Magnolol-mediated regulation of plasma triglyceride through affecting lipoprotein lipase activity in apolipoprotein A5 knock-in mice.

Hyperlipidemia is a risk factor of arteriosclerosis, stroke, and other coronary heart disease, which has been shown to correlate with single nucleotide polymorphisms of genes essential for lipid metabolism, such as lipoprotein lipase (LPL) and apolipoprotein A5 (APOA5). In this study, the effect of magnolol, the main active component extracted from Magnolia officinalis, on LPL activity was investigated. A dose-dependent up-regulation of LPL activity, possibly through increasing LPL mRNA transcription, was observed in mouse 3T3-L1 pre-adipocytes cultured in the presence of magnolol for 6 days. Subsequently, a transgenic knock-in mice carrying APOA5 c.553G>T variant was established and then fed with corn oil with or without magnolol for four days. The baseline plasma triglyceride levels in transgenic knock-in mice were higher than those in wild-type mice, with the highest increase occurred in homozygous transgenic mice (106 mg/dL vs 51 mg/dL, p<0.01). After the induction of hyperglyceridemia along with the administration of magnolol, the plasma triglyceride level in heterozygous transgenic mice was significantly reduced by half. In summary, magnolol could effectively lower the plasma triglyceride levels in APOA5 c.553G>T variant carrier mice and facilitate the triglyceride metabolism in postprandial hypertriglyceridemia.

Implementation and Quality Control of Lung Cancer EGFR Genetic Testing by MALDI-TOF Mass Spectrometry in Taiwan Clinical Practice.

Molecular diagnostics in cancer pharmacogenomics is indispensable for making targeted therapy decisions especially in lung cancer. For routine clinical practice, the flexible testing platform and implemented quality system are important for failure rate and turnaround time (TAT) reduction. We established and validated the multiplex EGFR testing by MALDI-TOF MS according to ISO15189 regulation and CLIA recommendation in Taiwan. Totally 8,147 cases from Aug-2011 to Jul-2015 were assayed and statistical characteristics were reported. The intra-run precision of EGFR mutation frequency was CV 2.15% (L858R) and 2.77% (T790M); the inter-run precision was CV 3.50% (L858R) and 2.84% (T790M). Accuracy tests by consensus reference biomaterials showed 100% consistence with datasheet (public database). Both analytical sensitivity and specificity were 100% while taking Sanger sequencing as the gold-standard method for comparison. EGFR mutation frequency of peripheral blood mononuclear cell for reference range determination was 0.002 ± 0.016% (95% CI: 0.000-0.036) (L858R) and 0.292 ± 0.289% (95% CI: 0.000-0.871) (T790M). The average TAT was 4.5 working days and the failure rate was less than 0.1%. In conclusion, this study provides a comprehensive report of lung cancer EGFR mutation detection from platform establishment, method validation to clinical routine practice. It may be a reference model for molecular diagnostics in cancer pharmacogenomics.

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.

Characterisation of factor IX with a glycine-to-valine missense mutation at residue 190 in a patient with severe haemophilia B.

A patient with severe haemophilia B with a glycine-to-valine missense mutation at residue 190 (c25, chymotrypsin numbering) in factor IX (FIX; FIX-G190V or FIX-FuChou) had <1% of normal FIX clotting activity and 36% of normal FIX antigen levels (cross-reacting material- reduced, CRMr). Residue 190 in the C-terminal protease domain of human FIX is highly conserved in mammalian species and the serine protease family, suggesting that it has an indispensable role in protein function. To explore the pathological mechanism by which this mutation contributes to dysfunction of the FIX molecule, we functionally characterised FIX-G190V in vitro and in vivo. Liver-specific FIX-G190V gene expression following hydrodynamic plasmid delivery into haemophilia B mice revealed a 5.7-fold reduction in specific clotting activity compared with FIX-WT (wild type) and a two-fold decrease in plasma FIX-G190V concentration. Pulse-chase analysis demonstrated that FIX-G190V was secreted at a significantly slower rate than was FIX-WT. Purified FIX-G190V and FIX-WT displayed normal calcium-dependent conformational changes as shown by intrinsic fluorescence quenching. The in vivo half-lives of FIX-G190V and FIX-WT were indistinguishable. FIX-G190V was, however, more readily degraded than FIX-WT, especially after being activated by the active form of FXI. The vulnerable sites were mapped to the peptide bonds at Arg¹¹6-Leu¹¹7, Lys²65-Tyr²66, Arg³²7-Val³²8, and Arg³³8-Ser³³9, which are in the exposed loops of the FIX molecule. Also, failure of FXIa-activated FIX-G190V to bind p-aminobenzamidine indicated an abnormal conformation of the active-site pocket. Thus, the mutation at residue 190 of FIX may result in protein misfolding that affects secretion, clotting function, and hydrolysis.

Endonuclease V-mediated deoxyinosine excision repair in vitro.

Deoxyinosine (dI) in DNA can arise from hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the nfi gene product, in Escherichia coli. Repair was studied in vitro using M13mp18 derived heteroduplexes containing a site-specific deoxyinosine. Unpaired dI/G mismatch resides within the recognition site for XhoI restriction endonucleases, permitting evaluation of repair occurring on deoxyinosine-containing DNA strand. Our results show that dI lesions were efficiently repaired in nfi(+)E. coli extracts but the repair level was much reduced in nfi mutant extracts. We subjected the deoxyinosine-containing heteroduplex to a purified system consisting of soluble endonuclease V fusion protein, DNA polymerase I, and DNA ligase, along with the four deoxynucleoside triphosphates. Interestingly we found these three proteins alone are sufficient to process the dI lesion efficiently. We also found that the 3'-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay.

FIX-Triple, a gain-of-function factor IX variant, improves haemostasis in mouse models without increased risk of thrombosis.

Engineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976-1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.

Heparin interference in the cerebrospinal fluid protein assay measured with a pyrogallol red-molybdate complex.

BACKGROUND: Increased cerebrospinal fluid (CSF) total protein is a useful indicator of meningeal or central nervous system disease. Occasionally the primary care physicians added heparin to CSF samples to avoid clotting. The aim of this study is to investigate the interference of heparin on CSF total protein measurement. METHODS: CSF specimens were collected from 230 in-patients with various diseases and analyzed by the Vitros 950 PROT slide and the Toshiba TBA-120FR assay. After adding 0, 0.0625, 0.25, 0.5, 0.75, 1, 2 and 4 IU/ml of heparin that was diluted in 20 microl of normal saline to 180 microl of CSF aliquots, CSF total protein concentrations were determined again by the 2 assay systems in the absence or presence of protamine. RESULTS: At low (<40 mg/dl) and mildly increased (40-or<100 mg/dl) CSF total protein, the measured protein concentrations significantly decreased up to 91% when 4 IU/ml of heparin was added to the samples before being analyzed by the Toshiba TBA-120FR assay. At moderately increased (100-or<200 mg/dl) and high (>or=200 mg/dl) CSF total protein, 62% and 27% decreases were found, respectively. Only 1-8% decline was found when 4 IU/ml of heparin was added to the samples before being analyzed by the Vitros 950 PROT assay. Addition of protamine partially reversed the interference of heparin. CONCLUSIONS: The interference of heparin in the CSF total protein assay is dependent on the reaction principle, especially when the CSF total protein level is normal to mildly elevated.

Proficiency testing of Hb A1c: a 4-year experience in Taiwan and the Asian Pacific region.

BACKGROUND: The correlation between hemoglobin A(1c) (Hb A(1c)) and risk for complications in diabetic patients heightens the need to measure Hb A(1c) with accuracy. We evaluated the current performance for measuring Hb A(1c) in the Asian and Pacific region by examining data submitted by laboratories participating in the Taiwan proficiency-testing program. METHODS: Five fresh-pooled blood samples were sent to participating laboratories twice each year. The results were evaluated against target values assigned by the National Glycohemoglobin Standardization Program network laboratories; a passing criterion of +/-7% of the target value was used. Measurement uncertainty at Hb A(1c) concentrations of 7.0% and 8.0% were determined. RESULTS: A total of 276 laboratories from 11 countries took part in the Hb A(1c) survey. At the Hb A(1c) concentrations tested method-specific interlaboratory imprecision (CVs) were 1.1%-13.9% in 2005, 1.3%-10.1% in 2006, 1.2%-8.2% in 2007, and 1.1%-6.1% in 2008. Differences between target values and median values from the commonly used methods ranged from -0.24% to 0.22% Hb A(1c) in 2008. In 2005 83% of laboratories passed the survey, and in 2008 93% passed. At 7.0% Hb A(1c), measurement uncertainty was on average 0.49% Hb A(1c). CONCLUSIONS: The use of accuracy-based proficiency testing with stringent quality criteria has improved the performance of Hb A(1c) testing in the Asian and Pacific laboratories during the 4 years of assessment.

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).

Common sequence variant in lipoprotein lipase gene conferring triglyceride response to fibrate treatment.

AIMS: Little is known about whether the variations in the lipoprotein lipase (LPL) and apolipoprotein gene cluster (APOA1/C3/A5) confer appreciable triglyceride lowering after fibrate treatment among patients with hypertriglyceridemia. MATERIALS & METHODS: We investigated whether variations in these genes confer a triglyceride lowering response after fibrate treatment among 145 patients with hypertriglyceridemia receiving 6 months of fibrate treatment. RESULTS: Overall triglycerides decreased from 746.2 mg/dl to 465.9 mg/dl and the mean percentage of triglyceride lowering was 50.7 +/- 38.6%. A total of two polymorphisms, LPL IVS8 +483T>G and APOA1 +2169G>C, were associated with a significant percentage of triglyceride lowering. Common haplotype effects of LPL on the triglyceride lowering percentage were significant (p = 0.002) and the percentage of variance explained by the LPL haplotype was 7.5%. One common LPL haplotype encompassing three polymorphisms was associated with a -45.40% change (p < 0.001) and risk of a 5.9-fold risk for developing a poor response (95% confidence interval: 1.11-31, p = 0.037), compared with the most frequent LPL haplotype. CONCLUSION: Our results indicate that the LPL gene variant may cause triglyceride lowering after fibrate treatment among patients with hypertriglyceridemia.

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.

Urine sediment examination: a comparison of automated urinalysis systems and manual microscopy.

BACKGROUND: Several automated instruments examining urine sediment have been introduced. We compared the performance of Sysmex UF-100 and Iris iQ200 with manual microscopy in urine sediment testing. METHODS: Four hundred and thirty-six urine samples were collected. The urine sediments were examined by manual microscopy and these 2 automated urinalysis systems. RESULTS: The within-run CVs for urine samples ranged from 3.4% to 22.3% for the iQ200, 1.6% to 24.2% for the UF-100 and 12.5% to 43.9% for manual microscopy. Between-run CVs on quality-control samples ranged from 6.1% to 32.4% for the iQ200 and 3.5% to 24.7% for the UF-100. The agreement between methods was good for red blood cells and white blood cells counts based on r values of 0.935 to 0.968. However, for epithelial cells, the values measured by different systems were poorly correlated (r=0.888-0.922). The Bland-Altman plot indicated a trend towards the automated cell count being greater than the manual microscopy as the epithelial cell count increased. Casts were difficulty differentiated by 2 automated systems. CONCLUSIONS: These 2 automated urinalysis systems demonstrated good concordance with each other in urine sediment examination. The automated process could be used as a screening procedure but some manual microscopy was still necessary.

Evaluation and improvement strategy of analytical turnaround time in the stat laboratory.

BACKGROUND/PURPOSE: Laboratory analytical turnaround time represents laboratory effectiveness. Our study aimed to evaluate laboratory analytical turnaround time to optimize workflow and shorten analytical turnaround time. METHODS: We used the laboratory information system in a 2000-bed teaching hospital to compute and analyze the 90th percentile turnaround time of the Stat Laboratory from 2001 to 2003. RESULTS: The overall 90th percentile turnaround time in the Stat Laboratory was 40-49 minutes and positively correlated with test volume. The daily test volume in the Stat Laboratory has grown significantly in the latter 2 half-years of the study as compared with the previous 2 half-years (p < 0.05 and p < 0.001, respectively). The daily longest turnaround time occurred in the early morning, and troponin-I testing contributed to the majority of incidences of prolongation of analytical turnaround time. We prioritized the performance of troponin-I testing, which resulted in a reduction of the analytical turnaround time by about 18 minutes (from 66 to 48 minutes) and no increment of overall turnaround time (42 to 44 minutes) despite continuously increasing test volume. CONCLUSION: These findings demonstrated that a dedicated means of process control was able to significantly improve laboratory efficiency.

Comparison of three automated urinalysis systems--Bayer Clinitek Atlas, Roche Urisys 2400 and Arkray Aution Max for testing urine chemistry and detection of bacteriuria.

BACKGROUND: Our study is aimed to determine the performance of 3 automated urinalysis systems-Clinitek Atlas, Urisys 2400 and Aution Max. METHODS: One thousand urine specimens were analyzed with the 3 automated systems. The results of the 3 assays were compared for testing urine chemistry and evaluating the capacity of leukocyte esterase and nitrite to detect bacteriuria. RESULTS: The correlation between the 3 instruments represented as within 1 grading difference was better between the Atlas and Aution Max systems for pH, blood, glucose, urobilinogen, ketone and specific gravity. For protein and nitrite, better correlation was observed between the Atlas and Urisys 2400, while the Aution Max and Urisys 2400 conveyed better correlation for bilirubin and white blood cells. The sensitivity and specificity of both the leukocyte esterase and nitrite in screening for significant bacteriuria were 71.7, 58.9, 70.8% and 99.1, 99.1 and 97.2%, for the Clinitek Atlas, Aution Max and Urisys 2400, respectively. CONCLUSIONS: The automated urinalysis systems demonstrate acceptable correlations with each other in urine chemistries, especially between the Clinitek Atlas and Aution Max systems on the majority of items. The specificity and negative predictive value of leukocyte esterase and nitrite of the 3 instruments for screening of significant bacteriuria were sufficient to avoid unnecessary urine culture.

Multinomial logistic regression approach to haplotype association analysis in population-based case-control studies.

BACKGROUND: The genetic association analysis using haplotypes as basic genetic units is anticipated to be a powerful strategy towards the discovery of genes predisposing human complex diseases. In particular, the increasing availability of high-resolution genetic markers such as the single-nucleotide polymorphisms (SNPs) has made haplotype-based association analysis an attractive alternative to single marker analysis. RESULTS: We consider haplotype association analysis under the population-based case-control study design. A multinomial logistic model is proposed for haplotype analysis with unphased genotype data, which can be decomposed into a prospective logistic model for disease risk as well as a model for the haplotype-pair distribution in the control population. Environmental factors can be readily incorporated and hence the haplotype-environment interaction can be assessed in the proposed model. The maximum likelihood estimation with unphased genotype data can be conveniently implemented in the proposed model by applying the EM algorithm to a prospective multinomial logistic regression model and ignoring the case-control design. We apply the proposed method to the hypertriglyceridemia study and identifies 3 haplotypes in the apolipoprotein A5 gene that are associated with increased risk for hypertriglyceridemia. A haplotype-age interaction effect is also identified. Simulation studies show that the proposed estimator has satisfactory finite-sample performances. CONCLUSION: Our results suggest that the proposed method can serve as a useful alternative to existing methods and a reliable tool for the case-control haplotype-based association analysis.

Association between the apolipoprotein E genotypes and breast cancer patients in Taiwanese.

To display the association between the apolipoprotein E (APOE) genotypes and breast cancer patients, a cross sectional study including 291 patients and 148 controls was performed. The APOE genotypes were measured in all participants, and the pathological diagnosis, estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER-2) among breast cancer patients were collected. The results showed the APOE allele frequency in breast cancer patients was 11.7% epsilon2 carriers, 74.6% epsilon3 carriers and 13.7% epsilon4 carriers, and there was no significant difference when they were compared with those of the control group (15.5% epsilon2 carriers, 74.3% epsilon3 carriers and 10.1% epsilon4 carriers; p=0.342). Among the patients in pre-menopause, showed a higher frequency of epsilon2 carriers had the cancer site on the left than that of the epsilon3 carriers (78.6% versus 40.3%; p=0.019). Among breast cancer patients, there was no significant association between the APOE genotypes and menopausal status, pathological diagnosis, estrogen receptor, progesterone receptor, and HER-2. Our findings demonstrated that the APOE genotypes were not associated with breast cancer patients, and epsilon2 allele tended to induce breast cancer on the left site among those patients in pre-menopause.

A two-stage design for multiple testing in large-scale association studies.

Modern association studies often involve a large number of markers and hence may encounter the problem of testing multiple hypotheses. Traditional procedures are usually over-conservative and with low power to detect mild genetic effects. From the design perspective, we propose a two-stage selection procedure to address this concern. Our main principle is to reduce the total number of tests by removing clearly unassociated markers in the first-stage test. Next, conditional on the findings of the first stage, which uses a less stringent nominal level, a more conservative test is conducted in the second stage using the augmented data and the data from the first stage. Previous studies have suggested using independent samples to avoid inflated errors. However, we found that, after accounting for the dependence between these two samples, the true discovery rate increases substantially. In addition, the cost of genotyping can be greatly reduced via this approach. Results from a study of hypertriglyceridemia and simulations suggest the two-stage method has a higher overall true positive rate (TPR) with a controlled overall false positive rate (FPR) when compared with single-stage approaches. We also report the analytical form of its overall FPR, which may be useful in guiding study design to achieve a high TPR while retaining the desired FPR.

Comparison of Abbott AxSYM and Roche Elecsys 2010 for measurement of BNP and NT-proBNP.

BACKGROUND: B-type natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) are small cardiac hormones released from the heart. They can be used as an important aid to diagnose congestive heart failure (CHF). METHODS: We compared the performances of the Abbott AxSYM and Roche Elecsys 2010 for the measurement of BNP and NT-proBNP. The first method uses a microparticle enzyme-linked immunoassay, whereas the other uses chemiluminescent immunometric assay. RESULTS: The CVs using pooled sera ranged from 3.7% to 12.7% for the AxSYM and 0.9% to 2.2% for the Elecsys 2010. The Passing and Bablok regression was Elecsys 2010 NT-proBNP=7.23xAxSYM BNP+2.53. The BNP in EDTA plasma was more stable than in serum. The immunoreactivity difference of NT-proBNP in serum or EDTA plasma was within 10% when stored at 4 degrees Celsius or 25 degrees Celsius for 72 h. Receiver operating characteristic (ROC) curves were different for both assays, and the areas under the curves were 0.704 and 0.841 for the AxSYM and Elecsys 2010 method, respectively. CONCLUSIONS: Both assays were not entirely specific for heart failure. The precision and stability for NT-proBNP was better than for BNP in serum. It is important to use method-appropriate reference ranges (or cutoff) for the BNP and NT-proBNP, respectively, in the assessment of CHF.

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.

Nick-directed repair of palindromic loop mismatches in human cell extracts.

Palindromic sequences present in DNA may form secondary structures that block DNA replication and transcription causing adverse effects on genome stability. It has been suggested that hairpin structures containing mispaired bases could stimulate the repair systems in human cells. In this study, processing of variable length of palindromic loops in the presence or absence of single-base mismatches was investigated in human cell extracts. Our results showed that hairpin structures were efficiently processed through a nick-directed mechanism. In a similar sequence context, mismatch-containing hairpins have higher repair efficiencies. We also found that shorter hairpins are generally better repaired. A strand break located either 3' or 5' to the loop is sufficient to activate hairpin repair on the nicked strand. The reaction requires Mg(2+), the four dNTPs and hydrolysis of ATP for efficient repair on both palindromic loop insertions and deletions. Correction of each of these heteroduplexes was abolished by aphidicolin but was relatively insensitive to the presence of ddTTP, suggesting involvement of polymerase(s) alpha and/or delta. These findings are most consistent with the nick-directed loop repair pathway being responsible for processing hairpin heterologies in human cells.