Folkhälsan Research Center, Helsinki, Finland.
Department of Medical Genetics, Medicum, University of Helsinki, Helsinki, Finland.
PLoS One. 2022 May 16;17(5):e0267793. doi: 10.1371/journal.pone.0267793. eCollection 2022.
The human genome contains repetitive regions, such as segmental duplications, known to be prone to copy number variation. Segmental duplications are highly identical and homologous sequences, posing a specific challenge for most mutation detection methods. The giant nebulin gene is expressed in skeletal muscle. It harbors a large segmental duplication region composed of eight exons repeated three times, the so-called triplicate region. Mutations in nebulin are known to cause nemaline myopathy and other congenital myopathies. Using our custom targeted Comparative Genomic Hybridization arrays, we have previously shown that copy number variations in the nebulin triplicate region are pathogenic when the copy number of the segmental duplication block deviates two or more copies from the normal number, which is three per allele. To complement our Comparative Genomic Hybridization arrays, we have established a custom Droplet Digital PCR method for the detection of copy number variations within the nebulin triplicate region. The custom Droplet Digital PCR assays allow sensitive, rapid, high-throughput, and cost-effective detection of copy number variations within this region and is ready for implementation a screening method for disease-causing copy number variations of the nebulin triplicate region. We suggest that Droplet Digital PCR may also be used in the study and diagnostics of other segmental duplication regions of the genome.
人类基因组包含重复区域，如片段重复，已知易发生拷贝数变异。片段重复是高度相同和同源的序列，这对大多数突变检测方法构成了特殊挑战。巨大的肌联蛋白基因在骨骼肌中表达。它含有一个由八个外显子重复三次组成的大片段重复区域，即所谓的三重复区域。肌联蛋白突变已知会导致杆状体肌病和其他先天性肌病。使用我们的定制靶向比较基因组杂交阵列，我们之前已经表明，当重复块的拷贝数偏离正常数量（每个等位基因三个）两个或更多拷贝时，肌联蛋白三重复区域的拷贝数变异是致病性的。为了补充我们的比较基因组杂交阵列，我们建立了一种用于检测肌联蛋白三重复区域内拷贝数变异的定制液滴数字 PCR 方法。定制的液滴数字 PCR 检测可灵敏、快速、高通量且具有成本效益地检测该区域内的拷贝数变异，并且已经准备好实施用于肌联蛋白三重复区域致病拷贝数变异的筛选方法。我们建议液滴数字 PCR 也可用于基因组其他片段重复区域的研究和诊断。
