Candidate gene analysis using genomic quantitative PCR: identification of ADAMTS13 large deletions in two patients with Upshaw-Schulman syndrome.
Candidate gene analysis using genomic quantitative PCR: identification of ADAMTS13 large deletions in two patients with Upshaw-Schulman syndrome.
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DOI：
10.1002/mgg3.64
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发表时间：
2014-05
影响因子：
2
通讯作者：
Miyata, Toshiyuki
中科院分区：
文献类型：
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作者：
Eura, Yuka;Kokame, Koichi;Takafuta, Toshiro;Tanaka, Ryojiro;Kobayashi, Hikaru;Ishida, Fumihiro;Hisanaga, Shuichi;Matsumoto, Masanori;Fujimura, Yoshihiro;Miyata, Toshiyuki
直接测序是发现导致遗传性疾病的候选基因突变的流行方法。然而，该方法可能会漏掉某种类型的突变。在这里，我们报告了一种全面的基因组定量聚合酶链反应（qPCR），以弥补直接测序的弱点。 Upshaw-Schulman 综合征 (USS) 是一种与血浆 ADAMTS13 活性严重缺乏相关的隐性遗传性疾病。我们之前使用直接测序分析了 47 名 USS 患者的 ADAMTS13，其中 44 名患者具有纯合子或复合杂合子突变。然后，我们试图揭示其余三名患者中 ADAMTS13 更广泛的缺陷。我们使用基因组 qPCR 量化了患者中每个 ADAMTS13 外显子的拷贝数。每个引物对被设计为至少包含直接测序中使用的两个引物中的一个，以避免遗漏任何外显子缺失。 qPCR 显示一名患者杂合外显子 7 和 8 杂合缺失，另一名患者杂合外显子 27 缺失，进一步分析分别显示 c.746_987+373del1782 和 c.3751_3892+587del729。基因组 qPCR 为识别靶基因的广泛缺陷提供了一种有效的方法。
Direct sequencing is a popular method to discover mutations in candidate genes responsible for hereditary diseases. A certain type of mutation, however, can be missed by the method. Here, we report a comprehensive genomic quantitative polymerase chain reaction (qPCR) to complement the weakness of direct sequencing. Upshaw-Schulman syndrome (USS) is a recessively inherited disease associated with severe deficiency of plasma ADAMTS13 activity. We previously analyzed ADAMTS13 in 47 USS patients using direct sequencing, and 44 of them had either homozygous or compound heterozygous mutations. Then, we sought to reveal more extensive defects of ADAMTS13 in the remaining three patients. We quantified copy numbers of each ADAMTS13 exon in the patients by using genomic qPCR. Each primer pair was designed to contain at least one of the two primers used in direct sequencing, to avoid missing any exonic deletions. The qPCR demonstrated heterozygous loss of exons 7 and 8 in one patient and exon 27 in the other, and further analysis revealed c.746_987+373del1782 and c.3751_3892+587del729, respectively. Genomic qPCR provides an effective method for identifying extensive defects of the target genes.