Clinical Genomics and Experimental Therapeutics

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I. Epigenome analyses for novel target developments in AUD: We conducted the largest DNA methylation epigenome-wide association study (EWAS) analyses currently available for individuals with AUD (total N = 625) and employed a top hit replication (N = 4798) using a cross-tissue/cross-phenotypic approach with the goal of identifying novel epigenetic targets relevant to AUD. Results show that a network of differentially methylated regions in glucocorticoid signaling and inflammation-related genes were associated with alcohol use behaviors. A top probe consistently associated across all cohorts was located in the long non-coding RNA growth arrest specific five gene (GAS5) (p < 10-24). GAS5 has been implicated in regulating transcriptional activity of the glucocorticoid receptor and has multiple functions related to apoptosis, immune function and various cancers. Endophenotypic analyses using peripheral cortisol levels and neuroimaging paradigms in human showed that methylomic variation in GAS5 network-related probes were associated with stress phenotypes. Postmortem human brain analyses documented increased GAS5 expression in the amygdala of individuals with AUD. Our data suggest that alcohol use is associated with differential methylation in the glucocorticoid system that might influence stress and inflammatory reactivity and subsequently risk for AUD (Lohoff et al., 2020). We also follow up on another top target that was previously identified using an EWAS approach, namely the gene encoding the enzyme Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) (Lohoff et al., 2017). PCSK9 is predominantly expressed in the liver where it is synthesized and secreted. It targets low-density lipoprotein cholesterol receptors (LDL-R) in the liver cells and interferes with the regulation of LDL cholesterol (LDL-C) in the blood. Given alcohols toxic effect on the liver, we investigated PCSK9 inhibition in a rat model of chronic alcohol consumption. PCSK9 inhibition with alirocumab attenuated alcohol-induced hepatic triglyceride accumulation through regulation of lipid metabolism (mRNA expression of modulators of fatty acid synthesis (FAS) and catabolism (PPAR and CPT1)), hepatocellular injury (ALT), hepatic inflammation (mRNA expression of pro-inflammatory cytokines/chemokines (TNFa, IL-1, IL-22, IL-33, IL-17, IL-2, MIP-2, and MCP-1), and neutrophil infiltration (myeloperoxidase staining)). Alirocumab treatment also attenuated alcohol-induced PCSK9 mRNA elevation and upregulated LDL-receptor (LDL-R) via modulation of the transcription factors (SREBP-1, SREBP-2, and E2F1) in liver. We demonstrated that chronic anti-PCSK9 treatment using the monoclonal antibody alirocumab attenuated alcohol-induced steatohepatitis in the rat model. Given the large unmet clinical need for effective and novel treatments for alcohol-associated liver disease (ALD), anti-PCSK9 treatment with the monoclonal antibody that spares liver metabolism is a viable new therapeutic possibility. Future studies are needed to elucidate the exact role of PCSK9 in ALD and AUD and to evaluate efficacy and safety of anti-PCSK9 treatment in clinical populations with ALD/AUD (Lee et al., 2019). Studies have shown that individuals with AUD die earlier than healthy controls and are at a significantly increased risk for all-cause mortality (Westman et al., 2015, Larame et al., 2015). Given the potential role of AUD in the aging process, it is important to understand this relationship on a molecular, epigenetic level. To better assess biological age, several epigenetic clocks that robustly correlate with chronological age have been developed. These clocks are algorithms that take the weighted average of methylation levels at specific CpG sites to calculate DNA methylation age (DNAm age), which is highly correlated with chronological age. We have previously shown that AUD is associated with epigenetic age acceleration (Rosen et al., 2018). To further investigate the potential role of AUD in aging processes, we employed Levine's epigenetic clock (DNAm PhenoAge) to estimate DNA methylation age in a large cohort of individuals with AUD and healthy controls (HC). We evaluated the effects of heavy, chronic alcohol consumption on epigenetic age acceleration (EAA) using clinical biomarkers, including liver function test enzymes (LFTs) and clinical measures. To characterize potential underlying genetic variation contributing to EAA in AUD, we performed genome-wide association studies (GWAS) on EAA, including pathway analyses. We followed up on relevant top findings with in silico expression quantitative trait loci (eQTL) analyses for biological function using the BRAINEAC database. There was a 2.22-year age acceleration in AUD compared to controls after adjusting for gender and blood cell composition (p = 1.85 10-5). This association remained significant after adjusting for race, body mass index, and smoking status (1.38 years, p = 0.02). Secondary analyses showed more pronounced EAA in individuals with more severe AUD-associated phenotypes, including elevated gamma-glutamyl transferase (GGT) and alanine aminotransferase (ALT), and higher number of heavy drinking days (all ps < 0.05). The genome-wide meta-analysis of EAA in AUD revealed a significant single nucleotide polymorphism (SNP), rs916264 (p = 5.43 10-8), in apolipoprotein L2 (APOL2) at the genome-wide level. The minor allele A of rs916264 was associated with EAA and with increased mRNA expression in hippocampus (p = 0.0015). Our data demonstrate EAA in AUD and suggest that disease severity further accelerates epigenetic aging. EAA was associated with genetic variation in APOL2, suggesting potential novel biological mechanisms for age acceleration in AUD (Luo et al., 2020). II. Negative emotionality in AUD Another interest of CGET is to investigate the molecular mechanisms of negative affective states and how they contribute to the addiction cycle in human. Negative emotional states contribute to worsening of the addiction cycle and increase risk for relapse (Ahmed and Koob, 1998, Koob, 2015, George et al., 2014). Better understanding of the underlying neurocircuitries and molecular mechanisms involved in negative emotion processing associated with alcohol addiction, including genetic and epigenetic contributors, is crucial for the development of novel interventions and effective prevention strategies. To investigate this, our sections human clinical protocol 15-AA-0127: (Epi)Genetic modulators of fear extinction in alcohol dependence was developed and is actively recruiting. This protocol aims to investigate underlying neurobiology and neurocircuitries of fear extinction in individuals with AUD with and without early life stress (ELS). First data was obtained and analyzed, including a 2-day fear learning paradigm in 43 healthy participants and 43 individuals with AUD. Main outcomes of this multimodal study included structural and functional brain magnetic resonance imaging, clinical measures, as well as skin conductance responses to confirm differential conditioning. The AUD group showed significantly reduced BOLD responses in the right amygdala during conditioning and in the left amygdala during fear renewal. Right amygdala activation during conditioning was significantly correlated with AUD severity, depressive symptoms, trait anxiety, and perceived stress. Our data suggest that individuals with AUD have dysregulated fear learning, in particular, dysregulated neural activation patterns, in the amygdala. Furthermore, amygdala activation during fear conditioning was associated with AUD-related clinical measures. This paradigm may be a promising tool to investigate structures involved in negative affect regulation, which might inform the development of novel treatment approaches for AUD (Muench et al., 2019).