Clin Rheumatol (2011) 30:1227–1233 DOI 10.1007/s10067-011-1741-2
ORIGINAL ARTICLE
Galectin-3 gene (LGALS3) +292C allele is a genetic predisposition factor for rheumatoid arthritis in Taiwan Chung-Yi Hu & Sheng-Kai Chang & Chien-Sheng Wu & Wei-I Tsai & Ping-Ning Hsu
Received: 30 September 2010 / Accepted: 21 March 2011 / Published online: 8 April 2011 # Clinical Rheumatology 2011
Abstract Galectin-3 is a beta-galactoside-binding lectin which is involved in modulating inflammation and apoptosis. Elevated expression of galectin-3 has been demonstrated in synovium of rheumatoid arthritis (RA). The aim of our study is to investigate the genetic polymorphisms of galectin-3 in association with RA. Polymorphisms of galectin-3 gene (LGALS3) were compared between 151 RA patients and 182 healthy subjects in Taiwan. Variants at two LGALS3 single nucleotide polymorphism (SNP) sites (rs4644 and rs4652, corresponding to LAGLS3 +191 and +292) were genotyped by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and sequence-specific oligonucleotide probe hybridization, respectively. The allelic carriage of LGALS3 +292C was increased in patients with RA (66.9% in RA vs. 52.7% in controls, odds ratio=1.8, 95% confidence interval=1.2–2.8, p=0.009). These results implicate that the genetic polyC.-Y. Hu : S.-K. Chang Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China C.-S. Wu Department of Internal Medicine, Far Eastern Memorial Hospital, Taipei, Taiwan, Republic of China C.-S. Wu : W.-I. Tsai : P.-N. Hsu Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China P.-N. Hsu (*) Graduate Institute of Immunology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd., Taipei, Taiwan, Republic of China e-mail:
[email protected]
morphisms in galectin-3 gene may contribute to development of RA. Keywords Galectin-3 . Genetic polymorphism . Rheumatoid arthritis
Introduction Rheumatoid arthritis (RA) is a systemic, multi-factorial autoimmune disease characterized by chronic inflammation and destruction of joints. Enhanced infiltration of lymphocytes and macrophages can be observed in inflamed synovium. Up-regulation of pro-inflammatory cytokines is evident in the synovial fluids [1]. It is speculated that both genetic and environmental factors contributed to the pathogenesis of RA. Galectins are beta-galactoside-binding lectins that contain one or more carbohydrate-recognition domains (CRDs). Fifteen members have been discovered in mammals [2]. Human galectin-3 gene (LGALS3) is located on chromosome 14q21. It is composed of six exons that encode a protein of 32 kDa [3]. Galectin-3 (also known as Mac-2/Epsilon binding protein) is the sole member of the chimera-type of galectins, which contains a single Cterminal CRD (for carbohydrate recognition) and an extralong, flexible N-terminal domain. The N-terminal domain plays a critical role in its secretion [4]. Galectin-3 has been detected in different cell types, such as activated macrophages [5], fibroblasts [6], dendritic cells [7], eosinophils [8], mast cells [9], chondrocytes, and osteoblasts [10, 11]. Previous studies showed that galectin-3 played diverse roles in multiple tissues, and exerted its function both intracellularly [12] and extracellularly [13, 14]. The intracellular galectin-3 was implicated
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in nuclear pre-mRNA splicing [15], cell growth regulation [16], and protecting cells from apoptotic death induced by Fas signaling [17, 18], chemotherapeutic agents, TNF, or radiation [19]. Galectin-3 inhibited cisplatin-induced apoptosis in breast carcinoma cell line [20]. The anti-apoptotic property of galectin-3 was attributed to the highly conserved sequence (NWGA) in the BH1motif of Bcl-2 family. Galectin-3 was critical for phagocytosis by macrophages [21]. It was reported that intracellular galectin-3 affected actin rearrangement upon Fc-gamma receptor cross-linkage. Galectin-3 deficient macrophages exhibited reduced phagocytosis of IgG-opsonized erythrocytes and apoptotic thymocytes as compared to the wild-type ones. Furthermore, galectin-3 was recently characterized as an opsonin that enhanced clearance of apoptotic neutrophils by macrophages [22]. Galectin-3 was also reported to be involved in morphogenesis and angiogenesis of endothelial cells [23]; both were considered important steps in panus formation in the inflamed synovium. Galectin-3 is believed to be associated with rheumatoid arthritis. Human galectin-3 messenger RNA and protein were stained throughout the synovial membrane as compared with sparse staining in synovium of osteoarthritic subjects and healthy controls [24]. The synovial proteome analysis shows differential expression of galectin-3 in fibroblast-like synovial cells derived from the synovium of rheumatoid arthritis patients [25]. In a rat model of collagen-induced arthritis, enhanced expression of galectin3 was noted in the peripheral monocytes, and plasma galectin-3 level was consistent with disease progression [26]. As galectin-3 exhibits diverse functions in modulation and fine-tuning immune reactions, the role of galectin-3 in pathogenesis of RA remains to be determined. To test whether genetic polymorphisms of galectin-3 confer predisposition to RA, galectin-3 genetic variations were investigated and compared between the RA patients and healthy subjects. We also analyzed serum galectin-3 level as well as the intracellular galectin-3 in peripheral blood monocyte-derived macrophages, and both were found to be correlated with the galectin-3 genotype.
Materials and methods Patients and controls A total of 151 subjects with RA (132 females and 19 males, age mean ± SD=55.2±14.2) were enrolled from National Taiwan University Hospital. The patients were diagnosed according to the 1987 revised criteria of American College of Rheumatology for RA [27]. A total of 182 subjects (113 females and 69 males, age mean ± SD=52.9±10.9) were recruited as control for this study. Sera samples of 80 RA
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patients were subjected to anti-cyclic citrullinated peptide (anti-CCP) antibody detection. The selected control subjects were grossly healthy, negative in HBsAg and antiHCV antibody, and normal in liver enzymes (aspartate aminotransferase and alanine aminotransferase) and renal function test (creatinine). All the subjects were Taiwanese. Informed consents were obtained from all the studied subjects, and this study has been approved by the Institutional Medical Ethics Committee in National Taiwan University Hospital. Genetic typing of LGALS3 single nucleotide polymorphism The genomic DNA was prepared from peripheral blood leukocytes using standard protocol [28]. Variants at two LGALS3 single nucleotide polymorphism (SNP) sites (rs4644 and rs4652) were genotyped using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and sequence-specific oligonucleotide probe hybridization (SSOPH), respectively. The SNP rs4644, LGALS3 +191 A>C, renders residue 64 of galectin3 changing from histidine to proline (Gal-3 64 His to Pro), whereas the SNP rs4652, LGALS3 +292 A>C, changes the threonine at residue 98 to a proline (Gal-3 98 Thr to Pro). The PCR was performed using primers: 5′-GTAT GTCTTTCTTTCCAGCTC-3′ and 5′-GCAGTTTTAT CAGTGCCTTC-3′. This PCR amplified DNA sequence spanning the exon 3 of LGALS3 gene and covered the above-mentioned SNPs. In a pilot survey, PCR products from 10 randomly selected study subjects (five RA patients and five controls) were purified and subjected to DNA sequencing analysis. Samples with confirmed DNA sequences were used as control materials to confirm the sensitivity and specificity in the later investigations. To investigate genetic polymorphism at LGALS3 +191, the 553-bp PCR product was incubated with 1 U of NcoI for 16 h and analyzed on 3% agarose gel. LGALS3 +191A allele showed three DNA fragments (254, 189, and 110 bp) compared to two DNA fragments (443 and 110 base pairs) in GALS3 +191C allele (Fig. 1a). The SNP at LGALS3 +292 was analyzed by SSOPH as previously described [28]. Briefly, PCR products of the test subjects and the positive/negative controls were denatured and dot-blotted onto nylon membranes in a 12×8 format, and biotinylated probes were used to distinguish LGALS3 +292A and +292C alleles (+292A probe—AAGTGCCACCGGAGC, +292C probe— AAGTGCCCCCGGAGC, and E3-all-probe—positive loading control, CCTGGAGCTTATCCC) (Fig. 1b). Enzyme-linked immunosorbent assay and immunoblotting Enzyme-linked immunosorbent assay (ELISA) was performed to detect human serum galectin-3 [24]. The
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a
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LGALS3 +191 genotype M
U
CC
AC AA
BL
proteins. Twenty micrograms of total protein was analyzed by immunoblotting. Mouse anti-human galectin-3 monoclonal antibody (Abcam) was served as detection antibody followed by HRP-conjugated goat anti-mouse IgG (R&D). ECL kits (Western lightning™ Chemiluminescence reagent plus-ECL, PerkinElmer) were used to detect the hybridization signals. Statistical analysis
b
LGALS3 +292 genotype B AA AC CC
+292 A probe +292 C probe E3-all probe Fig. 1 Genotyping of LGALS3 +191 (A/C) and +292 (A/C). a Restriction fragment length polymorphism analysis of LGALS3 +191. The 553-bp PCR product was treated with 1 U of NcoI for 16 h and analyzed on 3% agarose gel pre-stained with ethidium bromide. LGALS3 +191A allele showed three DNA fragments (254, 189, and 110 bp) compared to two DNA fragments (443 and 110 bp) in LGALS3 +191C allele. M 100-bp DNA marker, U uncut PCR product (553 bp), BL reagent blank control. b PCR/SSOPH analysis of genetic polymorphism at LGALS3 +292. PCR products were denatured and immobilized on nylon membranes. SNP at LGALS3 +292 were investigated by sequence-specific oligonucleotide probes, +292A and +292C. The E3-all-probe was used to confirm proper loading of amplified exon 3 of LGALS3
polyclonal anti-Gal-3 antibodies used in ELISA were kindly provided by Dr. Liu FT. Serum anti-cyclic citrullinated peptide (anti-CCP) antibody was detected using commercial ELISA kit (Quanta Lite™ CCP3 IgG ELISA, Medical Technology Promedt Consulting GmbH, Germany). Cellular galectin-3 expression was analyzed in macrophages derived from healthy subjects with different LGALS3 +292 genotypes. Briefly, peripheral blood mononuclear cells were separated by Histopaque (Sigma) gradient and incubated in RPMI-1640 with 10% FCS at 37°C for 2 h. After monocytes have adhered to tissue culture plate, cells in suspension were removed and the adherent cells were cultured further for 5 days. RIPA buffer containing 2 mM PMSF was used to collect cellular
The genotype frequency, allelic frequency as well as allelic carriage rate at the two SNPs in LGALS3 were compared between RA patients and non-rheumatic controls by chisquare test. The allelic carriage rate is the number of individuals carrying at least one copy of the allele divided by the number of all subjects. Odd ratios with 95% confidence interval were calculated. A p value less than 0.05 was considered to be statistically significant. Haplotype combinations of LGALS3 +191–+292 were estimated from genotypes at both SNP sites by expectation–maximization (EM)-based algorithm using EH program (Jurg Ott, Rockefeller University, NY, USA). Whether genotype distribution adhered to the Hardy–Weinberg equilibrium was tested by chi-square test. Serum Gal-3 levels in RA patients and controls were compared by non-parametric Wilcoxon rank-sum test. Statistical analysis was performed using SPSS ver. 13.0 (SPSS Inc., Chicago, IL, USA).
Results Identification of SNPs in the galectin-3 gene Two non-synonymous SNPs were identified based on DNA samples from ten Taiwanese subjects, LGALS3 +191A>C (SNP rs4644) and +292A>C (SNP rs4652) (the first nucleotide of start codon ATG was referred as +1). The genotype distribution adheres to Hardy–Weinberg equilibrium for both SNPs in RA cases and normal subjects. The LGALS3 +292C allele predisposed subjects to rheumatoid arthritis The genotype distribution at LGALS3 +191 was similar among the 151 RA patients and 182 normal controls. However, the genotype distribution at LGALS3 +292 showed significant difference between RA patients and normal control groups (Table 1). LGALS3 +292C allele predisposed subjects to RA in a dominant model. Subjects carried LGALS3 +292C allele (+292AC or CC genotypes) were more susceptible to RA compared to subjects with +292AA genotype (odds ratio=1.8, 95% confidence interval=1.2–2.8, p=0.009).
1230 Table 1 Genotype distribution and allele frequency of LGALS3 +191 and +292 in 151 RA patients and 182 healthy controls
OR (95% CI): odds ratio (95% confidence interval) a
SNP rs4644: LGALS3 +191 A> C renders residue 64 of galectin-3 changes from His to Pro
b
SNP rs4652: LGALS3 +292 A> C renders residue 98 of galectin-3 changes from Thr to Pro
Clin Rheumatol (2011) 30:1227–1233 LGALS3 polymorphism
OR (95% CI)
p value
8 (5.3) 39 (25.8) 104 (68.9)
8 (4.4) 43 (23.6) 131 (72.0)
1.0 0.9 (0.3–2.6) 0.8 (0.3–2.1)
0.858 0.655
50 (33.1) 72 (47.7)
86 (47.3) 70 (38.5)
1.0 1.8 (1.1–2.8)
0.019
29 (19.2) 101 (66.9)
26 (14.3) 96 (52.7)
1.9 (1.0–3.6) 1.8 (1.2–2.8)
0.043 0.009
C/C Carriage of LGALS3 +292C
The LGALS3 +191–+292 (C–C) haplotype was positively associated with RA Because LGALS3 +292A was exclusively linked to +191C and no subjects displayed a LGALS3 +191–+292 (A–A) haplotype, the other haplotypes could be derived easily. Using EH program, the haplotype frequencies estimated in all test subjects were: LGALS3 +191–+292 (A–C) (17.1%), (A–A) (0.0%), (C–A) (62.2%), and (C–
OR (95% CI): odds ratio (95% confidence interval)
Controls N=182 n (%)
LGALS3 +191a A/A A/C C/C LGALS3 +292b A/A A/C
Eighty of the RA patients had been checked for serum anti-CCP antibody level and 49 showed positive antiCCP. Twenty-eight patients were treated with anti-TNFalpha biologics due to poor response to traditional DMARDs treatment (DAS28>5.1, all with methotrexate dose higher than 15 mg weekly). There was no significant difference in LGALS3 +292 genotype distribution or allelic carriage between the anti-CCP positive and negative RA patients or RA patients refractory to and well controlled by traditional DMARDs, using chi-square analysis (data not shown).
Table 2 LGALS3 +191–+292 haplotype distribution in 151 RA patients and 182 healthy controls
RA patients N=151 n (%)
C) (20.7%). Above all, the LGALS3+191–+292 (C–C) haplotype was significantly more prevalent in RA patients than in the normal controls (43% versus 30.8%, OR = 1.7, 95% confidence interval = 1.1–2.7, p = 0.02, Table 2). LGALS3 +292C carriage was associated with a lower serum level of galectin-3 in RA patients Due to limited quantity of polyclonal anti-galectin-3 antibodies, serum galectin-3 levels were measured in 72 RA patients and 32 normal subjects. There was no significant difference in the serum galectin-3 level between RA and the normal control group (mean 10.8 ng/ml versus 10.3 ng/ml) (Fig. 2a). Serum galectin-3 levels were compared between RA patients with different LGALS3 +292 genotypes. It was noted that LGALS3 +292AC and CC genotypes (Gal-3 98Pro/Thr and Pro/Pro) were associated with lower galectin-3 serum level as compared to that of LGALS3 +292AA genotypes (Gal-3 98Thr/Thr) in RA patients (mean 9.5 ng/ml vs. 13.2 ng/ml, p=0.006) (Fig. 2b).
RA patients N=151 n (%) LGALS3 +191–+292 haplotype combination C–A/C–A 50 (33.1) C–A/C–C 44 (29.1) C–C/C–C 10 (6.6) A–C/C–C 11 (7.3) A–C/A–C 8 (5.3) A–C/C–A 28 (18.5) C–C haplotype Non-carriers 86 (57.0) Carriers 65 (43.0)
Controls N=182 n (%)
86 (47.3) 38 (20.9) 7 (3.8) 11 (6.0) 8 (4.3) 32 (17.6) 126 (69.2) 56 (30.8)
OR (95% CI)
p value
1.0 2.0 2.4 1.7 1.7 1.5
0.015 0.079 0.237 0.303 0.192
(1.1–3.4) (0.9–6.5) (0.7–4.2) (0.6–4.7) (0.8–2.8)
1.0 1.7 (1.1–2.7)
0.02
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a
during the culture period (Fig. 3a, b). PBMC-derived macrophages from subjects with three different LGALS3 +292 genotypes were analyzed. Cellular galectin-3 was found to be highest in the day 5 macrophages derived from PBMCs of LGALS3 +292CC genotype (Gal-3 98Pro/Pro) (Fig. 3c).
Discussion Our study revealed that carriage of LGALS3 +292C allele was more prevalent in RA patients than in the normal controls among Taiwanese population. Such result was (n=72)
b
(n=32)
a
Days in culture D1
D3
D5
D7
Gal-3
p=0.006
β-actin
Relative expression of Galectin-3 (Gal-3/β-actin)
b 2.0 1.5 1.0 0.5 0.0 D3
D1
D5
D7
Days in vitro culture AA (n=25)
LGALS3 +292 genotype Fig. 2 Serum galectin-3 level in RA patients and healthy control subjects. a Serum galectin-3 level was detected in 72 RA patients and 32 healthy control subjects by ELISA. There was no significant difference between these two groups by Wilcoxon rank-sum test. b Among the RA patients, galectin-3 serum level was compared between 25 subjects of LGALS3 +292AA genotype vs. 47 LGALS3 +292C carriers (+292AC or CC genotypes). Serum galectin-3 was higher in patients with LGALS3 +292AA genotype compared to patients with LGALS3 +292AC or CC genotypes (p=0.006). The horizon bar represents the mean serum galectin-3 level in each group
LGALS3 +292CC genotype (Galectin-3 98Pro/Pro) correlated to higher intracellular galectin-3 in peripheral blood monocyte-derived macrophages To test whether the LGALS3 +292 genotype is associated with the cellular level of galectin-3, the macrophages from healthy donors were obtained and subjected to immunoblotting analysis. Cellular galectin-3 expression increased
c Relative expression of Galectin-3 (Gal-3/β−actin)
AC/CC (n=47)
5
Day1 Day5
4 3 2 1 0
AA
AC
CC
LGALS3 +292 genotype
Fig. 3 Expression of galectin-3 in cultured monocytes derived from normal subjects. a Monocytes derived from a healthy subject were culture for 7 days. Western blotting analysis of cellular galectin-3 was performed in monocytes derived from PBMCs at the first, third, fifth, and seventh days of culture (D1 the cells prepared at the day of blood collection, without in vitro culture). The expression of β-actin was analyzed in parallel as protein loading control. b Cellular galectin-3 expression increased after culture. Relative level of cellular galectin-3 expression was normalized to that of the β-actin. c Relative galectin-3 expression of the day-1- and day-5-cultured PBMC-derived monocytes prepared from normal subjects with different LGALS3 +292 genotypes [+292AA (n=2), +292AC (n=2), +292CC (n=3)]
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biologically plausible because galectin-3 is involved in several important processes related to RA, including angiogenesis [23], cytokine and chemokine expression in synovial fibroblast [29], apoptosis of inflammatory cells [19, 20, 30], and osteoclastogenesis [31]. In a recent report, galectin-3 was shown to be a target for gene therapy in an animal model for RA [32]. We did not find a significant difference in LGALS3 +292C allelic prevalence among antiCCP antibody positive patients or patients who required anti-TNF alpha agents (etanercept or adalimumab) for controlling disease progression. Serum galectin-3 level in our RA patients was not significantly higher than that of the normal controls as reported by Ohshima et al. [24]. This might be due to that most patients in our study were in stable disease status. However, we found that carriage of LGALS3 +292C allele was associated with a lower serum galectin-3 level in RA patients. Product of LGALS3 +292C allele carries proline at galectin-3 residue 98, while the proline at the 98th residue is located in a critical protein transport determination region. Galectins exhibits non-classical secretion pathway because of lacking recognizable signal sequence for endoplasmic reticulum/Golgi pathway [33, 34]. The Nterminal motif within mammalian galectin-3 is highly conserved and might be critical for its excretion from the cell. A short segment of N-terminal sequence comprising residues 89–96 (Tyr-Pro-Ser-Ala-Pro-Gly-Ala-Tyr) on hamster galectin-3 was proved to play a critical role in its secretion. The proline to alanine substitutions made at residue 90 and 93 of hamster galectin-3 resulted in reduced or abolished secretion of the galectin-3-CAT fusion protein [4]. The 98th residue of human galectin-3 resides in one of the N-terminal repeated domains (YPSAPGAY, residue 94– 101), which is highly homologous to the YPSAPGAY (residue 89–96) fragment in the hamster galectin-3, suggesting that LGALS3 +292 allele may also be involved in the regulation of galectin-3 secretion. Our data revealed that cellular galectin-3 in day 5 macrophages of LGALS3 +292CC genotype (Gal-3 98Pro/ Pro ) tended to be higher than those of the other genotypes. Such finding may be important in RA pathogenesis for that intracellular galectin-3 has important function in macrophage. It was reported that increased galectin-3 expression is associated with defective monocyte apoptosis in juvenile inflammatory arthritis [35]. Although not tested in our study, intracellular galectin-3 was known to have antiapoptotic property in T cells and macrophages [12], thus it might play an important role in persisted inflammation in RA synovium. Whether the variation at the 98th residue determines intracellular galectin-3 level via influencing its secretion deserved further study. In conclusion, LGALS3 +292C polymorphism is associated with rheumatoid arthritis susceptibility but
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not anti-CCP autoantibody status or response to nonbiologic DMARDs in Taiwan. Because of the complexity of different cellular distribution and variable intra-/ extracellular roles, functional activities of galectin-3 in the pathogenesis of rheumatoid arthritis await further studies. Acknowledgments This study was supported by grants from the National Science Council (NSC-94-2320-B-002-085), National Taiwan University Hospital (VN 9801), and National Health Research Institute (NHRI-EX95-9532SI), Taiwan. Polyclonal galectin-3 antibodies used in detecting serum galectin-3 were kindly provided by Dr. Liu FT. Disclosures None.
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