Pi-Yueh Chang

Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers

BACKGROUND 8-hydroxydeoxyguanosine (8-OHdG) is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Urinary 8-OHdG is now considered as a biomarker of generalized, cellular oxidative stress and is linked to degenerative diseases including cancer. METHODS We developed a competitive enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG by coating BSA conjugated 8-hydroxyguanine (8-OHG) on a microplate. Urine specimens containing 8-OHdG and monoclonal anti-8-OHdG antibody were incubated together in the microwell. Final quantification of bound anti-8-OHdG antibody was estimated by the addition of HRP-conjugated sheep-anti-mouse antibody. RESULTS The concentration range of the calibration curve was 0-60 ng/ml. The sensitivity of the assay was 0.5 ng/ml. The within-day precision and day-to-day precision were <10%. The ELISA correlated well with a commercial kit (r=0.9). Our assay measured not only 8-OHdG but also 8-OHG and 8-hyroxyguanine in urine. Increased urinary concentration of 8-OHdG and its analogs were detected in both patients with bladder cancer (70.5+/-38.2 ng/mg creatinine) and prostate cancer (58.8+/-43.4 ng/mg creatinine) as compared to the healthy control (36.1+/-24.5 ng/mg creatinine). CONCLUSION Our preliminary data suggest that the competitive ELISA for 8-OHdG and its analogs appears to be a simple method for quantifying the extent of oxidative stress and may have potential for identifying cancer risk.

HLA-A*31:01 and different types of carbamazepine-induced severe cutaneous adverse reactions: an international study and meta-analysis

HLA-A*31:01 was reported to be associated with carbamazepine (CBZ)-induced severe cutaneous adverse reactions (SCAR), including drug reaction with eosinophilia and systemic symptoms (DRESS), Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). We conducted an international study using consensus diagnosis criteria to enroll a total of 93 patients with CBZ-SCAR from Europe or Asia. We found that HLA-A*31:01 showed a significant association with CBZ-DRESS in Europeans (P<0.001; odds ratio (OR) (95% confidence interval (CI))=57.6 (11.0–340)), and the strong association was also found in Chinese (P<0.001; OR (95% CI)=23.0 (4.2–125)). However, HLA-A*31:01 had no association with CBZ-SJS/TEN in neither Chinese nor Europeans. By comparison, HLA-B*15:02 showed a strong association with CBZ-SJS/TEN in Chinese (P<0.001, OR (95% CI)=58.1 (17.6–192)). A meta-analysis of this and other published studies confirmed that in all populations, HLA-A*31:01 had an extremely strong association with CBZ-DRESS (P<0.001, a pooled OR (95% CI)=13.2 (8.4–20.8)), but a much weaker association with CBZ-SJS/TEN (P=0.01, OR (95% CI)=3.94 (1.4–11.5)). Our data revealed that HLA-A*31:01 is a specific predictor for CBZ-DRESS but not for CBZ-SJS/TEN. More studies are needed to investigate the genetic determinant of CBZ-SJS/TEN in Europeans. Considering the potential clinical utility, the cost-effectiveness of the combined HLA-A*31:01 and HLA-B*15:02 genetic test to prevent CBZ-SCAR in Chinese needs further investigation.

Genotype-phenotype association between HLA and carbamazepine-induced hypersensitivity reactions: strength and clinical correlations.

BACKGROUND Increasing studies reported genetic susceptibility to drug hypersensitivity reactions, as exemplified by the HLA-A*31:01 and HLA-B*15:02 association with carbamazepine (CBZ)-induced hypersensitivity reactions, such as maculopapular exanthema (MPE), drug rash with eosinophilia and systemic symptoms (DRESS), and Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN). OBJECTIVE To carry out a comprehensive analysis on the clinical spectrum and HLA genotype-phenotype correlations in CBZ-induced hypersensitivity reactions. METHODS We analyzed the clinical information of 194 patients with CBZ hypersensitivity (51 MPE, 23 DRESS, 112 SJS/TEN, and 8 cases with other phenotypes), and 152 CBZ-tolerant controls. All are Han Chinese. We examined the HLA-A/HLA-B genotypes, gene dosage, and drug dosage effects. RESULTS CBZ-SJS/TEN showed the strongest association with the HLA-B*15:02 allele (Pc=5.8×10(-43); odds ratio (OR) (95% CI)=97.6(42.0-226.8)), in which HLA-B*15:02 was identified in all patients (25/25) with SJS/TEN with >5% body surface area (BSA) skin detachment, but lost its 100% association (85.1%, 74/87) in SJS with <5% BSA detachment. In contrast, HLA-B*40:01 showed negative association with CBZ-induced SJS/TEN ((Pc=8.3×10(-5); OR (95% CI)=0.22(0.1-0.4)). By comparison, CBZ-induced MPE/DRESS had no association with HLA-B*15:02, but linked to HLA-A*31:01 (Pc=2.7×10(-3); OR (95% CI)=6.86(2.4-19.9), and HLA-B*51:01 (Pc=0.01; OR (95% CI)=4.56(2.0-10.5)). No gene dosage or CBZ dosage effects was observed. CONCLUSION This study reported the different strength of HLA association with CBZ hypersensitivity in Han Chinese. With the increasing application of pharmacogenetic markers, the HLA genotype-phenotype correlations and the results of the test need to be carefully interpreted for CBZ-induced hypersensitivity reactions.

Risk and association of HLA with oxcarbazepine-induced cutaneous adverse reactions in Asians

Objective: To investigate the risk and genetic association of oxcarbazepine-induced cutaneous adverse reactions (OXC-cADRs), including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), in Asian populations (Chinese and Thai). Methods: We prospectively enrolled patients with OXC-cADRs in Taiwan and Thailand from 2006 to 2014, and analyzed the clinical course, latent period, drug dosage, organ involvement, complications, and mortality. We also investigated the carrier rate of HLA-B*15:02 and HLA-A*31:01 of patients with OXC-cADRs and compared to OXC-tolerant controls. The incidence of OXC-SJS/TEN was compared with carbamazepine (CBZ)–induced SJS/TEN according to the nationwide population dataset from the Taiwan National Health Insurance Research Database. Results: We enrolled 50 patients with OXC-cADRs, including 20 OXC-SJS/TEN and 6  drug reaction with eosinophilia and systemic symptoms, of Chinese patients from Taiwan and Thai patients from Thailand. OXC-cADRs presented with less clinical severity including limited skin detachment (all ≦5%) and no mortality. There was a significant association between HLA-B*15:02 and OXC-SJS (p = 1.87 × 10−10; odds ratio 27.90; 95% confidence interval [CI] 7.84–99.23) in Chinese and this significant association was also observed in Thai patients. The positive and negative predictive values of HLA-B*15:02 for OXC-SJS/TEN were 0.73% and 99.97%, respectively. HLA-A*31:01 was not associated with OXC-cADRs. The incidence and mortality of OXC-SJS/TEN was lower than CBZ-STS/TEN in new users (p = 0.003; relative risk 0.212; 95% CI 0.077–0.584). Conclusions: Our findings suggest that HLA-B*15:02 is significantly associated with OXC-SJS in Asian populations (Chinese and Thai). However, the severity and incidence of OXC-SJS/TEN are less than that of CBZ-SJS/TEN. The need for preemptive HLA-B*15:02 screening should be evaluated further.

MicroRNA-223 and microRNA-92a in stool and plasma samples act as complementary biomarkers to increase colorectal cancer detection

Aberrant levels of circulating miRNAs are potential biomarkers for the early detection of colorectal cancer (CRC). However, no previous systematic study has examined miRNAs in various specimen types from the same patient to evaluate their clinical utility. In this study, we compiled information from ∼450 articles published before 2012, and selected the 46 most frequently reported CRC-related miRNAs as candidates. We then established a 46-miRNA multiplex RT-qPCR method, and efficiently examined two clinically accessible samples: stool from fecal occult blood test and EDTA plasma. A total of 62 tissue, 447 stool, and 398 plasma samples were collected from CRC patients and healthy controls. Good correlations of detectable miRNAs were noticed in paired tumor tissues, stool, and plasma samples of 62 CRC patients. Using these 62 CRC patients and 62 matched healthy controls as the training set, 5 and 11 differentially expressed miRNAs achieved the area under the ROC curve (AUC) greater than 0.7 in stool and plasma samples, respectively. The selected miRNAs was subsequently validated using the remaining enrolled samples as the test cohort; 4 miRNAs in stool and 6 miRNAs in plasma were maintained discriminating powers for CRC patients. After examining the complementary effect, combined analysis of miR-223 and miR-92a, which were commonly present in stool and plasma samples, yielded the highest sensitivity of 96.8% and the specificity of 75% for CRC (AUC = 0.907). These results allowed us to establish a two-miRNA biosignature in two types of CRC clinical specimens with a high sensitivity for CRC detection.

Association of soluble intercellular adhesion molecule–1 with insulin resistance and metabolic syndrome in Taiwanese

Circulating concentrations of soluble cell adhesive molecules are useful predictors for the risk of development and progression of atherosclerosis. This study was initiated to investigate the association between soluble intercellular adhesive molecule-1 (sICAM-1) levels and traditional and emerging cardiovascular risk factors, as well as insulin resistance and metabolic syndrome, in a Taiwanese population. Six hundred nine unrelated individuals recruited during routine health examinations were enrolled for the analysis. In age- and sex-adjusted regression models, sICAM-1 levels were negatively associated with high-density lipoprotein cholesterol levels and positively associated with systolic, mean, and diastolic blood pressure; body mass index; waist circumference; waist-hip ratio; the homeostasis model assessment index; fasting serum insulin; triglyceride; and C-reactive protein levels. The sICAM-1 levels were also higher in subjects with current smoking (P = .001), diabetes mellitus (P = .004), insulin resistance (P < .001), and metabolic syndrome (P < .001). The sICAM-1 levels increased in a stepwise fashion with increasing Framingham risk score quartiles (P = .001) and with increasing number of metabolic syndrome components (P < .001). In subjects with metabolic syndrome, increased C-reactive protein levels were associated with increased sICAM-1 levels (P = .003). In stepwise linear regression models, sICAM-1 levels remained associated with current smoking, insulin resistance, and metabolic syndrome. In conclusion, our data revealed that insulin resistance and metabolic syndrome were associated with sICAM-1 levels in Taiwanese. These data provide further evidence of the mechanisms of sICAM-1 as a molecular marker for atherosclerosis.

Effect of obesity on the association between common variations in the HNF1A gene region and C-reactive protein level in Taiwanese.

BACKGROUND The level of C-reactive protein (CRP), an inflammatory biomarker that predicts future cardiovascular events, is a heritable trait that has been associated with variants of CRP and hepatic nuclear factor-1α (HNF1A) genes. Our aim was to test the statistical association between HNF1A genotypes/haplotypes and serum CRP level in Taiwanese. METHODS A sample population of 617 Taiwanese subjects (all Han-Chinese origin) was enrolled. Five HNF1A single nucleotide polymorphisms (SNPs) rs1920792, rs1169288, rs7310409, rs2464196, rs1169310 were genotyped and analyzed. RESULTS After adjusting for clinical covariates, minor alleles of all the 5 study SNPs were associated with decreased CRP level (P=0.0078, P=0.0107, P=0.0006, P=0.0004 and P=0.0003, respectively). A common haplotype (TGATA) tagged by the minor alleles of study SNPs was associated with significantly decreased CRP level (P=0.0112). Subgroup analysis revealed that the association between HNF1A genotypes and CRP level occurred only in non-obese subjects. CONCLUSIONS HNF1A polymorphisms are independently associated with CRP level in Taiwanese. Further, HNF1A genotypes interact with obesity to set CRP level, revealing that genetic determinants for CRP level may be different between obese and non-obese individuals.

Association of matrix metalloproteinase 9 genotypes and cardiovascular disease risk factors with serum matrix metalloproteinase 9 concentrations in Taiwanese individuals.

Abstract Background: Circulating concentrations of matrix metalloproteinase 9 (MMP-9) are associated with cardiovascular disease mortality in patients with coronary artery disease. We investigated the determinants of MMP-9 concentrations by analyzing MMP-9 genotypes and risk factors for cardiovascular disease. Methods: A total of 596 individuals were recruited for this study. Six single nucleotide polymorphisms (SNP) with coverage of the MMP-9 gene region were analyzed; and two genotypes, rs3918242-TT and rs2274756-AA, which were in nearly complete linkage disequilibrium, were associated with higher MMP-9 values (p=0.007 and p=0.008, respectively). In age- and gender-adjusted regression models, MMP-9 concentrations were positively associated with the homeostasis model assessment of insulin resistance (HOMA-IR) index and triglyceride concentrations, fasting serum insulin, fasting plasma glucose, C-reactive protein, fibrinogen, and serum amyloid A. By multivariate analysis, rs2274756 genotypes, age, smoking status, fibrinogen and fasting plasma glucose concentrations were all independently associated with MMP-9 (p=0.007, p=0.033, p=0.003, p=0.013, and p=0.012, respectively). Conclusions: The rs2274756-AA and rs3918242-TT genotypes, younger age, current smoking status and increased fasting plasma glucose, and fibrinogen concentrations were independently associated with high serum MMP-9 concentrations in Taiwanese individuals. Clin Chem Lab Med 2010;48:543–9.

Association between functional variants of the ICAM1 and CRP genes and metabolic syndrome in Taiwanese subjects

Although inflammation has been shown to play an important role in metabolic syndrome (MetS), the association between inflammatory marker gene polymorphisms and the risk of MetS has not been fully elucidated. This study was initiated to investigate the association between functional variants of inflammatory marker genes and the risk of MetS in Taiwanese adults. The sample population comprised 615 unrelated subjects, of which 22% had MetS. The single nucleotide polymorphisms rs5491 on the intercellular adhesive molecule 1 (ICAM1) gene and rs3091244 on C-reactive protein (CRP) were genotyped. The ICAM1 rs5491 polymorphism was significantly associated with the level of soluble intercellular adhesive molecule 1 (P < .001). Both the ICAM1 rs5491 and the CRP rs3091244 were shown to have significant association with MetS after adjustment for age, sex, smoking, and body mass index, but not after adjustment for levels of the respective serum marker. Independent associations between the combined ICAM1-CRP (rs5491 and rs3091244) genotypes and MetS were found by multivariate analysis (P = .005). In subgroup analysis, association of combined genotypes with insulin resistance and MetS mainly occurred in subjects with central obesity. In conclusion, inflammatory marker gene polymorphisms play an important role in modulating the risk of insulin resistance and MetS for subjects with central obesity. These findings will contribute toward a better understanding of the mechanism of association between inflammatory markers and the risk of developing atherosclerotic disease.

Development of multiple complications in type 2 diabetes is associated with the increase of multiple markers of chronic inflammation.

Patients with type 2 diabetes (T2DM) are known at risk for developing cardiovascular disease (CVD), nephropathy, and cancer. We were interested to find out whether multiple markers associated with chronic inflammation are detectable in patients with T2DM and are increased in patients with T2DM who developed additional clinical complications. A sequence of multiple risk markers for atherogenesis, associated with chronic inflammation, was measured in patients with T2DM before and after the development of clinical complications. We found that multiple clinical complications frequently developed simultaneously in patients with T2DM. At the early stage of T2DM, only low levels and low percent elevations of multiple risk markers were detected. However, both the level and the percent elevation of these markers were found to increase with disease progression and the development of clinical complications. We believe that chronic inflammation not only contributes to the pathogenesis of T2DM but also continues to increase in T2DM patients who are developing additional clinical complications. It appears that these multiple markers are potentially useful not only for monitoring the progression of T2DM but also predicting the risk of developing macro‐ and microvascular disease, nephropathy, and cancer. J. Clin. Lab. Anal. 22:6–13, 2008.