Jau-Tsuen Kao

Regression-Based Association Analysis with Clustered Haplotypes through Use of Genotypes

Haplotype-based association analysis has been recognized as a tool with high resolution and potentially great power for identifying modest etiological effects of genes. However, in practice, its efficacy has not been as successfully reproduced as expected in theory. One primary cause is that such analysis tends to require a large number of parameters to capture the abundant haplotype varieties, and many of those are expended on rare haplotypes for which studies would have insufficient power to detect association even if it existed. To concentrate statistical power on more-relevant inferences, in this study, we developed a regression-based approach using clustered haplotypes to assess haplotype-phenotype association. Specifically, we generalized the probabilistic clustering methods of Tzeng to the generalized linear model (GLM) framework established by Schaid et al. The proposed method uses unphased genotypes and incorporates both phase uncertainty and clustering uncertainty. Its GLM framework allows adjustment of covariates and can model qualitative and quantitative traits. It can also evaluate the overall haplotype association or the individual haplotype effects. We applied the proposed approach to study the association between hypertriglyceridemia and the apolipoprotein A5 gene. Through simulation studies, we assessed the performance of the proposed approach and demonstrate its validity and power in testing for haplotype-trait association.

The graded effect of hyperhomocysteinemia on the severity and extent of coronary atherosclerosis

It is not clear to what extent methylenetetrahydrofolate reductase (MTHFR) gene and hyperhomocysteinemia effect the severity and extent of coronary atherosclerosis in Asian populations. We examined the MTHFR genotypes and plasma homocysteine (HCY) concentrations in 192 Taiwanese and investigated their relationship with coronary artery disease (CAD), and the severity and extent of coronary atherosclerosis. The distribution of MTHFR genotypes was similar in 116 CAD patients and 76 non-CAD subjects. Homozygosity was noted in 8% of CAD patients and 13% of non-CAD subjects (P=0.33; 95% CI, 0. 2-1.6). The geometric mean of HCY values was higher in CAD patients (11.10+/-1.51 micromol/l) than in non-CAD subjects (9.21+/-1.55 micromol/l) (P=0.003). HCY levels were higher in patients with multi-vessel disease (P<0.05) or in patients with > or = 90% stenotic lesions (P=0.005), compared with non-CAD subjects. The CAD risks in the top two HCY quartiles (> or = 14.0 and 10.1-13.9 micromol/l) were 4.0 (95% CI, 1.7-9.2) and 3.2 (95% CI, 1.4-7.4) times higher than in the lowest quartile (< or = 7.9 micromol/l) (P=0.001 and 0.007, respectively). Linear regression analysis showed significant correlations between HCY concentrations and the severity and extent of atherosclerosis (P=0.0001 for both). In conclusion, hyperhomocysteinemia appears to have a graded effect on the risk of CAD as well as the severity and extent of coronary atherosclerosis. Our findings do not support the homozygous genotype of MTHFR as a genetic risk factor for CAD in this Taiwanese population. Perhaps a further study including assessment of vitamin status is needed to better clarify the relationship between MTHFR genotypes and CAD.

The effects of apolipoprotein E polymorphism on the distribution of lipids and lipoproteins in the Chinese population

The effects of apolipoprotein E (apo E) polymorphism on the distribution of serum levels of total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were studied in 546 unrelated Chinese in Taiwan. By isoelectric focusing and immunoblotting, three common alleles were demonstrated. The frequencies of the epsilon 2, epsilon 3 and epsilon 4 alleles were 0.076, 0.875 and 0.049, respectively. The relative frequency of the epsilon 3 allele was higher than that in Caucasians, whereas the frequency of epsilon 4 was lower in the Chinese population. Individuals with apo E4/4 phenotype had the highest serum TC and LDL-C concentrations, while the lowest levels were associated with apo E2/2 phenotype. There was a trend for individuals carrying the epsilon 2 allele to have a higher HDL-C level. No relationship between apo E phenotype and TG level was observed in this study. These data indicated that a given apo E allele acted in a relatively similar way in determining individual differences between Chinese and Caucasian populations in serum lipid and lipoprotein.

APOA1/C3/A5 haplotype and risk of hypertriglyceridemia in Taiwanese

BACKGROUND Apolipoprotein A5 gene (APOA5) has been shown to modulate plasma triglyceride concentrations. We investigated 2 distinct APOA1/C3/A5 haplotypes roles for hypertriglyceridemia. METHODS We recruited 308 cases of hypertriglyceridemia and 281 normal controls from a hospital. Twelve single nucleotide polymorphisms (SNPs) across the APOA1/C3/A5 gene region were genotyped. RESULTS One haplotype containing the minor alleles of the APOA5 (-1131T>C, c.553G>T) and APOA1 (-3013C>T,-75G>A) was more prevalent in cases than in controls (11.3% vs. 1.1%, respectively) and was statistically significantly associated with high triglycerides (adjusted odds ratio: 12.83, 95% confidence interval [CI]: 5.1-32.4, P<0.001). Another haplotype that was associated with hypertriglyceridemia (adjusted odds ratio 2.13, 95% CI, 1.37-3.29, P=0.001). Participants carrying both minor alleles of APOA5-1131CC and c.553TT had a 116% higher triglyceride concentration compared with those carrying common allele. CONCLUSIONS The APOA1/C3/A5 haplotype represents an important locus for predicting risk of hypertriglyceridemia among Taiwanese.

Characterization of apolipoprotein E genetic variations in Taiwanese: association with coronary heart disease and plasma lipid levels.

Apolipoprotein E (apoE, protein; APOE, gene) is important in lipoprotein metabolism. Three isoforms, apoE2 (Cys112 Cys158), apoE3 (Cys112 Arg158), and apoE4 (Arg112 Arg158), are present in the general population. This report investigates the frequency distribution of apoE isoforms and the association of APOE genotypes with plasma lipid profile and coronary heart disease (CHD) in a population of Taiwan. ApoE isoforms were determined genetically by polymerase chain reaction and HhaI restriction enzyme digestion in control and coronary heart disease (CHD) patients. Plasma lipid and lipoprotein concentrations were also determined. The control group exhibited frequencies of 84.6% APOE3, 7.9% APOE4, 7.5% APOE2, 70.6% APOE3E3, 14.4% APOE3E4, 13.6% APOE2E3, and 1.4% APOE2E4. Comparable frequencies were observed in the CHD group. In both APOE2 carrier and APOE3E3 groups, the CHD patients expressed abnormal lipid profiles while the control group expressed normal lipid profiles. The APOE4 carriers, however, expressed abnormal lipid profiles in both normal control and CHD groups. Extremely high apoE levels in the hypertriglyceridemic group (TG > 400 mg/dL) seemed to be undesirable and were often observed in CHD patients

Serum Lipids and Lipoprotein(a) Concentrations in Chinese NIDDM Patients: Relation to metabolic control

OBJECTIVE To compare serum blood lipids and lipoprotein(a) [Lp(a)] levels in Chinese non-insulin-dependent diabetes mellitus (NIDDM) patients and nondiabetic control subjects and also to determine the influence of diabetes control on serum Lp(a) concentration in Chinese individuals. RESEARCH DESIGN AND METHODS We compared the serum blood lipids and Lp(a) levels in NIDDM patients (n = 100) and age- and sex-matched nondiabetic subjects (n = 100) who participated in a case-control study. Comparisons of Lp(a) concentrations were made between a normal control group, a group of diabetic patients with HbA1c < 8.0%, and a group of diabetic patients with HbA1c of 8% or higher. RESULTS The diabetic patients had higher total triglyceride, apolipoprotein B (apo B), and apo B-to-apo AI ratios, but lower high-density lipoprotein (HDL) cholesterol and apo AI concentrations than nondiabetic controls (P < 0.001, P < 0.01, P < 0.001, P < 0.05, and P < 0.001, respectively). A similar pattern of distribution of Lp(a) levels according to the degree of metabolic control was seen in patients with NIDDM and nondiabetic controls. No correlation was observed between Lp(a) levels and total cholesterol, low-density lipoprotein cholesterol, HDL cholesterol, apo AI, apo B, and triglyceride levels in all diabetic patients. No difference in the Lp(a) levels was noted between diabetic patients and nondiabetic subjects, even in poorly controlled diabetic patients. CONCLUSIONS In conclusion, Lp(a) levels are not elevated in diabetic patients, even in poorly controlled metabolic conditions.

Effects of air bubbles and tube transportation on blood oxygen tension in arterial blood gas analysis.

BACKGROUND AND PURPOSE Pneumatic tube transport has been reported to aggravate the error in partial pressure of oxygen (PO(2)) measurements caused by air bubbles. The aim of this study was to clarify the effect of manual and pneumatic tube methods of sample transportation and different amounts of air bubbles on arterial blood gas analysis. METHODS Blood gas samples from 15 patients and a pooled wasted blood mixture with 3 different levels of PO(2) were analyzed to determine the effects of air bubbles and manual versus pneumatic tube transportation on PO(2) levels. RESULTS PO(2) increased significantly in samples containing 10% air bubbles and was exaggerated by pneumatic tube transport (from 115.63 +/- 9.31 mm Hg to 180.51 +/- 11.29 mm Hg, p < 0.001). In samples with low PO(2) ( approximately 30 mm Hg), the measurement was not aberrant regardless of the method of transportation or the amount of air bubbles contained in the specimen. However, in samples with medium and high PO(2) (> 70 mm Hg), aberrances in measurements were noted even with only 0.5% air bubbles and regardless of whether the sample was transported by manual methods or pressurized tube. The increments of PO(2) correlated positively with the amount of air introduced into the specimens. Thus, the measured PO(2) increased 8.13 and 31.77 mm Hg when 0.5% and 10% air bubbles were introduced, respectively, to samples with medium PO(2) (p < 0.05). The interaction between the amount of air bubbles and the method of transportation was significant (p < 0.001). CONCLUSIONS Trapped air in the syringe should be expelled as thoroughly as possible, since the presence of only 1% air bubbles can result in aberrance in PO(2) measurement. Samples for blood gas analysis should be carried in ambient pressure to the laboratory because pneumatic tube delivery systems significantly aggravate the air bubble-related aberrance in PO(2) measurement.

Association of single-nucleotide polymorphism 3 and c.553G > T of APOA5 with hypertriglyceridemia after treatment with highly active antiretroviral therapy containing protease inhibitors in HIV-infected individuals in Taiwan.

We investigated the relationship between hypertriglyceridemia and the single-nucleotide polymorphisms (SNPs) on APOA5 in human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy (HAART) in Taiwan. Receipt of protease inhibitor-based HAART, high baseline triglyceride levels, and carriage of APOA5 SNP3 or c.553G>T variants or APOA5 SNP1T/SNP2G/SNP3C/c.553T haplotype were statistically significantly associated with development of extreme hypertriglyceridemia (triglyceride level, >500 mg/dL).

Functional importance of apolipoprotein A5 185G in the activation of lipoprotein lipase

BACKGROUND Apolipoprotein A5 (APOA5) over-expression enhances lipolysis of triglyceride (TG) through stimulation of lipoprotein lipase (LPL) activity; however, an APOA5 G185C variant was found associated with hypertriglyceridemia. The aim of this study was, therefore, to explore the importance of APOA5 185GG in the activation of LPL. METHODS A fragment containing mature human APOA5 cDNA was obtained by RT-PCR and subcloned into pET-15b vector. Site-directed mutagenesis was performed to generate 19 variants. Recombinant human APOA5 wild type and variants were produced in Escherichia coli, and then activation of LPL was measured. RESULTS Activity of APOA5 variants on LPL-mediated 1,2-dimyristoyl-sn-glycero-3-phosphocholine hydrolysis was reduced by 17 to 74% in comparison to wild type APOA5 (P<0.0001). All variants also showed reduced activation (P<0.0001) of LPL-mediated hydrolysis of very low-density lipoprotein (VLDL); activation abilities of APOA5 variants ranged from 31 to 81% of wild-type APOA5. CONCLUSIONS APOA5 residue 185G is very important in LPL-mediated VLDL hydrolysis, and any mutation at this residue will decrease LPL activation and concomitant TG modulation.

Interference caused by the contents of serum separator tubes in the Vitros CRP assay

Background: We have observed discrepancies between C-reactive protein (CRP) measured in serum prepared from a serum separator tube (SST) and that obtained from a plain tube, when using the Vitros CRP assay. Our study aimed at elucidating the cause of these discrepancies. Methods: Eighty-seven specimens from hospitalized patients with various types of inflammatory disease were analysed using a fixed-point immuno-rate method on a Vitros CRP slide. The serum was prepared simultaneously in both vacuum and SSTs. We also performed mixing tests by adding 47 samples of serum prepared from plain tubes to SSTs and incubating for 15 min before CRP analysis. Results: Lower values of CRP were found in serum prepared from plain tubes than in serum from SSTs. Addition of serum prepared from plain tubes to SSTs and incubating for 15 min increased the CRP values significantly. The ratio of CRP measured in serum prepared from plain tubes and from SSTs did not differ significantly from the ratio obtained when serum was prepared in a plain tube then added to an SST. Discussion: We propose that SSTs can adsorb some macromolocules that form complexes with CRP. The addition of SST gel to serum results in the release of CRP molecules from these complexes, which enhances the antigen-antibody reaction on the Vitros CRP slide and increases the measured CRP concentrations.