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RNA sequencing and target long-read sequencing reveal an intronic transposon insertion causing aberrant splicing

Journal of Human Genetics volume 69, pages 91–99 (2024)Cite this article

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Abstract

More than half of cases with suspected genetic disorders remain unsolved by genetic analysis using short-read sequencing such as exome sequencing (ES) and genome sequencing (GS). RNA sequencing (RNA-seq) and long-read sequencing (LRS) are useful for interpretation of candidate variants and detection of structural variants containing repeat sequences, respectively. Recently, adaptive sampling on nanopore sequencers enables target LRS more easily. Here, we present a Japanese girl with premature chromatid separation (PCS)/mosaic variegated aneuploidy (MVA) syndrome. ES detected a known pathogenic maternal heterozygous variant (c.1402-5A>G) in intron 10 of BUB1B (NM_001211.6), a known responsive gene for PCS/MVA syndrome with autosomal recessive inheritance. Minigene splicing assay revealed that almost all transcripts from the c.1402-5G allele have mis-splicing with 4-bp insertion. GS could not detect another pathogenic variant, while RNA-seq revealed abnormal reads in intron 2. To extensively explore variants in intron 2, we performed adaptive sampling and identified a paternal 3.0 kb insertion. Consensus sequence of 16 reads spanning the insertion showed that the insertion consists of Alu and SVA elements. Realignment of RNA-seq reads to the new reference sequence containing the insertion revealed that 16 reads have 5’ splice site within the insertion and 3’ splice site at exon 3, demonstrating causal relationship between the insertion and aberrant splicing. In addition, immunoblotting showed severely diminished BUB1B protein level in patient derived cells. These data suggest that detection of transcriptomic abnormalities by RNA-seq can be a clue for identifying pathogenic variants, and determination of insert sequences is one of merits of LRS.

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Acknowledgements

We would like to thank the patients for participating in this work. This work was supported by the Japan Agency for Medical Research and Development (AMED) (JP23ek0109549 to TO and JP23ek01099674, JP23ek0109637 to HS), Grants-in-Aid for Scientific Research (B) (JP20H03641 and JP23H02875 to HS), Grant-in-Aid for Early-Career Scientists (23K14944 to TH), Grant-in-Aid for Research Activity Start-up (22K20852 to TH) from the Japan Society for the Promotion of Science (JSPS) KAKENHI, Japan Intractable Diseases (Nanbyo) Research Foundation (2020A02), and the Takeda Science Foundation.

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  1. These authors contributed equally: Ryota Kawakami, Takuya Hiraide.

Authors and Affiliations

  1. Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, Japan

    Ryota Kawakami, Takuya Hiraide, Kota Hira, Kazuyuki Komatsu, Hidetoshi Ishigaki, Kimiyoshi Sakaguchi & Isao Miyairi

  2. Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan

    Kazuki Watanabe, Sachiko Miyamoto, Kazuyuki Komatsu, Tsutomu Ogata & Hirotomo Saitsu

  3. Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan

    Masato Maekawa & Keita Yamashita

  4. Department of Hamamatsu Child Health and Developmental Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan

    Tokiko Fukuda

  5. Department of Pediatrics, Hamamatsu Medical Center, Hamamatsu, Japan

    Tsutomu Ogata

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  1. Ryota Kawakami
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Contributions

HS contributed to the conception and design of the study. RK, TH, KW, SM, KH, KK, HI, KS, MM, KY, TF, IM, TO, and HS contributed to the acquisition and analysis of data. RK, TH, and HS contributed to drafting the text and preparing the figure. All authors read and approved the final manuscript.

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Correspondence to Hirotomo Saitsu.

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Kawakami, R., Hiraide, T., Watanabe, K. et al. RNA sequencing and target long-read sequencing reveal an intronic transposon insertion causing aberrant splicing. J Hum Genet 69, 91–99 (2024). https://doi.org/10.1038/s10038-023-01211-8

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