Evidence for enzymatic backbone methylation of the main membrane lipids in the archaeon Methanomassiliicoccus luminyensis

Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers (BDGTs and PDGTs, respectively) are recently identified classes of archaeal membrane lipids that are prominent constituents in anoxic subseafloor sediments. These lipids are intriguing as they possess unusual backbones with four or five carbon atoms instead of the canonical three-carbon glycerol backbone. In this study, we examined the biosynthesis of BDGTs and PDGTs by the methanogen Methanomassiliicoccus luminyensis , the only available isolate known to produce these compounds, via stable isotope labeling with [ methyl - 13 C] methionine followed by mass spectrometry analysis. We show that their biosynthesis proceeds from transfer(s) of the terminal methyl group of methionine to the more common archaeal membrane lipids, i.e., glycerol dibiphytanyl glycerol tetraethers (GDGTs). As this methylation targets a methylene group, a radical mechanism involving a radical S-adenosylmethionine (SAM) enzyme is probable. Over the course of the incubation, the abundance of PDGTs relative to BDGTs, expressed as backbone methylation index, increased, implying that backbone methylation may be related to the growth shift to stationary conditions, possibly due to limited energy and/or substrate availability. The increase of the backbone methylation index with increasing sediment age in a sample set from the Mediterranean Sea adds support for such a relationship. Importance Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers are membrane lipids recently discovered in anoxic environments. These lipids differ from typical membrane-spanning tetraether lipids because they possess a non-glycerol backbone. The biosynthetic pathway and physiological role of these unique lipids are currently unknown. Here, we show that in the strain Methanomassiliicoccus luminyensis these lipids are the result of methyl transfer(s) from a S-adenosyl methionine (SAM) intermediate. We observed a relative increase of the doubly methylated compound, pentanetriol dibiphytanyl glycerol tetraether, in the stationary phase of M. luminyensis as well as in the subseafloor of the Mediterranean Sea and thus introduced a backbone methylation index, which could be used to further explore microbial activity in natural settings.

Plasma S-Adenosylmethionine Is Associated with Lung Injury in COVID-19

Objective. S-Adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are indicators of global transmethylation and may play an important role as markers of severity of COVID-19. Methods. The levels of plasma SAM and SAH were determined in patients admitted with COVID-19 ( n = 56 , mean age = 61 ). Lung injury was identified by computed tomography (CT) in accordance with the CT0-4 classification. Results. SAM was found to be a potential marker of lung damage risk in COVID-19 patients ( SAM > 80 nM ; CT3,4 vs. CT 0-2: relative ratio (RR) was 3.0; p = 0.0029 ). SAM / SAH > 6.0 was also found to be a marker of lung injury (CT2-4 vs. CT0,1: RR = 3.47 , p = 0.0004 ). There was a negative association between SAM and glutathione level ( ρ = − 0.343 , p = 0.011 ). Interleukin-6 (IL-6) levels were associated with SAM ( ρ = 0.44 , p = 0.01 ) and SAH ( ρ = 0.534 , p = 0.001 ) levels. Conclusions. A high SAM level and high methylation index are associated with the risk of lung injury in patients with COVID-19. The association of SAM with IL-6 and glutathione indicates an important role of transmethylation in the development of cytokine imbalance and oxidative stress in patients with COVID-19.

Polymorphisms of the AS3MT gene are associated with arsenic methylation capacity and damage to the P21 gene in arsenic trioxide plant workers

Epidemiological evidence suggests that the metabolic profiles of each individual exposed to arsenic (As) are related to the risk of cancer, coronary heart disease, and diabetes. The arsenite methyltransferase ( AS3MT) gene plays a key role in As metabolism. Several single nucleotide polymorphisms in the AS3MT gene may affect both enzyme activity and gene transcription. AS3MT polymorphisms are associated with the proportions of monomethylarsenic acid (MMA) and dimethylarsenic acid (DMA) in urine as well as the incidence of cancer. P21 protein is a cyclin-dependent kinase inhibitor. Mutations of the P21 gene have been found in cancer patients. In our study, we investigate whether polymorphisms of the AS3MT gene alter As methylation capacity and adversely affect the P21 gene in arsenic trioxide plant workers. The DNA damage was examined by the quantitative polymerase chain reaction. Restriction fragment length polymorphism was used to analyze the genotype of the AS3MT gene. The results showed that DNA damage in P21 gene fragments was greater in those individuals exposed to high levels of As. There was a strong positive correlation between the DNA damage to P21 gene fragments and the percentage of MMA in urine. However, DNA damage in P21 gene fragments was negatively associated with the percentage of DMA in urine (%uDMA), primary methylation index (PMI), and secondary methylation index. We found that subjects with the rs7085104 GG or GA allele were associated with higher %uDMA and PMI and less DNA damage. The subjects with the rs11191454 GG+GA or GA allele were also associated with higher %uDMA and PMI and less DNA damage. Our results suggest that rs1191454 and rs7085104 in the AS3MT gene affect the As-induced DNA damage by altering individual metabolic efficiency.

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.

BIOM-07. QUANTITATIVE MGMT PROMOTER METHYLATION INDEX INDICATES A NON-LINEAR PROGNOSTIC EFFECT IN GLIOBLASTOMA, SUGGESTING THAT USE OF OPTIMAL CUTOFF POINTS MAY BE CLINICALLY DISADVANTAGEOUS

BACKGROUND Epigenetic inhibition of the O6-methylguanine-DNA-methyltransferase (MGMT) gene has emerged as a clinically relevant prognostic marker in glioblastoma (GBM). Methylation of the MGMT promoter has been shown to increase chemotherapy efficacy. While traditionally reported as a binary marker, recent methodological advancements have led to quantitative approaches that measure methylation, providing clearer insights into methylation’s functional relationship with survival. METHODS A CLIA assay and bisulfite sequencing was utilized to develop a quantitative, 17-point MGMT promoter methylation index derived from the number of methylated CpG sites. Retrospective review of 240 newly diagnosed GBM patients was performed in order to discern how risk for mortality transforms as promoter methylation increases. Non-linearities were captured by fitting splines to Cox proportional hazard models, plotting smoothed residuals, and creating survival plots. Covariates included age, KPS, IDH1 mutation, and extent of resection. RESULTS Median follow-up time and progression free survival were 16 and 9 months, respectively. 176 subjects experienced death. A one-unit increase in CpG methylation on a scale of 1-17 resulted in a 4% reduction in hazard (95% CI 0.93–0.99, P< 0.005). Moreover, GBM patients with low-levels of methylation (1-6 CpG sites) fared markedly worse (HR=1.62, 95% CI 1.03-2.54, P< 0.036) than individuals who were unmethylated (reference group). Subjects with medium-levels of methylation (7-12 CpG sites) had the greatest reduction in hazard (HR=0.48, 95% CI 0.29-0.80, P< 0.004), followed by individuals in the highest methylation tertile (HR=0.62, 95% CI 0.40-0.97, P< 0.035). CONCLUSION This novel approach offers greater bisulfite conversion efficiency when compared to alternative methods, reducing the likelihood of false positives. Analysis of the resulting methylation index scores demonstrates a non-linear relationship between MGMT methylation and survival, suggesting conformation of the marker’s protective effect. These findings challenge the current understanding of MGMT’s functional form and underline why implementing an “optimal cutoff point” may be disadvantageous.

Plasma S-Adenosylmethionine is Associated with Lung Injury in COVID-19

ObjectiveS-Adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are indicators of global transmethylation and may play an important role as markers of severity of COVID-19.MethodsThe levels of plasma SAM and SAH were determined in patients admitted with COVID-19 (n = 56, mean age = 61). Lung injury was identified by computed tomography (CT) in accordance with the CT0-4 classification.ResultsSAM was found to be a potential marker of lung damage risk in COVID-19 patients (SAM > 80 nM; CT3,4 vs. CT 0-2: relative ratio (RR) was 3.0; p = 0.0029). SAM/SAH > 6.0 was also found to be a marker of lung injury (CT2-4 vs. CT0,1: RR = 3.47, p = 0.0004). Interleukin-6 (IL-6) levels were associated with SAM (ρ = 0.44, p = 0.01) and SAH (ρ = 0.534, p = 0.001) levels.ConclusionsHigh SAM levels and high methylation index are associated with the risk of lung injury in COVID-19 patients. The association of SAM and SAH with IL-6 indicates an important role of transmethylation in the development of cytokine imbalance in COVID-19 cases.

Somatotroph Tumors and the Epigenetic Status of the GNAS Locus

Background: Forty percent of somatotroph tumors harbor recurrent activating GNAS mutations, historically called the gsp oncogene. In gsp-negative somatotroph tumors, GNAS expression itself is highly variable; those with GNAS overexpression most resemble phenotypically those carrying the gsp oncogene. GNAS is monoallelically expressed in the normal pituitary due to methylation-based imprinting. We hypothesize that changes in GNAS imprinting of gsp-negative tumors affect GNAS expression levels and tumorigenesis. Methods: We characterized the GNAS locus in two independent somatotroph tumor cohorts: one of 23 tumors previously published (PMID: 31883967) and classified by pan-genomic analysis, and a second with 82 tumors. Results: Multi-omics analysis of the first cohort identified a significant difference between gsp-negative and gsp-positive tumors in the methylation index at the known differentially methylated region (DMR) of the GNAS A/B transcript promoter, which was confirmed in the larger series of 82 tumors. GNAS allelic expression was analyzed using a polymorphic Fok1 cleavage site in 32 heterozygous gsp-negative tumors. GNAS expression was significantly reduced in the 14 tumors with relaxed GNAS imprinting and biallelic expression, compared to 18 tumors with monoallelic expression. Tumors with relaxed GNAS imprinting showed significantly lower SSTR2 and AIP expression levels. Conclusion: Altered A/B DMR methylation was found exclusively in gsp-negative somatotroph tumors. 43% of gsp-negative tumors showed GNAS imprinting relaxation, which correlated with lower GNAS, SSTR2 and AIP expression, indicating lower sensitivity to somatostatin analogues and potentially aggressive behavior.

NGMA-6. Quantitative MGMT Promoter Methylation Index Indicates Non-Linear, Prognostic Effect in Glioblastoma

Background Epigenetic inhibition of the O6-methylguanine-DNA-methyltransferase (MGMT) gene has emerged as a clinically relevant prognostic marker in glioblastoma (GBM). Methylation of the MGMT promoter has been shown to increase chemotherapy efficacy. While traditionally reported as a binary marker, recent methodological advancements have led to quantitative methods of measuring methylation, allowing for clearer insights into methylation’s functional relationship with survival. Methods A CLIA assay and bisulfite sequencing was utilized to develop a quantitative, 17-point MGMT promoter methylation index derived from the number of methylated CpG sites. Retrospective review of 242 newly diagnosed GBM patients was performed in order to discern how risk for mortality transforms as promoter methylation increases. Non-linearities were captured by fitting splines to Cox proportional hazard models, plotting smoothed residuals, and creating survival plots. Covariates included age, KPS, IDH1 mutation, and extent of resection. Results Median follow-up time and progression free survival were 15.9 and 9 months, respectively. 176 subjects experienced death. A one-unit increase in CpG methylation resulted in a 4% reduction in hazard (95% CI 0.93–0.99, P<0.005). Moreover, GBM patients with low levels of methylation (1–6 CpG islands) fared markedly worse (HR=1.62, 95% CI 1.03–2.54, P<0.036) than individuals who were unmethylated (reference group). Subjects with medium levels of methylation (7–12 sites) had the greatest reduction in hazard (HR=0.48, 95% CI 0.29–0.80, P<0.004), followed by individuals in the highest methylation tertile (HR=0.62, 95% CI 0.40–0.97, P<0.035). Conclusion The extent of MGMT methylation shares a non-linear relationship with survival, suggesting conformation of the marker’s protective effect. This finding challenges the current understanding of MGMT and underlines the clinical importance of determining its prognostic utility. Potential limitations include censoring, sample size, and extraneous mutations. Future research is warranted to examine whether the location of CpG site methylation contributes to a reduction in mortality hazard.

Diet-Induced Mild Hyperhomocysteinemia in Mice Fed a Ketogenic Diet Does Not Alter the Development of Atherosclerosis nor Specific Epigenetic Content

Objectives Elevated plasma homocysteine (Hcy), or hyperhomocysteinemia (HHcy), is a risk factor for atherosclerosis by mechanisms still elusive. A possibility includes the alteration of specific epigenetic tags at lysine 27 of histone H3 (H3K27) due to hypomethylating stress. Similarly, ketogenic diets (KD), or very-low carbohydrate diets, which stimulate ketosis, and may also affect the epigenetic content on the H3K27 residue. Studies connecting the effects of dietary ketosis, mild HHcy, and specific epigenetic dysregulation are lacking. We hypothesize that diet-induced HHcy and ketosis will induce H3K27 hypomethylation combined with increased acetylation to produce a pro-atherogenic phenotype. Methods Seven-week-old male apoe−/− (apolipoprotein E-deficient) mice, a model for human atherosclerosis, were fed ad libitum a KD (in %kcal: fat, 81; carbohydrate, 1; protein, 18; n = 4–6) or HHcy-KD (same macronutrients, with added methionine and reduced methyl donors; n = 4). After 4, 8 and 12 wk of diet treatment, plasma was collected to quantify ketosis via beta-hydroxybutyrate levels (OH-But) by a colorimetric assay, and measure Hcy by HPLC. At the endpoint, mice were euthanized and aortas were collected for quantification of the vascular methylation index, S-adenosylmethionine to S-adenosylHcy ratio by LC-MS-MS; 3-D analysis of the atherosclerotic plaque burden by magnetic resonance imaging; and quantification of the epigenetic tags H3K27me3 and H3K27ac using immunohistochemistry. Results A sustained ketosis was detected through elevated OH-But levels in both KD and HHcy-KD mice. HHcy was mildly but significantly (P < 0.05) elevated in HHcy-KD-mice compared to KD-mice after 4 wk (19.5 ± 2.3 vs 4.5 ± 0.6 µM) and 12 wk (17.2 ± 2.1 vs 4.4 ± 0.9 µM). Nevertheless, no significant differences were observed in aortic methylation index, plaque accumulation, or content of the H3K27me3 or H3K27ac epigenetic tags between the two groups of mice. Conclusions While mild HHcy was achieved in HHcy-KD mice, this phenotype failed to induce vascular hypomethylation, atherosclerosis progression or specific epigenetic dysregulation, suggesting that a more severe Hcy accumulation may be necessary to cause vascular toxicity and specific epigenetic dysregulation. Funding Sources Huck Institutes of the Life Sciences

The Resistance Responses of Potato Plants to Potato Virus Y Are Associated with an Increased Cellular Methionine Content and an Altered SAM:SAH Methylation Index

Plant-virus interactions are frequently influenced by elevated temperature, which often increases susceptibility to a virus, a scenario described for potato cultivar Chicago infected with potato virus Y (PVY). In contrast, other potato cultivars such as Gala may have similar resistances to PVY at both normal (22 °C) and high (28 °C) temperatures. To elucidate the mechanisms of temperature-independent antivirus resistance in potato, we analysed responses of Gala plants to PVY at different temperatures using proteomic, transcriptional and metabolic approaches. Here we show that in Gala, PVY infection generally upregulates the accumulation of major enzymes associated with the methionine cycle (MTC) independently of temperature, but that temperature (22° C or 28° C) may finely regulate what classes accumulate. The different sets of MTC-related enzymes that are up-regulated at 22 °C or 28 °C likely account for the significantly increased accumulation of S-adenosyl methionine (SAM), a key component of MTC which acts as a universal methyl donor in methylation reactions. In contrast to this, we found that in cultivar Chicago, SAM levels were significantly reduced which correlated with the enhanced susceptibility to PVY at high temperature. Collectively, these data suggest that MTC and its major transmethylation function determines resistance or susceptibility to PVY.