done with SAS software (version 9.1.3; SAS Institute, Cary, NC, USA).

Results

Characteristics of the study population

Among 329 patients and 697 controls who provided DNA samples, genotyping for the BLK rs2248932 T/C polymorphism was successful in 322 (97.9 %) patients and 696 (99.9 %) controls. The demographic and clinical characteristics of all subjects are summarized in Table 1. Subjects were adequately matched for age and sex (p = 0.829 and 0.190, respectively). The genotype distributions of BLK rs2248932 T/C in all subjects are illustrated in Table 2. The observed genotype frequencies for the polymorphism in controls were in HWE for BLK rs2248932 T/C (p = 0.284).
Table 1

Patient demographics and risk factors in rheumatoid arthritis, all subjects

Variable

Cases (n = 329)

Controls (n = 697)

p

Age (years)

53.64 (±15.52)

53.45 (±11.35)

0.829

Female/male

247/82

496/201

0.190

Age at onset (years), Mean ± SD

44.93 (±12.55)

Disease duration (years), Mean ± SD

8.76 (±9.31)

Treatment duration (years), mean ± SD

7.07 (±7.38)

RF-positive, no. (%)

266 (80.9 %)

Anti-CCP-positive, no. (%)

163 (49.5 %)

CRP-positive, no. (%)

165 (50.2 %)

ESR (mm/h)

34.00 (±23.96)

DAS28

4.33 (±1.61)

Functional class, no. (%)

 

 I

49 (14.9 %)

 II

136 (41.3 %)

 III

116 (35.3 %)

 IV

28 (8.5 %)

RF Rheumatoid factor, Anti-CCP Anti-cyclic citrullinated peptide, CRP C-reactive protein, ESR Erythrocyte sedimentation rate, DAS28 RA disease activity score

Table 2

Logistic regression analysis of associations between BLK polymorphisms and risk of rheumatoid arthritis

Genotype

Casesa (n = 329)

Controls (n = 697)

OR (95 % CI)

p

n

%

n

%

BLK rs2248932 T/C

 TT

179

55.6

400

57.5

1.00

 TC

114

35.4

262

37.6

0.97 (0.73–1.29)

0.845

 CC

29

9.0

34

4.9

1.91 (1.13–3.23)

0.016

 TT + TC

293

91.0

662

95.1

1.00

 CC

29

9.0

34

4.9

1.93 (1.15–3.22)

0.012

 C allele

 

26.7

 

23.7

  

Bold values are statistically significant (p < 0.05)

aGenotyping was successful in 322 cases and 696 controls for BLK rs2248932 T/C

Associations between BLK rs2248932 T/C polymorphism and risk of RA

The genotype frequencies of the BLK rs2248932 T/C polymorphism were 55.6 % (TT), 35.4 % (TC) and 9.0 % (CC) in RA patients, and 57.5 % (TT), 37.6 % (TC) and 4.9 % (CC) in controls (p = 0.039) (Table 2). When the BLK rs2248932 TT homozygote genotype was used as the reference group, the CC genotype was associated with a significantly increased risk for RA (OR = 1.91, 95 % CI = 1.13–3.23, p = 0.016). In the recessive model, when the BLK rs2248932 TT/TC genotypes were used as the reference group, the CC homozygote genotype was associated with a significant 1.93-fold increased susceptibility to RA (OR = 1.93, 95 % CI = 1.15–3.22, p = 0.012).

Stratification analyses of BLK rs2248932 T/C polymorphisms and the risk of RA

Stratification analyses were done to evaluate the effects of BLK genotypes on RA risk according to age, sex, C-reactive protein (CRP) status, anti-CCP status, RF status, ESR, DAS28 and functional class (Table 3). A significantly increased risk for RA associated with the BLK rs2248932 CC genotype was evident among younger patients (OR = 2.58, 95 % CI = 1.30–5.12, p = 0.007), CRP-negative patients (OR = 2.30, 95 % CI = 1.25–4.23, p = 0.007) and anti-CCP-positive patients (OR = 2.15, 95 % CI = 1.16–4.00, p = 0.016) compared with the BLK rs2248932 TT/TC genotype.
Table 3

Stratified analyses between BLK polymorphisms and the risk of rheumatoid arthritis

Variable

BLK rs2248932 T/Ca (case/control)

OR (95 % CI)

p

TT + TC

CC

TT + TC

CC

Sex

 Male

74/194

7/7

1.00

2.62 (0.89–7.73)

0.081

 Female

219/468

22/27

1.00

1.74 (0.97–3.13)

0.063

Age (years)

 <55

141/326

19/17

1.00

2.58 (1.305.12)

0.007

 ≥55

152/336

10/17

1.00

1.30 (0.58–2.91)

0.522

CRP status

 Positive

149/662

12/34

1.00

1.57 (0.79–3.10)

0.196

 Negative

144/662

17/34

1.00

2.30 (1.254.23)

0.007

Anti-CCP status

 Positive

145/662

16/34

1.00

2.15 (1.164.00)

0.016

 Negative

148/662

13/34

1.00

1.71 (0.88–3.32)

0.113

RF status

 Positive

236/662

24/34

1.00

1.98 (1.153.41)

0.014

 Negative

57/662

5/34

1.00

1.71 (0.64–4.54)

0.283

ESR (mm/h)

 <26.00

125/662

13/34

1.00

2.03 (1.043.95)

0.038

 ≥26.00

168/662

16/34

1.00

1.85 (1.00–3.44)

0.050

DAS28

 <3.20

79/662

9/34

1.00

2.22 (1.034.80)

0.043

 ≥3.20

214/662

20/34

1.00

1.82 (1.033.23)

0.041

Functional class

 I + II

164/662

17/34

1.00

2.02 (1.103.70)

0.023

 III + IV

129/662

12/34

1.00

1.81 (0.91–3.59)

0.089

Bold values are statistically significant (p < 0.05)

CRP C-reactive protein, Anti-CCP Anti-cyclic citrullinated peptide, RF Rheumatoid factor, ESR Erythrocyte sedimentation rate, DAS28 RA disease activity score

aGenotyping was successful in 322 cases and 696 controls for BLK rs2248932 T/C

The risk was also significantly evident among RF-positive patients, patients with lower ESR levels, patients with lower or higher DAS28 score, and patients of a lower functional class (Table 3).

Discussion

We determined the association between the BLK rs2248932 T/C polymorphism and the risk of RA in a Chinese population. This was the first positive finding of the BLK rs2248932 T/C polymorphism and RA. We found that the BLK rs2248932 CC allele may increase the risk of RA, and that this effect was more evident among younger patients, CRP-negative patients and anti-CCP-positive patients. The risk was also significantly evident among RF-positive patients, patients with lower ESR levels, patients with lower or higher DAS28 score, and patients with a lower functional class.

BLK expression is highly restricted to B cells. The tyrosine kinase BLK is expressed mainly in B cells and is activated during B cell receptor signaling, which further activates nuclear transcription factors through a signaling cascade. Studies have indicated that the FAM167A-BLK region may be a shared genetic factor for several autoimmune diseases in multiple populations, but that the genetic contribution may be greater in Asian populations because of differences in the genetic background [10].

BLK is a Src kinase expressed in thymocytes in mice and which transduces signals downstream of the BCR [14, 15]. Association of BLK with SLE was also confirmed by the study of Harley et al. [6] on SNP rs2248932.

In the stratified analysis, the T > C variant of rs2248932 could significantly increase the risk of RA among younger patients, CRP-negative patients, patients with lower ESR levels and patients with a lower functional class. We think that the BLK rs2248932 polymorphism may have a role in the early stage of RA development. In the later stage, other cytokines may have roles.

BLK encodes Src family tyrosine kinase and also plays a part in B cell signal transduction. BLK SNPs rs2248932 and/or rs13277113 have been widely implicated with SLE susceptibility in European [6, 8], Japanese [16] and Chinese [7] populations. In a GWAS and replication study in a Korean population with RA, BLK rs1600249 and rs2736340 were associated with RA risk [17]. BLK has also been implicated in multiple sclerosis [18]. The associated SNP (rs13277113) lies in the intergenic region of FAM167A and BLK, and its risk allele has been shown to be associated with low mRNA expression levels of BLK [8]. In a case–control association study involving 603 patients and 492 healthy controls in Japan, the BLK rs13277113 A/G polymorphism was associated with RA risk [10]. In another investigation, no association was observed between RA and the BLK rs13277113 A/G polymorphism [19]. This may be due to the different ethnic populations studied.

Several limitations of the present study need to be addressed. First, this was a hospital-based case–control study, so selection bias was unavoidable and the subjects were not fully representative of the general population. Second, the polymorphisms we investigated, based on their functional considerations, may not offer a comprehensive view of the genetic variability of BLK. Third, a single case–control study is not sufficient to fully interpret the relationship between BLK polymorphisms and susceptibility to RA because of the relatively small number of patients evaluated. Larger numbers of subjects are necessary to confirm our findings. Finally, we did not obtain detailed information about RA severity and the outcomes of treatment, which restricted our analyses.

In conclusion, the present study provided strong evidence that BLK rs2248932 T/C functional polymorphisms may contribute to the risk of RA. However, our results were obtained from a moderate-sized sample, and therefore this is a preliminary conclusion. Validation in a larger study from a more diverse ethnic population is needed to confirm these findings.

Acknowledgments

We appreciate all patients who participated in this study.

Conflict of interest

We declare that we have no conflicts of interest.

Copyright information

© Springer Science+Business Media B.V. 2012