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Original research
RNA splicing analysis contributes to reclassifying variants of uncertain significance and improves the diagnosis of monogenic disorders
  1. Wen-Bin He1,2,3,4,
  2. Wen-Juan Xiao2,5,
  3. Cong-Ling Dai1,2,3,4,
  4. Yu-Rong Wang5,
  5. Xiu-Rong Li3,4,
  6. Fei Gong1,2,3,4,
  7. Lan-Lan Meng1,3,
  8. Chen Tan1,
  9. Si-Cong Zeng3,5,
  10. Guang-Xiu Lu2,3,4,5,
  11. Ge Lin1,2,3,4,
  12. Yue-Qiu Tan1,2,3,4,5,
  13. Hao Hu3,4,
  14. Juan Du1,2,3,4
  1. 1Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan, China
  2. 2National Engineering and Research Center of Human Stem Cells, Changsha, Hunan, China
  3. 3Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China
  4. 4Clinical Research Center For Reproduction and Genetics In Hunan Province, Changsha, China
  5. 5Hunan Guangxiu Hospital, Medical College of Hunan Normal University, Changsha, Hunan, China
  1. Correspondence to Dr Juan Du, Central South University, Changsha 410078, Hunan, China; tandujuan{at}; Dr Hao Hu, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, Hunan, China; leonhu97{at}


Background Numerous variants of uncertain significance (VUSs) have been identified by whole exome sequencing in clinical practice. However, VUSs are not currently considered medically actionable.

Objective To assess the splicing patterns of 49 VUSs in 48 families identified clinically to improve genetic counselling and family planning.

Methods Forty-nine participants with 49 VUSs were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Bioinformatic analysis was performed to preliminarily predict the splicing effects of these VUSs. RT-PCR and minigene analysis were used to assess the splicing patterns of the VUSs. According to the results obtained, couples opted for different methods of reproductive interventions to conceive a child, including prenatal diagnosis and preimplantation genetic testing (PGT).

Results Eleven variants were found to alter pre-mRNA splicing and one variant caused nonsense-mediated mRNA decay, which resulted in the reclassification of these VUSs as likely pathogenic. One couple chose to undergo in vitro fertilisation with PGT treatment; a healthy embryo was transferred and the pregnancy is ongoing. Three couples opted for natural pregnancy with prenatal diagnosis. One couple terminated the pregnancy because the fetus was affected by short-rib thoracic dysplasia and harboured the related variant. The infants of the other two couples were born and were healthy at their last recorded follow-up.

Conclusion RNA splicing analysis is an important method to assess the impact of sequence variants on splicing in clinical practice and can contribute to the reclassification of a significant proportion of VUSs. RNA splicing analysis should be considered for genetic disease diagnostics.

  • genetic testing
  • mutation
  • sequence analysis
  • RNA

Data availability statement

Data are available upon reasonable request.

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Data availability statement

Data are available upon reasonable request.

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  • W-BH, W-JX and C-LD contributed equally.

  • HH and JD contributed equally.

  • Contributors HH and JD designed the study. W-BH, WX, CD and Y-Q T analysed the data and wrote the paper. Y-RW and SZ performed the bioinformatic analysis. W-BH, WX, CD, CT and LM performed the study. XL, FG, G-XL and GL performed the clinical work. JD is responsible for the overall content of the manuscript acting as guarantor.

  • Funding This work was supported by grants from the National Key Research and Development Program of China (2018YFC1004901), the National Natural Science Foundation of China (81771645 and 81971447), the Hunan Provincial Natural Science Foundation of China (2019JJ51006, 2019JJ50397), the Hunan Provincial Grant for Innovative Province Construction (2019SK4012), the Key Grant of Prevention and Treatment of Birth Defect from Hunan Province (2019SK1012), and the Research Grant of CITIC-Xiangya (YNXM-201915, YNXM-201913, YNXM-201912, YNXM-202002).

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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