Resources for Comparative Mendelian Disease Genomics

 描述(申请人提供)：大约2,000-3,000万美国人受到孟德尔遗传疾病的影响，这些疾病具有广泛的临床后果，包括先天性心脏病、先天性骨骼疾病、遗传性皮肤病、遗传性神经疾病、遗传性癌症等。在过去的几年里，高通量、全外显子组测序已被用于分子诊断和作为发现新的致病基因的研究工具(S)。自从六年前首次成功应用这项技术以来，已经确定了100多种孟德尔疾病的基本遗传基础。尽管取得了这些进展，但通过全外显子组测序发现人类孟德尔病基因的总体成功率仍然略低于50%。与这些临床病例相比，在小鼠身上发现孟德尔病基因是由遗传定义的近交系背景、用于分离分析的大型血缘家系以及通过使用令人兴奋的新CRISPR/CAS9方法和更传统的基因工程技术进行疾病建模所推动的。在这些相关技术的配合下，近年来全外显子组测序的应用使小鼠的突变发现率提高了近十倍。尽管如此，与人类一样，在小鼠身上发现孟德尔病基因的成功率仅略高于50%。在小鼠和人类基因组中发现疾病基因的全外显子组测序可能的局限性包括变异调用工具的缺点，描述正常的基因组变异的资源不足，以及可能存在调节突变的存在，这些突变驻留在蛋白质编码区之外，通过外显子组测序逃脱检测。随着探索和克服这些限制的承诺，我们的长期目标是开发基因组资源，通过正向基因发现方法和反向基因验证方法，促进小鼠基因组编码和非编码部分的功能化。更具体地说，这项建议的目标是扩大基因发现的范围，超越外显子组强加的限制，并利用这些数据开发小鼠基因组变异数据库和其他优化资源，从而提高突变发现成功率。此外，我们将应用这些新技术来探索大量小鼠新生儿的从头突变和围产期致死表型，以及在已建立的、基因定义的孟德尔疾病模型中识别可遗传的分子损伤。在这两种情况下，我们将使用现代CRISPR/CAS9和其他基因工程方法来验证和模拟突变。

 DESCRIPTION (provided by applicant): Approximately 20-30 million Americans are affected by Mendelian genetic disorders with broad clinical consequences including congenital heart disease, congenital bone diseases, inherited skin diseases, hereditary neurological disorders, hereditary cancers, and others. Over the last several years, high-throughput, whole-exome sequencing has been used for molecular diagnosis and as a research tool for discovery of new disease-causative gene(s). Since the first successful application of this technology six years ago, the fundamental genetic bases for over 100 Mendelian diseases have been identified. Despite these advances, the overall success rate for human Mendelian disease gene discovery by whole-exome sequencing remains at slightly less than 50 percent. In contrast to these clinical cases, the discovery of Mendelian disease genes in mice is powered by genetically defined inbred strain backgrounds, large consanguineous pedigrees for segregation analysis, and disease modeling through the use of exciting new CRISPR/Cas9 approaches and more traditional genetic engineering techniques. With these allied technologies, the application of whole-exome sequencing in recent years has increased the rate of mutation discovery in mouse by nearly ten fold. Still, as in humans, the success rate for Mendelian disease gene discovery in the mouse is only slightly better than 50 percent. Possible limitations of whole-exome sequencing for disease gene discovery in both the mouse and human genomes include shortcomings of variant calling tools, insufficient resources describing `normal' genome variation, and the likely existence of regulatory mutations that, residing outside of protein-codin regions, escape detection by exome-sequencing. With the promise of exploring and surmounting these limitations, our long-term goal is to develop genomic resources that facilitate functionalization of both the coding and non-coding portions of the mouse genome through forward genetic discovery approaches and reverse genetic validation. More specifically, the objectives of this proposal are to expand the scope of gene discovery beyond exome-imposed limitations, and to use these data to develop a mouse genome variation database, and other optimized resources, that will deliver improved mutation discovery success rates. Moreover, we will apply these new technologies to explore de novo mutations and perinatal lethal phenotypes in a large population of mouse neonates, as well as identifying the heritable molecular lesions in established, genetically defined Mendelian disease models. In both settings, we will validate and model the mutations using modern CRISPR/Cas9 and other genetic engineering approaches.