Arunrat Romphruk

HLA class I and II alleles and haplotypes in ethnic Northeast Thais

Allele frequencies (AFs) and haplotypic associations of human leukocyte antigen (HLA) class I and II were investigated in 400 unrelated, healthy, ethnic Northeast Thais. HLA-A, -B, -Cw, -DRB1 and -DQB1 were typed by polymerase chain reaction-sequence specific primer, -sequence specific oligonucleotide probe and -single-strand conformation polymorphism methods. In this population, 17 HLA-A, 26 HLA-B, 15 HLA-Cw, 26 HLA-DRB1 and 13 HLA-DQB1 alleles (or groups of alleles) were found. AFs > 10% included A*11 (23.3%), 24 (18.8%), 0207 (14.4%), 33 (11.5%), 0203 (10.6%); B*4601 (13.9%); Cw*07(01-03) (18.5%), 01 (15.9%), 04 (12.0%), 0304 (10.6%); DRB1*1502 (18.5%), 1202 (13.4%); DQB1*0502 (20.3%), 0501 (16.3%), 0301 (14.1%) and 02 (10.9%). The most common of 2-locus haplotypes included A*0207-B*4601 (9.3%), B*4601-Cw*01 (13.5%), B*5801-DRB1*0301 (5.8%) and DRB1*1502-DQB1*0501 (14.1%). Of the 49 five-locus HLA haplotypes identified, 24 were confirmed in 31 family studies: the most common being; A*33-Cw*0302-B*5801-DRB1*0301-DQB1*02 (4.6%), A*0207-Cw*01-B*4601-DRB1*09-DQB1*0303 (3.4%) and A*33-Cw*07(01-03)-B*44-DRB1*07-DQB1*02 (2.6%). Apparently, the HLA-B*46-carrying haplotype is fragmented in ethnic Northeast Thais, including seven haplotypes with different HLA-A and HLA-DR/DQ combinations. One of these haplotypes (A*11-Cw*01-B*4601-DRB1*1202-DQB1*0502) has not been reported in other Asians. The results indicated that there were marked differences in the distribution of HLA alleles and haplotypes between ethnic Northeast Thais and other ethnic groups in Southeast and East Asia. These results also dictate that future studies of HLA alleles and diseases need precise identification of ethnically and geographically matched controls. The HLA allele and haplotype analyses in this large sample provide baseline information on ethnic Northeast Thais for anthropological studies and for determining HLA allele/haplotype frequencies when searching for HLA-compatible donors for unrelated bone marrow transplantation.

Haplotype associations of the major histocompatibility complex with psoriasis in Northeastern Thais

Background  To evaluate the distributions of the human leukocyte antigen (HLA) at class I and II loci that may contribute to the genetic susceptibility to psoriasis patients in the north‐eastern Thai population.

Genotype analysis of Burkholderia pseudomallei using randomly amplified polymorphic DNA (RAPD): indicative of genetic differences amongst environmental and clinical isolates.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease common in the tropics. Melioidosis is most prevalent in the northeastern part of Thailand. The diseases has diverse clinical manifestations ranging from mild localized to fatal septicemic forms. The bacterial genetic factors contributing to the severity of melioidosis have not been completely identified. We have developed a genotyping method based upon randomly amplified polymorphic DNA (RAPD) analysis. Eighteen deca-oligo nucleotide primers with 70% GC content, eight previously published 60%GC RAPD primers, and four random deca oligomers were tested on nine strains of B. pseudomallei isolated from five patients with localized and four with septicemic melioidosis. The RAPD patterns were analyzed by polyacrylamide gel electrophoresis using a laser based automated fragment analyzer, GS2000. Based upon the pattern complexity, seven pairs consisting of eight primers were chosen for further analysis. Six hundred and thirty-two samples, including duplicates/triplicates, of B. pseudomallei isolated from melioidosis patients and the environment were analyzed. Two controls were included in each run of the test samples. All the samples were tested and patterns analyzed by blinded technical staff. Apparently, the method is reproducible. This is indicated by the RAPD patterns of the two controls of between run assay. Interestingly, some RAPD patterns were more prevalent in the clinical isolates than the environmental specimens and vice versa. For example, Q162KKU4-0 and Q162KKU1-0 were found 3. 5 and 3.3 times more often in the clinical specimens (P<0.025). Likewise, Q162KKU1-1 and Q162KKU4-1 were found 18 and 37 times more often in the environment (P<0.0000001). In addition, there was a bias in the distribution of arabinose positive strains and particular RAPD patterns; RAPD patterns of B. pseudomallei that were found frequently in septicemic patients were less likely to be arabinose positive. The data suggest the existence of bacterial genetic differences between the clinical and environmental isolates of B. pseudomallei. Further analysis of the RAPD patterns searching for common polymorphic DNA fragments and systemic comparative genomic analysis of B. pseudomallei in accordance with the clinical data should reveal genetic factors involved in severity and bacterial pathogenesis of B. pseudomallei in melioidosis.

Polymorphisms of NKG2D ligands: diverse RAET1/ULBP genes in Northeastern Thais

Unique long 16 (UL-16)-binding proteins (ULBP) or retinoic acid early transcripts-1 (RAET1) are ligands to the activating receptor, NKG2D. The human RAET1/ULBP gene family is identified as ten members (RAET1E to N) with six loci encoding for potentially functional proteins. These are ULBP1 or RAET1I, ULBP2 or RAET1H, ULBP3 or RAET1N, and RAET1L, which are glycosylinositol phospholipid (GPI)-linked glycoproteins and ULBP4 or RAET1E and ULBP5 or RAET1G, which are transmembrane glycoproteins. The RAET1 products contain the α1 and α2 domains but lack the α3 domain and do not associate with β2-microglobulin. RAET1/ULBPs have tissue-specific expressions, and some of them are also polymorphic. In the present study, polymorphic exons 2 and 3 of the RAET1E, G, H, I, L, and N were analyzed using sequence-based typing. One hundred and seventy-six unrelated healthy Northeastern Thais were included in this study. For RAET1E, RAET1G, RAET1H, and RAET1L, there were seven, two, five, and four single nucleotide polymorphisms (SNPs), respectively. Six of these are new SNPs, which are rare in this population. Of these, six new SNPs, two of two in RAET1E, two of three in RAET1H, and none of one in RAET1L are nonsynonymous substitutions. Interestingly, although the RAET1N is polymorphic in Caucasians, RAET1N and RAET1I had no variation in Thais indicating diverse RAET1 genes in different ethnic groups. These data provide the important basis for future analysis on the role of RAET1 genes in immune responses especially in cancer and infectious diseases.

Polymorphic Alu insertions and their associations with MHC class I alleles and haplotypes in the northeastern Thais.

Polymorphic Alu insertions (POALINs) are known to contribute to the strong polymorphic nature of the Major Histocompatibility Complex (MHC). Previous population studies on MHC POALINs were limited to only Australian Caucasians and Japanese. Here, we report on the individual insertion frequency of the five POALINs within the MHC class I region, their HLA‐A and ‐B associations, and the three and four locus alpha block POALIN haplotype frequencies in the Northeastern (NE) Thai population. Of the five POALINs, the lowest frequency was 0.018 for AluyHF and the highest frequency was 0.292 for AluyHJ and AluyHG. The strongest positive associations between the POALINs and HLA class I alleles was between AluyMICB and HLA‐B*57, AluyHJ and HLA‐A*24 and HLA‐A*01, and AluyHG and HLA‐A*02, supporting previous findings in Caucasians and Japanese. Single POALIN haplotypes were found more frequently than multiple POALIN haplotypes. However, of the seven different POALIN haplotypes within the MHC alpha block, there were only two significant differences between the NE Thais, Caucasians and Japanese. This study confirms that the MHC POALINs are in linkage disequilibrium with HLA‐A and –B alleles and that there are significant frequency differences for some of the POALINs when compared between NE Thai, Caucasians and Japanese.

Associations of MICB with cervical cancer in north‐eastern Thais: identification of major histocompatibility complex class I chain‐related gene B motifs influencing natural killer cell activation

The expression of MICB, a member of the major histocompatibility complex class I chain‐related gene B family, is induced in response to cellular stress. It is one of the ligands to the NKG2D receptor. MICB is polymorphic, but the distribution of MICB polymorphism in north‐eastern Thais and their potential associations with cancer have not yet been elucidated. In this study, polymerase chain reaction–sequence‐specific primers were developed to identify 15 MICB alleles and one group of alleles. We performed MICB typing in 100 healthy north‐eastern Thai females (NETF) and 99 cervical cancer patients to evaluate the association of MICB polymorphisms and the risk of developing cervical cancer. Eight and nine alleles were detected in the NETF and cervical cancer respectively. MICB*00502 was associated negatively with a corrected P‐value of 0·0009, suggesting the existence of a protective allele in cervical cancer. Amino acid substitutions carried by this allele were investigated for their potential involvement in natural killer (NK) cell activation. Although lysine at amino acid position 80 (Lys80) and aspartic acid at position 136 (Asp136) were associated negatively with cervical cancer, only MICB carrying Asp136 could induce NK cell killing more efficiently than MICB‐Lys80 when the NK cells were blocked by anti‐NKG2D. This result suggested that aspartic acid at position 136 may affect NKG2D binding, leading to different degrees of immune cell activation.

HLA-B*15 subtypes in Burmese population by sequence-based typing.

Human leukocyte antigen (HLA)-B*15 encompasses an increasing number of subtypes of more than 150. Frequency studies and a strong genetic association between HLA subtypes and susceptibility to drug hypersensitivity have been reported in different ethnic populations. To identify HLA-B*15 subtypes in Burmese using sequence-based typing (SBT) method, we selected 65 HLA-B*15-positive samples from 170 unrelated healthy Burmese who were genotyped HLA-B* by polymerase chain reaction with the sequence-specific primer method. The frequency of HLA-B*15 in Burmese was found to be 38.2%. By the SBT method, results showed 10 alleles of HLA-B*15 subtypes. Four common alleles, B*1502 (45.2%), B*1532 (16.4%), B*1525 (12.3%), and B*1501 (8.2%), were found in 82.1% of HLA-B*15-positive Burmese. Whereas the B*1501 was the highest in the Caucasians, Koreans, and Japanese, the highest frequency of HLA-B*15 alleles in Burmese was B*1502 (45.2%) that is similar to the frequency found in northeastern Thais and Vietnamese. This study is the first report of HLA-B*15 subtypes in Burmese. These results will provide the basic data in the further study in transplantations, genetic association with diseases, and drug hypersensitivity.

Bystander T cells in human immune responses to dengue antigens

BackgroundPrevious studies of T cell activation in dengue infection have focused on restriction of specific T cell receptors (TCRs) and classical MHC molecules. However, bystander T cell activation, which is TCR independent, occurs via cytokines in other viral infections, both in vitro and in vivo, and enables T cells to bypass certain control checkpoints. Moreover, clinical and pathological evidence has pointed to cytokines as the mediators of dengue disease severity. Therefore, we investigated bystander T cell induction by dengue viral antigen.ResultsWhole blood samples from 55 Thai schoolchildren aged 13-14 years were assayed for in vitro interferon-gamma (IFN-γ) induction in response to inactivated dengue serotype 2 antigen (Den2). The contribution of TCR-dependent and independent pathways was tested by treatment with cyclosporin A (CsA), which inhibits TCR-dependent activation of T cells. ELISA results revealed that approximately 72% of IFN-γ production occurred via the TCR-dependent pathway. The major IFN-γ sources were natural killer (NK) (mean ± SE = 55.2 ± 3.3), CD4+T (24.5 ± 3.3) and CD8+T cells (17.9 ± 1.5), respectively, as demonstrated by four-color flow cytometry. Interestingly, in addition to these cells, we found CsA-resistant IFN-γ producing T cells (CD4+T = 26.9 ± 3.6% and CD8+T = 20.3 ± 2.1%) implying the existence of activated bystander T cells in response to dengue antigen in vitro. These bystander CD4+ and CD8+T cells had similar kinetics to NK cells, appeared after 12 h and were inhibited by anti-IL-12 neutralization indicating cytokine involvement.ConclusionsThis study described immune cell profiles and highlighted bystander T cell activation in response to dengue viral antigens of healthy people in an endemic area. Further studies on bystander T cell activation in dengue viral infection may reveal the immune mechanisms that protect or enhance pathogenesis of secondary dengue infection.

HLA-B*27 subtypes in Northern and Northeastern Thais, Karens, and Bamars determined by a high-resolution PCR-SSP technique

Human leukocyte antigens (HLA), class I, are a group of antigens expressed on most nucleated cell surfaces. They transport endogenous peptides to the cell surface for recognition by T-cell receptors. Their functions are involved in immune responses. Many diseases are associated with HLA alleles, especially HLA-B*27 that is strongly associated with ankylosing spondylitis (AS). HLA-B*27 consists of 42 subtypes. Different subtypes of HLA-B*27 were reported in different ethnic groups of AS patients. In this study, a high-resolution polymerase chain reaction-sequence-specific primer technique has been developed to define all the HLA-B*27 subtypes with a total of 29 primer mixtures. Two of the primer mixes were used to detect the HLA-B*27-specific group, and 27 primer mixes were used to identify 42 subtypes (B*2701-B*2721 and B*2723-B*2743). The HLA-B*27-group-specific primers have been tested in unrelated healthy subjects; 846 Northeastern Thais (NET), 334 Northern Thais (NT), 264 Karens, and 310 Bamars. Sixty-three NET (phenotype frequency, PF = 7.4%), 24 NT (PF = 7.1%), 5 Karens (PF = 1.8%), and 12 Bamars (PF = 3.9%) were positive for HLA-B*27. Only B*2704 was found in Karens, whereas B*2704, B*2705/37/39, B*2706, and B*2707 were found in NET and NT. In Bamars, B*2704, B*2705/37/39, B*2706, and B*2725 were found. The distribution of HLA-B*27 subtypes was compared with other studies in Asian and Caucasian populations. Significant differences of the distribution of HLA-B*27 subtypes were found in most of the populations. This study established a simple technology for HLA-B*27 subtyping and provided basic information for anthropology and further studies in disease associations.

Development of a multiplex polymerase chain reaction-sequence-specific primer method for NKG2D and NKG2F single-nucleotide polymorphism typing using isothermal multiple displacement amplification products

Natural killer group 2 member D (NKG2D) on immune effector cells recognizes multiple stress-inducible ligands. NKG2D single-nucleotide polymorphism (SNP) haplotypes were related to the levels of cytotoxic activity of peripheral blood mononuclear cells. Indeed, these polymorphisms were also located in NKG2F. Isothermal multiple displacement amplification (IMDA) is used for whole genome amplification (WGA) that can amplify very small genomic DNA templates into microgram with whole genome coverage. This is particularly useful in the cases of limited amount of valuable DNA samples requiring multi-locus genotyping. In this study, we evaluated the quality and applicability of IMDA to genetic studies in terms of sensitivity, efficiency of IMDA re-amplification and stability of IMDA products. The smallest amount of DNA to be effectively amplified by IMDA was 200 pg yielding final DNA of approximately 16 µg within 1.5 h. IMDA could be re-amplified only once (second round of amplification), and could be kept for 5 months at 4°C and more than a year at -20°C without loosing genome coverage. The amplified products were used successfully to setup a multiplex polymerase chain reaction-sequence-specific primer for SNP typing of the NKG2D/F genes. The NKG2D/F multiplex polymerase chain reaction (PCR) contained six PCR mixtures for detecting 10 selected SNPs, including 8 NKG2D/F SNP haplotypes and 2 additional NKG2D coding SNPs. This typing procedure will be applicable in both clinical and research laboratories. Thus, our data provide useful information and limitations for utilization of genome-wide amplification using IMDA and its application for multiplex NKG2D/F typing.