Long Jieyi, Long Tingting, Li Ying, Yuan Peihong, Liu Ke, Li Jiaoyuan, Cheng Liming
Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Front Oncol. 2021 Sep 6;11:698993. doi: 10.3389/fonc.2021.698993. eCollection 2021.
The 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.
To 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.
We 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.
Sixteen 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; = 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 ( < 0.001, = 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.
These 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.
全基因组关联研究（GWAS）鉴定出的疾病相关非编码变异在开放染色质区域（OCR）中富集，并与基因调控有关。因此，OCR中的遗传变异可能发挥调控功能，并导致非小细胞肺癌（NSCLC）易感性。
利用染色质可及性和组蛋白修饰数据精细定位GWAS位点中导致NSCLC易感性的潜在功能变异，并通过人群研究和生化实验分析探索其功能。
我们使用癌症基因组图谱（TCGA）中转座酶可及染色质测序（ATAC-seq）数据绘制肺组织的染色质可及区域，并通过与DNA元件百科全书（ENCODE）中染色质免疫沉淀测序（ChIP-seq）数据的组蛋白特征比对，对肺癌GWAS位点内的潜在调控变异进行优先级排序。开展一项包含1830例病例和2001例对照的两阶段病例对照研究，以探索中国人群中候选变异与NSCLC风险之间的关联。进行生物信息学注释和生化实验以进一步揭示显著变异的潜在功能。
从生物信息学分析中选择了16个潜在的功能性单核苷酸多态性（SNP）作为候选。在第1阶段病例对照研究的基因分型后，16个候选SNP中有3个变异留存下来，而在第2阶段病例对照研究分析中仅SNP rs13064999的结果得到成功验证。在联合分析中，rs13064999与NSCLC风险显著相关[加性模型；比值比（OR）=1.17；95%可信区间（CI），1.07 - 1.29；P = 0.001]。功能注释表明其具有潜在的增强子生物活性，双荧光素酶报告基因检测显示，与具有野生型G等位基因的重组质粒相比，具有rs13064999 A等位基因的重组质粒的荧光素酶活性显著增加（P < 0.001，t = 0.004）。进一步的电泳迁移率变动分析（EMSA）和超迁移分析证实HP1γ对含有SNP rs13064999 A等位基因的结合基序具有更强的亲和力。
这些发现表明，通过整合ATAC-seq和ChIP-seq数据鉴定出的功能性变异rs13064999通过影响HP1γ结合导致NSCLC易感性，而确切的生物学机制有待进一步探索。
