Development of ultra-deep targeted RNA sequencing for analyzing X-chromosome inactivation in female Dent disease

Shogo Minamikawa, Kandai Nozu, Yoshimi Nozu, Tomohiko Yamamura, Mariko Taniguchi-Ikeda, Keita Nakanishi, Junya Fujimura, Tomoko Horinouchi, Yuko Shima, Koichi Nakanishi, Masuji Hattori, Kyoko Kanda, Ryojiro Tanaka, Naoya Morisada, China Nagano, Nana Sakakibara, Hiroaki Nagase, Ichiro Morioka, Hiroshi Kaito, Kazumoto Iijima

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)589-595
Number of pages7
JournalJournal of Human Genetics
Issue number5
Publication statusPublished - 01-05-2018
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Genetics
  • Genetics(clinical)


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