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Analyzing the effects of BRCA1/2 variants on mRNA splicing by minigene assay

Abstract

As BRCA1/2 gene sequencing become more extensive, a large number VUS (variants of uncertain significance) emerge rapidly. Verifying the splicing effect is an effective means for VUS reclassification. The Minigene Assay platform was established and its reliability was verified in this article. 47 BRCA1 or BRCA2 variants were selected and performed to validate their effect on mRNA splicing. The results showed that, a total of 16 variants were experimentally proved to have effects on mRNA splicing, among which 14 variants were shown to cause truncated proteins by Sanger sequencing. While the other two variants, BRCA2 c.7976 + 3 A > G and BRCA1 c.5152 + 3_5152 + 4insT was analyzed to cause 57 bp and 26 bp base in-frame deletion, respectively. The remaining 31 variants were not shown to cause mRNA splicing abnormity, including several sites at the edge of exons, which were predicted to affect splicing of mRNA by multiple bioinformatic software. Based on our experimental results, 37 variants were reclassified by ACMG rules. Our study showed that experimental splicing analysis was effectual for variants classification, and multiple functional assay or clinical data were also necessary for comprehensive judgment of variants.

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Acknowledgements

We thank the Lifeint Co. Ltd. (Xiamen, China), and Zhan Huang (Amoy Diagnostics Co., Ltd., Xiamen, China) for technical support and proofreading the manuscript.

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Correspondence to Huaiyin Shi.

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Dong, Z., Wang, Y., Zhang, J. et al. Analyzing the effects of BRCA1/2 variants on mRNA splicing by minigene assay. J Hum Genet 68, 65–71 (2023). https://doi.org/10.1038/s10038-022-01077-2

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