A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimens.

BACKGROUND: KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain. METHODS: We conducted a two-site comparison of two commercial KRAS mutation kits - the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit - and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles. RESULTS: Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger. CONCLUSION: The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation.

Genetic profile of ischemic cerebrovascular disease and carotid stenosis.

OBJECTIVES: Carotid artery stenosis (CS) is a major risk factor for ischemic cerebrovascular disease (ICVD) and is therefore of interest in genetic investigating. Here we report the distribution of 100 polymorphisms in 47 suspected susceptibility genes for ICVD and its risk factors. MATERIALS AND METHODS: Previously published markers in suspected susceptibility genes were genotyped in ICVD patients and controls (928/602). Genotyping was performed using multiplex polymerase chain reaction (PCR) and linear immobilized probe array assays. ICVD cases were subtyped according to Trial of Org 10172 in Acute Stroke Treatment (TOAST) or subdivided into CS and non-CS patients by ultrasonography in a separate analysis. RESULTS: Three polymorphisms located in the lipoprotein lipase (LPL), angiotensinogen (AGT) and guanine nucleotide-binding protein beta-3 (GNB3) genes were significantly associated with ICVD after correction for age and gender. The strongest association was found for the protective LPL Ser447Term polymorphism. All the significant markers showed varying frequencies in different subphenotypes of ICVD. Factor VII, apolipoprotein E and two renin polymorphisms were differentially frequent in patients with evidence of CS compared with non-CS patients. CONCLUSIONS: We have found that some previously described susceptibility polymorphisms are weakly associated with ICVD and that subdivision of patients into CS and non-CS groups may help to identify new candidate polymorphisms.

Effects of 5' regulatory-region polymorphisms on paraoxonase-gene (PON1) expression.

Human HDL-associated paraoxonase (PON1) hydrolyzes a number of toxic organophosphorous compounds and reduces oxidation of LDLs and HDLs. These properties of PON1 account for its ability to protect against pesticide poisonings and atherosclerosis. PON1 also hydrolyzes a number of lactone and cyclic-carbonate drugs. Among individuals in a population, PON1 levels vary widely. We previously identified three polymorphisms in the PON1 regulatory region that affect expression levels in cultured human hepatocytes. In this study, we determined the genotypes of three regulatory-region polymorphisms for 376 white individuals and examined their effect on plasma-PON1 levels, determined by rates of phenylacetate hydrolysis. The -108 polymorphism had a significant effect on PON1-activity level, whereas the -162 polymorphism had a lesser effect. The -909 polymorphism, which is in linkage disequilibrium with the other sites, appears to have little or no independent effect on PON1-activity level in vivo. Other studies have found that the L55M polymorphism in the PON1-coding region is associated with differences in both PON1-mRNA and PON1-activity levels. The results presented here indicate that the L55M effect of lowered activity is not due to the amino acid change but is, rather, largely due to linkage disequilibrium with the -108 regulatory-region polymorphism. The codon 55 polymorphism marginally appeared to account for 15.3% of the variance in PON1 activity, but this dropped to 5% after adjustments for the effects of the -108 and Q192R polymorphisms were made. The -108C/T polymorphism accounted for 22.8% of the observed variability in PON1-expression levels, which was much greater than that attributable to the other PON1 polymorphisms. We also identified four sequence differences in the 3' UTR of the PON1 mRNA.

Polymorphisms in the human paraoxonase (PON1) promoter.

Paraoxonase (PON1) is a protein component of high-density lipoprotein (HDL) particles that protects against oxidative damage to both low-density lipoprotein and HDL and detoxifies organophosphorus pesticides and nerve agents. A wide range of expression levels of PON1 among individuals has been observed. We examined the promoter region of PON1 for genetic factors that might affect PON1 activity levels. We conducted a deletion analysis of the PON1 promoter region in transient transfection assays and found that cell-type specific promoter elements for liver and kidney are present in the first 200bp upstream of the coding sequence. Sequence analysis of DNA from a BAC clone and a YAC clone identified five polymorphisms in the first 1000 bases upstream of the coding region at positions -108, -126, -162, -832 and -909. Additionally, the promoter sequences of two individuals expressing high levels of PON1 and two individuals expressing low levels of PON1 were analysed. The two polymorphisms at -126 and -832 had no apparent effect on expression level in the reporter gene assay. The polymorphisms at position -909, -162 (a potential NF-I transcription factor binding site) and -108 (a potential SP1 binding site) each have approximately a two-fold effect on expression level. The expression level effects of the three polymorphisms appear not to be strictly additive and may depend on context effects.

Efficacies of lipophilic inhibitors of dihydrofolate reductase against parasitic protozoa.

Competitive inhibitors of dihydrofolate reductase (DHFR) are used in chemotherapy or prophylaxis of many microbial pathogens, including the eukaryotic parasites Plasmodium falciparum and Toxoplasma gondii. Unfortunately, point mutations in the DHFR gene can confer resistance to inhibitors specific to these pathogens. We have developed a rapid system for testing inhibitors of DHFRs from a variety of parasites. We replaced the DHFR gene from the budding yeast Saccharomyces cerevisiae with the DHFR-coding region from humans, P. falciparum, T. gondii, Pneumocystis carinii, and bovine or human-derived Cryptosporidium parvum. We studied 84 dicyclic and tricyclic 2,4-diaminopyrimidine derivatives in this heterologous system and identified those most effective against the DHFR enzymes from each of the pathogens. Among these compounds, six tetrahydroquinazolines were effective inhibitors of every strain tested, but they also inhibited the human DHFR and were not selective for the parasites. However, two quinazolines and four tetrahydroquinazolines were both potent and selective inhibitors of the P. falciparum DHFR. These compounds show promise for development as antimalarial drugs.

Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON1(192) or PON1(55) genotype.

The paraoxonase (PON1) PON1-Q192R and PON1-L55M polymorphisms have been inconsistently associated with vascular disease. Plasma PON1 activity phenotypes vary markedly within genotypes and were, therefore, expected to add to the informativeness of genotype for predicting vascular disease. The case-control sample included 212 age- and race-matched men (mean age 66.4 years). The 106 carotid artery disease (CAAD) cases had >80% carotid stenosis, and the 106 controls had <15%. Two PON1 substrate hydrolysis rates (paraoxon [POase] and diazoxon [DZOase]) were significantly lower in cases than in controls and were significant predictors of CAAD by use of logistic regression (POase, P=0.005; DZOase, P=0.019). DZOase predicted vascular disease independently of lipoprotein profile, high density lipoprotein subfractions, apolipoprotein A-I, and smoking. PON1-192 and PON1-55 genotypes or haplotypes did not predict case-control status unless the activity phenotype was also included as a predictor by use of logistic regression. When phenotype was included as a predictor, PON1-192 and PON1-55 genotypes or combined haplotypes were significant predictors (P<0.05). In conclusion, examining PON1-192 and/or PON1-55 genotypes alone may mistakenly lead to the conclusion that there is no role of PON1 in CAAD. These results support the benefit of a "level crossing" approach that includes intervening phenotypes in the study of complexly inherited disease.

The PON1 gene and detoxication.

It has been assumed since its discovery that serum paraoxonase (PON1) plays a major role in the detoxication of specific organophosphorus compounds. It was also assumed that individuals with low PON1 activity would be more susceptible to paraoxon/parathion poisoning than individuals with higher PON1 activity. Evidence supporting this hypothesis was provided by injection of rabbit PON1 into rodents. Injected PON1 protected against paraoxon toxicity in rats and chlorpyrifos oxon toxicity in mice. The recent availability of PON1 knockout mice has provided an in vivo system with which one can more closely examine the role of PON1 in detoxication. PON1 knockout mice demonstrated dramatically increased sensitivity to chlorpyrifos oxon and diazoxon and moderately increased sensitivity to the respective parent compounds. The PON1 knockout mutation also resulted in the elimination of liver PON1 activity, accounting for the dramatic increase in sensitivity to chlorpyrifos oxon and diazoxon. Totally unexpected was our finding that the PON1 knockout mice were not more sensitive to paraoxon. This was particularly surprising in light of the earlier enzyme injection experiments. Differences in the relative catalytic efficiencies of rabbit vs. mouse PON1 for the specific oxon forms explain these observations. Mouse PON1 has good catalytic efficiency for the hydrolysis of diazoxon and chlorpyrifos oxon, but a poor efficiency for paraoxon hydrolysis relative to rabbit PON1. The human PON1Q192 isoform has a catalytic efficiency similar to that of mice, whereas the human PON1R192 isoform has a much better catalytic efficiency, predicting that individuals expressing high levels of the PONIR192 isoform may have increased resistance to paraoxon toxicity.

Analysis of paraoxonase (PON1) L55M status requires both genotype and phenotype.

Paraoxonase (PON1) is tightly associated with high-density lipoprotein particles and is believed to contribute to the prevention of atherosclerosis by metabolizing oxidized lipids. PON1 also hydrolyses the bioactive oxon forms of organophosphorus pesticides such as parathion, diazinon and chlorpyrifos. Two common polymorphisms have been identified in the coding sequence of human PON1: L55M and R192Q. Several previous studies have found that the presence of the PON1R192 allele raises the risk of cardiovascular disease while others found no correlation. The studies, however, have focused on the genotype of PON1 and not the expression level of the protein. We found that the PON1 expression level in plasma, as determined by the rates of paraoxon and diazoxon hydrolysis, varies widely among individuals and within a genotype. Previous studies found that individuals having Met at PON155 have lower levels of both PON1 mRNA and activity. In this study, we determined the plasma activity levels of PON1 and examined the relationships between PON155 genotype and PON1 level. As with PON1192, we found considerable overlap in activity among the PON155 genotypes. Of the 317 individuals whose PON1 status was determined in this study, 48.9% were PON1Q192 homozygotes. Analysis of the PON1QQ192 population showed that while the average PON1 activity (diazoxon hydrolysis) was 12266 U/L for PON1LL55 and 7777 U/L for PON1MM55, a given PONMM55 individual could have more than twice the activity of a PON1LL55 individual. PON1 status, which includes PON1 level as well as PON1192 genotype, may be a better predictor for cardiovascular disease or organophosphate susceptibility than PON1 genotype alone.

Identification of Cryptosporidium parvum dihydrofolate reductase inhibitors by complementation in Saccharomyces cerevisiae.

There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium parvum. Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested against C. parvum. To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae. We expressed the DHFR genes of C. parvum, Plasmodium falciparum, Toxoplasma gondii, Pneumocystis carinii, and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinant C. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. parvum DHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis.

Expression of CD14 corrects the slow response to lipopolysaccharide in the 1B8 mutant of the B cell lymphoma 70Z/3.

B cells and macrophages both activate NF-kappaB/Rel in response to lipopolysaccharide (LPS), but differ in sensitivity to LPS and in downstream genes that are activated. CD14 is a high-affinity receptor for LPS found on macrophages, but not B cells. We expressed human CD14 (hCD14) in the mouse B lymphoma, 70Z/3, and a mutant, 1B8, which responds slowly to LPS, to test whether expression of hCD14 could correct or bypass the defect in 1B8 cells. We compared the timing and extent of known responses to LPS in 70Z/3 cells and the 1B8 mutants. The hCD14+ 1B8 and 70Z/3 cells responded more rapidly and were sensitive to 100-fold lower levels of LPS than their untransfected counterparts. Degradation of the IkappaB-alpha and -beta molecules and translocation of the NF-kappaB/Rel complexes into the nucleus were more rapid and the steady-state levels of Igk mRNA and mIgM on the cell surface were markedly increased in cells that expressed hCD14. The LPS response of the hCD14+ 1B8 and 70Z/3 cells showed subtle differences. In the 1B8 hCD14 cells, the p50/p50 complexes were never abundant in nuclear extracts, and degradation of IkappaB-beta was slower than in hCD14 70Z/3 cells. This partial correction of the 1B8 phenotype suggests that the defective component in 1B8 participates in the CD14 signaling pathway and could include the B-cell LPS receptor itself.

Yeast as a model system to study drugs effective against apicomplexan proteins.

Biochemical and genetic analyses are required to identify potential drug targets in apicomplexan parasites, but these studies have proved difficult in most parasite systems. We have developed methods based on expression of parasite proteins in the budding yeast, Saccharomyces cerevisiae, to rapidly screen drugs directed against particular parasite targets, to study the structure and function of these target molecules, and to identify mutations in the parasite genes that alter enzyme specificity or drug sensitivity. In this paper we outline the parameters that need to be considered to design yeast strains that function efficiently to assay function of parasite proteins. Basic protocols and methods are included. We detail some problems that might be encountered in the engineering of these yeast strains and suggest possible solutions.