背景:X连锁肌营养不良蛋白缺陷型肌营养不良(MD)是由DMD基因变异引起的MD形式。它是一种致命的疾病,其特征是骨骼肌进行性虚弱和退化。
目的:使用下一代测序仪通过全基因组测序(WGS)鉴定DMD中的有害遗传变异。
方法:一只受MD影响的猫,它的父母,和354只来自繁殖地的猫。
方法:我们将受影响的猫的WGS数据与国家生物技术信息中心数据库中的数据进行了比较,并通过计算机模拟分析搜索了候选的高影响变体。接下来,我们使用来自父母和繁殖群体的猫的样本通过Sanger测序确认了候选变体。我们使用了2个基因组组件,标准的felCat9(来自阿比西尼亚猫)和小说AnAms1.0(来自美国短毛猫),评估基因组组装差异。
结果:我们发现了2种新的高影响变体:felCat9中的1bp缺失和felCat9和AnAms1.0中的相同无义变体。全基因组和Sanger测序验证表明,由于组装错误,felCat9中的缺失是假阳性。在357只猫中,废话变体只在受影响的猫中发现,这表明它是一个从头变体。
结论:我们在受影响的猫中确定了从头变异,并且确定了整个DMD基因的基于下一代测序的基因分型对于受影响的猫是必要的,因为受影响的猫的父母没有风险变异。
BACKGROUND: X-linked dystrophin-deficient muscular dystrophy (MD) is a form of MD caused by variants in the DMD gene. It is a fatal disease characterized by progressive weakness and degeneration of skeletal muscles.
OBJECTIVE: Identify deleterious genetic variants in DMD by whole-genome sequencing (WGS) using a next-generation sequencer.
METHODS: One MD-affected cat, its parents, and 354 cats from a breeding colony.
METHODS: We compared the WGS data of the affected cat with data available in the National Center for Biotechnology Information database and searched for candidate high-impact variants by in silico analyses. Next, we confirmed the candidate variants by Sanger sequencing using samples from the parents and cats from the breeding colony. We used 2 genome assemblies, the standard felCat9 (from an Abyssinian cat) and the novel AnAms1.0 (from an American Shorthair cat), to evaluate genome assembly differences.
RESULTS: We found 2 novel high-impact variants: a 1-bp deletion in felCat9 and an identical nonsense variant in felCat9 and AnAms1.0. Whole genome and Sanger sequencing validation showed that the deletion in felCat9 was a false positive because of misassembly. Among the 357 cats, the nonsense variant was only found in the affected cat, which indicated it was a de novo variant.
CONCLUSIONS: We identified a de novo variant in the affected cat and next-generation sequencing-based genotyping of the whole DMD gene was determined to be necessary for affected cats because the parents of the affected cat did not have the risk variant.