In-vivo and ex-vivo tests for culprit drugs identification in severe cutaneous adverse drugs reactions.

Drug causality assessment in severe cutaneous adverse reactions (SCARs) remains challenging. We investigated the usefulness of in-vivo drug patch tests (PT), ex-vivo interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assay, and lymphocyte transformation test (LTT) in 30 SCARs patients within the past 36 months. Drug PT yielded a 20% positivity rate (n = 6), while IFN-γ ELISpot and LTT showed positive rates of 56.67% (n = 17) and 41.38% (n = 12), respectively. Combining the three tests resulted in an overall positive rate of 66.67% (n = 20) of cases. IFN-γ ELISpot offered additional positivity, especially with oxypurinol. Employing a combined diagnostic approach may enhance the chances of obtaining a positive result.

Correlation of plasma microRNA-21 expression and bone turnover markers in postmenopausal women.

OBJECTIVES: This study assessed the correlation of plasma microRNA-21 expression and bone turnover markers (BTMs: CTx, P1NP) in healthy Thai postmenopausal women. We secondarily compared microRNA-21 expression between participants with normal and low bone mineral density (BMD: osteopenia and osteoporosis). METHODS: Postmenopausal women who had never been diagnosed with fracture or never used anti-osteoporosis drugs were included in this study. Baseline characteristics were collected from all 195 participants. BTMs and plasma miR-21-5p were analyzed from blood collection at 8:00 and 9:00 am after overnight fasting for at least 8 h. RESULTS: There was no significant correlation between miR-21-5p and any of the BTMs (CTx, r = 0.094, p = 0.19; P1NP, r = 0.05, p = 0.485). Significant correlation between miR-21-5p and P1NP was found when participants were further categorized into those aged ≥70 years (r = 0.46, p = 0.05) and those having osteoporosis (r = 0.51, p = 0.06). Slight negative correlations were found between miR-21-5p and BMD. There was statistically significant higher expression of miR-21-5p in those with low BMD when compared to the normal BMD group (p < 0.01). CONCLUSION: In this study, we did not find significant correlation between plasma microRNA-21-5p expression and the BTMs. Nevertheless, there seemed to be higher expression of miR-21-5p in the low BMD participants.

Predictive role of serum HBsAg and HBcrAg kinetics in patients with HBeAg-negative chronic hepatitis B receiving pegylated interferon-based therapy.

OBJECTIVES: To investigate the role of serum hepatitis B core-related antigen (HBcrAg) kinetics in predicting long-term outcome of pegylated interferon (PEG-IFN)-based therapy in patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB). METHODS: A total of 121 Thai patients with HBeAg-negative CHB recruited from a previous randomized trial of 48-week PEG-IFN alone or combined with entecavir were enrolled. Hepatitis B surface antigen (HBsAg) and HBcrAg levels were serially examined. Paired biopsy samples taken at baseline and after treatment were assessed for intrahepatic covalently closed circular DNA (cccDNA). RESULTS: Persistent virologic remission (PVR, defined by persistent hepatitis B virus (HBV) DNA <2000 IU/mL) and HBsAg clearance at 3 years after treatment were 29% (35/121) and 9% (11/121) respectively. Baseline HBcrAg correlated with HBV DNA and cccDNA but not with HBsAg. Baseline HBsAg, as well as HBsAg and HBcrAg, declines were associated with PVR, while HBsAg decline was predictive of HBsAg clearance. High baseline antigen levels (HBsAg ≥3.4 log10 IU/mL plus HBcrAg ≥3.7 log10 U/mL) yielded high negative predictive values of PVR (45/50, 90%) and HBsAg clearance (50/50, 100%). At week 12, declines of HBsAg, HBcrAg and both antigens combined of <0.5 log10 yielded negative predictive values for PVR of 90% (71/79), 82% (61/74) and 96% (48/50) respectively. CONCLUSIONS: Quantitative HBcrAg was significantly associated with cccDNA in HBeAg-negative CHB. This novel antigen, together with HBsAg, could identify patients with low probability of PVR and HBsAg clearance in long-term follow-up.

The effect of missing KIR ligands, activating KIR genotype and haplotype on the outcome of T-cell-replete hematopoietic stem cell transplantation from HLA-identical siblings in Thai patients.

This study was a retrospective analysis of Thai patients undergoing T-replete hematopoietic stem cell transplant from human leukocyte antigen (HLA)-identical sibling donors. We investigated 66 patients, including 40 patients with acute myeloid leukemia (AML), 12 patients with acute lymphoblastic leukemia and 14 patients with chronic myeloid leukemia. Killer cell immunoglobulin-like receptor (KIR) genes and HLA ligands were typed by polymerase chain reaction-sequence specific oligonucleotide probes. We analyzed the effect of the number of missing KIR ligands (Bw4, C1 and C2) on clinical outcomes. A beneficial effect of missing KIR ligand was not observed in both univariate and multivariate analysis. When we analyzed the effect of specific missing KIR ligand on clinical outcomes, there was a trend that patients with missing A11 ligand had lower relapse rate (P = 0.076). Therefore, we also conducted the analysis by including the group with missing KIR ligands of Bw4, C1, C2 and A11. Patients with two or more than two missing KIR ligands had a trend for better clinical outcome including reduced relapse (P = 055) and statistically significant in terms of reduced acute graft-vs-host disease (aGVHD) rate (P = 0.013). In multivariate analysis, patients with two or more than two missing KIR ligands had a statistically significant better clinical outcome in terms of reduced aGVHD rate (HR = 0.155, 95%CI = 0.040-0.605, P = 0.007). The association between clinical outcome with KIR haplotypes, centromeric B haplotype and activating KIR was not observed here. Although the sample size in this study is rather limited, these data can later be subjected to meta-analysis to help reach the conclusion of the usefulness of this additional promising KIR genotyping in various hematopoietic stem cell transplantation types.

Elevated expressions of myeloid-related proteins-8 and -14 are danger biomarkers for lupus nephritis.

Myeloid-related proteins, MRP-8 and -14, which have been identified as molecules that mediate the danger signaling in innate immune response, are also known as the DAMPs (damage associated molecular pattern molecules). The proteins were found in infiltrating macrophages and neutrophils at inflammatory sites. Their expression was correlated with severe forms of glomerulonephritis. Therefore, this study examined whether or not MRP-8 and -14 can be used as biomarkers for identifying severely active lupus nephritis (LN). Total blood leukocyte samples and renal biopsy tissues from a prospective cohort of LN patients were used to determine mRNA and protein expression levels of MRP-8 and -14. The mRNA levels of MRP-8 and -14 in total blood leukocytes were significantly higher in active LN patients than quiescent LN patients and healthy controls. Moreover, the mRNA levels of MRP-8 and -14 in the total blood leukocytes and kidney tissues were significantly correlated with therapeutic response and the mRNA expression levels in the kidney were associated with an early loss of the kidney function. MRP-8 and -14 can be used as non-invasive prognostic biomarkers in patients with LN.

Gene polymorphisms of interleukin 28B and the risk to chronic hepatitis B virus infection in Thai.

In this study, we aimed to evaluate the effect of two single nucleotide polymorphisms (SNPs) of interleukin 28B (IL28B) (rs12979860C/T and rs8099917G/T) on chronic hepatitis B virus (CHB) infection in Thai population. We studied 375 subjects: 83 CHB with hepatocellular carcinoma (HCC) patients, 128 CHB without HCC and 164 individuals with self-limited HBV infection, by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan allelic discrimination assay. Results revealed significant risk of IL28B rs8099917T allele associated with CHB without HCC compared with self-limited HBV [odds ratio (OR) (95% confidence interval (CI)) = 2.56 (1.08-6.28), P = 0.019]. The effect of this T allele was similar to that of an autosomal recessive gene in the presence of TT genotype compared with GG and GT genotype [OR (95% CI) = 2.70 (1.11-6.77), P = 0.016, P (logistic regression) = 0.048]. The two locus haplotype analysis of the two IL28B SNP loci did not show any association with CHB (P > 0.05). In conclusion, these results suggested a IL28B rs8099917T allele predispose for susceptibility to chronic HBV infection but not leading to HCC in Thai population.

Association of IFNAR2 and IL10RB genes in chronic hepatitis B virus infection.

In this study, we investigated the effects of two functional polymorphisms, type I interferon receptor 2 gene (IFNAR2)-F8S and interleukin-10 receptor subunit beta gene (IL10RB)-K47E, on chronic hepatitis B virus (HBV) infection. We included 227 Thai patients with chronic HBV infection [100 with hepatocellular carcinoma (HCC) and 127 non-HCC], 170 individuals with self-limited HBV infection and 150 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyze these two single nucleotide polymorphisms (SNPs). In this study, the C allele of IFNAR2-F8S was found to be significantly increased in chronic HBV patients when compared with healthy controls [odds ratio, OR (95% confidence interval, CI)= 3.31 (2.11-5.21), P = 6.214 × 10(-9) and corrected P-value, P(c)= 1.864 × 10(-8)]. The effect of this allele was similar to that of an autosomal dominant gene in the presence of CC and CT genotype, when compared to TT with an OR of 4.02 (P = 4.631 × 10(-9) and P(c)= 1.389 × 10(-8)). Furthermore, AA genotype of IL10RB-K47E was found to be significantly decreased in chronic HBV patients compared with individuals with self-limited HBV infection (P = 0.006, P(c)= 0.018 and OR = 0.45). For haplotype analysis, we found CA and CG haplotypes were associated with susceptibility to chronic HBV (P = 0.014, OR = 6.84 and P = 0.002, OR = 3.75, respectively) when compared with healthy individuals. This study suggests that IFNAR2-F8S polymorphisms might be involved in the susceptibility to chronic HBV infection. Moreover, AA genotype of IL10RB-K47E may provide a protective effect in this disease. However, an association study using a larger sample size should be performed to confirm these findings.

Association of interferon-alpha gene polymorphisms with chronic hepatitis B virus infection.

In this study, the association between the risk of chronic hepatitis B virus infection and the polymorphisms within promoter regions of IFN-α1 and five genes was explored. This association study was performed on 180 Thai patients with chronic HBV infection [hepatocellular carcinoma (HCC) = 65 and non-HCC = 115], 173 individuals with self-limited HBV infection and 140 healthy controls. Our results showed that the A allele of -1823G/A SNP within IFNA1 gene was significantly associated with an increased risk of chronic HBV infection as compared to healthy individuals and self-limited HBV group [OR (95% CI) = 2.20 (1.51-3.19), P = 0.000014 and OR (95% CI) = 1.61 (1.12-2.33), P = 0.0073, respectively]. The effect of A allele was similar to autosomal recessive in which the presence of AA genotype when compared to GG and GA conferred the OR of 2.79 (95% CI = 1.72-4.52, P = 0.0000085). By multifactor dimensionality reduction analysis, we found the interaction between IFNA5 (-2529) and IFNA1 (-1823) genes that gave the risk to chronic HBV infection, with the OR (95% CI) of the high-risk to low-risk group was 2.79 (1.77-4.40), P < 0.0001. However, further study in functional significance is required.

A nested sequence-specific primer-polymerase chain reaction for the detection of HLA-B*15:02.

In this study, we reported a new technique in detecting HLA-B*15:02 by using a nested sequence-specific primer-polymerase chain reaction (SSP-PCR) that can be used on genomic DNA and whole blood for carbamazepine hypersensitivity prediction. We tested a total of 200 blind samples with known human leukocyte antigen (HLA)-B allelic types (44 positive for HLA-B*15:02 and 156 negative for HLA-B*15:02) with this new nested SSP-PCR technique and compared its efficacy to that of commercial sequence-specific oligonucleotide probe-polymerase chain reaction (SSOP-PCR). Using starting materials from DNA and whole blood, we were able to detect HLA-B*15:02 in 44 of our samples correctly. The test is very sensitive and is highly reproducible.

Association of CD247 with systemic lupus erythematosus in Asian populations.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with complex genetic inheritance. CD247 (CD3Z, TCRZ) plays a vital role in antigen recognition and signal transduction in antigen-specific immune responses, and is known to be involved in SLE pathogenesis. Weak disease association was reported for genetic variants in this gene in Caucasian studies for SLE, Crohn's disease and systemic sclerosis, but its role as a genetic risk factor was never firmly established. METHODS: In this study, using a collection of 612 SLE patients and 2193 controls of Chinese ethnicity living in Hong Kong in a genome-wide study, single nucleotide polymorphisms (SNPs) in and around CD247 were identified as being associated with SLE. The two most significant SNPs in this locus were selected for further replication using TaqMan genotyping assay in 3339 Asian patients from Hong Kong, Mainland China, and Thailand, as well as 4737 ethnically and geographically matched controls. RESULTS: The association of CD247 with SLE in Asian populations was confirmed (rs704853: odds ratio [OR] = 0. 81, p = 2.47 × 10(-7); rs858543: OR = 1.10, p = 0.0048). Patient-only analysis suggested that rs704853 is also linked to oral ulcers, hematologic disorders and anti-double-stranded DNA (dsDNA) antibody production. CONCLUSION: A significant association between variants in CD247 and SLE was demonstrated in Asian populations. Understanding the involvement of CD247 in SLE may shed new light on disease mechanisms and development of new treatment paradigms.

Distribution of cytokine gene polymorphisms in Thai population.

The distribution of 21 cytokine polymorphisms within 13 cytokine and cytokine receptor genes was analyzed in 102 healthy Thai individuals using the LIFECODES Cytokine SNP Typing kit. The TGFB codon25 marker is monomorphic in the Thai population. The IL1B+3962, IL6-174, and TNFA-238 are very rare polymorphisms, with only 0.01-0.04 minor allele frequency (MAF). The IL4-1098, IL1A-889, and IL10-1082 are found only 0.06-0.08 in Thai. Other cytokine polymorphisms (IL1B-511, IL1R pst1 1970, IL1RN mspa1 11100, IL4RA+1902, IL12B-1188, IFNG+874, TGFB codon10, TNFA-308, IL2-330, IL2+166, IL4-590, IL4-33, IL10-819, and IL10-592) in Thai have MAFs more than 0.10, ranging between 0.13 and 0.47. When comparing the allele and genotype frequencies with public single nucleotide polymorphism (SNP) database, most cytokine polymorphisms in Thai show similar distribution to Han Chinese and Japanese, but significantly different from Caucasian and African populations. Only a few markers, including IL4A+1902, TNFA-308, IL1B+3962, and IL2+166 show statistically different distribution among Thai and other Asian populations especially with the Japanese.

Tumour necrosis factor-alpha gene polymorphisms and susceptibility to oral lichen planus.

OBJECTIVE: This study is aimed to investigate the association between OLP susceptibility and clinical type in the Thai population and three polymorphisms within the promoter region of the TNF-α at positions -863, -308 and -238 which have putative functional significances. MATERIALS AND METHODS: Genomic DNA from 75 Thai patients with OLP and 154 healthy controls were genotyped for TNF-α polymorphisms-- -863(rs1800630), -308(rs1800629), and -238(rs361525)--using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: We found a higher proportion of TNF-alpha-308 AA genotype (high producer genotype) among OLP patients (5/75; 6.67%) when compared to healthy controls (1/154; 0.65%; OR = 10.93; 95% CI = 1.21-251.9). For other polymorphisms (-863 and -238), we did not find any significant association with OLP development; this was also the case with haplotype analysis (-863/-308/-238). CONCLUSION: TNF-α-308AA may play a relevant role in the susceptibility and severity of OLP in the Thai population. However, further investigation of this study is needed.

The association of single nucleotide polymorphism within vascular endothelial growth factor gene with systemic lupus erythematosus and lupus nephritis.

There were no statistically significant difference in allele and genotype frequency of the polymorphisms within the vascular endothelial growth factor (VEGF) gene (-460 and +405) between 193 systemic lupus erythematosus patients and 234 healthy controls. However, the +405 GG was significantly associated with lupus nephritis (LN) patients with low VEGF mRNA expression and LN with end-stage renal disease.

Major lupus organ involvement: severe lupus nephritis.

Lupus nephritis is a common and severe complication of systemic lupus erythematosus. A number of patients have nephritis as a presenting feature that, in its severe form, can shortly lead to end-stage renal disease and/or death. Renal flare usually occurs a few years after the first episode and is remarkably predominant in the Asian population. Frequent monitoring for renal flare enhances early recognition and timely treatment. The mainstay therapy continues to be the prolonged use of cytotoxic/immunosuppressive drugs that have a number of undesirable effects, particularly ovarian failure and development of opportunistic infections. This review will focus on the pathogenesis and the unique genetic factors found in Asian patients with lupus nephritis. Here, we propose an appropriate management scheme for the treatment of lupus nephritis in Asian patients.

Expression profile of HIN200 in leukocytes and renal biopsy of SLE patients by real-time RT-PCR.

HIN200 is a human IFN-inducible gene and homologous to murine IFI202 gene, which was identified as a candidate gene for SLE susceptibility in lupus mouse model. We determined these gene expressions in leukocytes from 20 SLE patients and 10 healthy controls and in renal biopsies from 29 SLE patients and 15 kidney donors using sensitive real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The expressions of MNDA, IFIX, IFI16 and AIM2 genes significantly increased in leukocytes but not in kidney biopsies from SLE patients as compared to the control individuals, with P = 0.0003, P = 0.0056, P = 0.0002 and P < 0.0001, respectively. We also assessed the expression profiles of IFIX and IFI16 isoforms using semi-quantitative RT-PCR. We found up-regulation of B isoform (short product) of IFI16 in SLE patients. In addition, the expression levels were analyzed in correlation with disease activity and clinical characteristics. Interestingly, higher expression of MNDA was observed in patients who were positive for anti-dsDNA antibodies than in patients who were negative (P = 0.0276). In conclusion, it is suggested that the HIN200 genes have a role in SLE pathogenesis. Our study also observed a possible important role of a specific short isoform of IFI16 as well as a link between MNDA and anti-dsDNA antibody production.

Population differences in SLE susceptibility genes: STAT4 and BLK, but not PXK, are associated with systemic lupus erythematosus in Hong Kong Chinese.

In this study, we compared the association of several newly discovered susceptibility genes for systemic lupus erythematosus (SLE) between populations of European origin and two Asian populations. Using 910 SLE patients and 1440 healthy controls from Chinese living in Hong Kong, and 278 SLE patients and 383 controls in Thailand, we studied association of STAT4, BLK and PXK with the disease. Our data confirmed association of STAT4 (rs7574865, odds ratio (OR) =1.71, P=3.55 x 10(-23)) and BLK (rs13277113, OR=0.77, P=1.34 x 10(-5)) with SLE. It was showed that rs7574865 of STAT4 is also linked to hematologic disorders and potentially some other subphenotypes of the disease. More than one genetic variant in STAT4 were found to be associated with the disease independently in our populations (rs7601754, OR=0.59, P=1.39 x 10(-9), and P=0.00034 when controlling the effect of rs7574865). With the same set of samples, however, our study did not detect any significant disease association for PXK, a risk factor for populations of European origin (rs6445975, joint P=0.36, OR=1.06, 95% confidence interval: 0.93-1.21). Our study indicates that some of the susceptibility genes for this disease may be population specific.

Genetic association of interferon-alpha subtypes 1, 2 and 5 in systemic lupus erythematosus.

In this study, the association between the systemic lupus erythematosus (SLE) susceptibility and the new candidate genes, IFNA1, IFNA2 and IFNA5 genes, major interferon-alpha subtypes, in responses to viral infection was investigated. Allele and genotype frequencies of each marker were compared between 150 SLE patients and 150 healthy control subjects. This study indicated that the A/A genotype of IFNA5 (-2529) and the G/G genotype of IFNA1 (-1823) were associated with the protection of SLE disease in a recessive model [P(c) = 0.03, P = 0.01, odds ratio (OR) = 0.4, 95% confidence interval (CI) = 0.2-0.8 and P(c) = 0.09, P = 0.03, OR = 0.5, 95% CI = 0.2-0.9, respectively). Multifactor dimensionality reduction analysis showed a marginal interaction between IFNA5 (-2529) and IFNA1 (-1823) gene, with a cross-validation consistency of 10 of 10 and a prediction error of 46% (permutation P-value = 0.05). This is the first report of positive association of IFNA gene in SLE, especially the role of specific subtypes IFNA1 and IFNA5.

Association of the CTG (-2578/-460/+405) haplotype within the vascular endothelial growth factor gene with early-onset psoriasis.

Polymorphism of vascular endothelial growth factor (VEGF) influences the VEGF production and is subjected to genetic susceptibility of many diseases including psoriasis. Three single nucleotide polymorphisms (SNPs) within the promoter and exon 1 region [-2578(C/A), -460(C/T) and +405(C/G)] were analyzed in 154 patients with chronic plaque psoriasis and in 234 ethnically matched healthy controls from Thailand. The CTG (-2578/-460/+405) haplotype frequency was higher in patients with early-onset psoriasis (44.12%) compared with healthy controls (33.33%) (odds ratio = 1.54, 95% confidence interval = 1.08-2.18, P = 0.016, corrected P value: P(c) = 0.048). The results suggest that the CTG haplotype can be used as a genetic marker for psoriasis especially the early-onset group of Thais.

Defects in Notch1 upregulation upon activation of T Cells from patients with systemic lupus erythematosus are related to lupus disease activity.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of autoantibodies and deposition of immune complexes in various organs. T cells play a central role in driving disease progression, and multiple defects in T cells from patients with SLE have been uncovered. Notch signalling is an evolutionarily well-conserved signalling cascade involved in the proliferation, differentiation and apoptosis of T lymphocytes during development and peripheral effector functions. In this study, we investigated the correlation between expression of Notch receptor and the severity of SLE disease. On the contrary to T lymphocytes from healthy controls (n=11), Tlymphocytes from patients with active SLE (n=12) failed to upregulate Notch1 upon in-vitro stimulation as quantified by quantitative real time RT-PCR (P

Azathioprine-induced fatal myelosuppression in systemic lupus erythematosus patient carrying TPMT*3C polymorphism.

Azathioprine (AZA) is a commonly used immunosuppressant for systemic lupus erythematosus (SLE). Myelosuppression is a serious adverse reaction due to AZA and its metabolites. Thiopurine S-methyltransferase (TPMT) is the rate-limiting enzyme. Variations of TPMT enzyme activity may be responsible for myelosuppression. However, a correlation between certain mutant alleles of low TPMT enzyme activity and myelotoxicity has also been suggested as a factor. We describe herein a case of AZA-induced severe myelosuppression associated with TPMT*3C heterozygous mutant allele in a SLE patient. The patient presented with pancytopenia, sepsis, typhlitis and disseminated intravascular coagulopathy after a short period of AZA therapy. The patient had low TPMT activity and TPMT*3C genotype. Measurement of TPMT activity and determination of TPMT variant allele may identify patients at risk for AZA-induced myelosuppression.