IMPE2023 Poster Presentations Adrenals and HPA Axis (16 abstracts)
Developing A Novel application using long read sequence for CYP21A2 gene analysis
Eriko Adachi 1 , Atsumi Tsuji-Hosokawa 1 , Ryuichi Nakagawa 1 , Maki Gau 1 , Shizuka Kirino 1 , Analia Yogi 1 , Hisae Nakatani 1 , Kei Takasawa 1 , Tomohiro Morio 1 , Osamu Ohara 2 & Kenichi Kashimada 1
1Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University (TMDU), Tokyo, Japan. 2Department of Applied Genomics, Kazusa DNA Research Institute, Chiba, Japan
Introduction: The genetic polymorphisms in CYP21A2, the responsible gene of 21-hydroxylase deficiency (21OHD), are mostly generated by intragenic recombination with the adjacently located pseudogene, CYP21A1P, which shares 98% homologous sequences with CYP21A2. Gene conversion of the CYP21A2 region cannot be identified by usual PCR and Sanger sequencing, because of complicated cross-contamination of CYP21A1P. Next-generation sequencing is not applicable either. The MLPA analysis whose probes are designed to detect copy number variations of CYP21A and TNX genes, and “the hot spot” sequence of CYP21A2 (p.P30L, p.G110fsX21, p.I172N, E6cluster, and p.Q318X mutations is widely accepted. However, uncommon sequence variants cannot be detected, requiring Sanger sequence with a complicated two-step nested PCR amplification. MLPA does not provide the allelic information, and trio-analysis with the parents’ samples is also essential. Recently developed long-read sequencing (LRS) technology, which routinely generates reads in excess of 10 kb, offers advantages over short-read sequencing. In this study, we successfully developed a novel and simple LRS platform for CYP21A2 analysis with a simplifying laboratory workflow.
Objective: Developing a novel quick and affordable methodology for the genetic diagnosis of 21OHD using the LRS technique.
Materials and Methods: Genomic DNA samples from fifty-six subjects of 21OHD were analyzed by two approaches, MLPA and LRS. For the MLPA analysis, we used the SALSA® MLPA® Probemix P050-D1 CAH. For the LRS analysis, we prepared the library by amplification of the CYP21A2 locus using PCR which was designed to cover the whole area of CYP21A2 and the 31-35 exon of the TNXB gene. For cost efficiency, we bar-coded the PCR product and enabled to analyze twenty four samples together for a single long read sequence assay. After the preparation of the library, we analyzed the samples on a MinION platform using a Frongle Flow Cell. The obtained data from LRS were compared to the results by classical genetic analysis, examining the performance of LRS.
Results: Of the 82 alleles from 41 subjects, the genotype of 20 alleles was not determined solely by MLPA, resulting in 75% of the diagnostic rate. On the other hand, one-step LRS identified the variants of all 82 alleles (100%). The cost for one sample was 70 and 30 USD in MLPA and LRS, respectively.
Discussion and Conclusion: Our newly developed LRS analysis is highly efficient for the genetic analysis of CYP21A2.
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