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Identification and characterization of two DMD pedigrees with large inversion mutations based on a long-read sequencing pipeline


Pathogenic large inversions are rarely reported on DMD gene due to the lack of effective detection methods. Here we report two DMD pedigrees and proposed a reliable pipeline to define large inversions in DMD patients. In the first pedigree, conventional approaches including multiplex ligation-dependent probe amplification, and whole-exome sequencing by next generation sequencing were failed to detect any pathologic variant. Then an advanced analysis pipeline which consists of RNA-seq, cDNA array capture sequencing, optical mapping, long-read sequencing was built. RNA-seq and cDNA capture sequencing showed a complete absence of transcripts of exons 3–55. Optical mapping identified a 55 Mb pericentric inversion between Xp21 and Xq21. Subsequently, long-read sequencing and Sanger sequencing determined the inversion breakpoints at 32,915,769 and 87,989,324 of the X chromosomes. In the second pedigree, long-read sequencing was directly conducted and Sanger sequencing was performed to verify the mutation. Long-read sequencing and Sanger sequencing found breakpoints at 32,581,576 and 127,797,236 on DMD gene directly. In conclusion, large inversion might be a rare but important mutation type in DMD gene. An effective pipeline was built in detecting large inversion mutations based on long-read sequencing platforms.

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Fig. 1: The muscle pathology, family pedigree and diagnostic PCR results of the first pedigree.
Fig. 2: The results of RNA sequencing and cDNA sequencing.
Fig. 3: The results of optical mapping of the pathogenic structural variation.
Fig. 4: The pathogenic inversion variant identified by long-read sequencing for two pedigrees.

Data availability

The data are available from the corresponding author on reasonable request.


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The authors express sincere gratitude to the patient and the family members who participated in the study. Special acknowledgements go to Dr Xiaoming (BGI Clinical Laboratory, Wuhan, China) for assistance for muscle tissue mRNA sequencing and to Ms Minming Zheng (Grandomics Biosciences, Beijing, China) for preparing the figures.


This study was funded by CAMS Innovation Fund for Medical Sciences (CIFMS) (No.2016-I2M-1-002) and Chinese National Basic Research Program (973) (No.2017YFC1001902).

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Authors and Affiliations



YD and JP designed the study. CG, CZ, YD, and JP collected clinical information of the pedigree. BZ and JH performed karyotype and PCR tests of family members. YZ and LC performed the pathology analysis of the muscle biopsy. PL, FL, and HJ conducted Bionano and ONT data analysis, YW and YRW performed Bionano and ONT sequencing and data collection, FY performed MLPA experiment. YD, JP LYC, DL, SL, DW, and LC supervised the project. CG, CZ, YT, MSP, YD, and JP drafted the paper with input from all authors.

Corresponding authors

Correspondence to Jing Peng or Yi Dai.

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The authors declare no competing interests.

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This study was approved by the ethics committee of the Peking Union Medical College Hospital (IRB #JS-1233). All participants and the parents of those <18years of age at the time of genetic testing gave written informed consent.

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Geng, C., Zhang, C., Li, P. et al. Identification and characterization of two DMD pedigrees with large inversion mutations based on a long-read sequencing pipeline. Eur J Hum Genet (2022).

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