Transcription profiling of mouse strains for eQTL CNV Analysis

Background; Haplotype association mapping is a recently developed method that can be used to identify expression Quantitative Trait Loci (eQTLs). The underlying genomic structure of the haplotype can be defined by single nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTRs) and copy number variations (CNVs). Microarray genotyping platforms are able to interrogate all three genomic polymorphisms simultaneously. Combined with gene expression data, these data allow for the elucidation of sites responsible for the regulation of transcription. Results; Thirty three inbred strains were genotyped on a microarray platform to assess possible contributions of polymorphic regions to gene expression. Over one hundred million statistical correlations were made between probe performance on genotyping and gene expression microarrays. This analysis provided 10,655 trans associations and 31 cis associations. Further investigation of one of the cis associations revealed that a 84kb region of MMU3 acts as a haplotype-specific locus control region for the glutathione-S-transferase mu family in multiple tissues. In the strains that share the minor haplotype, reductions in mRNA levels in members of the Gst mu family are observed. Conclusions; We have identified a haplotype containing a putative locus control region for the Gst mu family gene cassette. In the strains with affected haplotype, Gst mu expression is drastically reduced. The reduction in Gst mu levels has important relevance for pharmacology and toxicology studies. The reduction of Gst mu levels in general, and Gstm5 in particular, has implication in models of dopamine metabolism, Parkinson's disease, and chemical neurotoxicity. Experiment Overall Design: Samples GSM262266-GSM262267, GSM262270-GSM262272 representing liver gene expression Samples from 10 week old male mouse livers were analyzed in a confirmation study of an identified eQTL. Samples came from pools of three animals, and were compared across strains. Experiment Overall Design: The genotyping summary output file and associated .CEL files are linked below as supplementary files. Briefly: Experiment Overall Design: After hybridization and staining the arrays are scanned on an Affymetrix Scanner 3000 7G 4C using four distinct filters, one for each DNA base (A, C, G, T). These scanned images are saved at .dat files. A grid is superimposed on the .dat array image such that each element of the grid brackets a feature. Once the grid has been applied, GCOS computes one intensity value for each cell, and saves these values in a .CEL file. A .CEL file is generated for each possible base (A, C, G, T). Only the .CEL files are read by GTGS. Experiment Overall Design: The .CEL files computed by GCOS are then imported into GTGS and analyzed. The experiments are then clustered together and used to generate a normalized profile of each probe. From the clustered profiles a genotype call is made for every specific probe on an individual chip. These genotyped calls are then exported from GTGS into a .txt file containing assay id, sample id and relative intensity values for each DNA base over the entire set of SNPs analyzed.背景：单倍型关联定位（haplotype association mapping）是近年来发展出的一种可用于鉴定表达数量性状位点（expression Quantitative Trait Loci, eQTLs）的方法。单倍型的潜在基因组结构可通过单核苷酸多态性（single nucleotide polymorphisms, SNPs）、可变数目串联重复序列（variable number tandem repeats, VNTRs）以及拷贝数变异（copy number variations, CNVs）进行定义。基因分型微阵列平台可同时检测这三类基因组多态性。结合基因表达数据，上述数据能够阐明调控转录的相关位点。 结果：本研究在微阵列平台上对33个近交品系进行基因分型，以评估多态性区域对基因表达的潜在贡献。本研究在基因分型微阵列探针表现与基因表达微阵列数据之间开展了超过1亿次统计相关性分析，共鉴定得到10655个反式关联（trans associations）与31个顺式关联（cis associations）。针对其中一个顺式关联的进一步研究显示，小鼠3号染色体（MMU3）上一段84kb的区域可作为谷胱甘肽S-转移酶μ家族（glutathione-S-transferase mu family）的单倍型特异性位点控制区，在多种组织中发挥调控作用。在携带次要单倍型的品系中，可观察到Gstμ家族成员的mRNA水平出现下调。 结论：本研究鉴定出一段包含Gstμ家族基因簇推定位点控制区的单倍型。在携带该异常单倍型的品系中，Gstμ的表达量显著降低。Gstμ水平的下调与药理学及毒理学研究密切相关。其中，Gstμ整体水平下调，尤其是Gstm5的表达变化，与多巴胺代谢、帕金森病以及化学性神经毒性的相关模型具有潜在关联。 实验总体设计：样本GSM262266-GSM262267、GSM262270-GSM262272来自10周龄雄性小鼠肝脏，用于已鉴定eQTL的验证研究。样本由3只动物混合制备，并在不同品系间进行比较。 实验总体设计：基因分型汇总输出文件及关联的.CEL格式文件已作为补充材料附于文末。简要说明如下： 实验总体设计：阵列经过杂交与染色后，使用Affymetrix Scanner 3000 7G 4C扫描仪搭配4种分别对应4种DNA碱基（A、C、G、T）的滤光片进行扫描，扫描得到的图像以.dat格式文件保存。随后在.dat格式的阵列图像上叠加网格，使网格的每个单元框定一个探针位点。完成网格定位后，GCOS软件为每个单元计算一个强度值，并将这些数值保存至.CEL格式文件。每个DNA碱基对应生成一个.CEL格式文件，仅.CEL格式文件可被GTGS读取。 实验总体设计：将GCOS软件生成的.CEL格式文件导入GTGS并进行分析。随后对实验样本进行聚类，用于生成每个探针的标准化表达谱。基于聚类后的表达谱，为单张芯片上的每个特异性探针完成基因分型判定。最终将这些分型结果从GTGS导出至.txt格式文件，该文件包含本次分析的全部SNP对应的分析物ID、样本ID以及每种DNA碱基的相对强度值。