Siwalee Santon

HLA-B allele and haplotype diversity among Thai patients identified by PCR-SSOP: evidence for high risk of drug-induced hypersensitivity

Background: There are 3 classes of HLA molecules; HLA class I, II and III, of which different classes have different functions. HLA-B gene which belongs to HLA class I play an important role predicting drug hypersensitivity. Materials and Methods: Nine hundred and eighty-six Thai subjects who registered at a pharmacogenomics laboratory were determined for HLA-B genotype using a two-stage sequence-specific oligonucleotide probe system (PCR-SSOP). Results: In this study, HLA-B alleles did not deviate from Hardy-Weinberg equilibrium (P > 0.05). The most common HLA-B alleles observed in this population were HLA-B*46:01 (11.51%), HLA-B*58:01 (8.62%), HLA-B*40:01 (8.22%), HLA-B*15:02 (8.16%) and HLA-B*13:01 (6.95%). This finding revealed that HLA-B allele frequency in the Thai population was consistent with the Chinese population (p > 0.05), however, differed from the Malaysian population (p < 0.05). The top five HLA-B genotypes were HLA-B*40:01/46:01 (2.13%), HLA-B*46:01/46:01 (2.03%), HLA-B*40:01/58:01 (2.03%), HLA-B*46:01/58:01 (1.93%) and HLA-B*15:02/46:01 (1.83%). This study found that 15.92% of Thai subjects carry HLA-B*15:02, which has been associated with carbamazepine-induced severe cutaneous adverse drug reactions (SCARs). Moreover, 16.33% of Thai subjects carry the HLA-B*58:01 allele, which has been associated with allopurinol-induced SCARs. Conclusion: This study demonstrates a high diversity of HLA-B polymorphisms in this Thai population. The high frequency of HLA-B pharmacogenomic markers in the population emphasizes the importance of such screening to predict/avoid drug hypersensitivity.

CYP2C19 polymorphisms in the Thai population and the clinical response to clopidogrel in patients with atherothrombotic-risk factors

Genetic variation in the cytochrome P450 2C19 (CYP2C19) gene has been documented gradually as the determinant conversion and variability in the antiplatelet effect of clopidogrel. The aims of this study were to determine the prevalence of clinically relevant allele variants (CYP2C19*2, CYP2C19*3, and CYP2C19*17) in a Thai study population, and finally determine whether the allele distributes and predicts metabolic phenotypes in clopidogrel treated patients. A total of 1,051 Thai patients participated in this study. Genotypes for CYP2C19 polymorphisms were detected by the microarray-based technique. Furthermore, results of genotyping and platelet aggregation in 96 cardiovascular disease patients on 75 mg clopidogrel maintenance daily dose therapy also were analyzed. Among 1,051 samples, the allele frequencies of CYP2C19 *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *1/*17 were found in 428 (40.72%), 369 (35.10%), 72 (6.85%), 77 (7.32%), 59 (5.61%), and 45 (4.30%) of the patients, respectively. Homozygous CYP2C19 *3/*3 was found in one patient (0.10%). Therefore, 40.72% of the patients were predicted as extensive metabolizers, 41.95% as intermediate metabolizers, 13.03% as poor metabolizers, and 4.30% as ultra-rapid metabolizers. Among 96 patients, the frequency of poor metabolizers was significantly higher in the clopidogrel non-responder group than in the responder group (36.0% and 15.5%, respectively, P = 0.03). CYP2C19*1/*17 was observed in responders (n = 2; 2.8%). As a result, CYP2C19 variants were associated with clopidogrel non-responders. However, there is a need for further elucidation of the clinical importance and use of this finding to make firm and cost-effective recommendations for drug treatment in the future.

Polymorphisms of the ApoE (Apolipoprotein E) Gene and Their Influence on Dyslipidemia in HIV-1-Infected Individuals

The purpose of this retrospective case-control study was to investigate the frequency of Apolipoprotein E (ApoE) polymorphisms and their influence on antiretroviral therapy (ART)-induced lipodystrophy or dyslipidemia in HIV-infected Thai patients. The clinical characteristics and frequencies of ApoE genotypes were compared between the case (moderate to severe lipodystrophy, n = 67) and control (absent to mild lipodystrophy, n = 18) groups. The ApoE genotype frequencies among the 85 participants were 2.35% (n = 2) for E2/E2, 20% (n = 17) for E2/E3, 9.41% (n = 8) for E2/E4, 36.47% (n = 31) for E3/E3, 30.59% (n = 26) for E3/E4, and 1.18% (n = 1) for E4/E4. None of the ApoE genotypes showed association with ART-induced lipodystrophy. However, the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-cholesterol), and ApoB were lower in patients carrying the E2 allele but higher in E4 carriers. Interestingly, the ratios between TC and high-density lipoprotein (TC/HDL cholesterol ratio) and ApoB/ApoA-I ratio were significantly higher in the case group. Patients carrying the E2 allele displayed protective lipid profile, while those carrying E4 appeared to be at higher risk of dyslipidemia. In conclusion, ApoE polymorphisms were not associated with lipodystrophy in patients undergoing antiretroviral therapy but influenced lipid alteration.

Small-Dense LDL Cholesterol/Large-Buoyant LDL Cholesterol Ratio as an Excellent Marker for Indicating Lipodystrophy in HIV-Infected Patients

OBJECTIVES To examine whether the lipid parameters are predicting factors for human immunodeficiency virus (HIV)-associated lipodystrophy. METHODS Whole-body fat compositions of HIV-positive patients receiving stavudine-containing antiretroviral regimens (n = 79) were determined. Lipodystrophy was defined as a ratio of trunk fat mass/lower limb fat mass greater than 2.28. Blood samples were analyzed for total cholesterol (TC), triglycerides, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), small-dense LDL-C (sdLDL-C), apoAI, apoB, lipoprotein(a), and CD4 cell counts. Large-buoyant LDL-C (lbLDL-C) was calculated (LDL-C minus sdLDL-C). RESULTS Twenty-six patients were classified as having lipodystrophy. The mean values of triglycerides, HDL-C, sdLDL-C, apoB, TC/HDL-C, apolipoprotein (apo) B/apoAI, and sdLDL-C/lbLDL-C showed significant differences between patients with and without lipodystrophy (P < .02). Using logistic regression analysis, sdLDL-C/lbLDL-C was identified as a significant predictor of lipodystrophy (P < .001). At a ratio of 0.554, the odds ratio was 17.8 with a likelihood ratio of 5.5. CONCLUSIONS The sdLDL-C/lbLDL-C ratio is an excellent marker for indicating lipodystrophy in HIV-infected patients.

Association of HLA-A and HLA-B Alleles with Lamotrigine-Induced Cutaneous Adverse Drug Reactions in the Thai Population

Background: Lamotrigine (LTG) is commonly used for treatment of epilepsy and bipolar disorder. It is one of the common cause of cutaneous adverse drug reactions (CADR). Clinical symptoms of LTG-induced CADR range from maculopapular exanthema (MPE) to severe cutaneous adverse reactions (SCAR). This study aimed to determine the association of the LTG-induced CADR with human leukocyte antigen (HLA) alleles in Thai patients. Methods: Fifteen patients with LTG-induced CADR [10 MPE; 4 Stevens–Johnson syndrome; and 1 drug reaction with eosinophilia and systemic symptoms] and 50 LTG-tolerant controls were included in the study. HLA-A and HLA-B genotyping was performed using polymerase chain reaction-sequence-specific oligonucleotides probes. Results: The proportion of HLA-A∗02:07 and HLA-B∗15:02 allele carriers were significantly higher in the LTG-induced CADR group than in the tolerant controls [odds ratio (OR): 7.83; 95% confidence interval (CI): 1.60–38.25; P = 0.013, and OR: 4.89; 95% CI: 1.28–18.67; P = 0.014]. In addition, subjects with HLA-A∗33:03, HLA-B∗15:02, and HLA-B∗44:03 were significantly higher in the LTG-induced MPE group than in the tolerant controls (OR: 8.27; 95% CI: 1.83–37.41; P = 0.005, OR: 7.33; 95% CI: 1.63–33.02; P = 0.005; and OR: 10.29; 95% CI: 1.45–72.81; P = 0.029). In contrast to the LTG-induced MPE group, there were no significant differences between HLA alleles and LTG-induced SCAR group. Conclusion: HLA-A∗02:07 and HLA-B∗15:02 were associated with LTG-induced CADR in Thai patients. We also identified an association between HLA-A∗33:03, HLA-B∗15:02, and HLA-B∗44:03 and LTG-induced MPE in this population. These results suggest that these alleles could be useful screening markers for preventing CADR before LTG treatment in Thai patients, but further replication studies with larger sample sizes are needed.

Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC–MS/MS: An application for phenotypic and genotypic correlations

HighlightsThe determination of TPMT activity by LC–MS/MS from whole blood samples was optimized.The developed method was less labor and time consuming and required a smaller volume of whole blood (100 &mgr;l).This method had a shorter run time for the analysis and showed high precision and accuracy with a 6‐MMP‐d3 isotope internal standard.Finally, the TPMT activity resulting from the optimized method was correlated with genotyping analysis. Abstract A liquid chromatography‐tandem mass spectrometry (LC–MS/MS) method was developed for the determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on conversion of 6‐mercaptopurine (6‐MP) by TPMT to 6‐methylmercaptopurine (6‐MMP) using S‐adenosyl‐l‐methionine (SAM) as the methyl donor. This method was improved from the previous laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In addition, the TPMT incubation was optimized and the chromatography was performed in a short runtime of 7 min on a C18‐column by detection via triple quadrupole mass spectrometry. The MS/MS was optimally tuned to monitor mass to charge a ratio (m/z) for 6‐MMP 167.2 → 151.9 and the isotope 6‐MMP‐d3 with m/z of 170.5 → 152.2 were applied as an internal standard. The calibration curve covered the range of 2.5–360 ng/ml and the correlation coefficient was greater than 0.999. The accuracy of this method was determined in four concentrations of control of quality that ranged between 99.33 and 106.33%. The intra‐assay coefficient of variation (CV) was less than 4.41% and the inter‐assay was less than 5.43%. This method developed for measuring TPMT by LC–MS/MS is a reliable, safe, and simple with a small volume requirement (100 &mgr;l of whole blood EDTA). The assay was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1 nmol 6‐MMP/g Hb/h with means and median values of TPMT activity 55.9 ± 12.47 nmol 6‐MMP/g Hb/h and 54.2 nmol 6‐MMP/g Hb/h. The genotype‐phenotype association of TPMT was evaluated for common ethnic Thai single nucleotide polymorphisms (SNP) in 30 samples and demonstrated good concordance.

Development and Validation of Voriconazole Concentration by LC-MS-MS: Applied in Clinical Implementation

Voriconazole (VRZ) is a triazole antifungal used for treatment of invasive fungal infection, which is a life‐threatening condition. Therapeutic drug monitoring is recommended for identifying the optimal dose in patients who have hepatic/renal impairment or reduced function of the CYP2C19 metabolizing enzyme.

HLA-B*58:01 allele is strongly associated with allopurinol-induced severe cutaneous adverse reactions in a Thai population

Background Allopurinol has been reported as the most frequent causes of SCARs (severe cutaneous adverse reactions) in Thailand. Recent publications have shown that HLAB*58:01 allele is a strong marker for allopurinol-induced SJS/TEN (Stevens-Johnson syndrome/toxic epidermal necrolysis). The aim of this study was to clarify the association of allopurinol-induced SCARs with the HLA-B*58:01 allele in Thai patients. Method To investigate this relationship, we performed PCR-SSOP (sequence specific oligonucleotide probe) on allopurinoltolerant controls (n=56) and patients affected by allopurinol-induced SCARs (n=14). Among of allopurinol-induced SCARs, including 3 patients with allopurinol-induced DRESS (drug reaction with eosinophilia and systemic symptoms), 9 patients with SJS/TEN and 2 patients with MPE (maculo-papular exanthema) were included. The presence of HLA-B*58:01 allele were genotyped by PCRSSOP method at Laboratory for Pharmacogenomics, Ramathibodi Hospital. Results Of the 14 patients with allopurinol-induced SCARs, 13 (92.8%) patients (3 DRESS, 9 SJS/TEN and one severe MPE) had HLA-B*58:01 while only 6 (10.71%) of the allopurinol-tolerant controls had this allele. The risk of allopurinol-induced SCARs was significantly higher in the patients with HLA-B*58:01 with an OR (odd ratios) of 108.33 (95% CI 11.96-980.82, p<10-6). When compared with normal population, HLA-B*58:01 was associated with a higher risk of SCARs, both DRESS (OR: 80, 95% CI 3.42-372.87) and SJS/TEN (OR: 217.26, 95%CI 12.41-925.35). Conclusion In this study we confirmedthat HLA-B*58:01 allele is strongly associated with allopurinol-induced SCARs in Thai population. Therefore, screening tests for HLAB*58:01 allele in patients who will be treated with allopurinol will be clinically helpful in preventing the risk of developing SCARs.

HLA-B*15:02 genotype associated with hypersensitivity syndrome to lamotrigine in Thai population

Method A total of 133 patients, including 11 patients with lamotrigine-induced hypersensitivity syndrome (MPE; maculo-papular exanthema, SJS; Stevens-Johnson syndrome and TEN; toxic epidermal necrolysis), 9 lamotrigine-tolerant controls and 113 healthy controls were included in this study. HLA-B genotyping was performed. This case-control study was approved by the Ethics Committee of Ramathibodi Hospital.

Evaluation of a pharmacogenetic test in Thailand for abacavir hypersensitivity screening in human immunodeficiency virus infection

Abacavir hypersensitivity reaction (ABC-HSR) is associated with the presence of HLA-B*5701 allele. Alternative tests for ABC-HSR associated allele determination are needed where sequence-based HLA typing is not available, particularly in resource-limited settings or developing countries. This study focused on the development and evaluation of two HLA-B*5701 tagging SNPs (single nucleotide polymorphism) HCP5 (HLA complex P5) rs2395029 and TNF (tumor necrosis factor) rs3093726 genotyping assays. Two hundred and thirteen purified genomic DNA samples were used to evaluate the performance characteristics of a HLA-B*5701 screening method based on allele-specific polymerase chain reaction (AS-PCR) with melting curve analysis. HCP5 rs2395029 and TNF rs3093726 were also genotyped using simple probe real-time PCR assay. All samples were successfully genotyped wherein AS-PCR genotyping provided 100% sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) when compared with specific HLA-B status by sequencing based assay. When comparing the AS-PCR screening method with the HCP5 rs2395029 and TNF rs3093726 genotyping method, the former had 100% sensitivity, 100% specificity, 100% PPV and 100% NPV using a simple probe; while the latter one had 95.24% sensitivity, 100% specificity, 100% PPV and 99.48% NPV, respectively. In conclusion, our study lends support on a molecular tool for pharmacogenetic screening in resource-limited settings. Thus, serious drug hypersensitivity associated with ABC may potentially be reduced in Thailand by further evaluation of the proposed assay in clinical practice.