ACE2 and TMPRSS2 SARS-CoV-2 infectivity genes: deep mutational scanning and characterization of missense variants.

The human angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) proteins play key roles in the cellular internalization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus responsible for the coronavirus disease of 2019 (COVID-19) pandemic. We set out to functionally characterize the ACE2 and TMPRSS2 protein abundance for variant alleles encoding these proteins that contained non-synonymous single-nucleotide polymorphisms (nsSNPs) in their open reading frames (ORFs). Specifically, a high-throughput assay, deep mutational scanning (DMS), was employed to test the functional implications of nsSNPs, which are variants of uncertain significance in these two genes. Specifically, we used a 'landing pad' system designed to quantify the protein expression for 433 nsSNPs that have been observed in the ACE2 and TMPRSS2 ORFs and found that 8 of 127 ACE2, 19 of 157 TMPRSS2 isoform 1 and 13 of 149 TMPRSS2 isoform 2 variant proteins displayed less than ~25% of the wild-type protein expression, whereas 4 ACE2 variants displayed 25% or greater increases in protein expression. As a result, we concluded that nsSNPs in genes encoding ACE2 and TMPRSS2 might potentially influence SARS-CoV-2 infectivity. These results can now be applied to DNA sequence data for patients infected with SARS-CoV-2 to determine the possible impact of patient-based DNA sequence variation on the clinical course of SARS-CoV-2 infection.

IL17RB genetic variants are associated with acamprosate treatment response in patients with alcohol use disorder: A proteomics-informed genomics study.

Acamprosate is a Food and Drug Administration (FDA) approved medication for the treatment of alcohol use disorder (AUD). However, only a subset of patients achieves optimal treatment outcomes. Currently, no biological measures are utilized to predict response to acamprosate treatment. We applied our established pharmaco-omics informed genomics strategy to identify potential biomarkers associated with acamprosate treatment response. Specifically, our previous open-label acamprosate clinical trial recruited 442 patients with AUD who were treated with acamprosate for three months. We first performed proteomics using baseline plasma samples to identify potential biomarkers associated with acamprosate treatment outcomes. Next, we applied our established "proteomics-informed genome-wide association study (GWAS)" research strategy, and identified 12 proteins, including interleukin-17 receptor B (IL17RB), associated with acamprosate treatment response.​ A GWAS for IL17RB concentrations identified several genome-wide significant signals. Specifically, the top hit single nucleotide polymorphism (SNP) rs6801605 with a minor allele frequency of 38% in the European American population mapped 4 kilobase (Kb) upstream of IL17RB, and intron 1 of the choline dehydrogenase (CHDH) gene on chromosome 3 (p: 4.8E-20). The variant genotype (AA) for the SNP rs6801605 was associated with lower IL17RB protein expression. In addition, we identified a series of genetic variants in IL17RB that were associated with acamprosate treatment outcomes. Furthermore, the variantgenotypes for all of those IL17RB SNPs were protective for alcohol relapse. Finally, we demonstrated that the basal level of mRNA expression of IL17RB was inversely correlated with those of nuclear factor-κB (NF-κB) subunits, and a significantly higher expression of NF-κB subunits was observed in AUD patients who relapsed to alcohol use. In summary, this study illustrates that IL17RB genetic variants might contribute to acamprosate treatment outcomes. This series of studies represents an important step toward generating functional hypotheses that could be tested to gain insight into mechanisms underlying acamprosate treatment response phenotypes. (The ClinicalTrials.gov Identifier: NCT00662571).

Single cell transcriptomics reveals distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with opioid use disorder.

The opioid epidemic represents a national crisis. Oxycodone is one of the most prescribed opioid medications in the United States, whereas buprenorphine is currently the most prescribed medication for opioid use disorder (OUD) pharmacotherapy. Given the extensive use of prescription opioids and the global opioid epidemic, it is essential to understand how opioids modulate brain cell type function at the single-cell level. We performed single nucleus RNA-seq (snRNA-seq) using iPSC-derived forebrain organoids from three male OUD subjects in response to oxycodone, buprenorphine, or vehicle for seven days. We utilized the snRNA-seq data to identify differentially expressed genes following drug treatment using the Seurat integrative analysis pipeline. We utilized iPSC-derived forebrain organoids and single-cell sequencing technology as an unbiased tool to study cell-type-specific and drug-specific transcriptional responses. After quality control filtering, we analyzed 25787 cells and identified sixteen clusters using unsupervised clustering analysis. Our results reveal distinct transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with OUD. Specifically, buprenorphine displayed a significant influence on transcription regulation in glial cells. However, oxycodone induced type I interferon signaling in many cell types, including neural cells in brain organoids. Finally, we demonstrate that oxycodone, but not buprenorphine activated STAT1 and induced the type I interferon signaling in patients with OUD. These data suggest that elevation of STAT1 expression associated with OUD might play a role in transcriptional regulation in response to oxycodone. In summary, our results provide novel mechanistic insight into drug action at single-cell resolution.

TSPAN5 influences serotonin and kynurenine: pharmacogenomic mechanisms related to alcohol use disorder and acamprosate treatment response.

We previously reported that SNPs near TSPAN5 were associated with plasma serotonin (5-HT) concentrations which were themselves associated with selective serotonin reuptake inhibitor treatment outcomes in patients with major depressive disorder (MDD). TSPAN5 SNPs were also associated with alcohol consumption and alcohol use disorder (AUD) risk. The present study was designed to explore the biological function of TSPAN5 with a focus on 5-HT and kynurenine concentrations in the tryptophan pathway. Ethanol treatment resulted in decreased 5-HT concentrations in human induced pluripotent stem cell (iPSC)-derived neuron culture media, and the downregulation of gene expression of TSPAN5, DDC, MAOA, MAOB, TPH1, and TPH2 in those cells. Strikingly, similar observations were made when the cells were treated with acamprosate-an FDA approved drug for AUD therapy. These results were replicated in iPSC-derived astrocytes. Furthermore, TSPAN5 interacted physically with proteins related to clathrin and other vesicle-related proteins, raising the possibility that TSPAN5 might play a role in vesicular function in addition to regulating expression of genes associated with 5-HT biosynthesis and metabolism. Downregulation of TSPAN5 expression by ethanol or acamprosate treatment was also associated with decreased concentrations of kynurenine, a major metabolite of tryptophan that plays a role in neuroinflammation. Knockdown of TSPAN5 also influenced the expression of genes associated with interferon signaling pathways. Finally, we determined that TSPAN5 SNPs were associated with acamprosate treatment outcomes in AUD patients. In conclusion, TSPAN5 can modulate the concentrations of 5-HT and kynurenine. Our data also highlight a potentially novel pharmacogenomic mechanism related to response to acamprosate.

SLCO1B1: Application and Limitations of Deep Mutational Scanning for Genomic Missense Variant Function.

SLCO1B1 (solute carrier organic anion transporter family member 1B1) is an important transmembrane hepatic uptake transporter. Genetic variants in the SLCO1B1 gene have been associated with altered protein folding, resulting in protein degradation and decreased transporter activity. Next-generation sequencing (NGS) of pharmacogenes is being applied increasingly to associate variation in drug response with genetic sequence variants. However, it is difficult to link variants of unknown significance with functional phenotypes using "one-at-a-time" functional systems. Deep mutational scanning (DMS) using a "landing pad cell-based system" is a high-throughput technique designed to analyze hundreds of gene open reading frame (ORF) missense variants in a parallel and scalable fashion. We have applied DMS to analyze 137 missense variants in the SLCO1B1 ORF obtained from the Exome Aggregation Consortium project. ORFs containing these variants were fused to green fluorescent protein and were integrated into "landing pad" cells. Florescence-activated cell sorting was performed to separate the cells into four groups based on fluorescence readout indicating protein expression at the single cell level. NGS was then performed and SLCO1B1 variant frequencies were used to determine protein abundance. We found that six variants not previously characterized functionally displayed less than 25% and another 12 displayed approximately 50% of wild-type protein expression. These results were then functionally validated by transporter studies. Severely damaging variants identified by DMS may have clinical relevance for SLCO1B1-dependent drug transport, but we need to exercise caution since the relatively small number of severely damaging variants identified raise questions with regard to the application of DMS to intrinsic membrane proteins such as organic anion transporter protein 1B1. SIGNIFICANCE STATEMENT: The functional implications of a large numbers of open reading frame (ORF) "variants of unknown significance" (VUS) in transporter genes have not been characterized. This study applied deep mutational scanning to determine the functional effects of VUS that have been observed in the ORF of SLCO1B1(s olute carrier organic anion transporter family member 1B1). Several severely damaging variants were identified, studied, and validated. These observations have implications for both the application of deep mutational scanning to intrinsic membrane proteins and for the clinical effect of drugs and endogenous compounds transported by SLCO1B1.

Pharmacogenomic Next-Generation DNA Sequencing: Lessons from the Identification and Functional Characterization of Variants of Unknown Significance in CYP2C9 and CYP2C19.

CYP2C9 and CYP2C19 are highly polymorphic pharmacogenes; however, clinically actionable genetic variability in drug metabolism due to these genes has been limited to a few common alleles. The identification and functional characterization of less-common open reading frame sequence variation might help to individualize therapy with drugs that are substrates for the enzymes encoded by these genes. The present study identified seven uncharacterized variants each in CYP2C9 and CYP2C19 using next-generation sequence data for 1013 subjects, and functionally characterized the encoded proteins. Constructs were created and transiently expressed in COS-1 cells for the assay of protein concentration and enzyme activities using fluorometric substrates and liquid chromatography- tandem mass spectrometry with tolbutamide (CYP2C9) and (S)-mephenytoin (CYP2C19) as prototypic substrates. The results were compared with the SIFT, Polyphen, and Provean functional prediction software programs. Cytochrome P450 oxidoreductase (CPR) activities were also determined. Positive correlations were observed between protein content and fluorometric enzyme activity for variants of CYP2C9 (P < 0.05) and CYP2C19 (P < 0.0005). However, CYP2C9 709G>C and CYP2C19 65A>G activities were much lower than predicted based on protein content. Substrate intrinsic clearance values for CYP2C9 218C>T, 343A>C, and CYP2C19 337G>A, 518C>T, 556C>T, and 557G>A were less than 25% of wild-type allozymes. CPR activity levels were similar for all variants. In summary, sequencing of CYP2C9 and CYP2C19 in 1013 subjects identified low-frequency variants that had not previously been functionally characterized. In silico predictions were not always consistent with functional assay results. These observations emphasize the need for high-throughput methods for pharmacogene variant mutagenesis and functional characterization.

Molecular mechanisms involved in alcohol craving, IRF3, and endoplasmic reticulum stress: a multi-omics study.

Alcohol use disorder (AUD) is the most prevalent substance use disorder worldwide. Acamprosate and naltrexone are anti-craving drugs used in AUD pharmacotherapy. However, molecular mechanisms underlying their anti-craving effect remain unclear. This study utilized a patient-derived induced pluripotent stem cell (iPSC)-based model system and anti-craving drugs that are used to treat AUD as "molecular probes" to identify possible mechanisms associated with alcohol craving. We examined the pathophysiology of craving and anti-craving drugs by performing functional genomics studies using iPSC-derived astrocytes and next-generation sequencing. Specifically, RNA sequencing performed using peripheral blood mononuclear cells from AUD patients with extreme values for alcohol craving intensity prior to treatment showed that inflammation-related pathways were highly associated with alcohol cravings. We then performed a genome-wide assessment of chromatin accessibility and gene expression profiles of induced iPSC-derived astrocytes in response to ethanol or anti-craving drugs. Those experiments identified drug-dependent epigenomic signatures, with IRF3 as the most significantly enriched motif in chromatin accessible regions. Furthermore, the activation of IRF3 was associated with ethanol-induced endoplasmic reticulum (ER) stress which could be attenuated by anti-craving drugs, suggesting that ER stress attenuation might be a target for anti-craving agents. In conclusion, we found that craving intensity was associated with alcohol consumption and treatment outcomes. Our functional genomic studies suggest possible relationships among craving, ER stress, IRF3 and the actions of anti-craving drugs.

FKBP5 genetic variation: association with selective serotonin reuptake inhibitor treatment outcomes in major depressive disorder.

OBJECTIVES: FKBP51 (51 kDa immunophilin) acts as a modulator of the glucocorticoid receptor and a negative regulator of the Akt pathway. Genetic variation in FKBP5 plays a role in antidepressant response. The aim of this study was to comprehensively assess the role of genetic variation in FKBP5, identified by both Sanger and Next Generation DNA resequencing, as well as genome-wide single nucleotide polymorphisms (SNPs) associated with FKBP5 expression in the response to the selective serotonin reuptake inhibitor (SSRI) treatment of major depressive disorder. METHODS: We identified 657 SNPs in FKBP5 by Next Generation sequencing of 96 DNA samples from white patients, and 149 SNPs were selected for the genotyping together with 235 SNPs that were trans-associated with variation in FKBP5 expression in lymphoblastoid cells. A total of 529 DNA samples from the Mayo Clinic PGRN-SSRI Pharmacogenomic trial for which genome-wide SNPs had already been obtained were genotyped for these 384 SNPs, and associations with treatment outcomes were determined. The most significant SNPs were genotyped using 96 DNA samples from white non-Hispanic patients of the NIMH-supported Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study to attempt replication, followed by functional genomic studies. RESULTS: Genotype-phenotype association analysis indicated that rs352428 was associated with both 8-week treatment response in the Mayo study (odds ratio=0.49; P=0.003) and 6-week response in the STAR*D replication study (odds ratio=0.74; P=0.05). The electrophoresis mobility shift assay and the reporter gene assay confirmed the possible role of this SNP in transcription regulation. CONCLUSION: This comprehensive FKBP5 sequence study provides insight into the role of common genetic polymorphisms that might influence SSRI treatment outcomes in major depressive disorder patients.

Epigenetic regulation of GABA catabolism in iPSC-derived neurons: The molecular links between FGF21 and histone methylation.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) analogs have been tested as potential therapeutics for substance use disorders. Prior research suggests that FGF21 administration might affect alcohol consumption and reward behaviors. Our recent report showed that plasma FGF21 levels were positively correlated with alcohol use in patients with alcohol use disorder (AUD). FGF21 has a short half-life (0.5-2 h) and crosses the blood-brain barrier. Therefore, we set out to identify molecular mechanisms for both the naïve form of FGF21 and a long-acting FGF21 molecule (PF-05231023) in induced pluripotent stem cell (iPSC)-derived forebrain neurons. METHODS: We performed RNA-seq in iPSC-derived forebrain neurons treated with naïve FGF21 or PF-05231023 at physiologically relevant concentrations. We obtained plasma levels of FGF21 and GABA from our previous AUD clinical trial (n = 442). We performed ELISA for FGF21 in both iPSC-derived forebrain neurons and forebrain organoids. We determined protein interactions using co-immunoprecipitation. Finally, we applied ChIP assays to confirm the occupancy of REST, EZH2 and H3K27me3 by FGF21 using iPSC-derived forebrain neurons with and without drug exposure. RESULTS: We identified 4701 and 1956 differentially expressed genes in response to naïve FGF21 or PF-05231023, respectively (FDR < 0.05). Notably, 974 differentially expressed genes overlapped between treatment with naïve FGF21 and PF-05231023. REST was the most important upstream regulator of differentially expressed genes. The GABAergic synapse pathway was the most significant pathway identified using the overlapping genes. We also observed a significant positive correlation between plasma FGF21 and GABA concentrations in AUD patients. In parallel, FGF21 and PF-05231023 significantly induced GABA levels in iPSC-derived neurons. Finally, functional genomics studies showed a drug-dependent occupancy of REST, EZH2, and H3K27me3 in the promoter regions of genes involved in GABA catabolism which resulted in transcriptional repression. CONCLUSIONS: Our results highlight a significant role in the epigenetic regulation of genes involved in GABA catabolism related to FGF21 action. (The ClinicalTrials.gov Identifier: NCT00662571).

Gemcitabine metabolic pathway genetic polymorphisms and response in patients with non-small cell lung cancer.

BACKGROUND AND OBJECTIVE: Gemcitabine is widely used to treat non-small cell lung cancer (NSCLC). The aim of this study was to assess the pharmacogenomic effects of the entire gemcitabine metabolic pathway, we genotyped single nucleotide polymorphisms (SNPs) within the 17 pathway genes using DNA samples from patients with NSCLC treated with gemcitabine to determine the effect of genetic variants within gemcitabine pathway genes on overall survival (OS) of patients with NSCLC after treatment of gemcitabine. METHODS: Eight of the 17 pathway genes were resequenced with DNA samples from Coriell lymphoblastoid cell lines (LCLs) using Sanger sequencing for all exons, exon-intron junctions, and 5'-, 3'-UTRs. A total of 107 tagging SNPs were selected on the basis of the resequencing data for the eight genes and on HapMap data for the remaining nine genes, followed by successful genotyping of 394 NSCLC patient DNA samples. Association of SNPs/haplotypes with OS was performed using the Cox regression model, followed by functional studies performed with LCLs and NSCLC cell lines. RESULTS: Five SNPs in four genes (CDA, NT5C2, RRM1, and SLC29A1) showed associations with OS of those patients with NSCLC, as well as nine haplotypes in four genes (RRM1, RRM2, SLC28A3, and SLC29A1) with a P value of less than 0.05. Genotype imputation using the LCLs was performed for a region of 200 kb surrounding those SNPs, followed by association studies with gemcitabine cytotoxicity. Functional studies demonstrated that downregulation of SLC29A1, NT5C2, and RRM1 in NSCLC cell lines altered cell susceptibility to gemcitabine. CONCLUSION: These studies help in identifying biomarkers to predict gemcitabine response in NSCLC, a step toward the individualized chemotherapy of lung cancer.

Human liver methionine cycle: MAT1A and GNMT gene resequencing, functional genomics, and hepatic genotype-phenotype correlation.

The "methionine cycle" plays a critical role in the regulation of concentrations of (S)-adenosylmethionine (AdoMet), the major biological methyl donor. We set out to study sequence variation in genes encoding the enzyme that synthesizes AdoMet in liver, methionine adenosyltransferase 1A (MAT1A) and the major hepatic AdoMet using enzyme, glycine N-methyltransferase (GNMT), as well as functional implications of that variation. We resequenced MAT1A and GNMT using DNA from 288 subjects of three ethnicities, followed by functional genomic and genotype-phenotype correlation studies performed with 268 hepatic biopsy samples. We identified 44 and 42 polymorphisms in MAT1A and GNMT, respectively. Quantitative Western blot analyses for the human liver samples showed large individual variation in MAT1A and GNMT protein expression. Genotype-phenotype correlation identified two genotyped single-nucleotide polymorphisms (SNPs), reference SNP (rs) 9471976 (corrected p = 3.9 × 10(-10)) and rs11752813 (corrected p = 1.8 × 10(-5)), and 42 imputed SNPs surrounding GNMT that were significantly associated with hepatic GNMT protein levels (corrected p values < 0.01). Reporter gene studies showed that variant alleles for both genotyped SNPs resulted in decreased transcriptional activity. Correlation analyses among hepatic protein levels for methionine cycle enzymes showed significant correlations between GNMT and MAT1A (p = 1.5 × 10(-3)) and between GNMT and betaine homocysteine methyltransferase (p = 1.6 × 10(-7)). Our discovery of SNPs that are highly associated with hepatic GNMT protein expression as well as the "coordinate regulation" of methionine cycle enzyme protein levels provide novel insight into the regulation of this important human liver biochemical pathway.

Genome-wide association study for circulating FGF21 in patients with alcohol use disorder: Molecular links between the SNHG16 locus and catecholamine metabolism.

OBJECTIVE: Alcohol consumption can increase circulating levels of fibroblast growth factor 21 (FGF21). The effects of FGF21 in the central nervous system are associated with the regulation of catecholamines, neurotransmitters that play a crucial role in reward pathways. This study aims to identify genetic variants associated with FGF21 levels and evaluate their functional role in alcohol use disorder (AUD). METHODS: We performed a genome-wide association study (GWAS) using DNA samples from 442 AUD subjects recruited from the Mayo Clinic Center for the Individualized Treatment of Alcoholism Study. Plasma FGF21 levels were measured using Olink proximity extension immunoassays. Alcohol consumption at time of entry into the study was measured using the self-reported timeline followback method. Functional genomic studies were performed using HepG2 cells and induced pluripotent stem cell (iPSC)-derived brain organoids. RESULTS: Plasma FGF21 levels were positively correlated with recent alcohol consumption and gamma-glutamyl transferase levels, a commonly used marker for heavy alcohol use. One variant, rs9914222, located 5' of SNHG16 on chromosome 17 was associated with plasma FGF21 levels (p = 4.60E-09). This variant was also associated with AUD risk (ß: -3.23; p:0.0004). The rs9914222 SNP is an eQTL for SNHG16 in several brain regions, i.e., the variant genotype was associated with decreased expression of SNHG16. The variant genotype for the rs9914222 SNP was also associated with higher plasma FGF21 levels. Knockdown of SNHG16 in HepG2 cells resulted in increased FGF21 concentrations and decreased expression and enzyme activity for COMT, an enzyme that plays a key role in catecholamine metabolism. Finally, we demonstrated that ethanol significantly induced FGF21, dopamine, norepinephrine, and epinephrine concentrations in iPSC-derived brain organoids. CONCLUSIONS: GWAS for FGF21 revealed a SNHG16 genetic variant associated with FGF21 levels which are associated with recent alcohol consumption. Our data suggest that SNHG16 can regulate FGF21 concentrations and decrease COMT expression and enzyme activity which, in turn, have implications for the regulation of catecholamines. (The ClinicalTrials.gov Identifier: NCT00662571).

Genetic variants associated with acamprosate treatment response in alcohol use disorder patients: A multiple omics study.

BACKGROUND AND PURPOSE: Acamprosate is an anti-craving drug used for the pharmacotherapy of alcohol use disorder (AUD). However, only some patients achieve optimal therapeutic outcomes. This study was designed to explore differences in metabolomic profiles between patients who maintained sobriety and those who relapsed, to determine whether those differences provide insight into variation in acamprosate treatment response phenotypes. EXPERIMENTAL APPROACH: We previously conducted an acamprosate trial involving 442 AUD patients, and 267 of these subjects presented themselves for a 3-month follow-up. The primary outcome was abstinence. Clinical information, genomic data and metabolomics data were collected. Baseline plasma samples were assayed using targeted metabolomics. KEY RESULTS: Baseline plasma arginine, threonine, α-aminoadipic acid and ethanolamine concentrations were associated with acamprosate treatment outcomes and baseline craving intensity, a measure that has been associated with acamprosate treatment response. We next applied a pharmacometabolomics-informed genome-wide association study (GWAS) strategy to identify genetic variants that might contribute to variations in plasma metabolomic profiles that were associated with craving and/or acamprosate treatment outcome. Gene expression data for induced pluripotent stem cell-derived forebrain astrocytes showed that a series of genes identified during the metabolomics-informed GWAS were ethanol responsive. Furthermore, a large number of those genes could be regulated by acamprosate. Finally, we identified a series of single nucleotide polymorphisms that were associated with acamprosate treatment outcomes. CONCLUSION AND IMPLICATIONS: These results serve as an important step towards advancing our understanding of disease pathophysiology and drug action responsible for variation in acamprosate response and alcohol craving in AUD patients.

Plasma TNFSF10 levels associated with acamprosate treatment response in patients with alcohol use disorder.

Acamprosate is an anti-craving drug used in alcohol use disorder (AUD) pharmacotherapy. However, only a subset of patients achieves optimal treatment outcomes. The identification of predictive biomarkers of acamprosate treatment response in patients with AUD would be a substantial advance in addiction medicine. We designed this study to use proteomics data as a quantitative biological trait as a step toward identifying inflammatory modulators that might be associated with acamprosate treatment outcomes. The NIAAA-funded Mayo Clinic Center for the Individualized Treatment of Alcoholism study had previously recruited 442 AUD patients who received 3 months of acamprosate treatment. However, only 267 subjects returned for the 3-month follow-up visit and, as a result, had treatment outcome information available. Baseline alcohol craving intensity was the most significant predictor of acamprosate treatment outcomes. We performed plasma proteomics using the Olink target 96 inflammation panel and identified that baseline plasma TNF superfamily member 10 (TNFSF10) concentration was associated with alcohol craving intensity and variation in acamprosate treatment outcomes among AUD patients. We also performed RNA sequencing using baseline peripheral blood mononuclear cells from AUD patients with known acamprosate treatment outcomes which revealed that inflammation-related pathways were highly associated with relapse to alcohol use during the 3 months of acamprosate treatment. These observations represent an important step toward advancing our understanding of the pathophysiology of AUD and molecular mechanisms associated with acamprosate treatment response. In conclusion, applying omics-based approaches may be a practical approach for identifying biologic markers that could potentially predict alcohol craving intensity and acamprosate treatment response.

A novel application of pattern recognition for accurate SNP and indel discovery from high-throughput data: targeted resequencing of the glucocorticoid receptor co-chaperone FKBP5 in a Caucasian population.

The detection of single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) with precision from high-throughput data remains a significant bioinformatics challenge. Accurate detection is necessary before next-generation sequencing can routinely be used in the clinic. In research, scientific advances are inhibited by gaps in data, exemplified by the underrepresented discovery of rare variants, variants in non-coding regions and indels. The continued presence of false positives and false negatives prevents full automation and requires additional manual verification steps. Our methodology presents applications of both pattern recognition and sensitivity analysis to eliminate false positives and aid in the detection of SNP/indel loci and genotypes from high-throughput data. We chose FK506-binding protein 51(FKBP5) (6p21.31) for our clinical target because of its role in modulating pharmacological responses to physiological and synthetic glucocorticoids and because of the complexity of the genomic region. We detected genetic variation across a 160 kb region encompassing FKBP5. 613 SNPs and 57 indels, including a 3.3 kb deletion were discovered. We validated our method using three independent data sets and, with Sanger sequencing and Affymetrix and Illumina microarrays, achieved 99% concordance. Furthermore we were able to detect 267 novel rare variants and assess linkage disequilibrium. Our results showed both a sensitivity and specificity of 98%, indicating near perfect classification between true and false variants. The process is scalable and amenable to automation, with the downstream filters taking only 1.5h to analyze 96 individuals simultaneously. We provide examples of how our level of precision uncovered the interactions of multiple loci, their predicted influences on mRNA stability, perturbations of the hsp90 binding site, and individual variation in FKBP5 expression. Finally we show how our discovery of rare variants may change current conceptions of evolution at this locus.

Methionine adenosyltransferase 2A/2B and methylation: gene sequence variation and functional genomics.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major biological methyl donor. MAT1A and MAT2A encode two distinct MAT isoforms in mammals. MAT2A is expressed in nonhepatic tissues, whereas MAT1A is expressed in the liver. A third gene, MAT2B, encodes a MAT2A regulatory protein. We resequenced MAT2A and MAT2B exons, splice junctions, and flanking regions using 288 DNA samples from three ethnic groups and also imputed additional single nucleotide polymorphisms (SNPs) across both genes using data from the 1000 Genomes Project. For MAT2A, resequencing identified 74 polymorphisms, including two nonsynonymous (ns) SNPs. Functional genomic studies of wild type and the two MAT2A variant allozymes (Val11 and Val205) showed that the Val11 allozyme had approximately 40% decreases in levels of enzyme activity and immunoreactive protein after COS-1 cell transfection. For MAT2B, 44 polymorphisms, 2 nonsynonymous, were identified during resequencing. Neither of the two MAT2B nsSNPs displayed alterations in levels of protein. Imputation using 1000 Genomes Project data resulted in 1730 additional MAT2A and 1997 MAT2B polymorphisms within ± 200 kilobases of each gene, respectively. Coexpression of MAT2A and MAT2B in COS-1 cells resulted in significantly increased MAT enzyme activity that correlated with increased MAT2A and MAT2B immunoreactive protein, apparently as a result of decreased degradation. Finally, studies of mRNA expression in lymphoblastoid cells showed that 7 SNPs in MAT2A and 16 SNPs in MAT2B were significantly associated with mRNA expression with p < 0.01. These observations provide a foundation for future mechanistic and clinical translational pharmacogenomic studies of MAT2A/2B.

All-trans- and 9-cis-retinoic acids activate the human cyclooxynase-2 gene: a role for DR1 as RARE or RXRE.

The human COX-2 promoter contains a direct repeat 1 (DR1) which was shown to confer responsiveness to PPARs. We found that in AN(3)CA and F9 cells, this hCOX-2 DR1 mediates responsiveness to all-trans-retinoic acid (tRA) or 9-cis-retinoic acid (9cRA), but this effect was suppressed by PPARδ. Truncated PPARδ lacking the activation domain AF2 cannot suppress RA-induced activation of the hCOX-2 gene via DR1, suggesting that cofactor recruitment by AF2 is required for the suppression by PPARδ. Gel shift assay showed that PPAR/RXR, RARß/RXR, and RXR/RXR, bind to hCOX-2 DR1, revealing the promiscuity of this DR1. Particularly, RXR homodimer was able to bind to this DR1 only in the presence of 9cRA. Our results established that tRA and 9cRA are potent inducers of hCOX-2 and that the hCOX-2 DR1 could either serve as RARE or RXRE depending on cellular contexts.

Natriuretic peptide pharmacogenetics: membrane metallo-endopeptidase (MME): common gene sequence variation, functional characterization and degradation.

Membrane metallo-endopeptidase (MME), also known as neutral endopeptidase 24.11 (EC 3.4.24.11), is involved in the metabolism of natriuretic peptides that play a key role in modulating cardiac structure and function. Common genetic variation in MME has not been addressed by resequencing the gene using DNA from different ethnic populations. We set out to identify and functionally characterize common genetic variation in MME in three ethnic groups. DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence MME. Ninety polymorphisms, 65 novel, were identified, including 8 nonsynonymous single nucleotide polymorphisms (nsSNPs). Expression constructs for the nsSNPs were created and COS-1 cells were transfected with constructs for wild type (WT) and variant allozymes. Recombinant proteins were analyzed by quantitative Western blot analysis and by a one-step fluorometric assay. A significant reduction in enzyme activity (21% of WT) and immunoreactive protein (29% of WT) for the Val73 variant allozyme was observed. Proteasome-mediated degradation and autophagy participated in the degradation of this variant allozyme. The chaperone proteins, BiP and GRP94, were upregulated after transfection with Val73 MME, suggesting protein misfolding, compatible with conclusions based on the MME X-ray crystal structure. Multiple novel polymorphisms of MME were identified in three ethnic groups. The Val73 variant allozyme displayed a significant decrease in MME protein quantity and activity, with degradation mediated by both proteasome and autophagy pathways. This polymorphism could have a significant effect on the metabolism of natriuretic peptides.

Ecto-5'-nucleotidase and thiopurine cellular circulation: association with cytotoxicity.

Thiopurine drugs such as 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used to treat acute lymphoblastic leukemia of childhood. To test the hypothesis that variation in the expression of genes within the "thiopurine pathway" might influence 6-MP and 6-TG sensitivity, we generated basal gene expression profiles and IC(50) values for both of these thiopurine drugs using a model system consisting of 194 Human Variation Panel lymphoblastoid cell lines. Association analysis showed that thiopurine S-methyltransferase, ecto-5'-nucleotidase (NT5E), and multidrug resistance protein 4 (ABCC4) expression were correlated with thiopurine cytotoxicity. Those observations suggested the possible existence of a "thiopurine cellular circulation" involving nucleotide efflux by ABCC4, hydrolysis of thiopurine nucleotide monophosphates outside of the cell by NT5E, and subsequent transport of thiopurine nucleosides back into the cell by nucleoside transporters. The existence of this cellular circulation was confirmed by a series of functional experiments performed with cultured cells stably or transiently transfected with ABCC4 and/or NT5E. Because of the central role of NT5E in this cellular circulation, the NT5E gene was resequenced using 287 DNA samples from three ethnic groups, with the identification of 68 single nucleotide polymorphisms (SNPs), 46 of which were novel. Several SNPs in the 5'-flanking region of NT5E were highly correlated with expression, rs9450278 having the lowest p value (p = 2.4 × 10(-10), R = -0.376). The thiopurine cellular circulation and genetic polymorphisms for genes encoding the proteins involved should be incorporated into future studies of thiopurine drug therapy and effect.

CYP2C9 and CYP2C19: Deep Mutational Scanning and Functional Characterization of Genomic Missense Variants.

Single nucleotide variants in the open reading frames (ORFs) of pharmacogenes are important causes of interindividual variability in drug response. The functional characterization of variants of unknown significance within ORFs remains a major challenge for pharmacogenomics. Deep mutational scanning (DMS) is a high-throughput technique that makes it possible to analyze the functional effect of hundreds of variants in a parallel and scalable fashion. We adapted a "landing pad" DMS system to study the function of missense variants in the ORFs of cytochrome P450 family 2 subfamily C member 9 (CYP2C9) and cytochrome P450 family 2 subfamily C member 19 (CYP2C19). We studied 230 observed missense variants in the CYP2C9 and CYP2C19 ORFs and found that 19 of 109 CYP2C9 and 36 of 121 CYP2C19 variants displayed less than ~ 25% of the wild-type protein expression, a level that may have clinical relevance. Our results support DMS as an efficient method for the identification of damaging ORF variants that might have potential clinical pharmacogenomic application.