Donor Fractions of Cell-Free DNA Are Elevated During CLAD But Not During Infectious Complications After Lung Transplantation.

During the last few years, cell-free DNA (cfDNA) has emerged as a possible non-invasive biomarker for prediction of complications after lung transplantation. We previously published a proof-of-concept study using a digital droplet polymerase chain reaction (ddPCR)-based method for detection of cfDNA. In the current study, we aimed to further evaluate the potential clinical usefulness of detecting chronic lung allograft dysfunction (CLAD) using three different ddPCR applications measuring and calculating the donor fraction (DF) of cfDNA as well as one method using the absolute amount of donor-derived cfDNA. We analyzed 246 serum samples collected from 26 lung transplant recipients. Nine of the patients had ongoing CLAD at some point during follow-up. All four methods showed statistically significant elevation of the measured variable in the CLAD samples compared to the non-CLAD samples. The results support the use of ddPCR-detected cfDNA as a potential biomarker for prediction of CLAD. These findings need to be validated in a subsequent prospective study.

Absolute Quantification of Donor-Derived Cell-Free DNA in Pediatric and Adult Patients After Heart Transplantation: A Prospective Study.

In this prospective study we investigated a cohort after heart transplantation with a novel PCR-based approach with focus on treated rejection. Blood samples were collected coincidentally to biopsies, and both absolute levels of dd-cfDNA and donor fraction were reported using digital PCR. 52 patients (11 children and 41 adults) were enrolled (NCT03477383, clinicaltrials.gov), and 557 plasma samples were analyzed. 13 treated rejection episodes >14 days after transplantation were observed in 7 patients. Donor fraction showed a median of 0.08% in the cohort and was significantly elevated during rejection (median 0.19%, p < 0.0001), using a cut-off of 0.1%, the sensitivity/specificity were 92%/56% (AUC ROC-curve: 0.78). Absolute levels of dd-cfDNA showed a median of 8.8 copies/mL and were significantly elevated during rejection (median 23, p = 0.0001). Using a cut-off of 7.5 copies/mL, the sensitivity/specificity were 92%/43% for donor fraction (AUC ROC-curve: 0.75). The results support the feasibility of this approach in analyzing dd-cfDNA after heart transplantation. The obtained values are well aligned with results from other trials. The possibility to quantify absolute levels adds important value to the differentiation between ongoing graft damage and quiescent situations.

Cell-free DNA as a biomarker after lung transplantation: A proof-of-concept study.

BACKGROUND: Lung transplantation (LTx) is a lifesaving procedure burdened with limited long-term survival. The most common cause of death after LTx is chronic lung allograft dysfunction (CLAD). Today, useful biomarkers for the detection of CLAD are lacking. Circulating cell-free DNA (cfDNA) is released during cellular decay and can be detected using polymerase chain reaction (PCR). Thus, donor-derived cfDNA in recipient serum indicates cellular decay in the transplanted organ. In the current study, we explore the possibility of using a novel PCR method to detect cfDNA as a biomarker for clinical events, especially CLAD. METHODS: Four patients were retrospectively tested for levels of both donor and recipient-derived cfDNA using digital droplet PCR after targeted preamplification. The results were correlated to recorded clinical events. RESULTS: All available samples rendered results. Both patients that later developed CLAD showed a persistently elevated ratio between donor-and recipient-derived cfDNA. Also, the mean level of cfDNA was higher in the two patients who later developed CLAD than in patients who did not (p = .0015). CONCLUSIONS: This proof-of-concept study suggests that cfDNA quantified with PCR may be used as a biomarker of significant clinical events such as CLAD.

Was the C282Y mutation an Irish Gaelic mutation that the Vikings helped disseminate? HLA haplotype observations of hemochromatosis from the west coast of Sweden.

UNLABELLED: The HLA-related hemochromatosis mutation C282Y is thought to have originated in Ireland in a person with HLA-A3-B14 and was spread by Vikings. Irish people with two HLA-A3 alleles had a high risk of hemochromatosis. In this study, from west Sweden, we wanted to test these hypotheses. METHODS: HFE mutations in controls, bone marrow donors with HLA-A3/A3 and patients with hemochromatosis. HLA haplotypes, extended haplotype analysis and pedigree studies. RESULTS: The allelic C282Y frequency 0.04, (CI 0.01-0.07) was lower (P < 0.001) in Sweden than in Ireland 0.10 (CI 0.08-0.11), and Swedish bone marrow donors with HLA-A3/A3 (n = 77) had a low risk of hemochromatosis. HLA haplotypes available from 239/262 (91.5%) proband patients homozygous for C282Y showed a dominance of A3-B7 and A3-B14 both in linkage disequilibrium with controls (P < 0.001). Pedigree studies extended into the 17th century supported a local founder effect of A3-B14 in the county of Bohuslän. The A3-B14 haplotype may well be the original and A3-B7 the result of centromeric recombinations. The haplotype diversity and recombination events were not different from a Celtic series. These findings do not support the hypothesis of the C282Y mutation being of an Irish Celtic origin. CONCLUSIONS: The C282Y frequency shows a west to east decline from Ireland through the north of Europe. Vikings may have been involved in the spread of C282Y, but the mutation is probably older and may have been spread in Europe by earlier seafarers.

BCR-ABL1 transcript levels increase in peripheral blood but not in granulocytes after physical exercise in patients with chronic myeloid leukemia.

In chronic myeloid leukemia (CML) treatment response is determined by measurements of BCR-AB1L transcripts in peripheral blood by quantitative real-time PCR (qRT-PCR) and a 2-5 fold increase is considered a warning sign. The BCR-ABL1 gene is mainly expressed in myeloid cells whereas quantification of BCR-ABL1 is performed on the nucleated cell fraction of peripheral blood. Hence, leukocyte composition of the nucleated cell fraction may affect the result of BCR-ABL1 quantification. The aim of this study was to investigate if changes in leukocyte composition of peripheral blood had any effect on BCR-ABL1 transcript levels in CML patients. Six CML patients in complete cytogenetic remission (CCgR) performed a maximal physical exercise test. Blood samples were collected before exercise, at maximal exhaustion and after exercise. A biphasic increase in leukocyte count was observed and the relative proportion of granulocytes in peripheral blood changed significantly after exercise compared with baseline (p < 0.001). The BCR-ABL1 transcript level increased significantly following exercise, in nucleated cell fraction of peripheral blood (p < 0.05) but not in isolated granulocytes. In the nucleated cell fraction, the mean BCR-ABL1 transcript level was 3.3-fold (range 0.7-6.8) higher 180 min after exercise compared with baseline (p < 0.01). In conclusion, physical exercise induced significant increases in BCR-ABL1 transcript levels concomitant with changes in leukocyte content of peripheral blood. We therefore suggest that variations in leukocyte composition of peripheral blood, causing pre-analytic variations that affect BCR-ABL1 quantification, have to be accounted for. Consequently, small variations in BCR-ABL1 transcript levels should be interpreted cautiously in CML patients in CCgR.

Changes in expression of apoptosis-related genes are linked to the molecular response to imatinib treatment in chronic-phase chronic myeloid leukemia patients.

Most patients with a chronic phase of chronic myeloid leukemia (CML) treated with imatinib mesylate achieve a cytogenetic remission, but in the majority, residual disease is detectable by RT-PCR. The mechanisms by which residual leukemic cells survive imatinib treatment are unresolved. However, induction of apoptosis in leukemic stem cells and immunotherapy are currently under investigation. We studied the mRNA expression of apoptosis-related genes in peripheral blood mononuclear cells from chronic-phase CML patients before imatinib treatment. It was found that their BCL2 and BAD expression was significantly different compared to the normal controls, and a lower BAD expression was associated with a better molecular response to imatinib treatment at 12 months.

Glucose-dependent insulinotropic polypeptide is expressed in adult hippocampus and induces progenitor cell proliferation.

The hippocampal dentate gyrus (DG) is an area of active proliferation and neurogenesis within the adult brain. The molecular events controlling adult cell genesis in the hippocampus essentially remain unknown. It has been reported previously that adult male and female rats from the strains Sprague Dawley (SD) and spontaneously hypertensive (SHR) have a marked difference in proliferation rates of cells in the hippocampal DG. To exploit this natural variability and identify potential regulators of cell genesis in the hippocampus, hippocampal gene expression from male SHR as well as male and female SD rats was analyzed using a cDNA array strategy. Hippocampal expression of the gene-encoding glucose-dependent insulinotropic polypeptide (GIP) varied strongly in parallel with cell-proliferation rates in the adult rat DG. Moreover, robust GIP immunoreactivity could be detected in the DG. The GIP receptor is expressed by cultured adult hippocampal progenitors and throughout the granule cell layer of the DG, including progenitor cells. Thus, these cells have the ability to respond to GIP. Indeed, exogenously delivered GIP induced proliferation of adult-derived hippocampal progenitors in vivo as well as in vitro, and adult GIP receptor knock-out mice exhibit a significantly lower number of newborn cells in the hippocampal DG compared with wild-type mice. This investigation demonstrates the presence of GIP in the brain for the first time and provides evidence for a regulatory function for GIP in progenitor cell proliferation.

Epstein-Barr virus U leader exon contains an internal ribosome entry site.

Eukaryotic translation can be initiated either by a cap-dependent mechanism or by internal ribosome entry, a process by which ribosomes are directly recruited to structured regions of mRNA upstream of the initiation codon. Here we report the finding of an internal ribosome entry site (IRES) in the untranslated region of the Epstein-Barr nuclear antigen 1 (EBNA1) gene. EBNA1 is the only nuclear protein expressed in all known states of Epstein-Barr virus (EBV) latency and in the virus lytic cycle, and is required for the maintenance of the EBV episome. Using cDNA reporter constructs and in vitro transfection assays, we found that sequences contained in the 5' untranslated region (UTR) of the Fp and Qp initiated EBNA1 mRNA increased the expression level 4-14- fold in different Burkitt lymphoma cell lines. The U leader exon, located within the 5' UTR, included in all known EBNA1 transcripts and also contained in the EBNA3, 4 and 6 mRNAs, was demonstrated by bicistronic expression analyses to contain an IRES. The EBNA IRES initiates translation more efficiently than the encephalomyocarditis virus IRES in EBV-positive lymphoma cells. We propose that the EBNA IRES constitute a novel mechanism, whereby EBV regulates latent gene expression.

Tumor necrosis factor gene polymorphism and cardiac allograft vasculopathy.

BACKGROUND: Cardiac allograft vasculopathy (CAV) limits survival after cardiac transplantation. Tumor necrosis factor-alpha (TNF-alpha) may be a key factor in the development of CAV. Two bi-allelic polymorphisms associated with high TNF-alpha production have been identified in the TNF gene locus, TNFA1/2, at position -308 and TNFB1/2 at +252. We hypothesized that recipient TNFA2 and TNFB2 homozygosity is associated with the development of CAV after heart transplantation. METHODS: TNF gene polymorphisms were analyzed by multiplex fluorescent solid-phase mini-sequencing in 70 cardiac transplant recipients. Recipients homozygous for TNFA2 or TNFB2 (Group A, n = 29) were compared with recipients heterozygous or homozygous for TNFA1 and TNFB1 (Group B, n = 41). Coronary arteriography was performed annually or when indicated. Cumulative freedom from CAV and survival was calculated according to the Kaplan-Meier test. RESULTS: Mean follow-up was 3.8 +/- 0.3 years. In Group A, 11 of 29 recipients (38%) developed CAV compared with 9 of 41 (22%) in Group B (p = 0.12). Cumulative freedom from CAV at 3 years was 42% in Group A and 80% in Group B (p = 0.043). In Group A, 11 of 29 recipients (38%) died during follow-up compared with 4 of 41 (10%) in Group B (p = 0.006). Cumulative survival at 3 years was 72% in Group A and 93% in Group B (p = 0.003). CONCLUSIONS: The results suggest that TNFA2 and TNFB2 allele homozygosity is associated with cardiac allograft vasculopathy and mortality in heart transplant recipients.

Increased frequency of combined methylenetetrahydrofolate reductase C677T and A1298C mutated alleles in spontaneously aborted embryos.

The pathogenesis of spontaneous abortion is complex, presumably involving the interaction of several genetic and environmental factors. The methylenetetrahydrofolate reductase (MTHFR) gene C677T and A1298C polymorphisms are commonly associated with defects in folate dependent homocysteine metabolism and have been implicated as risk factors for recurrent embryo loss in early pregnancy. In the present study we have determined the prevalence of combined MTHFR C677T and A1298C polymorphisms in DNA samples from spontaneously aborted embryos (foetal death between sixth and twentieth week after conception) and adult controls using solid-phase minisequencing technique. There was a significant odds ratio of 14.2 (95% CI 1.78-113) in spontaneously aborted embryos comparing the prevalence of one or more 677T and 1298C alleles vs the wild type combined genotype (677CC/1298AA), indicating that the MTHFR polymorphisms may have a major impact on foetal survival. Combined 677CT/1298CC, 677TT/1298AC or 677TT/1298CC genotypes, which contain three or four mutant alleles, were not detected in any of the groups, suggesting complete linkage disequilibrium between the two polymorphisms. The present finding of high prevalence of mutated MTHFR genotypes in spontaneously aborted embryos emphasises the potential protective role of periconceptional folic acid supplementation.

The effects of hydroxyurea on PRV-1 expression in patients with essential thrombocythemia and polycythemia vera.

The study was designed to investigate the influence of hydroxyurea (HU) treatment on PRV-1 expression. Eighteen newly diagnosed patients with essential thrombocythemia (ET) or polycythemia vera (PV) were included. HU significantly increased PRV-1 gene expression in the early stage of treatment.

Influence of the apolipoprotein E epsilon4 allele on human embryonic development.

Human apolipoprotein E (apoE) exists in three major isoforms encoded by distinct alleles (APOE epsilon2, epsilon3 and epsilon4) and has important functions in nerve development and repair. Inheritance of the 4 allele is a major risk factor for the development of Alzheimer's disease. To investigate the role of APOE polymorphisms in embryonic development, we analyzed the APOE genotypes of 81 spontaneously aborted embryos and 110 adult controls using a solid-phase minisequencing technique. The epsilon4 allele was significantly less frequent in the spontaneous abortion group than in the control group (P=0.009), while the frequency of epsilon3 was significantly increased (P=0.005), suggesting that epsilon4 may have protective effects during embryogenesis. These protective effects might counterbalance the deleterious age-related effects of the epsilon4 allele in natural selection.

Serial monitoring of BCR-ABL transcripts in chronic myelogenous leukemia (CML) treated with imatinib mesylate.

Survival among chronic myelogenous leukemia (CML) patients can be linked to the reduction in leukemic cell burden. Treatment with imatinib mesylate results in a high frequency of complete cytogenetic response, which can be further stratified using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). We have serially monitored peripheral blood and bone marrow BCR-ABL transcripts using qRT-PCR in CML patients commencing imatinib therapy, and compared the results with bone marrow cytogenetics. Seventeen patients (aged 25-74 yr) with Philadelphia chromosome positive CML in first chronic phase were treated with imatinib targeting a dose of 400 mg/d. The median follow up is 30 mo (range 9-33 mo). Every third month the product of the BCR-ABL fusion gene was evaluated in both blood and bone marrow specimens by real-time RT-PCR using the TaqMan probe system. In 113 simultaneously obtained blood and bone marrow samples, the BCR-ABL transcript values agreed well with cytogenetic data. Blood and bone marrow specimens gave comparable values for BCR-ABL transcripts. Before start of imatinib therapy there was a considerable variation in BCR-ABL transcripts among the patients, ranging approximately one log (base 10). Similarly, patients with a complete cytogenetic response following imatinib therapy had variable BCR-ABL transcript levels, ranging at least three logs (base 10). The major decline in BCR-ABL transcripts occurred within 6 mo after start of imatinib therapy. The decline in BCR-ABL transcripts, following imatinib therapy, appears to level off at 12-15 mo. Two late responders were identified with a still decreasing level in BCR-ABL transcripts after 24 mo of treatment. It is concluded that BCR-ABL mRNA quantification in peripheral blood is suitable for routine monitoring of the response to treatment and long-term disease status in CML, especially in patients who have achieved a complete cytogenetic response. A plateau in BCR-ABL transcripts seems to have been reached after 12-15 mo of imatinib treatment; however, some "late responders" are seen.

EBNA1 expression in a lung transplant recipient with hypocomplementemic urticarial vasculitis syndrome.

This article describes a transplant recipient with underlying hypocomplementemic urticarial vasculitis syndrome who expressed persistently Epstein-Barr virus nuclear antigen 1 (EBNA1) in peripheral blood. The patient received a bilateral lung transplant and was subsequently followed with monitoring of EBV expression in peripheral blood. Evaluation of viral expression in peripheral blood, serum, and graft tissue was performed with RT-PCR, Q-PCR, indirect immunofluorescence, anti-peptide assays, and in situ hybridization; samples were collected at various time-points up to 91 days post-transplantation. The patient expressed EBNA1 in 8/10 (80%) of the peripheral blood samples tested during the post-transplantation period, and interestingly, even including the day of transplantation. After analyses of indicative EBV mRNA, EBNA1 expression was found mainly to be Qp-initiated EBNA1, known to be important for EBV maintenance. Anti-EBNA1 epitope mapping showed significantly higher and broader antibody responses to EBNA1 epitopes pre-transplantation when compared to normal controls and a matched lung transplant control. Post-transplantation this response was largely diminished but there were still epitopes significantly higher than controls. Our results show the presence of EBV-positive proliferating cells before onset of intensive immunosuppressive treatment. Although no previous connection between EBV and hypocomplementemic urticarial vasculitis syndrome has been reported, it is tempting to speculate that the continuous EBNA1 expression is not caused by immunosuppression or post-transplant lymphoproliferative disease, but may be a factor involved in the etiology of the autoimmune disease.

Cell specific internal translation efficiency of Epstein-Barr virus present in solid organ transplant patients.

The U leader exon in the 5' untranslated region of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene contains an internal ribosome entry site, the EBNA internal ribosome entry segment (IRES), which promotes cap-independent translation and increases the expression level of the EBNA1 protein. It was previously reported that immunosuppressed organ transplanted patients showed an alternatively spliced EBNA1 transcript, excluding the EBNA IRES element. To further investigate the function of the EBNA IRES, sequence analysis of the EBNA IRES mRNA was performed in samples from seven organ transplant patients. Two nucleotide changes, G --> A at position 67531 and C --> U at position 67585 were found in the EBNA IRES mRNA, relative to the corresponding genomic Epstein-Barr virus (EBV) sequence in all patients. Moreover, the patient derived EBNA IRES mRNA was shown to differ from the IRES mRNA derived from the cell line B95.8 at position 67531 and from the cell lines Rael and P3HR1 at positions 67531 and 67585. cDNA from the various EBNA IRES sequences were cloned into bicistronic vectors, respectively, and used in transient transfection experiments in six human cell lines. The patient specific sequence significantly decreased the IRES activity in T-cells, while the base changes had no significant impact on the activity in B- or in epithelial cells. The genetic mechanisms behind EBV-associated diseases are complex, involving gene regulation by alternative promoters, alternative splicing, and translational control. The nucleotide changes in the patient specific EBNA IRES transcript and its influence on the translational activity, might illustrate new strategies utilised by the EBV to adapt to the immune control in patients with EBV associated diseases.

Not all imatinib resistance in CML are BCR-ABL kinase domain mutations.

Point mutations within the ABL kinase domain of the BCR-ABL gene are associated with clinical resistance to imatinib mesylate in chronic myeloid leukemia (CML). To obtain more information about the association between BCR-ABL mutations and type of imatinib resistance, we studied 30 early chronic phase (CP) CML patients, commencing imatinib therapy, using a conventional sequencing technique. Seven patients treated in late CP and three patients treated in the accelerated phase were included for comparison. Blood samples were collected before and every third month during imatinib therapy. Mutations were not seen in any blood sample collected before start of therapy. During imatinib treatment, 2 of the 30 early CP patients acquired point mutations and both of them had other signs of imatinib resistance. None of the five early CP patients with a complete hematologic response (HR), but no cytogenetic response at 12 months, displayed any missense mutation. Likewise, none of 12 early CP patients with detectable BCR-ABL transcripts but in complete hematologic and cytogenetic remission at 12 months displayed any mutation. We conclude that screening early CP patients for BCR-ABL mutations before start of imatinib therapy is not cost-effective. BCR-ABL kinase domain mutations do not appear to explain cytogenetic or molecular (detectable BCR-ABL transcripts by polymerase chain reaction) disease persistence in patients otherwise in stable disease. However, in patients with signs of expanding disease burden, a search for BCR-ABL mutations is warranted.

Alternative EBNA1 expression in organ transplant patients.

In order to identify patients at risk for developing post-transplant lymphoproliferative disease (PTLD), a sensitive nested RT-PCR method for detection of EBNA1 gene expression in peripheral blood cells was used. EBNA1 expression in peripheral blood samples from 60 organ recipients was analyzed and compared with 24 healthy controls in a retrospective study. Overall, EBNA1-positive samples were detected at least once in 43% of the transplant patients with post-transplant lymphoproliferative disease, in 18% of the other transplant patients and in none of the healthy controls. The odds ratio for EBNA1 expression in patients with post-transplant lymphoproliferative disease was 3.42 (95% CI=1.02-11.54) compared to other transplant recipients. Together with normal EBV Q promoter initiated EBNA1 transcripts, an alternatively spliced form was expressed in peripheral blood cells in the above-mentioned transplant patients. This transcript lacks the U leader exon in the 5'-untranslated region (UTR). We have previously identified and characterized a functional internal ribosome entry site, the EBNA IRES, in the untranslated U leader exon of EBNA1. Transfection experiments with EBNA1 coding plasmids followed by Western blot showed that the EBNA IRES promotes cap-independent translation and increases the EBNA1 protein level. The alternative EBNA1 transcript lacking this function is expressed in the majority of the investigated EBNA1-positive patient samples as well as in some EBV-positive B-cell lines. Alternative splicing in this form gives EBV potential to regulate the translation of EBNA1 by modifying the 5' UTR. These findings indicate a new mechanism for EBNA1 expression in vivo.

Tumor necrosis factor gene polymorphisms and inflammatory response in coronary artery bypass grafting patients.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha), a key factor in the inflammatory cascade, has been implicated in coronary artery disease. Two biallelic polymorphisms in the TNF gene locus (TNFA at position -308 and TNFB at +252) may influence TNF-alpha production. Individuals with the rare TNFA2 allele or TNFB2 homozygosity have augmented TNF-alpha production. We investigated the genotypes associated with increased TNF-alpha production in coronary artery bypass grafting (CABG) patients and if these genotypes influence the magnitude of the postoperative inflammatory response. METHODS: TNF gene polymorphisms were analyzed by multiplex fluorescent solid-phase minisequencing in 86 CABG patients. Plasma concentrations of TNF-alpha, IL-6 and C3a and C-reactive protein (CRP) were analyzed before and after surgery in 45 of the patients and compared with genetically high and low TNF-alpha producers. RESULTS: Thirty percent of the patients carried the TNFA2 allele and 45% were TNFB2 homozygous. The allelic frequencies were TNFA1/TNFA2 = 0.84/0.16 and TNFB1/TNFB2 = 0.32/0.68. Pre- and postoperative levels of TNF-alpha, IL-6, C3a and CRP did not differ significantly between genetically high and low TNF-alpha producers. CONCLUSIONS: The frequency of high TNF-alpha producing genotypes in a CABG population was comparable to that previously reported from normal populations. Furthermore, we found no evidence that the investigated TNF-alpha gene polymorphisms influence postoperative inflammatory response after uncomplicated coronary surgery.

Tumor necrosis factor gene polymorphisms and inflammatory response in coronary artery bypass grafting patients.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha), a key factor in the inflammatory cascade, has been implicated in coronary artery disease. Two biallelic polymorphisms in the TNF gene locus (TNFA at position -308 and TNFB at +252) may influence TNF-alpha production. Individuals with the rare TNFA2 allele or TNFB2 homozygosity have augmented TNF-alpha production. We investigated the genotypes associated with increased TNF-alpha production in coronary artery bypass grafting (CABG) patients and if these genotypes influence the magnitude of the postoperative inflammatory response. METHODS: TNF gene polymorphisms were analyzed by multiplex fluorescent solid-phase minisequencing in 86 CABG patients. Plasma concentrations of TNF-alpha, IL-6 and C3a and C-reactive protein (CRP) were analyzed before and after surgery in 45 of the patients and compared with genetically high and low TNF-alpha producers. RESULTS: Thirty percent of the patients carried the TNFA2 allele and 45% were TNFB2 homozygous. The allelic frequencies were TNFA1/TNFA2=0.84/0.16 and TNFB1/TNFB2=0.32/0.68. Pre- and postoperative levels of TNF-alpha, IL-6, C3a and CRP did not differ significantly between genetically high and low TNF-alpha producers. CONCLUSIONS: The frequency of high TNF-alpha producing genotypes in a CABG population was comparable to that previously reported from normal populations. Furthermore, we found no evidence that the investigated TNF-alpha gene polymorphisms influence postoperative inflammatory response after uncomplicated coronary surgery.