Analysis of Candidate Colitis Genes in the Gdac1 Locus of Mice Deficient in Glutathione Peroxidase-1 and -2

BackgroundMice that are deficient for glutathione peroxidases 1 and 2 (GPX) show large variations in the penetrance and severity of colitis in C57BL/6J and 129S1/SvImJ backgrounds. We mapped a locus contributing to this difference to distal chromosome 2 (∼119–133 mbp) and named it glutathione peroxidase-deficiency-associated colitis 1 (Gdac1). The aim of this study was to identify the best gene candidates within the Gdac1 locus contributing to the murine colitis phenotype. Method/Principal FindingsWe refined the boundaries of Gdac1 to 118–125 mbp (95% confidence interval) by increasing sample size and marker density across the interval. The narrowed region contains 128 well-annotated protein coding genes but it excludes Fermt1, a human inflammatory bowel disease candidate that was within the original boundaries of Gdac1. The locus we identified may be the Cdcs3 locus mapped by others studying IL10-knockout mice. Using in silico analysis of the 128 genes, based on published colon expression data, the relevance of pathways to colitis, gene mutations, presence of non-synonymous-single-nucleotide polymorphisms (nsSNPs) and whether the nsSNPs are predicted to have an impact on protein function or expression, we excluded 42 genes. Based on a similar analysis, twenty-five genes from the remaining 86 genes were analyzed for expression-quantitative-trait loci, and another 15 genes were excluded. Conclusion/SignificanceAmong the remaining 10 genes, we identified Pla2g4f and Duox2 as the most likely colitis gene candidates, because GPX metabolizes PLA2G4F and DUOX2 products. Pla2g4f is a phospholipase A2 that has three potentially significant nsSNP variants and showed expression differences across mouse strains. PLA2G4F produces arachidonic acid, which is a substrate for lipoxygenases and, in turn, for GPXs. DUOX2 produces H2O2 and may control microbial populations. DUOX-1 and -2 control microbial populations in mammalian lung and in the gut of several insects and zebrafish. Dysbiosis is a phenotype that differentiates 129S1/SvImJ from C57BL/6J and may be due to strain differences in DUOX2 activity.背景 谷胱甘肽过氧化物酶1和2（glutathione peroxidases 1 and 2, GPX）缺陷型小鼠在C57BL/6J与129S1/SvImJ遗传背景下，其结肠炎的外显率与严重程度存在显著差异。本研究将导致该表型差异的基因座定位于2号染色体远端区域（约119~133 百万碱基对，megabase pairs, mbp），并将其命名为谷胱甘肽过氧化物酶缺陷相关结肠炎1（glutathione peroxidase-deficiency-associated colitis 1, Gdac1）。本研究旨在鉴定Gdac1位点内参与小鼠结肠炎表型的最优候选基因。 方法与主要发现 我们通过增加该区间内的样本量与遗传标记密度，将Gdac1的定位区间精细修正至118~125 mbp（95%置信区间）。该缩小后的区域包含128个已充分注释的蛋白编码基因，但排除了原本处于Gdac1原始定位区间内的人类炎症性肠病候选基因Fermt1。本研究鉴定的该基因座，可能与其他团队在白细胞介素10敲除（IL10-knockout）小鼠中定位的Cdcs3基因座一致。我们基于已发表的结肠组织表达数据、结肠炎相关通路的相关性、基因突变情况、非同义单核苷酸多态性（non-synonymous-single-nucleotide polymorphisms, nsSNPs）的存在及其对蛋白功能或表达的预测影响，对这128个基因进行in silico分析，最终排除了42个基因。基于类似的分析策略，我们对剩余86个基因中的25个进行了表达定量性状位点（expression-quantitative-trait loci, eQTLs）分析，并进一步排除了15个基因。 结论与意义 在剩余的10个基因中，我们鉴定出Pla2g4f与Duox2为最具潜力的结肠炎候选基因，原因在于GPX可代谢PLA2G4F与DUOX2的表达产物。Pla2g4f属于磷脂酶A2（phospholipase A2）家族，携带有3个具有潜在重要功能意义的nsSNP变异，且在不同小鼠品系中存在显著表达差异。PLA2G4F可催化生成花生四烯酸（arachidonic acid），而后者是脂氧合酶的底物，进而可作为GPX的代谢底物。DUOX2可生成过氧化氢（hydrogen peroxide, H2O2），并可能调控肠道菌群的种群结构。已有研究证实DUOX-1与DUOX-2可调控哺乳动物肺部以及多种昆虫与斑马鱼肠道内的菌群种群。菌群失调（dysbiosis）是区分129S1/SvImJ与C57BL/6J小鼠品系的表型特征之一，其发生可能与DUOX2活性的品系差异相关。