Yajie Gong

A functional polymorphism in lnc-LAMC2-1:1 confers risk of colorectal cancer by affecting miRNA binding

Genome-wide association studies (GWASs) have identified multiple susceptibility loci of colorectal cancer (CRC), however, causative polymorphisms have not been fully elucidated. Long non-coding RNAs (lncRNAs) are a recently discovered class of non-protein coding RNAs that involved in a wide variety of biological processes. We hypothesized that single nucleotide polymorphisms (SNPs) in lncRNA may associate with the CRC risk by influencing lncRNA functions. To evaluate the effects of SNPs on CRC susceptibility in Chinese populations, we first screened out all potentially functional SNPs in exons of lncRNAs located in CRC susceptibility loci identified by GWAS. Eight SNPs were selected and genotyped in 875 CRC cases and 855 controls and replicated in an independent case-control study consisting of 768 CRC cases and 768 controls. Analyses showed that CG and GG genotypes of the rs2147578 were significantly associated with increased risk for CRC occurrence in both case-control studies [combined analysis OR = 1.29; 95% confidence interval (CI) = 1.11-1.51, P = 0.001] compared to the rs2147578 CC genotype. Bioinformatics analyses showed that rs2147578 is located in the transcript of lnc-LAMC2-1:1 and could influence the binding of lnc-LAMC2-1:1/miR-128-3p. Further luciferase reporter assays demonstrated that the construct with the risk rs2147578G allele had relatively high expression activity compared with that of the rs2147578C allele. Expression quantitative trait loci analyses also showed that rs2147578 is correlated with the expression of a well established oncogene LAMC2 (laminin subunit gamma 2). These findings indicated that rs2147578 in lnc-LAMC2-1:1 might be a genetic modifier for the development of CRC.

A low-frequency variant in SMAD7 modulates TGF-β signaling and confers risk for colorectal cancer in Chinese population

The TGF‐β pathway plays an essential role in regulating cell proliferation and differentiation. GWASs and candidate approaches have identified a battery of genetic variants in the TGF‐β pathway contributing to colorectal cancer (CRC). However, most of the significant variants are common variants and their functions remain ambiguous. To identify causal variants with low‐frequency in the TGF‐β pathway contributing to CRC susceptibility in Chinese population, we performed targeted sequencing of 12 key genes in TGF‐β signaling in CRC patients followed by a two‐stage case‐control study with a total of 5109 cases and 5169 controls. Bioinformatic annotations and biochemical experiments were applied to reveal the potential functions of significant variants. Seven low‐frequency genetic variants were captured through targeted sequencing. The two stage association studies showed that missense variant rs3764482 (c. 83C>T; p. S28F) in the gene SMAD7 was consistently and significantly associated with CRC risk. Compared with the wild type, the ORs for variant allele were 1.37 (95%CI: 1.10‐1.70, P = 0.005), 1.55 (95%CI: 1.30‐1.86, P = 1.15 × 106), and 1.48 (1.29‐1.70, P = 2.44 × 10;8) in stage 1, stage 2, and the combined analyses, respectively. Functional annotations revealed that the minor allele T of rs3764482 was more effective than the major allele C in blocking the TGF‐β signaling and inhibiting the phosphorylation of receptor‐regulated SMADs (R‐SMADs). In conclusion, low‐frequency coding variant rs3764482 in SMAD7 is associated with CRC risk in Chinese population. The rs3764482 variant may block the TGF‐β signaling via impeding the activation of downstream genes, leading to cancer cell proliferation, thus contributing to CRC pathogenesis.

A functional polymorphism located at transcription factor binding sites, rs6695837 near LAMC1 gene, confers risk of colorectal cancer in Chinese populations.

Genome-wide association studies (GWASs) have identified multiple single nucleotide polymorphisms (SNPs) associated with colorectal cancer (CRC) susceptibility. However, the elucidation of causal SNPs and the biological mechanisms behind are still limited. In this study, we initially performed systematic bioinformatics analyses on CRC GWAS-identified loci to seek for potential functional SNPs located at transcription factor binding sites (TFBSs), and then a two-stage case-control study comprised of 1353 cases and 1448 controls of Chinese populations and functional analyses were conducted. As a result, only one SNP rs6695837 out of the nine candidate SNPs survived after two-stage analyses by Bonferroni correction. In combined analyses, rs6695837 exhibited significant associations with CRC risk (TT: CC, odds ratio (OR) = 1.31, 95% confidence interval (CI) = 1.06-1.63; dominant model, OR = 1.21, 95% CI = 1.03-1.43; additive model, OR = 1.15, 95% CI = 1.03-1.28). Functional annotations by RegulomeDB and rSNPBase indicated its biological role and dual-luciferase reporter assays revealed a significant increase in luciferase expression for the reconstructed plasmid with rs6695837T allele, compared with the one with C allele (PSW480 = 0.0002, PLovo = 0.0003). Further gene expression analyses demonstrated significantly higher expression of LAMC1 gene in CRC tumor tissues than that in adjacent non-cancerous tissues (P = 0.0004). These findings strongly suggest that the functional SNP located at TFBSs, rs6695837 might contribute to CRC susceptibility, and the exact biological mechanism awaits further research.

A polymorphic MYC response element in KBTBD11 influences colorectal cancer risk, especially in interaction with an MYC-regulated SNP rs6983267

Background MYC is a well-established cancer driver gene regulating the expression of numerous genes, indicating that polymorphisms in MYC response elements could affect tumorigenesis through altering MYC regulation. We performed integrative multistage study to evaluate the effects of variants in MYC response elements and colorectal cancer (CRC) risk. Patients and methods We systematically integrated ChIP-Seq, DNase-Seq and transcription factor motif data to screen variants with potential ability to affect the MYC binding affinity. Then, we conducted a two-stage case-control study, totally consisting of 4830 CRC cases and 4759 controls in Chinese population to identify risk polymorphisms and interactions. The effects of risk variants were confirmed by functional assays in CRC LoVo, SW480 and HCT15 cells. Results We identified a novel polymorphism rs11777210 in KBTBD11 significantly associated with CRC susceptibility (P = 2.43 × 10-12). Notably, we observed a significant interaction between rs11777210 and MYC nearby rs6983267 (P-multi = 0.003, P-add = 0.005), subjects carrying rs6983267 GG and rs11777210 CC genotypes showing higher susceptibility to CRC (2.83-fold) than those carrying rs6983267 TT and rs11777210 TT genotypes. We further demonstrated that rs6983267 T > G increased MYC expression, and MYC bound to and negatively regulated KBTBD11 expression when the rs11777210 C risk allele was present. KBTBD11 was downregulated in tumor tissues, and KBTBD11 knockdown promoted cell proliferation and inhibited cell apoptosis. Conclusion The rs11777210 is a potential predictive biomarker of CRC susceptibility, and KBTBD11 functions as a putative tumor suppressor in tumorigenesis. Our study highlighted the high CRC risk of people carrying rs6983267 G and rs11777210 C alleles, and provided possible biological mechanism of the interaction.

Genetic variants in the regulatory region of SLC10A1 are not associated with the risk of hepatitis B virus infection and clearance

The Na/taurocholate cotransporter NTCP (encoded by SLC10A1) was identified as a cellular entry receptor for the human hepatitis B virus (HBV), advancing our understanding of the molecular mechanism of HBV infection. An alternative hypothesis was put forward that regulatory variants in SLC10A1 might play an important role in HBV susceptibility by potentially influencing expression levels of NTCP. The three regulatory SNPs (rs8011311, rs7154439, rs111409076) were genotyped in 1023 HBV-persistent carriers, 735 subjects with HBV natural clearance and 732 HBV marker-negative subjects in a Han Chinese population. Real-time reverse transcription PCR analysis and luciferase assays have been performed to dissect the potential functionality. In logistic regression analysis, when subjects with HBV natural clearance were compared with HBV marker-negative subjects, no significant associations with the risk of HBV infection were observed for any of the three SNPs after adjusting for age, sex, smoking status and alcohol consumption (P>0.05). Similar negative results were also found for the three SNPs when HBV-persistent carriers were compared with HBV marker-negative subjects. Likewise, no significant associations with the risk of HBV clearance were observed when HBV-persistent carriers were compared with subjects with HBV natural clearance (P>0.05). Quantitative RT/PCR showed no significant difference in NTCP expression levels in normal liver tissue amongst individuals with different rs111409076 genotypes (P=0.317 for the general linear model). Moreover, no evident effect of the SLC10A1 rs111409076 AACA/- polymorphism on transcriptional activity was found by luciferase assay in either HepG2 (P=0.161) or Hep3b (P=0.129) cell lines. The present study indicated that the common variants in the regulatory region of SLC10A1 may not influence the expression of NTCP at the level of transcriptional regulation, and ultimately may not be associated with HBV susceptibility in this Chinese population.

Identification of a Potential Regulatory Variant for Colorectal Cancer Risk Mapping to Chromosome 5q31.1: A Post-GWAS Study

Large-scale genome-wide association studies (GWAS) have established chromosome 5q31.1 as a susceptibility locus for colorectal cancer (CRC), which was still lack of causal genetic variants. We searched potentially regulatory single nucleotide polymorphisms (SNPs) in the overlap region between linkage disequilibrium (LD) block of 5q31.1 and regulatory elements predicted by histone modifications, then tested their association with CRC via a case-control study. Among three candidate common variants, we found rs17716310 conferred significantly (heterozygous model: OR = 1.273, 95% confidence interval (95%CI) = 1.016–1.595, P = 0.036) and marginally (dominant model: OR = 1.238, 95%CI = 1.000–1.532, P = 0.050) increase risk for CRC in a Chinese population including 695 cases and 709 controls. This variation was suggested to be regulatory altering the activity of enhancer that control PITX1 expression. Using epigenetic information such as chromatin immunoprecipitation-sequencing (ChIP-seq) data might help researchers to interpret the results of GWAS and locate causal variants for diseases in post-GWAS era.

A Rare Variant P507L in TPP1 Interrupts TPP1–TIN2 Interaction, Influences Telomere Length, and Confers Colorectal Cancer Risk in Chinese Population

Background: Telomere dysfunction triggers cellular senescence and constitutes a driving force for cancer initiation. Genetic variants in genes involved in telomere maintenance may contribute to colorectal cancer susceptibility. Methods: In this study, we firstly captured germline mutations in 192 patients with colorectal cancer by sequencing the coding regions of 13 core components implicated in telomere biology. Five potential functional variants were then genotyped and assessed in a case–control set with 3,761 colorectal cancer cases and 3,839 healthy controls. The promising association was replicated in additional 6,765 cases and 6,906 controls. Functional experiments were used to further clarify the potential function of the significant variant and uncover the underlying mechanism in colorectal cancer development. Results: The two-stage association studies showed that a rare missense variant rs149418249 (c.C1520T and p.P507L) in the 11th exon of TPP1 (also known as ACD, gene ID 65057) was significantly associated with colorectal cancer risk with the ORs being 2.90 [95% confidence interval (CI), 1.04–8.07; P = 0.041], 2.50 (95% CI, 1.04–6.04; P = 0.042), and 2.66 (95% CI, 1.36–5.18; P = 0.004) in discovery, replication, and the combined samples, respectively. Further functional annotation indicated that the TPP1 P507L substitution interrupted TPP1–TIN2 interaction, impaired telomerase processivity, and shortened telomere length, which subsequently facilitated cell proliferation and promoted colorectal cancer development. Conclusions: A rare variant P507L in TPP1 confers increased risk of colorectal cancer through interrupting TPP1–TIN2 interaction, impairing telomerase processivity, and shrinking telomere length. Impact: These findings emphasize the important role of telomere dysfunction in colorectal cancer development, and provide new insights about the prevention of this type of cancer. Cancer Epidemiol Biomarkers Prev; 27(9); 1029–35. ©2018 AACR.

MAD1L1 Arg558His and MAD2L1 Leu84Met interaction with smoking increase the risk of colorectal cancer

The spindle assembly checkpoint (SAC) has been established as an important mechanism of driving aneuploidy, which occurs at a high frequency in the colorectal tumorigenesis. Two important components of SAC are MAD1L1 and MAD2L1, which function together in an interactive manner to initiate the checkpoint signal. We hypothesize that genetic variants in the binding domains of MAD1L1 and MAD2L1 may modulate protein structures and eventually contribute to CRC susceptibility. A case-control study including 710 CRC cases and 735 controls was performed to examine MAD1L1 Arg558His and MAD2L1 Leu84Met’s conferring susceptibility to CRC. Cytokinesis-block micronucleus cytome assays were applied to assess the effect of two functional variants on chromosomal instability (CIN). Significant associations with CRC risk were observed for MAD1L1 Arg558His (OR = 1.38,95% CI: 1.09–1.75) and MAD2L1 Leu84Met in a dominant model (OR = 1.48,95% CI: 1.09–2.01). Moreover, significant multiplicative gene-smoking interactions were found in MAD1L1 Arg558His (P = 0.019) and MAD2L184 Leu/Met (P = 0.016) to enhance CRC risk. Additionally, the frequencies of lymphocytic micro-nucleated binucleated cells for MAD1L1 Arg558His polymorphism were significantly different in the exposed group (P = 0.013), but not in the control group. The study emphasized that MAD1L1 Arg558His and MAD2L1 Leu84Met can significantly interact with smoking to enhance CRC risk, and the genetic effects of MAD1L1Arg558His on CIN need to be further clarified in follow-up studies.

A phosphorylation-related variant ADD1-rs4963 modifies the risk of colorectal cancer.

It is well-established that abnormal protein phosphorylation could play an essential role in tumorgenesis by disrupting a variety of physiological processes such as cell growth, signal transduction and cell motility. Moreover, increasing numbers of phosphorylation-related variants have been identified in association with cancers. ADD1 (α-adducin), a versatile protein expressed ubiquitously in eukaryotes, exerts an important influence on membrane cytoskeleton, cell proliferation and cell-cell communication. Recently, a missense variant at the codon of ADD1’s phosphorylation site, rs4963 (Ser586Cys), was reported to modify the risk of non-cardia gastric cancer. To explore the role of ADD1-rs4963 in colorectal cancer (CRC), we conducted a case-control study with a total of 1054 CRC cases and 1128 matched controls in a Chinese population. After adjustment for variables including age, gender, smoking and drinking, it was demonstrated that this variant significantly conferred susceptibility to CRC (G versus C: OR = 1.16, 95% CI = 1.03–1.31, P = 0.016; CG versus CC: OR = 1.25, 95% CI = 1.02–1.55, P = 0.036; GG versus CC: OR = 1.35, 95% CI = 1.06–1.72, P = 0.015). We further investigated the interaction of ADD1-rs4963 with smoking or drinking exposure, but found no significant result. This study is the first report of an association between ADD1 and CRC risk, promoting our knowledge of the genetics of CRC.

Association between bilirubin and risk of Non-Alcoholic Fatty Liver Disease based on a prospective cohort study.

The study aimed to assess the association between total, direct, and indirect bilirubin and nonalcoholic fatty live disease (NAFLD) risk given its high prevalence and serious clinical prognosis. Among 27,009 subjects who participated in a healthy screening program from the Dongfeng-Tongji cohort study in 2008, 8189 eligible subjects (aged 35–86 years; males, 43.95%) were ultimately enrolled. The incidence rates of NAFLD in 2013 were compared with respect to baseline bilirubin levels among subjects free of NAFLD, and the effect sizes were estimated by logistic regression analysis. During 5 years follow-up, we observed 1956 cases of newly developed NAFLD with the overall incidence of 23.88%. Direct bilirubin was presented to inversely associate with NAFLD risk. Compared with quartile 1 of direct bilirubin, the multivariable-adjusted ORs (95% CIs) for NAFLD of quartile 2 to 4 were 1.104 (0.867–1.187), 0.843 (0.719–0.989), and 0.768 (0.652–0.905), respectively, P for trend 0.002). Similarly, inverse effects of direct bilirubin on NAFLD incidence were also observed when stratified by sex and BMI. However, no significant associations were found between total, and indirect bilirubin and NAFLD risk. Direct bilirubin reduced NAFLD risk independent of possible confounders among middle-aged and elderly Chinese population, probably based on the endogenous antioxidation of bilirubin.