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已发表论文
利用下一代测序技术优化同源序列比对以鉴定 21-羟化酶缺乏症中 CYP21A2 基因变异
Authors Chen Y, Yu Q, Ge L, Weng L, Pan X, Zhou X, Zhou N, Wang Y, Jia J, Li H
Received 18 February 2025
Accepted for publication 26 July 2025
Published 16 September 2025 Volume 2025:18 Pages 3063—3078
DOI https://doi.org/10.2147/RMHP.S514355
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Haiyan Qu
Yibo Chen,1 Qi Yu,2 Lisha Ge,3,4 Lixin Weng,5 Xiaoli Pan,3,4 Xiaoxia Zhou,6 Nani Zhou,6 Yanjie Wang,6 Jia Jia,6 Haibo Li3,4,7
1Department of Clinical Laboratory, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China; 2Neonatal Screening Center, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China; 3The Central Laboratory of Birth Defects Prevention and Control, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China; 4Ningbo Key Laboratory of Genomic Medicine and Birth Defects Prevention, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China; 5Department of Clinical Laboratory, Ningbo Yinzhou District Maternity and Child Healthcare Hospital, Ningbo, 315000, People’s Republic of China; 6Shanghai Fujungenetics Biotechnology Co., Ltd, Shanghai, 201900, People’s Republic of China; 7Ningbo Key Laboratory for the Prevention and Treatment of Embryogenic Diseases, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China
Correspondence: Haibo Li, The Central Laboratory of Birth Defects Prevention and Control, Ningbo Key Laboratory of Genomic Medicine and Birth Defects Prevention, Ningbo Key Laboratory for the Prevention and Treatment of Embryogenic Diseases, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China, Tel +86 574 83882401, Tel +86 574 83882401, Email lihaibo-775@163.com Jia Jia, Shanghai Fujungenetics Biotechnology Co., Ltd, Shanghai, 201900, People’s Republic of China, Tel +86 18658176000, Tel +86 18658176000, Email jiajia@fujungenetics.com.cn
Objective: This study aimed to develop a novel homologous sequence analysis technique using high-throughput sequencing data to enhance CYP21A2 mutation detection. The approach leverages next-generation sequencing to overcome existing limitations and improve 21-hydroxylase deficiency diagnostic accuracy.
Methods: From April 21, 2022, to February 21, 2023, a total of 100 unrelated participants were enrolled at the Women and Children’s Hospital of Ningbo University, selected based on clinical manifestations and genetic testing results. The study used next-generation sequencing combined with a homologous sequence alignment (HSA) algorithm, which calculated the sequencing read ratios from homologous regions to identify pathogenic or likely pathogenic variants in the CYP21A2 gene. All detected variants were further validated using long-range PCR or multiplex ligation-dependent probe amplification. The accuracy of the HSA algorithm was systematically assessed.
Results: Among the 100 participants, 84 were identified as carriers of CYP21A2 mutations, while 16 were diagnosed with 21-hydroxylase deficiency. A total of 107 pathogenic mutations were detected using the homologous sequence alignment algorithm, comprising of 99 single nucleotide variants or insertions/deletions, 6 copy number variants, and 8 fusion mutations. Additionally, eight cases of CYP21A2-CYP21A1P gene conversions were identified based on HSA scores and confirmed through long-range PCR or multiplex ligation-dependent probe amplification. The algorithm demonstrated a positive predictive value of 96.26% for identifying mutations in CYP21A2. The most frequently observed mutations included c.955C > T, c.844G > T, c.293– 13C > G, c.518T > A, and exon-level deletions.
Conclusion: In genetic testing, particularly when addressing misalignment challenges associated with highly homologous genes such as CYP21A2, application of the HSA algorithm enables accurate mutation detection using commonly employed short-read sequencing methods. Through the characterization of homologous sequence features and optimization of the HSA algorithm, accurate mutation detection can be achieved in more homologous gene families (eg, HBA1/HBA2, SMN1/SMN2, GBA/GBAP1).
Keywords: CYP21A2, 21-hydroxylase deficiency, fusion mutations, highly homologous sequences, HSA algorithm, next generation sequencing
1Department of Clinical Laboratory, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China; 2Neonatal Screening Center, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China; 3The Central Laboratory of Birth Defects Prevention and Control, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China; 4Ningbo Key Laboratory of Genomic Medicine and Birth Defects Prevention, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China; 5Department of Clinical Laboratory, Ningbo Yinzhou District Maternity and Child Healthcare Hospital, Ningbo, 315000, People’s Republic of China; 6Shanghai Fujungenetics Biotechnology Co., Ltd, Shanghai, 201900, People’s Republic of China; 7Ningbo Key Laboratory for the Prevention and Treatment of Embryogenic Diseases, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China
Correspondence: Haibo Li, The Central Laboratory of Birth Defects Prevention and Control, Ningbo Key Laboratory of Genomic Medicine and Birth Defects Prevention, Ningbo Key Laboratory for the Prevention and Treatment of Embryogenic Diseases, Women and Children’s Hospital of Ningbo University, Ningbo, 315012, People’s Republic of China, Tel +86 574 83882401, Tel +86 574 83882401, Email lihaibo-775@163.com Jia Jia, Shanghai Fujungenetics Biotechnology Co., Ltd, Shanghai, 201900, People’s Republic of China, Tel +86 18658176000, Tel +86 18658176000, Email jiajia@fujungenetics.com.cn
Objective: This study aimed to develop a novel homologous sequence analysis technique using high-throughput sequencing data to enhance CYP21A2 mutation detection. The approach leverages next-generation sequencing to overcome existing limitations and improve 21-hydroxylase deficiency diagnostic accuracy.
Methods: From April 21, 2022, to February 21, 2023, a total of 100 unrelated participants were enrolled at the Women and Children’s Hospital of Ningbo University, selected based on clinical manifestations and genetic testing results. The study used next-generation sequencing combined with a homologous sequence alignment (HSA) algorithm, which calculated the sequencing read ratios from homologous regions to identify pathogenic or likely pathogenic variants in the CYP21A2 gene. All detected variants were further validated using long-range PCR or multiplex ligation-dependent probe amplification. The accuracy of the HSA algorithm was systematically assessed.
Results: Among the 100 participants, 84 were identified as carriers of CYP21A2 mutations, while 16 were diagnosed with 21-hydroxylase deficiency. A total of 107 pathogenic mutations were detected using the homologous sequence alignment algorithm, comprising of 99 single nucleotide variants or insertions/deletions, 6 copy number variants, and 8 fusion mutations. Additionally, eight cases of CYP21A2-CYP21A1P gene conversions were identified based on HSA scores and confirmed through long-range PCR or multiplex ligation-dependent probe amplification. The algorithm demonstrated a positive predictive value of 96.26% for identifying mutations in CYP21A2. The most frequently observed mutations included c.955C > T, c.844G > T, c.293– 13C > G, c.518T > A, and exon-level deletions.
Conclusion: In genetic testing, particularly when addressing misalignment challenges associated with highly homologous genes such as CYP21A2, application of the HSA algorithm enables accurate mutation detection using commonly employed short-read sequencing methods. Through the characterization of homologous sequence features and optimization of the HSA algorithm, accurate mutation detection can be achieved in more homologous gene families (eg, HBA1/HBA2, SMN1/SMN2, GBA/GBAP1).
Keywords: CYP21A2, 21-hydroxylase deficiency, fusion mutations, highly homologous sequences, HSA algorithm, next generation sequencing