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Identification of unique DNA methylation sites in Kabuki syndrome using whole genome bisulfite sequencing and targeted hybridization capture followed by enzymatic methylation sequencing



Kabuki syndrome (KS) is a congenital malformation syndrome caused by mutations in the KMT2D and KDM6A genes that encode histone modification enzymes. Although KS is considered a single gene disorder, its symptoms vary widely. Recently, disease-specific DNA methylation patterns, or episignatures, have been recognized and used as a diagnostic tool for KS. Because of various crosstalk mechanisms between histone modifications and DNA methylation, DNA methylation analysis may have high potential for investigations into the pathogenesis of KS.


In this study, we investigated altered CpG-methylation sites that were specific to KS to find important genes associated with the various phenotypes or pathogenesis of KS. Whole genome bisulfite sequencing (WGBS) was performed to select target CpG islands, and enzymatic conversion technology was applied after hybridization capture to confirm KS-specific episignatures of 130 selected differently methylated target regions (DMTRs) in DNA samples from the 65 participants, 31 patients with KS and 34 unaffected individuals, in this study. We identified 26 candidate genes in 22 DMTRs that may be associated with KS. Our results indicate that disease-specific methylation sites can be identified from a small number of WGBS samples, and hybridization capture followed by enzymatic methylation sequencing can simultaneously test the sites.


Although DNA methylation can be tissue-specific, our results suggest that methylation profiling of DNA extracted from peripheral blood may be a powerful approach to study the pathogenesis of diseases.

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Data availability

All data generated and analyzed during this study are included in this published article and its supplementary information files. The datasets used and analyzed during this study are available from the corresponding author on reasonable request.


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We thank all the participants in this study. We especially thank Hayashida C, Fukushima A, and Niiya A for their help with the experiments. We acknowledge all the facilities that provided samples. We thank Margaret Biswas, PhD, from Edanz ( for editing a draft of this manuscript.


This work was supported by the Practical Research Project for Rare/Intractable Diseases (No. 19ek0109234h0003, to KY) from the Agency for Medical Research and Development, and the Grant-in-Aid for Scientific Research (B) (to KY) from the Japan Society for the Promotion of Science.

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Authors and Affiliations



KY conceived the project and planned the study. YH conducted the experiments. YH, HM, TK, SS, and KH analyzed the data. YH and HM wrote the manuscript. AK and KY edited the manuscript. All authors read and approved the final manuscript.

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Correspondence to Koh-ichiro Yoshiura.

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The authors declare no competing interests.

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The experimental procedures were approved by the Committee for Ethical Issues on Human Genome and Gene Analysis at Nagasaki University (permission: #20140803). Written informed consent was obtained from all the participants.

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Hamaguchi, Y., Mishima, H., Kawai, T. et al. Identification of unique DNA methylation sites in Kabuki syndrome using whole genome bisulfite sequencing and targeted hybridization capture followed by enzymatic methylation sequencing. J Hum Genet 67, 711–720 (2022).

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