期刊
PEDIATRIC HEMATOLOGY AND ONCOLOGY
卷 38, 期 6, 页码 515-527出版社
TAYLOR & FRANCIS INC
DOI: 10.1080/08880018.2021.1887984
关键词
Diamond-Blackfan anemia; functional splicing analysis; intronic variant; minigene assay
This study presents functional splicing analysis results of deep intronic variants in the pathogenic gene RPS19 of Diamond-Blackfan anemia (DBA), with one variant leading to exon 6 skipping. Minigene assay analysis of three previously reported deep intronic variants showed no aberrant splicing. Therefore, the minigene assay is an effective method for functional splicing analysis of inherited diseases.
Diamond-Blackfan anemia (DBA) is mainly caused by pathogenic variants in ribosomal proteins and 22 responsible genes have been identified to date. The most common causative gene of DBA is RPS19 [NM_001022.4]. Nearly 180 RPS19 variants have been reported, including three deep intronic variants outside the splicing consensus sequence (c.72-92A > G, c.356 + 18G > C, and c.411 + 6G > C). We also identified one case with a c.412-3C > G intronic variant. Without conducting transcript analysis, the pathogenicity of these variants is unknown. However, it is difficult to assess transcripts because of their fragility. In such cases, in vitro functional splicing assays can be used to assess pathogenicity. Here, we report functional splicing analysis results of four RPS19 deep intronic variants identified in our case and in previously reported cases. One splicing consensus variant (c.411 + 1G > A) was also examined as a positive control. Aberrant splicing with a 2-bp insertion between exons 5 and 6 was identified in the patient samples and minigene assay results also identified exon 6 skipping in our case. The exon 6 skipping transcript was confirmed by further evaluation using quantitative RT-PCR. Additionally, minigene assay analysis of three reported deep intronic variants revealed that none of them showed aberrant splicing and that these variants were not considered to be pathogenic. In conclusion, the minigene assay is a useful method for functional splicing analysis of inherited disease.
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