Nuclease Assay Research Articles

A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin

Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATP-DnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATP-specific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATP-DnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.

Allelic Variants of Upstream Transcription Factor 1 Associate With Carotid Artery Intima-Media Thickness The Cardiovascular Risk in Young Finns Study

Polymorphisms of the upstream transcription factor 1 (USF1) have been associated with familial combined hyperlipidemia and coronary heart disease. The impact of this gene on subclinical atherosclerosis is unknown. Associations of 3 allelic variants of the USF1 gene and their haplotypes with carotid artery intima - media thickness (IMT), carotid artery compliance (CAC) and brachial artery flow mediated dilatation (FMD) were studied in a population of Finnish healthy young adults. The study population comprised 2,281 individuals participating in the Cardiovascular Risk in Young Finns study. IMT, CAC and FMD values were measured by ultrasound examination. Genotypes were analysed using the 5' nuclease assay. A significant difference in IMT was found for usf1s1 (rs3737787) and usf1s8 (rs2516838) genotypes (p-values 0.046 and 0.021, respectively). Moreover, there was a significant difference between groups in haplotype 1 and haplotype 2 for IMT (p-values 0.011 and 0.028 respectively). In multivariate stepwise linear regression models adjusted by age, sex, body mass index, systolic and diastolic blood pressures, smoking, C-reactive protein, glucose, high- and low-density lipoprotein-cholesterols and triglycerides there were significant associations for the usf1s1 minor genotype AA to predict low IMT (p=0.038) and usf1s8 minor genotype GG to predict high IMT (p=0.003). There was also a significant association for haplotype 2 to predict low IMT in the otherwise similar multivariate model (p=0.006). No associations were found for polymorphisms and CAC, FMD or serum lipids. The rs2516838 and rs3737787 polymorphisms of USF1 influence the carotid artery IMT, which is a new finding.

The Influence of Genetic Polymorphisms of GSTP1 on the Development of Asbestosis

Genetic factors play an important role in the development of asbestosis. The aim of this study was to investigate whether genetic polymorphisms of glutathione S-transferase (GST) P1 represent a risk factor for this disease. The study population included 262 workers with asbestosis and 265 matched controls. Information on cumulative asbestos exposure was available. A real-time PCR based on the 5' nuclease assay was designed for the analysis of GSTP1 Ile105Val and Ala114Val polymorphisms. Asbestosis was associated with GSTP1 genotype coding for an enzyme with high conjugation capacity versus genotypes resulting in intermediate and low enzyme activity (odds ratio = 1.49, confidence interval = 1.06-2.10). The key finding of the study was that GSTP1 genotype coding for an enzyme with high conjugation capacity significantly increases the risk of developing asbestosis.

The Glu228Ala polymorphism in the ligand binding domain of death receptor 4 is associated with increased risk for prostate cancer metastases

Death receptor 4, encoded by the TNFRSF10A gene, is an important mediator of apoptosis and its dysfunction may be related to cancer development and distant tumor spread. A single nucleotide polymorphism in TNFRSF10A (Glu228Ala, rs20576) within a conserved region of the extracellular cysteine-rich domain of death receptor 4 has been associated with an increased risk for a variety of tumor entities. Aim of the present study was to evaluate the role of the TNFRSF10A polymorphism in metastatic progression of prostate cancer after radiation therapy. We carried out a prospective study including 702 prostate cancer patients from the Austrian PROCAGENE (Prostate Cancer Genetics) study. Development of metastases was examined in regular follow-up investigations. TNFRSF10A genotypes were determined by a 5'-nuclease assay (TaqMan). Within a median follow-up time of 10 months (range 0-60 months), 24 (3.4%) patients developed metastases. In a Cox regression model including age at diagnosis and risk group as potential confounders, carriage of an 228Ala allele was associated with a relative risk of 2.47 (95% CI 1.10-5.54; P=0.028) for metastases. TNFRSF10A genotypes were not associated with tumor stage, grade, risk group or age at diagnosis. We conclude that the TNFRSF10A Glu228Ala polymorphism may be a novel independent risk factor for prostate cancer metastases after radiation therapy.

Population-specific effects of the Asn40Asp polymorphism at the μ-opioid receptor gene (OPRM1) on HPA-axis activation

Studies in European Americans (EAs) have shown that the hypothalamic-pituitary-adrenal (HPA)-axis activation by the opioid blockade is moderated by the single nucleotide polymorphism (SNP) A118G (Asn40Asp) at the mu-opioid receptor locus (OPRM1). We examined the effect of this, and of five intronic OPRM1 SNPs, on adrenocorticotropic hormone and cortisol concentrations, following the placebo-controlled administration of naloxone to healthy individuals who were of EA or Asian ancestry. We used a balanced, within-participant design with two test sessions to examine the hormonal responses to intravenous naloxone (an opioid antagonist) (125 microg/kg) or placebo in 29 healthy participants (62% men, 59% of Asian ancestry). DNA isolated from whole blood was PCR amplified and genotyped using a fluorogenic 5 nuclease assay (TaqMan) method. Consistent with earlier reports, participants with one or two Asp40 alleles (n=16) had a significantly greater cortisol response to naloxone than Asn40 homozygotes, but the effect was limited to EAs. Asians with the Asp40 allele did not show a greater increase in cortisol response compared with Asn40 homozygotes. None of the intronic SNPs was associated with cortisol response either directly or via an interaction effect with Asn40Asp. Effects of the Asn40Asp polymorphism at OPRM1 on HPA-axis activation seem to be population-specific. The association between the Asn40Asp and the HPA-axis response to naloxone cannot, therefore, be explained with reference only to the amino acid substitution encoded by that polymorphism. Further research to understand the basis for the observed association is warranted.

Toll-like receptor 4 gene (Asp299Gly) polymorphism associates with carotid artery elasticity: The cardiovascular risk in young Finns study

Objective Early subclinical markers of atherosclerosis, such as carotid artery intima media thickness (IMT) and elasticity predict future coronary events. The G allele of the Toll-like receptor 4 (TLR-4) gene Asp299Gly polymorphism has been previously associated with decreased development of atherosclerosis and with lower risk of myocardial infractions. We wanted to examine the association of this polymorphism with carotid IMT and compliance in a population of young Finnish Caucasian adults. Methods Carotid artery IMT and elasticity indices of 2201 study subjects who participated in a randomized multicenter study (cardiovascular risk in young Finns study) were measured with ultrasound. The genotyping was performed using the TaqMan 5′-nuclease assay. Results According to multivariate linear regression analysis adjusted with potential confounders, the G allele carriers had significantly higher carotid arterial compliance, measured in increase of luminal diameter percentage in response to blood pressure rise of 10 mmHg, than did the AA homozygotes ( β = 0.099 with 95% CI 0.029–0.169 and p = 0.006). The difference between AA homozygotes and GG homozygotes was even more pronounced ( β = 0.382 with 95% CI 0.119–0.644 and p = 0.004). Variation in the TLR-4 genotype was not related with IMT. The results of the two independent study cohorts of Eastern and Western Finland were in accordance with the results of the whole combined study population. Conclusion The G allele of the TLR-4 gene Asp299Gly polymorphism is associated with increased carotid artery compliance in young adults. This beneficial effect of the G allele may reduce the risk of future cardiovascular events.

Vascular endothelial growth factor gene polymorphisms in thyroid cancer

Vascular endothelial growth factor (VEGF) is a potent stimulator for angiogenesis. It has been implicated in growth and metastasis of thyroid cancer. Three functional single nucleotide polymorphisms (SNPs) of VEGF (-2578C/A, -634G/C, and +936C/T) are known to be related with VEGF expression. We conducted a case-control study to evaluate the genetic effects of these three functional SNPs on the development of thyroid cancer and lymph node metastasis. A total of 332 cases and 261 controls were recruited for this study. The genotypes were determined by the TaqMan 5'-nuclease assay. Hardy-Weinberg equilibrium (HWE) was tested for each SNP, and genetic effects were evaluated by the chi(2)-test and multiple logistic regression. We used Bonferroni correction to account for multiple testing, and a two-tailed P value <0.017 was considered statistically significant. All three SNPs were in HWE. The A allele of -2578C/A (i.e. SNP rs699947) increased a risk for thyroid cancer (adjusted OR=136, 95% CI=1.02-1.81, P=0.039). Haplotype analysis yielded a less significant result (an empirical P value of 0.07). There was a tendency of increasing the frequency of the risk allele from controls, patients without lymph node metastasis to patients with lymph node metastasis (P(trend)=0.019). Further analysis showed that the genetic effect was only in men (adjusted OR=1.97, 95% CI=1.16-3.37, P=0.013) but not in women (adjusted OR=1.15, 95% CI=0.81-1.62, P=0.435). The other two SNPs did not show significant results. The A allele of the SNP rs699947 increased the risk of thyroid cancer development and regional lymph node metastasis in men.

MSH2 −118T&gt;C and MSH6 −159C&gt;T promoter polymorphisms and the risk of colorectal cancer

The most important indicator of colorectal cancer (CRC) risk is the presence of family history of the disease. Inherited genetic changes, such as single nucleotide polymorphisms, in key candidate genes may contribute to CRC risk. We investigated whether promoter polymorphisms in DNA mismatch repair (MMR) genes MSH2 and MSH6 are associated with the risk of CRC. We genotyped 929 CRC patients and 1098 control subjects from Ontario, and 467 patients and 344 controls from Newfoundland and Labrador, for two promoter polymorphisms in the MMR genes MSH2 and MSH6 using the fluorogenic 5' nuclease assay. We used unconditional logistic regression to evaluate the association between each polymorphism and CRC after adjusting for age and sex. The associations between polymorphisms and tumor clinicopathological features were evaluated with a Pearson's chi-squared test or Fisher's exact test. All statistical tests were two sided. We observed strong associations between the MSH2 -118T>C polymorphism and family history of CRC based on the Amsterdam criteria I (P = 0.005) and Amsterdam criteria I and II (P = 0.036) among cases from Ontario. This association was especially evident among female CRC patients in Ontario (for Amsterdam criteria I, and I and II combined, P = 0.003 and P = 0.0001, respectively). The MSH2 -118T>C polymorphism was associated with strong family history of CRC in Ontario patients.

Dopamine Receptor D2 GeneTaq1A(C32806T) Polymorphism Modifies the Relationship Between Birth Weight and Educational Attainment in Adulthood: 21-Year Follow-up of the Cardiovascular Risk in Young Finns Study

Low birth weight is suggested to be a risk factor for a wide variety of negative outcomes, including low educational attainment, but the role of cognition-related genetic influences on this association remains unclear. The objective of this study was to study whether variation in the dopamine receptor gene (dopamine receptor D2 polymorphism, rs1800497) modifies the association between birth weight and educational attainment in adulthood. We studied the association between birth weight (range: 1440-4980 g) and educational attainment in 659 men and 832 women aged 27 to 39. Birth weight, gestational age, and parental education were assessed at ages 6 to 18. The genotyping was performed using TaqMan 5' nuclease assay. After adjustment for age, parental education, and gestational age, birth weight was associated with educational attainment in men with A1/A1 or A1/A2 (n = 245) genotype but not in men carrying A2/A2 (n = 414) genotype. In women, no moderating effect of dopamine receptor D2 polymorphism was found. Dopamine receptor D2 genotype is suggested to modify the association between birth weight and adulthood educational attainment over the whole birth weight range so that carriers of A1 allele capitalize on optimal birth weight, whereas a low birth weight seems to be a risk among them. These data support the hypothesis that the effect of birth weight on educational attainment depends on genetic influences. Gender-related difference may refer to an environmental effect (ie, to a better goodness-of-fit between girls' school behaving and expectations of school) that may mask a genetic effect.

Crystal Structure of Bacteriophage T4 5′ Nuclease in Complex with a Branched DNA Reveals How Flap Endonuclease-1 Family Nucleases Bind Their Substrates

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.

Comparative evaluation of in-house manual, and commercial semi-automated and automated DNA extraction platforms in the sample preparation of human stool specimens for a Salmonella enterica 5'-nuclease assay

In the present study, three methods (NucliSens miniMAG [bioMérieux], MagNA Pure DNA Isolation Kit III Bacteria/Fungi [Roche], and a silica-guanidiniumthiocyanate {Si-GuSCN-F} procedure for extracting DNA from stool specimens were compared with regard to analytical performance (relative DNA recovery and down stream real-time PCR amplification of Salmonella enterica DNA), stability of the extracted DNA, hands-on time (HOT), total processing time (TPT), and costs. The Si-GuSCN-F procedure showed the highest analytical performance (relative recovery of 99%, S. enterica real-time PCR sensitivity of 91%) at the lowest associated costs per extraction (€ 4.28). However, this method did required the longest HOT (144 min) and subsequent TPT (176 min) when processing 24 extractions. Both miniMAG and MagNA Pure extraction showed similar performances at first (relative recoveries of 57% and 52%, S. enterica real-time PCR sensitivity of 85%). However, when difference in the observed Ct values after real-time PCR were taken into account, MagNA Pure resulted in a significant increase in Ct value compared to both miniMAG and Si-GuSCN-F (with on average + 1.26 and + 1.43 cycles). With regard to inhibition all methods showed relatively low inhibition rates (< 4%), with miniMAG providing the lowest rate (0.7%). Extracted DNA was stable for at least 1 year for all methods. HOT was lowest for MagNA Pure (60 min) and TPT was shortest for miniMAG (121 min). Costs, finally, were € 4.28 for Si-GuSCN, € 6.69 for MagNA Pure and € 9.57 for miniMAG.

Association of single nucleotide polymorphisms (SNPs) of VEGF gene with diabetic retinopathy

Abstract Purpose: Vascular endothelial growth factor (VEGF) has an important role in the development of diabetic retinopathy. High concentration of vitreous VEGF and elevated VEGF expression has been found in diabetic retinopathy. Single nucleotide polymorphisms (SNPs) of VEGF gene have been shown to influence expression of VEGF. We studied the association of VEGF SNPs with severity of diabetic retinopathy. Methods: The study population consisted of 129 diabetic (type 1 or type 2) patients with laser‐treated retinopathy, 99 diabetic patients with mild or no retinopathy and 524 non‐diabetic controls. SNPs were chosen on structural basis at three different haploblocks of the VEGF gene covering the whole VEGF gene and in addition, polymorphisms previously known to affect VEGF production were selected. VEGF SNPs rs699947, rs2010963, rs2146323, rs3025033 and rs3025039 were genotyped using the 5' nuclease assay for allelic discrimination (TaqMan). The allele and genotype frequencies were analyzed with chi‐square test in SPSS. Results: Duration of diabetes was similar in patients with laser‐treated retinopathy and in patients with mild or no retinopathy (mean 22.8 vs. 22.7 years, respectively), but the laser‐treated diabetic group had somewhat poorer glycemic control (9.2 vs. 8.4 %). The distribution of genotypes and the allele frequencies did not differ significantly between the diabetics and controls, between patients with laser‐treated retinopathy and patients with mild or no retinopathy, or between patients with proliferative retinopathy and patients with non‐proliferative retinopathy. Conclusions: In conclusion, these findings suggest that VEGF gene SNPs studied are not associated with the severity of diabetic retinopathy.

Interleukin-18 promoter polymorphism associates with the occurrence of sudden cardiac death among Caucasian males: The Helsinki Sudden Death Study

Objective The increased plasma concentrations of pro-atherogenic and cardiomyocyte hypertrophic cytokine interleukin 18 (IL-18) predict mortality in patients with coronary heart disease (CHD) in addition to predicting the outcome of heart failure. The IL-18 gene has a functional −137 G/C polymorphism (rs187238) in the promoter region. The C allele carriage is associated with attenuated IL-18 production. The effect of IL-18 genotype on SCD is unknown. We studied the association of the IL-18 gene −137 G/C polymorphism with the occurrence of sudden cardiac death (SCD). Methods Using the TaqMan 5′ nuclease assay, we genotyped two independent consecutive and prospective autopsy series which were included in the Helsinki Sudden Death Study. Results Of the 663 men, 359 (54.1%) had the wild-type GG-genotype, 261 (39.4%) were heterozygotes (CG) and 43 (6.5%) were CC homozygotes. Compared to the GG homozygotes, the C allele carriers (i.e. subjects having CC or CG genotypes) had a lower adjusted risk for SCD from any cause (odds ratio [OR] 0.49; 95% confidence interval [CI], 0.31–0.77, p = 0.002), for SCD due to CHD (OR 0.51; 95% CI, 0.32–0.82, p = 0.005), and for SCD caused by non-coronary heart diseases (OR 0.34; 95% CI 0.13–0.90, p = 0.030). Conclusion IL-18 promoter −137 G/C polymorphism, which regulates the expression of IL-18, is an important predictor of SCD from any cause as well as SCD in patients with and without underlying CHD.

Association of Methylenetetrahydrofolate Reductase Gene Polymorphisms and Sex-Specific Survival in Patients With Metastatic Colon Cancer

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating intracellular folate levels, which affects DNA synthesis and methylation. Two MTHFR gene polymorphisms, C677T and A1298C, are linked to altered enzyme activity. Several studies have shown these two polymorphisms to be associated with response to fluorouracil (FU) -based treatment in advanced colon cancer patients, but data are inconsistent and contradictory. Meanwhile, epidemiologic studies demonstrated that these MTHFR polymorphisms were associated with cancer risk in a sex-specific manner. We tested the hypothesis of whether these two polymorphisms are associated with sex-specific clinical outcome in metastatic colon cancer patients treated with FU-based chemotherapy. This study included 318 patients (177 men and 141 women) with metastatic colon cancer treated between 1992 and 2003 at the University of Southern California/Norris Comprehensive Cancer Center or Los Angeles County/University of Southern California Medical Center. Peripheral blood samples were collected from each patient, and genomic DNA was extracted from WBCs. Two MTHFR gene polymorphisms (C677T and A1298C) were tested by fluorogenic 5'-nuclease assay. The A1298C polymorphism showed statistically significant differences in overall survival (OS) in female, but not male, patients with metastatic colon cancer (log-rank test, P = .038). Among females, OS was greater for patients with the A/A genotype (n = 67; median OS, 18.4 months) compared with patients with the A/C genotype (n = 50; median OS, 13.9 months) or C/C genotype (n = 10; median OS, 15.6 months). Although preliminary, these data support the role of the A1298C polymorphism in MTHFR as prognostic marker in female patients with metastatic colon cancer. Further studies are needed to confirm these findings.

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 10(10)R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

Polyunsaturated fat in the methionine-choline-deficient diet influences hepatic inflammation but not hepatocellular injury

Methionine-choline-deficient (MCD) diets that cause steatohepatitis in rodents are typically enriched in polyunsaturated fat. To determine whether the fat composition of the MCD formula influences the development of liver disease, we manufactured custom MCD formulas with fats ranging in PUFA content from 2% to 59% and tested them for their ability to induce steatohepatitis. All modified-fat MCD formulas caused identical degrees of hepatic steatosis and resulted in a similar distribution of fat within individual hepatic lipid compartments. The fatty acid composition of hepatic lipids, however, reflected the fat composition of the diet. Mice fed a PUFA-rich MCD formula showed extensive hepatic lipid peroxidation, induction of proinflammatory genes, and histologic inflammation. When PUFAs were substituted with more saturated fats, lipid peroxidation, proinflammatory gene induction, and hepatic inflammation all declined significantly. Despite the close relationship between PUFAs and hepatic inflammation in mice fed MCD formulas, dietary fat had no impact on MCD-mediated damage to hepatocytes. Indeed, histologic apoptosis and serum alanine aminotransferase levels were comparable in all MCD-fed mice regardless of dietary fat content. Together, these results indicate that dietary PUFAs promote hepatic inflammation but not hepatotoxicity in the MCD model of liver disease. These findings emphasize that individual dietary nutrients can make specific contributions to steatohepatitis.

Genetic polymorphisms in the vascular endothelial growth factor gene and breast cancer risk. The Austrian “tumor of breast tissue: incidence, genetics, and environmental risk factors” study

Vascular endothelial growth factor (VEGF) is a key regulator of tumor-induced angiogenesis and is required for growth of tumors. We tested the hypothesis that VEGF gene polymorphisms may be associated with breast cancer. We performed a case-control study including 804 female incident breast cancer patients and 804 female age-matched healthy control subjects. We selected seven VEGF candidate polymorphisms and determined genotypes by 5'-nuclease (TaqMan) assays. Furthermore, VEGF plasma levels and genotypes were analyzed in a group of 81 healthy volunteers (64 men and 17 women). Haplotype analysis showed two separate blocks of high-linkage disequilibrium, formed by five polymorphisms upstream of the coding sequence (promoter and 5' untranslated region) and two polymorphisms downstream of the coding sequence. None of the single polymorphisms or haplotypes was significantly associated with the presence of breast cancer. After Bonferroni correction for multiple testing, only one statistical signifcant association between VEGF genotypes and haplotypes and tumor characteristics was observed (-634C allele and small tumor size; p < 0.001). In a multivariate regression analysis including sex, age, VEGF genotypes, and haplotypes as covariates and VEGF plasma level as dependent variable, none of the VEGF polymorphism or haplotypes was a significant predictor of VEGF plasma levels. Our findings do not support the hypothesis that VEGF polymorphisms are associated with breast cancer risk. The association of the VEGF -634C allele with small tumor size is in clear contrast to a previous publication and should be interpreted with caution until replicated by additional studies.

Lack of association between endothelial nitric oxide synthase gene polymorphisms, microalbuminuria and endothelial dysfunction in hypertensive men

The Glu298Asp, T786C and 4a/4b genetic polymorphisms within the endothelial nitric oxide synthase (e-NOS) gene may predispose to hypertension, ischaemic heart disease and renal damage, possibly by reducing the generation of nitric oxide (NO), a fundamental substance in renal and cardiovascular biology. That same mechanism may contribute to raise albuminuria, a correlate of endothelial dysfunction and a marker of early kidney damage and poor cardiovascular prognosis in patients with hypertension. To assess that hypothesis, we evaluated the association of albuminuria with eNOS genotypes and their interacting potential with the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism. We also tested their impact on systemic NO availability, as reflected by endothelial-mediated forearm vasodilatation. Albuminuria (three overnight collections), blood pressure, body mass index, renal function, glucose, lipids and prevalence of the metabolic syndrome were measured in 235 genetically unrelated, never-treated, uncomplicated white men with essential hypertension. Endothelial function was assessed in a patient subgroup (n = 94) by measuring plethysmographic forearm blood flow vasodilatation in response to intra-arterial acetylcholine with sodium nitroprusside as a control. Polymerase chain reaction or a 5' nuclease assay were used to characterize the eNOS and ACE I/D variants. Albuminuria or microalbuminuria (albuminuria > or = 15 microg/min) showed no association with eNOS polymorphisms either per se or after accounting for the co-existing ACE I/D genetic configuration. Forearm responses to acetylcholine did not differ by eNOS polymorphisms. Cardiovascular, renal, metabolic parameters were homogeneously distributed across different genetic backgrounds. eNOS polymorphisms apparently play no role in promoting hypertensive renal damage, and do not influence endothelial-mediated vasodilatation in never-treated men with essential hypertension.

Polymorphism of XRCC1 predicts overall survival of gastric cancer patients receiving oxaliplatin-based chemotherapy in Chinese population

Pharmacogenetic advances in cancer chemotherapy have the potential to predict clinical benefit to particular regimens. Platinum agents have shown to be effective in the treatment of gastric cancer. We assessed whether single nucleotide polymorphisms (SNPs) in xeroderma pigmentosum group D (XPD), X-ray repair cross complementing group 1 (XRCC1) and glutathione S-transferase P1 (GSTP1) predicted overall survival in gastric cancer patients receiving oxaliplatin-based chemotherapy in Chinese population. SNPs of XPD-751, XRCC1-399 and GSTP1-105 in 62 gastric cancer patients were evaluated using the TaqMan 5' nuclease assay. Genotypes were correlated to survival. The median overall survival time was 322 days (range: 56-2058 days). The median survival times for patients with Arg/Arg or Arg/Gln genotypes of XRCC1 gene were significantly longer than others (P=0.03). For 58 patients with ECOG PS (Eastern Cooperative Oncology Group performance status)<or=2, more obvious significance was demonstrated (P=0.002). Patients with XRCC1-399 Gln/Gln genotype demonstrated a significant worse survival. No significant association was found between SNPs of XPD-751, GSTP1-105 and survival (P=0.125, 0.475, respectively). XRCC1 genotyping might make tailor chemotherapy possible for gastric cancer patients treated with oxaliplatin-based chemotherapy.

Evaluation of beta globin mRNA as an early marker of haemoglobin response to epoetin treatment

Approximately 60% of anaemic cancer patients respond to epoetin treatment. An early marker of response would be valuable in order to avoid ineffective treatment. We have previously shown that beta globin mRNA increases rapidly after epoetin beta treatment of healthy controls. In the present study we have evaluated whether a change of this marker during the first 2 weeks of epoetin treatment could predict later Hb response in anaemic cancer patients. Twenty cancer patients with Hb <11 g/dl received epoetin beta (NeoRecormon) 10,000 IU three times weekly during 6 weeks. Hb, reticulocytes and beta-globin mRNA were followed. The latter was measured quantitatively using PCR via the 5' nuclease assay. Eleven patients responded with a Hb increase of >1 g/dl, nine were nonresponders. All responders increased in beta-globin mRNA within 2 weeks, mean 7.7 x base-line. With a cut-off of an increase of 3 x base-line value, we obtained a specificity of 45% and a sensitivity of 91% for the prediction of a later increase of Hb >1 g/dl. With a cut-off of 4x base-line, the specificity increased to 66%, but the sensitivity decreased to 82%. Beta globin mRNA increases before Hb in all responding patients. However, some non-responding patients also show an increase, and there is a trade-off between specificity and sensitivity as the cut-off level is set at different levels. Compared to reticulocyte count, beta-globin mRNA is more reliable in the individual patient, but the clinical usefulness of the assay needs to be evaluated in further studies.