Discovering Splicing Defects in Human Genes

项目摘要/摘要 目前，小型研究小组对单个基因组进行测序是可行的，而较大的研究小组则可以 对数以万计的个体进行排序。不幸的是，我们识别影响表型的变异的能力 跟不上我们的测序能力。对于非编码变体来说尤其如此。这项建议 展示了超过32K的myCode和ClinVar变体的试点屏幕，这些变体建议1%-2%的外显子 突变会影响剪接。初步研究还显示，剪接突变并不均匀分布 跨越疾病基因，甚至在基因内。这项提案将在英国生物库的变种上继续这一努力， AllfU、ClinVar和GTEx定位于可操作的基因。剪接突变并不是在 外显子。该提案寻求绘制剪接突变的易感性图谱，并识别热点外显子(exons 特别容易受到剪接突变的影响)。除了鉴定剪接变异体的丢失之外， 剪接变异体(即可以激活伪外显子的单碱基突变)的获得问题研究很少 也将进行探索。初步研究定位了前信使核糖核酸中的许多内含子区域，这些内含子区域除了一个外都包含 剪接所需的大量顺式元件。除了定位对剪接突变的易感性外， 这些工作将有助于用高级方法进行组合信号识别的基础科学 这将定义剪接位点选择的机制。最后，我们提出了一种恢复剪接的原则性方法 使用反义寡核苷酸(ASO)削弱侧翼剪接位点的突变外显子。

Project Summary/Abstract It is currently feasible for small research groups to sequence individual genomes and for larger groups to sequence tens of thousands of individuals. Unfortunately, our ability to identify variants that impact phenotype has not kept pace with our sequencing capacity. This is particularly true of non-coding variants. This proposal presents a pilot screen of more than 32K variants from myCode and ClinVar that suggest 1-2% of exonic mutations affect splicing. The pilot study also revealed that splicing mutations are not uniformly distributed across disease genes or even within genes. This proposal will continue this effort on variants from UK biobank, AllofUs, ClinVar and GTEx that localize to actionable genes. Splicing mutations do not occur uniformly across exons. The proposal seeks to map susceptibility to splicing mutations and identify hotspot exons (exons unusually susceptible to splicing mutations) in the genome. In addition to identifying loss of splicing variants, the little studied problem of gain of splicing variants (i.e. single base mutations that can activate pseudoexons) will also be explored. Preliminary studies locate many intronic regions in pre-mRNA that contain all-but-one of the numerous cis-elements necessary for splicing. In addition to mapping susceptibility to splicing mutations, these efforts will contribute to the basic science of combinatorial signal recognition with a high-level approach that will define mechanisms of splice site selection. Finally, we present a principled method of restoring splicing to mutated exons using antisense oligonucleotides (ASO) that weaken flanking splice sites.