Exome screening to identify loss-of-function mutations in the rhesus macaque for development of preclinical models of human disease.
Exome screening to identify loss-of-function mutations in the rhesus macaque for development of preclinical models of human disease.
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DOI：
10.1186/s12864-016-2509-5
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发表时间：
2016-03-02
期刊：
影响因子：
4.4
通讯作者：
Norgren RB Jr
中科院分区：
文献类型：
--
作者：
Cornish AS;Gibbs RM;Norgren RB Jr
外显子组测序已用于鉴定与人类疾病相关的遗传变异。在恒河猴（Macaca mulatta）中用外显子组测序鉴定功能缺失突变可能会导致有价值的遗传疾病动物模型。已经尝试通过将外显子组数据与rheMac 2基因组草图比对来鉴定恒河猴中的变体。然而，由于与rheMac 2相关的不完整性和注释错误，这些努力受到了损害。我们希望确定是否可以将外显子组读数与我们新的改进的恒河猴基因组MacaM进行比对，以鉴定恒河猴中与人类疾病相关的高影响、功能丧失突变。我们比较了来自四只恒河猴、参考动物和三只无关动物的外显子组读数与rheMac 2和MacaM的比对。与rheMac 2相比，针对MacaM比对的读段实质上更多。我们遵循Broad Institute的变异发现最佳实践指南，该指南利用基因组分析工具包来识别高影响突变。当rheMac 2用作参考基因组时，鉴定出大量明显的假阳性。当MacaM被用作参考基因组时，假阳性的数量大大减少。在检查了以MacaM作为参考基因组进行的变异分析后，我们在与人类健康相关的基因中鉴定了两种假定的杂合状态的功能丧失突变。桑格测序证实了这些突变的存在。我们通过三代恒河猴跟踪了这些突变之一（丁酰硫代胆碱基因）的传播。此外，我们证明了丁酰硫代胆碱酯酶活性的功能性降低，类似于在同一基因中具有功能缺失突变的人类杂合子中观察到的。新的MacaM基因组可以有效地用于识别恒河猴中的功能丧失突变，而不会产生高水平的假阳性。在某些情况下，杂合子可以立即用作人类疾病的模型。对于需要纯合突变体的疾病，可以使用功能丧失杂合动物的定向育种来创建人类遗传疾病的恒河猴模型。我们在这里描述的方法可以应用于其他哺乳动物，但前提是它们的基因组已经超越了草案状态。本文的在线版本（doi：10.1186/s12864-016-2509-5）包含补充材料，可供授权用户使用。
Exome sequencing has been utilized to identify genetic variants associated with disease in humans. Identification of loss-of-function mutations with exome sequencing in rhesus macaques (Macaca mulatta) could lead to valuable animal models of genetic disease. Attempts have been made to identify variants in rhesus macaques by aligning exome data against the rheMac2 draft genome. However, such efforts have been impaired due to the incompleteness and annotation errors associated with rheMac2. We wished to determine whether aligning exome reads against our new, improved rhesus genome, MacaM, could be used to identify high impact, loss-of-function mutations in rhesus macaques that would be relevant to human disease. We compared alignments of exome reads from four rhesus macaques, the reference animal and three unrelated animals, against rheMac2 and MacaM. Substantially more reads aligned against MacaM than rheMac2. We followed the Broad Institute’s Best Practice guidelines for variant discovery which utilizes the Genome Analysis Toolkit to identify high impact mutations. When rheMac2 was used as the reference genome, a large number of apparent false positives were identified. When MacaM was used as the reference genome, the number of false positives was greatly reduced. After examining the variant analyses conducted with MacaM as reference genome, we identified two putative loss-of-function mutations, in the heterozygous state, in genes related to human health. Sanger sequencing confirmed the presence of these mutations. We followed the transmission of one of these mutations (in the butyrylthiocholine gene) through three generations of rhesus macaques. Further, we demonstrated a functional decrease in butyrylthiocholinesterase activity similar to that observed in human heterozygotes with loss-of-function mutations in the same gene. The new MacaM genome can be effectively utilized to identify loss-of-function mutations in rhesus macaques without generating a high level of false positives. In some cases, heterozygotes may be immediately useful as models of human disease. For diseases where homozygous mutants are needed, directed breeding of loss-of-function heterozygous animals could be used to create rhesus macaque models of human genetic disease. The approach we describe here could be applied to other mammals, but only if their genomes have been improved beyond draft status. The online version of this article (doi:10.1186/s12864-016-2509-5) contains supplementary material, which is available to authorized users.