Construction of a multilocus map of single nucleotide polymorphisms in pneumoconiosis susceptibility genes
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摘要:
目的 构建尘肺病易感基因单核苷酸多态性(single nucleotide polymorphisms, SNPs)多位点检测图谱。 方法 利用限制性片段长度多态性(restriction fragment length polymorphism, RFLP)技术，对已报道的易感基因SNPs位点进行整合，并根据基因位点设计不同的扩增片段和酶切片段，构建涵盖多个不同易感基因SNPs位点组成的多位点图谱，通过琼脂糖凝胶电泳的多个长短不一的条带快速实现基因分型；采用陕北某矿区煤工尘肺(coal workers′ pneumoconiosis, CWP)和接尘人群队列进行检测，通过遗传分化指数(fixation index, Fst)分析易感基因不同多态位点在队列遗传分化情况。 结果 成功构建5个易感基因IL-1β rs16944 T>C、IL-6 rs1800796 G>C、IL-8 rs4073 A>T、HSP70-1 rs562047 C>G、TGF-β rs1800469 T>C的SNPs图谱，可快速有效鉴别SNPs的基因型；Fst分析表明IL-1β rs16944、TGF-β rs1800469的Fst为＞0.05~0.15，即发生中等程度遗传分化。 结论 该易感基因SNPs多位点图谱可以有效实现基因分型，易感位点rs16944和rs1800469更易受到疾病选择压力的影响，与尘肺病的易感风险有关。 Abstract:Objective To construct a single nucleotide polymorphisms (SNPs) multilocus map of pneumoconiosis susceptibility genes. Methods Based on RFLP technology, we integrated the reported SNPs of susceptibility genes, and designed different amplification fragments and digestion fragments which cover several different susceptibility gene locus. Then the multiple bands of different lengths obtained by agarose gel electrophoresis were used to identify the genetic typing rapidly. In addition, the cohort of coal workers′ pneumoconiosis(CWP) and dust receiving population in a mining area in northern Shaanxi was used for detection, and the genetic differentiation of different polymorphic loci of susceptible genes in the cohort was analyzed by fixation index (Fst). Results The SNPs of five susceptible genes were successfully constructed, which included IL-1β rs16944 T>C, IL-6 rs1800796 G>C, IL-8 rs4073 A>T, HSP70-1 rs562047 C>G, TGF-β rs1800469 T>C. These SNPs genotypes can be identified effectively. Fst analysis showed that the Fst of IL-1β rs16944 and TGF-β rs1800469 was > 0.05-0.15, indicating that, moderate genetic differentiation occurred. Conclusions The susceptibility loci rs16944 and rs1800469 were more susceptible to disease selection pressure, which was related to the susceptibility risk of pneumoconiosis. -
Key words:
- Pneumoconiosis /
- Susceptibility genes /
- Single nucleotide polymorphisms /
- Multilocus map
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图 1 易感基因单核苷酸多态性多位点图谱
A：多位点图谱；B：CWP1~4琼脂糖凝胶电泳结果；C：CWP1~5基于图谱分析的基因型结果；a: IL-1 T; b: IL-1 C; c: IL-6 C; d: IL-6 G; e: IL-8 T; f: IL-8 A; g: HSP70-1 C; h: HSP70-1 G; i: HSP70-1 G; g: TGF-β T; k: TGF-β C。
Figure 1. Multilocus map of single nucleotide polymorphisms in susceptibility genes
A: multilocus map; B: results of CWP1-4 agarose gel electrophoresis; C: CWP1-5 genotype results based on multilocus map analysis; a: IL-1 T; b: IL-1 C; c: IL-6 C; d: IL-6 G; e: IL-8 T; f: IL-8 A; g: HSP70-1 C; h: HSP70-1 G; i: HSP70-1 G; g: TGF-β T; k: TGF-β C.
表 1 引物序列及扩增子长度
Table 1. Detail of Primer pairs
易感基因
Susceptibility genes引物(5′→3′)
Primer pairs(5′→3′)扩增长度/bp
Amplified fragment length/bpIL-1β F: CAAGAAAGGTCAATTTTCTCCT 648 R: ATTAAAGTCATACTTGAGCAATG IL-6 F: CCCCAACAAAGAGGTGAGTAGT 452 R: GCCTGGGATTATGAAGAAGGTA IL-8 F: TCAGGACACTAGGACATAAAGCCACC 1 800 R: CAGCATAGAAATTGCCTTCAGAAAGAA HSP70-1 F: CGCCCGTTGAAGAAGTC 1 297 R: CAAGCGGTCCGGATAAC TGF-β F: GTGAGTATCAGGGAGTGGGGAAT 841 R: GAAGAGGGTCTGTCAACATGGG 注：IL-1β，白介素1β；IL-6，白介素6；IL-8，白介素8；HSP70-1，热休克蛋白70-1；TGF-β，转化生长-因子。
Note：IL-1β, interleukin 1 β; IL-6, interleukin 6; IL-8, interleukin 8; HSP70-1, heat shock protein 70-1; TGF-β, transforming growth factor-β.
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表 2 易感基因位点信息和图谱片段长度
Table 2. The locus information and length of map fragment of susceptibility genes
易感基因
Susceptibility genesSNPs位点
SNPs locus内切酶
Endonuclease酶切基因型
Cutting site酶切后片段长度/bp
Digestion fragments length/bpIL-1β rs16944 T>C AvaI C T 648
C 597IL-6 rs1800796 G>C BsrBI G C 452
G 380IL-8 rs4073 A>T MfeI A A 900
T 1 800HSP70-1 rs562047 C>G BsaI C C 739&558
G 1 297TGF-β rs1800469 T>C Bsu36I C T 841
C 800注：1. SNPs, 单核苷酸多态性; IL-1β，白介素1β；IL-6，白介素6；IL-8，白介素8；HSP70-1，热休克蛋白70-1；TGF-β，转化生长因子。
2. 酶切后片段小于50 bp不列出。
Note: 1. SNPs, single nucleotide polymorphisms; IL-1β, interleukin 1 β; IL-6, interleukin 6; IL-8, interleukin 8; HSP70-1, heat shock protein 70-1; TGF-β, transforming growth factor-β.
2. Enzymatically cleaved fragments less than 50 bp are not listed.
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表 3 候选易感基因多态性位点统计
Table 3. Epidemiological investigation and statistics of susceptibility genes
易感基因
Susceptibility genesSNPs位点
SNPs locus位置
Location保护基因型
Protecting genotype中间型
Intermediate genotype易感基因型
Susceptible genotype相关性
RelevanceIL-1β rs16944 T>C 启动子Promoter CC TC TT 显著相关[4-5] Significantly relevant[4-5] IL-6 rs1800796 G>C 启动子Promoter GG CG CC 显著相关[6] Significantly relevant[6] IL-8 rs4073 A>T 启动子Promoter TT AT AA 显著相关[7-8] Significantly relevant[7-8] HSP70-1 rs562047 C>G 外显子Exons GG CG CC 显著相关[9] Significantly relevant[9] TGF-β rs1800469 T>C 启动子Promoter CC CT TT 显著相关[10-11] Significantly relevant[10-11] 注：IL-1β，白介素1β；IL-6，白介素6；IL-8，白介素8；HSP70-1，热休克蛋白70-1；TGF-β，转化生长因子。
Note: IL-1β, interleukin 1 β; IL-6, interleukin 6; IL-8, interleukin 8; HSP70-1, heat shock protein 70-1; TGF-β, transforming growth factor-β.
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表 4 候选SNPs的遗传多样性分析
Table 4. Genetic diversity based on SNPs of test population
SNPs位点
SNPs locus等位基因频率Allele frequency 基因型频率Genotype frequency 遗传分化指数
Fixation indexCK CD CWP CK CD CWP IL-1β rs16944 T 0.40 0.45 0.70 TT 0.20 0.30 0.40 0.093 C 0.60 0.55 0.30 TC 0.40 0.30 0.60 CC 0.40 0.40 0.00 IL-6 rs1800796 C 0.80 0.65 0.65 CC 0.00 0.30 0.40 0.024 G 0.20 0.35 0.35 CG 0.40 0.50 0.50 GG 0.50 0.10 0.10 IL-8 rs4073 T 0.60 0.65 0.55 TT 0.30 0.40 0.20 0.006 A 0.40 0.35 0.45 TA 0.60 0.50 0.70 AA 0.10 0.10 0.10 TGF-β rs1800469 C 0.55 0.40 0.75 CC 0.20 0.10 0.50 0.082 T 0.45 0.60 0.25 CT 0.70 0.70 0.50 TT 0.10 0.20 0.00 HSP70-1 rs562047 G 0.90 0.90 0.95 GG 0.80 0.80 0.90 0.004 C 0.10 0.10 0.05 GC 0.20 0.20 0.10 CC 0.00 0.00 0.00 注：SNPs, 单核苷酸多态性; IL-1β，白介素1β；IL-6，白介素6；IL-8，白介素8；HSP70-1，热休克蛋白70-1；TGF-β，转化生长因子。
Note: SNPs, single nucleotide polymorphisms; IL-1β, interleukin 1 β; IL-6, interleukin 6; IL-8, interleukin 8; HSP70-1, heat shock protein 70-1; TGF-β, transforming growth factor-β.
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