©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6762-6766 (2013) Frequencies of -308G/A (TNFA) and -509C/T (TGFB1) polymorphisms in sickle cell anemia patients from Brazil L.S. Torres1, E. Belini Junior1, D.G. Silva1, C.L. Lobo2, M.A. Ruiz3 and C.R. Bonini-Domingos1 1Departamento de Biologia, Laboratório de Hemoglobinas e Genética das Doenças Hematológicas, Universidade Estadual Paulista, São José do Rio Preto, SP, Brasil 2Instituto Estadual de Hematologia Arthur de Siqueira Cavalcanti, Hemorio, Rio de Janeiro, RJ, Brasil 3Hospital Beneficência Portuguesa, São José do Rio Preto, SP, Brasil Corresponding author: L.S. Torres E-mail: lidiane.unesp@gmail.com Genet. Mol. Res. 12 (4): 6762-6766 (2013) Received January 11, 2013 Accepted August 5, 2013 Published December 16, 2013 DOI http://dx.doi.org/10.4238/2013.December.16.1 ABSTRACT. Sickle cell anemia is an affection that causes chronic inflammation, with consequences for vaso-occlusion, oxidative stress and cytokine production. Genetic polymorphisms in markers involved in this process can modulate the inflammatory response, including polymorphisms -308G/A of TNFA (tumor necrosis factor alpha) and -509C/T of TGFB1 (transforming growth factor beta 1), reported to increase TNF-α and TGF-β1 production, respectively. Changes in the cytokine balance are important risk factors for clinical events; consequently, we examined the frequencies of these polymorphisms in 240 Brazilian sickle cell anemia patients from southeast Brazil. PCR- RFLP was used to detect these polymorphisms. The -509C/T (TGFB1) polymorphism was more frequent than -308G/A (TNFA), with allelic frequency of 0.3 for the mutant allele T (TGFB) agaist 0.1 for the mutant 6763 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6762-6766 (2013) TNFA and TGFB polymorphism frequencies allele A (TNFA). These allelic frequencies are similar to those known from populations with ethnicity similar to the Brazilian population. Inheritance of these polymorphisms does not seem to be associated with that of the Hb S mutation; however, this information could be useful in analyses of specific clinical characteristics of sickle cell anemia. Key words: Allelic frequency; Genetic polymorphisms; PCR-RFLP; Sickle cell disease; SNPs; Hemoglobin S INTRODUCTION Sickle cell anemia (SCA) is a hemolytic anemia caused by a mutation in the sixth codon of the beta globin chain (GAG → GTG), resulting in homozygous hemoglobin (Hb) S (Honig and Adams III, 1986; Frenette and Atweh, 2007). The inflammatory process that occurs in SCA patients may play an important role in vaso-occlusion, oxidative stress stimulation and cytokine production, contributing to the disease pathogenesis (Chiang and Frenette, 2005). Evidence shows the involvement of genetic polymorphisms in the inflammatory re- sponse in SCA patients, among them, some association studies suggest that TNFA (tumor necrosis factor alpha) and TGFB1 (transforming growth factor beta 1) genes interfere in the SCA clinical profile (Nolan et al., 2006; Cajado et al., 2010). TNF-α and TGF-β cytokine production may be genetically influenced by polymorphisms that affect gene regulation. The -308G/A (TNFA) (rs1800629) and -509C/T (TGFB1) (rs1800469) polymorphisms are reported to increase the production of TNF-α (Rodriguez-Rodriguez et al., 2011) and TGF-β1 (Bhayal et al., 2011), respectively. Changes in cytokine balance in SCA patients are important risk factors for clinical events, and thus, the objective of this study was to determine the frequencies of the -308G/A (TNFA) and -509C/T (TGFB1) polymorphisms in Brazilian SCA patients, and to compare our results with the frequencies reported in some recent studies in the literature. MATERIAL AND METHODS We analyzed 240 peripheral blood samples of SCA patients from southeast Brazil. All samples were submitted to a classical Hb diagnostic, including Hb electrophoresis at alkaline and acid pH (Marengo-Rowe, 1965; Vella, 1968) and high-performance liquid chromatogra- phy for Hb fraction quantification (VARIANT, Bio-Rad) (Bonini-Domingos, 2006). For mo- lecular analysis, genomic DNA was extracted by the phenol-chloroform method (Sambrook et al., 1989). Amplification of the segment that encodes Hb S was performed using specific primers, and fragments were cleaved with the restriction endonuclease FastDdeI (Fermentas, USA) (Belini et al., 2010). -308G/A (TNFA) and -509C/T (TGFB1) were determined by am- plification of the specific genomic segment, with subsequent treatment with specific restriction enzymes for mutations in the TNFA (FastNcoI; Fermentas) and TGFB1 (FastBsu36I; Fermen- tas) genes (Wilson et al., 1992; Silverman et al., 2004). Statistical analyses were performed for comparing the frequencies obtained against those of other studies and databases, using the Statistica 10.0 software with the chi-square test. The level of significance was set at P < 0.05. 6764 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6762-6766 (2013) L.S. Torres et al. RESULTS The genotypic and allelic frequencies for the polymorphisms evaluated in SCA pa- tients are shown in Table 1. The -509C/T (TGFB1) polymorphism was more frequent than -308G/A (TNFA), with the frequency of the mutant allele being 0.3 versus 0.1. Table 2 presents the literature frequencies for the -308G/A (TNFA) and -509C/T (TGFB1) polymorphisms ob- tained by other authors and in databases, as well as a comparison with our data, showing that our data were in accordance with other studies with similar populations. Polymorphism Genotype Genotypic frequency [N (%)] Allelic frequency TNFA GG 195 (81.25) G = 0.90 (-308G/A) GA 43 (17.92) A = 0.10 AA 2 (0.83) TGFB1 CC 109 (45.42) C = 0.70 (-509C/T) CT 116 (48.33) T = 0.30 TT 15 (6.25) Table 1. Genotypic and allelic frequency of the -308G/A (TNFA) and -509C/T (TGFB1) polymorphisms in sickle cell anemia patients. Population group N Allelic frequency P References G A -308G/A (TNFA) General population (Brazil) 200 0.86 0.14 0.1956 (Cajado et al., 2011) SCA (Brazil) 210 0.88 0.12 0.5175 (Cajado et al., 2011) SCA (Brazil) 49 0.92 0.08 0.6920 (Vicari et al., 2011) General population (India) 216 0.94 0.06 0.1200 (Ghosh et al., 2010) HapMap_CEU European 113 0.83 0.17 0.0678 ** HapMap_YRI African 113 0.91 0.09 0.6712 ** *SCA (Brazil) 240 0.90 0.10 - - -509C/T (TGFB1) General population (Korea) 352 0.52 0.48 <0.0001 (Kim et al., 2010) General population (Hungary) 30 0.58 0.42 0.1382 (Rovo et al., 2010) Severe asthma population (Brazil) 38 0.60 0.40 0.2419 (de Faria et al., 2008) HapMap_CEU European 113 0.71 0.29 0.8507 - HapMap_YRI African 113 0.79 0.21 0.0843 - *SCA (Brazil) 240 0.70 0.30 - - SCA = sickle cell anemia; CEU = Utah residents with ancestry from Northern and Western European; YRI = Yoruba in Ibadan, Nigeria. Statistical test: Chi-square test (α < 0.05). *Presente study; **available in dbSNP, NCBI [http:// www.ncbi.nlm.nih.gov/projects/SNP]. Table 2. Allelic frequency found to -308G/A (TNFA) and -509C/T (TGFB1) polymorphisms in some recent studies and database, comparing with the present study. DISCUSSION The frequencies found for the -308G/A (TNFA) and -509C/T (TGFB1) polymor- phisms are similar to those provided by databases for Caucasian and African populations from NCBI databases (http://www.ncbi.nlm.nih.gov/projects/SNP), reflecting the contribution of these groups to the ethnic composition of the Brazilian population. For the -308G/A (TNFA) polymorphism, the frequencies obtained were similar to those results of other SCA studies in Brazil (Cajado et al., 2010; Vicari et al., 2011). Our results also did not differ from those found for the Brazilian population without hemoglobinopathies (Cajado et al., 2011), suggesting that the inheritance of the polymorphisms studied is independent of the βS gene inheritance. In re- 6765 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6762-6766 (2013) TNFA and TGFB polymorphism frequencies lation to the Indian population, the frequencies found in our population did not differ (Ghosh et al., 2010), probably due to the ethnic heterogeneity of both. For the -509C/T (TGFB1) polymorphism, our data were similar to findings from a Brazilian study in severe asthma patients (de Faria et al., 2008). Regarding that study, there was no association between presence of polymorphism and risk of developing asthma, which is a chronic inflammatory disease. The similarity with the frequencies obtained in our study is probably due to resemblance of the two populations, but not to the chronic inflammatory pro- cess. Regarding a study in Hungary with healthy individuals (Rovo et al., 2010), a population with different ethnic characteristics from the Brazilian one, the absence of a difference can be explained by the small sample number (N = 30), which probably was not representative of the Hungarian population. Differences relative to Korean individuals (Kim et al., 2010), in turn, demonstrate the low influence of this ethnicity in the formation of the Brazilian population. In summary, the frequencies for the polymorphisms evaluated were not associated with inheritance of Hb S and were similar between populations with the same ethnic charac- teristics. Furthermore, the polymorphisms do not appear to be associated with other inflam- matory diseases. CONCLUSION Allelic frequencies found in this study for the -308G/A (TNFA) and -509C/T (TGFB1) polymorphisms are in agreement with the literature for population groups with similar ethnic- ity as compared to the Brazilian population. The inheritance of the two polymorphisms does not seem to be associated with the Hb S mutation, but knowledge about them can be useful in future studies involving specific clinical characteristics of SCA, since they are involved in the control of inflammation. ACKNOWLEDGMENTS Reasearch supported by Conselho Nacional de Desenvolvimento Científico e Tec- nológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Ministério da Saúde (#MS3072/2007), and Fundação Pró-Hemorio RJ (FUNDARJ). English text revised by Carolina Grünig Humberto da Silva. REFERENCES Belini JE, Cancado RD and Domingos CR (2010). The XmnI polymorphic site 5' to the gene G (gamma) in a Brazilian patient with sickle cell anaemia - fetal haemoglobin concentration, haematology and clinical features. Arch. Med. Sci. 6: 822-825. Bhayal AC, Prabhakar B, Rao KP, Penchikala A, et al. (2011). Role of transforming growth factor-beta1 -509 C/T promoter polymorphism in gastric cancer in south Indian population. Tumour. Biol. 32: 1049-1053. Bonini-Domingos CR (2006). Metodologias Laboratoriais para o Diagnóstico de Hemoglobinopatias e Talassemias. Inovação Distribuidora de Livros Ltda., São Paulo. Cajado CS, Cerqueira BAV, Barbosa CG and Lyra IM (2010). IL-8 e TNF-alfa: marcadores imunológicos no prognóstico da anemia falciforme. Gazeta Med. Bahia 80: 56-61. Cajado C, Cerqueira BA, Couto FD, Moura-Neto JP, et al. (2011). TNF-alpha and IL-8: serum levels and gene polymorphisms (-308G>A and -251A>T) are associated with classical biomarkers and medical history in children with sickle cell anemia. Cytokine 56: 312-317. 6766 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 12 (4): 6762-6766 (2013) L.S. Torres et al. Chiang EY and Frenette PS (2005). Sickle cell vaso-occlusion. Hematol. Oncol. Clin. North Am. 19: 771-84. de Faria IC, de Faria EJ, Toro AA, Ribeiro JD, et al. (2008). Association of TGF-beta1, CD14, IL-4, IL-4R and ADAM33 gene polymorphisms with asthma severity in children and adolescents. J. Pediatr. 84: 203-210. Frenette PS and Atweh GF (2007). Sickle cell disease: old discoveries, new concepts, and future promise. J. Clin. Invest. 117: 850-858. Ghosh J, Joshi G, Pradhan S and Mittal B (2010). Investigation of TNFA 308G > A and TNFB 252G > A polymorphisms in genetic susceptibility to migraine. J. Neurol. 257: 898-904. Honig GR and Adams III JG (1986). Human Hemoglobin Genetics. Springer-Verlag Wien New York, New York, 19-36. Kim JJ, Choi YM, Choung SH, Yoon SH, et al. (2010). Analysis of the transforming growth factor beta1 gene -509 C/T polymorphism in patients with advanced-stage endometriosis. Fertil. Steril. 93: 2121-2124. Marengo-Rowe AJ (1965). Rapid electrophoresis and quantitation of haemoglobins on cellulose acetate. J. Clin. Pathol. 18: 790-792. Nolan VG, Adewoye A, Baldwin C, Wang L, et al. (2006). Sickle cell leg ulcers: associations with haemolysis and SNPs in Klotho, TEK and genes of the TGF-beta/BMP pathway. Br. J. Haematol. 133: 570-578. Rodriguez-Rodriguez L, Gonzalez-Juanatey C, Palomino-Morales R, Vazquez-Rodriguez TR, et al. (2011). TNFA -308 (rs1800629) polymorphism is associated with a higher risk of cardiovascular disease in patients with rheumatoid arthritis. Atherosclerosis 216: 125-130. Rovo L, Szell M, Bella Z, Korsos A, et al. (2010). The -509 C/T genotype of TGFbeta1 might contribute to the pathogenesis of benign airway stenosis. Otolaryngol. Head Neck Surg. 142: 441-443. Sambrook J, Fritcsh EF and Manatis T (1989). Molecular Cloning: A Laboratory Manual. 2nd edn. Vols. 1, 2 and 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Silverman ES, Palmer LJ, Subramaniam V, Hallock A, et al. (2004). Transforming growth factor-beta1 promoter polymorphism C-509T is associated with asthma. Am. J. Respir. Crit. Care Med. 169: 214-219. Vella F (1968). Acid-agar gel electrophoresis of human hemoglobins. Am. J. Clin. Pathol. 49: 440-442. Vicari P, Silva GS, Nogutti MA, Neto FM, et al. (2011). Absence of association between TNF-alpha polymorphism and cerebral large-vessel abnormalities in adults with sickle cell anemia. Acta Haematol. 125: 141-144. Wilson AG, di Giovine FS, Blakemore AI and Duff GW (1992). Single base polymorphism in the human tumour necrosis factor alpha (TNF alpha) gene detectable by NcoI restriction of PCR product. Hum. Mol. Genet. 1: 353.