Chemistry

Total news: 39
Last news: June 23, 2007 18:22:18

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Background: Heteroduplex scanning techniques usually detect all heterozygotes, including common variants not of clinical interest. Methods: We conducted high-resolution melting analysis on the 24 exons of the ACVRL1 and ENG genes implicated in hereditary hemorrhagic telangiectasia (HHT). DNA in samples from 13 controls and 19 patients was PCR amplified in the presence of LCGreen® I, and all 768 exons melted in an HR-1® instrument. We used 10 wild-type controls to identify common variants, and the remaining samples were blinded, amplified, and analyzed by melting curve normalization and overlay. Unlabeled probes characterized the sequence of common variants. Results: Eleven common variants were associated with 8 of the 24 HHT exons, and 96% of normal samples contained at least 1 variant. As a result, the positive predictive value (PPV) of a heterozygous exon was low (31%), even in a population of predominantly HHT patients. However, all common variants produced unique amplicon melting curves that, when considered and eliminated, resulted in a PPV of 100%. In our blinded study, 3 of 19 heterozygous disease-causing variants were missed; however, 2 were clerical errors, and the remaining false negative would have been identified by difference analysis. Conclusions: High-resolution melting analysis is a highly accurate heteroduplex scanning technique. With many exons, however, use of single-sample instruments may lead to clerical errors, and routine use of difference analysis is recommended. Common variants can be identified by their melting curve profiles and genotyped with unlabeled probes, greatly reducing the false-positive results common with scanning techniques. - [Read more]

Background: CYP2C9 polymorphisms are associated with decreased S-warfarin clearance and lower maintenance dosage. Decreased expression of VKORC1 resulting from the –1639G>A substitution has also been implicated in lower warfarin dose requirements. We investigated the additional contribution of this polymorphism to the variance in warfarin dose. Methods: Sixty-five patients with stable anticoagulation were genotyped for CYP2C9 and VKORC1 with Tag-ItTM allele-specific primer extension technology. Plasma S-warfarin concentrations and warfarin maintenance dose were compared among patients on the basis of the VKORC1 –1639G>A genotype. Results: Eighty percent of CYP2C9*1/*1 patients stabilized on <4.0 mg/day warfarin had at least 1 VKORC1 –1639A allele. Mean warfarin doses (SD) were 6.7 (3.3), 4.3 (2.2), and 2.7 (1.2) mg/day for patients with the VKORC1 –1639GG, GA, and AA genotypes, respectively. Steady-state plasma concentrations of S-warfarin were lowest in patients with the VKORC1 –1639AA genotype and demonstrated a positive association with the VKORC1 –1639G allele copy number (trend P = 0.012). A model including VKORC1 and CYP2C9 genotypes, age, sex, and body weight accounted for 61% of the variance in warfarin daily maintenance dose. Conclusions: The VKORC1 –1639A allele accounts for low dosage requirements of most patients without a CYP2C9 variant. Higher plasma S-warfarin concentrations corresponding to increased warfarin maintenance dosages support a hypothesis for increased expression of the VKORC1 –1639G allele. VKORC1 and CYP2C9 genotypes, age, sex, and body weight account for the majority of variance in warfarin dose among our study population. - [Read more]

Background: The detection of circulating tumor cells (CTCs) may prove useful for screening, prognostication, and monitoring of response to therapy. However, given the large background of circulating cells, it is probably necessary to detect 1 cancer cell in >106 leukocytes. Although reverse transcription (RT)-PCR is potentially sensitive and specific enough to achieve this goal, success will require the use of appropriate mRNA markers. The goal of this study was to identify optimal marker combinations for detection of CTCs. Methods: An extensive literature and internet database survey was conducted to identify potential markers. We then used real-time quantitative RT-PCR to test for expression of selected potential markers in tissue samples from primary tumors of breast, colon, esophagus, head and neck, lung, and melanoma and normal blood samples. Markers with high expression in tumors and a median 1000-fold lower expression in normal blood were considered potentially useful for CTC detection and were tested further in an expanded sample set. Results: A total of 52 potential markers were screened, and 3–8 potentially useful markers were identified for each tumor type. The mRNAs for all but 2 markers were found in normal blood. Marker combinations were identified for each tumor type that had a minimum 1000-fold higher expression in tumors than in normal blood. Conclusions: Several mRNA markers may be useful for RT-PCR–based detection of CTCs from each of 6 cancer types. Quantification of these mRNAs is essential to distinguish normal expression in blood from that due to the presence of CTCs. Few markers provide adequate sensitivity individually, but combinations of markers may produce good sensitivity for detection of the presence of these 6 neoplasms. - [Read more]

Background: Definitive diagnosis of primary hyperoxaluria type 1 (PH1) requires analysis of alanine:glyoxylate aminotransferase (AGT) activity in the liver. We have previously shown that targeted screening for the 3 most common mutations in the AGXT gene (c.33_34insC, c.508G>A, and c.731T>C) can provide a molecular diagnosis in 34.5% of PH1 patients, eliminating the need for a liver biopsy. Having reviewed the distribution of all AGXT mutations, we have evaluated a diagnostic strategy that uses selected exon sequencing for the molecular diagnosis of PH1. Methods: We sequenced exons 1, 4, and 7 for 300 biopsy-confirmed PH1 patients and expressed the identified missense mutations in vitro. Results: Our identification of at least 1 mutation in 224 patients (75%) and 2 mutations in 149 patients increased the diagnostic sensitivity to 50%. We detected 29 kinds of sequence changes, 15 of which were novel. Four of these mutations were in exon 1 (c.2_3delinsAT, c.30_32delCC, c.122G>A, c.126delG), 7 were in exon 4 (c.447_454delGCTGCTGT, c.449T>C, c.473C>T, c.481G>A, c.481G>T, c.497T>C, c.424-2A>G), and 4 were in exon 7 (c.725insT, c.737G>A, c.757T>C, c.776 + 1G>A). The missense changes were associated with severely decreased AGT catalytic activity and negative immunoreactivity when expressed in vitro. Missense mutation c.26C>A, previously described as a pathological mutation, had activity similar to that of the wild-type enzyme. Conclusions: Selective exon sequencing can allow a definitive diagnosis in 50% of PH1 patients. The test offers a rapid turnaround time (15 days) with minimal risk to the patient. Demonstration of the expression of missense changes is essential to demonstrate pathogenicity. - [Read more]

Background: Drug metabolism is a multistep process by which the body disposes of xenobiotic agents such as therapeutic drugs. Genetic variation in the enzymes involved in this process can lead to variability in a patient’s response to medication. Methods: We used molecular-inversion probe technology to develop a multiplex genotyping assay that can simultaneously test for 1227 genetic variants in 169 genes involved in drug metabolism, excretion, and transport. Within this larger set of variants, we performed analytical validation of a clinically defined core set of 165 variants in 27 genes to assess accuracy, imprecision, and dynamic range. Results: In a test set of 91 samples, genotyping accuracy for the core set probes was 99.8% for called genotypes, with a 1.2% no-call (NC) rate. The majority of the core set probes (133 of 165) had ≤1 genotyping failure in the test set; a subset of 12 probes was responsible for the majority of failures (mainly NC). Genotyping results were reproducible upon repeat testing with overall within- and between-run variation of 1.1% and 1.4%, respectively—again, primarily NCs in a subset of probes. The assay showed stable genotyping results over a 6-fold range of input DNA. Conclusions: This assay generates a comprehensive assessment of a patient’s metabolic genotype and is a tool that can provide a more thorough understanding of patient-to-patient variability in pharmacokinetic responses to drugs. - [Read more]

Background: Soluble CD40 ligand (sCD40L) was suggested as a novel biomarker of cardiovascular risk. We examined the effect of preanalytical variation on the measurement of sCD40L concentration. Methods: From healthy control individuals (n = 20) and patients with acute coronary syndrome (ACS) (n = 20) or sepsis (n = 20), we obtained blood drawn into 5 tubes containing citrate or a mixture of citrate, theophylline, adenosine, and dipyridamole (CTAD). The tubes were incubated for 30 min at room temperature or 0 °C before a single or double centrifugation (15 min, 2500g) at room temperature or 4 °C, respectively. sCD40L, ß-thromboglobulin (ßTG), and platelet factor 4 (PF4) concentrations were measured using immunoassays. Results: Concentrations of sCD40L were very low in all CTAD and citrated samples maintained at 0 °C (median ≤0.076 µg/L). Although increased ßTG and PF4 confirmed disease-related in vivo platelet activation, sCD40L was not higher in patients than in controls. In contrast, if the samples were processed at room temperature, sCD40L was significantly higher in ACS patients than in controls (P <0.02 in CTAD and citrated plasma at room temperature). Moreover, the ßTG:PF4 ratio decreased in patient but not control CTAD samples, suggesting a greater susceptibility of patient platelets to in vitro activation. Conclusions: Increased sCD40L concentrations resulted from in vitro platelet activation during sample preparation. Disease-related in vivo activation did not contribute to sCD40L concentrations in plasma. Therefore, published studies of sCD40L demand cautious interpretation, because their preanalytical conditions were not standardized. - [Read more]

Background: The cell adhesion molecule P-selectin has an important role in the pathophysiology of thrombosis. The effect on venous thromboembolism (VTE) of increased circulating concentrations of soluble P-selectin (sP-selectin) and their association with the P-selectin variant Thr715Pro is still uncertain. Methods: This study was a case-control study of 116 patients with confirmed recurrent VTE and at least 1 event of unprovoked deep venous thrombosis or pulmonary embolism, and 129 age- and sex-matched healthy individuals. We measured sP-selectin by ELISA and P-selectin gene (SELP) variation by genotyping and sampled blood after a mean interval of 2.55 years after the most recent VTE event. Results: The mean (SD) sP-selectin concentration was higher in patients than in controls: 47.3 (15.0) µg/L vs 36.8 (11.0) µg/L, P <0.001. The unadjusted odds ratio (OR) for sP-selectin >55.1 µg/L, representing the 95th percentile for controls, was 8.5 (95% CI, 3.7–23.3; P <0.001) and increased after adjustment for factor V Leiden, the prothrombin G20210A variant, increased factor VIII, and hyperhomocysteinemia (OR, 10.6; 95% CI, 4.1–31.2; P <0.001). Pro715 carriers were more prevalent among controls than patients (21.7% vs 14.7%). sP-selectin concentrations were lower in this subgroup than in noncarriers: 31.3 (7.9) µg/L vs 44.1 (14.1) µg/L; P <0.001). Conclusions: Increased sP-selectin concentrations are associated with VTE and genotype status. sP-selectin concentrations are lower in individuals carrying the P-selectin Pro715 variant than in those without this variant. - [Read more]

Background: The complexity and diversity of the antibody immune response to the antigen repertoire of a pathogen has long been appreciated. Although it has been recognized that the detection of antibodies against multiple antigens dramatically improves the clinical sensitivity and specificity of diagnostic assays, the prognostic value of serum reactivity profiles against multiple microbial antigens in protection has not been investigated. Methods: Using malaria as a model we investigated whether antigen reactivity profiles in serum of children with different levels of clinical immunity to Plasmodium falciparum malaria correlated with protection. We developed a microarray immunoassay of 18 recombinant antigens derived from 4 leading blood-stage vaccine candidates for P. falciparum [merozoite surface protein 1 (MSP1), MSP2, MSP3, and apical membrane antigen (AMA)-1]. Associations between observed reactivity profiles and clinical status were sought using k-means clustering and phylogenetic networks. Results: The antibody immune response was unexpectedly complex, with different combinations of antigens recognized in different children. Serum reactivity to individual antigens did not correlate with immune status. By contrast, combined recognition of AMA-1 and allelic variants of MSP2 was significantly associated with protection against clinical malaria. This finding was confirmed independently by k-means clustering and phylogenetic networking. Conclusions: The analysis of reactivity profiles provides a wealth of novel information about the immune response against microbial organisms that would pass unnoticed in analysis of reactivity to antigens individually. Extension of this approach to a large fraction of the proteome may expedite the identification of correlates of protection and vaccine development against microbial diseases. - [Read more]

Background: The use of MALDI-TOF mass spectrometry (MS) in quantitative glycan profiling has not been reported. In this study, we attempted to establish a high-throughput quantitative assay for profiling serum N-glycome, and we applied the new assay to identifying serum N-glycans for diagnosis of liver fibrosis and cirrhosis. Methods: N-glycans from whole serum proteins in 2 µL serum were released by enzymatic digestion, cleaned up by hydrophilic chromatography, and subsequently quantitatively profiled with a linear MALDI-TOF MS system, which was originally designed for quantitative proteomic profiling. Serum N-glycome profiles from 46 patients with chronic hepatitis B infection and with different degrees of liver fibrosis were examined. Results: The intra- and interassay CVs of peak intensities of the standard N-glycans were <8% and <17%, respectively. When the assay was applied to the analysis of serum N-glycome profiles, 17 peaks were found to be potential biomarkers for detection of liver fibrosis/cirrhosis. Linear regression analysis revealed that 4 peaks of 1341.5, 1829.7, 1933.3, and 2130.3 m/z (all P <0.005) had complementary value in detecting liver fibrosis and included them, but not any serological markers, in the diagnostic model. Leave-one-out cross-validation showed the diagnostic model could identify significant fibrosis (Ishak score ≥3) and cirrhosis (Ishak score ≥5), both at 85% accuracy. Conclusion: This is the first study to illustrate the quantitative aspect of MALDI-TOF MS in N-glycome profiling and the first study to reveal the potential value of the serum N-glycan profile for identifying liver fibrosis. - [Read more]

Background: Plasma concentrations of B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are diagnostic and prognostic biomarkers of heart failure and are also increased in patients with chronic kidney disease (CKD). We examined the relevance of BNP and NT-proBNP as predictors of CKD progression. Methods: Of 227 nondiabetic patients with mild-to-moderate renal insufficiency, 177 patients ages 18–65 years were followed in a prospective multicenter cohort study for a period of ≤7 years. CKD progression was assessed by recording renal endpoints, defined as doubling of baseline serum creatinine or end-stage renal disease (ESRD) requiring renal replacement therapy. Results: BNP and NT-proBNP were significantly higher among 65 patients who reached the combined renal endpoint than among the 112 who did not [median (interquartile range) 61 (27–98) ng/L vs 39 (20–70) ng/L, P = 0.023, for BNP; 320 (117–745) ng/L vs 84 (44–176) ng/L, P <0.001, for NT-proBNP)]. Each increment of 1 SD in log-transformed BNP and NT-proBNP increased the risk of CKD progression by hazard ratios of 1.38 (95% CI 1.09–1.76, P = 0.009) and 2.28 (1.76–2.95, P <0.001), respectively. After adjustment for other established prognostic factors of CKD progression, NT-proBNP but not BNP remained a significant independent predictor of the combined renal endpoint. Conclusions: Increased BNP and NT-proBNP concentrations indicate an increased risk for accelerated progression of CKD to ESRD and may prove to be valuable biomarkers for the assessment of prognosis in patients with CKD. - [Read more]

Background: Microarray studies have identified different molecular subtypes of breast cancer with prognostic significance. To transition these classifications into the clinical laboratory, we have developed a real-time quantitative reverse transcription (qRT)-PCR assay to diagnose the biological subtypes of breast cancer from fresh-frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) tissues. Methods: We used microarray data from 124 breast samples as a training set for classifying tumors into 4 previously defined molecular subtypes: Luminal, HER2+/ER–, basal-like, and normal-like. We used the training set data in 2 different centroid-based algorithms to predict sample class on 35 breast tumors (test set) procured as FF and FFPE tissues (70 samples). We classified samples on the basis of large and minimized gene sets. We used the minimized gene set in a real-time qRT-PCR assay to predict sample subtype from the FF and FFPE tissues. We evaluated primer set performance between procurement methods by use of several measures of agreement. Results: The centroid-based algorithms were in complete agreement in classification from FFPE tissues by use of qRT-PCR and the minimized "intrinsic" gene set (40 classifiers). There was 94% (33 of 35) concordance between the diagnostic algorithms when comparing subtype classification from FF tissue by use of microarray (large and minimized gene set) and qRT-PCR data. We found that the ratio of the diagonal SD to the dynamic range was the best method for assessing agreement on a gene-by-gene basis. Conclusions: Centroid-based algorithms are robust classifiers for breast cancer subtype assignment across platforms and procurement conditions. - [Read more]

Background: TIMP-1 protein is a prognostic factor for recurrence-free and overall survival (OS) time in breast cancer. We evaluated the prognostic value of TIMP1 mRNA and a novel TIMP1 mRNA splice variant in 1301 primary breast cancer patients. Methods: We measured mRNA transcripts of full-length TIMP1 (TIMP1-v1) and the novel splice variant lacking exon 2 (TIMP1-v2) by use of real-time RT-PCR in frozen primary tumor samples. Transcript concentrations are correlated with histomorphological and biological factors, TIMP-1 protein, and distant metastasis-free survival (MFS) and OS time. Results: TIMP1-v1 and TIMP1-v2 alone were not informative with respect to predicting prognosis. However, the PCR assay designed to measure the combination of v1 + v2 showed that high concentrations of this combination were associated with good prognosis. In Cox multivariate regression analysis, which also included the traditional prognostic factors, increasing concentrations were independently associated with prolonged MFS (P = 0.004) and OS (P = 0.048). Including TIMP-1 protein and TIMP1-v1+v2 mRNA together in the multivariate model revealed that protein and mRNA were both independently associated with prognosis, with hazard ratios pointing in opposite directions. Conclusion: High concentrations of TIMP1-v1+2 mRNA are associated with good prognosis in patients with primary breast cancer. Since high concentrations of TIMP-1 protein are associated with poor prognosis, the presence of possible posttranscriptional mechanisms requires further investigation. - [Read more]

Background: We compared the diagnostic accuracy of brain natriuretic peptide (BNP) and amino-terminal proBNP (NT-proBNP) for diagnosis of preclinical and mild heart failure (HF). Methods: We assayed plasma NT-proBNP and BNP in 182 healthy controls and in a prospective cohort of 820 HF patients divided according to the American Heart Association/American College of Cardiology classification. These included 86 patients in stage A [mean (SE) ejection fraction 61% (1%); mean (SE) age 47 (2) years], 255 in stage B [65% (2%); 62 (1) years], 420 patients in stage C [35% (1%); 68 (1) years] and 59 in stage D [25% (1%); 74 (1) years]. Diagnostic accuracies of BNP and NT-proBNP were evaluated by ROC analysis, and a multivariate linear regression model was applied to predict HF staging. Results: Median BNP and NT-proBNP concentrations increased from stage A to D 57-fold and 107-fold, respectively. Both assays were accurate (P <0.001) in separating stage B from controls or stage A, and stage C from controls or stage A or B. NT-proBNP was more accurate (P <0.001) than BNP in differentiating stage C from stages A and B patients and controls and was a better predictor of HF classification in a model including age, sex, and renal function (P <0.001). Conclusions: Monitoring BNP or NT-proBNP enabled identification of asymptomatic patients at risk for the development of HF. NT-proBNP showed better accuracy than BNP for identifying mild HF. - [Read more]

Background: Increased concentrations of lipoprotein(a) [Lp(a)] have been considered a genetically determined risk factor for coronary artery and cerebrovascular disease. Only 2 small and conflicting studies have investigated the possibility of an association of peripheral arterial disease (PAD) with high serum Lp(a) concentrations and low molecular weight (LMW) phenotypes of apolipoprotein(a) [apo(a)]. Methods: We measured serum concentrations of Lp(a) and apo(a) phenotypes in 213 patients with symptomatic PAD and 213 controls matched for sex, age (within 2 years), and presence of diabetes. Results: Patients with PAD showed significantly higher median serum concentrations of Lp(a) (76 vs 47 mg/L; P = 0.003) and a higher frequency of LMW apo(a) phenotypes (41% vs 26%; P = 0.002) than controls. After adjustment for several potential confounders, increased Lp(a) concentrations (>195 mg/L, i.e., 75th percentile of the entire study sample) and LMW apo(a) phenotypes were significant predictors of PAD, with odds ratios of 3.73 (95% CI 2.08–6.67; P <0.001) and 2.21 (95% CI 1.33–3.67; P = 0.002), respectively. Conclusions: In this study sample, both increased serum concentrations of Lp(a) and the presence of LMW apo(a) phenotypes were associated with the presence of symptomatic PAD independent of traditional and nontraditional cardiovascular risk factors. Because PAD is considered an indicator of systemic atherosclerotic disease, our results suggest a possible role of Lp(a) as a genetically determined marker for systemic atherosclerosis. - [Read more]

Background: A prospective randomized trial in patients with Crohn disease studied whether 6-thioguanine nucleotide (6-TGN) concentration–adapted azathioprine (AZA) therapy is clinically superior to a standard dose of 2.5 mg/kg/day AZA. Methods: After 2 weeks of standard therapy, patients (n = 71) were randomized into standard (n = 32) or adapted-dose (n = 25) groups; 14 patients dropped out before randomization. In the adapted group, the AZA dose was adjusted to maintain 6-TGN concentrations between 250 and 400 pmol/8 x 108 erythrocytes (Ery). Response criteria were the number of patients in remission after 16 weeks without steroids (primary) and remission after 24 weeks, frequency of side effects, and quality of life (secondary). Results: After 16 weeks, 14 of 32 (43.8%) patients in the standard group vs 11 of 25 (44%) in the adapted group were in remission without steroids (intent-to-treat analysis). After 24 weeks, 43.8% vs 40% were in remission. No significant differences were found concerning quality of life, disease activity, 6-TGN concentrations, AZA dose, or dropouts due to side effects. Sixty-six patients had a wild-type thiopurine S-methyltransferase (TPMT) genotype, with TPMT activities of 8 to 20 nmol/(mL Ery x h). Five patients (dropouts after randomization) were heterozygous, with TPMT activities <8 nmol/(mL Ery x h). 6-Methyl mercaptopurine (6-MMP) concentrations >5700 pmol/8 x 108 Ery were not associated with hepatotoxicity. Conclusion: Standard and adapted dosing with the provided dosing scheme led to identical 6-TGN concentrations and remission rates. Adapted dosing had no apparent clinical benefit for patients with TPMT activity between 8 and 20 nmol/(mL Ery x h). Additionally, 6-MMP monitoring had no predictive value for hepatotoxicity. - [Read more]

Background: Plant-derived cardenolides reportedly possess anticancer properties in human leukemic cells via selective induction of apoptosis, cell cycle arrest, and differentiation. Selective induction of apoptosis with mammalian-derived digoxin-like immunoreactive factor (DLIF) could provide new strategies for anticancer drug development or the identification of biomarkers for cancer. We investigated whether DLIFs selectively induce apoptosis in human lymphoblastic leukemic cells. Methods: We compared the relative potencies of digoxin, ouabain, and DLIF on induction of programmed cell death in Jurkat cells (an acute T-leukemic cell line), K-562 (a myelogenous leukemia cell line), and nonpathologic human peripheral blood mononuclear cells (PBMCs). Apoptosis was measured by flow cytometry with the annexin V/propidium iodide method. Results: Digoxin and ouabain induced apoptosis in Jurkat cells [digoxin 50% inhibitory concentration (IC50), 24 nmol/L; ouabain IC50, 26 nmol/L]. Neither digoxin nor ouabain induced apoptosis in K-562 cells or PBMCs. DLIF was more potent (IC50, 1.9 nmol/L) and >2-fold more effective than digoxin or ouabain at inducing maximum apoptosis in Jurkat cells. The IC50 values in the apoptosis assays were >100-fold lower (DLIF) and 20-fold lower (digoxin and ouabain) than the IC50 required for Na+- and K+-dependent ATPase (DLIF, 200 nmol/L; digoxin, 910 nmol/L; ouabain, 600 nmol/L). Conclusion: DLIF selectively induces apoptosis in a human acute T-cell lymphoblastic leukemia cell line but not in K-562 cells or PBMCs. These data suggest a new physiological role for these endogenous hormone-like factors. - [Read more]