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Functional genomics
Original research
Strategic validation of variants of uncertain significance in ECHS1 genetic testing
  1. Yoshihito Kishita1,2,
  2. Ayumu Sugiura2,
  3. Takanori Onuki3,
  4. Tomohiro Ebihara4,
  5. Tetsuro Matsuhashi3,
  6. Masaru Shimura3,
  7. Takuya Fushimi3,
  8. Noriko Ichino2,
  9. Yoshie Nagatakidani1,
  10. Hitomi Nishihata1,
  11. Kazuhiro R Nitta2,
  12. Yukiko Yatsuka2,
  13. Atsuko Imai-Okazaki2,
  14. Yibo Wu5,6,
  15. Hitoshi Osaka7,
  16. Akira Ohtake8,9,
  17. Kei Murayama3,10,
  18. Yasushi Okazaki2,11
  1. 1 Department of Life Science, Faculty of Science and Engineering, Kindai University, Higashiosaka, Osaka, Japan
  2. 2 Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Juntendo University, Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan
  3. 3 Department of Metabolism, Chiba Children's Hospital, Midori-ku, Chiba, Japan
  4. 4 Department of Neonatology, Chiba Children's Hospital, Midori-ku, Chiba, Japan
  5. 5 Chemical Biology Mass Spectrometry Platform (CHEMBIOMS), Faculty of Sciences, University of Geneva, Geneve, Switzerland
  6. 6 YCI Laboratory for Next-Generation Proteomics, RIKEN Center of Integrative Medical Sciences, Yokohama, Kanagawa, Japan
  7. 7 Department of Pediatrics, Jichi Medical University, Shimotsuke, Tochigi, Japan
  8. 8 Department of Pediatrics & Clinical Genomics, Faculty of Medicine, Saitama Medical University, Moroyama, Saitama, Japan
  9. 9 Center for Intractable Diseases, Saitama Medical University Hospital, Moroyama, Saitama, Japan
  10. 10 Center for Medical Genetics, Chiba Children's Hospital, Midori-ku, Chiba, Japan
  11. 11 Laboratory for Comprehensive Genomic Analysis, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan
  1. Correspondence to Dr Yasushi Okazaki, Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Juntendo University, Graduate School of Medicine, Bunkyo-ku, Tokyo, Japan; ya-okazaki{at}juntendo.ac.jp

Abstract

Background Enoyl-CoA hydratase short-chain 1 (ECHS1) is an enzyme involved in the metabolism of branched chain amino acids and fatty acids. Mutations in the ECHS1 gene lead to mitochondrial short-chain enoyl-CoA hydratase 1 deficiency, resulting in the accumulation of intermediates of valine. This is one of the most common causative genes in mitochondrial diseases. While genetic analysis studies have diagnosed numerous cases with ECHS1 variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem.

Methods Here, we constructed an assay system to verify VUS function for ECHS1 gene. A high-throughput assay using ECHS1 knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. In parallel with the VUS validation system, a genetic analysis of samples from patients with mitochondrial disease was performed. The effect on gene expression in cases was verified by RNA-seq and proteome analysis.

Results The functional validation of VUS identified novel variants causing loss of ECHS1 function. The VUS validation system also revealed the effect of the VUS in the compound heterozygous state and provided a new methodology for variant interpretation. Moreover, we performed multiomics analysis and identified a synonymous substitution p.P163= that results in splicing abnormality. The multiomics analysis complemented the diagnosis of some cases that could not be diagnosed by the VUS validation system.

Conclusions In summary, this study uncovered new ECHS1 cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease.

  • RNA-Seq
  • genetic testing
  • genetic variation
  • molecular diagnostic techniques

Data availability statement

Data are available upon reasonable request. Raw data are available from the corresponding author on reasonable request. ECHS1 knockout cells can also be distributed. Some genomic information that could be used to identify individuals cannot be shared due to ethical restrictions.

https://doi.org/10.1136/jmg-2022-109027

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    Data availability statement

    Data are available upon reasonable request. Raw data are available from the corresponding author on reasonable request. ECHS1 knockout cells can also be distributed. Some genomic information that could be used to identify individuals cannot be shared due to ethical restrictions.

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    Footnotes

    • YK and AS are joint first authors.

    • Contributors YK, AS and YO wrote the manuscript. YK, AS, TK, TE, TM, MS, NI, YN, HN, YY and YW performed the experiments. YK, AS, TK, TE, TM, MS, NI, YN, HN, YY, AI-O, YW and YO analysed the data. TK, TE, TM, MS, TF, HO, AO and KM acquired clinical information. YK, AS, TF, KRN and AI-O did bioinformatics and statistical analysis. YK, AS and AO supervised the study. All authors discussed the results and commented on the manuscript. YO is responsible for the overall content as the guarantor.

    • Funding This work was supported by a grant for the Practical Research Project for Rare/Intractable Diseases from AMED to HO, KM, YO and AO (Fund ID: JP21im0210625, JP21ek0109511, JP22ek0109485, JP22ek0109468, JP19ek0109273), Program for Promoting Platform of Genomics based Drug Discovery to YO (Fund ID: JP22kk0305015), the Research Center Network for Realization of Regenerative Medicine (The Acceleration Program for Intractable Diseases Research utilizing Disease-specific iPS cells, JP21bm0804018), and JSPS KAKENHI JP19H03624 to YO and JP20H03648 to HO.

    • 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.