Blood Leukocyte DNA Research Articles

Temporal associations between leukocytes DNA methylation and blood lipids: a longitudinal study

BackgroundThe associations between blood lipids and DNA methylation have been investigated in epigenome-wide association studies mainly among European ancestry populations. Several studies have explored the direction of the association using cross-sectional data, while evidence of longitudinal data is still lacking.ResultsWe tested the associations between peripheral blood leukocytes DNA methylation and four lipid measures from Illumina 450 K or EPIC arrays in 1084 participants from the Chinese National Twin Registry and replicated the result in 988 participants from the China Kadoorie Biobank. A total of 23 associations of 19 CpG sites were identified, with 4 CpG sites located in or adjacent to 3 genes (TMEM49, SNX5/SNORD17 and CCDC7) being novel. Among the validated associations, we conducted a cross-lagged analysis to explore the temporal sequence and found temporal associations of methylation levels of 2 CpG sites with triglyceride and 2 CpG sites with high-density lipoprotein-cholesterol (HDL-C) in all twins. In addition, methylation levels of cg11024682 located in SREBF1 at baseline were temporally associated with triglyceride at follow-up in only monozygotic twins. We then performed a mediation analysis with the longitudinal data and the result showed that the association between body mass index and HDL-C was partially mediated by the methylation level of cg06500161 (ABCG1), with a mediation proportion of 10.1%.ConclusionsOur study indicated that the DNA methylation levels of ABCG1, AKAP1 and SREBF1 may be involved in lipid metabolism and provided evidence for elucidating the regulatory mechanism of lipid homeostasis.

Mild cognitive impairment is associated with low copy number of ribosomal genes in the genomes of elderly people.

Introduction: Mild cognitive impairments (MCI) accompanying aging are associated with oxidative stress. The ability of cells to respond to stress is determined by the protein synthesis level, which depends on the ribosomes number. Ribosomal deficit was documented in MCI. The number of ribosomes depends, together with other factors, on the number of ribosomal genes copies. We hypothesized that MCI is associated with low rDNA CN in the elderly person genome. Materials and Methods: rDNA CN and the telomere repeat (TR) content were determined in the DNA of peripheral blood leukocytes of 93 elderly people (61–91 years old) with MCI and 365 healthy volunteers (16–91 years old). The method of non-radioactive quantitative hybridization of DNA with biotinylated DNA probes was used for the analysis. Results: In the MCI group, rDNA CN (mean 329 ± 60; median 314 copies, n = 93) was significantly reduced (p < 10–15) compared to controls of the same age with preserved cognitive functions (mean 412 ± 79; median 401 copies, n = 168) and younger (16–60 years) control group (mean 426 ± 109; median 416 copies, n = 197). MCI is also associated with a decrease in TR DNA content. There is no correlation between the content of rDNA and TR in DNA, however, in the group of DNA samples with rDNA CN > 540, TR content range was significantly narrowed compared to the rest of the sample. Conclusion: Mild cognitive impairment is associated with low ribosomal genes copies in the elderly people genomes. A low level of rDNA CN may be one of the causes of ribosomal deficit that was documented in MCI. The potential possibilities of using the rDNA CN indicator as a prognostic marker characterizing human life expectancy are discussed.

Peripheral Blood DNA Methylation Profiles Do Not Predict Endoscopic Post-Operative Recurrence in Crohn's Disease Patients.

Prediction of endoscopic post-operative recurrence (POR) in Crohn’s disease (CD) patients following ileocolonic resection (ICR) using clinical risk factors alone has thus far been inadequate. While peripheral blood leukocyte (PBL) DNA methylation has shown promise as a tool for predicting recurrence in cancer, no data in CD patients exists. Therefore, this study explored the association and predictive value of PBL DNA methylation in CD patients following ICR. From a cohort of 117 CD patients undergoing ICR, epigenome-wide PBL methylation profiles from 25 carefully selected patients presenting either clear endoscopic remission (n = 12) or severe recurrence (n = 13) were assessed using the Illumina MethylationEPIC (850K) array. No statistically significant differentially methylated positions (DMPs) or regions (DMRs) associated with endoscopic POR were identified (FDR p ≤ 0.05), further evidenced by the low accuracy (0.625) following elastic net classification analysis. Nonetheless, interrogating the most significant differences in methylation suggested POR-associated hypermethylation in the MBNL1, RAB29 and LEPR genes, respectively, which are involved in intestinal fibrosis, inflammation and wound healing. Notably, we observed a higher estimated proportion of monocytes in endoscopic POR compared to remission. Altogether, we observed limited differences in the genome-wide DNA methylome among CD patients with and without endoscopic POR. We therefore conclude that PBL DNA methylation is not a feasible predictive tool in post-operative CD.

Relationship between miRNAs polymorphisms and peripheral blood leukocyte DNA telomere length in coke oven workers: A cross-sectional study

ObjectiveThe purpose of this study was to investigate the factors affecting telomere length (TL) in coke oven workers by analyzing the interaction between miRNAs polymorphisms and coke oven emissions (COEs) exposure. MethodsA total of 544 coke oven workers and 238 healthy controls were recruited. Peripheral blood was collected from the subjects, genomic DNA was extracted, leukocyte TL was detected by real-time quantitative polymerase chain reaction, and fifteen polymorphisms of eight miRNAs were genotyped by flight mass spectrometry. ResultsStatistical analysis showed that the peripheral blood DNA TL in the exposure group was shorter than that in the control group (P < 0.001). Generalized linear model found that COEs-exposure [β (95%CI) = -0.427 (−0.556, −0.299), P < 0.001], genotype CC+CT for miR-612 rs1144925 [β (95%CI) = −0.367 (−0.630, −0.104), P = 0.006], and the interaction of miR-181B1 rs12039395 TT genotype and COEs-exposure [β (95% CI) = 0.564 (0.108, 1.020), P = 0.015] were associated with the shortened TL. ConclusionCOEs-exposure and miR-612 rs1144925 TT could promote telomere shortening in coke oven workers. The interaction of miR-181B1 rs12039395 TT genotype and COEs-exposure could protect telomere. This provides clues for further mechanistic studies between miRNA and telomere damage.

Diagnostic value of Epstein – Barr virus DNA quantification in blood leukocytes in children with infectious mononucleosis

Aim. To determine the threshold value of the Epstein-Barr virus (EBV) viral load (VL) in blood leukocytes to improve the laboratory diagnostics of infectious mononucleosis in children.Materials and methods. EBV DNA quantification in blood leukocytes in children aged 1-17 years (n=163) was determined by real-time polymerase chain reaction. VL were compared in groups of EBV mononucleosis (n=67), non-EBV mononucleosis (n=25) and healthy donors (n=25). Threshold was determined based on VL data from children with active and latent EBV infection. The R program and the RStudio environment were used for satistic analysis.Results. EBV DNA is found in blood leukocytes in infectious mononucleosis not associated with EBV and in healthy virus carriers, however VL in these groups is significantly lower than in patients with EBV mononucleosis (p&lt;0.001). The threshold value was determined – 41 copies/105 cells (or 1.6 lg of EBV DNA/105 cells), which was characterized by acceptable values of specificity and sensitivity (0.90 and 0.85, respectively) of laboratory diagnostics. High EBV VL (equal to or above the set threshold) is associated with an 8.5-fold increased risk of detecting active EBV infection compared to children who have a low VL (below a set threshold) (RR 8.5; 95% CI: 3.7–19.7, p&lt;0.001).Conclusion. In general, the results obtained create prerequisites for more intensive implementation of quantitative studies of EBV DNA in blood leukocytes, both in the context of improving the early diagnosis of infectious mononucleosis and its etiological interpretation, and in terms of detailing the features of the course of EBV infection.

Telomere Length as a Marker of Suicidal Risk in Schizophrenia.

Schizophrenia and suicidal behavior are associated with shortening in the length of telomeres. The aim of the study was to compare the content (pg/mcg) of telomeric repeat in DNA isolated from peripheral blood cells in three groups of subjects: patients with schizophrenia and a history of suicide attempts, patients with schizophrenia without suicidal tendencies, and healthy control volunteers. Relapses according to gender and age were examined in 47 patients with schizophrenia with suicidal behavior, 47 patients without self-destructive conditions, and 47 volunteers with healthy control and maintenance for the content of telomeric and the number of copies of mitochondrial DNA (mtDNA) in peripheral blood leukocytes. Analysis of determining the content of telomeric repeat (TR) in the DNA of massive weight gain in the series: patients with schizophrenia and suicidal attempts - patients with schizophrenia without suicidal observations - healthy controls (225±28.4 (227 [190; 250]) vs. 243±21 (245 [228; 260]) vs. 255±17.9 (255 [242; 266]), p <0.005. The same trend is observed for the number of mtDNA copies (257±101.5 (250 [194; 297])) vs. 262.3±59.3 (254 [217; 312]) vs. 272±79.9 (274 [213; 304]); p=0.012), but no significant differences were recorded. For the first time, the phenomenon of telomere shortening was discovered in schizophrenics with suicidal risk. The length of the telomere corresponds to the parameter of a biological marker - an objectively measured indicator of normal or pathological processes, but gaining an idea of its reliability is still necessary for verification with an assessment of its sensitivity, specificity, and positive and negative predictive value. The telomere may be considered a putative predictive indicator of suicidal risk.

Association of DNA Methylation in Blood Pressure-Related Genes With Ischemic Stroke Risk and Prognosis.

BackgroundA genome-wide association study identified 12 genetic loci influencing blood pressure and implicated a role of DNA methylation. However, the relationship between methylation and ischemic stroke has not yet been clarified. We conducted a large-sample sequencing study to identify blood leukocyte DNA methylations as novel biomarkers for ischemic stroke risk and prognosis based on previously identified genetic loci.MethodsMethylation levels of 17 genes were measured by sequencing in 271 ischemic stroke cases and 323 controls, and the significant associations were validated in another independent sample of 852 cases and 925 controls. The associations between methylation levels and ischemic stroke risk and prognosis were evaluated.ResultsMethylation of AMH, C17orf82, HDAC9, IGFBP3, LRRC10B, PDE3A, PRDM6, SYT7 and TBX2 was significantly associated with ischemic stroke. Compared to participants without any hypomethylated targets, the odds ratio (OR) (95% confidence interval, CI) for those with 9 hypomethylated genes was 1.41 (1.33–1.51) for ischemic stroke. Adding methylation levels of the 9 genes to the basic model of traditional risk factors significantly improved the risk stratification for ischemic stroke. Associations between AMH, HDAC9, IGFBP3, PDE3A and PRDM6 gene methylation and modified Rankin Scale scores were significant after adjustment for covariates. Lower methylation levels of AMH, C17orf82, PRDM6 and TBX2 were significantly associated with increased 3-month mortality. Compared to patients without any hypomethylated targets, the OR (95% CI) for those with 4 hypomethylated targets was 1.12 (1.08–1.15) for 3-month mortality (P = 2.28 × 10−10).ConclusionThe present study identified blood leukocyte DNA methylations as potential factors affecting ischemic stroke risk and prognosis among Han Chinese individuals.

Association between single nucleotide genetic polymorphisms in ABC transporter genes with drug-resistant epilepsy in the Spanish population

Almost a third of all patients with epilepsy (30%) fail to respond to pharmacological treatment. The presence of single nucleotide polymorphisms (SNPs) in the individual may influence the variability of the response to drug treatment. The transporter hypothesis posits that the presence of SNPs in the genes encoding ABC proteins would affect the bioavailability of antiseizure drugs at the epileptogenic focus, giving rise to refractoriness. The aim of the present study was to evaluate the association of 13 polymorphisms in the ABCB1, ABCC2, ABCC5 and ABCG2 genes with drug-resistant epilepsy (DRE) in a Spanish population. A case-control study was conducted involving 327 patients with epilepsy: 227 resistant to drug therapy and 100 in whom their medication enabled them to control their symptoms, according to International League Against Epilepsy criteria. In the peripheral blood leukocyte DNA that was extracted, polymorphisms in the ABC transporter genes were studied. The iPlex® Gold and Mass ARRAY technology platform was used. The allele and genotypic frequencies of the case and control groups, p-value, odds ratio and 95% confidence intervals were compared. The allele and genotypic frequency of the present study was similar to that reported in population-based databases. For the SNPs studied, no significant differences (p > 0.05) were found in any of the inheritance models analysed. Our results suggest that there is no association between the polymorphisms analysed in the ABC genes and DRE in the Spanish population. Nevertheless, further studies will confirm or refute these results.

Diet, Sports, and Psychological Stress as Modulators of Breast Cancer Risk: Focus on OPRM1 Methylation.

Background: DNA methylation is influenced by environmental factors and contributes to adverse modification of cancer risk and clinicopathological features.Methods: A case-control study (402 newly diagnosed cases, 470 controls) was conducted to evaluate the effect of environmental factors and OPRM1 methylation in peripheral blood leukocyte (PBL) DNA on the risk of breast cancer. A case-only study (373 cases) was designed to evaluate the effects of environmental factors on OPRM1 methylation in tumor tissue and the relationship of methylation with clinicopathological features.Results: We found a significant association between hypermethylation of OPRM1 and the risk of breast cancer (OR = 1.914, 95%CI = 1.357–2.777). OPRM1 hypermethylation in PBL DNA combined with low intake of vegetable, garlic, soybean, poultry, and milk; high pork intake; less regular sports and a high psychological stress index significantly increased the risk of breast cancer. Soybean intake (OR = 0.425, 95%CI: 0.231–0.781) and regular sports (OR = 0.624, 95%CI: 0.399–0.976) were associated with OPRM1 hypermethylation in tumor DNA. OPRM1 hypermethylation in tumor tissue was correlated with estrogen receptor (ER) (OR = 1.945, 95%CI: 1.262–2.996) and progesterone receptor (PR) (OR = 1.611, 95%CI: 1.069–2.427) negative status; in addition, OPRM1 hypermethylation in PBL DNA was associated with human epidermal growth factor receptor 2 (HER-2) negative status (OR = 3.673, 95%CI: 1.411–9.564).Conclusion: A healthy diet, psychosocial adaptability, and regular sports are very beneficial for breast cancer prevention and progress, especially for OPRM1 hypermethylation carriers. Personalized treatment considering the correlation between OPRM1 hypermethylation and ER and PR status may provide a novel benefit for breast cancer patients.

Per-and polyfluoroalkyl substances, epigenetic ageand DNA methylation: across-sectional study of firefighters.

Background: Per- and polyfluoroalkyl substances (PFASs) are persistent chemicals that firefighters encounter. Epigenetic modifications, including DNA methylation, could serve as PFASstoxicity biomarkers. Methods: With a sample size of197 firefighters, we quantified theserum concentrations of nine PFASs, blood leukocyte DNA methylation and epigenetic age indicators via the EPIC array. We examined theassociations between PFASswith epigenetic age, site- and region-specific DNA methylation, adjusting for confounders. Results: Perfluorohexane sulfonate, perfluorooctanoate (PFOA)and the sum of branched isomers of perfluorooctane sulfonate (Sm-PFOS) were associated with accelerated epigenetic age. Branched PFOA, linear PFOS, perfluorononanoate, perfluorodecanoateand perfluoroundecanoate were associated with differentially methylated loci and regions. Conclusion: PFASs concentrations areassociated with accelerated epigenetic age and locus-specific DNA methylation. The implications for PFASs toxicity merit further investigation.

Epigenome-wide analysis of DNA methylation and coronary heart disease: a nested case-control study.

Identifying environmentally responsive genetic loci where DNA methylation is associated with coronary heart disease (CHD) may reveal novel pathways or therapeutic targets for CHD. We conducted the first prospective epigenome-wide analysis of DNA methylation in relation to incident CHD in the Asian population. We did a nested case-control study comprising incident CHD cases and 1:1 matched controls who were identified from the 10 year follow-up of the China Kadoorie Biobank. Methylation level of baseline blood leukocyte DNA was measured by Infinium Methylation EPIC BeadChip. We performed the single cytosine-phosphate-guanine (CpG) site association analysis and network approach to identify CHD-associated CpG sites and co-methylation gene module. After quality control, 982 participants (mean age 50.1 years) were retained. Methylation level at 25 CpG sites across the genome was associated with incident CHD (genome-wide false discovery rate [FDR] < 0.05 or module-specific FDR < 0.01). One SD increase in methylation level of identified CpGs was associated with differences in CHD risk, ranging from a 47 % decrease to a 118 % increase. Mediation analyses revealed 28.5 % of the excessed CHD risk associated with smoking was mediated by methylation level at the promoter region of ANKS1A gene (P for mediation effect = 0.036). Methylation level at the promoter region of SNX30 was associated with blood pressure and subsequent risk of CHD, with the mediating proportion to be 7.7 % (P = 0.003) via systolic blood pressure and 6.4 % (P = 0.006) via diastolic blood pressure. Network analysis revealed a co-methylation module associated with CHD. We identified novel blood methylation alterations associated with incident CHD in the Asian population and provided evidence of the possible role of epigenetic regulations in the smoking- and blood pressure-related pathways to CHD risk. This work was supported by National Natural Science Foundation of China (81390544 and 91846303). The CKB baseline survey and the first re-survey were supported by a grant from the Kadoorie Charitable Foundation in Hong Kong. The long-term follow-up is supported by grants from the UK Wellcome Trust (202922/Z/16/Z, 088158/Z/09/Z, 104085/Z/14/Z), grant (2016YFC0900500, 2016YFC0900501, 2016YFC0900504, 2016YFC1303904) from the National Key R&D Program of China, and Chinese Ministry of Science and Technology (2011BAI09B01).

Are dietary factors involved in the association of CDH4 methylation and breast cancer risk?

DNA methylation is one of the most important epigenetic modifications in breast cancer (BC) development, and long-term dietary habits can alter DNA methylation. Cadherin-4 (CDH4, a member of the cadherin family) encodes Ca2+-dependent cell-cell adhesion glycoproteins. We conducted a case-control study (380 newly diagnosed BC and 439 cancer-free controls) to explore the relationship of CDH4 methylation in peripheral blood leukocyte DNA (PBL DNA), as well as its combined and interactive effects with dietary factors on BC risk. A case-only study (335 newly diagnosed BC) was conducted to analyse the association between CDH4 methylation in breast tissue DNA and dietary factors. CDH4 methylation was detected using quantitative methylation-specific PCR. Unconditional logistic regressions were used to analyse the association of CDH4 methylation in PBL DNA and BC risk. Cross-over analysis and unconditional logistic regression were used to calculate the combined and interactive effects between CDH4 methylation in PBL DNA and dietary factors in BC. CDH4 hypermethylation was significantly associated with increased BC risk in PBL DNA (ORadjusted (ORadj) = 2·70, (95 % CI 1·90, 3·83), P < 0·001). CDH4 hypermethylation also showed significant combined effects with the consumption of vegetables (ORadj = 4·33, (95 % CI 2·63, 7·10)), allium vegetables (ORadj = 7·00, (95 % CI 4·17, 11·77)), fish (ORadj = 7·92, (95 % CI 3·79, 16·53)), milk (ORadj = 6·30, (95 % CI 3·41, 11·66)), overnight food (ORadj = 4·63, (95 % CI 2·69, 7·99)), pork (ORadj = 5·59, (95 % CI 2·94, 10·62)) and physical activity (ORadj = 4·72, (95 % CI 2·87, 7·76)). Moreover, consuming milk was significantly related with decreased risk of CDH4 methylation (OR = 0·61, (95 % CI 0·38, 0·99)) in breast tissue. Our findings may provide direct guidance on the dietary intake for specific methylated carriers to decrease their risk for developing BC.

Abstract 577: A unified pipeline to detect small mutations, structural variations and copy number alterations from targeted cell-free DNA sequencing in cancer

Abstract Introduction: Analysis of cell-free DNA (cfDNA) has recently emerged as a non-invasive modality for guiding cancer diagnostics and treatment decisions. However, efforts have predominantly focused on the analysis of single nucleotide variants (SNVs) and insertions/deletions (indels). Despite the clinical significance of many larger variants such as structural variations (SVs) and copy number alterations (CNAs), detecting them in cfDNA remains a challenge. Given the lack of existing tools we seek to develop an integrated bioinformatic pipeline for SV and CNA detection in cfDNA following targeted hybrid-capture next-generation sequencing (NGS), along with standard SNV and indel analysis. Methods: SVs were first detected using Manta, Lumpy and Delly in plasma cfDNA in comparison with matched peripheral blood leukocyte (PBL) DNA samples from cancer patients, then combined to identify consensus SVs and genotyped throughout samples from patients and healthy individuals. Next, consensus SVs were called somatic events if they were supported by split reads and discordant read pairs in cfDNA samples from patients but not in matched PBL or healthy donor cfDNA samples. For CNA analysis, the ratio of read depth between patient-derived plasma cfDNA and a panel of healthy controls was calculated across genomic bins using the CNVkit tool, followed by bias correction and recentralization using CNA negative control genes to account for read coverage imbalances in targeted NGS. Last, SNV and indel analysis was integrated from the CAPP-Seq pipeline. Results: We applied our pipeline to targeted hybrid-capture NGS data from 48 patients across two independent cohorts of metastatic castration resistant prostate cancer (mCRPC). The targeted panel covered the full-length AR gene body and a hotspot region of TMPRSS2-ERG fusion break points. Consistent with earlier whole genome studies, we confirmed known CNAs and SVs in tumor suppressors, oncogenes and regulatory elements including AR gene and AR enhancer duplications (22/48, 46% of patients), TMPRSS2-ERG gene fusions (9/48, 19%), PTEN and TP53 loss (8/48, 17%). Notably, our pipeline outperformed FACTERA which did not detect any TMPRSS2-ERG gene fusions or AR/enhancer tandem duplications. Subsequent analysis showed high concordance between plasma cfDNA and matched tumor biopsies, and our pipeline recapitulated the landscape of SVs and CNAs in an in silico cfDNA simulation from tumor biopsies. Finally, we showed that alterations of the AR/enhancer locus detected by our pipeline were strongly associated with treatment resistance, patient progression-free and overall survival in mCRPC. Conclusion: We developed a unified pipeline for detection of SVs, CNAs and small mutations in cfDNA targeted sequencing with potential applications in monitoring cancer progression and predicting patient treatment response. Citation Format: Ha X. Dang, Jace Webster, Pradeep S. Chauhan, Steven H. Hartman, Wenjia Feng, Elisa M. Ledet, Haley Ellis, Patrick J. Miller, Ellen B. Jaeger, Sydney A. Caputo, Peter K. Harris, A. Oliver Sartor, Russell K. Pachynski, Aadel A. Chaudhuri, Christopher A. Maher. A unified pipeline to detect small mutations, structural variations and copy number alterations from targeted cell-free DNA sequencing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 577.

Association between telomere length in the DNA of peripheral blood leukocytes and the propofol dose in anesthesia induction: an observational study

IntroductionPropofol is a widely used anesthetic and its dose is closely related to aging. Telomere length (TL) is a unique heritable trait, and emerging as a biomarker of aging, health and disease. Telomerase RNA component (TERC) plays an important role in maintaining TL. We proposed a hypothesis that propofol dose in general anesthesia can be predicted by measuring TL before operation, which greatly reduced the risk of anesthesia, especially the elderly. MethodsThe association between the propofol dose in anesthesia induction and: TL in the DNA of peripheral blood leukocytes; body weight; sex; difference of the Bispectral Index (BIS) before and after anesthesia induction in patients was evaluated by multivariable linear regression analyses. The mutation at the 5'end or 3'end of TERC was detected. We recruited 100 patients of elective surgery. ResultsWe found that propofol dose in anesthesia induction was clearly correlated significantly with TL (r = 0.78, p < 0.001), body weight (r = 0.84, p = 0.004), sex (r = 0.83, p= 0.84, p = 0.004), sex (r = 0.83, p = 0.004), and difference of BIS before and after anesthesia induction (r = 0.85, p = 0.029). By comparing the absolute values of standardized regression coefficients (0.58, 0.21, 0.19, and 0.12) of the four variables, it can be seen that TL contributes the most to the propofol dose in anesthesia induction. However, the mutation at the 5' end or 3' end of TERC was not found. ConclusionsThese findings provide preliminary evidence that the propofol dose in anesthesia induction was clearly correlated with genetically determined TL. TL may be a promising predictor of the propofol dose, which is beneficial to improve the safety of anesthesia and reduce perioperative complications.

Epigenome-wide scan identifies differentially methylated regions for lung cancer using pre-diagnostic peripheral blood

ABSTRACT Background: DNA methylation markers have been associated with lung cancer risk and may identify aetiologically relevant genomic regions, or alternatively, be markers of disease risk factors or biological processes associated with disease development. Methods: In a nested case–control study, we measured blood leukocyte DNA methylation levels in pre-diagnostic samples collected from 430 participants (208 cases; 222 controls) in the 1989 CLUE II cohort. We compared DNA methylation levels with case/control status to identify novel genomic regions, both single CpG sites and differentially methylated regions (DMRs), while controlling for known DNA methylation changes associated with smoking using a previously described pack-years-based smoking methylation score. Stratification analyses were conducted over time from blood draw to diagnosis, histology, and smoking status. Results: We identified 16 single CpG sites and 40 DMRs significantly associated with lung cancer risk (q < 0.05). The identified genomic regions were associated with genes including H19, HOXA3/HOXA4, RUNX3, BRICD5, PLXNB2, and RP13. For the single CpG sites, the strongest association was noted for cg09736286 in the DIABLO gene (OR [for 1 SD] = 2.99, 95% CI: 1.95–4.59, P-value = 4.81 × 10–7). We found that CpG sites in the HOXA3/HOXA4 region were hypermethylated in cases compared to controls. Conclusion: The single CpG sites and DMRs that we identified represented significant measurable differences in lung cancer risk, providing potential biomarkers for lung cancer risk stratification. Future studies will need to examine whether these regions are causally related to lung cancer.

Association of ZNF331 and WIF1 methylation in peripheral blood leukocytes with the risk and prognosis of gastric cancer

BackgroundPeripheral blood leukocyte (PBL) DNA methylation may serve as a surrogate marker to evaluate the susceptibility to and prognosis of gastric cancer (GC). In this study, blood-derived DNA methylation levels of two tumour-related genes, namely, ZNF331 and WIF1, and their impacts on the risk and prognosis of GC were evaluated.MethodsIn total, 398 GC cases and 397 controls were recruited for the study. Then, all cases were followed up for 5 years. ZNF331 and WIF1 promoter methylation status in PBLs was measured using a methylation-sensitive high-resolution melting method. Logistic and Cox regression models were used to analyse the correlation between gene methylation and the risk and prognosis of GC. Confounders were balanced through propensity score (PS) matching.ResultsHigh ZNF331 methylation significantly decreased GC risk after PS adjustment (OR = 0.580, 95% CI: 0.375–0.898, P = 0.015), which also presented in males (OR = 0.577, 95% CI: 0.343–0.970, P = 0.038). However, WIF1 methylation was not associated with GC risk. Additionally, significant combined effects between ZNF331 methylation and the intake of green vegetables and garlic were observed (OR = 0.073, 95% CI: 0.027–0.196, P < 0.001 and OR = 0.138, 95% CI: 0.080–0.238, P < 0.001, respectively). Furthermore, ZNF331 and WIF1 methylation had no impact on the prognosis of GC.ConclusionZNF331 methylation in PBLs may affect GC risk in combination with the consumption of green vegetables and garlic and may act as a potential biomarker of GC.

ЛЕЙКОЗ КРУПНОГО РОГАТОГО СКОТА — ДИАГНОСТИКА, ОЗДОРОВЛЕНИЕ, АНТРОПОЗООНОЗНЫЙ ПОТЕНЦИАЛ (ИСТОРИЯ ВОПРОСА) (обзор)

Of tumor diseases in the global animal husbandry industry, the greatest danger is bovine leukemia. Since the last century, this neoplastic disease has remained relevant for veterinary medicine and, in addition, more and more questions arise concerning the potential threat of the Bovine leukemia virus (BLV) for humans. The problem is being discussed (G.Yu. Kosovskiy et al., 2016) which additionally stimulates both fundamental research of the pathogen and the pathology, as well as the methods for detection to improve dairy cattle breeding and veterinary safety. A number of publications are devoted to BLV prevalence, peculiarities, the prospects for improving animal health and welfare based on selection and vaccines developed in the world (S.G. Hopkins et al., 1997; M.A. Juliarena et al., 2017). In our report, we retrospectively compare the experience of BLV eradication in the USSR, Russia and abroad, additionally focusing on the possible role of this pathogen in the occurrence of cancer in humans. In veterinary practice, serological tests, i.e., immunodiffusion (ID) assay in agar gel and enzyme-linked immunosorbent assay (ELISA) are mostly used to detect the BLV infection. G.C. Buehring et al. (2019) reported the detection of BLV proviral DNA in blood leukocytes in 38 % of patients examined by PCR and DNA sequencing. IgG antibodies to BLV were detected in 32 %, IgM — in 58 %, and IgA — in 32 % of the samples tested. Accumulated data indicate metabolic changes in the BLV-positive animals, e.g., disorder in the metabolism of tryptophan, a critical essential acid, leading to the accumulation of dangerous endogenous metabolites in the body. Particularly, free tryptophan, indole and anthranilic acid increased in level 4-8-fold are deposited in the organs of the immune and hematopoietic system (lymph nodes, spleen, liver), in the mammary gland, lungs, and kidneys. It has been established that the milk of leukemic animals differs from the milk of healthy cows in terms of physicochemical, bacteriological (lysozyme), technological parameters and mineral composition. In C57 mice fed with pasteurized milk and heat-treated meat from ID-positive cows, the blood cell profile changed. Available data drive to the unambiguous conclusion that the products derived from BLV-infected cattle pose an increased risk to humans. Despite the lack of convincing evidence of the BLV pathogenicity for humans, concerns about the etiological role of this virus in the occurance of cancer in humans additionally necessitates continuing research on the control and eradication of this common oncogenic retro-virus in livestock farms.

Bovine leukemia virus INFECTION — DIAGNOSTICS, ERADICATION, AND ANTHROPOZOONOTIC POTENTIAL (BACKGROUND) (review)

Of tumor diseases in the global animal husbandry industry, the greatest danger is bovine leukemia. Since the last century, this neoplastic disease has remained relevant for veterinary medicine and, in addition, more and more questions arise concerning the potential threat of the Bovine leukemia virus (BLV) for humans. The problem is being discussed (G.Yu. Kosovskiy et al., 2016) which additionally stimulates both fundamental research of the pathogen and the pathology, as well as the methods for detection to improve dairy cattle breeding and veterinary safety. A number of publications are devoted to BLV prevalence, peculiarities, the prospects for improving animal health and welfare based on selection and vaccines developed in the world (S.G. Hopkins et al., 1997; M.A. Juliarena et al., 2017). In our report, we retrospectively compare the experience of BLV eradication in the USSR, Russia and abroad, additionally focusing on the possible role of this pathogen in the occurrence of cancer in humans. In veterinary practice, serological tests, i.e., immunodiffusion (ID) assay in agar gel and enzyme-linked immunosorbent assay (ELISA) are mostly used to detect the BLV infection. G.C. Buehring et al. (2019) reported the detection of BLV proviral DNA in blood leukocytes in 38 % of patients examined by PCR and DNA sequencing. IgG antibodies to BLV were detected in 32 %, IgM — in 58 %, and IgA — in 32 % of the samples tested. Accumulated data indicate metabolic changes in the BLV-positive animals, e.g., disorder in the metabolism of tryptophan, a critical essential acid, leading to the accumulation of dangerous endogenous metabolites in the body. Particularly, free tryptophan, indole and anthranilic acid increased in level 4-8-fold are deposited in the organs of the immune and hematopoietic system (lymph nodes, spleen, liver), in the mammary gland, lungs, and kidneys. It has been established that the milk of leukemic animals differs from the milk of healthy cows in terms of physicochemical, bacteriological (lysozyme), technological parameters and mineral composition. In C57 mice fed with pasteurized milk and heat-treated meat from ID-positive cows, the blood cell profile changed. Available data drive to the unambiguous conclusion that the products derived from BLV-infected cattle pose an increased risk to humans. Despite the lack of convincing evidence of the BLV pathogenicity for humans, concerns about the etiological role of this virus in the occurance of cancer in humans additionally necessitates continuing research on the control and eradication of this common oncogenic retro-virus in livestock farms.

Assessment of HCMV-encoded microRNAs in plasma as potential biomarkers in pregnant women with adverse pregnancy outcomes.

BackgroundHuman cytomegalovirus (HCMV) is the most frequent cause of congenital infections and can lead to adverse pregnancy outcomes (APOs). HCMV encodes multiple microRNAs (miRNAs) that have been reported to be partially related to host immune responses, cell cycle regulation, viral replication, and viral latency, and can be detected in human plasma. However, the relevance for HCMV-encoded miRNAs in maternal plasma as an indicator for APOs has never been evaluated.MethodsExpression profiles of 22 HCMV-encoded miRNAs were first measured in plasma samples from 20 pregnant women with APOs and 28 normal controls using quantitative reverse-transcription polymerase chain reaction. Next, markedly changed miRNAs were validated in another independent validation set consisting of 20 pregnant women with APOs and 27 control subjects. Markedly changed miRNAs were further assessed in the placenta tissues. HCMV DNA in peripheral blood leukocytes (PBLs) and anti-HCMV immunoglobulin M (IgM) and anti-HCMV immunoglobulin G (IgG) in plasma were also examined in both training and validation sets. Diagnostic value and risk factors were compared between APO cohorts and normal controls.ResultsAnalysis of the training and validation data sets revealed that plasma concentrations of hcmv-miR-UL148D, hcmv-miR-US25-1-5p and hcmv-miR-US5-1 were significantly increased in pregnant women with APOs compared with normal controls. Hcmv-miR-US25-1-5p presented the largest area under the receiver-operating characteristic (ROC) curve (AUC) (0.735; 95% CI, 0.635–0.836), with a sensitivity of 68% and specificity of 71%. Furthermore, plasma levels of hcmv-miR-US25-1-5p and hcmv-miR-US5-1 correlated positively with APOs (P=0.029 and 0.035, respectively). Hcmv-miR-US25-1-5p in the placenta tissues were dramatically increased in APOs, and correlated with plasma hcmv-miR-US25-1-5p. Nevertheless, neither the concentration of HCMV DNA in PBLs nor the positivity rates of anti-HCMV IgM and anti-HCMV IgG in plasma showed a statistically significant correlation with APOs.ConclusionsWe identified a unique signature of HCMV-encoded miRNAs in pregnant women with APOs that may be useful as a potential noninvasive biomarker for predicting and monitoring APOs during HCMV infection.

The first detection of Anaplasma capra, an emerging zoonotic Anaplasma sp., in erythrocytes

ABSTRACT An emerging infectious disease caused by “Anaplasma capra” was reported in a 2015 survey of 477 hospital patients with a tick-bite history in China. However, the morphological characteristics and parasitic location of this pathogen are still unclear, and the pathogen has not been officially classified as a member of the genus Anaplasma. Anaplasma capra-positive blood samples were collected, blood cells separated, and DNA of whole blood cells, erythrocytes, and leukocytes extracted. Multiplex PCR detection assay was used to detect whole blood cell, erythrocytes and leukocytes, DNA samples, and PCR identification, nucleic acid sequencing, and phylogenetic analyses based on A. capra groEL, 16S rRNA, gltA, and msp4 genes. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), Wright–Giemsa staining, chromogenic in situ hybridization (CISH), immunocytochemistry, and indirect immunofluorescence assay (IFA) were used to identify the location and morphological characteristics of A. capra. Multiple gene loci results demonstrated that erythrocyte DNA samples were A. capra-positive, while leukocyte DNA samples were A. capra-negative. Phylogenetic analysis showed that A. capra is in the same clade with the A. capra sequence reported previously. SEM and TEM showed one or more pathogens internally or on the outer surface of erythrocytes. Giemsa staining, CISH, immunocytochemistry, and IFA indicated that erythrocytes were A. capra-positive. This study is the first to identify the novel zoonotic tick-borne Anaplasma sp., “Anaplasma capra,” in host erythrocytes. Based on our results, we suggest revision of Genus Anaplasma and formally name “A. capra” as Anaplasma capra sp. nov.