Genomic approaches to explore susceptibility and pathogenesis of alcohol use disorder and alcohol-associated liver disease.

Excessive alcohol use is a major risk factor for the development of an alcohol use disorder (AUD) and contributes to a wide variety of other medical illnesses, including alcohol-associated liver disease (ALD). Both AUD and ALD are complex and causally interrelated diseases, and multiple factors other than alcohol consumption are implicated in the disease pathogenesis. While the underlying pathophysiology of AUD and ALD is complex, there is substantial evidence for a genetic susceptibility of both diseases. Current genome-wide association studies indicate that the genes associated with clinical AUD only poorly overlap with the genes identified for heavy drinking and, in turn, neither overlap with the genes identified for ALD. Uncovering the main genetic factors will enable us to identify molecular drivers underlying the pathogenesis, discover potential targets for therapy, and implement patient care early in disease progression. In this review, we described multiple genomic approaches and their implications to investigate the susceptibility and pathogenesis of both AUD and ALD. We concluded our review with a discussion of the knowledge gaps and future research on genomic studies in these 2 diseases.

Integrated Transcriptomic and Proteomic Analysis Identifies Plasma Biomarkers of Hepatocellular Failure in Alcohol-Associated Hepatitis.

Alcohol-associated hepatitis (AH) is a form of liver failure with high short-term mortality. Recent studies have shown that defective function of hepatocyte nuclear factor 4 alpha (HNF4a) and systemic inflammation are major disease drivers of AH. Plasma biomarkers of hepatocyte function could be useful for diagnostic and prognostic purposes. Herein, an integrative analysis of hepatic RNA sequencing and liquid chromatography-tandem mass spectrometry was performed to identify plasma protein signatures for patients with mild and severe AH. Alcohol-related liver disease cirrhosis, nonalcoholic fatty liver disease, and healthy subjects were used as comparator groups. Levels of identified proteins primarily involved in hepatocellular function were decreased in patients with AH, which included hepatokines, clotting factors, complement cascade components, and hepatocyte growth activators. A protein signature of AH disease severity was identified, including thrombin, hepatocyte growth factor α, clusterin, human serum factor H-related protein, and kallistatin, which exhibited large abundance shifts between severe and nonsevere AH. The combination of thrombin and hepatocyte growth factor α discriminated between severe and nonsevere AH with high sensitivity and specificity. These findings were correlated with the liver expression of genes encoding secreted proteins in a similar cohort, finding a highly consistent plasma protein signature reflecting HNF4A and HNF1A functions. This unbiased proteomic-transcriptome analysis identified plasma protein signatures and pathways associated with disease severity, reflecting HNF4A/1A activity useful for diagnostic assessment in AH.

DEGnext: classification of differentially expressed genes from RNA-seq data using a convolutional neural network with transfer learning.

BACKGROUND: A limitation of traditional differential expression analysis on small datasets involves the possibility of false positives and false negatives due to sample variation. Considering the recent advances in deep learning (DL) based models, we wanted to expand the state-of-the-art in disease biomarker prediction from RNA-seq data using DL. However, application of DL to RNA-seq data is challenging due to absence of appropriate labels and smaller sample size as compared to number of genes. Deep learning coupled with transfer learning can improve prediction performance on novel data by incorporating patterns learned from other related data. With the emergence of new disease datasets, biomarker prediction would be facilitated by having a generalized model that can transfer the knowledge of trained feature maps to the new dataset. To the best of our knowledge, there is no Convolutional Neural Network (CNN)-based model coupled with transfer learning to predict the significant upregulating (UR) and downregulating (DR) genes from both trained and untrained datasets. RESULTS: We implemented a CNN model, DEGnext, to predict UR and DR genes from gene expression data obtained from The Cancer Genome Atlas database. DEGnext uses biologically validated data along with logarithmic fold change values to classify differentially expressed genes (DEGs) as UR and DR genes. We applied transfer learning to our model to leverage the knowledge of trained feature maps to untrained cancer datasets. DEGnext's results were competitive (ROC scores between 88 and 99[Formula: see text]) with those of five traditional machine learning methods: Decision Tree, K-Nearest Neighbors, Random Forest, Support Vector Machine, and XGBoost. DEGnext was robust and effective in terms of transferring learned feature maps to facilitate classification of unseen datasets. Additionally, we validated that the predicted DEGs from DEGnext were mapped to significant Gene Ontology terms and pathways related to cancer. CONCLUSIONS: DEGnext can classify DEGs into UR and DR genes from RNA-seq cancer datasets with high performance. This type of analysis, using biologically relevant fine-tuning data, may aid in the exploration of potential biomarkers and can be adapted for other disease datasets.

Transethnic meta-analysis of metabolic syndrome in a multiethnic study.

Genome-wide association studies (GWAS) have been used to establish thousands of genetic associations across numerous phenotypes. To improve the power of GWAS and generalize associations across ethnic groups, transethnic meta-analysis methods are used to combine the results of several GWAS from diverse ancestries. The goal of this study is to identify genetic associations for eight quantitative metabolic syndrome (MetS) traits through a meta-analysis across four ethnic groups. Traits were measured in the GENetics of Noninsulin dependent Diabetes Mellitus (GENNID) Study which consists of African-American (families = 73, individuals = 288), European-American (families = 79, individuals = 519), Japanese-American (families = 17, individuals = 132), and Mexican-American (families = 113, individuals = 610) samples. Genome-wide association results from these four ethnic groups were combined using four meta-analysis methods: fixed effects, random effects, TransMeta, and MR-MEGA. We provide an empirical comparison of the four meta-analysis methods from the GENNID results, discuss which types of loci (characterized by allelic heterogeneity) appear to be better detected by each of the four meta-analysis methods in the GENNID Study, and validate our results using previous genetic discoveries. We specifically compare the two transethnic methods, TransMeta and MR-MEGA, and discuss how each transethnic method's framework relates to the types of loci best detected by each method.

Gene expression profiling of fibroblasts in a family with LMNA-related cardiomyopathy reveals molecular pathways implicated in disease pathogenesis.

BACKGROUND: Intermediate filament proteins that construct the nuclear lamina of a cell include the Lamin A/C proteins encoded by the LMNA gene, and are implicated in fundamental processes such as nuclear structure, gene expression, and signal transduction. LMNA mutations predominantly affect mesoderm-derived cell lineages in diseases collectively termed as laminopathies that include dilated cardiomyopathy with conduction defects, different forms of muscular dystrophies, and premature aging syndromes as Hutchinson-Gilford Progeria Syndrome. At present, our understanding of the molecular mechanisms regulating tissue-specific manifestations of laminopathies are still limited. METHODS: To gain deeper insight into the molecular mechanism of a novel LMNA splice-site mutation (c.357-2A > G) in an affected family with cardiac disease, we conducted deep RNA sequencing and pathway analysis for nine fibroblast samples obtained from three patients with cardiomyopathy, three unaffected family members, and three unrelated, unaffected individuals. We validated our findings by quantitative PCR and protein studies. RESULTS: We identified eight significantly differentially expressed genes between the mutant and non-mutant fibroblasts, that included downregulated insulin growth factor binding factor protein 5 (IGFBP5) in patient samples. Pathway analysis showed involvement of the ERK/MAPK signaling pathway consistent with previous studies. We found no significant differences in gene expression for Lamin A/C and B-type lamins between the groups. In mutant fibroblasts, RNA-seq confirmed that only the LMNA wild type allele predominately was expressed, and Western Blot showed normal Lamin A/C protein levels. CONCLUSIONS: IGFBP5 may contribute in maintaining signaling pathway homeostasis, which may lead to the absence of notable molecular and structural abnormalities in unaffected tissues such as fibroblasts. Compensatory mechanisms from other nuclear membrane proteins were not found. Our results also demonstrate that only one copy of the wild type allele is sufficient for normal levels of Lamin A/C protein to maintain physiological function in an unaffected cell type. This suggests that affected cell types such as cardiac tissues may be more sensitive to haploinsufficiency of Lamin A/C. These results provide insight into the molecular mechanism of disease with a possible explanation for the tissue specificity of LMNA-related dilated cardiomyopathy.

A-Lister: a tool for analysis of differentially expressed omics entities across multiple pairwise comparisons.

BACKGROUND: Researchers commonly analyze lists of differentially expressed entities (DEEs), such as differentially expressed genes (DEGs), differentially expressed proteins (DEPs), and differentially methylated positions/regions (DMPs/DMRs), across multiple pairwise comparisons. Large biological studies can involve multiple conditions, tissues, and timepoints that result in dozens of pairwise comparisons. Manually filtering and comparing lists of DEEs across multiple pairwise comparisons, typically done by writing custom code, is a cumbersome task that can be streamlined and standardized. RESULTS: A-Lister is a lightweight command line and graphical user interface tool written in Python. It can be executed in a differential expression mode or generic name list mode. In differential expression mode, A-Lister accepts as input delimited text files that are output by differential expression tools such as DESeq2, edgeR, Cuffdiff, and limma. To allow for the most flexibility in input ID types, to avoid database installation requirements, and to allow for secure offline use, A-Lister does not validate or impose restrictions on entity ID names. Users can specify thresholds to filter the input file(s) by column(s) such as p-value, q-value, and fold change. Additionally, users can filter the pairwise comparisons within the input files by fold change direction (sign). Queries composed of intersection, fuzzy intersection, difference, and union set operations can also be performed on any number of pairwise comparisons. Thus, the user can filter and compare any number of pairwise comparisons within a single A-Lister differential expression command. In generic name list mode, A-Lister accepts delimited text files containing lists of names as input. Queries composed of intersection, fuzzy intersection, difference, and union set operations can then be performed across these lists of names. CONCLUSIONS: A-Lister is a flexible tool that enables the user to rapidly narrow down large lists of DEEs to a small number of most significant entities. These entities can then be further analyzed using visualization, pathway analysis, and other bioinformatics tools.

Variants Near CCK Receptors are Associated With Electrophysiological Responses to Pre-pulse Startle Stimuli in a Mexican American Cohort.

Neurophysiological measurements of the response to pre-pulse and startle stimuli have been suggested to represent an important endophenotype for both substance dependence and other select psychiatric disorders. We have previously shown, in young adult Mexican Americans (MA), that presentation of a short delay acoustic pre-pulse, prior to the startle stimuli can elicit a late negative component at about 400 msec (N4S), in the event-related potential (ERP), recorded from frontal cortical areas. In the present study, we investigated whether genetic factors associated with this endophenotype could be identified. The study included 420 (age 18-30 years) MA men (n = 170), and women (n = 250). DNA was genotyped using an Affymetrix Axiom Exome1A chip. An association analysis revealed that the CCKAR and CCKBR (cholecystokinin A and B receptor) genes each had a nearby variant that showed suggestive significance with the amplitude of the N4S component to pre-pulse stimuli. The neurotransmitter cholecystokinin (CCK), along with its receptors, CCKAR and CCKBR, have been previously associated with psychiatric disorders, suggesting that variants near these genes may play a role in the pre-pulse/startle response in this cohort.

Influence of cobalamin scarcity on diatom molecular physiology and identification of a cobalamin acquisition protein.

Diatoms are responsible for ~40% of marine primary production and are key players in global carbon cycling. There is mounting evidence that diatom growth is influenced by cobalamin (vitamin B(12)) availability. This cobalt-containing micronutrient is only produced by some bacteria and archaea but is required by many diatoms and other eukaryotic phytoplankton. Despite its potential importance, little is known about mechanisms of cobalamin acquisition in diatoms or the impact of cobalamin scarcity on diatom molecular physiology. Proteomic profiling and RNA-sequencing transcriptomic analysis of the cultured diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana revealed three distinct strategies used by diatoms to cope with low cobalamin: increased cobalamin acquisition machinery, decreased cobalamin demand, and management of reduced methionine synthase activity through changes in folate and S-adenosyl methionine metabolism. One previously uncharacterized protein, cobalamin acquisition protein 1 (CBA1), was up to 160-fold more abundant under low cobalamin availability in both diatoms. Autologous overexpression of CBA1 revealed association with the outside of the cell and likely endoplasmic reticulum localization. Cobalamin uptake rates were elevated in strains overexpressing CBA1, directly linking this protein to cobalamin acquisition. CBA1 is unlike characterized cobalamin acquisition proteins and is the only currently identified algal protein known to be implicated in cobalamin uptake. The abundance and widespread distribution of transcripts encoding CBA1 in environmental samples suggests that cobalamin is an important nutritional factor for phytoplankton. Future study of CBA1 and other molecular signatures of cobalamin scarcity identified here will yield insight into the evolution of cobalamin utilization and facilitate monitoring of cobalamin starvation in oceanic diatom communities.

Protective variant associated with alcohol dependence in a Mexican American cohort.

BACKGROUND: Mexican Americans, particularly those born in the United States, are at greater risk for alcohol associated morbidity and mortality. The present study sought to investigate whether specific genetic variants may be associated with alcohol use disorder phenotypes in a select population of Mexican American young adults. METHODS: The study evaluated a cohort of 427 (age 18 - 30 years) Mexican American men (n = 171) and women (n = 256). Information on alcohol dependence was obtained through interview using the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA). For all subjects, DNA was extracted from blood samples, followed by genotyping using an Affymetrix Axiom Exome1A chip. RESULTS: A protective variant (rs991316) located downstream from the ADH7 (alcohol dehydrogenase 7) gene showed suggestive significance in association with alcohol dependence symptom counts derived from DSM-III-R and DSM-IV criteria, as well as to clustered alcohol dependence symptoms. Additional linkage analysis suggested that nearby variants in linkage disequilibrium with rs991316 were not responsible for the observed association with the alcohol dependence phenotypes in this study. CONCLUSIONS: ADH7 has been shown to have a protective role against alcohol dependence in previous studies involving other ethnicities, but has not been reported for Mexican Americans. These results suggest that variants near ADH7 may play a role in protection from alcohol dependence in this Mexican American cohort.

Correlation analysis of genetic admixture and social identification with body mass index in a Native American community.

OBJECTIVES: Body mass index (BMI) is a well-known measure of obesity with a multitude of genetic and non-genetic determinants. Identifying the underlying factors associated with BMI is difficult because of its multifactorial etiology that varies as a function of geoethnic background and socioeconomic setting. Thus, we pursued a study exploring the influence of the degree of Native American admixture on BMI (as well as weight and height individually) in a community sample of Native Americans (n = 846) while accommodating a variety of socioeconomic and cultural factors. METHODS: Participants' degree of Native American (NA) ancestry was estimated using a genome-wide panel of markers. The participants also completed an extensive survey of cultural and social identity measures: the Indian Culture Scale (ICS) and the Orthogonal Cultural Identification Scale (OCIS). Multiple linear regression was used to examine the relation between these measures and BMI. RESULTS: Our results suggest that BMI is correlated positively with the proportion of NA ancestry. Age was also significantly associated with BMI, while gender and socioeconomic measures (education and income) were not. For the two cultural identity measures, the ICS showed a positive correlation with BMI, while OCIS was not associated with BMI. CONCLUSIONS: Taken together, these results suggest that genetic and cultural environmental factors, rather than socioeconomic factors, account for a substantial proportion of variation in BMI in this population. Further, significant correlations between degree of NA ancestry and BMI suggest that admixture mapping may be appropriate to identify loci associated with BMI in this population.

Single-cell transcriptomics of peripheral blood mononuclear cells indicates impaired immune and inflammatory responses in alcohol-associated hepatitis.

Alcohol-associated hepatitis (AH) is often diagnosed at advanced stages, and severe AH usually carries poor prognosis and high short-term mortality. In addition, it is challenging to understand the molecular mechanisms of immune dysregulation and inflammation in AH due to the cellular complexity and heterogeneity. Using single-cell RNA sequencing, previous studies found that AH causes dysfunctional innate immune response in monocytes, involving activation of pattern recognition receptors (PRRs) and cytokine signaling pathways. To better understand the coordinated systemic immune response in AH patients, we performed combined single-cell transcriptome, cell-surface protein, and lymphocyte antigen receptor analysis of peripheral blood mononuclear cell (PBMC) samples. Our results showed inflammatory cytokines and chemokines were highly expressed in AH, including IL-2, IL-32, CXC3R1 and CXCL16 in monocytes and NK cells, whereas HLA-DR genes were reduced in monocytes. In addition, we also found altered differentiation of T-helper cells (TH1 and TH17), which could further lead to neutrophil recruitment and macrophage activation. Lastly, our results also suggest impaired NK-cell activation and differentiation in AH with reduced gene expression of KLRC2 and increased gene expression of KLRG1. Our findings indicate different mechanisms may be involved in impaired immune and inflammatory responses for the cellular subtypes of the PBMCs in AH.

Heterogeneity in familial clustering of metabolic syndrome components in the multiethnic GENNID study.

OBJECTIVE: Metabolic syndrome (MetS) is defined by clustering of cardiometabolic components, which may be present in different combinations. The authors evaluated clustering in individuals and extended families within and across ancestry groups. METHODS: The prevalence of different combinations of MetS components (high fasting glucose, low high-density lipoprotein cholesterol, high triglycerides, high blood pressure, and abdominal obesity) was estimated in 1651 individuals (340 families) self-reporting as European American (EA), Hispanic/Mexican American (MA), African American (AA), and Japanese American (JA). Odds ratios were estimated using logistic regression with generalized estimating equations comparing individual MetS components, number, and combinations of components for each ancestry group versus EA. RESULTS: Clustering of all five components (Combination #16) was more prevalent in EA (29.9%) and MA (25.2%) individuals than in AA (18.7%) and JA (15.5%) individuals. Compared with EA individuals, AA individuals were 64% and 66% less likely to have high triglycerides and low high-density lipoprotein cholesterol, whereas JA individuals were 85% and 56% less likely to have abdominal obesity and high blood pressure, respectively. Compared with EA individuals, the odds of having two, four, or five components were at least 77% lower in JA individuals, whereas the odds of having three, four, or five components were at least 3.79 times greater in MA individuals. CONCLUSIONS: Understanding heterogeneity in MetS clustering may identify factors important in reducing health disparities.

Clinical and genetic risk factors for progressive fibrosis in metabolic dysfunction-associated steatotic liver disease.

BACKGROUND: Fibrosis-4 (FIB4) is a recommended noninvasive test to assess hepatic fibrosis among patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we used FIB4 trajectory over time (ie, "slope" of FIB4) as a surrogate marker of liver fibrosis progression and examined if FIB4 slope is associated with clinical and genetic factors among individuals with clinically defined MASLD within the Million Veteran Program Cohort. METHODS: In this retrospective cohort study, FIB4 slopes were estimated through linear regression for participants with clinically defined MASLD and FIB4 <2.67 at baseline. FIB4 slope was correlated with demographic parameters and clinical outcomes using logistic regression and Cox proportional hazard models. FIB4 slope as a quantitative phenotype was used in a genome-wide association analysis in ancestry-specific analysis and multiancestry meta-analysis using METAL. RESULTS: FIB4 slopes, generated from 98,361 subjects with MASLD (16,045 African, 74,320 European, and 7996 Hispanic), showed significant associations with sex, ancestry, and cardiometabolic risk factors (p < 0.05). FIB4 slopes also correlated strongly with hepatic outcomes and were independently associated with time to cirrhosis. Five genetic loci showed genome-wide significant associations (p < 5 × 10-8) with FIB4 slope among European ancestry subjects, including 2 known (PNPLA3 and TM6SF2) and 3 novel loci (TERT 5.1 × 10-11; LINC01088, 3.9 × 10-8; and MRC1, 2.9 × 10-9). CONCLUSIONS: Linear trajectories of FIB4 correlated significantly with time to progression to cirrhosis, with liver-related outcomes among individuals with MASLD and with known and novel genetic loci. FIB4 slope may be useful as a surrogate measure of fibrosis progression.

Identification of integrated proteomics and transcriptomics signature of alcohol-associated liver disease using machine learning.

Distinguishing between alcohol-associated hepatitis (AH) and alcohol-associated cirrhosis (AC) remains a diagnostic challenge. In this study, we used machine learning with transcriptomics and proteomics data from liver tissue and peripheral mononuclear blood cells (PBMCs) to classify patients with alcohol-associated liver disease. The conditions in the study were AH, AC, and healthy controls. We processed 98 PBMC RNAseq samples, 55 PBMC proteomic samples, 48 liver RNAseq samples, and 53 liver proteomic samples. First, we built separate classification and feature selection pipelines for transcriptomics and proteomics data. The liver tissue models were validated in independent liver tissue datasets. Next, we built integrated gene and protein expression models that allowed us to identify combined gene-protein biomarker panels. For liver tissue, we attained 90% nested-cross validation accuracy in our dataset and 82% accuracy in the independent validation dataset using transcriptomic data. We attained 100% nested-cross validation accuracy in our dataset and 61% accuracy in the independent validation dataset using proteomic data. For PBMCs, we attained 83% and 89% accuracy with transcriptomic and proteomic data, respectively. The integration of the two data types resulted in improved classification accuracy for PBMCs, but not liver tissue. We also identified the following gene-protein matches within the gene-protein biomarker panels: CLEC4M-CLC4M, GSTA1-GSTA2 for liver tissue and SELENBP1-SBP1 for PBMCs. In this study, machine learning models had high classification accuracy for both transcriptomics and proteomics data, across liver tissue and PBMCs. The integration of transcriptomics and proteomics into a multi-omics model yielded improvement in classification accuracy for the PBMC data. The set of integrated gene-protein biomarkers for PBMCs show promise toward developing a liquid biopsy for alcohol-associated liver disease.

Urinary glyphosate and AMPA levels in a cross-sectional study of postmenopausal women: Associations with organic eating behavior and dietary intake.

Animal and epidemiologic studies suggest that there may be adverse health effects from exposure to glyphosate, the most highly used pesticide in the world, and its metabolite aminomethylphosphonic acid (AMPA). Meanwhile, consumption of organic foods (presumably grown free of chemical pesticides) has increased in recent years. However, there have been limited biomonitoring studies assessing the levels of human glyphosate and AMPA exposure in the United States. We examined urinary levels of glyphosate and AMPA in the context of organic eating behavior in a cohort of healthy postmenopausal women residing in Southern California and evaluated associations with demographics, dietary intake, and other lifestyle factors. 338 women provided two first-morning urine samples and at least one paired 24-h dietary recall reporting the previous day's dietary intake. Urinary glyphosate and AMPA were measured using LC-MS/MS. Participants reported on demographic and lifestyle factors via questionnaires. Potential associations were examined between these factors and urinary glyphosate and AMPA concentrations. Glyphosate was detected in 89.9% of urine samples and AMPA in 67.2%. 37.9% of study participants reported often or always eating organic food, 30.2% sometimes, and 32.0% seldom or never. Frequency of organic food consumption was associated with several demographic and lifestyle factors. Frequent organic eaters had significantly lower urinary glyphosate and AMPA levels, but not after adjustment for covariates. Grain consumption was significantly associated with higher urinary glyphosate levels, even among women who reported often or always eating organic grains. Soy protein and alcohol consumption as well as high frequency of eating fast food were associated with higher urinary AMPA levels. In conclusion, in the largest study to date examining paired dietary recall data and measurements of first-void urinary glyphosate and AMPA, the vast majority of subjects sampled had detectable levels, and significant dietary sources in the American diet were identified.

Differentiating between liver diseases by applying multiclass machine learning approaches to transcriptomics of liver tissue or blood-based samples.

Background & Aims: Liver disease carries significant healthcare burden and frequently requires a combination of blood tests, imaging, and invasive liver biopsy to diagnose. Distinguishing between inflammatory liver diseases, which may have similar clinical presentations, is particularly challenging. In this study, we implemented a machine learning pipeline for the identification of diagnostic gene expression biomarkers across several alcohol-associated and non-alcohol-associated liver diseases, using either liver tissue or blood-based samples. Methods: We collected peripheral blood mononuclear cells (PBMCs) and liver tissue samples from participants with alcohol-associated hepatitis (AH), alcohol-associated cirrhosis (AC), non-alcohol-associated fatty liver disease, chronic HCV infection, and healthy controls. We performed RNA sequencing (RNA-seq) on 137 PBMC samples and 67 liver tissue samples. Using gene expression data, we implemented a machine learning feature selection and classification pipeline to identify diagnostic biomarkers which distinguish between the liver disease groups. The liver tissue results were validated using a public independent RNA-seq dataset. The biomarkers were computationally validated for biological relevance using pathway analysis tools. Results: Utilizing liver tissue RNA-seq data, we distinguished between AH, AC, and healthy conditions with overall accuracies of 90% in our dataset, and 82% in the independent dataset, with 33 genes. Distinguishing 4 liver conditions and healthy controls yielded 91% overall accuracy in our liver tissue dataset with 39 genes, and 75% overall accuracy in our PBMC dataset with 75 genes. Conclusions: Our machine learning pipeline was effective at identifying a small set of diagnostic gene biomarkers and classifying several liver diseases using RNA-seq data from liver tissue and PBMCs. The methodologies implemented and genes identified in this study may facilitate future efforts toward a liquid biopsy diagnostic for liver diseases. Lay summary: Distinguishing between inflammatory liver diseases without multiple tests can be challenging due to their clinically similar characteristics. To lay the groundwork for the development of a non-invasive blood-based diagnostic across a range of liver diseases, we compared samples from participants with alcohol-associated hepatitis, alcohol-associated cirrhosis, chronic hepatitis C infection, and non-alcohol-associated fatty liver disease. We used a machine learning computational approach to demonstrate that gene expression data generated from either liver tissue or blood samples can be used to discover a small set of gene biomarkers for effective diagnosis of these liver diseases.

Association of mammographic density with blood DNA methylation.

BACKGROUND: Altered DNA methylation may be an intermediate phenotype between breast cancer risk factors and disease. Mammographic density is a strong risk factor for breast cancer. However, no studies to date have identified an epigenetic signature of mammographic density. We performed an epigenome-wide association study of mammographic density. METHODS: White blood cell DNA methylation was measured for 385 postmenopausal women using the Illumina Infinium MethylationEPIC BeadChip array. Differential methylation was assessed using genome-wide, probe-level, and regional analyses. We implemented a resampling-based approach to improve the stability of our findings. RESULTS: On average, women with elevated mammographic density exhibited DNA hypermethylation within CpG islands and gene promoters compared to women with lower mammographic density. We identified 250 CpG sites for which DNA methylation was significantly associated with mammographic density. The top sites were located within genes associated with cancer, including HDLBP, TGFB2, CCT4, and PAX8, and were more likely to be located in regulatory regions of the genome. We also identified differential DNA methylation in 37 regions, including within the promoters of PAX8 and PF4, a gene involved in the regulation of angiogenesis. Overall, our results paint a picture of epigenetic dysregulation associated with mammographic density. CONCLUSION: Mammographic density is associated with differential DNA methylation throughout the genome, including within genes associated with cancer. Our results suggest the potential involvement of several genes in the biological mechanisms behind differences in breast density between women. Further studies are warranted to explore these potential mechanisms and potential links to breast cancer risk.

Association of Glyphosate Exposure with Blood DNA Methylation in a Cross-Sectional Study of Postmenopausal Women.

BACKGROUND: Glyphosate is the most commonly used herbicide in the world and is purported to have a variety of health effects, including endocrine disruption and an elevated risk of several types of cancer. Blood DNA methylation has been shown to be associated with many other environmental exposures, but to our knowledge, no studies to date have examined the association between blood DNA methylation and glyphosate exposure. OBJECTIVE: We conducted an epigenome-wide association study to identify DNA methylation loci associated with urinary glyphosate and its metabolite aminomethylphosphonic acid (AMPA) levels. Secondary goals were to determine the association of epigenetic age acceleration with glyphosate and AMPA and develop blood DNA methylation indices to predict urinary glyphosate and AMPA levels. METHODS: For 392 postmenopausal women, white blood cell DNA methylation was measured using the Illumina Infinium MethylationEPIC BeadChip array. Glyphosate and AMPA were measured in two urine samples per participant using liquid chromatography-tandem mass spectrometry. Methylation differences at the probe and regional level associated with glyphosate and AMPA levels were assessed using a resampling-based approach. Probes and regions that had an false discovery rate q<0.1 in ≥90% of 1,000 subsamples of the study population were considered differentially methylated. Differentially methylated sites from the probe-specific analysis were combined into a methylation index. Epigenetic age acceleration from three epigenetic clocks and an epigenetic measure of pace of aging were examined for associations with glyphosate and AMPA. RESULTS: We identified 24 CpG sites whose methylation level was associated with urinary glyphosate concentration and two associated with AMPA. Four regions, within the promoters of the MSH4, KCNA6, ABAT, and NDUFAF2/ERCC8 genes, were associated with glyphosate levels, along with an association between ESR1 promoter hypomethylation and AMPA. The methylation index accurately predicted glyphosate levels in an internal validation cohort. AMPA, but not glyphosate, was associated with greater epigenetic age acceleration. DISCUSSION: Glyphosate and AMPA exposure were associated with DNA methylation differences that could promote the development of cancer and other diseases. Further studies are warranted to replicate our results, determine the functional impact of glyphosate- and AMPA-associated differential DNA methylation, and further explore whether DNA methylation could serve as a biomarker of glyphosate exposure. https://doi.org/10.1289/EHP10174.

Genome-wide association analysis of metabolic syndrome quantitative traits in the GENNID multiethnic family study.

BACKGROUND: To identify genetic associations of quantitative metabolic syndrome (MetS) traits and characterize heterogeneity across ethnic groups. METHODS: Data was collected from GENetics of Noninsulin dependent Diabetes Mellitus (GENNID), a multiethnic resource of Type 2 diabetic families and included 1520 subjects in 259 African-American, European-American, Japanese-Americans, and Mexican-American families. We focused on eight MetS traits: weight, waist circumference, systolic and diastolic blood pressure, high-density lipoprotein, triglycerides, fasting glucose, and insulin. Using genotyped and imputed data from Illumina's Multiethnic array, we conducted genome-wide association analyses with linear mixed models for all ethnicities, except for the smaller Japanese-American group, where we used additive genetic models with gene-dropping. RESULTS: Findings included ethnic-specific genetic associations and heterogeneity across ethnicities. Most significant associations were outside our candidate linkage regions and were coincident within a gene or intergenic region, with two exceptions in European-American families: (a) within previously identified linkage region on chromosome 2, two significant GLI2-TFCP2L1 associations with weight, and (b) one chromosome 11 variant near CADM1-LINC00900 with pleiotropic blood pressure effects. CONCLUSIONS: This multiethnic family study found genetic heterogeneity and coincident associations (with one case of pleiotropy), highlighting the importance of including diverse populations in genetic research and illustrating the complex genetic architecture underlying MetS.

Genetic signature of human longevity in PKC and NF-κB signaling.

Gene variants associated with longevity are also associated with protection against cognitive decline, dementia and Alzheimer's disease, suggesting that common physiologic pathways act at the interface of longevity and cognitive function. To test the hypothesis that variants in genes implicated in cognitive function may promote exceptional longevity, we performed a comprehensive 3-stage study to identify functional longevity-associated variants in ~700 candidate genes in up to 450 centenarians and 500 controls by target capture sequencing analysis. We found an enrichment of longevity-associated genes in the nPKC and NF-κB signaling pathways by gene-based association analyses. Functional analysis of the top three gene variants (NFKBIA, CLU, PRKCH) suggests that non-coding variants modulate the expression of cognate genes, thereby reducing signaling through the nPKC and NF-κB. This matches genetic studies in multiple model organisms, suggesting that the evolutionary conservation of reduced PKC and NF-κB signaling pathways in exceptional longevity may include humans.