Abstract
The pattern of X-chromosome inactivation (XCI) can affect the clinical severity of X-linked disorders in females. XCI pattern analysis has been conducted mainly by HUMARA assay, a polymerase chain reaction-based assay using a methylation-sensitive restriction enzyme. However, this assay examines the XCI ratio of the androgen receptor gene at the genomic DNA level and does not reflect the ratio of either targeted gene directly or at the mRNA level. Here, we report four females with Dent disease, and we clarified the correlation between XCI and female cases of Dent disease using not only HUMARA assay but also a novel analytical method by RNA sequencing. We constructed genetic analysis for 4 female cases showing high level of urinary low-molecular-weight proteinuria and their parents. Their XCI pattern was analyzed by both HUMARA assay and an ultra-deep targeted RNA sequencing of the CLCN5 gene using genomic DNA and mRNA extracted from both leukocytes and urine sediment. All four cases possessed pathogenic variants of the CLCN5 gene. XCI analysis revealed skewed XCI in only two cases, while the other two showed random XCI. All assay results of HUMARA and targeted RNA sequencing in both leukocytes and urinary sediment were clearly identical in all four cases. We developed a novel XCI analytical assay of ultra-deep targeted RNA sequencing and revealed that skewed XCI explains the mechanism of onset of female Dent disease in only half of such cases.
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Acknowledgements
The authors gratefully acknowledge the cooperation of the attending patients and physicians in this study. The authors also thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
Funding
All phases of this study were supported by a grant from the Ministry of Health, Labour and Welfare (Japan) for Research on Rare Intractable Diseases in Kidney and Urinary Tract [H24-nanchitou (nan)-ippan-041 to Kazumoto Iijima] in the Research on Measures for Intractable Diseases Project and a Grant-in-Aid from the Ministry of Culture, Sports, Science and Technology [KAKENHI] (Subject ID: 15K09691 to Kandai Nozu, 26293203 to Kazumoto Iijima and 17K16087 to Shogo Minamikawa).
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Minamikawa, S., Nozu, K., Nozu, Y. et al. Development of ultra-deep targeted RNA sequencing for analyzing X-chromosome inactivation in female Dent disease. J Hum Genet 63, 589–595 (2018). https://doi.org/10.1038/s10038-018-0415-1
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DOI: https://doi.org/10.1038/s10038-018-0415-1
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