WBC DNA Research Articles

PATH-24. DETECTION OF POINT MUTATIONS AND GENE FUSIONS FROM CIRCULATING CELL-FREE DNA (CFDNA) OF GLIOBLASTOMA (GBM) PATIENTS

Abstract BACKGROUND GBM is characterized by intratumoral heterogeneity. Tumor heterogeneity, clonal diversity and mutation acquisition hamper the ability to tailor personalized therapy for GBM. Tumor sampling has limited ability to accurately capture the molecular landscape of the tumor and to disclose acquired molecular aberrations. Mutation analysis of cfDNA is a non-invasive procedure which may overcome these limitations as it may reflect the real composition of the tumor and track the molecular evolution. We sequenced cfDNA of GBM patients and assessed mutation patterns and fusion genes. METHODS We collected blood and respective tumor samples from 27 GBM patients and blood samples from 14 healthy controls. Tumor DNA, cfDNA and WBC DNA were sequenced using deep sequencing procedures. The data were analyzed for detection of single nucleotide polymorphism (SNPs) and gene-gene fusions. RESULTS GBM cfDNA concentrations were significantly elevated (median: 23.63 ng/mL; range 12.6–137) compared to healthy controls (median 2.06; range 1.68–7.62) (p < 0.0001). We identified unique SNPs in each glioma patient’s cfDNA and the corresponding tumor DNA including the top-10 most frequently mutated genes in GBM. For example, mutation of TP53 was detected in18.75%; EGFR in 37.5%; NF1-12.5%; LRP1B-25% and IRS4 in 25%. For gene-gene fusion we used the in-house fusion gene database, ChiTaRS 5.0, and identified fusions in cfDNA and tumor DNA. Thus, KMT2A-FLNA was the most frequent fusion found in 16.4% of samples. BCR-ABL1 in 8.82% and FGFR1-BCR in 2.94%. Other fusions included COL1A1-PDGFB (5.88%), NIN-PDGFRB (5.88%), KIF5B-RET (5.88%) and also TPM3-ROS1(2.94%), TFG-ALK(2.94%), MSN-ALK (2.94%) and NPM1-ALK (2.94%) which may be targeted by brain penetrating drugs that are ROS1 and ALK inhibitors. CONCLUSIONS Our study suggests that plasma cfDNA analysis may help to uncover real time mutational and gene fusion status of GBM by a non-invasive procedure. It may identify drug targets based on personalized gene-gene fusions.

Clinical significance of pathogenic variants in germline BRCA wild type patients at risk for hereditary breast cancer.

e13127 Background: Approximately, 10% of breast cancer (BC) are related to inherited germline mutations. BRCA1/2, the most recognized and tested genes, are responsible for 50% of hereditary BC. Genetic testing for hereditary BC has changed significantly. Increasing evidence suggests parallel multigene testing. Methods: NGS-based germline BRCA status assessment was performed on 209 high risk BC patients with at least one of the following risk factors: triple negative BC, early onset ( < -45), with a family history of BC, bilateral BC and male BC. Multigene-panel testing was subsequently offered to patients with at least 2 of the risk factors and WT germline BRCA. Capture-base targeted sequencing was performed on white blood cells using a panel consisting of 53 hereditary cancer-related genes, spanning 229kb of human genome. Results: Among the 209 patients screened, only 12 patients had pathogenic BRCA1/2 mutation. Next, we investigated the prevalence of non- BRCA pathogenic germline pathogenic mutations in patients with at least 2 risk factors. Thirty-seven patients met the criteria and only 23 patients had sufficient WBC DNA for sequencing. This cohort had a median age of 42, with a majority carrying infiltrating ductal carcinoma. Except for one bilateral BC patient who had stage IV disease, all other patients had early stage disease. We identified 5 pathogenic mutations from 5 patients spanning 4 genes: PALB2, PTEN, ATM and WRN, resulting in a prevalence rate of 20% for pathogenic germline mutations in high risk germline BRCA WT BC patients. Two patients carried mutations in PALB2, one with a frameshift and another with a splice mutation. Mutation types for PTEN, ATM and WRN were splice mutation, stop gain mutation and frameshift mutation, respectively. All 5 patients were diagnosed with BC before the age of 40. Three of them had bilateral BC; one had triple negative BC and another patient had a family history of BC. Conclusions: Our study confirms the clinical significance of testing non- BRCA genes, and suggests multigene panel testing for patients at risk for hereditary BC. Such approach increases the identification of hereditary BC, thus impacting clinical decision-making.

Cell-free DNA analysis in renal cell carcinoma: Comparison with tumor sequencing and correlation with response to therapy.

655 Background: Massively parallel sequencing (MPS) of circulating-free DNA (cfDNA) is seeing increasing use in multiple cancer types. There is little data on its use in metastatic renal cell carcinoma (mRCC) as a tool for prognostication and disease monitoring. Methods: cfDNA was extracted from 63 blood samples of 40 metastatic RCC patients (pts). Serial samples were obtained in 12 of 40 (30%) pts (median = 1, range = 1-7). cfDNA was used for targeted MPS using a custom bait-set of 27 genes commonly mutated in RCC. Variants observed in at least 3 reads, in both read directions, and at an allele frequency (AF) of ≥0.5% for single nucleotide variants (SNV), or in 2 reads and AF of ≥0.2% for small indels, were candidate variants validated by Sanger sequencing or amplicon MPS (aMPS). All mutations identified in cfDNA were also assessed in matched patient WBC DNA using aMPS and Sanger sequencing. Tumor specimens from 23 pts were also sequenced in parallel using our institutional OncoPanel assay that assesses 275-447 cancer-associated genes and results were compared with those seen in the cfDNA. Results: Thirty-one of 38 (82%) candidate variants were validated in 17 of 40 pts. Ten of those (32%) from 10 pts were also detected in WBC DNA, 3 of which were germline and 7 were at low mosaic frequency and likely reflected clonal hematopoiesis (CH). The remaining 21 variants validated in cfDNA were in TP53 (6), PBRM1 (3), SETD2 (3), VHL (2), ATM (2), NF2 (2), PTEN (1), PIK3CA (1), and MTOR (1). Two of 17 (12%) pts without tumor mutation analysis had 4 validated variants seen in cfDNA only. 10 of 23 (43%) pts with tumor mutation analysis had one or more variants seen in both tumor DNA and cfDNA. Three of the 23 had mutations seen only in cfDNA. Pts with any mutation in cfDNA (n = 14) had a significantly shorter overall survival in comparison to those without a finding (p < 0.001). Among 12 pts with serial samples, 5 had cfDNA variants identified. Response to therapy correlated with variant prevalence in all 5, including 2 with significant partial responses. Conclusions: This study suggests that paired tumor–cfDNA analysis has value in the assessment of response to therapy in RCC. Further analysis is proceeding.

Relationships between Global DNA Methylation in Circulating White Blood Cells and Breast Cancer Risk Factors.

It is not yet clear whether white blood cell DNA global methylation is associated with breast cancer risk. In this review we examine the relationships between multiple breast cancer risk factors and three markers of global DNA methylation: LINE-1, 5-mdC, and Alu. A literature search was conducted using Pubmed up to April 1, 2016, using combinations of relevant outcomes such as “WBC methylation,” “blood methylation,” “blood LINE-1 methylation,” and a comprehensive list of known and suspected breast cancer risk factors. Overall, the vast majority of reports in the literature have focused on LINE-1. There was reasonably consistent evidence across the studies examined that males have higher levels of LINE-1 methylation in WBC DNA than females. None of the other demographic, lifestyle, dietary, or health condition risk factors were consistently associated with LINE-1 DNA methylation across studies. With the possible exception of sex, there was also little evidence that the wide range of breast cancer risk factors we examined were associated with either of the other two global DNA methylation markers: 5-mdC and Alu. One possible implication of the observed lack of association between global WBC DNA methylation and known breast cancer risk factors is that the association between global WBC DNA methylation and breast cancer, if it exists, is due to a disease effect.

DNA methylation as a promising landscape: A simple blood test for breast cancer prediction.

Breast cancer is the most common malignancy among women worldwide. Risk assessment is one of the main services delivered by cancer clinics. Biomarker analysis on different tissues including the peripheral blood can provide crucial information. One of the potential epigenetic biomarkers (epimarkers) is introduced as the peripheral blood DNA methylation pattern. This study was conducted to evaluate the potential value of peripheral blood epimarkers as an accessible tool to predict the risk of breast cancer development. WBC's DNA was the focus of several case-control studies at both genome wide and candidate gene levels to reveal epigenetic changes accounting for predisposition to breast cancer, leading to suggest that ATM, TITF1, SFRP1, NUP155, NEUROD1, ZNF217, DBC2, DOK7 and ESR1 genes and the LINE1, Alu and Sat2 DNA elements could be considered as the potential epimarkers. To address that by which mechanisms WBC's DNA methylation patterns could be linked to the propensity to breast cancer, several contemplations have been offered. Constitutional epimutation during embryonic life, and methylation changes secondary to either environmental exposures or tumor-mediated immune response, are the two main mechanisms. One can deduce that epimarkers based on their potential properties or regulatory impacts on cancer-related genes may be employed for risk prediction, prognosis, and survival inferences that are highly required for breast cancer management toward personalized medicine.

Parameters Associated with Significant Liver Histological Changes in Patients with Chronic Hepatitis B

This study aimed to evaluate factors associated with significant liver histological changes. Liver biopsies from 157 CHB patients were retrospectively analyzed. Only ALB was significantly correlated with advanced liver necroinflammatory (P = 0.001). Age, ALB, GLOB, AST, PLT, and PT were independent predictors of significant fibrosis (P = 0.002, P < 0.001, P = 0.001, P = 0.048, P < 0.001, and P = 0.001, resp.). AST, WBC, and HBV DNA were significantly correlated with advanced fibrosis in normal ALT patients (P < 0.001, P = 0.041, and P = 0.012, resp.) and age, ALB, GLOB, PLT, and PT in patients with abnormal ALT (P = 0.003, P < 0.001, P = 0.004, P < 0.001, and P = 0.002, resp.). Age, AST, GGT, PLT, and PT were significantly associated with advanced fibrosis in HBeAg+ patients (P = 0.01, P = 0.016, P = 0.027, P = 0.016, and P = 0.009, resp.) and ALB, GLOB, WBC, PLT, and PT in HBeAg− patients (P < 0.001, P = 0.004, P = 0.005, P < 0.001, and P = 0.035, resp.). PLT was an excellent predictor for cirrhosis (P < 0.001 and AUROC = 0.805). ALT was not predictive of advanced fibrosis for patients with HBeAg+ or HBeAg− (P = 0.273 and P = 0.599, resp.). PLT was an excellent predictor for cirrhosis in CHB patients. Liver histopathology can be recommended for chronic HBV carriers of older age, with normal ALT, lower PLT, and lower ALB.

Quantitative Analysis of Tissue and Developmental Stage-Specific Differences in DNA Methylation throughout the β-Globin Gene Locus by DNA MassARRAY,

Abstract 3196DNA methylation plays a fundamental role in developmental regulation of γ-globin gene expression. The baboon is an excellent animal model to study the role of DNA methylation in developmental globin gene regulation because 1) the sequence and arrangement of genes within the β-globin locus are highly conserved between baboon and man, 2) only simian primates exhibit true fetal-stage γ-globin expression, 3) large numbers of highly purified erythroid cells can be isolated from baboon fetuses derived from timed pregnancies at varying stages of gestation and from the bone marrows of adult baboons, and 4) treatment of baboons with DNA methyltransferase inhibitors induce abundant γ-globin expression in adult baboons. Bisulfite sequence analysis data has shown that DNA hypomethylation within the 5’ promoter regions of the ε- and γ-globin genes is associated with expression during the embryonic and fetal stages of development (Lavelle et al Blood Cell Mol Dis 36:269, 2006), and developmentally regulated, large extended domains of DNA hypomethylation surrounding expressed genes have been also reported (Lathrop et al Exp Hematol 37:807, 2009). Although analysis of DNA methylation by bisulfite sequence analysis is considered to be the “gold standard”, quantitative analysis by this method is laborious and limited by the ability to perform DNA sequence analysis of large numbers of clones isolated following PCR amplification and bisulfite modification. Recently, a new quantitative method, MassARRAY, has been developed that allows direct quantitative analysis of cytosine methylation within defined PCR amplicons, thus eliminating the need for the laborious sequence analysis of large numbers of clones and allowing more precise quantitative analysis. To develop MassARRAY technology as a tool for studies of DNA methylation of the β-globin gene locus we compared the ability of MassARRAY and bisulfite sequencing to quantitate differences in DNA methylation at 13 specific CpG sites spanning the baboon β-globin gene cluster from HS4 within the LCR to the 5’ β-globin gene region in peripheral WBC, primitive nucleated erythrocytes isolated from a baboon fetus at 40 d gestation, purified erythroid precursors from pre-switch fetal liver, post-switch fetal bone marrow (BM) and adult BM pre- and post-treatment with decitabine administered by either subcutaneous injection (0.5mg/ml sc; 10d) or orally (0.25mg/kg/2d per week; 8 weeks) in combination with tetrahydrouridine. PCR amplification of specific regions of the baboon β-globin gene locus from bisulfite-converted DNA was performed using primers containing the 5’ T7 and 10mer tags selected using Sequenom Epidesigner software. PCR conditions were optimized to yield single bands by agarose gel analysis in all reactions. PCR products were treated with shrimp alkaline phosphatase and in vitro transcription and RNase digestion performed, followed by analysis using the Maldi-TOF MassARRAY system (Sequenom). Quantitation of DNA methylation of specific CpG residues was performed using EpiTYPER software. To compare results obtained by MassARRAY and traditional bisulfite sequence analysis, PCR products from bisulfite-converted DNA were also cloned in the PCR4 vector and sequence analysis of at least 10 clones performed for each amplicon. Results of MassARRAY and bisulfite sequence analysis were in close agreement. Analysis of all sites in all samples yielded a mean difference of 12.3 + 9.2% dmC between these two techniques. Four CpG sites within the LCR region were highly methylated in WBC DNA compared to erythroid tissues. Developmental differences in DNA methylation in the 5’ ε- and γ-globin regions were associated with stage-specific expression of these genes. Modest differences (10–15% dmC) in DNA methylation of four CpG sites in the 5’ γ-globin gene region in BM erythroid cells isolated from baboons treated with oral low-dose decitabine-tetrahydrouridine in combination detected by DNA MassARRAY were consistent with the modest 15–20% elevations of fetal hemoglobin (HbF). In conclusion, quantitative analysis of DNA methylation by MassARRAY technology provides an alternative, less laborious, sensitive quantitative methodology for analysis of differences in DNA methylation throughout the β-globin gene locus during development and following treatment with HbF-inducing agents. Disclosures:No relevant conflicts of interest to declare.

Effect of WBC BRCA1 promoter methylation on ovarian cancer risk.

5029 Background: Recently, some studies have reported BRCA1 WBC hypermethylation to be associated with increased risk of breast cancer. We assessed WBC BRCA1 promoter methylations in ovarian (OC) and breast cancer (BC) patients and healthy controls. In an OC subgroup harboring WBC BRCA1 methylation, we confirmed promoter methylation status in normal tissue. Methods: WBC DNA from 899 OC patients, 425 BC patients and 719 healthy controls were analyzed for BRCA1 promoter methylation by methylation specific PCR. In addition, we analyzed WBC DNA from 256 OC and 393 BC patients in blood samples drawn prior to diagnosis in a population-based study. We also analyzed 24 BC patients with a family history (BRCAPRO scores > 80%; Manchester score >40) without BRCA1/2 mutations. Paraffin-embedded normal tissue from 5 OC patients harboring WBC BRCA1 methylation was analyzed for BRCA1 methylation status. Finally, to determine potential risk factors in cis, we investigated BRCA1 haplotype status in a sub-cohort of 10 individuals with WBC methylated BRCA1and 13 controls. Results: We detected WBC BRCA1 promoter hypermethylation in 2.4% of healthy controls and 3.1% of BC patients (all BC individuals). No difference in BRCA1 methylation incidence was recorded between BC patients with blood samples drawn before (4.1%) or after (2.1%) diagnosis. In contrast, we detected WBC BRCA1 methylation in 10.3% of OC patients having blood samples drawn at diagnosis. Among OC patients with blood sampling 0-13 years (median 4.6 y) prior to diagnosis, BRCA1 promoter methylation was detected in 6.6%. BRCA1 promoter methylation was associated with a non-significant elevated risk of BC (HR 1.302; 95% CI 0.697-2.431) but a significantly increased risk of OC in the cohort of patients with blood samples drawn at time of (OR 4.765; CI 2.814-8.069) or prior to (OR 2.937; CI 1.476-5.845) diagnosis. We confirmed BRCA1 promoter methylation in normal tissue from all 5 individuals analyzed, excluding WBC promoter methylation being due to circulating DNA contamination. No association between BRCA1methylation and promoter haplotype was found. Conclusions: WBC BRCA1 promoter methylation is associated with increased risk of ovarian cancer.This finding has clinical as well as biological implications..

Abstract 295: Detection of hypomethylation of LINE1 on peripheral white blood cells and plasma DNA in US hepatocellular carcinoma patients

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and is increasing in the United States. Epigenetic changes, including genomic DNA hypomethylation, have been implicated in cancer development by inducing chromosomal instability. In the present study, methylation of long interspersed nucleotide element-1(LINE1), a surrogate marker of global hypomethylation, in peripheral white blood cells and plasma DNA in 47 US HCC patients was detected by pyrosequencing. Correlations between LINE1 methylation, and clinical data and other biomarkers were also analyzed. Methylation of LINE1 in WBC DNA is statistically significantly correlated with that in plasma DNA (R=0.32, p=0.04). WBC DNA global hypomethylation was significantly decreased in subjects with hepatitis C (HCV) infection (p=0.05). Plasma folate levels were not correlated with plasma DNA LINE1 methylation (R=0.13, p=0.41) or with WBC LINE1 methylation (R=−0.19, p=0.24). However, WBC folate levels were statistically significantly associated with patients’ survival. HCC patients who survived at least 3 months after diagnosis had lower WBC folate compared to patients who died within 3 months (p=0.004). We are currently expanding the HCC cohort and also analyzing biomarkers of chemical exposure (polycyclic aromatic hydrocarbons and aflatoxin B1) to investigate associations with methylation changes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2011-295

CoQ10 conveys protection from oxidative stress in plasma but not skeletal muscle

CoQ10 conveys protection from oxidative stress in plasma but not skeletal muscle Annateresa Papazzo1, Louise Lexis2, Paul Lewandowski11Molecular Nutrition Unit, School of Medicine, Deakin University, Victoria, Australia; 2School of Human Biosciences, La Trobe University, Victoria, AustraliaAbstract: Coenzyme Q10 (CoQ10) is commonly consumed as an antiaging supplement at doses of 30&ndash;210 mg/day. The aim of the study was to determine if CoQ10 alters markers of antioxidant status, oxidative damage, and gene expression in aging skeletal muscle. Female guinea pigs aged 26 months were supplemented for 6 weeks with CoQ10 at a human equivalent dose of 10 mg/kg/day. Body weight, plasma CoQ10 concentration, and WBC DNA abasic sites were measured at weeks 0, 2, 4, and 6 of the supplementation period. At the end of supplementation, concentrations of skeletal muscle CoQ10, glutathione, malondialdehyde, protein carbonyls, DNA abasic sites, activities of catalase and glutathione peroxidase, and the gene expression of cyctochrome c oxidase subunits were measured. Dietary supplementation with CoQ10 elevated plasma CoQ10 levels (pre 73 &plusmn; 3 nmol/L, post 581 &plusmn; 15 nmol/L, P &lt; 0.05) and decreased abasic sites in WBC DNA (pre 16.8 &plusmn; 0.5 Ap/100000 bp, post 9.7 &plusmn; 0.4 Ap/100000 bp, P &lt; 0.05). In contrast, all of the measures made in skeletal muscle were not different between groups (P &gt; 0.05). These results indicate that dietary supplementation with CoQ10 at a dose of 10 mg/kg/day may be capable of increasing antioxidant protection and reducing oxidative damage in the plasma, but may have no effect in skeletal muscle.Keywords: coenzyme Q10, aging, skeletal muscle, oxidative damage, gene expression

A Prospective Study of Telomere Length Measured by Monochrome Multiplex Quantitative PCR and Risk of Non-Hodgkin Lymphoma

Telomere length plays an important role in the maintenance of chromosomal stability and in tumorigenesis. We hypothesized that telomere length in peripheral WBC DNA obtained from healthy individuals would be a predictor of future risk of developing non-Hodgkin lymphoma. Using a new assay to measure relative telomere length, monochrome multiplex quantitative PCR, which strongly correlates with telomere length measured by Southern blot (Spearman r = 0.91, P < 0.0001) and has high precision (coefficient of variation = 7%), we compared telomere length in peripheral WBC DNA in 107 incident male non-Hodgkin lymphoma cases and 107 matched controls within the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study cohort. Median (10th, 90th percentile) telomere length was 1.10 (0.79, 1.43) in cases and 1.02 (0.78, 1.26) in controls (P = 0.0017, Wilcoxon sign test). There was a strong dose-response relationship between quartiles of telomere length and risk of non-Hodgkin lymphoma overall [odds ratios (95% confidence intervals) by quartile: 1.0; 1.1 (0.4-2.7); 1.8 (0.7-4.9); and 3.6 (1.4-8.9); P trend = 0.003], and this association was similar across the most common non-Hodgkin lymphoma subtypes present in this study. These results suggest that longer telomere length may be a potential predictor for future risk of non-Hodgkin lymphoma.

Six-Year Follow-Up of Inhibitor Prone Hemophilia B Dogs Treated with Muscle and Liver-Directed AAV2 Mediated Factor IX Gene Therapy.

Hemophilia B, caused by factor IX (FIX) deficiency, is intensively studied as a model for gene therapy. Preclinical studies in mice, dogs and initial clinical trials have documented the feasibility of adeno-associated virus (AAV) mediated gene therapy for hemophilia B. The purpose of this study is to report 6 year follow-up of inhibitor prone hemophilia B dogs treated with muscle or liver directed AAV2 FIX gene therapy. Short term, one year follow-up of these dogs (n=6) has been previously reported. The dogs treated with liver directed AAV2 FIX have not had a single bleed requiring FIX replacement in the 6 years since vector administration. The dogs undergoing muscle directed gene therapy have continued to have a bleed frequency similar to untreated FIX deficient dogs. The whole blood clotting time (WBCT), activated clotting time (ACT), and activated partial thromboplastin time (APTT) have remained at the upper limits of the normal ranges for the six year follow-up in the two liver treated dogs. The FIX activity has remained stable between 4–10% in both liver treated dogs but undetectable in the dogs undergoing muscle directed therapy. The vector/FIX sequences have persisted in liver biopsies but were undetectable in WBC and sperm DNA. Administration of purified canine FIX protein resulted in an immune response to FIX in the muscle treated dogs but not the dogs treated with liver directed gene therapy, suggesting FIX tolerance had been established in the hemophilia B dogs with liver directed gene therapy. A complete clinical evaluation of the dogs undergoing liver directed gene therapy including CBC, serum chemistries, bile acid profile, hepatic MRI and CT scans and liver biopsy was normal with no evidence for tumor formation. These results demonstrate that AAV mediated liver directed gene therapy completely corrects the hemophilia phenotype without toxicity or inhibitor development in the inhibitor prone null mutation dogs for more than six years.

Outdoor air pollution and lung cancer: recent epidemiologic evidence.

Outdoor air pollution and lung cancer: recent epidemiologic evidence.

Risk assessment of environmental carcinogenesis using WBC DNA adducts

Environmental carcinogens are enzymatically activated to form intermediates that can react with cellular DNA and form DNA adducts. Several kind of carcinogens bind to several sites of DNA. The measurement of WBC DNA adducts is a useful indicator for environmental carcinogen exposure monitoring. The 32P-Postlabeling method is a most popular and very sensitive method for DNA adduct analysis. We can analyze 1 adduct/10(8) nucleotides. In this review, I show some data of DNA adduct analysis for PAH exposure. These results demonstrated that inter-individual variation was very large. There were some confounding factors, such as metabolism or repair variations. I also showed some limitations of DNA adduct analysis. The method of adduct analysis is very complicated with several steps. We need to improve the accuracy. Do the data from WBC explain the target organ, such as the lung or liver? Almost all previous studies have been cross-sectional. We need a large-sized cohort study to evaluate whether adducts are a predictor of cancer. DNA adducts should be an important factor in gene-environment interaction.

CD177 polymorphisms: correlation between high-frequency single nucleotide polymorphisms and neutrophil surface protein expression.

Human neutrophil antigen-2a (NB1) is located on NB1 glycoprotein, which is expressed on subpopulations of neutrophils. PRV-1 is a gene that is over-expressed in neutrophils from patients with polycythemia rubra vera. The gene encoding NB1 differs from PRV-1 at four nucleotides. The purpose of this study was to determine if PRV-1 and NB1 are alleles of the same gene or two separate genes; and, moreover, if they are alleles, to explore potential correlations to NB1 glycoprotein expression. Primer pairs were used to amplify WBC DNA in the regions surrounding the four NB1 polymorphic sites. The four resulting amplicons were sequenced. The size of the neutrophil population in each donor that stained brightly with CD177 antibody was assessed by flow cytometry. If PRV-1 and NB1 are separate genes, then all people should be heterozygous for the PRV-1/NB1 polymorphisms. Because 11 of 23 donors were homozygous for PRV-1 polymorphisms at all four sites, PRV-1 and NB1 are alleles of the same gene, CD177. When the sequenced exons were compared with PRV-1, 13 single nucleotide polymorphisms (SNPs) that result in amino acid changes were found. The G42C NB1 polymorphism, found in 10 donors, was the most common SNP. The size of the CD177 bright-staining neutrophil population was greater in 42C/C donors than in 42G/G donors (66 +/- 20% vs. 41 +/- 21%, p = 0.004). PRV-1 and NB1 are alleles of the polymorphic gene CD177. The most common SNP in bp 42 predicted an amino acid change that may have an effect on protein expression.

DNA adducts as markers of exposure to carcinogens and risk of cancer

DNA adducts as markers of exposure to carcinogens and risk of cancer

Formation and persistence of N7-methylguanine DNA adducts in the target pyloric tissue following chronic exposure to N-methyl-N′-nitro-N-nitrosoguanidine

Outbred 7-week old male Wistar rats were exposed for 21 days to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) via the drinking water and N7-methyl deoxyguanosine 3'-monophosphate (N7-MedGp) levels in DNA from the pyloric mucosa (target tissue) and white blood cells (wbc: non-target tissue) were determined by 32P-postlabelling. Exposure to MNNG resulted in the non-linear, dose-related formation of N7-medGp in both tissues. Adduct levels in the pyloric mucosa were determined to be 1058, 5.4 and 1.1 μmole N7-medGp mole-1 deoxyguanosine 3'-monophosphate (dGp) after exposure to 4.1, 0.62 and 0.006 mg MNNG kg-1 day-1 respectively whereas adduct levels in the wbc DNA were lower at 5.2, 0.52 and 0.68 μmoles N7-medGp mole-1 dGp after exposure to 4.1, 0.62 and 0.062 mg MNNG kg-1 day-1 respectively. In addition, the persistence of N7-medGp was investigated. Loss of adduct occurred rapidly, with a decrease of 87 and 97% respectively in target tissue and wbc DNA by 48 h after cessation of 4.1 mg MNNG kg-1 day-1 exposure; 14 days post-MNNG treatment, however, N7-medGp was still detectable (0.46 μmole N7-medGp mole-1 dGp) in pyloric mucosal DNA. The quantitation of N7-medGp after exposure to low doses of carcinogen, i.e. 0.006 mg MNNG kg-1 day-1, approaching environmentally relevant levels has not been previously reported, and indicates that the 32P-postlabelling assay developed here possesses sufficient sensitivity to quantitate N7- medGp in human DNA arising from environmental exposure to methylating agents.

P196 産業分子疫学へのバイオマーカーの応用に関する研究 : 1. コークス炉作業者の白血球DNA付加体と代謝酵素多型の関係

P196 産業分子疫学へのバイオマーカーの応用に関する研究 : 1. コークス炉作業者の白血球DNA付加体と代謝酵素多型の関係

Modulation of DNA adduct levels in human mononuclear white blood cells and granulocytes by CYP1A1, CYP2D6 and GSTM1 genetic polymorphisms

The CYP1A1, CYP2D6 and GSTM1 genes encode biotransforming enzymes involved in activation and detoxification of xenobiotics. Metabolically activated chemical compounds may interact with DNA and form adducts. In this study, the effect of the GSTM1, CYP1A1 exon 7 and CYP2D6 polymorphisms on DNA adduct levels was studied in 170 healthy volunteers. DNA adducts levels were measured by 32P-postlabelling in mononuclear white blood cells (WBC, lymphocytes and monocytes) and granulocytes collected in summer and winter. The influence of the genotype on the level of DNA adducts in both types of WBCs was observed only in summer samples. Individuals with GSTM1 deficient (null) genotype had significantly elevated level of adducts in mononuclear WBCs (p=0.045) and granulocytes (p=0.031) compared to GSTM1 positives. Higher adduct levels in carriers of combined GSTM1(null)/CYP1A1-Ile/Val genotype were found in both types of WBCs when compared to GSTM1(+)/CYP1A1-Ile/Ile genotype carriers (p=0.046 in granulocytes, p=0.092 in mononuclear WBCs). CYP2D6 wild-type homozygotes (EMs) and heterozygotes (HEMs) were shown to have significantly higher mononuclear WBC DNA adduct levels than mutant homozygotes (PMs) (p=0.037 and p=0.014). When confounding factors associated with PAH exposure were taken into account a statistically significant effect of CYP1A1 exon 7 polymorphism on DNA adduct levels was found (p=0.012 in mononuclear WBCs, p=0.043 in granulocytes). In a subgroup of current smokers (n=95) high DNA adduct levels in granulocytes were associated with GSTM1-(null) genotype, and increased adduct levels in mononuclear WBCs correlated with CYP2D6 EM and HEM genotypes. In winter samples the association between the genotype and DNA adduct levels was not observed.

Assignment of human thyrotropin-releasing hormone (TRH) receptor gene to chromosome 8.

The human gene encoding thyrotropin-releasing hormone receptor was assigned to chromosome 8, using human-Chinese hamster ovary somatic cell hybrids, analyzed by Southern hybridizations. Hybridization was carried out with a 32P-labeled fragment of the human TRH-R genomic DNA. Hybridization of this probe to a human specific 10.5-kb DNA fragment of EcoRI-digested WBC DNA was used to localize the human TRH-R gene. No hybridization, by contrast, was seen with this probe and hamster DNA after EcoRI treatment. Results from 18 somatic cell hybrids corroborated unequivocally that the human TRH-R gene can be assigned to human chromosome 8.