DNA methylation in the inflammatory genes after neurosurgery and diagnostic ability of post-operative delirium

The pathophysiological mechanisms of postoperative delirium (POD) are still not clear, and no reliable biomarker is available to differentiate those with and without POD. Pre- and post-surgery blood from epilepsy subjects undergoing neurosurgery were collected. DNA methylation (DNAm) levels of the TNF gene, IL1B gene, and IL6 gene by the Illumina EPIC array method, and DNAm levels of the TNF gene by pyrosequencing, were analyzed. Blood from 37 subjects were analyzed by the EPIC array method, and blood from 27 subjects were analyzed by pyrosequencing. Several CpGs in the TNF gene in preoperative blood showed a negative correlation between their DNAm and age both in the POD group and in the non-POD group. However, these negative correlations were observed only in the POD group after neurosurgery. Neurosurgery significantly altered DNAm levels at 17 out of 24 CpG sites on the TNF gene, 8 out of 14 CpG sites on the IL1B gene, and 4 out of 14 CpG sites on the IL6 gene. Furthermore, it was found that the Inflammatory Methylation Index (IMI), which was based on the post-surgery DNAm levels at the selected five CpG sites, can be a potential detection tool for delirium with moderate accuracy; area under the curve (AUC) value was 0.84. The moderate accuracy of this IMI was replicated using another cohort from our previous study, in which the AUC was 0.79. Our findings provide further evidence of the potential role of epigenetics and inflammation in the pathophysiology of delirium.

BIOM-43. CROSS-PLATFORM ROBUSTNESS IN THE GLUCOCORTICOID RESPONSE PHARMACODYNAMIC BIOMARKER

The neutrophil dexamethasone methylation index (NDMI) is an algorithm-based biomarker to assess individuals’ exposures to dexamethasone, a synthetic glucocorticoid commonly administered for inflammation. Cortisol is the main endogenous glucocorticoid that controls vital processes including the immune response and lipid and carbohydrate metabolism. Variations in the NDMI score reflect individuals’ sensitivities of exposures to both exogenous and endogenous glucocorticoids, and this biomarker was trained using elastic net regression on Illumina’s most recent DNA methylation beadarray, the EPIC array, which contains 850,000 cytosine-guanine (CpG) sites. While technology for microarray research continues to advance over time, researchers are capable of conducting more comprehensive epigenome-wide association studies (EWAS). However, many studies are still run and archived using Illumina’s historical 450K platform with approximately 450,000 CpGs, and there are fewer published databases using the 850K EPIC array. To evaluate the cross-platform bioinformatic comparability, we performed elastic net regression modeling using predictors available in the 450K to train the NDMI. Among the 135 pre-surgery glioma cases from the UCSF Immune Profiles Study (IPS), NDMI scores between the 450K and 850K model were strongly correlated (r = 0.99, p < 0.0001). In the 311 controls from the UCSF Adult Glioma Study (AGS), similar correlations were observed (r = 0.96, p < 0.0001). We observe that NDMI remains a robust tool using historical 450K data and conclude that this algorithmic tool is capable of detecting the variations in individuals’ responses to dexamethasone.

PATH-48. RAPID-CNS2: RAPID COMPREHENSIVE ADAPTIVE NANOPORE-SEQUENCING OF CNS TUMORS, A PROOF OF CONCEPT STUDY

BACKGROUND The WHO classification 2021 includes multiple molecular markers for routine diagnostics in addition to histology. Sequencing setup for complete molecular profiling requires considerable investment, while batching samples for sequencing and methylation profiling can delay turnaround time. We introduce RAPID-CNS2, a nanopore adaptive sequencing pipeline that enables comprehensive mutational, methylation and copy number profiling of CNS tumours with a single third generation sequencing assay. It can be run for single samples and offers highly flexible target selection requiring no additional library preparation. METHODS Utilising ReadFish, a toolkit enabling targeted nanopore sequencing, we sequenced DNA from 22 diffuse glioma patient samples on a MinION device. Target regions comprised our Heidelberg brain tumour NGS panel and pre-selected CpG sites for methylation classification by an adapted random forest classifier. Pathognomonic alterations, copy number profiles, and methylation classes were called using a custom bioinformatics pipeline. Results were compared to their corresponding NGS panel-seq and EPIC array outputs. RESULTS Complete concordance with the EPIC array was found for copy number profiles from RAPID-CNS2. 94% pathognomonic mutations were congruent with NGS panel-seq. MGMT promoter status was correctly identified in all samples. Methylation families were detected with 96% congruence. Among the alterations decisive for rendering a classification-compatible integrated diagnosis, 97% of the alterations were consistent over the entire cohort (completely congruent in 19/22 cases and sufficient for unequivocal diagnosis in all). CONCLUSIONS RAPID-CNS2 provides a swift and highly flexible alternative to conventional NGS and array- based methods for SNV/Indel analysis, detection of copy number alterations and methylation classification. The turnaround time of ~4 days can be further shortened to < 12h by altering target sizes. It offers a low-capital approach that would be cost-efficient for low throughput settings and invaluable in cases requiring immediate diagnoses.

Per- and polyfluoroalkyl substances, epigenetic age and DNA methylation: a cross-sectional study of firefighters

Background: Per- and polyfluoroalkyl substances (PFASs) are persistent chemicals that firefighters encounter. Epigenetic modifications, including DNA methylation, could serve as PFASs toxicity biomarkers. Methods: With a sample size of 197 firefighters, we quantified the serum concentrations of nine PFASs, blood leukocyte DNA methylation and epigenetic age indicators via the EPIC array. We examined the associations between PFASs with 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, perfluorodecanoate and perfluoroundecanoate were associated with differentially methylated loci and regions. Conclusion: PFASs concentrations are associated with accelerated epigenetic age and locus-specific DNA methylation. The implications for PFASs toxicity merit further investigation.

Characterising sex differences of autosomal DNA methylation in whole blood using the Illumina EPIC array

Sex differences are known to play a role in disease etiology, progression and outcome. Previous studies have revealed autosomal epigenetic differences between males and females in some tissues, including differences in DNA methylation patterns. Here, we report for the first time an analysis of autosomal sex differences in DNAme using the Illumina EPIC array in human whole blood (n=1171). We identified 554 sex-associated differentially methylated CpG sites (saDMPs) with the majority found to be hypermethylated in females (70%). These saDMP's are enriched in CpG islands and CpG shores and located preferentially at 5'UTRs, 3'UTRs and enhancers. Additionally, we identified 311 significant sex associated differentially methylated regions (saDMRs). Transcription factor binding site enrichment revealed enrichment of transcription factors related to critical developmental processes and sex determination such as SRY and SOX9. Our study reports a reliable catalogue of sex associated CpG sites and elucidates several characteristics of these sites.

P07.04 Rapid-CNS2: Rapid comprehensive adaptive nanopore-sequencing of CNS tumors, a proof of concept study

BACKGROUND The WHO classification 2021 includes multiple molecular markers for routine diagnostics in addition to histology. Sequencing setup for complete molecular profiling requires considerable investment, while batching samples for sequencing and methylation profiling can delay turnaround time. We introduce RAPID-CNS2, a nanopore adaptive sequencing pipeline that enables comprehensive mutational, methylation and copy number profiling of CNS tumours with a single third generation sequencing assay. It can be run for single samples and offers highly flexible target selection requiring no additional library preparation. MATERIAL AND METHODS Utilising ReadFish, a toolkit enabling targeted nanopore sequencing, we sequenced DNA from 22 diffuse glioma patient samples on a MinION device. Target regions comprised our Heidelberg brain tumour NGS panel and pre-selected CpG sites for methylation classification by an adapted random forest classifier. Pathognomonic alterations, copy number profiles, and methylation classes were called using a custom bioinformatics pipeline. Results were compared to their corresponding NGS panel-seq and EPIC array outputs. RESULTS Complete concordance with the EPIC array was found for copy number profiles from RAPID-CNS2. 94% pathognomonic mutations were congruent with NGS panel-seq. MGMT promoter status was correctly identified in all samples. Methylation families were detected with 96% congruence. Among the alterations decisive for rendering a classification-compatible integrated diagnosis, 97% of the alterations were consistent over the entire cohort (completely congruent in 19/22 cases and sufficient for unequivocal diagnosis in all). CONCLUSION RAPID-CNS2 provides a swift and highly flexible alternative to conventional NGS and array-based methods for SNV/Indel analysis, detection of copy number alterations and methylation classification. The turnaround time of ~4 days can be further shortened to <12h by altering target sizes. It offers a low-capital approach that would be cost-efficient for low throughput settings and invaluable in cases requiring immediate diagnoses.

Distinct Associations of BMI and Fatty Acids With DNA Methylation in Fasting and Postprandial States in Men

We have previously shown that blood global DNA methylation (DNAm) differs between postprandial state (PS) and fasting state (FS) and is associated with BMI and polyunsaturated fatty acid (PUFA) (negatively and positively, respectively) in 12 metabolically healthy adult Mexican men (AMM cohort) equally distributed among conventional BMI classes. Here, we detailed those associations at CpG dinucleotide level by exploiting the Infinium methylation EPIC array (Illumina). We sought differentially methylated CpG (dmCpG) that were (1) associated with BMI (BMI-dmCpG) and/or fatty acids (FA) (FA-dmCpG) in FS or PS and (2) different across FS and PS within a BMI class. BMI-dmCpG and FA-dmCpG were more numerous in FS compared to PS and largely prandial state-specific. For saturated and monounsaturated FA, dmCpG overlap was higher across than within the respective saturation group. Several BMI- and FA-dmCpG mapped to genes involved in metabolic disease and in some cases matched published experimental data sets. Notably, SETDB1 and MTHFS promoter dmCpG could explain the previously observed associations between global DNAm, PUFA content, and BMI in FS. Surprisingly, overlap between BMI-dmCpG and FA-dmCpG was limited and the respective dmCpG were differentially distributed across functional genomic elements. BMI-dmCpG showed the highest overlap with dmCpG of the saturated FA palmitate, monounsaturated C20:1 and PUFA C20:2. Of these, selected promoter BMI-dmCpG showed opposite associations with palmitate compared to C20:1 and C20:2. As for the comparison between FS and PS within BMI classes, dmCpG were strikingly more abundant and variably methylated in overweight relative to normoweight or obese subjects (∼70–139-fold, respectively). Overweight-associated dmCpG-hosting genes were significantly enriched in targets for E47, SREBP1, and RREB1 transcription factors, which are known players in obesity and lipid homeostasis, but none overlapped with BMI-dmCpG. We show for the first time that the association of BMI and FA with methylation of disease-related genes is distinct in FS and PS and that limited overlap exists between BMI- and FA-dmCpG within and across prandial states. Our study also identifies a transcriptional regulation circuitry in overweight that might contribute to adaptation to that condition or to transition to obesity. Further work is necessary to define the pathophysiological implications of these findings.

Extensive Placental Methylation Profiling in Normal Pregnancies

The placental methylation pattern is crucial for the regulation of genes involved in trophoblast invasion and placental development, both key events for fetal growth. We investigated LINE-1 methylation and methylome profiling using a methylation EPIC array and the targeted methylation sequencing of 154 normal, full-term pregnancies, stratified by birth weight percentiles. LINE-1 methylation showed evidence of a more pronounced hypomethylation in small neonates compared with normal and large for gestational age. Genome-wide methylation, performed in two subsets of pregnancies, showed very similar methylation profiles among cord blood samples while placentae from different pregnancies appeared very variable. A unique methylation profile emerged in each placenta, which could represent the sum of adjustments that the placenta made during the pregnancy to preserve the epigenetic homeostasis of the fetus. Investigations into the 1000 most variable sites between cord blood and the placenta showed that promoters and gene bodies that are hypermethylated in the placenta are associated with blood-specific functions, whereas those that are hypomethylated belong mainly to pathways involved in cancer. These features support the functional analogies between a placenta and cancer. Our results, which provide a comprehensive analysis of DNA methylation profiling in the human placenta, suggest that its peculiar dynamicity can be relevant for understanding placental plasticity in response to the environment.

Novel DNA methylation signatures of tobacco smoking with trans-ethnic effects

Background Smoking remains one of the leading preventable causes of death. Smoking leaves a strong signature on the blood methylome as shown in multiple studies using the Infinium HumanMethylation450 BeadChip. Here, we explore novel blood methylation smoking signals on the Illumina MethylationEPIC BeadChip (EPIC) array, which also targets novel CpG-sites in enhancers. Method A smoking-methylation meta-analysis was carried out using EPIC DNA methylation profiles in 1407 blood samples from four UK population-based cohorts, including the MRC National Survey for Health and Development (NSHD) or 1946 British birth cohort, the National Child Development Study (NCDS) or 1958 birth cohort, the 1970 British Cohort Study (BCS70), and the TwinsUK cohort (TwinsUK). The overall discovery sample included 269 current, 497 former, and 643 never smokers. Replication was pursued in 3425 trans-ethnic samples, including 2325 American Indian individuals participating in the Strong Heart Study (SHS) in 1989–1991 and 1100 African-American participants in the Genetic Epidemiology Network of Arteriopathy Study (GENOA). Results Altogether 952 CpG-sites in 500 genes were differentially methylated between smokers and never smokers after Bonferroni correction. There were 526 novel smoking-associated CpG-sites only profiled by the EPIC array, of which 486 (92%) replicated in a meta-analysis of the American Indian and African-American samples. Novel CpG sites mapped both to genes containing previously identified smoking-methylation signals and to 80 novel genes not previously linked to smoking, with the strongest novel signal in SLAMF7. Comparison of former versus never smokers identified that 37 of these sites were persistently differentially methylated after cessation, where 16 represented novel signals only profiled by the EPIC array. We observed a depletion of smoking-associated signals in CpG islands and an enrichment in enhancer regions, consistent with previous results. Conclusion This study identified novel smoking-associated signals as possible biomarkers of exposure to smoking and may help improve our understanding of smoking-related disease risk.

A validation of Illumina EPIC array system with bisulfite-based amplicon sequencing

The Illumina Infinium® MethylationEPIC BeadChip system (hereafter EPIC array) is considered to be the current gold standard detection method for assessing DNA methylation at the genome-wide level. EPIC arrays are often used for hypothesis generation or pilot studies, the natural conclusion to which is to validate methylation candidates and expand these in a larger cohort, in a targeted manner. As such, an accurate smaller-scale, targeted technique, that generates data at the individual CpG level that is equivalent to the EPIC array, is needed. Here, we tested an alternative DNA methylation detection technique, known as bisulfite-based amplicon sequencing (BSAS), to determine its ability to validate CpG sites detected in EPIC array studies. BSAS was able to detect differential DNA methylation at CpG sites to a degree which correlates highly with the EPIC array system at some loci. However, BSAS correlated less well with EPIC array data in some instances, and most notably, when the magnitude of change via EPIC array was greater than 5%. Therefore, our data suggests that BSAS can be used to validate EPIC array data, but each locus must be compared on an individual basis, before being taken forward into large scale screening. Further, BSAS does offer advantages compared to the probe-based EPIC array; BSAS amplifies a region of the genome (∼500 bp) around a CpG of interest, allowing analyses of other CpGs in the region that may not be present on the EPIC array, aiding discovery of novel CpG sites and differentially methylated regions of interest. We conclude that BSAS offers a valid investigative tool for specific regions of the genome that are currently not contained on the array system.