Peripheral Blood Leukocyte DNA Research Articles

1844MO Association of peripheral blood leukocyte DNA methylation profiles with the development of depressive symptoms in lung cancer patients undergoing systemic therapy

1844MO Association of peripheral blood leukocyte DNA methylation profiles with the development of depressive symptoms in lung cancer patients undergoing systemic therapy

Proteomic and Global DNA Methylation Modulation in Lipid Metabolism Disorders with a Marine-Derived Bioproduct.

Dyslipidemia is a significant risk factor for cardiovascular disease and stroke. Our recent findings showed that RCI-1502, a bioproduct derived from the muscle of the European S. pilchardus, has lipid-lowering effects in the liver and heart in high-fat diet (HFD) fed mice. In the present follow-up study, we investigated the therapeutic potential of RCI-1502 on gene expression and DNA methylation in HFD-fed mice and in patients with dyslipidemia. Using LC-MS/MS, we identified 75 proteins in RCI-1502 that are primarily involved in binding and catalytic activity and which regulate pathways implicated in cardiovascular diseases. In HFD-fed mice, RCI-1502 treatment significantly reduced the expression of cardiovascular disease-related genes, including vascular cell adhesion molecule and angiotensin. RCI-1502 also decreased DNA methylation levels, which were elevated in HFD-fed mice, to levels similar to those in control animals. Furthermore, peripheral blood leukocyte DNA from dyslipidemic patients exhibited higher DNA methylation levels than healthy individuals, suggesting a potential association with cardiovascular risk. Serum analysis also revealed that RCI-1502 treatment regulated cholesterol and triglyceride levels in patients with dyslipidemia. Our findings appear to suggest that RCI-1502 is an epigenetic modulator for the treatment of cardiovascular diseases, specifically in individuals with dyslipidemia.

Sex-specific variations in global DNA methylation levels with age: a population-based exploratory study from North India.

Purpose: Aging is one of the most important risk factors for a number of human diseases. Epigenetic alterations, including changes in DNA methylation patterns, have been reported to be one of the hallmarks of aging. Being a malleable process, the role of site-specific DNA methylation in aging is being extensively investigated; however, much less attention has been given to alterations in global DNA methylation with aging at the population level. The present study aims to explore overall and sex-specific variations in global DNA methylation patterns with age. Methods: A total of 1,127 adult individuals (792 females) aged 30-75years belonging to Haryana, North India, were recruited. Socio-demographic data was collected using a pretested interview schedule. Global DNA methylation analysis, of peripheral blood leucocyte (PBL) DNA, was performed using the ELISA-based colorimetric technique. Results: Though the overall correlation analysis revealed a weak inverse trend between global DNA methylation and age, the adjusted regression model showed no significant association between global DNA methylation and age. In age-stratified analysis, global DNA methylation levels were found to be fairly stable until 60years of age, followed by a decline in the above-60 age group. Further, no significant difference in DNA patterns methylation pattern was observed between males and females. Conclusion: Overall, the study suggests a lack of association between global DNA methylation and age, especially until 60years of age, and a similar DNA methylation pattern between males and females with respect to age.

Characteristics of plasma Epstein-Barr virus DNA in children with primary infection

Objective: To explore the characteristics of plasma Epstein-Barr virus (EBV) DNA in primary infection in pediatric cases. Methods: The laboratory and clinical data of 571 children diagnosed with EBV primary infection in Children's Hospital of Fudan University during September 1st, 2017 to September 30th, 2018 were retrospectively analyzed. According to the results of plasma EBV DNA, they were divided into positive group and negative group. According to the EBV DNA, they were devided into high plasma virol load group and low plasma virol load group. The Chi-square test, Wilcoxon rank sum test were used to compare the differences between groups. Results: Among the 571 children with EBV primary infection, 334 were males and 237 were females. The age of first diagnosis was 3.8 (2.2, 5.7) years. There were 255 cases in positive group and 316 cases in negative group. The percentage of cases with fever,hepatomegaly and (or) splenomegaly, elevated transaminase in the positive group were higher than those in the negative group (235 cases (92.2%) vs. 255 cases (80.7%), χ2=15.22, P<0.001; 169 cases (66.3%) vs. 85 cases (26.9%), χ2=96.80, P<0.001; and 144 cases (56.5%) vs. 120 cases (38.0%), χ2=18.27, P<0.001; respectively).In the positive group, 70 cases were followed up for 46 (27, 106) days, 68 cases (97.1%) turned negative within 28 days, with the exception of 2 cases (2.9%) developed chronic active EBV infection by follow-up revision.There were 218 cases in high plasma viral DNA copies group and 37 cases in low copies group. More cases presented with elevated transaminases in the high plasma viral DNA copies group than those in the low group (75.7% (28/37) vs. 56.0%(116/207), χ2=5.00, P=0.025).Both the positive rate of EBV DNA in peripheral blood leukocytes (84.2% (266/316) vs. 44.7% (255/571), χ2=76.26, P<0.001) and the copies of EBV DNA (7.0×107 (1.3×107, 3.0×108) vs. 3.1×106 (1.6×106, 6.1×106) copies /L, Z=15.23, P<0.001) were higher than that of plasma. Conclusions: In immunocompetent pediatric cases diagnosed as EBV primary infection, cases with positive plasma EBV DNA were prone to have fever, hepatomegaly and (or) splenomegaly, and elevated transaminase than those with negative plasma viral DNA. The plasma EBV DNA usually turns negative within 28 days after initial diagnosis.Most cases with high viral load in plasma showed elevated aminotransferase.

Long-term Temporal Stability of Peripheral Blood DNA Methylation Profiles in Patients With Inflammatory Bowel Disease

There is great current interest in the potential application of DNA methylation alterations in peripheral blood leukocytes (PBLs) as biomarkers of susceptibility, progression, and treatment response in inflammatory bowel disease (IBD). However, the intra-individual stability of PBL methylation in IBD has not been characterized. Here, we studied the long-term stability of all probes located on the Illumina HumanMethylation EPIC BeadChip array. We followed a cohort of 46 adult patients with IBD (36 Crohn's disease [CD], 10 ulcerative colitis [UC]; median age, 44 years; interquartile range [IQR] 27-56 years; 50% female) that received standard care follow-up at the Amsterdam University Medical Centers. Paired PBL samples were collected at 2 time points with a median of 7 years (range, 2-9 years) in between. Differential methylation and intra-class correlation (ICC) analyses were used to identify time-associated differences and temporally stable CpGs, respectively. Around 60% of all EPIC array loci presented poor intra-individual stability (ICC <0.50); 78.114 (≈9%) showed good (ICC, 0.75-0.89), and 41.274 (≈5%) showed excellent (ICC ≥0.90) stability, between both measured time points. Focusing on previously identified consistently differentially methylated positions indicated that 22 CD-, 11 UC-, and 24 IBD-associated loci demonstrated high stability (ICC ≥0.75) over time; of these, we observed a marked stability of CpG loci associated to the HLA genes. Our data provide insight into the long-term stability of the PBL DNA methylome within an IBD context, facilitating the selection of biologically relevant and robust IBD-associated epigenetic biomarkers with increased potential for independent validation. These data also have potential implications in understanding disease pathogenesis.

Susceptibility study on the germline rare variants of bromodomain and extraterminal domain protein family-encoding genes and patients with cancer living in some regions of China

Objective: To explore the relationship between germline rare variants of bromodomain and extraterminal domain (BET) protein family-encoding genes and susceptibility to cancer in some regions of China. Methods: Capturing probes were designed for bromodomain-containing protein 2 (BRD2), BRD3 and BRD4 genes, and Illumina high-throughput sequencing platform was used to conduct targeted sequencing of genomic DNA of peripheral blood leukocytes from 1 673 patients with cancer and 1 661 individuals without cancer recruited between October 2015 and July 2018 from Chinese PLA General Hospital, the Second Affiliated Hospital of Guangxi Medical University, People's Hospital of Macheng City, Hubei Province and Geneplus-Beijing Co. Ltd. Mutation detection and analysis were carried out according to the genome analysis toolkit (GATK) best practice guidelines, ANNOVAR and VEP software were used for annotation, and germline rare variants in BET family were screened. To determine potential pathogenic germline rare variants, clinical and experimental evidence was obtained from the ClinVar database and SIFT and Polyphen-2 softwares were used to predict pathogenicity. Fisher's exact test was used to compare the difference of the carrying rate of variants in the case group and the control group, and multivariate regression analysis was performed with the SKAT software with sex and age used as covariates. Results: Among the 1 673 cancer patients, 911 were males and 762 were females, with the mean age was (57.9±11.7) years. There were 1, 111 cases (66.4%) of lung cancer, 266 cases (15.9%) of colorectal cancer, 186 cases of breast cancer (11.1%), and 110 cases (6.6%) of esophagus or gastric cancer. In the same period 1, 661 non-tumor control individuals were recruited, including 821 males and 840 females, with the mean age was (44.5±13.9) years. It was observed that there were 4 potential pathogenic germline rare variants in BRD2 gene carried by 17 patients with cancer, 5 potential pathogenic germline rare variants in BRD3 gene and 8 potential pathogenic germline rare variants in BRD4 gene. The carrying rate of potential pathogenic germline rare variants in BRD2 gene in cancer patients was 1.02% (17/1 673), significantly higher than that in controls without cancer [0 (0/1 661); OR=+∞, 95%CI: 4.81-+∞, P<0.001]. The carrying rate of potential pathogenic germline rare variants in BRD3 gene in cancer patients was 0.24% (4/1 673), and the difference was not statistically significant compared with controls without cancer [0.12% (2/1 661); OR=1.99, 95%CI: 0.46-10.47, P=0.690]. The carrying rate of potential pathogenic germline rare variants in BRD4 gene in cancer patients was 0.18% (3/1 673), and the difference was not statistically significant compared with controls without cancer [0.36% (6/1 661); OR=0.50, 95%CI: 0.14-2.08, P=0.340]. Furthermore, the dataset of whole exome sequencing of Chinese individuals in "Huabiao Project" was used as an additional control, and the rate of carrying BRD2 rare variants in cancer patients was 17/3 346 (0.51%), significantly higher than that in controls without cancer [0.07% (3/4 154); OR=7.07, 95%CI: 2.32-22.83, P<0.001]. Among the 17 patients carrying 4 potentially pathogenic germline rare variants of BRD2 gene, 9 were patients with lung cancer, 6 were patients with colorectal cancer, 1 was patient with breast cancer, and 1 was patients with esophagus or gastric cancer. The carrying rate of potential pathogenic germline rare variants in BRD2 gene in lung cancer patients was 0.81 (9/1 111), significantly higher than that in controls without cancer [0(0/1 661); OR=+∞, 95%CI: 3.95-+∞,P<0.001]. The carrying rate of potential pathogenic germline rare variants in BRD2 gene in patients with colorectal cancer was 2.26% (6/266), significantly higher than that in controls without cancer [0(0/1 661); OR=+∞, 95%CI: 9.03-+∞, P<0.001]. Wilcoxon rank-sum test results showed that patients with colorectal cancer carrying BRD2 rare variants had an earlier age at diagnosis [(47.0±7.4) vs (57.2±12.1) years old, P=0.017]. Conclusions: BRD2 gene may be served as a candidate genetic susceptibility gene for lung cancer and colorectal cancer. Carrying BRD2 potential pathogenic germline rare variants is associated with higher risk of lung cancer and colorectal cancer, and with earlier age of colorectal cancer.

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.

Telomere Length as a Marker of Suicidal Risk in Schizophrenia

BACKGROUND: 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.&#x0D; METHODS: 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.&#x0D; RESULTS: 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 (22528.4 (227 [190; 250]) vs. 24321 (245 [228; 260]) vs. 25517.9 (255 [242; 266]), p 0.005. The same trend is observed for the number of mtDNA copies (257101.5 (250 [194; 297])) vs. 262.359.3 (254 [217; 312]) vs. 27279.9 (274 [213; 304]); p=0.012), but no significant differences were recorded.&#x0D; CONCLUSIONS: 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 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.

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.

The Potential Use of DNA Methylation Patterns from Peripheral Blood Leukocytes as a Surrogate for Stress Axis Tissues in Mature Brahman Cows

Abstract DNA methylation (DNAm) patterns are tissue specific and aid in tissue specific gene expression changes. The use of DNAm patterns from peripheral blood leukocytes (PBL) as a surrogate for patterns in other tissues is common, especially in longitudinal studies when sampling of tissues is not plausible. Thus, the objective of this study was to investigate the suitability of using DNAm patterns of PBL as a surrogate for the DNAm patterns in neuroendocrine tissues responsible for stress responses and energy metabolism. Samples from the paraventricular region of the hypothalamus, anterior pituitary gland, adrenal cortex, and the adrenal medulla were harvested from 5-yr-old Brahman cows (n = 8) and DNA was extracted from each sample. Methylation was assessed using reduced representation sodium bisulfite sequencing and differentially methylated regions (DMR) between the PBL DNA and tissue DNA were identified using EdgeR from Bioconductor, R. Analysis revealed over 15,000 DMRs located within promoter regions of genes in each tissue, with the majority of the sites having increased methylation in the PBL (Table 1). To further evaluate the use of PBL DNA as a surrogate, Pearson correlation values were calculated for genes (n = 20) pertinent to each respective tissue using the mean methylation of the specific gene in the PBL and in the tissue (Table 2). Three correlations were significant (P ≤ 0.05), two of which were negative. The sizable differences indicate that DNA methylation patterns from PBL do not compare well to patterns from hypothalamic, pituitary, adrenal cortex, and adrenal medulla tissues from 5-yr-old Brahman cows. This is especially the case for the majority of the specific genes examined in this study. Whether DNAm in the surrogate PBL will shift in a direction similar to that of specific tissues of Brahman cows exposed to stressful stimuli during developmental periods remains to be determined.

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.

Serotyping versus genotyping in infected sheep and goats with small ruminant lentiviruses

Despite SRLV infection being endemic in Mexico, there is little information regarding which genotypes are present. We compared serotyping and PCR-sequencing results from sheep and goats infected with SRLV. We separated plasma and peripheral blood leukocytes (PBL) from 1940 blood samples from sheep and goats from 12 states across Mexico. To detect SRLV infection, we tested plasma samples using two commercial ELISA kits (VMRD and Eradikit SRLV Screening). Then, we serotyped the infecting virus (A/ B) using Eradikit SRLV Genotyping. PBL DNA was used to detect the proviral genome via PCR. Positive amplicons were sequenced to identify viral genotypes using a phylogenetic analysis. Also, we analysed for residues differences in the sequences of a capsid epitope between genotypes. The serological results indicated a higher detection of seropositive animals using the VMRD ELISA compared to Eradikit, with 21 % and 15.3 % more in sheep and goats respectively. Only 25.7 % of the ELISA serotyping results matched those from PCR-sequencing. PCR-sequencing was able to identify genotype A, B and coinfections in animals classified as indeterminate by the ELISA test. This lack of sensitivity may be related to the lack of epitopes from the matrix and transmembrane peptides used by ELISA screening. Sequences analysis revealed that SRLVs found in sheep cluster with genetic subtypes A2 and B1, while those in goats cluster with subtypes A1 and B1. Serotyping did not prove to be an adequate method for predicting the viral genotype (A and / or B) in infections caused by SRLV.

Clinical Features and Survival of Chinese Children With Trilateral Retinoblastoma During 2006-2019: A Retrospective Multicenter Study

To summarize the clinical features and survival of Chinese patients with trilateral retinoblastoma (TRb), which may help guide early diagnosis and more effective treatments. Retrospective case series. Clinical records of patients with TRb were reviewed to identify clinical characteristics and outcomes. TRb was diagnosed mainly based on imaging findings of an enlarged solid pineal or sellar mass. Mutation screening was performed using peripheral blood leucocyte DNA from 3 patients. Fourteen patients with TRb were identified from among 3,789 patients with retinoblastoma (0.4%). Thirteen patients had bilateral retinoblastoma and 1 patient had unilateral disease. The follow-up results revealed that 2 patients survived, 3 patients were lost to follow-up, and 9 patients died. The mean overall survival was 9.8months (95% confidence interval: 2.3-17.2), and the 2-year survival rate was 18.8% (95% confidence interval: 2.9-45.1) based on Kaplan-Meier estimates. Cox regression multivariate analysis showed metastasis at TRb diagnosis was an independent variable of overall survival (hazard ratio: 15.8; 95% confidence interval: 0.24-5.29; P= .032). Three germline mutations in the RB1 gene were detected via next-generation sequencing. TRb is a rare intracranial mid-line neuroblastic disease. Increased awareness of this diseasecould guide early detection, which has been associated with improved outcomes.