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ORIGINAL RESEARCH article

Front. Genet.
Sec. Genetics of Common and Rare Diseases
Volume 15 - 2024 | doi: 10.3389/fgene.2024.1472516
This article is part of the Research Topic Insights in Genetics of Common and Rare Diseases 2024 View all articles

Comparison of long-read sequencing and MLPA combined with long-PCR sequencing of CYP21A2 mutations in patients with 21-OHD

Provisionally accepted
Tian Lan Tian Lan 1JIN WANG JIN WANG 2*HUI YAO HUI YAO 1*Xiaohong C. Chenxhsci123 Xiaohong C. Chenxhsci123 1BI K. CHEN BI K. CHEN 1*RU J. ZHANG RU J. ZHANG 3*
  • 1 Department of Endocrinology, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
  • 2 Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
  • 3 Grandomics Biosciences Co.,Ltd., Changping, Beijing, China

The final, formatted version of the article will be published soon.

    Background 21-Hydroxylase deficiency (21-OHD) is caused by mutations in the CYP21A2 gene. Genetic testing for 21-OHD is currently facing challenges. Moreover, there are no comparative studies on detecting CYP21A2 mutations by both secondgeneration sequencing and long-read sequencing .Objective To detect CYP21A2 variations in 21-OHD patients using targeted capture with LRS method based on the PacBio Sequel II platform.Methods A total of 67 patients with 21-OHD. The full sequence of CYP21A2 gene was analyzed by targeted capture combined with LRS based on the PacBio Sequel II platform. The results were compared with those of long-polymerase chain reaction (Long-PCR) combined with multiplex ligation probe amplification (MLPA) detection. The correlation between different genotype groups and clinical typing was observed.The study analyzed a total of 67 patients. Among them, 44 (65.67%) were males and 23 (34.33%) were females, with a male-to-female ratio of approximately 1.9:1. A total of 27 pathogenic variants were identified in the 67 patients, of which micro-conversion accounted for 61.9%, new variants of CYP21A2 accounted for 8.2%; deletion accounted for 22.4% (CYP21A2 single deletion and chimeric TNXA/TNXB accounted for 12.7%, chimeric CYP21A1P/CYP21A2 accounted for 9.7%); and duplication accounted for 3.0% (CYP21A2 Gene Duplication). I2G was the most common variant (26.9%). Targeted capture LRS and MLPA combined with Long-PCR detection of CYP21A2 mutations showed 30 detection results with differences. The overall genotype-phenotype correlation was 82.1%. The correlation coefficient rs between the severity of the phenotype and the genotype group was 0.682 (P < 0.05).Targeted capture combined with LRS is an integrated approach for detecting CYP21A2 mutations, allowing precise determination of connected sites for multiple deletions/insertions and cis/trans configurations without analyzing parental genomic samples. The overall genotype-phenotype correlation for 21-OHD is generally strong, with higher associations observed between genotype and phenotype for group Null, A, and B mutations, and larger genotype-phenotype variation in group C mutations. Targeted capture with LRS sequencing offers a new method for genetic diagnosis in 21-OHD patients.

    Keywords: 21-Hydroxylase Deficiency(21-OHD), CYP21A2, Long-read Sequencing(LRS), Targeted capture, multiplex ligation probe amplification (MLPA), genotypes

    Received: 29 Jul 2024; Accepted: 14 Oct 2024.

    Copyright: © 2024 Lan, WANG, YAO, Chenxhsci123, CHEN and ZHANG. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    JIN WANG, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
    HUI YAO, Department of Endocrinology, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
    BI K. CHEN, Department of Endocrinology, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
    RU J. ZHANG, Grandomics Biosciences Co.,Ltd., Changping, Beijing, China

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