A multivariate genetic analysis confirms rs5010528 in the human leucocyte antigen-C locus as a significant contributor to Stevens-Johnson syndrome/toxic epidermal necrolysis susceptibility in a Mozambique HIV population treated with nevirapine

A multivariate genetic analysis confirms rs5010528 in the human leucocyte antigen-C locus as a... Abstract Objectives Nevirapine is used in developing countries for the treatment of HIV infection, but its use is associated with rare serious adverse reactions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Recently, an association between rs5010528 in the human leucocyte antigen (HLA)-C locus and SJS/TEN susceptibility has been described in sub-Saharan populations. Our aim was to verify this association in a population of nevirapine-treated patients from Mozambique. Methods The rs5010528 SNP was analysed by direct sequencing in 27 patients who had developed SJS/TEN and 75 patients who did not develop adverse reactions after nevirapine treatment. A case–control association study was conducted. A multivariate analysis was performed in order to evaluate the role of HLA-C also in relation to other susceptibility genetic factors (CYP2B6, TRAF3IP2, HCP5, PSORS1C1 and GSTM1 genes). Results rs5010528 was significantly associated with a higher risk of developing SJS/TEN; the variant allele was more frequent in cases than in controls, conferring a high risk of developing this adverse reaction in carriers (OR = 5.72 and P = 0.0002 at genotype level, OR = 3.51 and P = 0.0002 at allelic level). The multivariate analysis showed that the HLA-C SNP, CYP2B6 (rs28399499), TRAF3IP2 (rs76228616) and GSTM1 (null genotype) can explain 25% of the susceptibility to this reaction, with the HLA-C SNP as the most significant contributor (P = 0.02 and OR = 5.64). Conclusions Our study confirmed the association of the rs5010528 SNP in the HLA-C region with susceptibility to developing SJS/TEN in a population from Mozambique, suggesting that it could be a good genomic biomarker for SJS/TEN susceptibility in different sub-Saharan populations. Introduction Nevirapine is a potent NNRTI that has been used in first-line treatment of HIV infection in the last decade.1 This drug has been widely used in developing countries, both for its efficacy in reducing mother-to-child virus transmission and for its low cost,2 and continues to be used even if not in first-line treatment. However, its use is associated with common serious and sometimes life threatening adverse drug reactions.3 Indeed, some individuals treated with this drug can develop liver toxicity and severe cutaneous adverse events such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN).4–6 Approximately 5% of all patients treated with nevirapine developed symptomatic hepatic events associated with systemic symptoms such as jaundice, coagulopathy and markedly elevated ALT.7,8 Rarer and uncommon adverse effects are skin manifestations; SJS is a severe and potentially fatal hypersensitivity reaction characterized by skin tenderness and erythema of the skin and mucosa, followed by extensive cutaneous and mucosal epidermal necrosis and sloughing that may progress into the more serious form of TEN.9 Several features of nevirapine hypersensitivity suggest that genetic factors may play an important predisposing/protective role and, in recent years, several studies have demonstrated the involvement of the human leucocyte antigen (HLA) alleles in hypersensitivity.10,11 In our previous studies, in a population from Mozambique, we have identified polymorphisms in CYP2B6, TRAF3IP2, HCP5 and GSTM1 genes to be associated with a higher risk of developing SJS/TEN.12–15 Recently, we participated in a collaborative genome-wide association study (GWAS), in which the rs5010528 polymorphism in the HLA-C locus was found to predispose to nevirapine-induced SJS/TEN in a sub-Saharan African population.16 In particular, the association was confirmed in a replication cohort of HIV patients from Malawi, Uganda and Mozambique. Regarding the Mozambique population, 27 SJS/TEN patients and only 8 controls were included in the study (67 controls had been excluded because the DNA aliquots did not pass the quality control necessary for the technology used in that study). The aim of the current study was to replicate the association of the HLA-C polymorphism in the same Mozambique cohort (expanding the number of controls to a total of 75 subjects, the total number of control DNA available in our laboratory), by Sanger sequencing, which does not require a high standard of DNA quality, and to perform a multivariate analysis in order to evaluate the role of HLA-C in relation to other SJS/TEN susceptibility genetic factors that we had previously identified. Methods Patient recruitment Subjects were selected from patients accessing nevirapine-based combination treatment (zidovudine/stavudine plus lamivudine and nevirapine) according to the reported criteria.2 The recruitment was conducted in Mozambique in three DREAM (Drug Resource Enhancement against AIDS and Malnutrition) Programme Centres, during the years 2008–09. We recruited 27 patients who had developed SJS/TEN and 75 ethnically matched controls (treated patients who did not develop any kind of reaction). The study protocol was authorized by the Minister of Health of Mozambique (approval number 1195/C13–5/GMS/2008) and all procedures were performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. The clinical diagnosis of SJS/TEN was based on the development of exanthema, mainly starting on the trunk, involving more than 10% of the body surface, with mucosal involvement. In these patients, the skin lesions had not given pruritus and a skin detachment had been observed by the clinicians who made the diagnosis. Patients’ baseline clinical characteristics were previously described.12 No significant difference between cases and controls was observed at the beginning of nevirapine treatment regarding age, BMI, CD4 cell count, viral load, and transaminase levels. All subjects (cases and controls) were females [mean age 32 years (IQR 27–36)]. DNA extraction and genotyping DNA was extracted, stored and analysed at the Genetic Laboratory of University of Rome Tor Vergata, Rome, Italy. Genomic DNA was extracted from peripheral blood by Wizard® Genomic DNA Purification Kit (Promega, WI, USA). rs5010528 in the HLA-C locus was analysed by direct sequencing. Genotyping or sequencing of CYP2B6, HCP5, PSORS1C1, GSTM1 and TRAF3IP2 SNPs were previously described.12–15,17 Statistical analysis The Hardy–Weinberg equilibrium was verified by the Pearson χ2 test. Differences in genotypic frequencies between cases and controls were evaluated by Pearson χ2 test. ORs with 95% CIs were calculated. The cut-off for statistical significance was P < 0.05. Since this study is based on the same patient cohort of our previous studies,12–15 other genes (CYP2B6, TRAF3IP2, HCP5 and GSTM1) and relative SNPs have also been included in multivariate analyses. All statistical analyses were performed using the SPSS program (SSPS Inc., IL, USA). Results Genotypes and allele frequencies of rs5010528 are reported in Table 1. The variant allele (G) frequency in our controls is in agreement with the frequency observed in the Malawi population (discovery cohort of Carr et al.16). We observed a significant difference in genotype and allele distribution between cases with SJS/TEN and controls (P = 0.0002 and P = 0.0003, respectively). The variant G allele is more frequent in cases than in controls (with an allele frequency equal to 38.9% versus 15.3%, respectively), conferring a high risk of developing the adverse reaction in carriers (OR = 5.72 at the genotype level, OR = 3.51 at the allelic level). Table 1. Case–control association analysis in SJS/TEN Gene    Controls, n = 75  SJS/TEN, n = 27  P  OR (95% CI)  HLA-C rs5010528 (A>G)  AA  53 (70.7%)  8 (29.6%)  0.0002a  5.72 (2.18–15.00)  AG  21 (28%)  17 (63%)  GG  1 (1.3%)  2 (7.4%)  A  127 (84.7%)  33 (61.1%)  0.0003  3.51 (1.74–7.11)  G  23 (15.3%)  21 (38.9%)  Gene    Controls, n = 75  SJS/TEN, n = 27  P  OR (95% CI)  HLA-C rs5010528 (A>G)  AA  53 (70.7%)  8 (29.6%)  0.0002a  5.72 (2.18–15.00)  AG  21 (28%)  17 (63%)  GG  1 (1.3%)  2 (7.4%)  A  127 (84.7%)  33 (61.1%)  0.0003  3.51 (1.74–7.11)  G  23 (15.3%)  21 (38.9%)  a Heterozygotes (AG) and variant homozygotes (GG) were considered together. Table 1. Case–control association analysis in SJS/TEN Gene    Controls, n = 75  SJS/TEN, n = 27  P  OR (95% CI)  HLA-C rs5010528 (A>G)  AA  53 (70.7%)  8 (29.6%)  0.0002a  5.72 (2.18–15.00)  AG  21 (28%)  17 (63%)  GG  1 (1.3%)  2 (7.4%)  A  127 (84.7%)  33 (61.1%)  0.0003  3.51 (1.74–7.11)  G  23 (15.3%)  21 (38.9%)  Gene    Controls, n = 75  SJS/TEN, n = 27  P  OR (95% CI)  HLA-C rs5010528 (A>G)  AA  53 (70.7%)  8 (29.6%)  0.0002a  5.72 (2.18–15.00)  AG  21 (28%)  17 (63%)  GG  1 (1.3%)  2 (7.4%)  A  127 (84.7%)  33 (61.1%)  0.0003  3.51 (1.74–7.11)  G  23 (15.3%)  21 (38.9%)  a Heterozygotes (AG) and variant homozygotes (GG) were considered together. We performed a multivariate analysis to evaluate the relative contribution given by the rs5010528 SNP and by the other associated SNPs. Age, viral load, BMI and CD4 count were also included as covariates in the analysis; with a stepwise approach, only the genetic factors were significantly associated with SJS/TEN susceptibility. Therefore, we repeated the analysis including only significant variables and the final model (Table 2) showed that the HLA-C SNP is the most significant factor in the SJS/TEN predisposition in our population (P = 0.02 and OR = 5.64). However, CYP2B6 (rs28399499), TRAF3IP2 (rs76228616) and GSTM1 (null genotype) also significantly contribute to conferring a high risk of developing this reaction, with an OR = 4.63, 3.97 and 3.56, respectively. Our model overall explains 25% of the susceptibility to this reaction. Table 2. Final model of multiple regression analysis Variable  βa  P  OR (95% CI)  R2 (Cox and Snell)  CYP2B6 (rs3745274)  1.09  0.11  2.98 (0.77–11.48)  0.25  CYP2B6 (rs28399499)  1.53  0.04  4.63 (1.09–19.63)  TRAF3IP2 (rs76228616)  1.38  0.04  3.97 (1.08–14.64)  GSTM1 (null genotype)  1.27  0.04  3.56 (1.07–11.82)  HLA-C (rs5010528)  1.73  0.02  5.64 (1.93–16.45)  Variable  βa  P  OR (95% CI)  R2 (Cox and Snell)  CYP2B6 (rs3745274)  1.09  0.11  2.98 (0.77–11.48)  0.25  CYP2B6 (rs28399499)  1.53  0.04  4.63 (1.09–19.63)  TRAF3IP2 (rs76228616)  1.38  0.04  3.97 (1.08–14.64)  GSTM1 (null genotype)  1.27  0.04  3.56 (1.07–11.82)  HLA-C (rs5010528)  1.73  0.02  5.64 (1.93–16.45)  Significant associations are shown in bold. a Indicates the β coefficient that represents the degree of change in the outcome variable. Table 2. Final model of multiple regression analysis Variable  βa  P  OR (95% CI)  R2 (Cox and Snell)  CYP2B6 (rs3745274)  1.09  0.11  2.98 (0.77–11.48)  0.25  CYP2B6 (rs28399499)  1.53  0.04  4.63 (1.09–19.63)  TRAF3IP2 (rs76228616)  1.38  0.04  3.97 (1.08–14.64)  GSTM1 (null genotype)  1.27  0.04  3.56 (1.07–11.82)  HLA-C (rs5010528)  1.73  0.02  5.64 (1.93–16.45)  Variable  βa  P  OR (95% CI)  R2 (Cox and Snell)  CYP2B6 (rs3745274)  1.09  0.11  2.98 (0.77–11.48)  0.25  CYP2B6 (rs28399499)  1.53  0.04  4.63 (1.09–19.63)  TRAF3IP2 (rs76228616)  1.38  0.04  3.97 (1.08–14.64)  GSTM1 (null genotype)  1.27  0.04  3.56 (1.07–11.82)  HLA-C (rs5010528)  1.73  0.02  5.64 (1.93–16.45)  Significant associations are shown in bold. a Indicates the β coefficient that represents the degree of change in the outcome variable. Discussion In this study our goal was to evaluate the overall and relative contribution of rs5010528 SNP and other studied genetic risk factors to SJS/TEN susceptibility in a population from Mozambique. Part of our study cohort was included in the study of Carr et al.16 (replication cohort) that identified rs5010528 in the HLA-C gene as being associated with SJS/TEN. However, in that study only a small number (n = 8) of our controls from Mozambique was examined, therefore we have extended the analysis of rs5010528 to the whole cohort of our controls (n = 75). With the case–control association study, we confirmed the association of the HLA-C SNP with susceptibility to SJS/TEN in the Mozambique population. Moreover, we have carried out a multivariate analysis to evaluate the overall contribution of the genetic risk factors so far identified, including the HLA-C SNP. The multivariate analysis confirmed the importance of genetic factors to SJS/TEN susceptibility in the Mozambique population, highlighting that HLA-C gives an important contribution together with the other genetic factors (CYP2B6, TRAF3IP2 and GSTM1). Although the role of the rs5010528 SNP needs to be clarified, docking studies showed that nevirapine could bind the B pocket of HLA-C: 04: 01.16 Replication studies in different populations are essential to understand the contribution of genetic variants to adverse drug reactions. Indeed, as it is known, some associations could be specific only for certain populations. In fact, some genetic variants can be associated with a toxic effect in a population, but not in other populations, or even not be present. For example, the HLA-B*15: 02 allele is a marker for carbamazepine-induced SJS/TEN only in southeast Asian populations,9 showing that ethnicity could play a role in susceptibility to adverse drug reaction. Using a control population from Mozambique, our study confirmed and strengthened the results of Carr et al.,16 suggesting that rs5010528 SNP in the HLA-C region could be a good genomic biomarker for SJS/TEN susceptibility in different sub-Saharan populations. In addition, our multivariate analysis confirmed that several genetic factors could contribute to this specific nevirapine adverse reaction in the population from Mozambique. Of course, the identification of new specific genetic markers for different populations will be of great utility to establish a personalized therapeutic approach and to prevent the occurrence of adverse reactions. Acknowledgements We thank all people collaborating in the DREAM Programme in Mozambique centres. Funding This study was supported by internal funding. Transparency declarations None to declare. References 1 van Leth F, Phanuphak P, Ruxrungtham K et al.   Comparison of first-line antiretroviral therapy with regimens including nevirapine, efavirenz, or both drugs, plus stavudine and lamivudine: a randomised open-label trial, the 2NN Study. Lancet  2004; 363: 1253– 63. Google Scholar CrossRef Search ADS PubMed  2 Marazzi MC, Germano P, Liotta G et al.   Safety of nevirapine-containing antiretroviral triple therapy regimens to prevent vertical transmission in an African cohort of HIV-1-infected pregnant women. HIV Med  2006; 7: 338– 44. Google Scholar CrossRef Search ADS PubMed  3 Gozalo C, Gérard L, Loiseau P et al.   Pharmacogenetics of toxicity, plasma trough concentration and treatment outcome with nevirapine-containing regimen in anti-retroviral-naïve HIV-infected adults: an exploratory study of the TRIANON ANRS 081 trial. Basic Clin Pharmacol Toxicol  2011; 109: 513– 20. Google Scholar CrossRef Search ADS PubMed  4 Stern JO, Robinson PA, Love J et al.   A comprehensive hepatic safety analysis of nevirapine in different populations of HIV infected patients. J Acquir Immune Defic Syndr  2003; 34 Suppl 1: S21– 33. Google Scholar CrossRef Search ADS PubMed  5 Aaron E, Kempf MC, Criniti S et al.   Adverse events in a cohort of HIV infected pregnant and non-pregnant women treated with nevirapine versus non-nevirapine antiretroviral medication. PLoS One  2010; 5: e12617. Google Scholar CrossRef Search ADS PubMed  6 Coffie PA, Tonwe-Gold B, Tanon AK et al.   Incidence and risk factors of severe adverse events with nevirapine-based antiretroviral therapy in HIV-infected women. MTCT-Plus program, Abidjan, Côte d'Ivoire. BMC Infect Dis  2010; 10: 188. Google Scholar CrossRef Search ADS PubMed  7 Shakil AO, Kramer D, Mazariegos GV et al.   Acute liver failure: clinical features, outcome analysis, and applicability of prognostic criteria. Liver Transpl  2000; 6: 163– 9. Google Scholar PubMed  8 Shubber Z, Calmy A, Andrieux-Meyer I et al.   Adverse events associated with nevirapine and efavirenz-based first-line antiretroviral therapy: a systematic review and meta-analysis. AIDS  2013; 27: 1403– 12. Google Scholar CrossRef Search ADS PubMed  9 Rufini S, Ciccacci C, Politi C et al.   Stevens-Johnson syndrome and toxic epidermal necrolysis: an update on pharmacogenetics studies in drug-induced severe skin reaction. Pharmacogenomics  2015; 16: 1989– 2002. Google Scholar CrossRef Search ADS PubMed  10 Gao S, Gui XE, Liang K et al.   HLA-dependent hypersensitivity reaction to nevirapine in Chinese Han HIV-infected patients. AIDS Res Hum Retroviruses  2012; 28: 540– 3. Google Scholar CrossRef Search ADS PubMed  11 Likanonsakul S, Rattanatham T, Feangvad S et al.   HLA-Cw*04 allele associated with nevirapine-induced rash in HIV-infected Thai patients. AIDS Res Ther  2009; 6: 22. Google Scholar CrossRef Search ADS PubMed  12 Ciccacci C, Di Fusco D, Marazzi MC et al.   Association between CYP2B6 polymorphisms and Nevirapine-induced SJS/TEN: a pharmacogenetics study. Eur J Clin Pharmacol  2013; 69: 1909– 16. Google Scholar CrossRef Search ADS PubMed  13 Borgiani P, Di Fusco D, Erba F et al.   HCP5 genetic variant (RS3099844) contributes to Nevirapine-induced Stevens Johnsons syndrome/toxic epidermal necrolysis susceptibility in a population from Mozambique. Eur J Clin Pharmacol  2014; 70: 275– 8. Google Scholar CrossRef Search ADS PubMed  14 Ciccacci C, Rufini S, Mancinelli S et al.   A pharmacogenetics study in Mozambican patients treated with nevirapine: full resequencing of TRAF3IP2 gene shows a novel association with SJS/TEN susceptibility. Int J Mol Sci  2015; 16: 5830– 8. Google Scholar CrossRef Search ADS PubMed  15 Ciccacci C, Latini A, Politi C et al.   Impact of glutathione transferases genes polymorphisms in nevirapine adverse reactions: a possible role for GSTM1 in SJS/TEN susceptibility. Eur J Clin Pharmacol  2017; 73: 1253– 9. Google Scholar CrossRef Search ADS PubMed  16 Carr DF, Bourgeois S, Chaponda M et al.   Genome-wide association study of nevirapine hypersensitivity in a sub-Saharan African HIV-infected population. J Antimicrob Chemother  2017; 72: 1152– 62. Google Scholar PubMed  17 Ciccacci C, Borgiani P, Ceffa S et al.   Nevirapine-induced hepatotoxicity and pharmacogenetics: a retrospective study in a population from Mozambique. Pharmacogenomics  2010; 11: 23– 31. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. 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A multivariate genetic analysis confirms rs5010528 in the human leucocyte antigen-C locus as a significant contributor to Stevens-Johnson syndrome/toxic epidermal necrolysis susceptibility in a Mozambique HIV population treated with nevirapine

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Abstract

Abstract Objectives Nevirapine is used in developing countries for the treatment of HIV infection, but its use is associated with rare serious adverse reactions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Recently, an association between rs5010528 in the human leucocyte antigen (HLA)-C locus and SJS/TEN susceptibility has been described in sub-Saharan populations. Our aim was to verify this association in a population of nevirapine-treated patients from Mozambique. Methods The rs5010528 SNP was analysed by direct sequencing in 27 patients who had developed SJS/TEN and 75 patients who did not develop adverse reactions after nevirapine treatment. A case–control association study was conducted. A multivariate analysis was performed in order to evaluate the role of HLA-C also in relation to other susceptibility genetic factors (CYP2B6, TRAF3IP2, HCP5, PSORS1C1 and GSTM1 genes). Results rs5010528 was significantly associated with a higher risk of developing SJS/TEN; the variant allele was more frequent in cases than in controls, conferring a high risk of developing this adverse reaction in carriers (OR = 5.72 and P = 0.0002 at genotype level, OR = 3.51 and P = 0.0002 at allelic level). The multivariate analysis showed that the HLA-C SNP, CYP2B6 (rs28399499), TRAF3IP2 (rs76228616) and GSTM1 (null genotype) can explain 25% of the susceptibility to this reaction, with the HLA-C SNP as the most significant contributor (P = 0.02 and OR = 5.64). Conclusions Our study confirmed the association of the rs5010528 SNP in the HLA-C region with susceptibility to developing SJS/TEN in a population from Mozambique, suggesting that it could be a good genomic biomarker for SJS/TEN susceptibility in different sub-Saharan populations. Introduction Nevirapine is a potent NNRTI that has been used in first-line treatment of HIV infection in the last decade.1 This drug has been widely used in developing countries, both for its efficacy in reducing mother-to-child virus transmission and for its low cost,2 and continues to be used even if not in first-line treatment. However, its use is associated with common serious and sometimes life threatening adverse drug reactions.3 Indeed, some individuals treated with this drug can develop liver toxicity and severe cutaneous adverse events such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN).4–6 Approximately 5% of all patients treated with nevirapine developed symptomatic hepatic events associated with systemic symptoms such as jaundice, coagulopathy and markedly elevated ALT.7,8 Rarer and uncommon adverse effects are skin manifestations; SJS is a severe and potentially fatal hypersensitivity reaction characterized by skin tenderness and erythema of the skin and mucosa, followed by extensive cutaneous and mucosal epidermal necrosis and sloughing that may progress into the more serious form of TEN.9 Several features of nevirapine hypersensitivity suggest that genetic factors may play an important predisposing/protective role and, in recent years, several studies have demonstrated the involvement of the human leucocyte antigen (HLA) alleles in hypersensitivity.10,11 In our previous studies, in a population from Mozambique, we have identified polymorphisms in CYP2B6, TRAF3IP2, HCP5 and GSTM1 genes to be associated with a higher risk of developing SJS/TEN.12–15 Recently, we participated in a collaborative genome-wide association study (GWAS), in which the rs5010528 polymorphism in the HLA-C locus was found to predispose to nevirapine-induced SJS/TEN in a sub-Saharan African population.16 In particular, the association was confirmed in a replication cohort of HIV patients from Malawi, Uganda and Mozambique. Regarding the Mozambique population, 27 SJS/TEN patients and only 8 controls were included in the study (67 controls had been excluded because the DNA aliquots did not pass the quality control necessary for the technology used in that study). The aim of the current study was to replicate the association of the HLA-C polymorphism in the same Mozambique cohort (expanding the number of controls to a total of 75 subjects, the total number of control DNA available in our laboratory), by Sanger sequencing, which does not require a high standard of DNA quality, and to perform a multivariate analysis in order to evaluate the role of HLA-C in relation to other SJS/TEN susceptibility genetic factors that we had previously identified. Methods Patient recruitment Subjects were selected from patients accessing nevirapine-based combination treatment (zidovudine/stavudine plus lamivudine and nevirapine) according to the reported criteria.2 The recruitment was conducted in Mozambique in three DREAM (Drug Resource Enhancement against AIDS and Malnutrition) Programme Centres, during the years 2008–09. We recruited 27 patients who had developed SJS/TEN and 75 ethnically matched controls (treated patients who did not develop any kind of reaction). The study protocol was authorized by the Minister of Health of Mozambique (approval number 1195/C13–5/GMS/2008) and all procedures were performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. The clinical diagnosis of SJS/TEN was based on the development of exanthema, mainly starting on the trunk, involving more than 10% of the body surface, with mucosal involvement. In these patients, the skin lesions had not given pruritus and a skin detachment had been observed by the clinicians who made the diagnosis. Patients’ baseline clinical characteristics were previously described.12 No significant difference between cases and controls was observed at the beginning of nevirapine treatment regarding age, BMI, CD4 cell count, viral load, and transaminase levels. All subjects (cases and controls) were females [mean age 32 years (IQR 27–36)]. DNA extraction and genotyping DNA was extracted, stored and analysed at the Genetic Laboratory of University of Rome Tor Vergata, Rome, Italy. Genomic DNA was extracted from peripheral blood by Wizard® Genomic DNA Purification Kit (Promega, WI, USA). rs5010528 in the HLA-C locus was analysed by direct sequencing. Genotyping or sequencing of CYP2B6, HCP5, PSORS1C1, GSTM1 and TRAF3IP2 SNPs were previously described.12–15,17 Statistical analysis The Hardy–Weinberg equilibrium was verified by the Pearson χ2 test. Differences in genotypic frequencies between cases and controls were evaluated by Pearson χ2 test. ORs with 95% CIs were calculated. The cut-off for statistical significance was P < 0.05. Since this study is based on the same patient cohort of our previous studies,12–15 other genes (CYP2B6, TRAF3IP2, HCP5 and GSTM1) and relative SNPs have also been included in multivariate analyses. All statistical analyses were performed using the SPSS program (SSPS Inc., IL, USA). Results Genotypes and allele frequencies of rs5010528 are reported in Table 1. The variant allele (G) frequency in our controls is in agreement with the frequency observed in the Malawi population (discovery cohort of Carr et al.16). We observed a significant difference in genotype and allele distribution between cases with SJS/TEN and controls (P = 0.0002 and P = 0.0003, respectively). The variant G allele is more frequent in cases than in controls (with an allele frequency equal to 38.9% versus 15.3%, respectively), conferring a high risk of developing the adverse reaction in carriers (OR = 5.72 at the genotype level, OR = 3.51 at the allelic level). Table 1. Case–control association analysis in SJS/TEN Gene    Controls, n = 75  SJS/TEN, n = 27  P  OR (95% CI)  HLA-C rs5010528 (A>G)  AA  53 (70.7%)  8 (29.6%)  0.0002a  5.72 (2.18–15.00)  AG  21 (28%)  17 (63%)  GG  1 (1.3%)  2 (7.4%)  A  127 (84.7%)  33 (61.1%)  0.0003  3.51 (1.74–7.11)  G  23 (15.3%)  21 (38.9%)  Gene    Controls, n = 75  SJS/TEN, n = 27  P  OR (95% CI)  HLA-C rs5010528 (A>G)  AA  53 (70.7%)  8 (29.6%)  0.0002a  5.72 (2.18–15.00)  AG  21 (28%)  17 (63%)  GG  1 (1.3%)  2 (7.4%)  A  127 (84.7%)  33 (61.1%)  0.0003  3.51 (1.74–7.11)  G  23 (15.3%)  21 (38.9%)  a Heterozygotes (AG) and variant homozygotes (GG) were considered together. Table 1. Case–control association analysis in SJS/TEN Gene    Controls, n = 75  SJS/TEN, n = 27  P  OR (95% CI)  HLA-C rs5010528 (A>G)  AA  53 (70.7%)  8 (29.6%)  0.0002a  5.72 (2.18–15.00)  AG  21 (28%)  17 (63%)  GG  1 (1.3%)  2 (7.4%)  A  127 (84.7%)  33 (61.1%)  0.0003  3.51 (1.74–7.11)  G  23 (15.3%)  21 (38.9%)  Gene    Controls, n = 75  SJS/TEN, n = 27  P  OR (95% CI)  HLA-C rs5010528 (A>G)  AA  53 (70.7%)  8 (29.6%)  0.0002a  5.72 (2.18–15.00)  AG  21 (28%)  17 (63%)  GG  1 (1.3%)  2 (7.4%)  A  127 (84.7%)  33 (61.1%)  0.0003  3.51 (1.74–7.11)  G  23 (15.3%)  21 (38.9%)  a Heterozygotes (AG) and variant homozygotes (GG) were considered together. We performed a multivariate analysis to evaluate the relative contribution given by the rs5010528 SNP and by the other associated SNPs. Age, viral load, BMI and CD4 count were also included as covariates in the analysis; with a stepwise approach, only the genetic factors were significantly associated with SJS/TEN susceptibility. Therefore, we repeated the analysis including only significant variables and the final model (Table 2) showed that the HLA-C SNP is the most significant factor in the SJS/TEN predisposition in our population (P = 0.02 and OR = 5.64). However, CYP2B6 (rs28399499), TRAF3IP2 (rs76228616) and GSTM1 (null genotype) also significantly contribute to conferring a high risk of developing this reaction, with an OR = 4.63, 3.97 and 3.56, respectively. Our model overall explains 25% of the susceptibility to this reaction. Table 2. Final model of multiple regression analysis Variable  βa  P  OR (95% CI)  R2 (Cox and Snell)  CYP2B6 (rs3745274)  1.09  0.11  2.98 (0.77–11.48)  0.25  CYP2B6 (rs28399499)  1.53  0.04  4.63 (1.09–19.63)  TRAF3IP2 (rs76228616)  1.38  0.04  3.97 (1.08–14.64)  GSTM1 (null genotype)  1.27  0.04  3.56 (1.07–11.82)  HLA-C (rs5010528)  1.73  0.02  5.64 (1.93–16.45)  Variable  βa  P  OR (95% CI)  R2 (Cox and Snell)  CYP2B6 (rs3745274)  1.09  0.11  2.98 (0.77–11.48)  0.25  CYP2B6 (rs28399499)  1.53  0.04  4.63 (1.09–19.63)  TRAF3IP2 (rs76228616)  1.38  0.04  3.97 (1.08–14.64)  GSTM1 (null genotype)  1.27  0.04  3.56 (1.07–11.82)  HLA-C (rs5010528)  1.73  0.02  5.64 (1.93–16.45)  Significant associations are shown in bold. a Indicates the β coefficient that represents the degree of change in the outcome variable. Table 2. Final model of multiple regression analysis Variable  βa  P  OR (95% CI)  R2 (Cox and Snell)  CYP2B6 (rs3745274)  1.09  0.11  2.98 (0.77–11.48)  0.25  CYP2B6 (rs28399499)  1.53  0.04  4.63 (1.09–19.63)  TRAF3IP2 (rs76228616)  1.38  0.04  3.97 (1.08–14.64)  GSTM1 (null genotype)  1.27  0.04  3.56 (1.07–11.82)  HLA-C (rs5010528)  1.73  0.02  5.64 (1.93–16.45)  Variable  βa  P  OR (95% CI)  R2 (Cox and Snell)  CYP2B6 (rs3745274)  1.09  0.11  2.98 (0.77–11.48)  0.25  CYP2B6 (rs28399499)  1.53  0.04  4.63 (1.09–19.63)  TRAF3IP2 (rs76228616)  1.38  0.04  3.97 (1.08–14.64)  GSTM1 (null genotype)  1.27  0.04  3.56 (1.07–11.82)  HLA-C (rs5010528)  1.73  0.02  5.64 (1.93–16.45)  Significant associations are shown in bold. a Indicates the β coefficient that represents the degree of change in the outcome variable. Discussion In this study our goal was to evaluate the overall and relative contribution of rs5010528 SNP and other studied genetic risk factors to SJS/TEN susceptibility in a population from Mozambique. Part of our study cohort was included in the study of Carr et al.16 (replication cohort) that identified rs5010528 in the HLA-C gene as being associated with SJS/TEN. However, in that study only a small number (n = 8) of our controls from Mozambique was examined, therefore we have extended the analysis of rs5010528 to the whole cohort of our controls (n = 75). With the case–control association study, we confirmed the association of the HLA-C SNP with susceptibility to SJS/TEN in the Mozambique population. Moreover, we have carried out a multivariate analysis to evaluate the overall contribution of the genetic risk factors so far identified, including the HLA-C SNP. The multivariate analysis confirmed the importance of genetic factors to SJS/TEN susceptibility in the Mozambique population, highlighting that HLA-C gives an important contribution together with the other genetic factors (CYP2B6, TRAF3IP2 and GSTM1). Although the role of the rs5010528 SNP needs to be clarified, docking studies showed that nevirapine could bind the B pocket of HLA-C: 04: 01.16 Replication studies in different populations are essential to understand the contribution of genetic variants to adverse drug reactions. Indeed, as it is known, some associations could be specific only for certain populations. In fact, some genetic variants can be associated with a toxic effect in a population, but not in other populations, or even not be present. For example, the HLA-B*15: 02 allele is a marker for carbamazepine-induced SJS/TEN only in southeast Asian populations,9 showing that ethnicity could play a role in susceptibility to adverse drug reaction. Using a control population from Mozambique, our study confirmed and strengthened the results of Carr et al.,16 suggesting that rs5010528 SNP in the HLA-C region could be a good genomic biomarker for SJS/TEN susceptibility in different sub-Saharan populations. In addition, our multivariate analysis confirmed that several genetic factors could contribute to this specific nevirapine adverse reaction in the population from Mozambique. Of course, the identification of new specific genetic markers for different populations will be of great utility to establish a personalized therapeutic approach and to prevent the occurrence of adverse reactions. Acknowledgements We thank all people collaborating in the DREAM Programme in Mozambique centres. Funding This study was supported by internal funding. Transparency declarations None to declare. 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Liver Transpl  2000; 6: 163– 9. Google Scholar PubMed  8 Shubber Z, Calmy A, Andrieux-Meyer I et al.   Adverse events associated with nevirapine and efavirenz-based first-line antiretroviral therapy: a systematic review and meta-analysis. AIDS  2013; 27: 1403– 12. Google Scholar CrossRef Search ADS PubMed  9 Rufini S, Ciccacci C, Politi C et al.   Stevens-Johnson syndrome and toxic epidermal necrolysis: an update on pharmacogenetics studies in drug-induced severe skin reaction. Pharmacogenomics  2015; 16: 1989– 2002. Google Scholar CrossRef Search ADS PubMed  10 Gao S, Gui XE, Liang K et al.   HLA-dependent hypersensitivity reaction to nevirapine in Chinese Han HIV-infected patients. AIDS Res Hum Retroviruses  2012; 28: 540– 3. Google Scholar CrossRef Search ADS PubMed  11 Likanonsakul S, Rattanatham T, Feangvad S et al.   HLA-Cw*04 allele associated with nevirapine-induced rash in HIV-infected Thai patients. AIDS Res Ther  2009; 6: 22. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: May 11, 2018

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