Non-coding Single Nucleotide Polymorphisms Research Articles

Chromatin interaction maps of human arterioles reveal new mechanisms for the genetic regulation of blood pressure.

Arterioles are small blood vessels located just upstream of capillaries in nearly all tissues. The constriction and dilation of arterioles regulate tissue perfusion and are primary determinants of systemic blood pressure (BP). Abnormalities in arterioles are central to the development of major diseases such as hypertension, stroke, and microvascular complications of diabetes. Despite the broad and essential role of arterioles in physiology and disease, current knowledge of the functional genomics of arterioles is largely absent, partly because it is challenging to obtain and analyze human arteriole samples. Here, we report extensive maps of chromatin interactions, single-cell expression, and other molecular features in human arterioles and uncover new mechanisms linking human genetic variants to gene expression in vascular cells and the development of hypertension. Compared to large arteries, arterioles exhibited a higher proportion of pericytes which were strongly associated with BP traits. BP-associated single nucleotide polymorphisms (SNPs) were enriched in chromatin interaction regions in arterioles, particularly through enhancer SNP-promoter interactions, which were further linked to gene expression specificity across tissue components and cell types. Using genomic editing in animal models and human induced pluripotent stem cells, we discovered novel mechanisms linking BP-associated noncoding SNP rs1882961 to gene expression through long-range chromatin contacts and revealed remarkable effects of a 4-bp noncoding genomic segment on hypertension in vivo. We anticipate that our rich data and findings will advance the study of the numerous diseases involving arterioles. Moreover, our approach of integrating chromatin interaction mapping in trait-relevant tissues with SNP analysis and in vivo and in vitro genome editing can be applied broadly to bridge the critical gap between genetic discoveries and physiological understanding.

Semi-supervised learning with pseudo-labeling compares favorably with large language models for regulatory sequence prediction.

Predicting molecular processes using deep learning is a promising approach to provide biological insights for non-coding single nucleotide polymorphisms identified in genome-wide association studies. However, most deep learning methods rely on supervised learning, which requires DNA sequences associated with functional data, and whose amount is severely limited by the finite size of the human genome. Conversely, the amount of mammalian DNA sequences is growing exponentially due to ongoing large-scale sequencing projects, but in most cases without functional data. To alleviate the limitations of supervised learning, we propose a novel semi-supervised learning (SSL) based on pseudo-labeling, which allows to exploit unlabeled DNA sequences from numerous genomes during model pre-training. We further improved it incorporating principles from the Noisy Student algorithm to predict the confidence in pseudo-labeled data used for pre-training, which showed improvements for transcription factor with very few binding (very small training data). The approach is very flexible and can be used to train any neural architecture including state-of-the-art models, and shows in most cases strong predictive performance improvements compared to standard supervised learning. Moreover, small models trained by SSL showed similar or better performance than large language model DNABERT2.

Zibotentan in Microvascular Angina: A Randomized, Placebo-Controlled, Crossover Trial.

Background: Microvascular angina is associated with dysregulation of the endothelin system and impairments in myocardial blood flow, exercise capacity, and health-related quality of life. The G allele of the noncoding single nucleotide polymorphism RS9349379 enhances expression of the endothelin-1 gene (EDN1) in human vascular cells, potentially increasing circulating concentrations of Endothelin-1 (ET-1). Whether zibotentan, an oral ET-A receptor selective antagonist, is efficacious and safe for the treatment of microvascular angina is unknown. Methods: Patients with microvascular angina were enrolled in this double-blind, placebo-controlled, sequential crossover trial of zibotentan (10 mg daily for 12 weeks). The trial population was enriched to ensure a G allele frequency of 50% for the RS9349379 single nucleotide polymorphism. Participants and investigators were blinded to genotype. The primary outcome was treadmill exercise duration (seconds) using the Bruce protocol. The primary analysis estimated the mean within-participant difference in exercise duration after treatment with zibotentan versus placebo. Results: A total of 118 participants (mean ±SD; years of age 63.5 [9.2 ]; 71 [60.2% ] females; 25 [21.2% ] with diabetes) were randomized. Among 103 participants with complete data, the mean exercise duration with zibotentan treatment compared with placebo was not different (between-treatment difference, -4.26 seconds [95 ] CI, -19.60 to 11.06] P=0.5871). Secondary outcomes showed no improvement with zibotentan. Zibotentan reduced blood pressure and increased plasma concentrations of ET-1. Adverse events were more common with zibotentan (60.2%) compared with placebo (14.4%; P<0.001). Conclusions: Among patients with microvascular angina, short-term treatment with a relatively high dose (10 mg daily) of zibotentan was not beneficial. Target-related adverse effects were common.

Abstract 45: Deletion of a 4-bp noncoding genomic segment orthologous to human rs1882961 results in sex-divergent changes in salt-induced hypertension in Dahl salt-sensitive rats

Most single nucleotide polymorphisms (SNPs) associated with human blood pressure (BP) are in noncoding regions of the genome and have small effect sizes in the general population. Functional studies in a controlled model system are essential to ascertain the effect of these noncoding loci on blood pressure. Such direct testing is challenging and largely absent, contributing to a critical gap between genetic discoveries and physiological understanding. We reasoned that physiological studies in genetically engineered and phenotypically permissive animal models would help to address this critical gap. We tested the hypothesis by studying the noncoding SNP rs1882961. The T allele of rs1882961, which has a frequency of approximately 30% in the European population, is associated with 0.23 mmHg higher systolic blood pressure in humans. We mapped the human rs1882961 SNP to the rat genome and used CRISPR/Cas9 to delete a 4-bp segment encompassed the rs1882961 orthologous site in the genome of the Dahl salt-sensitive (SS) rat (SS-Δrs1882961). Male SS-Δrs1882961 -/- rats exhibited significantly higher mean arterial pressure (MAP) and systolic and diastolic blood pressures (SBP and DBP) than wild-type littermates after the rats were fed a high-salt (4% NaCl) diet for 14 days (156.6 ± 5.9 vs 149.3 ± 5.1 mmHg for MAP, 179.4 ± 7.8 vs 171.9 ± 6.0 mmHg for SBP, and 133.2 ± 4.6 vs 125.1 ± 4.5 mmHg for DBP; n=7 for SS-Δrs1882961 -/- and 9 for wild-type, p&lt;0.05). In contrast, MAP, SBP, DBP, and pulse pressure of female SS-Δrs1882961 -/- rats were significantly lower than wild-type littermates on a high-salt diet (139.8 ± 1.8 vs 149.5 ± 4.7 mmHg for MAP, 159.3 ± 2.5 vs 172.3 ± 6.0 mmHg for SBP, 116.8 ± 1.5 vs 124.5 ± 3.7 mmHg for DBP, and 42.5 ± 1.6 vs 47.7 ± 2.3 mmHg for pulse pressure; n=8 for SS-Δrs1882961 -/- and 7 for wild-type, p&lt;0.05). The difference in systolic blood pressure reached more than 10 mmHg in female rats. Urinary albumin and protein levels of SS-Δrs1882961 -/- rats after 14 days on a high-salt diet tended to be higher in male and lower in female compared with wild-type littermates. The remarkable effects on BP as well as the sex-divergent changes resulted from a 4-bp noncoding segment deletion around rs1882961 orthologous site indicate that noncoding genomic segment may have profound impact on phenotypes on permissive genomic background and with informative stressors.

Blood pressure-associated non-coding single nucleotide polymorphism rs217727 modulates gene expression in human endothelial cells

Majority of Blood Pressure (BP)-associated single nucleotide polymorphisms (SNPs) are in intronic or intergenic regions. Allele G of the noncoding SNP rs217727 is associated with decreased systolic, diastolic and pulse pressure. MRPL23 is the closest protein-coding gene to rs217727, and the transcriptional start site of MRPL23 is 48.3kb away from rs217727. rs217727 is an expression quantitative trait locus for MRPL23 and TNNT3 (76.1kb away). We performed a series of studies to experimentally identify genes modulated by rs217727 and to begin to understand the mechanism. We differentiated a human induced pluripotent stem cell (iPSC) line to endothelial cells (iECs). The iECs were purified using VE-cadherin sorting. Chromatin conformation analysis using Micro-C identified several genes including MRPL23, IGF2 (141.6kb away) and TNNT3 as sharing a chromatin loop with rs217727. We used CRISPR-Cas9 to generate isogenic iPSC lines with either homozygous low BP (G) or high BP allele (A) of rs217727. The screening of several hundred clones led to the establishment of three and four clones with low BP and high BP genotypes. The edited iPSC lines were subjected to six rounds of differentiation to iECs for targeted real-time PCR analysis of genes near rs217727, including the genes mentioned above. We found significantly (p&lt;0.05) decreased expression of TNNT3 (0.44±0.04 fold), LSP1 (142.6kb away; 0.12±0.02 fold) and IGF2 (0.78±0.06 fold) and slightly increased expression of CD81 (380.5kb away; 1.09±0.03 fold) in iECs with the high BP allele (n≥43) compared to the low BP allele (n≥34) of rs217727 (p&lt;0.05). TNNT3 encodes troponin T3 fast skeletal type, LSP1 encodes lymphocyte specific protein 1 and IGF2 encodes insulin-like growth factor 2. The closest gene MRPL23, CTSD (231.8kb away), and MRPS6, a gene on a different chromosome, were not significantly differentially expressed. In summary, the results indicate that rs217727 influences the expression of several local genes in an allele-specific manner. The results provide new insights into the function of a BP-associated noncoding SNP. The work is supported by NHLBI P01 HL149620. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1102: Post Gwas Functional Genomics Analysis To Identify Non-coding Single Nucleotide Polymorphisms Controlling The Risk Of Spontaneous Coronary Artery Dissection

Background: Spontaneous Coronary Artery Dissection (SCAD) is a non-atherosclerotic coronary artery disease and the leading cause of acute myocardial infarction in young females. SCAD results from the combination of genetic susceptibility and acquired triggering factors including inflammation and sex hormones. Three loci have been revealed by two large-scale genome-wide association studies (GWAS) in SCAD. However, GWAS only reveals the statistical associations between the genetic loci and diseases, and the biological mechanisms remain unknown. Methods: GWAS-reported tag single nucleotide polymorphisms (SNPs) and the linked haplotype SNPs were collected. Synthesized oligonucleotides representing the SNP alleles were subjected to electrophoretic mobility shift assays (EMSA) with nuclear proteins extracted from vascular endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts. Hi-C analysis was performed to predict the potential target gene(s) based on chromatin interactions. The functionality of the SNP-associated target genes was examined in cultured vascular cells by RNA silencing. Results: The risk allele of tag SNPs in three genetic loci (chr1q21.2, 6p24.11 and 12q13.1) were associated with 1.5 to 1.8-fold higher risk of SCAD. Intronic, non-tag SNP rs7549396 (G/A) in chr1q21.2 locus was identified as the functional SNP in EMSA, given its consistent allele-imbalanced gel shifting pattern in ECs, SMCs and fibroblasts. Hi-C analysis in human aortic tissue identified a 490kb long-range chromatin interaction between SNP rs7549396 and the promoter of Annexin A9 (ANXA9), an extracellular matrix (ECM) and an inflammation-related protein potentially associated with the pathogenesis of SCAD. The knockdown of ANXA9 in vascular cells showed significant changes in inflammatory makers (IL-1β, vCAM-1 and CCL-2) and ECM degradation enzyme (MMP-1). Conclusions: Leveraging our post-GWAS functional genomics approach, we identified functional non-coding SNPs in disease-associated genetic locus and predicted a novel potential pathogenic gene ANXA9 for SCAD. Further studies on the functions of this SNP and its associated target gene ANXA9 will provide genetics and mechanistic explanation for this mysterious disease.

MicroRNA-Mediated Suppression of Glial Cell Line-Derived Neurotrophic Factor Expression Is Modulated by a Schizophrenia-Associated Non-Coding Polymorphism.

Plasma levels of glial cell line-derived neurotrophic factor (GDNF), a pivotal regulator of differentiation and survival of dopaminergic neurons, are reportedly decreased in schizophrenia. To explore the involvement of GDNF in the pathogenesis of the disease, a case-control association analysis was performed between five non-coding single nucleotide polymorphisms (SNP) across the GDNF gene and schizophrenia. Of them, the 'G' allele of the rs11111 SNP located in the 3' untranslated region (3'-UTR) of the gene was found to associate with schizophrenia. In silico analysis revealed that the rs11111 'G' allele might create binding sites for three microRNA (miRNA) species. To explore the significance of this polymorphism, transient co-transfection assays were performed in human embryonic kidney 293T (HEK293T) cells with a luciferase reporter construct harboring either the 'A' or 'G' allele of the 3'-UTR of GDNF in combination with the hsa-miR-1185-1-3p pre-miRNA. It was demonstrated that in the presence of the rs11111 'G' (but not the 'A') allele, hsa-miR-1185-2-3p repressed luciferase activity in a dose-dependent manner. Deletion of the miRNA binding site or its substitution with the complementary sequence abrogated the modulatory effect. Our results imply that the rs11111 'G' allele occurring more frequently in patients with schizophrenia might downregulate GDNF expression in a miRNA-dependent fashion.

A regulatory variant impacting TBX1 expression contributes to basicranial morphology in Homo sapiens

Changes in gene regulatory elements play critical roles in human phenotypic divergence. However, identifying the base-pair changes responsible for the distinctive morphology of Homo sapiens remains challenging. Here, we report a noncoding single-nucleotide polymorphism (SNP), rs41298798, as a potential causal variant contributing to the morphology of the skull base and vertebral structures found in Homo sapiens. Screening for differentially regulated genes between Homo sapiens and extinct relatives revealed 13 candidate genes associated with basicranial development, with TBX1, implicated in DiGeorge syndrome, playing a pivotal role. Epigenetic markers and in silico analyses prioritized rs41298798 within a TBX1 intron for functional validation. CRISPR editing revealed that the 41-base-pair region surrounding rs41298798 modulates gene expression at 22q11.21. The derived allele of rs41298798 acts as an allele-specific enhancer mediated by E2F1, resulting in increased TBX1 expression levels compared to the ancestral allele. Tbx1-knockout mice exhibited skull base and vertebral abnormalities similar to those seen in DiGeorge syndrome. Phenotypic differences associated with TBX1 deficiency are observed between Homo sapiens and Neanderthals (Homo neanderthalensis). In conclusion, the regulatory divergence of TBX1 contributes to the formation of skull base and vertebral structures found in Homo sapiens.

Non-coding autoimmune risk variant defines role for ICOS in T peripheral helper cell development

Fine-mapping and functional studies implicate rs117701653, a non-coding single nucleotide polymorphism in the CD28/CTLA4/ICOS locus, as a risk variant for rheumatoid arthritis and type 1 diabetes. Here, using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated risk allele is functional, reducing affinity for the inhibitory chromosomal regulator SMCHD1 to enhance expression of inducible T-cell costimulator (ICOS) in memory CD4+ T cells from healthy donors. Higher ICOS expression is paralleled by an increase in circulating T peripheral helper (Tph) cells and, in rheumatoid arthritis patients, of blood and joint fluid Tph cells as well as circulating plasmablasts. Correspondingly, ICOS ligation and carriage of the rs117701653 risk allele accelerate T cell differentiation into CXCR5-PD-1high Tph cells producing IL-21 and CXCL13. Thus, mechanistic dissection of a functional non-coding variant in human autoimmunity discloses a previously undefined pathway through which ICOS regulates Tph development and abundance.

Mutagenic primer-based novel multiplex PCR-RFLP technique to genotype BECN1 SNPs rs10512488 and rs11552192.

Single Nucleotide Polymorphisms (SNPs) in candidate autophagy gene BECN1 could influence its functions thereby autophagy process. BECN1 noncoding SNPs were found to be significantly associated with neurodegenerative disease and type 2 diabetes mellitus. This study aimed to develop a simultaneous genotyping technique for two BECN1 SNPs (rs10512488 and rs11552192). A mutagenic primer-based approach was used to introduce a NdeI restriction site to genotype rs10512488 by Artificial-Restriction Fragment Length Polymorphism (A-RFLP) along with rs11552192 by Polymerase Chain Reaction (PCR)-RFLP. Multiplexing PCR and restriction digestion reactions were set up for simultaneous genotyping of both SNPs in 100 healthy individuals. Genotypic and allele frequencies were manually calculated, and the Hardy-Weinberg Equilibrium was assessed using the chi-square test. We successfully developed PCR and RFLP conditions for the amplification and restriction digestion of both SNPs within the same tube for genotyping. The results of genotyping by newly developed multiplexing PCR-RFLP technique were concordant with the genotypes obtained by Sanger sequencing of samples. Allelic frequencies of rs10512488 obtained were 0.15 (A) and 0.85 (G), whereas allelic frequencies of rs11552192 were 0.16 (T) and 0.84 (A). The newly developed technique is rapid, cost-effective and time-saving for large-scale applications compared to sequencing methods and would play an important role in low-income settings. For the first time, allelic frequencies of rs10512488 and rs11552192 were reported among the North Indian population.

Evaluating the regulatory function of non-coding autism-associated single nucleotide polymorphisms on gene expression in human brain tissue.

Common variants account for most of the estimated heritability associated with autism spectrum disorder (autism). Although several replicable single nucleotide polymorphisms (SNPs) for the condition have been detected using genome-wide association study (GWAS) methodologies, their pathophysiological relevance remains elusive. Examining this is complicated, however, as all detected loci are situated within non-coding regions of the genome. It is therefore likely that they possess roles of regulatory function as opposed to directly affecting gene coding sequences. To bridge the gap between SNP discovery and mechanistic insight, we applied a comprehensive bioinformatic pipeline to functionally annotate autism-associated polymorphisms and their non-coding linkage disequilibrium (i.e., non-randomly associated) partners. We identified 82 DNA variants of probable regulatory function that may contribute to autism pathogenesis. To validate these predictions, we measured the impact of 11 high-confidence candidates and their GWAS linkage disequilibrium partners on gene expression in human brain tissue from Autistic and non-Autistic donors. Although a small number of the surveyed variants exhibited measurable influence on gene expression as determined via quantitative polymerase chain reaction, these did not survive correction for multiple comparisons. Additionally, no significant genotype-by-diagnosis effects were observed for any of the SNP-gene associations. We contend that this may reflect an inability to effectively capture the modest, neurodevelopmental-specific impact of individual variants on biological dysregulation in available post-mortem tissue samples, as well as limitations in the existing autism GWAS data.

Comprehensive characterization of coding and non-coding single nucleotide polymorphisms of the Myoneurin (MYNN) gene using molecular dynamics simulation and docking approaches.

Genome-wide association studies (GWAS) identified a coding single nucleotide polymorphism, MYNN rs10936599, at chromosome 3q. MYNN gene encodes myoneurin protein, which has been associated with several cancer pathogenesis and disease development processes. However, there needed to be a more detailed characterization of this polymorphism's (and other coding and non-coding polymorphisms) structural, functional, and molecular impact. The current study addressed this gap and analyzed different properties of rs10936599 and non-coding SNPs of MYNN via a thorough computational method. The variant, rs10936599, was predicted functionally deleterious by nine functionality prediction approaches, like SIFT, PolyPhen-2, and REVEL, etc. Following that, structural modifications were estimated through the HOPE server and Mutation3D. Moreover, the mutation was found in a conserved and active residue, according to ConSurf and CPORT. Further, the secondary structures were predicted, followed by tertiary structures, and there was a significant deviation between the native and variant models. Similarly, molecular simulation also showed considerable differences in the dynamic pattern of the wildtype and mutant structures. Molecular docking revealed that the variant binds with better docking scores with ligand NOTCH2. In addition to that, non-coding SNPs located at the MYNN locus were retrieved from the ENSEMBL database. These were found to disrupt the transcription factor binding regulatory regions; nonetheless, only two affect miRNA target sites. Again, eight non-coding variants were detected in the testes with normalized expression, whereas HaploReg v4.1 unveiled annotations for non-coding variants. In summary, in silico comprehensive characterization of coding and non-coding single nucleotide polymorphisms of MYNN gene will assist researchers to work on MYNN gene and establish their association with certain types of cancers.

Computational Investigation of Regulatory Region SNPs of Autophagy Gene BECN1

The autophagy process plays a cytoprotective role and ensures the healthy survival of a cell. The role of autophagy has been implicated in various diseases, making it an essential candidate for therapeutic interventions. Beclin 1, a candidate autophagy protein, plays a critical role during autophagy initiation and maturation by interacting with various other autophagy proteins. Beclin1 has been reported to be involved in various human diseases. This study uses a computational approach to study the effect of non-coding region single nucleotide polymorphisms (SNPs) of gene encoding beclin1. RegulomeDB, SNP2TFBS, and PROMO ALLGEN were used to predict the effect of promoter region variants on transcription factor binding sites. SNPs located within 3'UTR were analyzed by miRdSNP, PolymiRTS Database 3.0, miRNASNP-V3, MicroSNIPER, and miRmap. Nine promoter region variants that alter the transcription factor binding sites and 4 variants in 3'UTR were identified that either create a new target site for miRNA or disrupt an existing one. The functional analysis of these identified SNPs could be done experimentally to unravel their relation with a particular disease and the genetic predisposition of human subjects for a disease.

Single cell multi‐omics implicates immune and vascular cells in Alzheimer’s etiology

AbstractBackgroundUnderstanding the genetic etiology of Alzheimer’s disease (AD) is critical to inform effective therapies but remains a challenge. Genetic heritability of late‐onset AD is ∼60‐80%, and genome‐wide association studies (GWAS) have uncovered dozens of noncoding single nucleotide polymorphisms (SNPs) that influence AD risk. Two challenges for the functional interpretation of AD genetic risk are (1) determining the cell types in which SNPs operate and (2) identifying the genes they dysregulate to drive AD pathogenesis. Single‐nucleus sequencing studies of human neurons, microglia, and other brain cell types have begun addressing these challenges. Yet, many AD risk SNPs remain unmapped. One possibility is that they are expressed in immunovascular brain cell types largely missed by current methods.MethodHere, we optimized Vessel Isolation and Nuclei Extraction for Sequencing (VINE‐seq) to capture multimodal gene expression and chromatin accessibility assays across all major brain cell types from 30 cognitively normal, MCI, and AD human prefrontal cortex samples.ResultWe discover early gene dysregulation in immune and vascular cell types along AD progression. We highlight the gene regulatory networks mediating this dysregulation and implicate processes associated with AD pathology and cognitive decline. Finally, we map noncoding AD risk variants to diverse immune and vascular cell types, thereby expanding our understanding of AD etiology.ConclusionDysfunction of diverse human brain immune and vascular cell types mediate the genetic risk for Alzheimer’s disease.

Abstract 16731: Post-GWAS Functional Analysis of CBLN2 Locus Reveals the Role of CUX1 in the Inflammatory Pathogenesis of Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a complex and deadly pulmonary vascular disease highly relevant to genetic susceptibility and acquired inflammatory triggers. Chr18q22.3 near cerebellin 2 precursor (CBLN2) gene is the first genetic locus identified by Genome-wide Association Study (GWAS) that is significantly associated with PAH risk in the European population. This locus includes closely linked non-coding intergenic single nucleotide polymorphisms (SNPs) downstream of CBLN2. Although CBLN2 is upregulated in PAH and causes pulmonary vascular dysfunction, it remains unknown how these non-coding SNPs remotely affect CBLN2 and in turn, alter the genetic susceptibility of PAH. Methods and Results: We combined Hi-C chromatin interaction analysis, electrophoretic mobility shift assay, and SNP genotype-PAH correlation analysis with the All of Us database (N=20,343). In doing so, we identified rs11151770(A/G) as a PAH-associated functional SNP (fSNP), among 19 haplotype SNPs in linkage disequilibrium with the associated CBLN2 locus. The genomic region containing rs11151770(A/G) exhibited long-range chromatin interaction with the CBLN2 promotor. Proteomic analysis revealed that nuclear transcription factor Cut-like Homeobox 1 (CUX1) binds specifically to the risk allele A of rs11151770 but not to the non-risk allele G, regulating the expression of CBLN2 as an activator in pulmonary arterial endothelial cells. CUX1-CBLN2 was upregulated by acquired inflammatory PAH triggers IL-1β and IL-6. SNP genotyping in PAH patients (N=159) and non-PAH controls (N=87) showed a 2.2-fold enrichment of the risk allele A only in patients with PAH associated with connective tissue disorders (N=77, p=0.036). These findings supported a pathogenic mechanism of an inflammation-sensitive pathway (CUX1-CBLN2) controlled by an fSNP (risk allele A) underlying the reported genetic association of the CBLN2 locus with PAH risk. Conclusion: Our post-GWAS functional genomics analysis defined a pathogenic CUX1-mediated, fSNP rs11151770(A/G)-controlled, and inflammation-responsive pathway that provides a mechanistic explanation for the GWAS-identified association between genetic variants in CBLN2 locus and PAH susceptibility.

Transcription factor-binding k-mer analysis clarifies the cell type dependency of binding specificities and cis-regulatory SNPs in humans

BackgroundTranscription factors (TFs) exhibit heterogeneous DNA-binding specificities in individual cells and whole organisms under natural conditions, and de novo motif discovery usually provides multiple motifs, even from a single chromatin immunoprecipitation-sequencing (ChIP-seq) sample. Despite the accumulation of ChIP-seq data and ChIP-seq-derived motifs, the diversity of DNA-binding specificities across different TFs and cell types remains largely unexplored.ResultsHere, we applied MOCCS2, our k-mer-based motif discovery method, to a collection of human TF ChIP-seq samples across diverse TFs and cell types, and systematically computed profiles of TF-binding specificity scores for all k-mers. After quality control, we compiled a set of TF-binding specificity score profiles for 2,976 high-quality ChIP-seq samples, comprising 473 TFs and 398 cell types. Using these high-quality samples, we confirmed that the k-mer-based TF-binding specificity profiles reflected TF- or TF-family dependent DNA-binding specificities. We then compared the binding specificity scores of ChIP-seq samples with the same TFs but with different cell type classes and found that half of the analyzed TFs exhibited differences in DNA-binding specificities across cell type classes. Additionally, we devised a method to detect differentially bound k-mers between two ChIP-seq samples and detected k-mers exhibiting statistically significant differences in binding specificity scores. Moreover, we demonstrated that differences in the binding specificity scores between k-mers on the reference and alternative alleles could be used to predict the effect of variants on TF binding, as validated by in vitro and in vivo assay datasets. Finally, we demonstrated that binding specificity score differences can be used to interpret disease-associated non-coding single-nucleotide polymorphisms (SNPs) as TF-affecting SNPs and provide candidates responsible for TFs and cell types.ConclusionsOur study provides a basis for investigating the regulation of gene expression in a TF-, TF family-, or cell-type-dependent manner. Furthermore, our differential analysis of binding-specificity scores highlights noncoding disease-associated variants in humans.

Causal role of a promoter polymorphism in natural variation of the Arabidopsis floral repressor gene FLC

Noncoding polymorphism frequently associates with phenotypic variation, but causation and mechanism are rarely established. Noncoding single-nucleotide polymorphisms (SNPs) characterize the major haplotypes of the Arabidopsis thaliana floral repressor gene FLOWERING LOCUS C (FLC). This noncoding polymorphism generates a range of FLC expression levels, determining the requirement for and the response to winter cold. The major adaptive determinant of these FLC haplotypes was shown to be the autumnal levels of FLC expression. Here, we investigate how noncoding SNPs influence FLC transcriptional output. We identify an upstream transcription start site (uTSS) cluster at FLC, whose usage is increased by an A variant at the promoter SNP-230. This variant is present in relatively few Arabidopsis accessions, with the majority containing G at this site. We demonstrate a causal role for the A variant at -230 in reduced FLC transcriptional output. The G variant upregulates FLC expression redundantly with the major transcriptional activator FRIGIDA (FRI). We demonstrate an additive interaction of SNP-230 with an intronic SNP+259, which also differentially influences uTSS usage. Combinatorial interactions between noncoding SNPs and transcriptional activators thus generate quantitative variation in FLC transcription that has facilitated the adaptation of Arabidopsis accessions to distinct climates.

Machine Learning Study of SNPs in Noncoding Regions to Predict Non-small Cell Lung Cancer Susceptibility

Non-small cell lung cancer (NSCLC) is the most common pathological subtype of lung cancer. Both environmental and genetic factors have been reported to impact the lung cancer susceptibility. We conducted a genome-wide association study (GWAS) of 287 NSCLC patients and 467 healthy controls in a Chinese population using the Illumina Genome-Wide Asian Screening Array Chip on 712,095 SNPs (single nucleotide polymorphisms). Using logistic regression modeling, GWAS identified 17 new noncoding region SNP loci associated with the NSCLC risk, and the top three (rs80040741, rs9568547, rs6010259) were under a stringent p-value (<3.02e-6). Notably, rs80040741 and rs6010259 were annotated from the intron regions of MUC3A and MLC1, respectively. Together with another five SNPs previously reported in Chinese NSCLC patients and another four covariates (e.g., smoking status, age, low dose CT screening, sex), a predictive model by machine learning methods can separate the NSCLC from healthy controls with an accuracy of 86%. This is the first time to apply machine learning method in predicting the NSCLC susceptibility using both genetic and clinical characteristics. Our findings will provide a promising method in NSCLC early diagnosis and improve our understanding of applying machine learning methods in precision medicine.

Abstract P386: Rat Remap Initiative At The Rat Genome Database (RGD): A Resource Of Integrated Transcriptomic And Epigenomic Data For Laboratory Rats

Complex traits including the genetic control of blood pressure have been associated with both coding and non-coding single nucleotide polymorphisms (SNPs) at more than a thousand loci across the human genome. Many of these haplotypes are found between and distant from genes, and their mechanisms leading to blood pressure variation are largely unknown. Attempts to compare and translate these non-coding mechanisms to animal model systems such as the laboratory rat are difficult, but even further complicated by the difficulty of accessing already existing large-scale genome-wide datasets including transcriptomic and epigenomic (e.g. chromatin accessibility, transcription factor binding, and DNA methylation) data that are currently distributed across a variety of public data repositories and supplementary datasets. These datasets are often annotated to previous and less complete reference genome assemblies, further hampering their broad use without time consuming reanalysis. Collectively, these challenges impede discovery and leveraging of these datasets across the broader research community toward understanding mechanisms controlling complex traits such as blood pressure. To address these challenges, the Rat ReMap Initiative seeks to establish a framework for a publicly accessible resource for rat genomic, transcriptomic and epigenomic data to housed within RGD, with analysis updated to the current reference genome assembly. Users will be able to explore and find these datasets, link to the original raw data, and access analyzed summary data files. Remapped datasets will be linked to existing genomic features, strain, and genetic variation annotations to link with disease, pathways, phenotypes, and models portals already annotated and maintained in the Rat Genome Database (RGD). Here we report our initial efforts to standardize metadata and remap a targeted set of publicly available rat transcriptome, chromatin accessibility and transcription factor binding datasets, some across multiple ages and both sexes, onto the newest rat reference assembly (mRatBN7.2), and to integrate them into an updated genome browser (JBrowse2) and into a comparative genome map browser (VCMap) at RGD.

Abstract 076: The Blood Pressure-associated Non-coding Single Nucleotide Polymorphism Rs28451064 Influences Gene Expression In Human Endothelial Cells

Most Blood Pressure (BP)-associated single nucleotide polymorphisms (SNPs) are in intronic or intergenic regions. For the noncoding SNP rs28451064, allele A is associated with elevated diastolic BP as well as increased risk of myocardial infarction and angina pectoris. rs28451064 is mapped to the nearby protein coding gene KCNE2 and is an expression quantitative trait locus for RCAN1 . We performed a series of studies to experimentally identify genes influenced by rs28451064 and to begin to understand the mechanism. We differentiated a human induced pluripotent stem cell (iPSC) line to endothelial cells (iECs). The iECs were purified by sorting with VE-cadherin. Chromatin conformation analysis using Micro-C identified several genes including MRPS6 and SLC5A3 as sharing a chromatin loop with rs28451064. We used CRISPR-Cas9 to generate isogenic iPSC lines with either homozygous low BP (G) or high BP allele (A) of rs28451064. Three clones of iPSCs with each genotype were established by screening several hundred clones. The edited iPSCs were differentiated into iECs for RNA-seq analysis, which showed that several genes that are genomically close to rs28451064 tended to be differentially expressed, including KCNE2 , SMIM34 , and RCAN1 . The edited iPSC lines were subjected to six rounds of differentiation to iECs for targeted real-time PCR analysis of genes near rs28451064, including the genes mentioned above. The results indicated significantly increased expression of SMIMI11 (1.93±0.25 fold) and SMIM34 (1.45±0.13 fold) (n=34) and decreased expression of RCAN1 (0.90 ± 0.03 fold) and SLC5A3 (0.90 ± 0.03 fold) (n=36) in iECs with the high BP allele compared to the low BP allele of rs28451064 (p&lt;0.05). SMIM11 and SMIM34 encode small integral membrane proteins with unknown functions. RCAN1 encodes regulator of calcineurin 1, and SLC5A3 encodes sodium/myo-inositol cotransporter 1. Interestingly, the closest genes, MRPS6 and KCNE2 , along with MRPL23 , a control gene on a different chromosome, were not significantly differentially expressed. In summary, we have shown definitively that rs28451064 influences the expression of several local genes in an allele-specific manner. The results provide new insights into the function of a BP-associated noncoding SNP.