Functional Genomics in Mice by Exome Sequencing

The mapping of mutations underlying monogenic phenotypes in the laboratory mouse has proven to be an extraordinarily powerful paradigm, yielding functional insights into thousands of genes, as well as mouse strains that are highly effective for modeling human disease. Although massively parallel sequencing extraordinary potential to accelerate the gene discovery process in mice, the complete sequencing of mouse genomes remains burdensome and expensive for many laboratories. Targeted sequencing of genetically concordant intervals represents a more cost-effective approach, but requires substantial genetic mapping and custom capture reagents. In contrast, targeted sequencing of the “exome”, i.e. all coding sequences, potentially allows for the comprehensive, cost-effective identification of protein-altering mutations in mouse mutants with minimal mapping data and utilizing a standardized protocol. To this end, we developed reagents for in solution, hybridization based capture of the mouse exome, comprising 54 Mb of annotated coding sequence (C57BL/6J, NCBI37/mm9). We describe the application of these reagents to sequence the exomes of 18 strains of mice, including 15 novel mutants and 3 controls, and identify strong candidate genes for the majority of these mutants including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis. Moreover, these data provide novel functional associations for several poorly characterized genes including Lhfpl2 and Pfas. Whole exome sequencing is a robust method for mutation discovery in the mouse genome, and will be useful for completing the genetic analysis of the thousands of established mouse mutants exhibiting clinically relevant phenotypes.

在实验小鼠中解析单基因表型背后的突变，已被证实为极具影响力的研究范式，不仅为数千个基因的功能解析提供了关键认知，还获得了可高效模拟人类疾病的小鼠品系。尽管大规模平行测序（massively parallel sequencing）在加速小鼠基因发现进程中具备巨大潜力，但对多数实验室而言，完整测序小鼠基因组依然耗时费力且成本高昂。针对遗传一致性区间的靶向测序是一种更具成本效益的方案，但需要开展大量遗传定位工作并使用定制捕获试剂。相比之下，对"外显子组（exome）"——即全部编码序列——开展靶向测序，则有望依托标准化流程，仅需少量定位数据，即可全面且经济高效地鉴定小鼠突变体中引发蛋白质序列改变的突变。为此，我们开发了基于液相杂交的小鼠外显子组捕获试剂，覆盖C57BL/6J参考基因组（NCBI37/mm9）中54 Mb的注释编码序列。本研究将该试剂应用于18株小鼠的外显子组测序，其中包含15株新构建的突变体与3株对照品系，并为其中绝大多数突变体鉴定到了高可信度候选基因，涵盖口面裂、泌尿生殖系统畸形、脊柱后凸以及自身免疫性肝炎的新型疾病模型。此外，本研究的数据还为Lhfpl2、Pfas等数个功能尚未得到充分阐释的基因提供了全新的功能关联线索。全外显子组测序是小鼠基因组突变发现的可靠技术手段，将有助于完成数千株已构建、携带临床相关表型的小鼠突变体的遗传分析工作。