Human Single Nucleotide Polymorphisms Research Articles

Functional Captures of Multiple Human Quantitative Trait Loci Regulating Blood Pressure with the Use of Orthologs in Genetically Defined Rat Models

BackgroundMost signals from human genome-wide association studies (GWAS) for blood pressure (BP) are single-nucleotide polymorphisms (SNPs). It was unknown if such SNPs can functionally affect BP. Because BP is similar between humans and rodents, unraveling basic mechanisms from rodents can reveal the same BP-modulating mechanisms in humans originating from their common ancestors while overcoming limitations in human epidemiology. MethodsFor the first time, we used quantitative trait loci (QTLs) from Dahl salt-sensitive (DSS) rats as functional surrogates to capture human BP QTLs. ResultsA total of 107 human GWAS genes may be classified into 2 common pathways of hypertension pathogeneses. Among them, 4 DSS BP QTLs correspond to 4 human GWAS genes. Each of them independently showed a major impact on BP in vivo and thus functional redundancy. BP was altered by each of these 4 QTLs, but human GWAS SNPs marking these QTLs do not exist in the rat. They cannot be responsible for physiological changes in BP caused by these QTLs and are genome signposts marking positions of the QTLs nearby, rather than being QTLs themselves. These SNPs appeared during primate evolution, independently of BP regulation. Because the functional dosage of QTLs, not their gene dose, determined hypertension pathogenesis, a role for the noncoding GWAS SNPs in BP via regulating gene expressions can be discounted. ConclusionsThe human QTLs may function in a common pathway, with each involved in a different step in the pathway leading to BP control. These results may be conceptually paradigm shifting.

Genetic Architecture Associated With Familial Short Stature.

Human height is an inheritable, polygenic trait under complex and multilocus genetic regulation. Familial short stature (FSS; also called genetic short stature) is the most common type of short stature and is insufficiently known. To investigate the FSS genetic profile and develop a polygenic risk predisposition score for FSS risk prediction. The FSS participant group of Han Chinese ancestry was diagnosed by pediatric endocrinologists in Taiwan. The genetic profiles of 1163 participants with FSS were identified by using a bootstrapping subsampling and genome-wide association studies (GWAS) method. Genetic profile, polygenic risk predisposition score for risk prediction. Ten novel genetic single nucleotide polymorphisms (SNPs) and 9 reported GWAS human height-related SNPs were identified for FSS risk. These 10 novel SNPs served as a polygenic risk predisposition score for FSS risk prediction (area under the curve: 0.940 in the testing group). This FSS polygenic risk predisposition score was also associated with the height reduction regression tendency in the general population. A polygenic risk predisposition score composed of 10 genetic SNPs is useful for FSS risk prediction and the height reduction tendency. Thus, it might contribute to FSS risk in the Han Chinese population from Taiwan.

Combined Effect of HPV and Several Gene SNPs in Laryngeal Cancer.

Background and objectives: Laryngeal squamous cell carcinoma (LSCC) is one of the most common head and neck tumors. The molecular mechanism of LSCC remains unclear. The aim of this study was to evaluate the prevalence of Human papillomavirus (HPV) and single nucleotide polymorphisms (SNPs) of TP53, MDM2, MDM4, MTHFR, CASP8, and CCR5 genes in LSCC, and to assess their correlations with patient survival. Materials and Methods: 49 LSCC patients were enrolled in this study. PCR and qRT-PCR were used to detect, identify, and quantify HPV. SNPs were genotyped using PCR and PCR-RFLP. Results: By analyzing the interactions of the SNPs of the genes with clinical parameters, the majority of patients with lymph node status (N1,2) were identified as carriers of MDM2 T/G, CASP8 ins/del, CCR5 wt/wt SNP. Cluster analysis showed that patients with MDM2 T/T SNP survive longer than patients identified as CASP8 ins/ins, MTHFR C/C, and MDM4 A/A variant carriers; meanwhile, LSCC patients with MDM2 T/T polymorphic variant had the best survival. Multivariate analysis showed that HPV-positive patients without metastasis in regional lymph nodes (N0) and harboring CASP8 ins/del variant had the best survival. Meanwhile, HPV-negative patients with identified metastasis in lymph nodes (N1 and N2) and CASP8 ins/del variant had poor survival. Conclusions: This finding suggests patients survival prognosis and tumor behavior are different according HPV status, SNP variants, and clinical characteristics of the LSCC.

Analysis of association between common variants of uncoupling proteins genes and diabetic retinopathy in a Chinese population

BackgroundThe aim of this study was to explore the association between diabetic retinopathy (DR) and the variants of uncoupling proteins (UCPs) genes in a Chinese population of type 2 diabetes, in total and in patients of different glycemic status separately.MethodsThis case-control study included a total of 3107 participants from two datasets, among which 662 were DR patients (21.31%). Eighteen tag single nucleotide polymorphisms (SNPs) of UCP1, UCP2, and UCP3 were selected as genetic markers. TaqMan probes, Sequenom MassARRAY MALDI-TOF mass spectrometry platform and Affymetrix Genome-Wide Human SNP Array were used for genotyping. Online SHEsis software was used for association analysis. Bonferroni correction was used for multiple comparisons correction.ResultsThree SNPs of UCP1: rs7688743 (A allele, OR = 1.192, p = 0.013), rs3811787 (T allele, OR = 0.863, p = 0.023), and rs10011540 (G allele, OR = 1.368, p = 0.004) showed association with DR after the adjustment of glucose, but only rs10011540 was marginally significantly associated with DR when Bonferroni correction was strictly applied (padj = 0.048). In patients with uncontrolled glucose, rs7688743 (A allele, p = 0.012, OR = 1.309), rs10011540 (G allele, p = 0.033, OR = 1.432), and rs3811787 (T allele, p = 0.022, OR = 0.811) were associated with DR, while in participants with well controlled glucose, the rs2734827 of UCP3 was associated with DR (A allele, p = 0.017, OR = 0.532). Rs3811787 of UCP1 showed a protective effect to sight threatening DR (T allele, p = 0.007, OR = 0.490), and the association existed after the adjustment for environmental factors and the correction. In patients with uncontrolled glucose, the rs3811787 of UCP1 (T allele, p = 0.017, OR = 0.467) and the rs591758 of UCP3 (C allele, p = 0.026, OR = 0.103) were associated with STDR. While in those with well controlled glucose, only the rs7688743 of UCP1 showed a protective effect (A allele, p = 0.024, OR = 0.049). None of the associations remain significant when Bonferroni correction was strictly applied (all p < 0.05).ConclusionsThe rs10011540 and rs3811787 of the UCP1 gene was marginally significantly associated with DR in Chinese type 2 diabetes patients. There might be different mechanisms of DR development in patients with different glycemic status.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Pharmosome: an integrative and collective database for exploration and analysis of single nucleotide polymorphisms associated with disease

Current single nucleotide polymorphism (SNP) databases are limited to a narrow set of SNPs, which has led to a lack of interactivity between different databases, limited tools to analyze and manipulate the already existing data, and complexity in the graphical user interface. Here we introduce Pharmosome, a web-based, user-friendly and collective database for more than 30,000 human disease-related SNPs, with dynamic pipelines to explore SNPs associated with disease development, drug response and the pathways shared between different genes related to these SNPs. Pharmosome implements several tools to design primers to detect SNPs in large genomes and facilitates analysis of different SNPs to determine relationships between them by aligning sequences, constructing phylogenetic trees, and providing consensus sequences illustrating the connections between SNPs. Pharmosome was written in the Python programming language using the Django web framework in combination with HTML, CSS, and JavaScript to receive user inputs, and process and export the sorted result to the interface. Pharmosome is available from: https://pharmosome.herokuapp.com/.

A Comprehensive In-Silico Analysis of Deleterious Missense SNPs in Human Trehalase Gene: Gaining an Interactome Insights into Type 2 Diabetes Mellitus

Introduction: Trehalase (TREH), a glycoside hydrolase enzyme that catalyses the conversion of trehalose to glucose in sugar metabolism. In spite of severe health threats caused by diabetes worldwide, no systematic and programmed study on human TREH Single Nucleotide Polymorphism (SNPs) and its functional role in Type 2 Diabetes Mellitus (T2DM) has been performed. Aim: This study aimed to identify pathogenic missense SNPs in the human TREH gene. Materials and Methods: A series of different bioinformatic tools including Sorting Intolerant from Tolerant (SIFT), Polyphen, I-mutant, Variant Effect Predictor, Project Hope and GeneMANNIA were used for this study. At all stages, a p-value of 0.05 was considered as statistically significant. Results: This study demonstrated 10 potential mutations out of 241 missense human TREH SNPs from the SNP Database (dbSNP) database of National Center for Biotechnology Information (NCBI), three of which confirmed to have damaging effects on protein function. Out of these three, rs535722007 had the most deleterious effect that altered secondary properties and tertiary structure of the experimental TREH protein and decreased the stability. Further analysis showed a strong connection among TREH, Insulin (INS) and other genes of carbohydrate metabolism associated with T2DM. Gene expression studies found the down-regulation of TREH in all of the experimental studies linked toT2DM. Conclusion: As the probability of the disease predisposition increases with SNPs in primary or co-expressed gene(s), therefore, characterisation of TREH SNPs from human and its gene networking analysis can aid in better understanding of genetic variations and signalling pathways as well as to elucidate the effective diagnostic and treatment strategies.

Incorporation of Agouti-Related Protein (AgRP) Human Single Nucleotide Polymorphisms (SNPs) in the AgRP-Derived Macrocyclic Scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-dPro] Decreases Melanocortin-4 Receptor Antagonist Potency and Results in the Discovery of Melanocortin-5 Receptor Antagonists.

While the melanocortin receptors (MCRs) are known to be involved in numerous biological pathways, the potential roles of the MC5R have not been clearly elucidated in humans. Agouti-related protein (AgRP), an MC3R/MC4R antagonist and MC4R inverse agonist, contains an exposed β-hairpin loop composed of six residues (Arg-Phe-Phe-Asn-Ala-Phe) that is imperative for binding and function. Within this active loop of AgRP, four human missense polymorphisms were deposited into the NIH Variation Viewer database. These polymorphisms, Arg111Cys, Arg111His, Phe112Tyr, and Ala115Val (AgRP full-length numbering), were incorporated into the peptide macrocycles c[Pro1-Arg2-Phe3-Phe4-Xaa5-Ala6-Phe7-dPro8], where Xaa was Dap5 or Asn5, to explore the functional effects of these naturally occurring substitutions in a simplified AgRP scaffold. All peptides lowered potency at least 10-fold in a cAMP accumulation assay compared to the parent sequences at the MC4Rs. Compounds MDE 6-82-3c, ZMK 2-82, MDE 6-82-1c, ZMK 2-85, and ZMK 2-112 are also the first AgRP-based chemotypes that antagonize the MC5R.

SMaSH: Sample matching using SNPs in humans

BackgroundInadvertent sample swaps are a real threat to data quality in any medium to large scale omics studies. While matches between samples from the same individual can in principle be identified from a few well characterized single nucleotide polymorphisms (SNPs), omics data types often only provide low to moderate coverage, thus requiring integration of evidence from a large number of SNPs to determine if two samples derive from the same individual or not.MethodsWe select about six thousand SNPs in the human genome and develop a Bayesian framework that is able to robustly identify sample matches between next generation sequencing data sets.ResultsWe validate our approach on a variety of data sets. Most importantly, we show that our approach can establish identity between different omics data types such as Exome, RNA-Seq, and MethylCap-Seq. We demonstrate how identity detection degrades with sample quality and read coverage, but show that twenty million reads of a fairly low quality RNA-Seq sample are still sufficient for reliable sample identification.ConclusionOur tool, SMASH, is able to identify sample mismatches in next generation sequencing data sets between different sequencing modalities and for low quality sequencing data.

The pathogenicity, structural and functional exploration of human HMGB1 single nucleotide polymorphisms using in silico study

The human HMGB1 gene mutations have a major impact on several immune-related diseases and cancer. The detrimental effect of non-synonymous mutations of HMGB1 has not been investigated yet, hence the present study aims to examine single nucleotide polymorphisms and their implications on the structure-function of human HMGB1. The multifaceted HMGB1 protein acts as pleiotropic cytokine and regulates essential genes for coordinated cellular functions. The mutational effect on HMGB1 was analyzed by sequence-based homology methods, supervised learning methods, and structure-based methods. The study identified 58 non-synonymous mutations in human HMGB1, out of which only 2 mutations; R10T (rs61742222) and F103C (rs61733675) were classified as the SNPs with highest deleterious and disease-causing mutants. The effect of these mutations in structure of HMGB1 was scrutinized and the R10T mutant found to have a distinct structural behaviour in the B-box domain. In addition, R10T mutant predicted that it affects the MoRF function of HMGB1 and it could disrupt the DNA binding or/and protein partner interaction activity by HMGB1. F103C mutation takes place at the TLR binding and cytokine inducing region of HMGB1, hence it could affect the protein binding activity which involves in many cellular signaling. The study identified potent mutations R10T (a cancer-causing somatic mutation) and F103C (a novel mutation) and these mutations either directly or indirectly hinder DNA binding activity and TLR and cytokine binding of HMGB1. These findings will help in understanding the molecular basis of these promising mutations and functional role of human HMGB1 in cancer and immunological diseases.AbbreviationsAGERAdvanced glycosylation end product-specific receptorCXCLChemokine (C-X-C motif) liganddbSNPThe single nucleotide polymorphism databaseHMGB1High mobility group box 1LINCSLINear Constraint SolverMDSMolecular dynamics simulationMoRFMolecular recognition featuresNPTNumber of particle, Pressure and TemperatureNVTNumber of particle, Volume and TemperaturensSNPNon-synonymous SNPPBCPartial boundary conditionPCAPrincipal component analysisPMEPartial mesh EwaldRMSDRoot mean square deviationRMSFRoot mean square fluctuationSNPSingle nucleotide polymorphismSPCSingle-point chargeTLRToll-like receptorUTRUn-translated RegionCommunicated by Ramaswamy H. Sarma

Redistribution of EC-SOD resolves bleomycin-induced inflammation via increased apoptosis of recruited alveolar macrophages.

A human single nucleotide polymorphism (SNP) in the matrix-binding domain of extracellular superoxide dismutase (EC-SOD), with arginine to glycine substitution at position 213 (R213G), redistributes EC-SOD from the matrix into extracellular fluids. We reported that, following bleomycin (bleo), knockin mice harboring the human R213G SNP (R213G mice) exhibit enhanced resolution of inflammation and protection against fibrosis, compared with wild-type (WT) littermates. In this study, we tested the hypothesis that the EC-SOD R213G SNP promotes resolution via accelerated apoptosis of recruited alveolar macrophage (AM). RNA sequencing and Ingenuity Pathway Analysis 7 d postbleo in recruited AM implicated increased apoptosis and blunted inflammatory responses in the R213G strain exhibiting accelerated resolution. We validated that the percentage of apoptosis was significantly elevated in R213G recruited AM vs. WT at 3 and 7 d postbleo in vivo. Recruited AM numbers were also significantly decreased in R213G mice vs. WT at 3 and 7 d postbleo. ChaC glutathione-specific γ-glutamylcyclotransferase 1 (Chac1), a proapoptotic γ-glutamyl cyclotransferase that depletes glutathione, was increased in the R213G recruited AM. Overexpression of Chac1 in vitro induced apoptosis of macrophages and was blocked by administration of cell-permeable glutathione. In summary, we provide new evidence that redistributed EC-SOD accelerates the resolution of inflammation through redox-regulated mechanisms that increase recruited AM apoptosis.-Allawzi, A., McDermott, I., Delaney, C., Nguyen, K., Banimostafa, L., Trumpie, A., Hernandez-Lagunas, L., Riemondy, K., Gillen, A., Hesselberth, J., El Kasmi, K., Sucharov, C. C., Janssen, W. J., Stenmark, K., Bowler, R., Nozik-Grayck, E. Redistribution of EC-SOD resolves bleomycin-induced inflammation via increased apoptosis of recruited alveolar macrophages.

The association of integration patterns of human papilloma virus and single nucleotide polymorphisms on immune- or DNA repair-related genes in cervical cancer patients

The present study investigated the association between single nucleotide polymorphisms (SNPs) in immune- or DNA repair-related genes and the integration pattern of human papillomavirus (HPV), a promising prognostic marker in cervical cancer. The HPV integration patterns of cervical cancer patients were determined by polymerase chain reaction and in situ hybridization, and categorized as episomal (group A), single-copy or multi-copy tandem repetition integrated (group B), and undetectable HPV types (group C). After sample and SNP quality control, 166,505 SNPs in 161 samples (38, 111, and 12 patients in groups A, B, and C, respectively) were examined. None of the SNPs reached genome-wide significance, and several candidate SNPs for future study were selected, including rs10999435 on chromosome 10q22, rs1322054 on chromosome 9q32-33, and rs10902171 on chromosome 11p15. Luciferase assay identified rs1322054 as the primary functional variant to regulate gene expression in immune cell. Further studies are needed to determine the genetic background of different integration patterns of HPV in cervical cancer patients.

Abstract 040: Comparative Genomic Analysis and Functional Insights for Blood Pressure-Associated Single Nucleotide Polymorphisms

Genetic factors play an important role in the regulation of blood pressure (BP) and the development of hypertension. Recent genome-wide association studies (GWAS) in large cohorts have identified associations of hundreds of single nucleotide polymorphisms (SNPs) with human BP. Understanding the molecular function of these SNPs and linking them to BP regulation are among the most pressing challenges in BP research. Animal models, including mouse models, are essential tools for studying BP regulatory mechanisms. In this study, we performed a thorough analysis of the most comprehensive set of human BP-associated SNPs to date and experimental validation. We mapped 26,585 human BP-associated SNPs in 1,071 linkage disequilibrium (LD) regions to 9,447 syntenic regions in the mouse genome. 6,551 of these sites have the same nearest protein-coding genes in human and mouse. 87.0% (932) of the 1,071 LD regions do not contain any nonsynonymous SNP, including 21.8% (234) that are at least 10 kb from any protein-coding gene. Approximately 300 BP-associated SNPs are expression quantitative trait loci for a few dozen known BP physiology genes in tissues including specific kidney regions that are physiologically important for BP regulation. CCCTC-binding factors (CTCF) are essential for regulating long-distance actions of enhancers. Using a newly developed deep learning model, we identified 5 BP-associated SNPs that might influence the conservation of CTCF binding across 40 cell types, and two of them are predicted to have strong effect on CTCF binding. Using SpliceAI, a deep neural network model for predicting the effect of SNPs on mRNA splicing, we identified 19, 9, 22, and 13 SNPs that have a delta value of more than 0.2 for acceptor gain, acceptor loss, donor gain, and donor loss, respectively. Several SNPs are predicted to influence secondary structure of mRNAs and long noncoding RNAs. We experimentally validated that synonymous SNP rs9337951, predicted to influence the secondary structure of its host mRNA JCAD, influences JCAD protein expression. These findings provide novel insights into the potential functional significance of human BP-associated SNPs and form an extensive basis for developing experiments to test such significance in human cells and animal models.

Non-invasive prenatal paternity testing using a standard forensic genetic massively parallel sequencing assay for amplification of human identification SNPs.

Prenatal paternity testing often relies on invasive procedures that cause risk to both the mother and the foetus. Non-invasive, prenatal paternity testing by investigating paternally inherited single nucleotide polymorphisms (SNPs) in cell-free foetal DNA (cffDNA) in maternal plasma was performed at consecutive time points during early gestation. Plasma from 15 pregnant women was investigated at consecutive time points from gestational weeks (GWs) 4-20. The Precision ID Identity Panel and an Ion S5 Sequencer was used to analyse the cffDNA. Paternally inherited foetal SNP alleles were detected from GW7. The median foetal fractions were 0%, 3.9%, 5.1%, 5.2%, and 4.7% at GWs 4, 7, 12, 16, and 20, respectively. The corresponding median numbers of detected paternally inherited foetal autosomal SNP alleles were 0, 3, 9, 10, and 12, respectively. The typical (i.e. geometric mean) paternity indices at GW12 and GW20 were 24 (range 0.0035-8389) and 199 (range 5.1-30,137), respectively. The method is very promising. However, the method can be improved by shortening the lengths of the PCR amplicons and increasing the number of SNPs. To our knowledge, this is the first study to successfully identify paternally inherited foetal SNP alleles at consecutive time points in early gestation independently of the foetal gender.

Donor human leukocyte antigen-G single nucleotide polymorphisms are associated with post-lung transplant mortality.

Human leukocyte antigen (HLA)-G is a non-classical HLA that inhibits immune responses. Its expression is modified by single nucleotide polymorphisms (SNPs), which are associated with transplant outcomes. Our aim was to investigate the association of donor and recipient HLA-G SNPs with chronic lung allograft dysfunction (CLAD) and mortality after lung transplantation.In this single-centre study, we examined 11 HLA-G SNPs in 345consecutive recipients and 297donors of a first bilateral lung transplant. A multivariable Cox proportional hazards model assessed associations of SNPs with death and CLAD. Transbronchial biopsies (TBBx) and bronchoalveolar lavage (BAL) samples were examined using quantitative PCR, ELISA and immunofluorescence.Over a median of 4.75 years, 142patients (41%) developed CLAD; 170 (49%) died. Multivariable analysis revealed donor SNP +3142 (GG+CG versus CC) was associated with increased mortality (hazard ratio 1.78, 95% CI 1.12-2.84; p=0.015). In contrast, five donor SNPs, -201(CC), -716(TT), -56(CC), G*01:03(AA) and 14 bp INDEL, conferred reduced mortality risk. Specific donor-recipient SNP pairings reduced CLAD risk. Predominantly epithelial HLA-G expression was observed on TBBx without rejection. Soluble HLA-G was present in higher concentrations in the BAL samples of patients who later developed CLAD.Specific donor SNPs were associated with mortality risk after lung transplantation, while certain donor-recipient SNP pairings modulated CLAD risk. TBBx demonstrated predominantly epithelial, and therefore presumably donor-derived, HLA-G expression in keeping with these observations. This study is the first to demonstrate an effect of donor HLA-G SNPs on lung transplantation outcome.

Tetraspanin CD82 Organizes Dectin-1 into Signaling Domains to Mediate Cellular Responses to Candida albicans.

Tetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other receptors and signaling proteins, resulting in functional complexes called "tetraspanin microdomains." Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate β-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of Candida albicans-containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased C. albicans viability within macrophages, and decreased cytokine production (TNF-α, IL-1β) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to C. albicans as compared with wild-type mice. Furthermore, patient C. albicans-induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to C. albicans.

Synaptic Regulation by OPRM1 Variants in Reward Neurocircuitry.

Mu-opioid receptors (MORs) are the primary site of action of opioid drugs, both licit and illicit. Susceptibility to opioid addiction is associated with variants in the gene encoding the MOR, OPRM1 Varying with ethnicity, ∼25% of humans carry a single nucleotide polymorphism (SNP) in OPRM1 (A118G). This SNP produces a nonsynonymous amino acid substitution, replacing asparagine (N40) with aspartate (D40), and has been linked with an increased risk for drug addiction. While a murine model of human OPRM1 A118G (A112G in mouse) recapitulates most of the phenotypes reported in humans, the neuronal mechanisms underlying these phenotypes remain elusive. Here, we investigated the impact of A118G on opioid regulation of synaptic transmission in mesolimbic VTA dopaminergic neurons. Using electrophysiology, we showed that both inhibitory and excitatory inputs to VTA dopaminergic neurons projecting to the NAc medial shell were suppressed by the MOR agonists DAMGO and morphine, which caused a shift in the excitatory/inhibitory balance and an increased action potential firing rate. Mice carrying the 112G/G allele exhibited lower sensitivity to DAMGO and morphine compared with major allele carriers (112A/A). Paradoxically, DAMGO produced facilitatory effects on mEPSCs, which were mediated by presynaptic GABAB receptors. However, this was only prominent in homozygous major allele carriers, which could explain a stronger shift in action potential firing in 112A/A mice. This study provides a better understanding on the neurobiological mechanisms that may underlie risk of addiction development in carriers of the A118G SNP in OPRM1SIGNIFICANCE STATEMENT The pandemic of opioid drug abuse is associated with many socioeconomic burdens. The primary brain target of opioid drugs is the μ-opioid receptor (MOR), encoded by the OPRM1 gene, which is highly polymorphic in humans. Using a mouse model of the human OPRM1 A118G single nucleotide polymorphism (SNP) (A112G in mice), we demonstrated that MOR and GABAB signaling coordinate in regulating mesolimbic dopamine neuronal firing via presynaptic regulation. The A118G SNP affects MOR-mediated suppression of GABA and glutamate release, showing weaker efficacy of synaptic regulation by MORs. These results may shed light on whether MOR SNPs need to be considered for devising effective therapeutic interventions.

TAGOOS: genome-wide supervised learning of non-coding loci associated to complex phenotypes.

Genome-wide association studies (GWAS) associate single nucleotide polymorphisms (SNPs) to complex phenotypes. Most human SNPs fall in non-coding regions and are likely regulatory SNPs, but linkage disequilibrium (LD) blocks make it difficult to distinguish functional SNPs. Therefore, putative functional SNPs are usually annotated with molecular markers of gene regulatory regions and prioritized with dedicated prediction tools. We integrated associated SNPs, LD blocks and regulatory features into a supervised model called TAGOOS (TAG SNP bOOSting) and computed scores genome-wide. The TAGOOS scores enriched and prioritized unseen associated SNPs with an odds ratio of 4.3 and 3.5 and an area under the curve (AUC) of 0.65 and 0.6 for intronic and intergenic regions, respectively. The TAGOOS score was correlated with the maximal significance of associated SNPs and expression quantitative trait loci (eQTLs) and with the number of biological samples annotated for key regulatory features. Analysis of loci and regions associated to cleft lip and human adult height phenotypes recovered known functional loci and predicted new functional loci enriched in transcriptions factors related to the phenotypes.In conclusion, we trained a supervised model based on associated SNPs to prioritize putative functional regions. The TAGOOS scores, annotations and UCSC genome tracks are available here: https://tagoos.readthedocs.io.

Human IL10 promoter haplotypes confer differential outcomes to experimental colitis in humanized BAC mice

Abstract Most genetic variants associated with complex diseases map to non-coding regions of the genome, but determining their phenotypic consequences remains challenging. We devised a bacterial artificial chromosome (BAC) transgenic approach to model the effects of human non-coding single nucleotide polymorphisms (ncSNPs) on complex disease outcomes. As proof-of-concept, we focus on human IL10, a potent anti-inflammatory cytokine and key mediator of gut homeostasis. ncSNPs in the IL10 locus have been strongly associated with a variety of disease risks, including colitis susceptibility, but their biological functions are unclear. We created two genetically humanized IL10BAC transgenic mouse lines based on two common and well-defined −1082/−819/−592 IL10 promoter haplotypes (“ATA” and “GCC”) on a mouse Il10-deficient background. Although both IL10BAC strains were protected from spontaneous colitis, only ATA-bearing mice were resistant to dextran sulfate sodium (DSS)-induced acute colitis, while GCC and WT mice had high rates of morbidity and mortality. We found that ATAs had different kinetics of cell-type-specific IL-10 production pre- and post-DSS treatment compared to GCCs and WTs, which influenced the expression profiles of other cytokines and led to distinct immunophenotypes. The skewed responses culminated in the inability of GCCs and WTs to control DSS-induced bacteremia, resulting in death. Our data suggest that human IL10 ncSNPs impart allele-specific differences in spatiotemporal regulation of IL-10 expression, leading to differential outcomes to experimental colitis. Humanized BAC mice may serve as an important tool in modeling the potential impact of human non-coding variants on complex biological outcomes.

Abstract 162: Human Genetics in Vascular Mechanotransduction and Metabolism

Biomechanical cues dynamically control cellular processes but whether genetic variants actively participate in mechano-sensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. GWAS have identified chromosome 1p32.2 strongly associated with CAD/IS; however, the causal mechanism remains unknown. Employing statistical analyses, ATAC-seq, ChIP-seq, CRISPR/Cas9-based assays, expression QTL, chromatin accessibility QTL, and allelic imbalance assay, our results demonstrated that CAD/IS protective allele at rs17114036 confers an increased endothelial enhancer activity that is dynamically regulated by transcription factor KLF2 and unidirectional shear stress, leading to elevated expression of phospholipid phosphatase 3 (PLPP3) to promote endothelial quiescence (PNAS, In Press). Our unpublished data further showed that flow-sensitive PLPP3 causatively regulates endothelial metabolism. In addition, our recent unpublished analyses identified additional CAD SNPs located in flow-sensitive enhancers in vascular endothelium. These results demonstrate for the first time that a human single-nucleotide polymorphism contributes to critical endothelial mechanotransduction and metabolism mechanisms and suggest that human haplotypes and related cis-regulatory elements provide a previously unappreciated layer of regulatory control in cellular mechano-sensing mechanisms.