CPM基因区域遗传变异与尘肺病易感性的精细定位研究

Pneumoconiosis is the most serious occupational disease in China. Individual variation in the susceptibility to pneumoconiosis was observed among the workers with similar exposure at a given time. A genome-wide significant (P < 5.0 × 10-8) association for rs73329476 has been identified by our previous pneumoconiosis genome-wide association study (GWAS). In addition, rs73329476 was significantly associated with the expression levels of CPM. It’s reported that CPM is involved in monocyte to macrophage differentiation, while low expression of which may be associated with lung fibrosis. Therefore, a two-stage case-control study matched by cumulative dust exposure and age at first dust exposure will be designed to fine-map CPM, confirming the associations between genetic variants across which and the risk of pneumoconiosis. Meanwhile, molecular biology techniques will be performed to clarify the biological mechanism of the SNPs with P < 0.05, determining the causal variants. The study results may provide new insights into the pathogenesis of pneumoconiosis, and may also have some clinical utility for risk prediction for pneumoconiosis and high-risk population screening for workers with occupational silica exposure.

尘肺病是当前我国危害最广泛而严重的职业病。在特定时间相似的粉尘暴露情况下，具有不同遗传背景的个体对尘肺病的易感性不同。申请人前期开展的尘肺病全基因组关联研究（GWAS）发现一个达到GWAS显著性水准（P<5×10-8）的标签位点rs73329476，且该位点与其上游CPM基因表达显著相关。CPM基因参与调节单核细胞转化为巨噬细胞，其低表达可能与肺纤维化发生密切相关。本项目拟在前期研究基础上，采用两阶段病例对照研究设计，严格匹配病例组和对照组累积粉尘暴露量（CDE）和初次接触粉尘年龄（AFDE），进一步对CPM基因开展精细定位研究，以确定CPM基因区域尘肺病遗传标志物及其效应值，同时通过分子生物学技术，初步阐述阳性SNP的生物学机制，确定致病位点。研究结果有望对识别和评价尘肺病的遗传易感因素，阐明尘肺病的机制奠定基础，其发现的遗传易感标志物也有望为粉尘暴露高危人群的筛检及预防提供科学依据。

我们之前所开展的中国人群中SiO2相关尘肺病的全基因组关联研究（GWAS）发现位于染色体12q15区域的遗传变异rs73329476能显著增加SiO2相关尘肺病的发病风险。然而该区域真正致病位点还未发现。为了寻找该区域的真正致病位点，我们采用病例-对照研究设计（包含177例病例和204名接触粉尘的健康对照），以之前中国人群尘肺病GWAS报道的易感位点rs73329476为marker-SNP，对其所在的染色体12q15区域CPM和CPSF6基因区域选取8个潜在功能性SNPs进行基因型检测，发现位于CPM基因启动子区的rs12812500与尘肺病的发病风险显著相关，后续相关的荧光素酶报告基因实验显示rs12812500危险等位基因G能显著增加CPM基因表达,提示该位点可能是该区域的致病位点。同时，我们所开展的4例矽肺病人和4例接触SiO2粉尘的健康对照外周血淋巴细胞全转录组测序（RNA-seq）结果显示，CPM在矽肺病人外周血淋巴细胞中表达显著上调，进一步我们在60例矽肺病人和60例接触SiO2粉尘的健康对照外周血淋巴细胞中开展扩大样本的实时荧光定量PCR（qRT-PCR）验证，结果证实CPM在矽肺病人外周血淋巴细胞中表达显著上调。SiO2诱导的肺泡基底上皮细胞A549和支气管上皮细胞16HBE的细胞实验显示，随着SiO2浓度的升高及细胞培养时间的增加，A549和16HBE细胞活性显著下降，随着SiO2浓度的升高，CPM在A549和16HBE中表达均显著升高。生物信息学预测结果显示，rs12812500的不同等位基因能影响转录因子ETV2与CPM基因的结合。综上所述，rs12812500可能通过影响CPM基因的表达进而影响尘肺病的易感性。

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