The following is a summary of “A MinION-based Long-Read Sequencing Application With One-Step PCR for the Genetic Diagnosis of 21-Hydroxylase Deficiency,” published in the March 2024 issue of Endocrinology by Adachi, et al.

The study explored the potential of long-read sequencing (LRS) technology for analyzing the CYP21A2 gene associated with 21-hydroxylase deficiency (21OHD). While LRS has been considered promising for genetic analysis, its clinical application for CYP21A2 analysis has been limited. Researchers sought to develop an efficient and cost-effective LRS system for screening the CYP21A2 gene, aiming to improve diagnosis and understanding of 21OHD and other genetic diseases caused by structural rearrangements.

A DNA fragment library was prepared in a single polymerase chain reaction (PCR), covering the entire CYP21A2 gene and known junctions d+ue to TNXB gene structural rearrangements. This yielded an 8-kb product of CYP21A2 or chimeric CYP21A1P/CYP21A2. Barcoding was introduced, and the PCR products were sequenced using a MinION-based platform with Flongle Flow Cells R9.4.1 and R10.4.1.

Reference genotypes of 55 patients with 21OHD were established using conventional methods with multiplex ligation-dependent probe amplification (MLPA) and nested PCR. LRS with Flongle Flow Cell R9.4.1 provided consistent results. Additionally, LRS “duplex” analysis with Flongle Flow Cell R10.4.1 accurately sequenced a variant in a homopolymer region. The introduction of a barcode system reduced costs that were comparable to conventional analysis. A novel single-nucleotide variation, c.940-1G > C, was discovered at the acceptor site of intron 7. A subtype of the classical chimeric junction CH2, “CH2a,” was also identified.

The study successfully established a low-cost and highly accurate LRS system for 21OHD genetic analysis. It demonstrated the feasibility of using LRS for diagnosing 21OHD and other genetic diseases caused by structural rearrangements. The approach could improve diagnostic accuracy and understanding of complex genetic disorders.

Reference: academic.oup.com/jcem/article-abstract/109/3/750/7296056