Peripheral Blood Cell DNA Research Articles

Dominance of intrinsic genetic factors in shaping the human immunoglobulin V λ repertoire

The expressed human immunoglobulin V λ repertoire demonstrates a strong bias in the use of individual V λ segments. Mechanisms that underlie such biases can be divided into two categories: intrinsic genetic processes that lead to the preferential rearrangement and/or expression of certain segments; and selection following light chain expression. Here, we have used two approaches to investigate the factors that shape the human V λ repertoire. Firstly, we characterised 136 V λ rearrangements (59 productive and 77 non-productive) amplified from the human genomic DNA of peripheral blood cells. Secondly, we analysed V λ segment use in a library of 2000 cDNA clones from a transgenic mouse containing a 380 kb region (including 15 functional V λ segments) from the human immunoglobulin λ locus. By hybridisation and sequencing we found that the patterns of use of human V λ segments in the transgenic mouse were similar to those found in the expressed human peripheral blood repertoire and in productive and non-productive genomic DNA rearrangements. These data indicate the importance of intrinsic genetic factors in shaping the human V λ repertoire and highlight the remarkable conservation of the molecular mechanisms involved in the production of the antibody repertoire in mouse and man. Therefore, transgenic mice represent a good model for analysis of the human antibody repertoire and for the production of human antibodies.

The factor V Leiden mutation in Japanese couples with recurrent spontaneous abortion.

Thrombosis of placental vessels can be a major cause of recurrent spontaneous abortion (RSA). The factor V Leiden (FVL) mutation, a single point mutation in the factor V gene, is the most common genetic predisposition to thrombosis in European countries and the United States. However, even among Caucasian populations, the association between the FVL mutation and RSA is still controversial. The objectives of the present study were to investigate the prevalence of the FVL mutation in Japanese women who have experienced RSA and to clarify the contribution of the FVL mutation to recurrent miscarriages. A total of 52 Japanese women with a history of three or more consecutive idiopathic first trimester miscarriages and 41 of their male partners were studied. The control group consisted of 55 parous women without obstetric complications. Peripheral blood cell DNA was examined for the presence of the FVL alleles by polymerase chain reaction with Mnl I restriction fragment length polymorphisms. None of the 52 women with RSA and the 41 partners carried the mutation. We also found no subject carrying the FVL alleles in the control group. These results suggest that the FVL alleles are not concentrated in women with RSA at least to clinically significant levels and that there is no apparent association between the FVL mutation and RSA in our Japanese population.

Lymphoproliferative responses to human herpesvirus-6 variant A and variant B in healthy adults

Human herpesvirus-6 (HHV-6) isolates can be classified into variants A and B, and over 95% of people older than 2 years of age are seropositive for either or both variants. However, the prevalence of the two HHV-6 variants is still not defined since the serological methods used at present cannot discriminate one variant from the other. Lymphoproliferative responses to glycine extracted cellular antigens from human herpes-virus-6 (HHV-6) GS strain (variant A)- and Z 29 strain (variant B)-infected T-lymphoid cell lines were examined in healthy Swedish and Japanese adults. Nine of 36 (25%) persons had responses to the GS antigen, while 21/36 (58%) had responses to the Z 29 antigen (P=0.008). Individuals with low anti-HHV-6 IgG titers (< or = 320) were more likely to respond to the Z 29 antigen than to the GS antigen (P=0.006), while there was no difference in those with high anti-HHV-6 IgG titers (> or =1280). Three of 7 Japanese adults had lymphoproliferative responses to the GS antigen compared with 6/29 Swedes (not significant), and 7/7 Japanese had lymphoproliferative responses to the Z 29 antigen compared with 14/29 Swedes (P=0.03). Lymphoproliferative responses were neither related with the presence of HHV-6 DNA nor related with the presence of HHV-7 DNA in peripheral blood cells. These results suggest a higher prevalence of HHV-6 variant B than variant A in both Swedes and Japanese adults, and possibly a difference in either the HHV-6 virus strains and/or the nature of immune response of Swede and Japanese.

Thiopurine methyltransferase genotype and the toxicity of azathioprine in Japanese.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that preferentially catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including Azathioprine (AZA). It has been reported that the level of AZA toxicity is dependent on the TPMT genotypes in Caucasian individuals; we thus investigated this relationship in Japanese. The TPMT genotype was determined using peripheral blood cell DNA obtained from 36 Japanese patients with rheumatic diseases who were treated with AZA, by polymerase chain reaction (PCR) technique. Duration of AZA administration, white blood cell counts before and after AZA administration, and side effects were investigated in each subject, and were compared between the patients with or without TPMT mutation. The TPMT allelotype was TPMT*1/TPMT*1 in 33 (91.7%) and TPMT*1/TPMT*3C in 3 (8.3%) individuals. All 3 patients (100%) with the mutant TPMT allele (TPMT*3C) discontinued AZA treatment due to leucopenia while only 4 patients (12%) without mutant TPMT alleles showed leucopenia (p=0.0049, Fisher's exact test). However, leucopenia developed relatively late in patients with mutant TPMT. The TPMT mutant allele, TPMT*3C, also exists in Japanese individuals, and the bone marrow toxicity of AZA is likely stronger in patients with this mutant allele.

Impact of CYP2E1 genotype in renal cell and urothelial cancer patients

Genetic polymorphisms of enzymes involved in carcinogen metabolism have been found to influence susceptibility to cancer. Ethanol-inducible CYP2E1 is an enzyme of major toxicological interest because it metabolizes several drugs, precarcinogens and solvents to reactive metabolites. In the present study, we investigated the cytochrome P450 2E1 genetic polymorphism in renal cell/urothelial cancer patients from two German regions, Jena and Halle, different with respect to their environmental pollution degree in comparison with healthy controls from the same regions. DNA of peripheral white blood cells was isolated both from 224 renal cell/urothelial cancer patients and 304 controls. We focussed on polymorphisms in the promoter region and intron 6 of the CYP2E1 gene. The polymorphisms were identified as RFLP's by amplification of the appropriate DNA fragment and subsequent digestion with the restriction enzymes PstI, RsaI and DraI. In Jena as well as in Halle, the frequency distributions of the PstI/RsaI, DraI and combined DraI + PstI/RsaI genotypes showed no significant differences between controls and renal cell/urothelial cancer patients. We did not find significant differences between Jena and Halle. 86.2% of all subjects with a homozygote PstI/RsaI genotype also carried a heterozygote DraI genotype, whereas 5.1% of the subjects with a heterozygote PstI/RsaI genotype also carried a heterozygote DraI genotype. Renal cell cancer as well as urothelial cancer risk was not elevated in patients with heterozygote DraI, PstI/RsaI and combined DraI + PstI/RsaI genotypes (odds ratios slightly insignificantly increased). Interestingly enough, an association between these polymorphisms and renal cell cancer risk was found in the female subgroup but not in the male subgroup. The basis of these sex-specifically increased risks are different frequencies concerning heterozygote and homozygote genotypes in controls and cancer patients. In controls, the heterozygote genotype frequency was lower in females than in males. In renal cell cancer patients, the results were quite the contrary. Summing up, our results demonstrate an lack between CYP2E1 genetic polymorphism and renal cell/urothelial cancer risk.

Exclusion of the First EGF Domain of Factor VII by a Splice Site Mutation Causes Lethal Factor VII Deficiency

We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5′ splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor–like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the ▵EGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans.© 1998 by The American Society of Hematology.

Exclusion of the First EGF Domain of Factor VII by a Splice Site Mutation Causes Lethal Factor VII Deficiency

AbstractWe have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5′ splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor–like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the ▵EGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans.© 1998 by The American Society of Hematology.

An oncogenic fusion product of the phosphatidylinositol 3-kinase p85beta subunit and HUMORF8, a putative deubiquitinating enzyme.

Peripheral blood cell DNA from a patient with a chronic myeloproliferative disorder was tested in the tumorigenicity assay. Upon tumor induction in nude mice we isolated a human oncogene by means of genomic cloning, exon trap analysis and cDNA cloning. Sequence analysis revealed a fusion product of the p85beta subunit of phosphatidylinositol (PI) 3-kinase and HUMORF8, a putative deubiquitinating enzyme, which has been generated during the DNA transfection process. Application of the tumorigenicity assay to various p85beta and HUMORF8 cDNA constructs indicated that the recombination of both genes rather than the truncation of one of the fusion partners renders the chimeric protein tumorigenic. Moreover, sequence analysis of human wildtype p85beta revealed an alanine for serine substitution at a site important for the regulation of the lipid kinase activity of PI 3-kinase in human p85alpha. This variation may relate to differences in the mode of signal transduction from both p85 isoforms.

Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations.

Molecular analysis of T cell receptor (TCR) genes is frequently used to prove or exclude clonality and thereby support the diagnosis of suspect T cell proliferations. PCR techniques are more and more being used for molecular clonality studies. The main disadvantage of the PCR-based detection of clonal TCR gene rearrangements, is the risk of false-positive results due to 'background' amplification of similar rearrangements in polyclonal reactive T lymphocytes. Therefore, PCR-based clonality assessment should include analyses that discern between PCR products derived from monoclonal and polyclonal cell populations. One such method is heteroduplex analysis, in which homo- and heteroduplexes resulting from denaturation (at 94 degrees C) and renaturation (at lower temperatures) of PCR products, are separated in non-denaturing polyacrylamide gels based on their conformation. After denaturation/renaturation, PCR products of clonally rearranged TCR genes give rise to homoduplexes, whereas in case of polyclonal cells heteroduplexes with heterogeneous junctions are formed. We studied heteroduplex PCR analysis of TCR gene rearrangements with respect to the time and temperature of renaturation and the size of the PCR products. Variation in time did not have much influence, but higher renaturation temperatures (>30 degrees C) clearly showed better duplex formation. Nevertheless, distinction between monoclonal and polyclonal samples was found to be more reliable at a renaturation temperature of 4 degrees C, using relatively short PCR products. To determine the sensitivity of heteroduplex analysis with renaturation at 4 degrees C, (c)DNA of T cell malignancies with proven clonal rearrangements was serially diluted in (c)DNA of polyclonal mononuclear peripheral blood cells and amplified using V and C primers (TCRB genes) or V and J primers (TCRG and TCRD genes). Clonal TCRB and TCRD gene rearrangements could be detected with a sensitivity of at least 5%, whereas the sensitivity for TCRG genes was somewhat lower (10-15%). The latter could be improved by use of Vgamma member primers instead of Vgamma family primers. We conclude from our results that heteroduplex PCR analysis of TCR gene rearrangements is a simple, rapid and cheap alternative to Southern blot analysis for detection of clonally rearranged TCR genes.

聚氯乙烯工人GST T1，GST M1及CYP2E1基因型與其肝功能之相關研究

Vinyl chloride monomer (VCM) is hepatotoxic as well as carcinogenic in humans. There are reports that exposure to VCM seems to induce abnormal liver function, liver fibrosis, cirrhosis, and angiosarcoma of the liver. In vivo, VCM is metabolized by cytochrome P450 2E1 (CYP2E1) to form chloroethylene oxide (CEO) and chloroactetaldehyde (CAA), both electrophilic metabolites, which may either cause cell damage or are further metabolized and detoxified by glutathione S-transferases (GSTs). This study investigated whether or not the genotypes, CYP2E1, glutathione S-transferase θ (GST T1) and μ (GST M1), correlated the abnormal liver function in vinyl chloride exposed workers. We enrolled 251 workers from 5 polyvinyl chloride plants for this study. The workers who were exposed to VCM were classified into two, high and low exposure groups and the degree of exposure was determined based on their job titles and airborne VCM concentration. The enzyme activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were used as the parameters of liver function. The genotypes, CYP2E1, GST T1 and GST M1, were determined by performing PCR and RFLP on peripheral white blood cell DNA, Other potential risk factors were also ascertained and the confounding effect was adjusted accordingly. Stratified analyses were used to explore the correlation between the alteration of liver function and the genotypes, CYP2E1, GST T1 and GST M1, among the workers exposed to different levels of VCM. The results obtained showed: (1)at low VCM exposure, the odds ratio (OR) of GST T1 non-null genotype on abnormal ALT was 3.8 (95% CI=1.2-14.5) but the CYP2E1 genotype was not associated with abnormal ALT; (2)at high VCM exposure, on the other band, a c2c2 CYP2E1 genotype was associated with increased OR on abnormal ALT (OR=4.8, 95% CI=0.6-34.8) but the GST T1 non-null genotype was not associated with abnormal ALT (OR=0.9, 95% CI=0.2-3,8); and (3)multiple logistic regression also showed strong interactions of the VCM exposure to CYP2E1 as well as to the GST T1 genotype. Thus, These observations suggest that the two genotypes, CYP2E1 and GST T1, may play important roles in the biotransformation of VCM, whose effect leads to liver damage.

The GST T1 and CYP2E1 genotypes are possible factors causing vinyl chloride induced abnormal liver function.

Vinyl chloride monomer (VCM) is hepatotoxic as well as carcinogenic in humans. There are reports that exposure to VCM seems to induce abnormal liver function, liver fibrosis, cirrhosis, portal hypertension, and angiosarcoma of the liver. In vivo, VCM is metabolized by cytochrome P450 2E1 (CYP2E1) to form the electrophilic metabolites, chloroethylene oxide (CEO) and chloroacetaldehyde (CAA), which may either cause cell damage or be further metabolized and detoxified by glutathione S-transferases (GSTs). This study investigated whether or not the genotypes CYP2E1, glutathione S-transferase theta (GST T1) and mu (GST M1) correlated with abnormal liver function found in vinyl chloride exposed workers. For this study, 251 workers from five polyvinyl chloride plants were enrolled. The workers were classified into two exposure groups (high and low) and the degree of exposure was determined based on their job titles and airborne VCM concentration. The activity of serum alanine aminotransferase (ALT) was used as the parameter of liver function. The genotypes CYP2E1, GST T1 and GST M1 were determined by polymerase chain reaction and restriction fragment length polymorphism on peripheral white blood cell DNA. Other potential risk factors were also ascertained and the confounding effect was adjusted accordingly. Stratified analyses were used to explore the correlation between the alteration of liver function and the genotypes CYP2E1, GST T1 and GST M1 among the workers exposed to different levels of VCM. The following results were obtained (1) at low VCM exposure, the odds ratio (OR) of positive GST T1 on abnormal ALT was 3.8 (95% CI 1.2-14.5) but the CYP2E1 genotype was not associated with abnormal ALT. (2) At high VCM exposure, a c2c2 CYP2E1 genotype was associated with increased OR on abnormal ALT (OR 5.4, 95% CI 0.7-35.1) and positive GST T1 was significantly associated with decreased OR on abnormal ALT (OR 0.3, 95% CI 0.1-0.9). (3) Multiple linear and logistic regression also showed strong interactions of the VCM exposure to CYP2E1 as well as to the GST T1 genotype. These observations suggest that the two genotypes, CYP2E1 and GST T1, may play important roles in the biotransformation of VCM, the effect of which leads to liver damage.

Alcoholism and alcoholic organ damage and genetic polymorphisms of alcohol metabolizing enzymes in Chinese patients

It is still not clear why some alcoholic patients acquire certain organ-specific complications of alcoholism whereas other alcoholic patients acquire different ones. As we know the liver alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and cytochrome P4502E1 (P4502E1) are polymorphic at the ADH2, ADH3, and ALDH2 loci and the 5'-flanking region of the P4502E1. The aim of this study was to investigate the differences between Chinese alcoholic patients with cirrhosis and acute pancreatitis by studying the genetic polymorphisms of ADH2, ADH3, ALDH2, and P4502E1. Genotyping of ADH2, ADH3, ALDH2, and P4502E1 was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods on peripheral white blood cell DNA from 75 alcoholic cirrhotic patients, 48 acute alcoholic pancreatitis patients, 19 heavy drinkers without liver disease or pancreatitis, and 235 controls. The results showed that the frequencies of the alleles ADH2*1 and ALDH2*1 in the alcoholic cirrhotic patients were significantly higher than those in the nonalcoholic controls. In acute alcoholic pancreatitis patients, only the frequency of allele ALDH2*1, not ADH2*1 was significantly higher than in the nonalcoholic controls. The allele frequency of ADH2*1 in acute pancreatitis patients was significantly lower (P < .01) than in alcoholic cirrhotic patients. The daily amount of alcohol consumption was significantly lower in patients with acute pancreatitis than in patients with cirrhosis (P < .0005). The genotype distributions of P4502E1, detected by RsaI and PstI, were not different among alcoholic cirrhotic patients, alcoholic pancreatitis patients, heavy drinker, and nonalcoholic controls. In conclusion, ALDH2*1 is the most important alcohol metabolizing gene affecting predisposition to alcoholism whereas the ADH2*2 gene may influence susceptibility to acute alcoholic pancreatitis. The patients with alcohol-induced cirrhosis and with alcohol-induced acute pancreatitis are of two different subpopulations.(Hepatology 1997 Jan;25(1):112-7)

Alcoholism and alcoholic organ damage and genetic polymorphisms of alcohol metabolizing enzymes in Chinese patients.

It is still not clear why some alcoholic patients acquire certain organ-specific complications of alcoholism whereas other alcoholic patients acquire different ones. As we know the liver alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and cytochrome P4502E1 (P4502E1) are polymorphic at the ADH2, ADH3, and ALDH2 loci and the 5'-flanking region of the P4502E1. The aim of this study was to investigate the differences between Chinese alcoholic patients with cirrhosis and acute pancreatitis by studying the genetic polymorphisms of ADH2, ADH3, ALDH2, and P4502E1. Genotyping of ADH2, ADH3, ALDH2, and P4502E1 was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods on peripheral white blood cell DNA from 75 alcoholic cirrhotic patients, 48 acute alcoholic pancreatitis patients, 19 heavy drinkers without liver disease or pancreatitis, and 235 controls. The results showed that the frequencies of the alleles ADH2*1 and ALDH2*1 in the alcoholic cirrhotic patients were significantly higher than those in the nonalcoholic controls. In acute alcoholic pancreatitis patients, only the frequency of allele ALDH2*1, not ADH2*1 was significantly higher than in the nonalcoholic controls. The allele frequency of ADH2*1 in acute pancreatitis patients was significantly lower (P < .01) than in alcoholic cirrhotic patients. The daily amount of alcohol consumption was significantly lower in patients with acute pancreatitis than in patients with cirrhosis (P < .0005). The genotype distributions of P4502E1, detected by RsaI and PstI, were not different among alcoholic cirrhotic patients, alcoholic pancreatitis patients, heavy drinker, and nonalcoholic controls. In conclusion, ALDH2*1 is the most important alcohol metabolizing gene affecting predisposition to alcoholism whereas the ADH2*2 gene may influence susceptibility to acute alcoholic pancreatitis. The patients with alcohol-induced cirrhosis and with alcohol-induced acute pancreatitis are of two different subpopulations.

Mitochondrial DNA mutations in pancreatic biopsy specimens from IDDM patients

We investigated the contribution of mitochondrial DNA mutations to the pathogenesis of IDDM by analyzing mitochondrial DNA in pancreatic biopsy specimens and peripheral blood cells from 18 patients with newly-diagnosed IDDM. All patients presented with typical abrupt onset of diabetes and ketosis on initial examination. Point mutations at nucleotides 3243, 3271 and 8344 were assayed by polymerase chain reaction and restriction fragment length polymorphism analysis or by mismatch-primer analysis. A common large deletion from nucleotides 8483–13459 was analyzed by a primer shift method. All of these mutations are known to be pathogenic mutations. However, none of the mitochondrial DNA mutations were detected in any of 18 IDDM patients. Several types of mitochondrial DNA mutation have been identified in the peripheral blood cells in some patients with non-insulin-dependent diabetes mellitus as well as in some with IDDM, however, our results suggest that abrupt-onset IDDM does not correlate with any of the known mitochondrial DNA mutations.

Cytochrome P450 2E1 and glutathione S-transferase M1 polymorphisms and susceptibility to hepatocellular carcinoma

Background & Aims : Genetic polymorphisms in enzymes involved in carcinogen metabolism have been found to influence susceptibility to cancer. The aim of this study was to examine whether cytochrome P450 2E1 (CYP2E1) and/or glutathione S-transferase M1 (GSTM1) genetic polymorphisms were related to susceptibility to hepatocellular carcinoma (HCC). Methods : Genotyping of CYP2E1 and GSTM1 was performed using the polymerase chain reaction on peripheral white blood cell DNA from 30 patients with HCC and 150 controls nested in a cohort study. Results : The c1/c1 genotype of CYP2E1, detected by Pstl or Rsal digestion, was found in 83.3% of patients with HCC and in 63.3% of controls ( P = 0.034). Homozygosity for the c1/c1 genotype significantly increased the risk of developing HCC in cigarette smokers ( P = 0.001) but posed no increased risk in those who never smoked. The HCC risk associated with cumulative exposure to cigarette smoke was also more striking in individuals who carried the c1/c1 genotype. Habitual alcohol drinking modified the HCC risk of cigarette smoking among those with the c1/c1 genotype. No association with the risk of HCC was observed for the Dral polymorphism of CYP2E1 or for the GSTM1-null genotype. Conclusions : Polymorphisms of CYP2E1 may play an important role in cigarette smoking-related hepatocarcinogenesis.

Increased number of single-LTR HIV-1 DNA junctions correlates with HIV-1 antigen expression and CD4+ cell decline in vivo.

Human immunodeficiency type 1 (HIV-1) DNA in peripheral blood cells of HIV-1 infected individuals may be present as integrated and/or unintegrated DNA. Several reports have indicated that a major proportion of HIV-1 DNA in the asymptomatic phase is linear, full-length, and unintegrated and in the symptomatic phase either circular unintegrated or integrated in the host genome. We developed a quantitative polymerase chain reaction (PCR) technique to detect single-LTR HIV-1 DNA junctions, reflecting the presence of unintegrated single-LTR circles. In vitro infection of a CD4+ T-cell line resulted first in the increase of single-LTR junctions followed by syncytium formation and a rise of p24 antigen production. The number of single-LTR HIV-1 DNA junctions was further studied in two acutely infected individuals and in 21 long-term infected individuals. The number of single-LTR junctions was significantly correlated with CD4+ cell decline, p24 antigen expression, and total HIV-1 DNA content of peripheral blood mononuclear cells (PBMC). Single-LTR HIV-1 DNA junctions were absent from PBMC containing other forms of HIV-1 DNA in four of nine non/slow progressors relative to 2 of 12 rapid progressors/AIDS patients. We conclude from our data that quantitative detection of single-LTR HIV-1 DNA junctions can be used as an early DNA marker of the transition from clinical latency to active replication in the peripheral blood.

Genotoxic risk for humans due to work place exposure to ethylene oxide: remarkable individual differences in susceptibility.

Single strand breaks of DNA of peripheral mononuclear blood cells from 97 male and female workers occupationally exposed to ethylene oxide were analysed by the alkaline elution method. These individuals were occupied with the sterilization of medical devices in hospitals and in commercial plants. Ethylene oxide in the air of the working areas was detected up to a maximal concentration of 16.5 mg/m3 calculated as 4-h time-weighted average (4h TWA). Mean value was 1.47 +/- 0.52 mg/m3 (1 mg/m3 = 0.55 ppm). Compared to the mean elution rate of the DNA from non-smoking workers exposed to air concentrations of ethylene oxide below the detection limit of 0.1 mg/m3 (4h TWA) the non-smokers working in rooms with a concentration of ethylene oxide between 0.5 mg/m3 and 2 mg/m3 showed a statistically significant (P < 0.05) 119% higher mean elution rate and even for the non-smokers exposed to 0.1-0.5 mg/m3 of ethylene oxide a statistically significant (P < 0.05) 53% higher mean elution rate was observed. For smokers a similar tendency was found but the increase in elution rates in response to the external exposure was smaller than in non-smokers and no statistical significance was obtained. According to their sensitivity to ethylene oxide the non-smoking workers could be classified into two subpopulations. In the majority of the non-smokers (67%) approximately 5-fold more DNA strand breaks were induced by ethylene oxide than in the other non-smokers. A lowest detectable effect level could only be specified for non-smokers. (ABSTRACT TRUNCATED AT 250 WORDS)

Point mutations of the N‐ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia

Oncogene mutations are frequently found in several tumour types and, among these, point mutations of the ras gene are particularly significant. A predominance of N-ras mutations has been found in the bone marrow DNA of patients with myelodysplastic syndrome (MDS) or acute myelogenous leukaemia (AML). On the other hand, increased levels of plasma DNA have previously been observed in patients suffering from various malignant diseases. In the present work we have investigated, by polymerase chain reaction (PCR), point mutations of the N-ras gene in the DNA of plasma, blood cells and bone marrow of 10 patients suffering from AML or MDS. The different ras mutations detected in five cases were always present in the plasma DNA while sometimes absent in the DNA of peripheral blood cells or bone marrow. This indicates that a bone marrow biopsy or aspiration does not necessarily contain all the malignant clones involved in the disease. Plasma could thus prove to be an easily accessible and useful material for detection and monitoring of myeloid disorders.

APC GENE-MUTATIONS IN FAMILIAL ADENOMATOUS POLYPOSIS-COLI IDENTIFIED AT THE DNA AND PROTEIN LEVEL

Peripheral blood cell DNA of patients suffering from Familial Adenomatous Polyposis coli (FAP) were subjected to SSCP screening analyses. Here, we report on a patient, who was diagnosed at the age of 31 years suffering from a severe form of adenomatous polyposis coli. The germline mutation was identified to result from a 5-bp germline microdeletion surrounding Adenomatous Polyposis Coli (APC) codon 1309. Examination of DNA derived from pooled adenomas of the same patient confirmed the inherited mutation. Western blot analysis with an APC N-terminus specific antiserum (anti-APC123) applied to tumor-derived proteins identified a polypeptide chain of 140 kD corresponding to the truncation induced by the APC germline mutation. We further examined DNA isolated individually from eight adenomas of the same patient. The analysis revealed the surprising observation, that three out of eight tumor samples analyzed, gave rise to a predominant PCR-amplified band of normal size, implicating that the allele containing the germline deletion had been lost, and instead hemi-/homozygosity for the somatic, not for the germline mutation had been aquired as a tertiary event. A similar mutational mechanism has been reported for the Rb-1 tumor suppressor gene in the retinoblastoma model.

Human herpesvirus 6 DNA in peripheral blood cells and saliva from immunocompetent individuals

Human herpesvirus 6 (HHV-6) genome equivalents were quantitated in peripheral blood mononuclear cells (PBMCs) and saliva from 20 healthy individuals by the polymerase chain reaction (PCR). Nineteen of 20 subjects (95%) harbored HHV-6 DNA: 18 (90%) had HHV-6 in their PBMCs and 18 had HHV-6 in their saliva. Quantitative PCR revealed HHV-6 DNA levels ranging from negative to 4,000 HHV-6 genome equivalents per 10(6) PBMCs and from negative to 200,000 HHV-6 genome equivalents per ml of saliva. Longitudinal saliva samples from 15 HHV-6-seropositive subjects revealed salivary HHV-6 DNA persistence in 13 subjects. HHV-6 antibodies were detected in 17 of 19 subjects, with titers ranging from 1:400 to 1:51,200 (geometric mean titer, 1:2,500). Antibody titers did not correlate with HHV-6 DNA levels in PBMCs or saliva (P = 0.27 and P = 0.44, respectively). One subject with persistent HHV-6 DNA lacked detectable HHV-6 antibodies. The high prevalence of HHV-6 DNA in PBMCs and saliva supports the concept that HHV-6 exists at these sites in normal individuals.