Association of the chemokine stromal-derived factor-1 (SDF-1) genetic polymorphism with transaminitis in HIV-infected Thais who are on combined anti-retroviral therapy T. Chiraunyanann 1,2 , K. Changsri 3 , W. Sretapunya 4 , K. Yuenyongchaiwai 5 , C. Akekawatchai 3 1 Bureau of AIDS, TB & STIs, Department of Disease Control, Ministry of Public Health, Nonthaburi, Thailand, 2 Graduate Program in Medical Technology, 3 Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand, 4 Nakhon Nayok Hospital, Nakhon Nayok, Thailand, 5 Department of Physical Therapy, Faculty of Allied Health Sciences, Pathumthani, Thailand Table 1. Characteristics and clinical features of HIV-1 patients classified according to SDF-1 genotypes. Table 2. Univariate and multivariate analysis of risks for trans- aminitis in HIV patients. PRESENTED AT THE 21 ST INTERNATIONAL AIDS CONFERENCE - DURBAN, SOUTH AFRICA Characteristic All N (%) SDF-1 p-value AA vs GA N (%) GG N (%) Patients 164 (100) 71 (43.3) 93 (56.7) Age ≤30 27 (16.5) 15 (9.1) 12 (7.3) 31-40 65 (39.6) 26 (15.9) 39 (23.8) 41-50 42 (25.6) 19 (11.6) 23 (14.0) >50 30 (18.3) 11 (6.7) 19 (11.6) 0.468 Gender Male 90 (54.9) 44 (26.8) 46 (28.0) Female 74 (45.1) 27 (16.5) 47 (28.7) 0.111 CD4+ T cell count (n=141) <200 (n, %) 32 (22.7) 17 (12.1) 15 (10.6) 200-349.9 (n, %) 35 (24.8) 13 (9.2) 22 (15.6) ≥350 (n, %) 74 (52.5) 29 (20.6) 45 (31.9) 0.332 Duration of ARV treatment (n=117) <3 months 7 (6.0) 2 (1.7) 5 (4.3) 3-6 months 6 (5.1) 2 (1.7) 4 (3.4) >6 months 104 (88.9) 44 (37.6) 60 (51.3) 0.717 ARV drugs Naive to ARV treatment 47 (28.7) 23 (14.0) 24 (14.6) Lamivudine/Stavudine/Nevirapine 11 (6.7) 4 (2.4) 7 (4.3) Lamivudine/Zidovudine/Nevirapine 57 (34.8) 22 (13.4) 35 (21.3) Lopinavir/Ritonavir combination 10 (6.1) 3 (1.8) 7 (4.3) Others (n, %) 39 (23.8) 19 (11.6) 20 (12.2) 0.640 Nevirapine experience (n= 117 ) Nevirapine-based combination 74 (63.2) 30 (25.6) 44 (37.6) Other nevirapine combination (n, %) 43 (36.8) 18 (15.4) 25 (21.4) 0.889 Platelet count (n= 147 ) Platelet count (10 9 /L)(mean, SD) 276 (±105) 267 (89) 283 (115) 0.534 Severity of thrombocytopenia (n=147) Grade 0 ( >151,000) 137 (93.2) 56 (38.1) 81 (55.1) Grade 1 (75,000 - 150,000) 8 (5.4) 5 (3.4) 3 (2.0) Grade 2 (50,000 - <75,000) 2 (1.4) 0 2 (1.4) Grade 3 (25,000 - <50,000) 0 0 0 Grade 4 (<25,000) 0 0 0 0.235 HBV and HCV coinfection (n=132) HIV monoinfection 108 (81.8) 46 (34.8) 62 (47.0) HIV-HBV coinfection 12 (9.1) 6 (4.5) 6 (4.5) HIV-HCV coinfection 11 (8.3) 6 (4.5) 5 (3.8) HIV-HBV-HCV tripleinfection 1 (0.8) 0 (0) 1 (0.8) 0.672 AST:ALT ratio (n= 143 ) ≤ 1 (n, %) 56 (39.2) 24 (16.8) 32 (22.4) >1 (n, %) 87 (60.8) 35 (24.5) 52 (36.4) 0.755 Transaminitis (n=143 ) AST and/or ALT £ULN(40 U/L) 103 (72.0) 36 (25.2) 67 (46.9) AST and/or ALT >ULN(40 U/L) 40 (28.0) 23 (16.1) 17 (11.9) 0.014* a Data shown as mean value ± S.D. * Data shown as P value <0.05 Characteristics En- rolled pa- tients Patients with transam- initis N (%) TRANSAMINITIS Crude OR (95%CI) p-value Adjusted OR (95%CI) p-value Gender Female 90 81 (56.6) 1 Male 74 62 (43.4) 3.7 (1.6-8.4) 0.002* CD4+ T cell count (cells/ml) ≥350 74 71 (51.8) 1 200-349.9 35 35 (25.5) 1.5 (0.6-3.6) 0.412 <200 32 31 (22.6) 1.7 (0.7-4.4) 0.232 HBV and HCV coinfec- tion HIV monoinfection 108 93 (81..6) 1 1 HIV-HBV coinfection 12 11 (9.6) 1.1 (0.3-4.7) 0.854 0.8 (0.2-3.7) 0.823 HIV-HCV coinfection 11 10 (8.8) 12.2 (2.4-61.5) 0.002* 11.5 (2.2-61.6) 0.004* SDF-1 genotype GG 93 84 (58.7) 1 AA vs GA 71 59 (41.3) 2.5 (1.2-5.3) 0.015* ARV drugs Naive to ARV treatment 47 31 (21.7) 1 Lamivudine/Stavudine/ Nevirapine 11 10 (7.0) 3.4 (0.8-15.3) 0.107 Lamivudine/Zidovudine/ Nevirapine 57 56 (39.2) 1.0 (0.4-3.0) 0.946 Lopinavir/Ritonavir combi nation 10 9 (6.3) 1.7 (0.3-8.7) 0.515 Others 39 37 (25.9) 1.6 (0.6-4.9) 0.369 Nevirapine experi ence Naive to ARV treat ment 47 31 (21.7) 1 Nevirapine-based combination 74 72 (50.3) 1.3 (0.5-3.5) 0.583 Others 43 40 (28.0) 1.7 (0.6-4.8) 0.359 Data was adjusted for gender, CD4+ T cell count, HBV and HCV coinfection, SDF-1 genotype, ARV in the model. * P<0.05; OR: Odds ratio; CI: Confidence interval Introduction Liver disease has emerged as one of the major causes of death in HIV-infected patients in the era of highly active antiretroviral thera- py (HAART). Multiple factors associated with liver complications in HIV-infected individuals have been reported such as coinfection with hepatitis B or C viruses, HAART, immune reconstitution and host genetic factors. The chemokine stromal-derived factor 1 alpha (SDF-1), the primary ligand for seven transmembrane G-protein couple receptor CXCR4, plays a role in homing of immune cells to the liver. Increased expression of SDF-1 by stellate, endothelial and biliary epithelial cells promotes hematopoietic stem cell recruit- ment to the hepatic area and nearly 50% of infiltrating immune cells are CXCR4 positive. Previously, SDF-1 gene polymorphism (G to A) at the position 801 in 3’ UTR was shown to be associated with progression of AIDS and hepatitis. The mRNA containing 3’A was stable than the 3’G variant leading to increased levels of SDF- 1 mRNA and protein. Increased serum SDF-1 levels have been as- sociated with acute and chronic liver injury (Saiman Y, 2012). Our recent study has reported high prevalence of an elevation of liver enzymes in HIV-infected Thais receiving HAART (Akekawatchai C, 2015). Herein, this study aimed to examine frequency of the 3’ UTR G801A SDF-1 gene polymorphism, and its association with an increase of serum liver enzymes, aspartate ami- notransferase or alanine aminotransferase above the upper normal limits, termed transaminitis, in HIV-infected Thais on a HAART program. Conclusion Our data indicated a significant association of the G801A SDF-1 gene polymorphism with transaminitis and the AA/GA genotypes, together with male gender and HCV coinfection, are potential pre- dictive factors of progression to liver abnormality in Thai HIV pa- tients mostly receiving long-term suppressive HAART. This obser- vation supports the role of host genetic factors in development of liver disease in HIV patients ongoing HAART. Materials and Methods Patients and Clinical Data Collection With the approval of the human Ethics Committees of Thammasat University and Nakhon Nayok Hospital, one hundred and sixty four patients in ART clinic in Nakorn Nayok hospital in Thialand were recruited in this study. Patients were older than 15 years-old, docu- mented as HIV-infected person, provided written informed consent. Patients with opportunistic infection, alcohol, steroid drug and herbal consumptions were excluded from the study. All clinical data were collected from medical records. Blood samples were collect- ed and tested routinely for levels of AST, ALT, complete blood count, and HBsAg and anti-HCV. The G801A SDF-1 gene poly- morphisms were examined by tetra-primer and amplification muta- tion system. Genotype Analysis for SDF tetra-primer ARMS PCR Genomic DNA was isolated from peripheral blood by NucleoSpin® kits. DNA was then analyzed by means of tetra-primer and amplifi- cation refractory mutation system (tetra-primer ARMS PCR). The specific primers contains SDF 3’ UTR-F inner primer (sense 5’- CATCCACATGGGAGCCA-3’), SDF 3’UTR-R inner primer (antisense 5’-CCAGAAGAGGCAGACCC-3’), SDF 3’ UTR-F outer primer (sense 5’-GCTCTGAAACCAGTGTTAGG-3’) and SDF 3’UTR-R outer primer (antisense 5’- ACATTGGTCACAGAGGAG- GA-3’) (Ye S, 2001) . DNA Electrophoresis PCR products were subjected to electrophoresis in 2% agarose gels. SDF-1 wild-type alleles (SDF1-wt) yielded 495 and 826-bp products, while SDF1-3’A alleles yield a 364-bp and 826-bp pro- ducts. Statistical Analysis The major characteristics of the study population and the different parameters evaluated are expressed with a sample size (n), medi- an, and qualitative variables have been summarized with frequen- cy distributions and percentages. Associations between SDF-1 genotypes and different qualitative parameters were explored us- ing λ2 test or Fishers’s exact test as appropriate. Comparisons be- tween groups were carried out using nonparametric analysis of variance (Kruskal Walis test). Univariable and multivariable (logistic regression) analyses were used to determine a risk of liver complication defined as transaminitis. Odds ratio (OR) with 95% confidence interval (CI) and P value were calculated. All p-values ≤ 0.05 were considered as statistically significant. Results Of 164 HIV patients, rate of transaminitis was 28% and prevalence of hepatitis B virus (HBV) coinfection, hepatitis C virus (HCV) coin- fection and HBV/HCV triple infection were 9.1%, 8.3% and 0.8% respectively. Genotype frequencies of AA, GA and GG in the stud- ied group were 6.5%, 36.1%, 57.1% respectively. Acknowledgments We would like to thank all patients who voluntarily participated in the study. We thank nurses and medical technologists, in Nakhon Nayok hospital for collecting specimens. We also thank Dr. Doung- napa Kingkeow, Chiang Mai University Research Institute for Health Sciences (CMU-RIHES) for providing control materials of the genotypes for G801A SDF-1 polymorphism. The authors sin- cerely thank Dr. Sumet Ongwandee, director of Bureau of AIDS, TB, and STIs (BATs) Thailand for supporting the participation in this conference. This study was financially supported by Gradu- ate Research Fellowships, National Research Council of Thailand and Graduate Research Fellowships, Thammasat University, Thai- land References 1. Saiman Y, Friedman SL. The role of chemokines in acute liver injury. Frontiers in physiology. 2012;3:213. 2. Akekawatchai C. Hepatitis B or C virus coinfection in and risks for transaminitis in human immunodeficiency virus-infected Thais on com- bined antiretroviral therapy. Asian Biomed. 2015;9(3):353. 3. Ye S, Dhillon S, Ke X, Collins AR, Day IN. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic acids research. 2001;29(17):E88-8. Fig 1. Genotyping analysis for SDF-1 gene by tetra-primer-PCR. The 826, 495 and 364 bp product were from the amplification us- ing internal control, G allele specific and A allele specific primers respectively. Lane 1, 2, 3, 5, 6, 7, 8, 9, 10 show the products from G allele. Lane 4 show the products from A allele. Lane 11 show the product from A and G allele. AA AG and GG lane are positive con- trol. The last two lanes are negative controls (no template) and DNA marker respectively. Chi-square test demonstrated a significant association between AA/GA genotypes were significant risk factors of transaminitis (crude odd ratio; 95%CI; p-value: 2.5; 1.2-5.3; p=0.015), together with male gender and hepatitis C virus (HCV) coinfection. Howev- er, multivariate analysis indicated only coinfection with hepatitis C virus (HCV) as a significant risk of transaminitis in this studied group.
Association of the chemokine stromal 1 (SDF 1)programme.aids2016.org/PAGMaterial/eposters/0_1821.pdf(SDF-1), the primary ligand for seven transmembrane G-protein couple receptor CXCR4,
