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.


INTRODUCTION
Human leukocyte antigen (HLA) genes are located in the short arm of chromosome 6, are part of a large family of genes, and are inherited from both parents, one of paternal origin and one of maternal origin for each of the loci (Alper et al., 2006). HLA proteins are present at the cellular surface of antigen presenting cells. There are 3 classes of HLA. Class I loci (HLA-A, -B, -C) present endogenous antigens such as viral protein or tumor antigen to CD8 + T cells. Class II loci (HLA-DR, -DQ, -DP) present exogenous antigens to CD4 + T cells. Class III (Bf, C2, C4A) are involved in the complement system. HLA genes are highly polymorphic, which becomes important in associations of susceptibility or resistance to disease, development of tumor processes, molecular vaccine development (Dawson et al., 2001) and determination of organ or tissue transplant survival (Dhaliwal et al., 2003;Avila-Rios et al., 2009). In addition, HLA allele frequency might be useful in anthropological studies.
HLA-B alleles have been used as a marker for predicting druginduced adverse reactions (Sukasem et al., 2014a) and are a major contributor to hypersensitivity reactions involving direct stimulation of immune effector cells and imitating an allergic reaction. Several studies reported that allopurinol-induced severe cutaneous adverse reactions (SCARs) are strongly associated with the HLA-B * 58:01 allele in Han Chinese and Thais (Hung et al., 2005;Tassaneeyakul et al., 2009;Jantararoungtong et al., 2014). The HLA-B * 15:02 allele was strongly related to carbamazepine-induced Steven-Johnson syndrome (SJS)-toxic epidermal necrolysis (TEN) in Taiwanese and Thai populations (Tassaneeyakul et al., 2010;Chen et al., 2011;Sukasem et al., 2014b). Importantly, Thais showed cutaneous adverse reactions associated with HLA-B * 35:05 (Chantarangsu et al., 2009(Chantarangsu et al., , 2011. In addition, the HLA-B * 57:01 allele is involved in abacavir-induced hypersensitivity reaction (Mallal et al., 2008). Consequently, screening of HLA-B genotyping before prescription of medication may reduce the risk of SCARs and drug hypersensitivity reaction.
Presently, HLA-B genotyping is available in clinical practices before commencing therapy with the aforementioned drugs in Thailand (Sukasem et al., 2014a). This study aimed to determine the distribution of HLA-B haplotypes, and to establish the most common HLA haplotype-associated drug induced hypersensitivity using routine pharmacogenomic laboratory procedures. Over a period of 3 years, HLA-B allele genotype was analyzed in 986 Thai people registered at the Laboratory for Pharmacogenomics, Ramathibodi Hospital, Thailand. The frequency of HLA-B alleles and HLA-B genotypes were determined and used to define the probability associated with a Pharmacogenomic marker indicating adverse drug reaction.

SUBJECTS AND CHARACTERISTICS
A descriptive, observational, cross-sectional, retrospective study was conducted in 986 unrelated individuals. Data were collected at the Laboratory for Pharmacogenomics, Somdech Phra Debaratana Medical Center (SDMC), Ramathibodi Hospital, Bangkok, Thailand during August 2011-June 2014. Among these patients, there were 590 (59.83%) males and 396 (40.17%) females. The mean age of patients at recruitment was 41.98 ± 4.40 years. The study was approved by the Ethics Committee of the Faculty of Medicine, Ramathibodi Hospital, Mahidol University. The protocol number is ID 04-56-24.

GENOMIC DNA EXTRACTION
Blood samples were collected into EDTA tubes. DNA was isolated using the MagNA Pure automated extraction system (Roche diagnostics, USA), which uses magnetic-bead technology with a lysis buffer and proteinase K. Nucleic acids bind to the surface of the magnetic glass particles. Cellular debris was removed by several washing steps and the purified nucleic acids were eluted. From the 1 ml input volume of EDTA-whole blood, 200 µL output volume of extracted genomic DNA product was obtained.
The quality of genomic DNA was assessed by using Nano Drop ND-1000. Genomic DNA was detected by measuring absorbance at 260 nm. Purity of the sample was evaluated by calculation of the optical density (OD) ratio, 260/280 nm. The recommended purified genomic DNA template for this study was 20 ng, and the OD ratio greater than 1.7. All DNA was aliquotted and stored at −20 • C before analysis.

HLA-B TYPING
HLA-B genotyping was carried out using the Luminex™ Multiplex Technology (Luminex®IS 100, USA) based on Polymerase Chain Reaction-sequence specific oligonucleotides probe (PCR-SSOP) principles. Briefly, the PCR products were hybridized against a panel of oligonucleotide probes coated onto polystyrene microspheres. Probe sequences were complementary to stretches of polymorphic sequence within the target HLA-B alleles. The amplicon-probe complex was visualized using a colorimetric reaction and fluorescence detection technology. Data analyses for the HLA-B assays were performed with HLA fusion™2.0 software.

STATISTICAL ANALYSIS
The HLA-B allele frequencies of the samples were assayed by direct counting and, subsequently, by dividing the total number of occurrences of that allele by the total number of alleles at that locus in the population. The samples were also evaluated for Hardy-Weinberg equilibrium using the Chi-square or Fisher's exact test if the number in any cell of the 2 × 2 contingency tables was less than five.    01%). None of these genotypes were present in more than 5% of the population. Table 2 shows the frequency of the top 10 genotypes the Thai population in this study.

DISCUSSION
The HLA-B allele frequencies of 968 Thai subjects were investigated. This is the first report of HLA-B genotyping in the Thai population using the Luminex HLA-SSOP method. Our data showed that the Thai population has extensive diversity at the HLA-B locus. One hundred and sixteen allele types were identified and the frequencies of HLA-B alleles did not significantly deviate from Hardy-Weinberg equilibrium.  (Jinam et al., 2010;Kupatawintu et al., 2010;Romphruk et al., 2010). This may indicate that Thais have a closer relationship with their Chinese neighbors than with Malaysia. Previous studies of HLA alleles in the Thai population had larger subject numbers, but reported no pharmacogenomic data, which we present in the current study. HLA-B * 15:02 allele is found in high prevalence among people in South-East Asian countries. The frequency of the HLA-B allele varies among different populations suggesting that different alleles may also function in drug hypersensitivity. Currently, there has been an increase of publications associated with HLA-B alleles and drug hypersensitivity (Sukasem et al., 2014a).
In the present study, we found that 16.33% of Thai people carry the HLA-B * 58:01 allele, which is associated with allopurinol hypersensitivity in gout treatment (Tassaneeyakul et al., 2009;Saokaew et al., 2014). Beside drug hypersensitivity, other studies of HLA-B alleles, such as HLA-B * 46:01, report a significant increase in severity of cerebral malaria compared with patients with mild CNS symptoms (Hirayama et al., 1998). Moreover, HLA-B * 40:01 is associated with lipodystrophy in Thai HIV-1positive patients who received antiretroviral therapy containg stavudine (Wangsomboonsiri et al., 2010). HLA-B * 13:01 has been suggested as a genetic marker for dermal hypersensitivity associated with Trichloroethylene, an industrial solvent (Li et al., 2007).
Our findings demonstrated the diversity of HLA-B alleles in the Thai population, and many more HLA associated with ADR. Other classes of HLA may be observed in upcoming studies. The subjects enrolled in the study were mostly from central Thailand. This narrow sample may restrict our calculations of HLA-B allele distribution in Thai populations. Future studies should consider this limitation of our survey.
HLA-associated drug hypersensitivity is dependent upon the different populations studied, and variation in genetic backgrounds. Recently, HLA-B genotyping has become accessible in clinical practice, providing appropriate clinical monitoring, patient counseling and recommendations for treatment. Interestingly, "pharmacogenetic tests" and "pharmacogenomic cards" (Figure 1) have been successfully implemented in clinical practice in Thailand at the Laboratory for Pharmacogenomics, Somdech Phra Debaratana Medical Center, Ramathibodi Hospital (Sukasem et al., 2014a). Recently, HLA-B * 15:02 screening has been preliminarily introduced by the National Health Security Office (NHSO), Thailand, to screen patients who may be at risk of carbamazepine-induced SJS/TEN.
In conclusion, our data suggest that HLA-B genotype should be screened before medication is prescribed to reduce the incidence of drug hypersensitivity. These findings provided useful information in the study of pharmacogenomics and HLA-B polymorphisms in the Thai population. The frequency of the HLA-B alleles associated with drug hypersensitivity will help in designing alternative treatment regimens or better therapy for affected individuals. Understanding of the mechanism underlying drug hypersensitivity reactions requires further investigation.