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

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. Access through your institution Buy or subscribe This is a preview of subscription

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references 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. AUTHOR INFORMATION Author notes * These authors contributed equally: Ryota Kawakami, Takuya Hiraide. AUTHORS AND AFFILIATIONS *

Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, Japan Ryota Kawakami, Takuya Hiraide, Kota Hira, Kazuyuki Komatsu, Hidetoshi Ishigaki, Kimiyoshi Sakaguchi &

 Isao Miyairi * Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan Kazuki Watanabe, Sachiko Miyamoto, Kazuyuki Komatsu, Tsutomu Ogata & Hirotomo Saitsu

* Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan Masato Maekawa & Keita Yamashita * Department of Hamamatsu Child Health and Developmental

Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan Tokiko Fukuda * Department of Pediatrics, Hamamatsu Medical Center, Hamamatsu, Japan Tsutomu Ogata Authors * Ryota

Kawakami View author publications You can also search for this author inPubMed Google Scholar * Takuya Hiraide View author publications You can also search for this author inPubMed Google

Scholar * Kazuki Watanabe View author publications You can also search for this author inPubMed Google Scholar * Sachiko Miyamoto View author publications You can also search for this author

inPubMed Google Scholar * Kota Hira View author publications You can also search for this author inPubMed Google Scholar * Kazuyuki Komatsu View author publications You can also search for

this author inPubMed Google Scholar * Hidetoshi Ishigaki View author publications You can also search for this author inPubMed Google Scholar * Kimiyoshi Sakaguchi View author publications

You can also search for this author inPubMed Google Scholar * Masato Maekawa View author publications You can also search for this author inPubMed Google Scholar * Keita Yamashita View

author publications You can also search for this author inPubMed Google Scholar * Tokiko Fukuda View author publications You can also search for this author inPubMed Google Scholar * Isao

Miyairi View author publications You can also search for this author inPubMed Google Scholar * Tsutomu Ogata View author publications You can also search for this author inPubMed Google

Scholar * Hirotomo Saitsu View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Hirotomo Saitsu. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests.

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Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 09 August 2023 * Revised: 28 November 2023 *

Accepted: 01 December 2023 * Published: 15 December 2023 * Issue Date: February 2024 * DOI: https://doi.org/10.1038/s10038-023-01211-8 SHARE THIS ARTICLE Anyone you share the following link

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