Discovery Cohort Research Articles

PATH-24. NOVEL MOLECULAR SUBTYPES IDENTIFIED IN INTRACRANIAL GERM CELL TUMORS SHOWED UNIQUE WHOLE-GENOME METHYLATION PROFILE

Abstract BACKGROUND By integrating whole-genome sequencing (WGS) and RNA-seq data, we found that intracranial germ cell tumors (IGCTs) can be divided into three distinct molecular subtypes: immune-hot, MYC/E2F, and SHH, each characterized by a unique profile of genomic abnormalities. To further define these subtypes, we subsequently performed whole-genome bisulfate sequencing (WGBS) on IGCT tumors METHODS Within our prior discovery cohort (n=110), DNA from 84 samples was available for profiling methylation levels using Whole Genome Bisulfite Sequencing (WGBS). Fastq data was aligned to the human reference genome (hg19), with duplicates removed, and DNA methylation was extracted using bismark pipeline (ver0.24.0). After quality control, weight methylation level was calculated for each CpG island as the sum of methylated reads divided by coverage. To identify IGCTs molecular groups, we applied hierarchical consensus k-means clustering to the most variant methylated CpG island (n=1000) across 84 IGCTs tumor tissues. The methylation level of RNA-seq subtypes was visualized to further clarify the molecular subtypes of IGCTs RESULTS Unsupervised clustering revealed that IGCTs can be divided into three groups based on the CpG island methylation levels: hypomethylation group (n=32), moderate methylation group (n=27), and hyper methylation group (n=25). The hypomethylation and moderate methylation group are predominantly observed in germinoma (>90%). In contrast, hypermethylation group comprises the majority of non-germinomatous germ cell tumors. Furthermore, these subgroups derived from methylation analysis are highly consistent with the molecular subtypes previously defined through gene transcription profile analysis. We observed that the majority of MYC/E2F subtype (24/33) belonged to the hypomethylation group, whereas the majority of immune-hot subtype (14/23) fell into the moderate methylation group. The SHH subtype was the most common within the hypermethylation group (18/26) CONCLUSIONS The three molecular IGCT subtypes showed unique methylation profile. These results indicate different mechanisms of tumorigenesis among the subtypes and pave the way for the clinical application of methylation-based subtyping.

The protective role of vitamin d in nasopharyngeal carcinoma: insights from Mendelian randomization and meta-analysis

BackgroundIn recent years, the anti-tumor effects of vitamin D have garnered increasing attention. However, previous epidemiological studies on the relationship between vitamin D and nasopharyngeal carcinoma (NPC) have yielded inconsistent results. This study aims to further explore whether vitamin D helps reduce the risk of NPC through Mendelian randomization (MR) and meta-analysis.MethodsBased on the core assumption of MR study, instrumental variables (IVs) for vitamin D, serving as genetic proxies, were obtained from summary data of large genome-wide association study (GWAS). Inverse-variance weighted (IVW) was utilized as the primary MR analytical method to explore the causal relationship between vitamin D and NPC. Sensitivity analyses included heterogeneity testing and horizontal pleiotropy testing. To further validate the robustness of the result, meta-analysis was employed to obtain pooled effects from databases of different sources.ResultsIn the discovery cohort, the IVW result suggest that vitamin D is a potential protective factor against NPC (odds ratio (OR) = 0.35, 95% confidence interval (CI): 0.13–0.89, P = 0.028). The finding was further corroborated by two independent replication cohorts [OR = 0.32, 95% CI: 0.13–0.80, P = 0.018 (ukb-d-30890_irnt); OR = 0.34, 95% CI: 0.13–0.90, P = 0.029(ebi-a-GCST90025967)]. Subsequent meta-analysis indicated that vitamin D markedly reduces the risk of NPC (OR = 0.34, 95% CI: 0.19–0.58, P < 0.001). Multiple sensitivity analyses, including heterogeneity analysis and horizontal pleiotropy tests, did not reveal any significant findings (all P > 0.05).ConclusionThis study provides robust evidence that vitamin D significantly reduces the risk of NPC. Through MR and meta-analysis, we have demonstrated a protective role of vitamin D in NPC development. These findings suggest that maintaining adequate vitamin D levels may be a potential strategy for reducing NPC. Further research is warranted to confirm these results and explore the underlying mechanisms involved.

Thyroid hormones and prognosis in adults with status epilepticus: a retrospective study

ObjectivesThyroid hormone levels have been indicated to be associated with the functional outcome in critical illness. However, the studies on thyroid hormones and status epilepticus (SE) are rare. This study aimed to evaluate the predictive value of serum thyroid hormone levels on admission for unfavorable outcome in adult patients with SE.MethodsWe investigated and validated the predictive value of serum thyroid hormone levels on admission for the prognosis of adult SE patients. We extracted the clinical information and outcomes of patients. Modified Rankin scale (mRS) scores were applied to assess the patients’ functional outcome, and mortality at 30 days after SE onset was identified. Serum levels of thyroid hormones including free thyroxin (FT4), free triiodothyronine (FT3) and thyroid-stimulating hormone (TSH) were detected on admission.ResultsWe first analyzed the discovery cohort of 87 patients with SE. We found that 35.6% (31/87) of the patients had a poor outcome at discharge, and 18.4% (16/87) of the patients died during hospital stay and at 30-day follow up. The serum FT3 levels in the non-survivors group were significantly lower than those in the survivors group. Low T3 syndrome occurred in 29.9% (26/87) of SE cases and patients with low T3 syndrome were more likely to have unfavorable outcomes. Furthermore, we observed similar results in the external cohort, which validated our findings.ConclusionsSerum FT3 levels measured on admission are independently associated with 30-day mortality in SE patients. Additionally, low T3 syndrome may be a promising candidate for predicting SE prognosis.

Effect of NK cell receptor genetic variation on allogeneic stem cell transplantation outcome and in vitro NK cell cytotoxicity

Natural killer (NK) cells recognize and may kill malignant cells via their cell surface receptors. Killer cell immunoglobulin-like receptor (KIR) genotypes of donors have been reported to adjust the risk of relapse after allogeneic stem cell transplantation (HSCT), particularly in patients with acute myeloid leukemia. To test whether non-KIR NK cell receptors have a similar effect, we screened 1,638 genetic polymorphisms in 21 non-KIR NK cell receptor genes for their associations with relapse and graft-versus-host disease (GVHD) after HSCT in 1,491 HSCT donors (from Finland, the UK, Spain, and Poland), divided into a discovery and replication cohort. Eleven polymorphisms regulating or located in CD226, CD244, FCGR3A, KLRD1, NCR3, and PVRIG were associated with the risks for relapse and GVHD. These associations could not be confirmed in the replication cohort. Blood donor NK cells carrying alleles showing genetic protection for relapse had a higher in vitro NK cell killing activity than non-carriers whereas those with alleles genetically protective for GVHD had lower cytotoxicity, potentially indicating functional effects. Taken together, these results show no robust effects of genetic variation in the tested non-KIR NK cell receptors on the outcome of HSCT.

Home Mechanical Ventilation and Risk of Hospitalization in Obesity Hypoventilation Syndrome - The Population-based DISCOVERY Study.

Patients with obesity hypoventilation syndrome (OHS) have high risk of hospitalization, which might be decreased by home mechanical ventilation (HMV). To evaluate annualized hospitalization rate (AHR) and change in AHR in patients with OHS starting home mechanical ventilation (HMV), and explore if there were any difference in AHR by starting HMV acutely or electively. Population-based longitudinal study of patients with OHS starting HMV in the Swedish DISCOVERY cohort between 1996 and 2018, cross-linked with the National Patient Registry for national data on hospital admissions. AHR was calculated for each patient for three years before (Year -3, -2, -1) and three years after (Year 1, 2, 3) the year of starting HMV (Year 0; start date ± 6 months). Differences in AHR were analyzed using Wilcoxon signed-rank test (between years) and Mann-Whitney U test (between acute/elective). Proportion of patients hospitalized in each year was analyzed and comparison between years was done with McNemar´s test. Factors associated with change in AHR were identified using multivariate linear regression models. In total, 2,445 patients were included: 47% females, mean age 62.3 ± 12.2 years, and 1,418 (58%) started HMV electively. Overall, AHR decreased with 0.88 (95%CI 0.74-1.02) hospitalization/year after start of HMV and starting treatment acutely was associated with greater decrease in AHR. There was no statistically significant difference in AHR in Year 1 between acute and elective start (P=0.199). The year after start of HMV, proportion of patients hospitalized decreased from 84% to 54% (P<0.05). Initiation of HMV was associated with reduced hospitalization rate in patients with OHS, irrespective of acute or elective start. Majority of patients with OHS are hospitalized in the year of HMV initiation.

Identifying causal relationships between 35 blood and urine biomarkers and urologic cancers: MR-meta combined with Bayesian colocalization Mendelian randomization analysis

BackgroundBlood and urine biomarkers have been associated with urologic tumors, but their causal relationship with urologic tumors is unclear.MethodsWe performed a bidirectional Mendelian randomization (MR) analysis of the association between 35 blood and urine biomarkers and urological tumors in our discovery cohort. A Bayesian weighting approach was used to validate the positive results identified in the discovery cohort, and steiger filtering analysis was used to distinguish causality from reverse causality. Bayesian colocalization analysis was used to analyze which single nucleotide polymorphisms (SNPs) specifically co-located between the positive blood and urine biomarkers and the disease phenotypes were driven, and MR-positive results from the discovery cohort and the validation cohort were combined using the MR-meta method.ResultsSeveral blood and urine biomarkers were found to be significantly and causally associated with urologic cancers. Notably, calcium (OR: 1.34, 95%CI 1.10–1.63, P = 0.0040) and sex hormone-binding globulin (SHBG) (OR: 0.81, 95%CI 0.70–0.95, P = 0.0092) were associated with bladder cancer; gamma glutamyl transferase (GGT) (OR: 0.91, 95%CI 0.83–0.99, P = 0.0209), lipoprotein A (Lp(a)) (OR: 1.12, 95%CI 1.01–1.24, P = 0.0399), and insulinlike growth factor 1 (IGF 1) (OR: 1.10, 95%CI 1.01–1.20, P = 0.0220) were linked to prostate cancer (PCa); non albumin protein (OR: 0.78, 95%CI 0.65–0.93, P = 0.0065) and total protein (OR: 0.80, 95%CI 0.64–0.99, P = 0.0380) were linked to renal cancer; and apolipoprotein A (Apo-A) (OR: 0.56, 95%CI 0.32–0.98, P = 0.0426) and urate (OR:1.89, 95%CI 1.03–3.47, P = 0.0399) were associated with renal pelvis cancer. These associations were validated in an independent cohort, with GGT, IGF 1, and Lp(a) being consistently linked to PCa.ConclusionThis study identified significant biomarkers associated with urological cancers in blood and urine. These include GGT, IGF 1, and Lp(a), which are strong biomarkers for PCa. In addition, the findings of this study provide evidence for a handful of risk and protective factors for the development of urologic cancers.

Rare variation in LMNA underlies polycystic ovary syndrome (PCOS) pathogenesis in two independent cohorts.

Polycystic ovary syndrome (PCOS) is a common, heritable endocrinopathy that is a common cause of anovulatory infertility in reproductive age women. Variants in LMNA cause partial lipodystrophy, a syndrome with overlapping features to PCOS. We tested the hypothesis that rare variation in LMNA contributes to PCOS pathogenesis and selects a lipodystrophy-like subtype of PCOS. We sequenced LMNA by targeted sequencing a discovery cohort of 811 PCOS patients and 164 healthy controls. We then analyzed LMNA from whole-exome sequencing (WES) of a replication cohort of 718 PCOS patients and 281 healthy controls. Variation in the LMNA gene, hormone and lipid profiles of participants. In the discovery cohort, we identified 8 missense variants in 15/811 cases, and 1 variant in 1/172 reproductively healthy controls. There is strong evidence for association between the variants and PCOS compared to gnomAD non-Finnish European population controls (χ2=17, p=3.7x10-5, OR=2.9). In the replication cohort, we identified 11 unique variants in 15/718 cases, and 1 variant in 281 reproductively healthy controls. Again, there is strong evidence for association with population controls (χ2=30.5, p=3.4x10-8, OR= 4.0). In both the discovery and replication cohorts, variants in LMNA identify women with PCOS with high triglycerides and extreme insulin resistance. Rare missense variation in LMNA is reproducibly associated with PCOS and identifies some individuals with lipodystrophy-like features. The overlap between this PCOS phenotype and genetic partial lipodystrophy syndromes warrants further investigation into additional lipodystrophy genes and their potential in PCOS etiology.

A blood-based biomarker panel for non-invasive diagnosis of metabolic dysfunction-associated steatohepatitis.

The current diagnosis of metabolic dysfunction-associated steatotic liver disease (MASLD) and its severe form, metabolic dysfunction-associated steatohepatitis (MASH), is suboptimal. Here, we recruited 700 individuals, including 184 from Hong Kong as a discovery cohort and 516 from San Diego, Wenzhou, and Hong Kong as three validation cohorts. A panel of 3 parameters (C-X-C motif chemokine ligand 10 [CXCL10], cytokeratin 18 fragments M30 [CK-18], and adjusted body mass index [BMI]) was formulated (termed N3-MASH), which discriminated patients with MASLD from healthy controls with an area under the receiver operating characteristic (AUROC) of 0.954. Among patients with MASLD, N3-MASH could identify patients with MASH with an AUROC of 0.823, achieving 90.0% specificity, 62.9% sensitivity, and 88.6% positive predictive value. The diagnostic performance of N3-MASH was confirmed in three validation cohorts with AUROC of 0.802, 0.805, and 0.823, respectively. Additionally, N3-MASH identifies patients with MASH improvement with an AUROC of 0.857. In summary, we developed a robust blood-based panel for the non-invasive diagnosis of MASH, which might help clinicians reduce unnecessary liver biopsies.

Exploring potential therapeutic targets for asthma: a proteome-wide Mendelian randomization analysis

BackgroundAsthma poses a significant global health challenge, characterized by high rates of morbidity and mortality. Despite available treatments, many severe asthma patients remain poorly managed, highlighting the need for novel therapeutic strategies. This study aims to identify potential drug targets for asthma by examining the influence of circulating plasma proteins on asthma risk.MethodsThis study employs summary-data-based Mendelian randomization (MR) and two-sample MR methods to investigate the association between 2940 plasma proteins from the UK Biobank study and asthma. The analysis includes discovery (FinnGen cohort) and replication (GERA cohort) phases, with Bayesian colocalization used to validate the relationships between proteins and asthma. Furthermore, protein–protein interaction and druggability assessments were conducted on high-evidence strength protein biomarkers, and candidate drug prediction and molecular docking were performed for proteins without targeted drugs. Given the complexity of asthma pathogenesis, the study also explores the relationships between plasma proteins and asthma-related endpoints (e.g., obesity-related asthma, infection-related asthma, childhood asthma) to identify potential therapeutic targets for different subtypes.ResultsIn the discovery cohort, 75 plasma proteins were associated with asthma, including IL1RAP, IL1RL1, IL6, CXCL5, and CXCL8. Additionally, 6 proteins (IL4R, LTB, CASP8, MAX, PCDH12, and SCLY) were validated through co-localization analysis and validation cohort. The assessment of drug targetability revealed potential drug targets for IL4R, CASP8, and SCLY, while candidate drugs were predicted for LTB and MAX proteins. MAX exhibited strong binding affinity with multiple small molecules indicating a highly stable interaction and significant druggability potential. Analysis of the 75 proteins with 9 asthma-related endpoints highlighted promising targets such as DOK2, ITGAM, CA1, BTN2A1, and GZMB.ConclusionThese findings elucidate the link between asthma, its related endpoints, and plasma proteins, advancing our understanding of molecular pathogenesis and treatment strategies. The discovery of potential therapeutic targets offers new insights into asthma drug target research.

Exercise-changed gut mycobiome as a potential contributor to metabolic benefits in diabetes prevention: an integrative multi-omics study

ABSTRACT Background The importance of gut microbes in mediating the benefits of lifestyle intervention is increasingly recognized. However, compared to the bacterial microbiome, the role of intestinal fungi in exercise remains elusive. With our established randomized controlled trial of exercise intervention in Chinese males with prediabetes (n = 39, ClinicalTrials.gov:NCT03240978), we investigated the dynamics of human gut mycobiome and further interrogated their associations with exercise-elicited outcomes using multi-omics approaches. Methods Clinical variations and biological samples were collected before and after training. Fecal fungal composition was analyzed using the internal transcribed spacer 2 (ITS2) sequencing and integrated with paired shotgun metagenomics, untargeted metabolomics, and Olink proteomics. Results Twelve weeks of exercise training profoundly promoted fungal ecological diversity and intrakingdom connection. We further identified exercise-responsive genera with potential metabolic benefits, including Verticillium, Sarocladium, and Ceratocystis. Using multi-omics approaches, we elucidated comprehensive associations between changes in gut mycobiome and exercise-shaped metabolic phenotypes, bacterial microbiome, and circulating metabolomics and proteomics profiles. Furthermore, a machine-learning algorithm built using baseline microbial signatures and clinical characteristics predicted exercise responsiveness in improvements of insulin sensitivity, with an area under the receiver operating characteristic (AUROC) of 0.91 (95% CI: 0.85–0.97) in the discovery cohort and of 0.79 (95% CI: 0.74–0.86) in the independent validation cohort (n = 30). Conclusions Our findings suggest that intense exercise training significantly remodels the human fungal microbiome composition. Changes in gut fungal composition are associated with the metabolic benefits of exercise, indicating gut mycobiome is a possible molecular transducer of exercise. Moreover, baseline gut fungal signatures predict exercise responsiveness for diabetes prevention, highlighting that targeting the gut mycobiome emerges as a prospective strategy in tailoring personalized training for diabetes prevention.

No genetic causality between appendectomy and gastrointestinal cancers: a Mendelian randomization study and meta-analysis in European population

The impact of appendectomy on the risk of gastrointestinal cancers remains unknown. We aimed to systematically estimate the causal relationship between appendectomy and gastrointestinal cancers in the European population using two-sample Mendelian randomization (TSMR) study methods and meta-analysis. As part of the discovery cohort analysis, we identified independent genetic variants strongly associated with appendectomy from the UK Biobank (50,105 cases) to serve as instrumental variables (IVs). Summary-level data for gastrointestinal cancers were obtained from the FinnGen study. As the replication cohort, IVs associated with appendectomy were extracted in the FinnGen study (28,601 cases). The data for gastrointestinal cancers were obtained from the UK Biobank. Finally, meta-analyses were conducted to evaluate the combined causal effects of the MR results. We found no causal relationship between appendectomy and gastrointestinal cancers in both the discovery and replication cohorts. Finally, the meta-analysis revealed no causal association between appendectomy and gastrointestinal cancers. Our findings suggest no causal relationship exists between appendectomy and gastrointestinal cancers in the European population. This genetic evidence supports the conclusion from other observational studies that appendectomy does not affect the risk of gastrointestinal cancers in the European population.

Developing a Reproducible Radiomics Model for Diagnosis of Active Crohn's Disease on CT Enterography Across Annotation Variations and Acquisition Differences.

To systematically identify radiomics features on CT enterography (CTE) scans which can accurately diagnose active Crohn's disease across multiple sources of variation. Retrospective study of CTE scans curated between 2013 and 2015, comprising 164 subjects (65 male, 99 female; all patients were over the age of 18) with endoscopic confirmation for the presence or absence of active Crohn's disease. All patients had three distinct sets of scans available (full and reduced dose, where the latter had been reconstructed via two different methods), acquired on a single scanner at a single institution. Radiomics descriptors from annotated terminal ileum regions were individually and systematically evaluated for resilience to different imaging variations (changes in dose/reconstruction, batch effects, and simulated annotation differences) via multiple reproducibility measures. Multiple radiomics models (by accounting for each source of variation) were evaluated in terms of classifier area under the ROC curve (AUC) for identifying patients with active Crohn's disease, across separate discovery and hold-out validation cohorts. Radiomics descriptors selected based on resiliency to multiple sources of imaging variation yielded the highest overall classification performance in the discovery cohort (AUC = 0.79 ± 0.04) which also best generalized in hold-out validation (AUC = 0.81). Performance was maintained across multiple doses and reconstructions while also being significantly better (p < 0.001) than non-resilient descriptors or descriptors only resilient to a single source of variation. Radiomics features can accurately diagnose active Crohn's disease on CTE scans across multiple sources of imaging variation via systematic analysis of reproducibility measures. Clinical utility and translatability of radiomics features for diagnosis and characterization of Crohn's disease on CTE scans will be contingent on their reproducibility across multiple types and sources of imaging variation.

Novel protein-based biomarkers of out-of-hospital sudden cardiac death after myocardial infarction

Abstract Background Risk stratification of out-of-hospital sudden cardiac death (SCD) patients after acute myocardial infarction (AMI) is crucial for timely therapeutic interventions. However, left ventricular ejection fraction (LVEF) as a traditional clinical tool has major limitations, necessitating improved risk stratification strategies. High-throughput proteomic offers the potential to detect novel biomarkers to improve risk assessment. Purpose To identifiy optimal circulating protein combination predicting out-of-hospital SCD post-AMI. Methods We conducted a two-center retrospective nested case-control study of cases with out-of-hospital SCD and controls with AMI who discharged alive. In a discovery cohort, we carried out plasma proteomics profiling of 6,592 peptides and 522 proteins using mass spectrometry. The proteins associated to SCD were further assessed using targeted quantitative proteomics, and the optimal protein combination for predicting out-of-hospital SCD post-AMI was identified by predictor selection classifier. Finally, ELISA validated its predictive value by comparing with a clinical stratification tool (LVEF ≤35%) and two reported risk stratification models within discovery and two independent validation cohorts. Results In the discovery cohort (Figure 1, 210 participants including 105 SCD cases [median age: 68.0 years, 59.05% men]), mass spectrometry discovered 44 differential proteins not previously associated to SCD. Targeted proteomics quantified these proteins and identified optimal protein combination, SCD-warning three protein combination (SCD-W3P), including coronin-1A, haptoglobin, and complement factor D (CFD). A model based on expression levels of SCD-W3P outperformed LVEF alone (Figure 2, C-statistic: 0.752 vs. 0.548, P&amp;lt;0.001), improving its risk prediction (ΔC-statistic = 0.281, P&amp;lt;0.001) and net reclassification improvement (9.5%, P&amp;lt;0.001). Similar incremental discrimination metrics were observed in two reported stratification models combined with SCD-W3P, particularly within the LVEF-preserved population. These findings were repeatably validated in two independent cohorts (Figure 1 and 2, validation 1: 160 participants including 40 SCD cases [median age: 72.5 years, 67.50% men]; validation 2: 96 participants including 24 SCD cases [median age: 74.5 years, 79.17% men]). Finally, CFD inhibition protection for mortality and pro-malignant arrhythmias in AMI mice supported biological plausibility of the critical protein in SCD-W3P. Conclusions This two-center retrospective nested case-control study with two independent validation serves as the largest-scale investigation with the most comprehensive proteomics profiling in out-of-hospital SCD after AMI, identifying an optimal protein combination which may contribute to enhance early identification for timely intensive management. These findings suggest pivotal proteins for improving understanding SCD pathophysiology.Methods overview.Comparison of model performances for pre

Methylome of circulating cell free DNA as a novel biomarker tool: from acute coronary syndrome to broader risk stratification of cardiovascular disease

Abstract Background Cell death during tissue injury leads to release of chromosomal DNA which is promptly degraded. However, short DNA fragments wrapped around nucleosomes survive longer in the bloodstream and can be isolated as circulating cell-free DNA (ccfDNA). Oncology field pioneered the use of cfDNA as clinical biomarker by identifying sequence modifications induced in cancer cells. Importantly, ccfDNA also retains the same epigenetic information as the tissue of origin. Despite major advances for disease diagnosis and therapy, coronary artery diseases (CAD) and associated conditions such acute myocardial infarction (AMI) or heart failure (HF) are responsible for leading number of deaths worldwide. Preventive and surveillance strategies with non-invasive testing are needed. Purpose Evaluation of ccfDNA methylation profiles as prognostic non-invasive biomarker for risk stratification of acute coronary syndromes and their potential application in preventive and surveillance strategies in other cardiovascular disorders. Methods CcfDNA isolated from citrate-plasma collected from our discovery cohort, comprising healthy controls, STEMI, NSTEMI and unstable angina (UA) patients, was analyzed applying Whole Genome Bisulfite Sequencing (WGBS) using a low-input BS-seq (PBAT) protocol. Computational analysis generated differentially methylated regions (DMRs) by tiling CpG areas and setting a threshold of minimal 25% methylation difference compared to the healthy group. To assess the validity of our findings, a selection of disease-specific DMRs were tested using targeted methylation sequencing protocol in a new set of patients. This procedure consisted in enzymatic conversion of ccfDNA methylated sites followed by probe-specific enrichment libraries and sequencing at a Novaseq 6000 platform. Results The study identified a total of 1941 DMRs and from those 486 were STEMI, 223 NSTEMI and 684 UA specific. From the identified DMRs, 74.13% from STEMI, 72.68% from NSTEMI and 61.62% from UA annotated with heart-related diseases using DisGeNET gene-disease associations. Interestingly, most of identified DMRs located within intronic regions (~60%) and also annotated as enhancers and lncRNAs. The independent validation with targeted methylation sequencing identified 566, 306 and 248 DMRs, respectively, and reduced the number of disease-specific DMRs to 171 in STEMI, 115 in NSTEMI and 82 in UA. Conclusion and Future Approaches Methylation profiles of ccfDNA showed potential as a biomarker to stratify the severity of acute coronary syndromes, a novel non-invasive detection methodology that could be extrapolated to risk stratification in other cardiovascular disease such as heart failure or coronary artery disease.

Deciphering the Multifaceted Immune Landscape of Unresectable Primary Liver Cancer to Predict Immunotherapy Response.

Immunotherapies employing PD-1/PD-L1 immune checkpoint inhibitors (ICIs) are vital for primary liver cancer (PLC), but response rates remain unsatisfying. Accurate differentiation of responders from non-responders to immunotherapy is imperative. Here, single-cell-scaled mass cytometry analysis on sequential peripheral blood mononuclear cells (PBMCs) from ICI-treated PLC patients is conducted, and tissue residence of immune subpopulations is assessed via multiplex immunohistochemistry. In the discovery cohort (n = 24), responders have lower baseline B cell and HLA-DR+CD8+T cell, and higher CD14+CD16- classical monocyte (CM) proportions. CMs decrease more in responders PBMCs, while HLA-DR+CD8+T cells conformably amplify after ICI-exposure. Responsive individuals display upregulated exhaustion and activation markers in peripheral immune lineages. In the expanded cohort of 77 patients, the augment of the B cells in non-responders is re-confirmed. Responders demonstrate much higher enrichment of B cells or tertiary lymphoid structures in tumor compared to non-responders. A prospective model that excelled in early discrimination of responders is developed using generalized linear model and achieves a satisfactory AUC over 0.9 in all three independent cohorts. Integratedly, the study unveils dynamic immune landscapes in PLC patients undergoing ICI-based therapy, aiding in PLC patient stratification for ICI-based treatment and fostering new response monitoring strategies.

Fecal microbial marker panel for aiding diagnosis of autism spectrum disorders.

Accumulating evidence suggests that gut microbiota alterations influence brain function and could serve as diagnostic biomarkers and therapeutic targets. The potential of using fecal microbiota signatures to aid autism spectrum disorder (ASD) detection is still not fully explored. Here, we assessed the potential of different levels of microbial markers (taxonomy and genome) in distinguishing children with ASD from age and gender-matched typically developing peers (n = 598, ASD vs TD = 273 vs 325). A combined microbial taxa and metagenome-assembled genome (MAG) markers showed a better performance than either microbial taxa or microbial MAGs alone for detecting ASD. A machine-learning model comprising 5 bacterial taxa and 44 microbial MAG markers (2 viral MAGs and 42 bacterial MAGs) achieved an area under the receiving operator curve (AUROC) of 0.886 in the discovery cohort and 0.734 in an independent validation cohort. Furthermore, the identified biomarkers and predicted ASD risk score also significantly correlated with the core symptoms measured by the Social Responsiveness Scale-2 (SRS-2). The microbiome panel showed a superior classification performance in younger children (≤6 years old) with an AUROC of 0.845 than older children (>6 years). The model was broadly applicable to subjects across genders, with or without gastrointestinal tract symptoms (constipation and diarrhea) and with or without psychiatric comorbidities (attention deficit and hyperactivity disorder and anxiety). This study highlights the potential clinical validity of fecal microbiome to aid in ASD diagnosis and will facilitate studies to understand the association of disturbance of human gut microbiota and ASD symptom severity.

MiRNAs profile as a signature for cardiovascular disease in long-term breast cancer female survivors: a pilot study

Abstract Background/Introduction Cardiovascular diseases (CVD) is a matter of immediate concern among long term breast cancer (BC) survivors. An early identification of those patients who are at higher risk of developing CVD is warranted to tailor prevention strategies and improve their quality of life. Purpose To identify whether a specific miRNA signature exists in breast cancer female survivors who develop CVD in a long term follow up. Methods We performed cross-sectional comparison of miRNA expression between female BC survivors (i.e. individual with a diagnosis of invasive ductal carcinoma who were free from cancer at 10-year follow up and without known CVD before BC diagnosis) with CVD (n=11) and without CVD (n=11) matched for age, BC grade, treatment received and traditional cardiovascular risk factors. CVD was defined as the incidence of established atherosclerotic cardio- or cerebro-vascular diseases (i.e., ischemic heart disease, carotid endarterectomy, stroke, heart failure), atrial fibrillation or venous thromboembolism. Thirteen miRNAs were selected since they were involved in either BC and CVD. On plasma derived samples, miRNAs expression profile analysis was performed by retro-transcription and real-time PCR assays, using miRNA-specific probes. Results The BC female survivors (mean age 73 years, 70% hypertension, 60% histological grade 2 and 3) were treated with surgery. The majority (76%) received hormone therapy after surgery according to the BC receptor expression. In half of the cohort, radiotherapy was performed. Those female BC survivors developing CVD experienced more commonly cerebral or cardiovascular atherosclerotic events (70%). As reported in Figure 1, some miRNAs analyzed (miR-19a; miR-21; miR-24; miR-125b; miR-195) are upregulated in BC survivors who developed CVD. Of note miR-19a, miR-21 (and miR-195) increase is so significantly relevant to consider these miRNAs as candidate biomarkers. Conclusion(s) In this discovery cohort, a specific miRNA profile signature identified female BC survivors experiencing CVD. Validation of such signatures is needed in a larger cohort as well as mechanistic studies to understand what is the pathophysiological link between the miRNAs identified and vascular health. Nevertheless these miRNAs in both cancer and cardiac diseases are mainly associated with promotion of cell proliferation and inhibition of apoptotic processes. The clinical implications of using a specific miRNA profile as a likely early biomarker of adverse cardiovascular events are indeed of great interest to better preserve BC survivors' health.Figure 1

Causal association of serum calcium, phosphate, vitamin D, parathyroid hormone, and FGF23 with the risk of aortic stenosis

Abstract Aim Disorders of mineral metabolism, including elevated levels of serum calcium, phosphate, 25-hydroxyvitamin D (25OH-VitD), parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23), have been reported in patients with calcific aortic valve stenosis (CAVS). However, evidence of the causal role of mineral metabolism in CAVS is still lacking. We aimed to investigate the causality between mineral metabolism and CAVS. Methods A systematic pipeline combining Mendelian randomization (MR), Steiger directionality test, colocalization analysis, protein-protein network, and enrichment analysis was applied to investigate the causal effect. Genome-wide association study (GWAS) and protein quantitative trait loci data for mineral metabolism markers were extracted from large-scale meta-analyses. Summary statistics for CAVS were obtained from two independent GWAS datasets as discovery and replication cohorts (n=374,277 and 653,867). Results In MR analysis, genetic mimicry of serum FGF23 elevation was associated with increased CAVS risk [ORdiscovery=3.081 (1.649-5.760), Pdiscovery=4.21×10-4; ORreplication=2.280 (1.461-3.558), Preplication=2.82×10-4] without evidence of reverse causation (Psteiger=7.21×10-98). Strong colocalisation association with CAVS was observed for FGF23 expression in the blood (PP.H4 = 0.96). Additionally, we identified some protein-protein interactions between FGF23 and known CAVS causative genes. Serum calcium, phosphate, 25OH-VitD, and PTH failed to show causal effects on CAVS at Bonferroni-corrected significance (all P&amp;gt;0.05/5=0.01). Conclusions Elevated serum FGF23 level is a causal risk factor for CAVS, and its mechanism of action in CAVS development may be independent of its function in regulating mineral metabolism. Hence, FGF23 may serve as a circulating marker and a promising preventive target for CAVS, warranting further investigation.

Plasma levels of sCASP3 predicts carotid intima media thickness progression

Abstract Background Studies on human atherosclerotic plaques have shown that apoptosis is associated with rupture prone plaques and that apoptosis is a key process for fast plaque progression. In line with this, we showed that circulating levels of soluble Caspase-3 (sCaspase-3) predict atherosclerotic events. However, the relationship between circulating levels of apoptosis markers and plaque progression remains to be explored. Purpose Here we aimed to investigate if circulating levels of apoptosis markers could predict mean intima-media thickness in the common carotid artery (IMT-CCA) progression among individuals with atherosclerotic carotid plaques. Methods The Malmö Diet and Cancer (MDC) cardiovascular cohort was used. MDC is a prospective population-based cohort study. Baseline variables (blood, clinical information and carotid ultrasound measurements) were collected between 1991-1994 and the right carotid ultrasound re-examinations were performed between 1994-2001. IMT was measured at by ultrasound at baseline as well as at the re-examinations. The Olink Proximity Extension Assay (SciLifeLab, Uppsala, Sweden) was used to analyze plasma levels of sCaspase-3, sFADD, sCaspase-8, sTRAIL-R2 and sTNFR1 at baseline. Correlation and multiple linear regression analyses, adjusted for age, sex, and cardiovascular risk factors, were employed to evaluate the relationships between apoptosis biomarkers and IMT-CCA as well as average annual change in IMT-CCA. Results In total, 1929 participants (median age 60 years) with an ultrasound confirmed carotid plaque (focal IMT &amp;gt;1.2mm) at baseline were included. Subjects who underwent ultrasound IMT re-examination over a period of &amp;gt;4.5 years were divided into a discovery (n=406) and a validation (n=355) cohort. sCaspase-3 was identified as the marker with the strongest correlation to IMT-CCA at baseline (p=0.02) and upon re-examination (p=0.04), among the five apoptosis markers examined, in both the discovery and validation cohorts. sCaspase-3 also showed a correlation with the average annual change of IMT-CCA (r=0.12, p=8×10-4) in both cohorts. Furthermore, when diving the cohort into tertiles based on plasma levels of sCapase-3, subjects with high levels of sCaspase-3 (3rd tertile) had greater annual changes in IMT-CCA compared to the 1st tertile (p=0.03). Multiple linear regression analyses confirmed that sCapase-3 was associated with the average annual IMT-CCA change in both the discovery (beta=0.14, 95% confidence interval (CI) 0.04–0.24) cohort and the validation cohort (beta=0.11, 95%CI 0.01–0.21). Conclusion Using a large, population-based study with repeated IMT measurements, we demonstrated that circulating levels of sCapase-3 were associated with increasing IMT in the CCA. While further studies are warranted, these results suggest that sCapase-3 levels could serve as potential screening marker to identify individuals at higher risk for plaque progression.

Expression of cardiomyopathy-related genes in mononuclear blood cells predicts all-cause mortality in patients presenting with acute versus chronic coronary syndrome requiring revascularisation

Abstract Objective Despite advances in the revascularisation techniques their net clinical benefit differs substantially between patients presenting with myocardial infarction (ACS) and chronic coronary syndrome (CCS) due to obstructive coronary atherosclerosis. Considering that leukocytes represents a key pathophysiological component of myocardial ischaemia, exploration of mononuclear blood cell (PBMC) gene expression profile in CCS and ACS setting could reveal clinically relevant transcriptomic signals. The aim of the present study was to identify differences in PBMC transcriptome between ACS and CCS patients which are linked to mortality after revascularisation. Methods and Results We analysed PBMC transcriptomes from719 ACS and 486 CCS patients creating discovery cohort. The results of transcriptomic analysis were verified in replication cohort (ACS: N=682 for CCS: N=489) generated based on propensity score matching. Median time from revascularisation to blood collection (IQR) was 15.68 (-4.52, 23.23) hrs. Patients with ACS showed consistent differential expression of 8173 genes (4544 upregulated, 3629 downregulated) when compared with CCS patients. The subsequent pathway analysis also showed significant enrichment of several canonical pathways involved in major cardiovascular pathologies i.e. atherosclerosis (p = 1.22-06, enrichment = 3.39), vascular endothelial growth factor production (p = 1.16-06, enrichment = 16.73), positive regulation of vascular development (p = 2.78-06, enrichment = 5.30) and heterotopic cell adhesion including gene desmocollin 2 (DSC2, p = 3.64-04, enrichment = 6.30). In der multivariate survival analysis with differentially expressed genes, increased expression of DCS2, lamin A/C (LMNA), and UGGT2, and decreased expression of SNRNP70 predicted poor survival over the median follow-up 11.35 yrs. Consistently, controlling the false discovery rate at ≤ 5% these genes enriched pathways involved in cardiomyopathies such as arrhythmogenic right-ventricular cardiomyopathy (p = 4.19x10-4, enrichment = 58.9) and collagen disease (p = 2.29x10-3, enrichment = 23.8). Conclusion We found altered PBMC expression of multiple genes in patients requiring revascularisation for ACS versus CCS. Several differentially expressed genes included in cardiomyopathy pathways predicted long-term all-cause mortality in these patients.