A MinION-based Long-Read Sequencing Application With One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency
Abstract
Context: Recently developed long-read sequencing (LRS) technology has been considered an option for CYP21A2 analysis. However, the clinical use of LRS for CYP21A2 analysis is limited. Objective: This study's objective is to develop an efficient and low-cost LRS system for CYP21A2 screening. Methods: A DNA fragment library was prepared in a single polymerase chain reaction (PCR) that covers the entire CYP21A2 gene and all known junctions caused by TNXB gene structural rearrangements, yielding a single 8-kb product of CYP21A2 or CYP21A1P/CYP21A2 chimera. After barcoding, the PCR products were sequenced on a MinION-based platform with Flongle Flow Cell R9.4.1 and R10.4.1. Results: The reference genotypes of 55 patients with 21-hydroxylase deficiency (21OHD) were established using the conventional method with multiplex ligation-dependent probe amplification (MLPA) and nested PCR. LRS using Flongle Flow Cell R9.4.1 yielded consistent results. Additionally, the recently updated LRS “duplex” analysis with Flongle flow cell R10.4.1 was tested to reveal an advantage of accurately sequencing a variant located on the homopolymer region. By introducing a barcode system, the cost was reduced to be comparable to that of conventional analysis. A novel single-nucleotide variation was discovered at the acceptor site of intron 7, c.940-1G > C. We also identified a subtype of the classical chimeric junction CH2, “CH2a,” in the region from the latter part of intron 5 to exon 6. Conclusion: We successfully established a novel low-cost and highly accurate LRS system for 21OHD genetic analysis. Our study provides insight into the feasibility of LRS for diagnosing 21OHD and other genetic diseases caused by structural rearrangements.
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CYP21A2 genotyping may be complicated by large deletions and gene conversions that generate TNXA/TNXB or CYP21A1P/CYP21A2 chimeric alleles through non-allelic homologous recombination.
“Beyond the effect of individual variants, CYP21A2 genotyping may be complicated by large deletions and gene conversions that generate TNXA/TNXB or CYP21A1P/CYP21A2 chimeric alleles through non-allelic homologous recombination.”
From Evolution of molecular diagnostic strategies for 21-hydroxylase deficiency: from classical methods to advanced genomic techniques (2026) · cites this paper as [97]
Cites
Congenital Adrenal Hyperplasia—Current Insights in Pathophysiology, Diagnostics, and Management
Claahsen - van der Grinten, H. L., Speiser, P. W., Ahmed, S. F., Arlt, W., Auchus, R. J., Falhammar, H., Flück, C. E., Guasti, L., Huebner, A., Kortmann, B. B. M., et al. (2022) · Endocrine Reviews · doi:10.1210/endrev/bnab016 · Cited by 556 · see citing works
New, M. I., Abraham, M., Gonzalez, B., Dumic, M., Razzaghy-Azar, M., Chitayat, D., Sun, L., Zaidi, M., Wilson, R. C., & Yuen, T. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1300057110 · Cited by 365 · see citing works
Characterization of frequent deletions causing steroid 21-hydroxylase deficiency
White, P. C., Vitek, A., Dupont, B., & New, M. I. (1988) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.85.12.4436 · Cited by 229 · see citing works
Falhammar, H., Frisén, L., Hirschberg, A. L., Norrby, C., Almqvist, C., Nordenskjöld, A., & Nordenström, A. (2015) · The Journal of Clinical Endocrinology & Metabolism · doi:10.1210/jc.2015-2093 · Cited by 204 · see citing works
Nonsense mutation causing steroid 21-hydroxylase deficiency
Globerman, H., Amor, M., Parker, K. L., New, M. I., & White, P. C. (1988) · Journal of Clinical Investigation · doi:10.1172/jci113562 · Cited by 130 · see citing works
Junction Site Analysis of Chimeric CYP21A1P/CYP21A2 Genes in 21-Hydroxylase Deficiency
Chen, W., Xu, Z., Sullivan, A., Finkielstain, G. P., Van Ryzin, C., Merke, D. P., & McDonnell, N. B. (2012) · Clinical Chemistry · doi:10.1373/clinchem.2011.174037 · Cited by 86 · see citing works
Tardy, V., Menassa, R., Sulmont, V., Lienhardt-Roussie, A., Lecointre, C., Brauner, R., David, M., & Morel, Y. (2010) · The Journal of Clinical Endocrinology & Metabolism · doi:10.1210/jc.2009-1202 · Cited by 73 · see citing works
Liu, S.-Y., Lee, C.-T., Tung, Y.-C., Chien, Y.-H., Hwu, W.-L., & Tsai, W.-Y. (2018) · Journal of the Formosan Medical Association · doi:10.1016/j.jfma.2017.03.008 · Cited by 18 · see citing works
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Miranda, M. C. D., Oliveira, A. C. M. D., Soares, G. J., Batatinha, J. A. P., Latronico, A. C., Mendonca, B. B., & Bachega, T. A. S. S. (2026) · Frontiers in Endocrinology · doi:10.3389/fendo.2026.1770274 · Cited by 2 · see citing works
Claps, A., Kolomenski, J. E., Fernández, F., Macchiaroli, N., Ingravidi, M. L., Delea, M., Fernández, C., Castro, T., Laiseca, J., Kamenetzky, L., et al. (2025) · Scientific Reports · doi:10.1038/s41598-025-03799-7 · Cited by 3 · see citing works
- Authors
- Adachi, E., Nakagawa, R., Tsuji-Hosokawa, A., Gau, M., Kirino, S., Yogi, A., Nakatani, H., Takasawa, K., Yamaguchi, T., Kosho, T., Murakami, M., Tajima, T., Hasegawa, T., Yamada, T., Morio, T., Ohara, O., & Kashimada, K.
- Affiliations
- Tokyo Medical and Dental University (JP)
- Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University (TMDU) , Tokyo 113-8519 , Japan
- Shinshu University (JP)
- Jichi Medical University (JP)
- Keio University (JP)
- Kazusa DNA Research Institute (JP)
- Corresponding author
- Kashimada, Kenichi
- ORCID
- Venue
- The Journal of Clinical Endocrinology & Metabolism
- Publisher
- The Endocrine Society
- Published
- 2023-10-06
- Volume
- 109
- Issue
- 3
- Pages
- 750-760
- ISSN
- 0021-972X (ISSN-L)
- 1945-7197
- OpenAlex
- W4387421026
- Cited by
- 16
- References
- 8
- Citations by year
- 2026: 8 · 2025: 6 · 2024: 2
- FWCI
- 1.42
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The Journal of Clinical Endocrinology & Metabolism (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Ministry of Health, Labour and Welfare of Japan (No. 20FC1020)
- Japan Agency for Medical Research and Development (No. 23ek0109630h0001)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sexual Differentiation and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Keywords
- 21-hydroxylase deficiency, CYP21A2, long-read sequencing, Oxford Nanopore MinION, structural rearrangement, duplex sequencing
- MeSH terms
- Adrenal Hyperplasia, Congenital, Genotype, Humans, Mutation, Steroid 21-Hydroxylase, Multiplex Polymerase Chain Reaction
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