Tumor Necrosis Factor-alpha Gene G-308A and G-238A Polymorphisms are not associated with Rheumatic Heart Disease in Taiwan

全文

(1)149. ORIGINAL ARTICLE. Tumor Necrosis Factor-α Gene G-308A and G-238A Polymorphisms are not Associated with Rheumatic Heart Disease in Taiwan 1,2,3,4. Hsiang-Tai Chou, Fuu-Jen Tsai 1. 2. 3. Division of Cardiology, Department of Medicine, Department of Pediatrics, Medical Research and Medical 4. Genetics, China Medical University Hospital; Graduate Institute of Chinese Medical Science, College of Chinese Medicine, China Medical University, Taichung, Taiwan.. P u r p o s e . The aim of this study was to test whether tumor necrosis factor (TNF)-α gene polymorphisms could be used as markers of susceptibility or severity of rheumatic heart disease (RHD) among the Chinese population in Taiwan. Methods. A group of 115 patients with RHD diagnosed by echocardiography, and another group of 103 age and sex-matched normal control subjects were studied. TNF-α gene G-308A and G-238A polymorphisms were identified by polymerase chain reaction-based restriction analysis. R e s u l t s . There was no significant difference in the distribution of genotypes and allelic frequencies of the TNF-α gene G-308A and G-238A polymorphisms between RHD patients and controls. Further categorization of RHD patients into mitral valve disease and combined valve disease subgroups also revealed no statistical difference in these gene polymorphisms when compared with controls. Conclusions. These findings suggest that the TNF-α gene G-308A and G-238A polymorphisms are not suitable genetic markers for RHD in Taiwan Chinese. ( Mid Taiwan J Med 2006;11:14954 ). Key words Taiwan, TNF-α gene polymorphisms, rheumatic heart disease. INTRODUCTION. Rheumatic heart disease (RHD) is an autoimmune sequel of beta hemolytic group A streptococcal infection of the pharynx complicated by rheumatic fever (RF) [1]. Autoimmunity induced by antigenic mimicry between the streptococcal glycoprotein and human cardiac myosin and laminin may be responsible for the pathogenesis of rheumatic carditis [2,3]. Some RF patients exhibit progressive fibrosis of the valves and develop chronic valvular disease; however, the factors Received : 30 March 2006. Revised : 25 April 2006. Accepted : 4 May 2006. Address reprint requests to : Hsiang-Tai Chou, Division of Cardiology, Department of Medicine, China Medical University Hospital, 2 Yuh-Der Road, Taichung 404, Taiwan.. leading to continued fibrosis and subsequent valve disease remain unclear. It has been shown that TNF-α might be involved in the pathogenesis of RHD. For example, the production of tumor necrosis factor-α (TNF-α) is higher during active rheumatic carditis [4,5]. Furthermore, recent data revealed that RHD was associated with TNF-α gene polymorphisms in a Mexican population [6]. The pro-inflammatory cytokine TNF-α plays an important role in inflammatory processes. The gene for TNF-α is located on chromosome six in the class III region of the major histocompatibility complex [7]. Different polymorphisms have been described, including the TNF-α G-308A polymorphism and the G238A polymorphism. Both are located within the.

(2) TNF-α Gene Polymorphisms in RHD. 150. Figure. PCR-based restriction analysis of TNF-α gene G-308A (upper panel) and G-238A (lower panel) polymorphisms shown on 3% agarose electrophoresis.. promoter region [8,9]. It has been demonstrated that these different allelic forms have functional implications. The TNF-α G-308A A allelic form results in two-fold greater transcription than the TNF-α G-308A G allelic form in PMAstimulated Jurkat T cells and U937 pre-monocytic cells [10]. To test if these polymorphisms might serve as markers of susceptibility or severity of RHD, the distribution of genotypes and allelic frequencies in a normal healthy control population was compared with that in a prospective cohort of 115 patients with RHD. MATERIALS AND METHODS. Between May 2000 and April 2002, a total. of 115 unrelated patients (31 men and 84 women; age range, 28 to 80 yr; mean age, 51.0 Ų 12.2 yr) with echocardiographically-documented predominant rheumatic mitral stenosis (MS) were enrolled in this study. Valve lesions were diagnosed by echocardiography, catheterization and cineangiography, or both. The patients were categorized into either a mitral valve disease group (MVD; n = 53; 13 men and 40 women; mean age, 51.1 Ų 12.4 yr) or a combined valvular disease group (CVD; n = 62; 18 men and 44 women; mean age, 50.9 Ų 12.1 yr). The MVD group consisted of patients with MS (n = 25) and those with MS and mitral regurgitation (n = 28). Patients with predominant mitral regurgitation and those with aortic or tricuspid valvular disease alone were excluded from this study. The control group comprised 103 age- and sex-matched unrelated healthy volunteers (28 men and 75 women; age range, 26 to 79 yr; mean age, 49.7 Ų 16.5 yr) who were free of autoimmune diseases, had normal echocardiography findings and no family history of RHD. All participants were ethnic Han Chinese residing in Taiwan. The study was approved by our hospital's internal review committee, and informed consent was obtained from each participant. The genomic DNA was prepared from peripheral blood leukocytes using a genomic DNA isolation kit (Blossom, Taipei, Taiwan). Polymerase chain reactions (PCRs) were carried out to a total volume of 50 µL containing genomic DNA, 2-6 pmol of each primer, 1X Taq polymerase buffer (1.5 mmol/L MgCl2), and 0.25 unit of Pro-Taq DNA polymerase (Pro-Tech, Taipei, Taiwan). The TNF-α gene G-308A and G-238A polymorphisms were typed by the restriction fragment length polymorphism (RFLP) method [9,10]. Primers for G-308A polymorphism were (forward) 5'-AGGCAA TAGGTTTTGAGGGCCAT-3' and (backward) 5'ACACTCCCCATCCTCCCTGCT-3'; those for G238A polymorphism were (forward) 5'-AAACAG ACCACAGACCTGGTC-3' and (backward) 5'CTCACACTCCCCATCCTCCCGGATC-3'. The PCR amplifications were performed in a GeneAmp PCR System 2400 programmable thermal cycler (PerKin-Elmer). The cycling.

(3) Hsiang-Tai Chou, et al.. 151. α genotypes in patients with rheumatic heart disease (RHD) (n = 115) and healthy Table 1. Distribution of TNF-α control patients (n = 103) RHD patients Controls Gene polymorphism Genotype p χ2 n (%) n (%) 101 (88) 89 (86) G-308A GG 13 (11) 14 (14) GA 0.57 1 (1) 0 (0) 1.14 AA 0.91 0.93/0.07 0.93/0.07 0.01 G/A 112 (97) 100 (97) G-238A GG 3 (3) 3 (3) GA 0.89 0 (0) 0 (0) 0.02 AA 0.89 0.99/0.01 0.99/0.01 0.02 G/A Genotype frequencies are indicated in absolute values (values in parentheses are percentages). Allelic frequencies are indicated in fractions. TNF-α = tumor necrosis factor-α gene.. conditions were set as follows: one cycle at 94˚C for 5 minutes, 35 cycles at 94˚C for 30 seconds, 60˚C for 30 seconds, 72˚C for 45 seconds, and one final cycle of extension at 72˚C for 7 minutes. The G-308A polymorphism in the promoter region of the TNF-α gene was categorized by digestion of PCR products with NcoI restriction enzyme followed by 3% agarose gel electrophoresis. The non-digested fragment was a single band of 117 bp (AA); the homozygous G allele was digested into a 97 bp and a 20 bp band (GG); heterozygotes (GA) showed three fragments of 117, 97 and 20 bp (Figure, upper panel). The homozygous A allele of the TNF-α G-238A polymorphism appeared as a single 155 bp band; the G allele was digested by BamHI restriction enzyme, resulting in a 130 bp and a 25 bp band (Figure, lower panel). Differences in genotype distribution between patients with RHD and control subjects were tested by the χ 2 test with 2 degrees of freedom (df). For statistical analysis of the allelic frequency distribution in the polymorphisms, the two groups were compared by the χ2 test with 1 df. Allelic frequencies were calculated from genotype frequencies in patients with RHD and control patients. The differences among the MVD, CVD and control groups were estimated by the χ2 test with 4 df. All statistical analyses were performed by NCSS 2000 Software (Kaysville, Utah, USA). A value of p < 0.05 was considered statistically significant. RESULTS. The χ 2 test confirmed that the genotype. proportions of TNF-α polymorphisms G-308A and G-238A fit the Hardy-Weinberg equilibrium. The distribution of the TNF-α genotypes and allelic frequencies among patients with RHD and control patients are shown in Table 1. There was no significant difference in the distribution of the G-308A polymorphism (χ2 = 1.14, p = 0.57) between patients and controls. The allelic frequencies of the TNF-α G-308A polymorphism did not differ significantly between patients and controls (χ2 = 0.01, p = 0.91). There was no statistically significant difference in genotype distribution of the TNF-α G-238A polymorphism between patients with RHD and control patients (χ2 = 0.02, p = 0.89). The allelic frequency of the TNF-α G-238A polymorphism did not differ significantly between patients and controls (χ 2 = 0.02, p = 0.89). No statistically significant difference in the G-308A (χ2 = 3.37, p = 0.50) polymorphism was observed among the MVD and CVD subgroups and the control group. Likewise, there was no significant difference in the distribution of G238A (χ2 = 0.52, p = 0.77) polymorphism among the MVD and CVD subgroups and the control group (Table 2). DISCUSSION. Several studies have suggested that genetic susceptibility to RF and RHD is linked to HLA class II alleles/haplotypes (HLA-DR2 in AfricanAmericans, HLA-DR4 in American Caucasians, HLA-DQA1 in Chinese southern Hans, HLADQA1*0104 and DQB1*05031 in Japanese, and.

(4) 152. TNF-α Gene Polymorphisms in RHD. α genotypes in patients with mitral valve disease (MVD) (n = 53) and combined Table 2. Distribution of TNF-α valvular disease (CVD) (n = 62), and healthy control patients (n = 103) MVD CVD Controls Gene polymorphism Genotype n (%) n (%) n (%) 55 (89) 89 (86) 46 (87) G-308A GG 7 (11) 14 (14) 6 (11) GA 0 (0) 0 (0) 1 (2) AA 2 χ = 3.37, p = 0.50 Significance* 61 (98) 51 (96) 100 (97) GG G-238A 1 (2) 2 (4) 3 (3) GA 0 (0) 0 (0) 0 (0) AA χ2 = 0.52, p = 0.77 0.99/0.01 Significance* Genotype frequencies are indicated in absolute values (values in parentheses are percentages). *Comparison among "MVD", "CVD" and "control" groups. TNF-α = tumor necrosis factor-α gene.. HLA-DRB1*1602 allele and HLA-DRB1*1602DQA1*0501-DQB1*0301 haplotype in Mexicans) [11-14]. However, the results were inconsistent as well as discrepant among the different populations studied. It has been suggested that a persistent inflammatory process occurs in the heart tissue of RHD patients, in the absence of infectious agents. Rheumatic involvement is present in 99% of stenotic mitral valves excised at the time of mitral valve replacement [15]. Guedez et al [16] reported that MVD patients had 5-fold fewer episodes of acute recurrent RF compared with multivalvular lesion patients; the mean rates of RF recurrence in MVD patients was 0.7 while that of multivalvular lesion patients was 3.3. The extent of original inflammation and recurrence of RF are not the only predictors of the crippling process. Ultimately, the deformed valve is subject to nonspecific fibrosis and calcification. The anatomic changes in severe MS and aortic stenosis may result from the combined effects of a smoldering rheumatic process and constant trauma to the mitral vlave or aortic valve by turbulent flow [17]. A recent study has reported that the ACE DD genotype is associated with an increased risk of subsequent heart valve damage in patients with RF [18]. However, we found an association between the ACE II genotype and RHD. An increased risk of RHD has been reported to be associated with ACE I allele. The difference might be caused by ethnic factors [19]. We also found that patients with RHD had a lower frequency of transforming growth factor-β1 (TGF-β1) C-509T CC genotype and a higher. frequency of T869C T allele, which supported the role of the TGF-β1 gene C-509T and T869C polymorphisms in determining the risk/protection of RHD in Taiwan Han Chinese [20]. Previous studies have shown a high production of interleukin (IL)-1, IL-2 and TNF-α by peripheral and mitral valve-infiltrating T cells in patients with RF and RHD [5,6,21], suggesting these inflammatory cytokines may play a role in the pathogenesis of these diseases. We did not find an association between IL-1β, IL-1receptor antagonist, IL-4 or IL-10 gene polymorphisms and RHD in our population [22]. An association between TNF-α G-238A polymorphism and RHD in a Mexican population has been reported. The G allele and GG genotype frequencies were both significantly increased in RHD patients when compared with healthy controls [7]. However, we did not find an association between TNF-α G238A polymorphism and RHD in our population. The difference might be caused by ethnic factors. In this case-controlled study, no association was found between the TNF-α gene G-308A and G-238A polymorphisms and RHD. Our results show no evidence of an association between these polymorphisms and the pattern of valve damage seen in RHD patients. Other single nucleotide polymorphisms on the TNF-α gene at positions – 1031, – 863, – 857, – 376, and +489 need to be studied to clarify the relationship with RHD [23,24]. We conclude that the TNF-α gene G-308A and G-238A polymorphisms are not suitable genetic markers for RHD among the Han Chinese.

(5) Hsiang-Tai Chou, et al.. population in Taiwan. Further studies are needed to clarify the pathogenesis of progression of RHD. REFERENCES. 1. Stollermann GH. Rheumatic fever. [Review] Lancet 1997;349:935-42. 2. Zabriskie JB. Rheumatic fever: a model for the pathological consequences of microbial-host mimicry. Clin Exp Rheumatol 1986;4:65-73. 3. Cunningham M. Molecular mimicry between group a streptococci and myosin in the pathogenesis of acute rheumatic fever. In: Narula J, Virmani R, Reddy KS, et al, eds. Rheumatic fever. Washington, DC: Armed Forces Institute of Pathology, 1999:135-65. 4. Narin N, Kutukculer N, Ozyurek R, et al. Lymphocytes subsets and plasma IL-1 alpha, IL-2, and TNF-alpha concentrations in acute rheumatic fever and chronic rheumatic heart disease. Clin Immunol Immunopathol 1995;77:172-6. 5. Yegin O, Coskun M, Ertug H. Cytokines in acute rheumatic fever. Eur J Pediatr 1997;156:25-9. 6. Hernandez-Pacheco G, Flores-Dominguez C, Rodriguez-Perez JM, et al. Tumor necrosis factoralpha promoter polymorphisms in Mexican patients with rheumatic heart disease. J Autoimmun 2003;21: 59-63. 7. Spies T, Morton CC, Nedospasov SA, et al. Genes for the tumor necrosis factors alpha and beta are linked to the human major histocompatibility complex. Proc Natl Acad Sci U S A 1986;83:8699-704. 8. D'Alfonso S, Richiardi PM. A polymorphic variation in a putative regulation box of the TNF alpha promoter region. Immunogenetics 1994;39:150-4. 9. Wilson AG, Symons JA, McDowell TL, et al. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci U S A 1997;94:3195-9. 10. Kroeger KM, Carville KS, Abraham LJ. The -308 tumor necrosis factor-alpha promoter polymorphism effects transcription. Mol Immunol 1997;34:391-9. 11. Carlquist JF, Ward RH, Meyer KJ, et al. Immune response factors in rheumatic heart disease: metaanalysis of HLA-DR associations and evaluation of additional class II alleles. J Am Coll Cardiol 1995;26: 452-7. 12. Gu J, Yu B, Zhou J. HLA-DQA1 genes involved in genetic susceptibility to rheumatic fever and rheumatic heart disease in southern Hans. Zhonghua Nei Ke Za Zhi 1997;36:308-11. (In Chinese; English abstract). 153. 13. Koyanagi T, Koga Y, Nishi H, et al. DNA typing of HLA class II genes in Japanese patients with rheumatic heart disease. J Mol Cell Cardiol 1996;28: 1349-53. 14.Hernandez-Pacheco G, Aguilar-Garcia J, FloresDominguez C, et al. MHC class II alleles in Mexican patients with rheumatic heart disease. Int J Cardiol 2003;92:49-54. 15. Olson LJ, Subramanian R, Ackerman DM, et al. Surgical pathology of the mitral vlave: a study of 712 cases spanning 21 years. Mayo Clin Proc 1987;62:2234. 16. Guedez Y, Kotby A, El-Demellawy M, et al. HLA class II association with rheumatic heart disease are more evident and consistent among clinically homogeneous patients. Circulation 1999;99:2784-90. 17. Schoen FJ, St. John Sutton M. Contemporary pathologic considerations in valvular disease. In: Virmani B, Atkinson JB, Feuoglio JJ, eds. Cardiovascular Pathology. Philadelphia: Saunders, 1991:334-53. 18.Atalar E, Tokgozoglu SL, Alikasifoglu M, et al. Angiotensin-converting enzyme genotype predicts valve damage in acute rheumatic fever. J Heart Valve Dis 2003;12:7-10. 19.Chou HT, Tsai CH, Tsai FJ. Association between angiotensin I-converting enzyme gene insertion/ deletion polymorphism and risk of rheumatic heart disease. Jpn Heart J 2004;45:949-57. 20. Chou HT, Chen CH, Tsai CH, et al. Association between transforming growth factor-beta1 gene C509T and T869C polymorphisms and rheumatic heart disease. Am Heart J 2004;148:181-6. 21. Guilherme L, Cunha-Neto E, Tanaka AC, et al. Heartdirected autoimmunity: the case of rheumatic fever. J Autoimmun 2001;16:363-7. 22.Chou HT, Tsai CH, Chen WC, et al. Lack of association of genetic polymorphisms in the interleukin-1beta, interleukin-1 receptor antagonist, interleukin-4, and interleukin-10 genes with risk of rheumatic heart disease in Taiwan Chinese. Int Heart J 2005;46:397-406. 23.Herrmann SM, Ricard S, Nicaud V, et al. Polymorphisms of the tumor necrosis factor-alpha gene, coronary heart disease and obesity. Eur J Clin Invest 1998;28:59-66. 24. Kucukaycan M, Van Krugten M, Pennings HJ, et al. Tumor necrosis factor-alpha +489G/A gene polymorphism is associated with chronic obstructive pulmonary disease. Respir Res 2002;3:29..

(6) 154. ϥ‫ݭ‬ཚሳᗼѪЯ̄ૄЯG-308Ä́G-238A̝к‫ّݭ‬ᄃ ࢲᒅّ͕᝙ঽ൑࠹ᙯّ ‫׹‬ശέ ችᅃ̥. 1,2,3,4. ઼̚ᗁᘽ̂ጯ‫ܢ‬నᗁੰā̰ࡊొ͕᝙ࡊā‫ొࡊ׊‬. 1. ᗁࡁొ. ઼̚ᗁᘽ̂ጯā̚ᗁጯੰ઼̚ᗁጯࡁտٙ. ϫ۞. 2. ૄЯᗁጯొ. 3. 4. ࢲᒅّ͕᝙ঽߏͽၙّ൴‫ۆ‬࿅඀ࠎ‫ܑ׎‬ᇈĂ௟ࡪ፬৵ϥ‫ݭ‬ཚሳᗼѪЯ̄дၙ. ّ൴‫ͅۆ‬ᑕ˯ፉЇࢦࢋ֎ҒĄώࡁտ۞ϫ۞дଣ੅ϥ‫ݭ‬ཚሳᗼѪЯ̄ૄЯк‫ߏّݭ‬ ӎਕ༊үέ៉ˠࢲᒅّ͕᝙ঽঽଈຏ‫ّٕצ‬Пᐍّ۞ᇾ੃Ą. ͞‫ڱ‬. ͽ 115 Ҝགྷ͕᝙෹ࢰ‫گ‬෧ᕝࠎࢲᒅّ͕᝙ঽঽଈ̈́ 103 Ҝѐ᛬ّҾ࠹੨۞ϒ૱. ˠࠎ၆෪ĂϡჸЪāాᗆͅᑕࢨ‫ט‬ā̶‫ࡁڱژ‬տࢲᒅّ͕᝙ঽଈ̈́ϒ૱ˠ۞ϥ‫ݭ‬ཚሳ ᗼѪЯ̄ૄЯ G-308A ̈́ G-238A ̝к‫ّݭ‬Ą. ඕ‫ڍ‬. ࢲᒅّ͕᝙ঽଈ̈́ϒ૱ˠม۞ϥ‫ݭ‬ཚሳᗼѪЯ̄ૄЯ G-308A ̈́ G-238A ̝к. ‫ૄّݭ‬Я‫̈́ݭ‬၆ઊૄЯᐛத̶Ҷ֭൑मளĄ૟ࢲᒅّ͕᝙ঽঽଈ̶ј˟ыᘝ়ঽ̈́к ᘝቯ়ঽ‫׌‬௡Ă˵Ϗ൴ன‫׌‬௡ม۞ϥ‫ݭ‬ཚሳᗼѪЯ̄ૄЯ G-308A ̈́ G-238A ̝к‫ّݭ‬ ૄЯ‫̈́ݭ‬၆ઊૄЯᐛத̶Ҷѣ௚ࢍጯ˯۞मளĄ. ඕኢ. ϥ‫ݭ‬ཚሳᗼѪЯ̄ૄЯ G-308A ̈́ G-238A к‫ّ̙ݭ‬ዋЪ༊үέ៉ˠࢲᒅّ͕. ᝙ঽ۞ᇾ੃ĄĞ̚έ៉ᗁᄫ 2006;11:149-54ğ ᙯᔣෟ έ៉Ăϥ‫ݭ‬ཚሳᗼѪЯ̄ૄЯк‫ّݭ‬Ăࢲᒅّ͕᝙ঽ. ᓑඛү۰Ĉ‫׹‬ശέ г ӬĈ 404 έ̚ξΔડֈᇇྮ 2 ཱི ઼̚ᗁᘽ̂ጯ‫ܢ‬నᗁੰā̰ࡊొ͕᝙ࡊ ࣒Լ͟ഇĈ 2006 ѐ 4 ͡ 25 ͟ ќ͛͟ഇĈ 2006 ѐ 3 ͡ 30 ͟ ତ‫͟צ‬ഇĈ 2006 ѐ 5 ͡ 4 ͟.

(7)