Potential Functional Single Nucleotide Polymorphisms Research Articles

219 Identification of Functional Single Nucleotide Polymorphisms Responsible for Alternative Splicing Affecting Gastrointestinal Nematodes Resistance in Grazing Sheep

Abstract Sheep infected with gastrointestinal nematodes (GIN) can show weight loss, anemia, diarrhea, decrease appetite, and hypoproteinemia, which can sometimes lead to death. These signs of infection decrease productivity and cause economic losses. Economic losses can also be associated with treatment without efficacy due to GIN resistance to most anthelmintic classes. Combined approaches need to be used to reduce the reliance on anthelmintic as the only control method to GIN. The aim of this study is the identification of potential functional single nucleotide polymorphisms (SNPs) responsible for alternate splicing in Rideau X Dorset crossbred sheep with different immune profiles using RNA-Sequencing (RNA-Seq). Animals were exposed to GIN while grazing, after that those with the greatest and least gastrointestinal tract parasite burdens were selected for liver RNA extracting. These animals can be separated into groups of high (H; n = 5) and medium (M; n = 6) innate immune responses. In addition, liver RNA was extracted for GIN-unexposed control lambs (U; n = 4). RNA-Seq analysis was performed using the CLC genomics workbench software, version 20.0.4 (CLC Bio, Aarhus, Denmark). Quality control analyses, including guanine-cytosine (GC) content, ambiguous base content, Phred score, base coverage, nucleotide contributions, and over-represented sequences parameters, were performed on fastq files. Sequence reads were aligned to the annotated Oar_rambouillet_v.1 (release 109) ovine reference genome. After filtering steps, mRNA isoforms with multiple (³ 2) expressed mRNA isoforms with at least one mRNA isoform differently expressed (DE) between H- and M-innate immune response and GIN-unexposed control groups (FDR < 0.05 and |FC| > 2) were keep. A total of 1, 75, and 58 DE mRNA isoforms were identified for M versus H, U versus M, and U versus H comparisons, respectively. Gene ontology (GO) analysis including the biological process, molecular function, and cellular component GO categories and metabolic pathways analysis were performed using STRING-db software. Enriched biological processes and metabolic pathways related to the immune system, as well as cholesterol and other sterol metabolism (e.g., sterol biosynthesis, steroid metabolism, cholesterol biosynthesis, cholesterol metabolism, ascorbate and aldarate metabolism, drug metabolism, and terpenoid biosynthesis) were identified (FDR < 0.05). Next, the list of DE mRNA isoforms identified will be deeply studied by performing SNP discovery analysis to validate if specific functional SNPs are responsible for the alternative splicing affecting GIN resistance in grazing sheep. The complete results will help to better understand the biological mechanisms that may be regulating the immune response to GIN. Candidate functional SNPs have the potential to be included in a commercial SNP panel for selection of more resistant animals to improve GIN resistance in sheep flocks.

Potentially functional genetic variants in RPS6KA4 and MAP2K5 in the MAPK signaling pathway predict HBV-related hepatocellular carcinoma survival.

Hepatocellular carcinoma (HCC) ranks the third leading cause of cancer deaths with a dismal 5-year survival rate. The mitogen-activated protein kinase (MAPK) signaling pathway is abnormally activated in HCC to promote growth and aggressive metastatic potential of cancer cells. Therefore, genetic variants in the MAPK signaling pathway may serve as potential predictors of Hepatitis B virus (HBV)-related HCC survival. In the present study, we performed a two-stage survival analysis to evaluate the associations between 10,912 single nucleotide polymorphisms (SNPs) in 79 MAPK signaling pathway genes and the overall survival (OS) of 866 HBV-related HCC patients, followed by functional annotation. In combined datasets, we identified two novel and potential functional SNPs (RPS6KA4 rs600377 T>G and MAP2K5 rs17300363 A>C) as prognostic factors for HBV-related HCC, with adjusted allelic hazards ratios of 1.24 (95% confidence interval [CI] = 1.05-1.46, p = 0.010) and 1.48 (1.15-1.91, p = 0.001), respectively. Furthermore, their combined risk genotypes also predicted a poor survival in a dose-response manner in the combined data set (Ptrend < 0.001). Additional functional analysis showed that RPS6KA4 rs600377 G and MAP2K5 rs17300363 C alleles were associated with elevated mRNA expression levels of the corresponding genes in normal tissues. These results provide new insights into the role of genetic variants in the MAPK signaling pathway genes in HBV-related HCC survival.

Association of ERCC1 and XPF polymorphisms with pediatric glioma susceptibility.

Association of ERCC1 and XPF polymorphisms with pediatric glioma susceptibility.

Variant rs8400 enhances ALKBH5 expression through disrupting miR-186 binding and promotes neuroblastoma progression.

AlkB homolog 5 (ALKBH5) has been proven to be closely related to tumors. However, the role and molecular mechanism of ALKBH5 in neuroblastomas have rarely been reported. The potential functional single-nucleotide polymorphisms (SNPs) in ALKBH5 were identified by National Center for Biotechnology Information (NCBI) dbSNP screening and SNPinfo software. TaqMan probes were used for genotyping. A multiple logistic regression model was used to evaluate the effects of different SNP loci on the risk of neuroblastoma. The expression of ALKBH5 in neuroblastoma was evaluated by Western blotting and immunohistochemistry (IHC). Cell counting kit-8 (CCK-8), plate colony formation and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays were used to evaluate cell proliferation. Wound healing and Transwell assays were used to compare cell migration and invasion. Thermodynamic modelling was performed to predict the ability of miRNAs to bind to ALKBH5 with the rs8400 G/A polymorphism. RNA sequencing, N6-methyladenosine (m6A) sequencing, m6A methylated RNA immunoprecipitation (MeRIP) and a luciferase assay were used to identify the targeting effect of ALKBH5 on SPP1. ALKBH5 was highly expressed in neuroblastoma. Knocking down ALKBH5 inhibited the proliferation, migration and invasion of cancer cells. miR-186-3p negatively regulates the expression of ALKBH5, and this ability is affected by the rs8400 polymorphism. When the G nucleotide was mutated to A, the ability of miR-186-3p to bind to the 3'-UTR of ALKBH5 decreased, resulting in upregulation of ALKBH5. SPP1 is the downstream target gene of the ALKBH5 oncogene. Knocking down SPP1 partially restored the inhibitory effect of ALKBH5 downregulation on neuroblastoma. Downregulation of ALKBH5 can improve the therapeutic efficacy of carboplatin and etoposide in neuroblastoma. We first found that the rs8400 G>A polymorphism in the m6A demethylase-encoding gene ALKBH5 increases neuroblastoma susceptibility and determines the related mechanisms. The aberrant regulation of ALKBH5 by miR-186-3p caused by this genetic variation in ALKBH5 promotes the occurrence and development of neuroblastoma through the ALKBH5-SPP1 axis.

The allelic regulation of tumor suppressor ADARB2 in papillary thyroid carcinoma.

Papillary thyroid cancer (PTC) is one of the histological subtypes of thyroid cancer which is the most common endocrine malignancy in the world. The disrupted balance of the adenosine-to-inosine (A-to-I) RNA editing due to dysregulation of the editing genes exists in thyroid cancer. However, it is still largely unknown how functional single nucleotide polymorphisms (SNPs) in the A-to-I RNA editing genes contribute to PTC genetic susceptibility. In this study, we systematically annotated and investigated the role of 28 potential functional SNPs of ADAR, ADARB1, ADARB2 and AIMP2 in PTC. We identified ADARB2 rs904957 and rs1007147 genetic variants which are associated with significantly elevated PTC risk in two case-control sets consisting of 2020 PTC cases and 2021 controls. Further investigations disclosed that ADARB2 could inhibit cell viability and invasion capabilities of PTC cells as a novel tumor suppressor. The ADARB2 rs904957 T-to-C polymorphism in gene 3'-untranslated region enhances miR-1180-3p binding affinity and represses ADARB2 expression through an allele-specific manner. In line with this, carriers with the rs904957 C allele correlated with decreased tumor suppressor ADARB2 expression in tissue specimens showed notably increased risk to develop PTC compared to the T allele carriers. Our findings highlight that the A-to-I RNA editing gene ADARB2 SNPs confer PTC risk. Importantly, these insights would improve our understanding for the general roles of RNA editing and editing genes during cancer development.

Association and functional analysis of angiotensin-converting enzyme 2 genetic variants with the pathogenesis of pre-eclampsia.

The aim of this study was to investigate the relationship between potential functional single-nucleotide polymorphisms (SNPs) of the angiotensin-converting enzyme 2 (ACE2) gene and the pathogenesis of pre-eclampsia (PE) in Guangxi, China. A case-control study was conducted involving 327 PE cases and 591 age-matched, normal, singleton pregnant women. Potential functional ACE2 gene variants (rs2106809 A>G, rs6632677 G>C, and rs2074192 C>T) were selected and genotyped using kompetitive allele-specific PCR. The strength of the associations between the studied genetic variants and the risk of PE were evaluated using odds ratios (ORs) and corresponding 95% confidence intervals (CIs). After adjusting for age and body mass index (BMI), unconditional logistic regression analysis showed that rs2106809 A>G was significantly associated with PE risk (AG vs. AA, OR = 1.43, 95% CI = 1.03-1.99, p = 0.034; AG/GG vs. AA, OR = 1.45, 95% CI = 1.06-1.99, p = 0.019), especially with severe PE (AG vs. AA, adjusted OR = 1.70, 95% CI = 1.10-2.61; AG/GG vs. AA, adjusted OR = 1.71, 95% CI = 1.14-2.57). Further stratified analysis showed that rs2106809 was even more pronounced in subjects in the pre-pregnancy BMI (pre-BMI) >23 kg/m2 (adjusted OR = 2.14, 95% CI = 1.32-3.45) and triglyceride (TG) >2.84 mmol/L subgroups (adjusted OR = 1.81, 95% CI = 1.09-3.01) under the dominant genetic model. We also found that rs2106809 interacted with pre-BMI (p interaction = 0.040), thereby affecting an individual's genetic susceptibility to PE. Multiple dimension reduction analysis demonstrated that rs2106809 made the best one-locus model, and the three-locus model was the best interaction model for predicting PE risk. Functional analysis suggested that rs2106809 A>G causes a change in the reliability of classifications of two putative splice sites in the ACE2 gene, potentially regulating the expression of functional genes (PIR, ACE2, and CLTRN) in multiple tissues and cell lines (p< 0.05). The ACE2 gene rs2106809 A>G variant is significantly associated with the risk of PE via individual locus effects and/or complex gene-gene and gene-environment interactions. Regulating the expression of functional genes such as PIR, ACE2, and CLTRN may be the molecular mechanism by which rs2106809 increases an individual's susceptibility to PE.

Potentially functional genetic variants of the notch signaling pathway genes predict survival of Chinese patients with esophageal squamous cell carcinoma.

The Notch signaling pathway is involved in the progression of esophageal squamous cell carcinoma (ESCC), although the roles of single nucleotide polymorphisms (SNPs) of the Notch signaling pathway genes in the process remain unknown. The present study included 1009 patients with histopathologically diagnosed ESCC at Fudan University Shanghai Cancer Center. Two-stage multivariate Cox proportional hazards regression analysis was used to estimate associations between 13,248 SNPs in 103 Notch signaling pathway genes and overall survival of the patients. We found that overall survival of the patients was significantly associated with genotypes of HDAC9 rs1729318 (AT+TT vs. AA: hazard ratio =1.44, 95% confidence interval =1.16-1.80, pcombined =0.001) and HDAC9 rs1339555498 (GT + TT vs. GG: hazard ratio =1.38, 95% confidence interval =1.10-1.74, pcombined =0.005). Further receiver operator characteristic (ROC) curve analysis indicated that the model with both available clinical factors and these two SNPs improved the area under the ROC curve compared to the model with clinical factors only (1-year: 0.66 vs. 0.64, p= 0.034). Additional expression quantitative trait loci analysis showed that the rs1729318 T variant genotypes were associated with increased mRNA expression levels of HDAC9 in normal esophageal muscular tissue (p= 0.003). The results suggest that these two potential functional SNPs on HDAC9 may serve as biomarkers for predicting survival of ESCC patients. However, further studies are needed to confirm these findings.

Identification of functional single nucleotide polymorphisms in the porcine SLC6A4 gene associated with aggressive behavior in weaned pigs after mixing.

Variation in genes of the serotonergic system influences aggressive behavior by affecting serotonin levels in the central and cortical nervous system. SLC6A4 (serotonin transporter) is a master regulator of 5-HT signaling and involved in the regulation of aggressive behavior in humans and rodents. To identify potential functional single nucleotide polymorphisms (SNPs) for the porcine SLC6A4 gene associated with aggressive behavior, a total of 500 pigs (268 barrows and 232 gilts) were selected and mixed in 51 pens. Their behavior was recorded and observed for 72 h after mixing. Based on a composite aggressive score (CAS), the most aggressive and the least aggressive pigs within each pen were selected separately (a total of 204 pigs). Ear tissue was sampled to extract genomic DNA. Eight SNPs in the 5'-flanking region, coding region, and 3'-untranslated region (3'-UTR) of SLC6A4 were genotyped, of which 6 SNPs had significant differences (P < 0.05) in allele frequency between the most aggressive and least aggressive pigs. Luciferase activity was greater in plasmids of genotype GG than plasmids of genotype CC of rs345058216 (P < 0.01). Computational analysis nominated MAZ as putative transcription factor (TF) with higher probability to bind the SLC6A4 promoter at the SNP (rs345058216) site. Also, we demonstrated that MAZ overexpression modulates SLC6A4 promoter activity in allele-specific manner with an in vitro assay. In addition, we demonstrated that SLC6A4 was a direct target of miR-671-5p. The dual luciferase reporter gene assay and cell transfection were performed to examine the role of miR-671-5p in regulating SLC6A4 expression. The luciferase assays revealed that the SNP rs332335871 affects regulation of miR-671-5p in SLC6A4 expression. After overexpression of miR-671-5p in porcine primary neural cells, the SLC6A4 mRNA levels can be significantly reduced. In conclusion, we here found that miR-671-5p and MAZ mediated porcine SLC6A4 expression level, which provides the possible molecular mechanism of aggressive behavior.

Variants of Toll-like receptor 6 associated with tuberculosis susceptibility in the Chinese Tibetan population

BackgroundOur investigation attempted to understand the role of innate immunity related genes played in tuberculosis. The relationship between single-nucleotide polymorphisms (SNPs) of three innate immunity-related genes (TLR6, MyD88, and TIRAP) and tuberculosis (TB) risk in two Chinese populations were explored. MethodsTotally 1185 Chinese Han, consisting of 580 active TB cases and 605 healthy controls (HCs), and 1216 Chinese Tibetan individuals including 613 TB patients and 603 HCs were enrolled to conduct two case-control studies. TagSNPs of the three genes were selected based on the HapMap database and genotyped by the SNPscan™ Kit. Haploview software 4.2 was applied to perform linkage disequilibrium (LD) analysis and online software SHEsis was used to discover significant haplotype block. RegulomeDB and HaploReg were applied to predict potential functional SNPs of the three genes. ResultsThe results showed that minor alleles of rs5743808 and rs5743827 of TLR6 were related with increased TB risk (p = 0.001, OR 95%CI = 1.51 (1.18–1.95) and p = 0.002, OR 95%CI = 1.42 (1.14–1.77)), and significant association was also observed between rs5743827 and TB risk in male subgroup (p = 0.003, OR 95%CI = 1.67 (1.91–2.35)) in the Tibetan population. For the Tibetan population, frequency of haplotype ACGT of rs1039559-rs3775073-rs5743808-rs5743827 of TLR6 was significantly higher in the TB group (p = 0.0008), while haplotype ATAC was significantly higher in the control group (p = 0.0002). The above associations remained after permutation and Bonferroni correction. No significant association was found in the Han population. Probable functions of tagSNPs of TLR6 and some other linked variants were discovered after bioinformatic analysis. ConclusionsThis study suggested that variants of TLR6 might be associated with TB risk in the Tibetan population, while not in the Han population. The difference between Chinese Han and Tibetan people will provide better understanding of tuberculosis.

A Functional Polymorphism in Accessible Chromatin Region Confers Risk of Non-Small Cell Lung Cancer in Chinese Population.

BackgroundThe disease-associated non-coding variants identified by genome-wide association studies (GWASs) were enriched in open chromatin regions (OCRs) and implicated in gene regulation. Genetic variants in OCRs thus may exert regulatory functions and contribute to non-small cell lung cancer (NSCLC) susceptibility.ObjectiveTo fine map potential functional variants in GWAS loci that contribute to NSCLC predisposition using chromatin accessibility and histone modification data and explore their functions by population study and biochemical experimental analyses.MethodsWe mapped the chromatin accessible regions of lung tissues using data of assay for transposase-accessible chromatin using sequencing (ATAC-seq) in The Cancer Genome Atlas (TCGA) and prioritized potential regulatory variants within lung cancer GWAS loci by aligning with histone signatures using data of chromatin immunoprecipitation assays followed by sequencing (ChIP-seq) in the Encyclopedia of DNA Elements (ENCODE). A two-stage case–control study with 1,830 cases and 2,001 controls was conducted to explore the associations between candidate variants and NSCLC risk in Chinese population. Bioinformatic annotations and biochemical experiments were performed to further reveal the potential functions of significant variants.ResultsSixteen potential functional single-nucleotide polymorphisms (SNPs) were selected as candidates from bioinformatics analyses. Three variants out of the 16 candidate SNPs survived after genotyping in stage 1 case–control study, and only the results of SNP rs13064999 were successfully validated in the analyses of stage 2 case–control study. In combined analyses, rs13064999 was significantly associated with NSCLC risk [additive model; odds ratio (OR) = 1.17; 95%CI, 1.07–1.29; p = 0.001]. Functional annotations indicated its potential enhancer bioactivity, and dual-luciferase reporter assays revealed a significant increase in luciferase activity for the reconstructed plasmid with rs13064999 A allele, when compared to the one with wild-type G allele (pA549 < 0.001, pSK-MES-1 = 0.004). Further electrophoretic mobility shift assays (EMSA) and super-shift assays confirmed a stronger affinity of HP1γ for the binding motif containing SNP rs13064999 A allele.ConclusionThese findings suggested that the functional variant rs13064999, identified by the integration of ATAC-seq and ChIP-seq data, contributes to the susceptibility of NSCLC by affecting HP1γ binding, while the exact biological mechanism awaits further exploration.

Genome-wide gene-smoking interaction study identified novel susceptibility loci for non-small cell lung cancer in Chinese populations.

Gene-smoking interactions play important roles in the development of non-small cell lung cancer (NSCLC). To identify single-nucleotide polymorphisms (SNPs) that modify the association of smoking behavior with NSCLC risk, we conducted a genome-wide gene-smoking interaction study in Chinese populations. The genome-wide interaction analysis between SNPs and smoking status (ever- versus never-smokers) was carried out using genome-wide association studies of NSCLC, which included 13 327 cases and 13 328 controls. Stratified analysis by histological subtypes was also conducted. We used a genome-wide significance threshold of 5 × 10-8 for identifying significant gene-smoking interactions and 1 × 10-6 for identifying suggestive results. Functional annotation was performed to identify potential functional SNPs and target genes. We identified three novel loci with significant or suggestive gene-smoking interaction. For NSCLC, the interaction between rs2746087 (20q11.23) and smoking status reached genome-wide significance threshold [odds ratio (OR) = 0.63, 95% confidence interval (CI): 0.54-0.74, P = 3.31 × 10-8], and the interaction between rs11912498 (22q12.1) and smoking status reached suggestive significance threshold (OR = 0.72, 95% CI: 0.63-0.82, P = 8.10 × 10-7). Stratified analysis by histological subtypes identified suggestive interactions between rs459724 (5q11.2) and smoking status (OR = 0.61, 95% CI: 0.51-0.73, P = 7.55 × 10-8) in the risk of lung squamous cell carcinoma. Functional annotation indicated that both classic and novel biological processes, including nicotine addiction and airway clearance, may modulate the susceptibility to NSCLC. These novel loci provide new insights into the biological mechanisms underlying NSCLC risk. Independent replication in large-scale studies is needed and experimental studies are warranted to functionally validate these associations.

Genetic impacts on thermostability of onco-lncRNA HOTAIR during the development and progression of endometriosis.

HOTAIR is a well-known long non-coding RNA (lncRNA) involved in various cellular signaling, whereas its functional impacts on endometriosis development are still largely unknown. To this end, six potential functional single nucleotide polymorphisms (SNPs) in HOTAIR, with minor allele frequencies more than 10% in Han population and altered net energy of RNA structures larger than 0.5 kcal/mol, were selected for genotyping study. The study included 207 endometriosis patients and 200 healthy women. Genetic substitutions at rs1838169 and rs17720428 were frequently found in endometriosis patients, and rs1838169 showed statistical significance (p = 0.0174). The G-G (rs1838169-rs17720428) haplotype showed the most significant association with endometriosis (p < 0.0001) with enhanced HOTAIR stability, and patients who harbor such haplotype tended to show higher CA125. Data mining further revealed higher mRNA HOTAIR levels in the endometria of patients with severe endometriosis which consistently showed reduced HOXD10 and HOXA5 levels. HOTAIR knockdown with specific shRNAs down-regulated cell proliferation and migration with the induction of HOXD10 and HOXA5 expression in human ovarian clear cancer cells. Our study therefore provided evidence to indicate a prominent role of HOTAIR in promoting endometriosis, which could be used as a potential target for clinical applications.

Association studies of dopamine synthesis and metabolism genes with multiple phenotypes of heroin dependence

BackgroundHeroin dependence is a complex disease with multiple phenotypes. Classification of heroin users into more homogeneous subgroups on the basis of these phenotypes could help to identify the involved genetic factors and precise treatments. This study aimed to identify the association between genetic polymorphisms of DA synthesis and metabolism genes, including tyrosine hydroxylase (TH), DOPA decarboxylase (DDC), solute carrier family 6 member 3 (SLC6A3) and DA beta-hydroxylase (DBH), with six important phenotypes of heroin dependence.MethodsA total of 801 heroin dependent patients were recruited and fourteen potential functional single nucleotide polymorphisms (SNPs) were genotyped by SNaPshot. Associations between SNPs with six phenotypes were mainly assessed by binary logistic regression. Generalized multifactor dimensionality reduction was used to analyze the gene-by-gene and gene-by-environment interactions.ResultsWe found that DBH rs1611114 TT genotype had a protective effect on memory impairment after heroin dependence (P = 0.002, OR = 0.610). We also found that the income-rs12666409-rs129915-rs1611114 interaction yielded the highest testing balance accuracy and cross-validation consistency for memory change after heroin dependence.ConclusionsOur results suggest that the memory change after heroin dependence was a result of a combination of genetics and environment. This finding could lead to a better understanding of heroin dependence and further improve personalized treatment.

κ Opioid Receptor 1 Single Nucleotide Polymorphisms were Associated with the Methadone Dosage.

Background: Heroin use disorder (HUD) is a complex brain disease that includes multiple phenotypes. Heroin acts primarily as a mu-opioid receptor (OPRM1) agonist. The κ opioid receptor 1 (OPRK1) is critically involved in abstinence and remission. Multiple studies confirm that the OPRM1 and OPRK1 genes are associated with HUD. However, their relationship with the addictive phenotype is still unclear. This study was designed to identify the genetic polymorphisms within OPRM1 and OPRK1 with six HUD phenotypes. Methods: A total of 801 patients with HUD were recruited from the Methadone Maintenance Treatment Program in Xi'an. We identified eight potential functional single nucleotide polymorphisms (SNPs) in the two genes that were genotyped using SNaPshot SNP technology. We then performed a case-control association analysis, investigated particular disease phenotypes, and assessed the extent of epistasis among the variants of the two genes. Results: The OPRK1 rs3802279, rs3802281, and rs963549 genotypes were significantly associated with methadone dosage analyzed by Pearson's chi-square test or binary logistic regression to correct for covariates. The rs3802279 CC, rs3802281 TT, and rs963549 CC genotype carriers required a lower methadone maintenance dose per day. Multifactor dimensionality reduction analysis indicated strong interactions between sex and OPRK1 rs963549. The results of the OPRM1 genotyping did not reveal any associations with the various HUD phenotypes. Conclusion: These findings support an important role of the OPRK1 polymorphism in determining the daily methadone dose and may guide future studies in identifying additional genetic risk factors for HUD.

The single nucleotide variant rs2868371 associates with the risk of mortality in non-small cell lung cancer patients: A multicenter prospective validation

Background and purposeDefinitive radiation therapy (RT) with or without chemotherapy has become the standard treatment for non-metastatic unresectable non-small cell lung cancer (NSCLC). However, treatment outcomes can differ substantially and patients’ genetic background could play a crucial role. Potential associations between single-nucleotide polymorphisms (SNP) in Heat shock protein beta-1 (HSPB1) and survival have been reported in prior single-institution retrospective reports. Materials and methodsThe current assay aims to validate such connection in a prospective multicenter study in a European cohort including 181 NSCLC patients. Median follow-up time for all patients was 13 months (range, 3–57 months). ResultsThe results obtained show an association between the rs2868371 GG genotype and better overall survival (HR: 0.35; 95%CI: 0.13–0.96; p = 0.042) in multivariate analysis. Two-year overall survival rate was 72% for patients carrying the rs2868371 GG genotype versus 36% for those patients harboring the rs2868371 CC/CG genotypes (p = 0.013). Additionally, the rs2868371 GG genotype was found to be associated with better disease-free survival in the multivariate analysis (HR: 0.36; 95%CI: 0.13–0.99; p = 0.048). In silico analysis of the potential functional SNP suggested significant difference in the affinity of the Glucocorticoid Receptor binding site between alternative allelic variants, confirmed by chromatin immunoprecipitation analysis displaying stronger affinity for the risk allele (C). Furthermore, our findings indicate that the rs2868371 influences (mRNA) HSPB1 expression, offering insight into the regulation of HSPB1 transcription. ConclusionThe functional HSPB1 rs2868371 promoter variant may affect lung cancer survival by regulation of HSPB1 expression levels through glucocorticoid receptor interaction.

A Functional 5'-UTR Polymorphism of MYC Contributes to Nasopharyngeal Carcinoma Susceptibility and Chemoradiotherapy Induced Toxicities.

MYC is a transcription factor acting as a pivotal regulator of genes involved in cell cycle progression, apoptosis, differentiation and metabolism. In this study, we evaluated the association of MYC polymorphisms with nasopharyngeal carcinoma (NPC) risk and chemoradiotherapy induced toxicities among Chinese population. By using bioinformatic tools, five potential functional single nucleotide polymorphisms of MYC were genotyped in a case-control study with 684 NPC patients and 823 healthy controls. We found two SNPs rs4645948 (C>T) and rs2071346 (G>T) were significantly associated with increased risk of developing NPC (TT+CT vs CC, OR=1.557, P=3.34×10-4; TT+GT vs GG, OR=1.361, P=0.007, respectively). In addition, rs4645948 (C>T) was conferred with increased risk of anemia (CT vs CC, OR=2.152, P=0.001) and severe leukopenia (CT vs CC, OR=1.893, P=0.034) for NPC patients receiving chemoradiotherapy. We also found rs2071346 (G>T) variant genotype carriers were subjected to higher risk of anemia (GT vs GG, OR=1.665, P=0.022) and thrombocytopenia (GT vs GG, OR=1.685, P=0.035). Our results demonstrated that the relative expression of MYC was dramatically higher in NPC tissues compared to rhinitis tissues. Over-expression of MYC was positively correlated with advanced T stage, N stage, and late clinical stage. Notably, the expression of MYC in rs4645948 CT and TT genotypes carriers were significantly higher than CC genotype carriers. Luciferase reporter assay indicated that the T allele of rs4645948 led to significantly higher transcription activity of MYC compared to the C allele. These findings suggested that individual carrying the rs4645948 T allele may be at greater risk for NPC due to an increase of MYC transcriptional activity and an augment of MYC expression.

Detection of Putative Functional Single Nucleotide Polymorphisms in Blood Pressure Loci and Validation of Association Between Single Nucleotide Polymorphism in WBP1L and Hypertension in the Chinese Han Population.

We have performed a gene-based association study and detected several important blood pressure (BP)-associated genes. In this study, we explored functional variants in these genes by bioinformatics analysis and validated the associations between the functional single nucleotide polymorphisms (SNPs) and hypertension with public data and our in-house data of 857 cases and 927 controls. We found various functional variants in the BP-associated genes, including missense mutations and phosphorylation-related SNPs. Most of these SNPs were associated with expressions of the local genes. Some of these SNPs were associated with coronary artery disease or ischemic stroke. The associations between 12 functional SNPs in 7 genes and BP were validated (P < 5 × 10). The intronic SNP rs176185, which may influence promoter histone, enhancer histone, DNase and regulatory motifs and showed cis-eQTL effect on WBP1L, was associated with hypertension in the Chinese Han population (P = 0.0119). Our study detected plenty of potential functional SNPs in the BP-associated genes and demonstrated that rs176185 may be associated with hypertension in the Chinese Han population.

XPG rs873601 G>A contributes to uterine leiomyoma susceptibility in a Southern Chinese population.

XPG gene contributes to DNA repair defects and genomic instability, which may lead to the initiation of uterine leiomyoma. We hypothesized that genetic variants of XPG gene may alter the carriers’ susceptibility to leiomyoma. The association between five potential functional single nucleotide polymorphisms (SNPs), i.e. rs2094258 C>T, rs751402 C>T, rs2296147 T>C, rs1047768 T>C, rs873601 G>A, and uterine leiomyoma risk in Chinese, was investigated in this case–control study, which included 398 incident leiomyoma cases and 733 controls. We found that rs873601 was significantly associated with tumor risk in a recessive genetic model after being adjusting for age and menopause. When compared with rs873601 GG/GA genotypes, the AA genotype had an increased leiomyoma risk (adjusted OR = 1.59, 95% CI = 1.16–2.18, P=0.004; Bonferroni adjusted P=0.040). Furthermore, stratified analysis revealed that the association between the rs873601 AA genotype and leiomyoma risk was more evident among subjects younger than 40 years old (adjusted OR = 1.58, 95% CI = 1.06–2.35, P=0.023) and patients who had more than three myomas (adjusted OR = 2.05, 95% CI = 1.24–3.41, P=0.006). Yet, no significant association between the other four polymorphisms and leiomyoma risk was observed. To sum up, the present study reported on the association between XPG gene polymorphisms and myoma risk. The observed data indicated that SNP rs873601 G>A contributes to uterine leiomyoma susceptibility in a Southern Chinese population.

Genetic association analyses and meta-analysis of Dynorphin-Kappa Opioid system potential functional variants with heroin dependence

Prodynorphin (PDYN) binds to k-opioid receptors (KOPr; encoded by OPRK1) and is known to regulate dopaminergic tone, making this system important for drugs addiction. Dynorphin (Dyn)/KORr system are powerful effectors of stress-induced alterations in reward processing and dysphoric states. Thus, We identified 11 potential functional SNPs and one variable number of tandem repeat (VNTR) in this system, performed a case-control association analysis, investigated particular disease phenotypes, assessed the joint effect of variants in two genes, carried out a meta-analysis to analyze the association between this VNTR and Heroin dependence (HD) risk. Eleven single-nucleotide polymorphisms (SNPs) were genotyped using SNaPshot SNP technology. Participants included 566 healthy controls and 541 patients with HD. We found that PDYN polymorphisms modulate the susceptibility to HD. An increased risk of HD was significantly associated with H alleles of PDYN VNTR (χ2 = 10.824, p = 0.001, OR = 1.419, 95% CI = 1.151–1.748). In addition, the results revealed the patients with the HH genotype showed greater number of withdrawal instances (F(2538) = 7.987, p = 0.0004) compared to the patients with the LL genotype. The Meta-analysis showed the pooled effect of the H allele at this locus is a risk factor for HD in Chinese Han. Gene-gene interaction analysis indicated strong interactions between PDYN rs3830064, 68-bp VNTR and OPRK1 rs16918842, rs3802279. These findings support the important role of PDYN polymorphism in HD, and may guide future studies to identify genetic risk factors for HD.

Multiple Functional Variants at 13q14 Risk Locus for Osteoporosis Regulate RANKL Expression Through Long-Range Super-Enhancer

RANKL is a key regulator involved in bone metabolism, and a drug target for osteoporosis. The clinical diagnosis and assessment of osteoporosis are mainly based on bone mineral density (BMD). Previous powerful genomewide association studies (GWASs) have identified multiple intergenic single-nucleotide polymorphisms (SNPs) located over 100 kb upstream of RANKL and 65 kb downstream of AKAP11 at 13q14.11 for osteoporosis. Whether these SNPs exert their roles on osteoporosis through RANKL is unknown. In this study, we conducted integrative analyses combining expression quantitative trait locus (eQTL), genomic chromatin interaction (high-throughput chromosome conformation capture [Hi-C]), epigenetic annotation, and a series of functional assays. The eQTL analysis identified six potential functional SNPs (rs9533090, rs9594738, r8001611, rs9533094, rs9533095, and rs9594759) exclusively correlated with RANKL gene expression (p < 0.001) at 13q14.11. Co-localization analyses suggested that eQTL signal for RANKL and BMD-GWAS signal shared the same causal variants. Hi-C analysis and functional annotation further validated that the first five osteoporosis SNPs are located in a super-enhancer region to regulate the expression of RANKL via long-range chromosomal interaction. Particularly, dual-luciferase assay showed that the region harboring rs9533090 in the super-enhancer has the strongest enhancer activity, and rs9533090 is an allele-specific regulatory SNP. Furthermore, deletion of the region harboring rs9533090 using CRISPR/Cas9 genome editing significantly reduced RANKL expression in both mRNA level and protein level. Finally, we found that the rs9533090-C robustly recruits transcription factor NFIC, which efficiently elevates the enhancer activity and increases the RANKL expression. In summary, we provided a feasible method to identify regulatory noncoding SNPs to distally regulate their target gene underlying the pathogenesis of osteoporosis by using bioinformatics data analyses and experimental validation. Our findings would be a potential and promising therapeutic target for precision medicine in osteoporosis. © 2018 American Society for Bone and Mineral Research.