当前位置: X-MOL 学术Eur. J. Hum. Genet. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease
European Journal of Human Genetics ( IF 5.2 ) Pub Date : 2020-11-09 , DOI: 10.1038/s41431-020-00751-3
Atze J Bergsma 1, 2, 3 , Stijn L M In 't Groen 1, 2, 3 , Fabio Catalano 1, 2, 3 , Manjiro Yamanaka 1, 4 , Satoru Takahashi 5 , Toshika Okumiya 6 , Ans T van der Ploeg 2, 3 , W W M Pim Pijnappel 1, 2, 3
Affiliation  

DNA variants affecting mRNA expression and processing in genetic diseases are often missed or poorly characterized. We previously reported a generic assay to identify variants that affect mRNA expression and splicing in Pompe disease, a monogenic disorder caused by deficiency of acid α-glucosidase (GAA). However, this assay could miss mRNA that is subjected to degradation. Here, we inhibited mRNA degradation using cycloheximide and performed unbiased splicing analysis of all GAA exons using exon flanking RT-PCR and exon internal RT-qPCR. In four patients that were suspected of harboring splicing variants but for which aberrant splicing could not be detected in normally growing cells, we detected a total of 10 novel splicing events in cells treated with cycloheximide. In addition, we found that sequences of GAA introns 6 and 12 were naturally included in a subset of transcripts from patients and healthy controls, indicating inefficient canonical splicing. Identification of aberrant splicing caused by the common Asian variant c.546G>T allowed the development of an antisense oligonucleotide that promoted canonical GAA pre-mRNA splicing and elevated GAA enzymatic activity. Our results indicate that this extended generic splicing assay allows the detection of aberrant splicing in cases of mRNA degradation to enable functional analysis of unknown splicing variants and the development of targeted treatment options.



中文翻译:

一种用于识别在庞贝病中诱导无义介导衰变的剪接变体的通用测定法

影响遗传疾病中 mRNA 表达和加工的 DNA 变体经常被遗漏或表征不佳。我们之前报道了一种通用测定法,用于鉴定影响庞贝病中 mRNA 表达和剪接的变体,庞贝病是一种由酸性 α-葡萄糖苷酶 ( GAA )缺乏引起的单基因疾病。然而,该测定可能会遗漏受降解的 mRNA。在这里,我们使用放线菌酮抑制 mRNA 降解并对所有GAA进行了无偏剪接分析外显子使用外显子侧翼 RT-PCR 和外显子内部 RT-qPCR。在怀疑存在剪接变体但在正常生长的细胞中无法检测到异常剪接的四名患者中,我们在用放线菌酮处理的细胞中检测到总共 10 个新的剪接事件。此外,我们发现 GAA内含子 6 和 12 的序列自然地包含在来自患者和健康对照的转录子集中,表明规范剪接效率低下。由常见的亚洲变体 c.546G>T 引起的异常剪接的鉴定允许开发促进规范GAA的反义寡核苷酸pre-mRNA 剪接和 GAA 酶活性升高。我们的结果表明,这种扩展的通用剪接测定允许在 mRNA 降解的情况下检测异常剪接,从而能够对未知剪接变体进行功能分析并开发靶向治疗方案。

更新日期:2020-11-09
down
wechat
bug