Biomarkers
ISSN: 1354-750X (Print) 1366-5804 (Online) Journal homepage: http://www.tandfonline.com/loi/ibmk20
EGF rs11568835 G/A polymorphism is associated with increased risk of rheumatoid arthritis Liqun Wang, Lin Bo, Ting Yan, Hui Zhang, Guoxin Zhou & Ruiping Liu To cite this article: Liqun Wang, Lin Bo, Ting Yan, Hui Zhang, Guoxin Zhou & Ruiping Liu (2014) EGF rs11568835 G/A polymorphism is associated with increased risk of rheumatoid arthritis, Biomarkers, 19:7, 563-566 To link to this article: http://dx.doi.org/10.3109/1354750X.2014.946450
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Date: 12 September 2015, At: 09:51
http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2014; 19(7): 563–566 ! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2014.946450
RESEARCH ARTICLE
EGF rs11568835 G/A polymorphism is associated with increased risk of rheumatoid arthritis Liqun Wang1*#, Lin Bo2*#, Ting Yan3, Hui Zhang3, Guoxin Zhou3, and Ruiping Liu3,4# School of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou, China, 2Department of Rheumatology, The Second Affiliated Hospital of Soochow University, Suzhou, China, 3Department of Orthopedic Trauma, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou, China, and 4Central Laboratory, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou, China
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1
Abstract
Keywords
Rheumatoid arthritis (RA) is an autoimmune rheumatological disease thought to have substantial genetic contributions. Epidermal growth factor (EGF) can activate DNA synthesis and cellular proliferation. Early RA synovial fluid was characterized by significantly elevated levels stromal cell and macrophage-related cytokines including EGF. We therefore hypothesized that EGF polymorphisms may contribute to RA susceptibility in the Chinese population. We studied EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms in 520 patients with RA and 520 controls in a Chinese population. When the EGF rs11568835 GG homozygote genotype was used as the reference group, the AA genotype was associated with an increased risk for RA (AA versus GG, odds ratio [OR] ¼ 3.64, 95% confidence interval [CI] ¼ 1.19–11.17, p ¼ 0.024), the GA or GA/AA genotype was not associated with the risk for RA (GA versus GG, OR ¼ 0.99, 95% CI ¼ 0.75–1.31, p ¼ 0.931; GA + AA versus GG, OR ¼ 1.06, 95% CI ¼ 0.81–1.40, p ¼ 0.659). EGF rs3756261 T/C was not associated with susceptibility to RA. These results provide the first positive evidence for an association between EGF rs11568835 G/A polymorphism and RA.
EGF, molecular epidemiology, polymorphisms, rheumatoid arthritis
Introduction Rheumatoid arthritis (RA) is a systemic, inflammatory and autoimmune disease caused by a failure of immune selftolerance. RA is characterized by chronic synovial joint inflammation, overgrowth of synoviocytes, progressive erosions and cartilage destruction (FitzGerald et al., 1991; Iguchi & Ziff, 1986). Untreated RA patients have a progressive course, resulting in either short-term or long-term disability. The etiologies and pathogeneses of RA are complex and remain unresolved. Genetic and environmental risk factors and their interaction contribute to RA pathogenesis (Mahdi et al., 2009). RA is thought to have substantial genetic contributions (Orozco et al., 2011). The cytokines epidermal growth factor (EGF) and vascular endothelial growth factor (VEGFA) are powerful mitogens; EGF can activate DNA synthesis and cellular proliferation (Levin, 2003; Yarden, 2001) and is also involved in angiogenesis of epidermal tissue (Harari, 2004;
*These authors contributed equally to this work. #Liqun Wang, Lin Bo and Ruiping Liu are responsible for statistical design and analysis. E-mail: [email protected] (L. Wang), [email protected] (L. Bo) and [email protected] (R. Liu) Address for correspondence: Dr. Ruiping Liu, Department of Orthopedic Trauma, Changzhou Second People’s Hospital, Changzhou 213003, China. E-mail: [email protected]
History Received 21 June 2014 Accepted 16 July 2014 Published online 4 August 2014
Yarden & Sliwkowski, 2001). Early RA synovial fluid was characterized by significantly elevated levels stromal cell and macrophage-related cytokines including EGF (Raza et al., 2005). The EGF gene contains an atypical TATA box, polypurinerich motifs and consensus binding sequences for many transcription factors (Fenton et al., 1996; Mullhaupt et al., 2000). EGF expression and cell division was suggested to be regulated by EGF promoter (Mullhaupt et al., 2000). Two single nucleotide polymorphisms (SNPs) in the promoter region of EGF (G-1380A, rs11568835 and A-1744G, rs3756261) (Jin et al., 2007; Wang et al., 2008), with a minor allele frequency 0.05 in Chinese population, were also selected from the public SNP database. The variant alleles of the EGF promoter, 1380A and 1744G, were also associated with a decreased risk of gastric cancer with borderline significance (Jin et al., 2007). However, although EGF plays an important part in the immune response, few studies have focused on the influences of EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms in the susceptibilities to RA. Functional variations in EGF genes may contribute to the development of RA. We previously conducted a hospital-based case–control study to evaluate VEGFA rs699947 C/A, rs2010963 G/C and rs3025039 C/T polymorphisms and RA risk (Zhang et al., 2013); we now genotyped EGF polymorphisms in a cohort of 520 RA patients and 520 controls.
564
L. Wang et al.
Patients and methods
Biomarkers, 2014; 19(7): 563–566
Table 1. Patient demographics and risk factors in rheumatoid arthritis.
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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. Five-hundred twenty RA patients who fulfilled the criteria for RA set by the American College of Rheumatology classification in 1987 (Arnett et al., 1988) 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 May 2012. The controls were patients without RA, matched RA 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 a custom-by-design 48-Plex SNPscanä Kit Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediamine tetra-acetic acid. Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genotyping was performed using a custom-by-design 48-Plex SNPscanÔ Kit (Genesky Biotechnologies Inc., Shanghai, China) as previously described (Ren et al., 2014; Yin et al., 2014a, b). This kit was developed according to patented SNP genotyping technology by Genesky Biotechnologies Inc., which was based on double ligation and multiplex fluorescence PCR. For quality control, repeated analyses were performed for 4% of randomly selected samples with high DNA quality. Statistical analysis Differences in demographics, variables and genotypes of the EGF rs11568835 G/A and EGF rs3756261 T/C polymorphism variants were evaluated using a chi-squared test. The associations between EGF rs11568835 G/A and EGF rs3756261 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. Differences in genotypes of EGF polymorphism variants were evaluated using Student’s t-test. Haplotype frequencies were obtained from the PHASE 2.0 program based on the observed EGF genotypes. All statistical analyses were done with SAS software, version 9.1.3 (SAS Institute, Cary, NC).
Results Characteristics of the study population Among 1040 DNA samples (520 RA patients and 520 controls), the EGF rs11568835 G/A polymorphism was
Variablea Age (years) Female/male Age at onset, years, mean ± SD Disease duration, years, mean ± SD Treatment duration, years, mean ± SD RF-positive, no. (%) ACPA-positive, no. (%) CRP-positive, no. (%) ESR, mm/h DAS28 Functional class, no. (%) I II III IV a
RA cases (n ¼ 520)
Controls (n ¼ 520)
p
54.72 (± 15.27) 390/130
54.17 (± 10.50) 385/135
0.496 0.722
46.43 (± 13.28)
–
–
8.34 (± 9.40) 7.03 (± 7.93)
– –
– –
(79.6%) (51.7%) (42.3%) (± 29.41) (± 1.53)
– – – – –
– – – – –
(13.1%) (45.4%) (35.2%) (6.3%)
– – – –
– – – –
414 269 220 36.93 4.55 68 236 183 33
RF: rheumatoid factor; ACPA: anti-cyclic citrullinated peptide antibody; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; and DAS28: RA disease activity score.
successful in 507 (97.5%) RA patients and 519 (99.8%) controls. Genotyping for the EGF rs3756261 T/C polymorphism was successful in 508 (97.7%) RA patients and 520 (100.0%) controls. The concordance rates of repeated analyses were 100%. The demographic and clinical characteristics of all subjects are listed in Table 1. Subjects were adequately matched for age and sex (p ¼ 0.496 and 0.722, respectively) for RA patients and controls (Table 1). The primary information for two genotyped SNPs is mentioned in Table S1. The genotype distributions of EGF rs11568835 G/A and EGF rs3756261 T/C in all subjects are illustrated in Table 2. Associations between EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms and risk of RA The genotype frequencies of the EGF rs11568835 G/A polymorphism were 71.79% (GG), 25.44% (GA) and 2.76% (AA) in RA patients, and 73.03% (GG), 26.20% (GA) and 0.77% (AA) in controls (p ¼ 0.052) (Table 2). When the EGF rs11568835 GG homozygote genotype was used as the reference group, the AA genotype was associated with an increased risk for RA (AA versus GG, OR ¼ 3.64, 95% CI ¼ 1.19–11.17, p ¼ 0.024), the GA or GA/AA genotype was not associated with the risk for RA (GA versus GG, OR ¼ 0.99, 95% CI ¼ 0.75–1.31, p ¼ 0.931; GA + AA versus GG, OR ¼ 1.06, 95% CI ¼ 0.81–1.40, p ¼ 0.659). In the recessive model, when the EGF rs11568835 GG/GA genotypes were used as the reference group, the AA homozygote genotype was associated with susceptibility to RA (OR ¼ 3.66, 95% CI ¼ 1.20–11.18, p ¼ 0.023). The genotype frequencies of the EGF rs3756261 T/C polymorphism were 65.35% (TT), 31.10% (TC) and 3.54% (CC) in RA patients, and 66.92% (TT), 27.12% (TC) and 5.96% (CC) in controls (p ¼ 0.098) (Table 2). When the EGF rs3756261 TT homozygote genotype was used as the reference group, the TC or CC or TC/CC genotype was not associated with the risk for RA (TC versus TT, OR ¼ 1.18,
EGF rs11568835 G/A polymorphism and RA
DOI: 10.3109/1354750X.2014.946450
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Table 2. Logistic regression analysis of associations between EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms and risk of rheumatoid arthritis.
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Genotype EGF rs11568835 G/A GG GA AA AA versus GA versus GG GA + AA GG + GA AA G allele A allele EGF rs3756261 T/C TT TC CC CC versus TC versus TT TC + CC TT + TC CC T allele C allele
Casesa (n ¼ 520)
Controls (n ¼ 520)
n
%
n
%
OR (95% CI)
p
364 129 14
71.79 25.44 2.76
379 136 4
73.03 26.20 0.77
1.00 0.99 (0.75–1.31) 3.64 (1.19–11.17)
143 493 14 857 157
28.21 97.24 2.76 84.52 15.48
140 515 4 894 144
26.97 99.23 0.77 86.13 13.87
1.06 (0.81–1.40) 1.00 3.66 (1.20–11.18) 1.00 1.14 (0.89–1.45)
— 0.931 0.024 0.052 0.659 — 0.023 — 0.303
332 158 18
65.35 31.10 3.54
348 141 31
66.92 27.12 5.96
1.00 1.18 (0.90–1.54) 0.61 (0.33–1.11)
176 490 18 822 194
34.65 96.46 3.54 80.91 19.09
172 489 31 837 203
33.08 94.04 5.96 80.48 19.52
1.07 (0.83–1.39) 1.00 0.58 (0.32–1.05) 1.00 0.97 (0.78–1.21)
— 0.247 0.105 0.098 0.595 — 0.072 — 0.807
a
Genotyping was successful in 507 RA cases and 519 controls for EGF rs11568835. G/A; genotyping was successful in 508 RA cases and 520 controls for EGF rs3756261 T/C. Bold values are statistically significant (p50.05).
Table 3. EGF haplotype frequencies (%) in cases and controls and risk of rheumatoid arthritis. Cases (n ¼ 1040) Haplotypes EGF EGF EGF EGF
Trs3756261Grs11568835 Crs3756261Grs11568835 Trs3756261Ars11568835 Crs3756261Ars11568835
Controls (n ¼ 1040)
n
%
n
%
Crude OR (95%CI)
p
689 190 154 7
66.250 18.269 14.808 0.673
693 203 144 0
66.635 19.519 13.846 0
1.00 0.94 (0.75–1.18) 1.08 (0.84–1.38) –
0.598 0.568 0.975
With the order of EGF rs3756261 T/C and EGF rs11568835 G/A in gene position.
95% CI ¼ 0.90–1.54, p ¼ 0.247; CC versus TT, OR ¼ 0.61, 95% CI ¼ 0.33–1.11, p ¼ 0.105; TC + CC versus TT, OR ¼ 1.07, 95% CI ¼ 0.83–1.39, p ¼ 0.595). In the recessive model, when the EGF rs3756261 TT/TC genotypes were used as the reference group, the CC homozygote genotype was not associated with susceptibility to RA (OR ¼ 0.58, 95% CI ¼ 0.32–1.05, p ¼ 0.072). Haplotype analysis of EGF polymorphisms and susceptibility of RA As shown in Table 3, haplotype analysis was performed, and haplotypes were derived from the observed genotypes of these two EGF polymorphisms; no association was observed between EGF haplotypes and RA (Table 3).
Discussion Early RA synovial fluid was characterized by significantly elevated levels of stromal cell and macrophage-related cytokines including EGF (Raza et al., 2005). The EGF gene contains an atypical TATA box, polypurine-rich motifs and consensus binding sequences for many transcription factors. The variant alleles of the EGF promoter, 1380A
(rs11568835) and 1744G (rs3756261), were also associated with a decreased risk of gastric cancer with borderline significance (Jin et al., 2007). This study determined the association between the EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms and the risks of RA in a Chinese population, and provided the first positive evidence for a relationship between EGF rs11568835 G/A polymorphism and RA. The EGF rs11568835 AA allele appeared to increase the risk of RA. However, no positive associations were found between the EGF rs3756261 T/C polymorphism and RA risk. In a case–control study involving 248 psoriatic arthritis (PsA) patients and 154 controls, the EGF polymorphisms was not associated with PsA risk. This study, however, found positive association between RA and the EGF rs11568835 G/A polymorphism (Butt et al., 2007). Based on EGF rs11568835 G/A mutant alleles in the control group, case samples and control samples, the power of our study (a ¼ 0.05) was 1.000 in 507 RA cases and 519 controls with an OR of 3.66. Several limitations of this study need to be addressed. First, this was a hospital-based case–control study, and selection bias was therefore unavoidable, meaning that the
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subjects were not fully representative of the general population. Second, the polymorphisms investigated, based on their functional considerations, may not offer a comprehensive view of the genetic variability of EGF. Third, a single case– control study is not sufficient to fully interpret the relationship between EGF polymorphisms and susceptibility to RA because of the relatively moderate number of patients evaluated. Studies with larger numbers of subjects are necessary to confirm our findings. Finally, environmental factors differ between Chinese populations and others; because RA risk is likely to be influenced by gene–gene and gene–environment interactions, the EGF gene may be associated with different degrees of genetic risk in different ethnic groups and under different environmental exposures. In conclusion, this study provided evidence that the EGF rs11568835 G/A functional polymorphism may contribute to the risk of RA, whereas EGF rs3756261 T/C polymorphisms may not be associated with the risk of RA. However, further studies are needed to confirm the results of this preliminary study.
Declaration of interest None of the authors has any potential financial conflict of interest related to this manuscript. This study was supported, in part, by the National Natural Science Foundation of China (81371927) and Nanjing Medical University Foundation for Development of Science and Technology (2012NJMU128).
References Arnett FC, Edworthy SM, Bloch DA, et al. (1988). The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315–24. Butt C, Lim S, Greenwood C, et al. (2007). VEGF, FGF1, FGF2 and EGF gene polymorphisms and psoriatic arthritis. BMC Musculoskelet Disord 8:1. Fenton SE, Groce NS, Lee DC. (1996). Characterization of the mouse epidermal growth factor promoter and 50 -flanking region. Role for an atypical TATA sequence. J Biol Chem 271:30870–8.
Fitzgerald O, Soden M, Yanni G, et al. (1991). Morphometric analysis of blood vessels in synovial membranes obtained from clinically affected and unaffected knee joints of patients with rheumatoid arthritis. Ann Rheum Dis 50:792–6. Harari PM. (2004). Epidermal growth factor receptor inhibition strategies in oncology. Endocr Relat Cancer 11:689–708. Iguchi T, Ziff M. (1986). Electron microscopic study of rheumatoid synovial vasculature. Intimate relationship between tall endothelium and lymphoid aggregation. J Clin Invest 77:355–61. Jin G, Miao R, Deng Y, et al. (2007). Variant genotypes and haplotypes of the epidermal growth factor gene promoter are associated with a decreased risk of gastric cancer in a high-risk Chinese population. Cancer Sci 98:864–8. Levin ER. (2003). Bidirectional signaling between the estrogen receptor and the epidermal growth factor receptor. Mol Endocrinol 17:309–17. Mahdi H, Fisher BA, Kallberg H, et al. (2009). Specific interaction between genotype, smoking and autoimmunity to citrullinated alphaenolase in the etiology of rheumatoid arthritis. Nat Genet 41:1319–24. Mullhaupt B, Feren A, Jones A, et al. (2000). DNA sequence and functional characterization of the human and rat epidermal growth factor promoter: regulation by cell growth. Gene 250:191–200. Orozco G, Eyre S, Hinks A, et al. (2011). Study of the common genetic background for rheumatoid arthritis and systemic lupus erythematosus. Ann Rheum Dis 70:463–8. Raza K, Falciani F, Curnow SJ, et al. (2005). Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin. Arthritis Res Ther 7:R784–95. Ren Z, Li M, Liu R, et al. (2014). Interleukin 17A rs3819024 A4G polymorphism is associated with an increased risk of gastric cardia adenocarcinoma in a Chinese population. Biomarkers 19:411–16. Wang Y, Tian T, Hu Z, et al. (2008). EGF promoter SNPs, plasma EGF levels and risk of breast cancer in Chinese women. Breast Cancer Res Treat 111:321–7. Yarden Y. (2001). The EGFR family and its ligands in human cancer. Signalling mechanisms and therapeutic opportunities. Eur J Cancer 37:S3–8. Yarden Y, Sliwkowski MX. (2001). Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2:127–37. Yin J, Tang W, Shao A, et al. (2014a). Caspase8 rs1035142 G4T polymorphism was associated with an increased risk of esophageal cancer in a Chinese population. Mol Biol Rep 41:2037–43. Yin J, Wang X, Wei J, et al. (2014b). Interleukin 12B rs3212227 T4G polymorphism was associated with an increased risk of gastric cardiac adenocarcinoma in a Chinese population. Dis Esophagus. doi: 10.1111/dote.12189. [Epub ahead of print]. Zhang Y, Qiu H, Zhang H, et al. (2013). Vascular endothelial growth factor A (VEGFA) polymorphisms in Chinese patients with rheumatoid arthritis. Scand J Rheumatol 42:344–8.
Supplementary material available online Supplementary Table S1.
ISSN: 1354-750X (Print) 1366-5804 (Online) Journal homepage: http://www.tandfonline.com/loi/ibmk20
EGF rs11568835 G/A polymorphism is associated with increased risk of rheumatoid arthritis Liqun Wang, Lin Bo, Ting Yan, Hui Zhang, Guoxin Zhou & Ruiping Liu To cite this article: Liqun Wang, Lin Bo, Ting Yan, Hui Zhang, Guoxin Zhou & Ruiping Liu (2014) EGF rs11568835 G/A polymorphism is associated with increased risk of rheumatoid arthritis, Biomarkers, 19:7, 563-566 To link to this article: http://dx.doi.org/10.3109/1354750X.2014.946450
View supplementary material
Published online: 04 Aug 2014.
Submit your article to this journal
Article views: 38
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ibmk20 Download by: [University of Georgia]
Date: 12 September 2015, At: 09:51
http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2014; 19(7): 563–566 ! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2014.946450
RESEARCH ARTICLE
EGF rs11568835 G/A polymorphism is associated with increased risk of rheumatoid arthritis Liqun Wang1*#, Lin Bo2*#, Ting Yan3, Hui Zhang3, Guoxin Zhou3, and Ruiping Liu3,4# School of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou, China, 2Department of Rheumatology, The Second Affiliated Hospital of Soochow University, Suzhou, China, 3Department of Orthopedic Trauma, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou, China, and 4Central Laboratory, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou, China
Downloaded by [University of Georgia] at 09:51 12 September 2015
1
Abstract
Keywords
Rheumatoid arthritis (RA) is an autoimmune rheumatological disease thought to have substantial genetic contributions. Epidermal growth factor (EGF) can activate DNA synthesis and cellular proliferation. Early RA synovial fluid was characterized by significantly elevated levels stromal cell and macrophage-related cytokines including EGF. We therefore hypothesized that EGF polymorphisms may contribute to RA susceptibility in the Chinese population. We studied EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms in 520 patients with RA and 520 controls in a Chinese population. When the EGF rs11568835 GG homozygote genotype was used as the reference group, the AA genotype was associated with an increased risk for RA (AA versus GG, odds ratio [OR] ¼ 3.64, 95% confidence interval [CI] ¼ 1.19–11.17, p ¼ 0.024), the GA or GA/AA genotype was not associated with the risk for RA (GA versus GG, OR ¼ 0.99, 95% CI ¼ 0.75–1.31, p ¼ 0.931; GA + AA versus GG, OR ¼ 1.06, 95% CI ¼ 0.81–1.40, p ¼ 0.659). EGF rs3756261 T/C was not associated with susceptibility to RA. These results provide the first positive evidence for an association between EGF rs11568835 G/A polymorphism and RA.
EGF, molecular epidemiology, polymorphisms, rheumatoid arthritis
Introduction Rheumatoid arthritis (RA) is a systemic, inflammatory and autoimmune disease caused by a failure of immune selftolerance. RA is characterized by chronic synovial joint inflammation, overgrowth of synoviocytes, progressive erosions and cartilage destruction (FitzGerald et al., 1991; Iguchi & Ziff, 1986). Untreated RA patients have a progressive course, resulting in either short-term or long-term disability. The etiologies and pathogeneses of RA are complex and remain unresolved. Genetic and environmental risk factors and their interaction contribute to RA pathogenesis (Mahdi et al., 2009). RA is thought to have substantial genetic contributions (Orozco et al., 2011). The cytokines epidermal growth factor (EGF) and vascular endothelial growth factor (VEGFA) are powerful mitogens; EGF can activate DNA synthesis and cellular proliferation (Levin, 2003; Yarden, 2001) and is also involved in angiogenesis of epidermal tissue (Harari, 2004;
*These authors contributed equally to this work. #Liqun Wang, Lin Bo and Ruiping Liu are responsible for statistical design and analysis. E-mail: [email protected] (L. Wang), [email protected] (L. Bo) and [email protected] (R. Liu) Address for correspondence: Dr. Ruiping Liu, Department of Orthopedic Trauma, Changzhou Second People’s Hospital, Changzhou 213003, China. E-mail: [email protected]
History Received 21 June 2014 Accepted 16 July 2014 Published online 4 August 2014
Yarden & Sliwkowski, 2001). Early RA synovial fluid was characterized by significantly elevated levels stromal cell and macrophage-related cytokines including EGF (Raza et al., 2005). The EGF gene contains an atypical TATA box, polypurinerich motifs and consensus binding sequences for many transcription factors (Fenton et al., 1996; Mullhaupt et al., 2000). EGF expression and cell division was suggested to be regulated by EGF promoter (Mullhaupt et al., 2000). Two single nucleotide polymorphisms (SNPs) in the promoter region of EGF (G-1380A, rs11568835 and A-1744G, rs3756261) (Jin et al., 2007; Wang et al., 2008), with a minor allele frequency 0.05 in Chinese population, were also selected from the public SNP database. The variant alleles of the EGF promoter, 1380A and 1744G, were also associated with a decreased risk of gastric cancer with borderline significance (Jin et al., 2007). However, although EGF plays an important part in the immune response, few studies have focused on the influences of EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms in the susceptibilities to RA. Functional variations in EGF genes may contribute to the development of RA. We previously conducted a hospital-based case–control study to evaluate VEGFA rs699947 C/A, rs2010963 G/C and rs3025039 C/T polymorphisms and RA risk (Zhang et al., 2013); we now genotyped EGF polymorphisms in a cohort of 520 RA patients and 520 controls.
564
L. Wang et al.
Patients and methods
Biomarkers, 2014; 19(7): 563–566
Table 1. Patient demographics and risk factors in rheumatoid arthritis.
Downloaded by [University of Georgia] at 09:51 12 September 2015
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. Five-hundred twenty RA patients who fulfilled the criteria for RA set by the American College of Rheumatology classification in 1987 (Arnett et al., 1988) 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 May 2012. The controls were patients without RA, matched RA 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 a custom-by-design 48-Plex SNPscanä Kit Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediamine tetra-acetic acid. Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genotyping was performed using a custom-by-design 48-Plex SNPscanÔ Kit (Genesky Biotechnologies Inc., Shanghai, China) as previously described (Ren et al., 2014; Yin et al., 2014a, b). This kit was developed according to patented SNP genotyping technology by Genesky Biotechnologies Inc., which was based on double ligation and multiplex fluorescence PCR. For quality control, repeated analyses were performed for 4% of randomly selected samples with high DNA quality. Statistical analysis Differences in demographics, variables and genotypes of the EGF rs11568835 G/A and EGF rs3756261 T/C polymorphism variants were evaluated using a chi-squared test. The associations between EGF rs11568835 G/A and EGF rs3756261 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. Differences in genotypes of EGF polymorphism variants were evaluated using Student’s t-test. Haplotype frequencies were obtained from the PHASE 2.0 program based on the observed EGF genotypes. All statistical analyses were done with SAS software, version 9.1.3 (SAS Institute, Cary, NC).
Results Characteristics of the study population Among 1040 DNA samples (520 RA patients and 520 controls), the EGF rs11568835 G/A polymorphism was
Variablea Age (years) Female/male Age at onset, years, mean ± SD Disease duration, years, mean ± SD Treatment duration, years, mean ± SD RF-positive, no. (%) ACPA-positive, no. (%) CRP-positive, no. (%) ESR, mm/h DAS28 Functional class, no. (%) I II III IV a
RA cases (n ¼ 520)
Controls (n ¼ 520)
p
54.72 (± 15.27) 390/130
54.17 (± 10.50) 385/135
0.496 0.722
46.43 (± 13.28)
–
–
8.34 (± 9.40) 7.03 (± 7.93)
– –
– –
(79.6%) (51.7%) (42.3%) (± 29.41) (± 1.53)
– – – – –
– – – – –
(13.1%) (45.4%) (35.2%) (6.3%)
– – – –
– – – –
414 269 220 36.93 4.55 68 236 183 33
RF: rheumatoid factor; ACPA: anti-cyclic citrullinated peptide antibody; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate; and DAS28: RA disease activity score.
successful in 507 (97.5%) RA patients and 519 (99.8%) controls. Genotyping for the EGF rs3756261 T/C polymorphism was successful in 508 (97.7%) RA patients and 520 (100.0%) controls. The concordance rates of repeated analyses were 100%. The demographic and clinical characteristics of all subjects are listed in Table 1. Subjects were adequately matched for age and sex (p ¼ 0.496 and 0.722, respectively) for RA patients and controls (Table 1). The primary information for two genotyped SNPs is mentioned in Table S1. The genotype distributions of EGF rs11568835 G/A and EGF rs3756261 T/C in all subjects are illustrated in Table 2. Associations between EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms and risk of RA The genotype frequencies of the EGF rs11568835 G/A polymorphism were 71.79% (GG), 25.44% (GA) and 2.76% (AA) in RA patients, and 73.03% (GG), 26.20% (GA) and 0.77% (AA) in controls (p ¼ 0.052) (Table 2). When the EGF rs11568835 GG homozygote genotype was used as the reference group, the AA genotype was associated with an increased risk for RA (AA versus GG, OR ¼ 3.64, 95% CI ¼ 1.19–11.17, p ¼ 0.024), the GA or GA/AA genotype was not associated with the risk for RA (GA versus GG, OR ¼ 0.99, 95% CI ¼ 0.75–1.31, p ¼ 0.931; GA + AA versus GG, OR ¼ 1.06, 95% CI ¼ 0.81–1.40, p ¼ 0.659). In the recessive model, when the EGF rs11568835 GG/GA genotypes were used as the reference group, the AA homozygote genotype was associated with susceptibility to RA (OR ¼ 3.66, 95% CI ¼ 1.20–11.18, p ¼ 0.023). The genotype frequencies of the EGF rs3756261 T/C polymorphism were 65.35% (TT), 31.10% (TC) and 3.54% (CC) in RA patients, and 66.92% (TT), 27.12% (TC) and 5.96% (CC) in controls (p ¼ 0.098) (Table 2). When the EGF rs3756261 TT homozygote genotype was used as the reference group, the TC or CC or TC/CC genotype was not associated with the risk for RA (TC versus TT, OR ¼ 1.18,
EGF rs11568835 G/A polymorphism and RA
DOI: 10.3109/1354750X.2014.946450
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Table 2. Logistic regression analysis of associations between EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms and risk of rheumatoid arthritis.
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Genotype EGF rs11568835 G/A GG GA AA AA versus GA versus GG GA + AA GG + GA AA G allele A allele EGF rs3756261 T/C TT TC CC CC versus TC versus TT TC + CC TT + TC CC T allele C allele
Casesa (n ¼ 520)
Controls (n ¼ 520)
n
%
n
%
OR (95% CI)
p
364 129 14
71.79 25.44 2.76
379 136 4
73.03 26.20 0.77
1.00 0.99 (0.75–1.31) 3.64 (1.19–11.17)
143 493 14 857 157
28.21 97.24 2.76 84.52 15.48
140 515 4 894 144
26.97 99.23 0.77 86.13 13.87
1.06 (0.81–1.40) 1.00 3.66 (1.20–11.18) 1.00 1.14 (0.89–1.45)
— 0.931 0.024 0.052 0.659 — 0.023 — 0.303
332 158 18
65.35 31.10 3.54
348 141 31
66.92 27.12 5.96
1.00 1.18 (0.90–1.54) 0.61 (0.33–1.11)
176 490 18 822 194
34.65 96.46 3.54 80.91 19.09
172 489 31 837 203
33.08 94.04 5.96 80.48 19.52
1.07 (0.83–1.39) 1.00 0.58 (0.32–1.05) 1.00 0.97 (0.78–1.21)
— 0.247 0.105 0.098 0.595 — 0.072 — 0.807
a
Genotyping was successful in 507 RA cases and 519 controls for EGF rs11568835. G/A; genotyping was successful in 508 RA cases and 520 controls for EGF rs3756261 T/C. Bold values are statistically significant (p50.05).
Table 3. EGF haplotype frequencies (%) in cases and controls and risk of rheumatoid arthritis. Cases (n ¼ 1040) Haplotypes EGF EGF EGF EGF
Trs3756261Grs11568835 Crs3756261Grs11568835 Trs3756261Ars11568835 Crs3756261Ars11568835
Controls (n ¼ 1040)
n
%
n
%
Crude OR (95%CI)
p
689 190 154 7
66.250 18.269 14.808 0.673
693 203 144 0
66.635 19.519 13.846 0
1.00 0.94 (0.75–1.18) 1.08 (0.84–1.38) –
0.598 0.568 0.975
With the order of EGF rs3756261 T/C and EGF rs11568835 G/A in gene position.
95% CI ¼ 0.90–1.54, p ¼ 0.247; CC versus TT, OR ¼ 0.61, 95% CI ¼ 0.33–1.11, p ¼ 0.105; TC + CC versus TT, OR ¼ 1.07, 95% CI ¼ 0.83–1.39, p ¼ 0.595). In the recessive model, when the EGF rs3756261 TT/TC genotypes were used as the reference group, the CC homozygote genotype was not associated with susceptibility to RA (OR ¼ 0.58, 95% CI ¼ 0.32–1.05, p ¼ 0.072). Haplotype analysis of EGF polymorphisms and susceptibility of RA As shown in Table 3, haplotype analysis was performed, and haplotypes were derived from the observed genotypes of these two EGF polymorphisms; no association was observed between EGF haplotypes and RA (Table 3).
Discussion Early RA synovial fluid was characterized by significantly elevated levels of stromal cell and macrophage-related cytokines including EGF (Raza et al., 2005). The EGF gene contains an atypical TATA box, polypurine-rich motifs and consensus binding sequences for many transcription factors. The variant alleles of the EGF promoter, 1380A
(rs11568835) and 1744G (rs3756261), were also associated with a decreased risk of gastric cancer with borderline significance (Jin et al., 2007). This study determined the association between the EGF rs11568835 G/A and EGF rs3756261 T/C polymorphisms and the risks of RA in a Chinese population, and provided the first positive evidence for a relationship between EGF rs11568835 G/A polymorphism and RA. The EGF rs11568835 AA allele appeared to increase the risk of RA. However, no positive associations were found between the EGF rs3756261 T/C polymorphism and RA risk. In a case–control study involving 248 psoriatic arthritis (PsA) patients and 154 controls, the EGF polymorphisms was not associated with PsA risk. This study, however, found positive association between RA and the EGF rs11568835 G/A polymorphism (Butt et al., 2007). Based on EGF rs11568835 G/A mutant alleles in the control group, case samples and control samples, the power of our study (a ¼ 0.05) was 1.000 in 507 RA cases and 519 controls with an OR of 3.66. Several limitations of this study need to be addressed. First, this was a hospital-based case–control study, and selection bias was therefore unavoidable, meaning that the
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subjects were not fully representative of the general population. Second, the polymorphisms investigated, based on their functional considerations, may not offer a comprehensive view of the genetic variability of EGF. Third, a single case– control study is not sufficient to fully interpret the relationship between EGF polymorphisms and susceptibility to RA because of the relatively moderate number of patients evaluated. Studies with larger numbers of subjects are necessary to confirm our findings. Finally, environmental factors differ between Chinese populations and others; because RA risk is likely to be influenced by gene–gene and gene–environment interactions, the EGF gene may be associated with different degrees of genetic risk in different ethnic groups and under different environmental exposures. In conclusion, this study provided evidence that the EGF rs11568835 G/A functional polymorphism may contribute to the risk of RA, whereas EGF rs3756261 T/C polymorphisms may not be associated with the risk of RA. However, further studies are needed to confirm the results of this preliminary study.
Declaration of interest None of the authors has any potential financial conflict of interest related to this manuscript. This study was supported, in part, by the National Natural Science Foundation of China (81371927) and Nanjing Medical University Foundation for Development of Science and Technology (2012NJMU128).
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Supplementary material available online Supplementary Table S1.
