A MinION-based Long-Read Sequencing Application With One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency
October 13, 2023
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.
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.
Journal Article
JOURNAL：Journal of Clinical Endocrinology and Metabolism
TITLE：A MinION-Based Long-Read Sequencing Application with One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency
DOI：https://doi.org/10.1210/clinem/dgad577
TITLE：A MinION-Based Long-Read Sequencing Application with One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency
DOI：https://doi.org/10.1210/clinem/dgad577
Correspondence to
Kenichi Kashimada ,Associate Professor
Department of Pediatrics and Developmental Biology,
Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University (TMDU)
E-mail：kkashimada.ped (at) tmd.ac.jp
＊Please change (at) in e-mail addresses to @ on sending your e-mail to contact personnels.
Department of Pediatrics and Developmental Biology,
Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University (TMDU)
E-mail：kkashimada.ped (at) tmd.ac.jp
＊Please change (at) in e-mail addresses to @ on sending your e-mail to contact personnels.