Rapid Identification of a Disease Allele in Mouse Through Whole Genome Sequencing and Bulk Segregation Analysis
Rapid Identification of a Disease Allele in Mouse Through Whole Genome Sequencing and Bulk Segregation Analysis
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DOI：
10.1534/genetics.110.124586
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发表时间：
2011-03-01
期刊：
影响因子：
3.3
通讯作者：
Beutler, Bruce
中科院分区：
文献类型：
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作者：
Arnold, Carrie N.;Xia, Yu;Beutler, Bruce
在一个由诱变剂N-乙基-N-亚硝基脲（ENU）诱导的种系突变纯合子C57 BL/6 J小鼠谱系中，许多动物在6至7月龄之间在无特定病原体（SPF）条件下死亡。死亡是由肾炎综合征引起的，后者进展为与局灶节段性肾小球硬化相关的肾衰竭。为了确定导致肾脏疾病的突变，我们使用Applied Biosystems SOLiD测序平台对受影响动物的基因组DNA进行了测序。在小鼠基因组中，约74%的包含编码序列和剪接点的核苷酸被覆盖至少三次。在基因组的这一部分中，64个差异被标记为潜在的纯合突变，82个被标记为潜在的杂合突变。通过毛细管测序验证了这些调用中的总共10个，全部为纯合的。其中一个经验证的突变破坏了Col 4a 4转录物的剪接。通过批量分离分析进行的遗传作图排除了所有突变，但这一突变是Aoba小鼠肾脏疾病的原因。Col 4a 4在小鼠中尚未被靶向，这种被命名为Aoba的菌株代表了该物种中第一个功能无效的等位基因。我们的研究证明了全基因组测序结合低分辨率减数分裂作图作为鉴定ENU诱导的致病突变的一种手段的速度和实用性。
In a pedigree of C57BL/6J mice homozygous for germline mutations induced by the mutagen N-ethyl-N-nitrosourea (ENU), numerous animals died under specific pathogen-free (SPF) conditions between 6 and 7 months of age. Death was caused by nephritic syndrome, which progressed to renal failure associated with focal segmental glomerulosclerosis. To identify the mutation responsible for renal disease, we sequenced genomic DNA from an affected animal using the Applied Biosystems SOLiD sequencing platform. Approximately 74% of the nucleotides comprising coding sequences and splice junctions in the mouse genome were covered at least three times. Within this portion of the genome, 64 discrepancies were flagged as potential homozygous mutations and 82 were flagged as potential heterozygous mutations. A total of 10 of these calls, all homozygous, were validated by capillary sequencing. One of the validated mutations disrupted splicing of the Col4a4 transcript. Genetic mapping by bulk segregation analysis excluded all mutations but this one as the cause of renal disease in Aoba mice. Col4a4 has not been targeted in the mouse, and this strain, named Aoba, represents the first functionally null allele in this species. Our study demonstrates the speed and utility of whole genome sequencing coupled with low resolution meiotic mapping as a means of identifying causative mutations induced by ENU.