Independent Case-control Sets Research Articles

Improving Specificity for Ovarian Cancer Screening Using a Novel Extracellular Vesicle–Based Blood Test: Performance in a Training and Verification Cohort

The low incidence of ovarian cancer (OC) dictates that any screening strategy needs to be both highly sensitive and highly specific. This study explored the utility of detecting multiple colocalized proteins or glycosylation epitopes on single tumor-associated extracellular vesicles (EVs) from blood. The novel OC Test employs immunoaffinity capture of tumor-associated EVs followed by proximity-ligation qPCR to detect combinations of up to three biomarkers to maximize specificity and measures multiple combinations to maximize sensitivity. A high-grade serous carcinoma (HGSC) case-control training set of EDTA plasma samples from 397 women was used to lock down the test design, the data interpretation algorithm, and the cut-off between cancer and non-cancer. Performance was verified and compared to CA125 in an independent blinded case-control set of serum samples from 390 women (132 controls, 66 HGSC, 83 non-HGSC OC, 109 benign). In the verification study, the OC Test showed a specificity of 97.0% (128/132; 95% CI: 92.4%-99.6%), a HGSC sensitivity of 97.0% (64/66; 95% CI: 87.8%-99.2%), and an AUC of 0.97 (95% CI 0.93-0.99) and also detected 73.5% (61/83; 95% CI: 62.7%-82.6%) of the non-HGSC OC cases. This test exhibited fewer false positives in subjects with benign ovarian tumors, non-ovarian cancers, and inflammatory conditions when compared to CA125. The combined sensitivity and specificity of this new test suggests it may have potential in OC screening.

Common Genetic Variants Contribute to Risk of Transposition of the Great Arteries.

Dextro-transposition of the great arteries (D-TGA) is a severe congenital heart defect which affects approximately 1 in 4,000 live births. While there are several reports of D-TGA patients with rare variants in individual genes, the majority of D-TGA cases remain genetically elusive. Familial recurrence patterns and the observation that most cases with D-TGA are sporadic suggest a polygenic inheritance for the disorder, yet this remains unexplored. We sought to study the role of common single nucleotide polymorphisms (SNPs) in risk for D-TGA. We conducted a genome-wide association study in an international set of 1,237 patients with D-TGA and identified a genome-wide significant susceptibility locus on chromosome 3p14.3, which was subsequently replicated in an independent case-control set (rs56219800, meta-analysis P=8.6x10-10, OR=0.69 per C allele). SNP-based heritability analysis showed that 25% of variance in susceptibility to D-TGA may be explained by common variants. A genome-wide polygenic risk score derived from the discovery set was significantly associated to D-TGA in the replication set (P=4x10-5). The genome-wide significant locus (3p14.3) co-localizes with a putative regulatory element that interacts with the promoter of WNT5A, which encodes the Wnt Family Member 5A protein known for its role in cardiac development in mice. We show that this element drives reporter gene activity in the developing heart of mice and zebrafish and is bound by the developmental transcription factor TBX20. We further demonstrate that TBX20 attenuates Wnt5a expression levels in the developing mouse heart. This work provides support for a polygenic architecture in D-TGA and identifies a susceptibility locus on chromosome 3p14.3 near WNT5A. Genomic and functional data support a causal role of WNT5A at the locus.

An SNP reducing SNORD105 and PPAN expression decreases the risk of hepatocellular carcinoma in a Chinese population.

BackgroundWith hepatocellular carcinoma (HCC) becoming a heavy disease burden in China, it is particular to reveal its pathological mechanism. Recent researches have indicated that small nucleolar RNAs (snoRNAs) may be involved in various cancers including HCC. Polymorphisms within snoRNAs may affect its function or expression level, and even its host gene, then produce series of effects related to itself or its host gene.MethodsThe association of the single nucleotide polymorphism (SNP) rs2305789 in SNORD105 with HCC susceptibility was evaluated in two independent case‐control sets (712 HCC and 801 controls). The contribution of rs2305789 to HCC risk was investigated using case‐control, genotype‐phenotype correlation analysis, and functional assays.ResultsThe SNP rs2305789 was significantly associated with a decreased risk of HCC in both case‐control sets (OR = 0.80, 95% CI: 0.69–0.93, p = 0.003). Compared with the AA genotype, the GG genotype was significantly correlated with lower expression of both SNORD105 and PPAN (p < 0.01). Furthermore, the overexpressed SNORD105 up‐regulated PPAN expression level (p < 0.05). Finally, the in vivo experiment showed that the overexpressed SNORD105 increased cell viability and motility in both HepG2 and Huh7 cell lines (p < 0.05).ConclusionsTo sum up, our results suggested that rs2305789 decreased the risk of HCC by reducing the expression of both SNORD105 and PPAN, which reduced HCC cell viability and motility.

TIM-3 polymorphism is involved in the progression of esophageal squamous cell carcinoma by regulating gene expression.

The T-cell immunoglobulin and mucin domain containing molecule 3 (TIM-3), a crucial immune regulatory molecule, is an emerging immune checkpoint target for cancer therapy. Our study aimed to investigate the association between TIM-3 polymorphisms (rs10053538 C > A, rs10515746 C > A, and rs1036199 A > C) and the susceptibility and prognosis of esophageal squamous cell carcinoma (ESCC). We further detect the effects of polymorphisms on TIM-3 expression. Two independent case-control sets (population-based and hospital-based sets) were performed in total 994 ESCC patients and 998 controls. TIM-3 polymorphisms were genotyped by polymerase chain reaction-ligase detection reaction (PCR). Survival data were available for 198 patients who received platinum-based chemotherapy after surgery. The regulation on TIM-3 expression by the polymorphisms was investigated in 35 patients using real-time quantitative PCR. The association between mRNA level of TIM-3 and survival was detected by using Kaplan-Meier plotter database. We found that for rs10053538 C > A polymorphisms, A allele was associated with significant increased risk of ESCC (odds ratios [OR] = 1.34, 95%CI = 1.05-1.72), and CA/AA genotypes enhanced susceptibility to ESCC for smokers (adjusted OR = 1.61, 95%CI = 1.00-2.59). The patients with AA genotypes had significantly poor prognosis (adjusted HR = 4.98, 95%CI = 1.14-21.71). The patients carrying CA/AA genotypes had significantly higher mRNA levels of TIM-3 than those carrying the CC genotype. Furthermore, high mRNA level of TIM-3 had a shorter overall survival in patients (HR = 2.56, 95%CI = 1.04-6.28). For rs10515746 C > A and rs1036199 A > C polymorphisms, there were no statistical correlation with the progression of ESCC. These data demonstrate that rs10053538 C > A polymorphisms may be associated with the susceptibility and prognosis of ESCC patients through regulating expression of TIM-3.

MiR-1262 suppresses gastric cardia adenocarcinoma via targeting oncogene ULK1.

Gastric cardia adenocarcinoma (GCA) is one of two main gastric cancer subtypes and has its own epidemiological, pathogenic and clinical characteristics. Genetic polymorphisms locating in a microRNA (miRNA) gene enhancer could transcriptionally regulates miRNA expression via impacting binding of transcriptional factors. It is still unclear how miR-1262 and a potential regulatory rs12740674 polymorphism mapping to a strong enhancer region of miR-1262 contribute to GCA development. We genotyped miR-1262 rs12740674 in two independent case-control sets consisting of 1,024 GCA patients and 1,118 controls, and found that the rs12740674 CT or TT genotype carriers had a 0.69-fold decreased risk to develop GCA compared to the CC genotype carriers (95% confidence interval=0.57-0.84, P=2.1×10-4). In the genotype-phenotype correlation analyses of 21 pairs of GCA-normal tissues, the rs12740674 CT or TT genotype was associated with significantly increased levels of miR-1262. Cell proliferation, wound healing and transwell assays elucidated that miR-1262 is a novel GCA tumor suppressor. Consistently, a significantly down-regulated level of miR-1262 exists in GCA specimens compared to normal tissues. Furthermore, multiple lines of evidences indicated that oncogene ULK1 is the target gene of miR-1262 in GCA. Our findings demonstrate miR-1262 transcriptionally modulated by an enhancer genetic variant suppresses GCA via targeting oncogene ULK1. Our data highlight miR-1262 as a promising diagnostic marker and therapeutic target for GCA.

Anti‐N‐methyl‐D‐aspartate receptor (NMDAR) encephalitis is associated with IRF7, BANK1 and TBX21 polymorphisms in two populations

Autoantibodies targeting the GluN1(NR1) subunit of the anti-N-methyl-D-aspartate receptor (NMDAR) cause encephalitis. Although it has been shown that anti-NMDAR encephalitis is associated with human leukocyte antigen (HLA) loci, susceptibility genes for the disease outside the HLA loci remain unidentified. In this study, we aimed to explore the association of anti-NMDAR encephalitis with non-HLA genes. Two Chinese anti-NMDAR encephalitis cohorts from Han populations were recruited for this study. The North Chinese case-control set consisted of 98 patients and 460 controls, while the South Chinese case-control set included 78 patients and 541 controls. All participants were genotyped for 28 single nucleotide polymorphisms that are associated with autoimmune disorders or infectious diseases. In two independent case-control sets, we identified significant associations of anti-NMDAR encephalitis with IRF7 rs1131665 (odds ratio [OR] 3.34, 95% confidence interval [CI] 1.99-5.63; P<0.000001, Padjusted =0.00004), BANK1 rs4522865 (OR1.44, 95% CI 1.15-1.82; P=0.0017, Padjusted =0.0149), and TBX21 rs17244587 (OR2.03, 95% CI 1.35-3.05; P=0.00051, Padjusted =0.0066). Furthermore, analysis of the three polymorphisms with clinical features of the disease revealed that the IRF7 rs1131665 was associated with tumor status. The present study has for the first time identified non-HLA susceptibility genes for anti-NMDAR encephalitis. The association of IRF7, BANK1 and TBX21 with anti-NMDAR encephalitis suggests that B-cell activation, Th1 responses, virus infection and the type I interferon signaling pathway are involved in the pathogenesis of the disease.

Genome-Wide Association Study (GWAS) Identifies a Significant Acute Myeloid Leukemia (AML) Susceptibility Locus Near BICRA

Introduction: AML is the most common form of acute leukemia in adults, but hereditary predisposition to AML has been insufficiently studied. To date, investigations of AML heritability have focused on families with a clear history of AML and identified rare highly penetrant germline mutations that segregate with disease. However, the role of common polymorphisms in susceptibility to AML is not clear. We therefore conducted the first large-scale GWAS to identify risk alleles for adult de novo AML.Methods: Whole-genome genotyping was performed on two independent American case-control sets, using Illumina Infinium Omni-1 Quad-bead arrays. GWAS quality control measures consisted of excluding samples with <94% genotyping yield or <90% European ancestry, and variants with <94% yield, differences among batches or significant (P<10-6) deviation from Hardy-Weinberg equilibrium. Phasing and imputation yielded a total of 16,058,266 autosomal variants. Association tests were performed using logistic regression including age, sex, and ten principal components as co-variates. Selected variants were genotyped in a German validation case-control set using an Agena MassARRAY System. Results were combined using METAL with fixed effects. P<5x10-8 was considered the threshold for genome-wide significance.Results: Two separate GWASs were performed in a total of 1,183 American AML patients from the Alliance for Clinical Trials in Oncology and 2,369 population- and age-matched controls from independent case-control sets (USA1: 894 cases and 1,714 controls; USA2: 289 cases and 655 controls). After combining the results from these two data sets in a meta-analysis, five independent loci with a total of 11 polymorphisms were suggestively associated with AML (P<10-6), located at: 1q41 (P=7.2x10-7), 13q22.1 (P=3.1x10-7), 18q22.3 (P=7.4x10-7), 19p13.11 (P=4.0x10-7) and 19q13.33 (P=3.0x10-7) (Figure 1). Six variants with the lowest P-values from three loci (13q22.1, 19p13.11 and 19q13.33) were selected and genotyped in an additional 350 German patient samples and compared with 1,600 population- and age-matched controls from the German LIFE-Adult study. In the German sample set alone, both the 19p13.11 and the 19q13.33 loci showed support for association with AML (P<0.05). Results from the three independent sample sets were combined in an overall meta-analysis to calculate the final odds ratios (ORs) and significance (Table 1). The overall P-value for the 19q13.33 variant, rs75797233, was 4.15x10-8 (OR=2.28), which meets the threshold for genome-wide significance (P<5x10-8), making it the first reported common germline variant significantly associated with AML risk, with a risk allele frequency (RAF) in the cases of 3.1% compared to a RAF of 1.8% in the controls.rs75797233 is located 13kb 5′ of the BICRA gene, which encodes a key component in a newly described SWI/SNF chromatin remodeling complex that can impact on cancer cell proliferation. GTEx expression data revealed that individuals who have the rs75797233 AML risk allele (thymine [T]) have significantly lower expression of BICRA in whole blood samples compared with individuals who are homozygous for the non-risk rs75797233 allele (adenine [A]; P=0.0002). As rs75797233 is located in a region of open chromatin marked by histone H3 lysine 4 mono-methylation, we examined ENCODE chromatin immunoprecipitation sequencing (ChIP-seq) data for transcription factor binding. Indeed, the GATA2 transcription factor showed a clear ChIP-seq binding peak that encompassed rs75797233 and the surrounding region. Analysis of transcription factor motifs using MotifbreakR indicated that when the non-risk allele of rs75797233 (A) is present it is in a critical position of a GATA2 binding site; however, GATA2 is not able to bind to this site when the rs75797233 risk allele (T) is present (Figure 2).Conclusions: Our study sheds light on the lower-penetrance heritability of AML and identifies rs75797233 as the first common AML risk allele. rs75797233 might associate with AML risk through mechanisms involving regulation of BICRA by GATA2, and ongoing characterization of the risk locus could yield novel insights into AML disease susceptibility. DisclosuresKolitz:Magellan Health: Consultancy, Honoraria. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Wang:Amgen: Consultancy; Amgen: Consultancy; Jazz: Speakers Bureau; Jazz: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Niederwieser:Miltenyi: Speakers Bureau; Novartis: Research Funding.

DNA Repair Genes May Influence Cognitive Outcomes in Adult Patients with Hematologic Malignancies (HM) Treated with Blood or Marrow Transplantation (BMT)

Introduction: We recently demonstrated that cognitive impairment (CI) is prevalent and persistent in HM patients treated with BMT (Sharafeldin et al., J Clin Oncol, 2018). While the impact of genetic factors on cognition in the non-oncology realm is well-recognized, limited information exists in HM patients treated with BMT. We tested the hypothesis that genetic variants in blood-brain barrier (BBB), drug transporter, telomere homeostasis, neurotransmission, neural repair and DNA repair are associated with CI in HM patients treated with BMT. We previously presented preliminary results of the Discovery cohort (Sharafeldin et al., Blood, 2016); here we present additional analyses (at the gene level) on the Discovery cohort, and replication of salient findings in an independent Validation cohort.Methods: The Discovery cohort consisted of prospective, longitudinally-assessed patients undergoing autologous or allogeneic BMT for HM at a single center. Cognitive function was assessed objectively using a battery of standardized tests prior to BMT, and at 6mo, 1y, 2y, and 3y post-BMT; germline DNA was collected pre-BMT. Global Deficit Score (GDS) was used as an indicator of CI (GDS ≥0.5 indicated CI). We employed a candidate gene approach, relying on biologically-plausible hypotheses for gene selection. Standard quality control procedures yielded 985 SNPs in 68 genes with a total genotyping call rate of 99% in 277 BMT recipients. Generalized estimating equation models were fitted to examine association of individual SNPs with CI. Linkage disequilibrium-based pruning yielded 326 independent tests, with an overall p-value threshold of 1.53x10-4 using Bonferroni correction for multiple testing. We also conducted gene-level analysis considering each gene as a SNP-set using the machine learning approach of logic regression, with cross-validation, to identify SNP-SNP interactions at the gene-level (Bonferroni p ≤ 7.94x10-4). An independent Validation case-control set from the BMT Survivor Study was used to replicate significant findings from the Discovery cohort, using conditional logistic regression models. Cases (n=141) were BMT survivors who reported: i) presence of memory/learning problems; and ii) severity of memory/learning problems. Controls (n=258) were BMT survivors without cognitive problems, matched (1-2/case) on race/ethnicity, HM, and time from BMT.Results:Discovery: Among the 277 patients in the Discovery cohort, mean age at BMT was 50y, 59% were males, 69% were non-Hispanic whites and 47% received an allogeneic BMT. Male sex (OR=3.2, 95%CI, 1.5-6.7, p=0.002) and lower pre-BMT cognitive reserve (OR=4.6, 95%CI, 2.2-9.6, p <0.0001) were associated with CI. Risk of CI was increased in the younger patients (<50y) if they received TBI (age*TBI interaction OR=3.86, 95%CI, 1.0-15.1, p=0.05). Final genetic models were adjusted for relevant variables with significant independent main effects including BMT type, age at BMT, sex, race/ethnicity, and cognitive reserve. We identified significant associations between CI and BBB, telomere homeostasis and DNA repair SNPs using the single-SNP (Fig 1) and gene-level analyses. Replication: The mean age at BMT was 44y, 54% were males, 88% were non-Hispanic whites and 5% received an allogeneic BMT. Replication models were adjusted for age at BMT, sex, race, and education and income levels. Of the 69 SNPs identified for replication, DNA repair SNPs from the single-SNP analysis rs13006837 on XRCC5 (OR=5.8, 95%CI, 2.7-12.3, p=2.0 x 10-5) and rs293796 on OGG1 gene (OR=11.4, 95%CI, 4.6-28.5, p-value=1.08 x 10-6) were successfully replicated. Five SNPs on DNA repair genes from the gene-level analysis showed individual statistically-significant associations in the Validation cohort: rs238417 on ERCC2 (OR=0.48, 95%CI, 0.25-0.91, p=0.03); rs2526698 on EXO1 (OR=0.57, 95%CI: 0.33-0.98, p=0·04); rs3793906 on MGMT (OR=1.96, 95%CI: 1.2-3.3, p=0.01); rs4725016 on RPA3 (OR=0.20, 95%CI, 0.05-0.92, p=0.04); and rs1479569 on XRCC4 (OR=2.3, 95%CI, 1.3-4.1, p=0.005).Conclusion: Our study provides genetic characterization of adult HM patients at increased risk of cognitive impairment following BMT; associations with DNA repair variants were independently replicated. These findings could have implications towards the personalized management of patients undergoing BMT. DisclosuresForman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

Plasma amino acid profile in major depressive disorder: Analyses in two independent case-control sample sets

Some amino acids act as neurotransmitters themselves, or are precursors of neurotransmitters. Previous studies reported inconsistent results regarding their changes in blood in major depressive disorder (MDD), which prompted us to examine plasma levels of amino acids and related molecules in two independent case-control sample sets. In total, 511 subjects were recruited. Sample set A consisted of 164 patients with MDD (147 currently depressed [dMDD]; 17 in remission, DSM-IV) and 217 healthy controls. Sample set B consisted of 65 patients (51 dMDD; 14 in remission) and 65 controls. Plasma amino acid levels were measured using high-performance liquid chromatography for set A and liquid chromatography/mass spectrometry for set B. We further analyzed the relationships between plasma amino acid levels and clinical variables. In sample set A, plasma asparagine, histidine+1-methylhistidine, methionine, phenylalanine, tryptophan, and tyrosine levels were decreased, while plasma glutamate and phosphoethanolamine were elevated in dMDD compared to controls (all P < 0.0005), even after correcting for multiple testing. Plasma leucine levels were associated with “psychic anxiety.” In sample set B, glutamate and methionine levels were also altered in the same direction to that in sample set A (both P < 0.05). In the integrative analysis, plasma glutamate and methionine levels were found to be significantly associated with the diagnosis of MDD with small to medium effect sizes (both P < 1.0E-6). In conclusion, several amino acids and related molecules were altered in patients with MDD. Decreased methionine and increased glutamate levels were found consistently in the two sample sets, suggesting their involvement in MDD. Further investigations are warranted on the possible role of amino acids in the pathophysiology of MDD.

Predicting outcome: role of gene signatures

Pathological assessments, such as Gleason grading, which is a strong clinical predictor of prostate cancer progression 1, have a role to play in predicting the outcome of a patient's response to therapy. The addition of PSA and TNM staging are further used to inform appropriate treatment strategies in patients who are at low, intermediate and high risk of disease progression. Unfortunately, approximately 30% of men with intermediate-risk prostate cancer will fail to be cured by surgery or radiation therapy approaches. This is not surprising as primary prostate cancer represents a complex heterogeneous disease that is clearly not fully explained by the current clinical prognostic factors, and further molecular characterization is required. There is now significant emerging evidence that the molecular characterization of tumours is important to enable us to stratify prostate cancer patients by their response to primary therapy and to identify the next appropriate steps in their treatment pathway. Long et al. 2 have identified gene signatures that can define different genomic subtypes of prostate cancer and are predictive of biochemical recurrence. Erho et al. 3 discovered and validated a 22-gene signature, which they termed a genomic classifier for the prediction of early metastasis after radical prostatectomy, while Penny et al. 4 have identified an mRNA expression signature of Gleason grade which is predictive of lethal prostate cancer. Long et al. 2 identified a 24-gene signature, which they discovered through RNA sequencing analysis of 100 formalin-fixed paraffin-embedded prostatectomy samples. They went on to validate their findings in a publically available independent gene expression microarray dataset of 140 patients. This 24-gene signature forms the basis of the present study by Pellegrini et al. 5, who refer to this gene signature as Sig24. In their study, they firstly undertook to determine if Sig24 was associated with pathological Gleason score as a marker of tumour aggressiveness, and then if it had prognostic value for the identification of patients at risk of metastasis or prostate cancer-specific mortality after radical prostatectomy. The Gleason score association study was carried out using the data from three independent case–control sets, including 182 patients from the Cleveland Clinic and two cohorts (cohort I, n = 545; cohort II, n = 235) from the Mayo Clinic, for which gene expression analysis had previously been conducted by Genome Dx using the Affymetrix Human 1.0 ST Genechip platform. The Sig24 score was calculated for each patient and higher Gleason score was associated with significantly higher Sig24 scores. The association studies for metastatic disease and prostate cancer-specific mortality, however, were only carried out in the Mayo Clinic cohort II. For both clinical endpoints the Sig24 score combined with the clinical model outperformed the clinical model alone of PSA, Gleason score and tumour stage. For metastatic disease the area under the curve for the clinical model alone was 0.69 (0.62–0.77) compared with 0.73 (0.66–0.78) for the clinical model combined with Sig24. For the prostate cancer-specific mortality endpoint, the area under the curve for the clinical model alone was 0.69 (0.67–0.87) compared with 0.74 (0.63–0.85) for the clinical model combined with Sig24. These gene signatures have significant potential for predicting the progression of disease rather than waiting for PSA relapse, which is currently used to identify disease recurrence, with interval to biochemical failure being the best univariate factor predicting prostate cancer mortality and overall survival 6. This would allow the initiation of additional therapies before recurrence and a better outcome for the patient. This concept has been demonstrated in a study in which the use of the Decipher gene signature was shown to improve the identification of patients who could benefit from adjuvant radiotherapy and thus only these patients were targeted for therapy 7. Incorporating these gene signatures in robust clinical assays and integrating them into clinical decision-making is the next essential step in order for these strategies to have an impact on patient outcomes. None declared.

Genome-wide association study identifies HLA-DR variants conferring risk of HBV-related acute-on-chronic liver failure

ObjectiveAcute-on-chronic liver failure (ACLF) is an extreme condition after severe acute exacerbation of chronic hepatitis B; however, the underlying genetic factors involved in its onset and progression are currently unclear.DesignWe...

An indel polymorphism within pre-miR3131 confers risk for hepatocellular carcinoma.

Polymorphisms in pre-miRNAs may affect its expression, then have effect on its target mRNAs and be associated with cancer susceptibility. In this study, we evaluated the association of an indel polymorphism rs57408770 in pre-miR-3131 with hepatocellular carcinoma (HCC) susceptibility in a Chinese population. The contribution of rs57408770 to HCC risk was investigated in two independent case-control sets (1051 HCC and 1058 controls). Logistic regression analysis showed that the insertion allele of rs57408770 was significantly associated with an increased risk for HCC occurrence in both case-control studies. Moreover, the results of genotype-phenotype correlation analysis from both in vivo and in vitro experiments showed that the insertion allele was significantly correlated with higher expression of mature miR-3131 comparing with the deletion allele. The RNA-Binding Protein Immunoprecipitation assay results indicated that rs57408770 could affect the expression level of mature miR-3131 probably through disturbing the binding of splicing factor SRp20 with pre-miR-3131. Furthermore, overexpression of miR-3131 displayed a proliferation promoting and anti-apoptosis effect on HCC cell lines, suggesting that miR-3131 may act as a proto-oncogene in HCC. Finally, human genome-wide gene expression profile assay was used to screen the targets of miR-3131. The overexpressed miR-3131 could lead to a significant decrease of DTHD1 and XAF1 mRNA level. Taken together, our findings provided evidence that rs57408770 may play a functional role in the carcinogenesis of HCC by affecting SRp20 binding with pre-miR-3131 and affecting the expression of mature miR-3131, subsequently affecting the expression of DTHD1 and XAF1, thus confers risk for HCC.

Association of the GLB1 rs4678680 genetic variant with risk of HBV-related hepatocellular carcinoma.

Accumulated evidences demonstrated that GLB1 is involved in cell senescence and cancer development. The GLB1 rs4678680 single nucleotide polymorphism (SNP) has been identified as a hepatocellular carcinoma (HCC) susceptibility polymorphism by a genome-wide association study in Korean population previously. However, little or nothing was known about its involvement and functional significance in hepatitis B viruses (HBV)-related HCC in Chinese. Therefore, we investigated the association between the GLB1 rs4678680 SNP and HBV-related HCC risk as well as its biological function in vivo. Genotypes were determined in two independent case-control sets from two medical centers of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The potential regulation role the rs4678680 genetic variant on GLB1 expression was examined with HCC and normal liver tissues. We found that The rs4678680 G allele was showed to be risk allele; individuals with the TG genotype had an OR of 1.51 (95% CI = 1.10–2.07, P = 0.010, Shandong set) or 1.49 (95% CI = 1.11–1.99, P = 0.008, Jiangsu set) for developing HBV-related HCC, respectively, compared with individuals with the TT genotype. This association was more pronounced in males, individuals aged older than 57 years and drinkers (all P < 0.05). In the genotype-phenotype correlation analyses of fifty-six human liver tissue samples, rs4678680 TG or GG was associated with a statistically significant increase of GLB1 mRNA expression (P < 0.05). Our data indicated that the GLB1 rs4678680 SNP contributes to susceptibility to develop HBV-related HCC, highlighting the involvement of GLB1 and cell senescence in etiology of HCC.

Erratum: Functional BCL-2 regulatory genetic variants contribute to susceptibility of esophageal squamous cell carcinoma

B-cell lymphoma-2 (BCL-2) prevents apoptosis and its overexpression could promote cancer cell survival. Multiple functional BCL-2 genetic polymorphisms, such as rs2279115, rs1801018 and rs1564483, have been identified previously and might be involved in cancer development through deregulating BCL-2 expression. Therefore, we examined associations between these three polymorphisms and esophageal squamous cell carcinoma (ESCC) susceptibility as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1588 ESCC patients and 1600 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. The impact of the rs2279115 polymorphism on BCL-2 expression was detected using esophagus tissues. Our results demonstrated that the BCL-2 rs2279115 AA genotype was significantly associated with decreased ESCC risk compared with the CC genotype (OR = 0.72, 95% CI = 0.57–0.90, P = 0.005), especially in nonsmokers (OR = 0.42, 95% CI = 0.29–0.59, P = 0.001) or nondrinkers (OR = 0.44, 95% CI = 0.32–0.62, P = 0.002). Genotype-phenotype correlation studies demonstrated that subjects with the rs2279115 CA and AA genotypes had a statistically significant decrease of BCL-2 mRNA expression compared to the CC genotype in both normal and cancerous esophagus tissues. Our results indicate that the BCL-2 rs2279115 polymorphism contributes to ESCC susceptibility in Chinese populations.

Association of the functional BCL-2 rs2279115 genetic variant and small cell lung cancer.

As a well-known oncogene, B cell lymphoma-2 (BCL-2) can promote cancer cell survival through preventing their apoptosis. Several functional BCL-2 single nucleotide polymorphisms (SNPs), such as rs2279115, rs1801018, and rs1564483, have been identified and might contribute to cancer susceptibility. However, the involvement of these SNPs in small cell lung cancer (SCLC) was still unclear. As a result, we investigated associations between these three genetic variants and SCLC risk in a case-control design. Genotypes were determined in two independent case-control sets consisted of 520 SCLC patients and 1040 controls from two medical centers. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated utilizing unconditional logistic regression. We found that only BCL-2 rs2279115 genetic variant significantly contributed to decreased SCLC risk in Chinese Han populations, with the rs2279115 A allele as the protective allele. Stratified analyses of association between BCL2 rs2279115 SNP and SCLC risk indicated that the functional polymorphism was only significantly associated with decreased risk of the limited stage SCLC but not the extensive stage disease. Our results indicate that the BCL-2 rs2279115 genetic variant was associated with SCLC risk in Chinese populations and support the hypothesis that SNPs in regulatory regions of oncogenes might contribute to cancer susceptibility.

A Regulatory MDM4 Genetic Variant Locating in the Binding Sequence of Multiple MicroRNAs Contributes to Susceptibility of Small Cell Lung Cancer.

A functional rs4245739 A>C single nucleotide polymorphism (SNP) locating in the MDM43’-untranslated (3’-UTR) region creates a miR-191-5p or miR-887-3p targeting sites. This change results in decreased expression of oncogene MDM4. Therefore, we examined the association between this SNP and small cell lung cancer (SCLC) risk as well as its regulatory function in SCLC cells. Genotypes were determined in two independent case-control sets consisted of 520SCLC cases and 1040 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. The impact of the rs4245739 SNP on miR-191-5p/miR-887-3p mediated MDM4 expression regulation was investigated using luciferase reporter gene assays. We found that the MDM4 rs4245739AC and CC genotypes were significantly associated with decreased SCLC susceptibility compared with the AA genotype in both case-control sets (Shandong set: OR = 0.53, 95% CI = 0.32–0.89, P = 0.014; Jiangsu set: OR = 0.47, 95% CI = 0.26–0.879, P = 0.017). Stratified analyses indicated that there was a significantly multiplicative interaction between rs4245739 and smoking (P interactioin = 0.048). After co-tranfection of miRNAs and different allelic-MDM4 reporter constructs into SCLC cells, we found that the both miR-191-5p and miR-887-3p can lead to significantly decreased MDM4 expression activities in the construct with C-allelic 3’-UTR but not A-allelic 3’-UTR, suggesting a consistent genotype-phenotype correlation. Our data illuminate that the MDM4rs4245739SNP contributes to SCLC risk and support the notion that gene 3’-UTR genetic variants, impacting miRNA-binding, might modify SCLC susceptibility.

Association between indel polymorphism in the promoter region of lncRNA GAS5 and the risk of hepatocellular carcinoma.

The growth arrest special 5 (GAS5) is known to be involved in various cancers. However, its expression regulation remains unclear. Polymorphisms in the promoter region of GAS5 may affect its expression and be associated with cancer susceptibility. In this research, we first evaluated the association of a 5-base pair indel polymorphism (rs145204276) in the promoter region of GAS5 with hepatocelluar carcinoma (HCC) susceptibility in Chinese populations. Logistic regression analysis showed that the deletion allele of rs145204276 significantly increased the risk of HCC in two independent case control sets (1034 HCC and 1054 controls). Further genotype-phenotype association analysis revealed that the deletion allele was markedly correlated with higher expression of GAS5 in HCC tissues. The luciferase activity analysis in an in vitro reporter gene system suggested that the deletion allele improved an increased expression of GAS5 in three hepatoma cell lines. Intriguingly, overexpression of GAS5 displayed an anti-apoptosis effect in HCC cell lines, GAS5 knockdown could partially revert this anti-apoptosis effect, suggesting that GAS5 may act as a proto-oncogene in HCC, in contrast with its inhibitory role in other cancers. Further pyrosequencing revealed that the genotypes of rs145204276 were associated with methylation status of GAS5 promoter region. Taken together, our findings provided evidence that rs145204276 may contribute to hepatocarcinogenesis by affecting methylation status of the GAS5 promoter and subsequently its transcriptional activity thus serving as a potential therapy target for HCC.

Functional BCL-2 regulatory genetic variants contribute to susceptibility of esophageal squamous cell carcinoma.

B-cell lymphoma-2 (BCL-2) prevents apoptosis and its overexpression could promote cancer cell survival. Multiple functional BCL-2 genetic polymorphisms, such as rs2279115, rs1801018 and rs1564483, have been identified previously and might be involved in cancer development through deregulating BCL-2 expression. Therefore, we examined associations between these three polymorphisms and esophageal squamous cell carcinoma (ESCC) susceptibility as well as its biological function in vivo. Genotypes were determined in two independent case-control sets consisted of 1588 ESCC patients and 1600 controls from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression. The impact of the rs2279115 polymorphism on BCL-2 expression was detected using esophagus tissues. Our results demonstrated that the BCL-2 rs2279115 AA genotype was significantly associated with decreased ESCC risk compared with the CC genotype (OR = 0.72, 95% CI = 0.57–0.90, P = 0.005), especially in nonsmokers (OR = 0.42, 95% CI = 0.29–0.59, P = 0.001) or nondrinkers (OR = 0.44, 95% CI = 0.32–0.62, P = 0.002). Genotype-phenotype correlation studies demonstrated that subjects with the rs2279115 CA and AA genotypes had a statistically significant decrease of BCL-2 mRNA expression compared to the CC genotype in both normal and cancerous esophagus tissues. Our results indicate that the BCL-2 rs2279115 polymorphism contributes to ESCC susceptibility in Chinese populations.

A functional lncRNA HOTAIR genetic variant contributes to gastric cancer susceptibility.

Long noncoding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) acts as an oncogene in gastric cancer development. HOTAIR could induce genome-wide retargeting of polycomb-repressive complex 2, trimethylates histone H3 lysine-27 (H3K27me3) and deregulation of multiple downstream genes. Additionally, as the ceRNA of miR-331-3p, HOTAIR may modulate HER2 deregulation in gastric cancer cells. We hypothesized that the functional single nucleotide polymorphisms (SNP) in HOTAIR may affect HOTAIR expression and/or its function and, thus, gastric cancer risk. We examined the association between three haplotype-tagging SNPs (htSNP) across the whole HOTAIR locus and gastric cancer risk as well as the functional relevance of a gastric cancer susceptibility SNP rs920778. Genotypes were determined in two independent hospital-based case-control sets that consisted of 800 gastric cancer patients and 1600 controls. The allele-specific regulation on HOTAIR expression by the rs920778 SNP was examined in vitro and in vivo. We found that the HOTAIR rs920778 TT carriers had a 1.66- and 1.87-fold increased gastric cancer risk in Jinan and Huaian populations compared with the CC carriers (P = 4.2 × 10(-4) and 6.5 × 10(-5)). During inspecting functional relevance of the rs920778 SNP, we observed an allelic regulation of rs920778 on HOTAIR expression in both gastric cancer cell lines and tissue samples, with higher HOTAIR expression among T allele carriers. These findings elucidate that functional genetic variants influencing lncRNA expression may explain a portion of gastric cancer genetic basis.

Leukocyte telomere length-related rs621559 and rs398652 genetic variants influence risk of HBV-related hepatocellular carcinoma.

Recent genome-wide association studies (GWAS) have identified eleven leukocyte telomere length (LTL)-related single nucleotide polymorphisms (SNPs). Since LTL has been associated with risk of many malignancies, LTL-related SNPs may contribute to cancer susceptibility. To test this hypothesis in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), we genotyped these eleven LTL-related SNPs in a case-control set including 1186 HBV-related HCC cases, 508 chronic HBV carriers and 1308 healthy controls at the discovery stage. The associations of HCC risk with these SNPs were further confirmed in an independent case-control set. We found that 1p34.2 rs621559 and 14q21 rs398652 were significantly associated with HBV-related HCC risk (both P<0.005 after Bonferroni corrections). There was no significant difference of either rs621559 or rs398652 genotypes between chronic HBV carriers and healthy controls, demonstrating that the association was not due to predisposition to HBV infection. In the pooled analyses (1806 HBV-related HCC cases and 1954 controls), we observed a decreased HCC risk, 0.72-times, associated with the 1p34.2 rs621559 AA genotype compared to the GG genotype (P = 1.6×10−6). Additionally, there was an increased HCC risk, 1.27-fold, associated with the rs398652 GG genotype (P = 3.3×10−6). A statistical joint effect between the rs621559 GG and rs398652 GG genotypes may exist in elevating risk of HBV-related HCC. We show, for the first time, that rs398652 and rs621559 might be marker genetic variants for risk of HBV-related HCC in the Chinese population.