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CYP21A2 mutation analysis in Korean patients with congenital adrenal hyperplasia using complementary methods: sequencing after long-range PCR and restriction fragment length polymorphism analysis with multiple ligation-dependent probe amplification assay.
Ann Lab Med. 2015 Sep; 35(5):535-9.AL

Abstract

CYP21A2 mutation analysis of congenital adrenal hyperplasia (CAH) is challenging because of the genomic presence of a homologous CYP21A2 pseudogene and the significant incidence of pseudogene conversion and large deletions. The objective of this study was to accurately analyze the CYP21A2 genotype in Korean CAH patients using a combination of complementary methods. Long-range PCR and restriction fragment length polymorphism analyses were performed to confirm valid amplification of CYP21A2 and to detect large gene conversions and deletions before direct sequencing. Multiple ligation-dependent probe amplification (MLPA) analysis was conducted concurrently in 14 CAH-suspected patients and six family members of three patients. We identified 27 CYP21A2 mutant alleles in 14 CAH-suspected patients. The c.293-13A>G (or c.293-13C>G) was the most common mutation, and p.Ile173Asn was the second, identified in 25% and 17.9% of alleles, respectively. A novel frame-shift mutation of c.492delA (p.Glu 164Aspfs*24) was detected. Large deletions were detected by MLPA in 10.7% of the alleles. Mutation studies of the six familial members for three of the patients aided in the identification of haplotypes. In summary, we successfully identified CYP21A2 mutations using both long-range PCR and sequencing and dosage analyses. Our data correspond relatively well with the previously reported mutation spectrum analysis.

Authors+Show Affiliations

Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. nayadoo@hanmail.net.Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea.Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

26206692

Citation

Hong, Geehay, et al. "CYP21A2 Mutation Analysis in Korean Patients With Congenital Adrenal Hyperplasia Using Complementary Methods: Sequencing After Long-range PCR and Restriction Fragment Length Polymorphism Analysis With Multiple Ligation-dependent Probe Amplification Assay." Annals of Laboratory Medicine, vol. 35, no. 5, 2015, pp. 535-9.
Hong G, Park HD, Choi R, et al. CYP21A2 mutation analysis in Korean patients with congenital adrenal hyperplasia using complementary methods: sequencing after long-range PCR and restriction fragment length polymorphism analysis with multiple ligation-dependent probe amplification assay. Ann Lab Med. 2015;35(5):535-9.
Hong, G., Park, H. D., Choi, R., Jin, D. K., Kim, J. H., Ki, C. S., Lee, S. Y., Song, J., & Kim, J. W. (2015). CYP21A2 mutation analysis in Korean patients with congenital adrenal hyperplasia using complementary methods: sequencing after long-range PCR and restriction fragment length polymorphism analysis with multiple ligation-dependent probe amplification assay. Annals of Laboratory Medicine, 35(5), 535-9. https://doi.org/10.3343/alm.2015.35.5.535
Hong G, et al. CYP21A2 Mutation Analysis in Korean Patients With Congenital Adrenal Hyperplasia Using Complementary Methods: Sequencing After Long-range PCR and Restriction Fragment Length Polymorphism Analysis With Multiple Ligation-dependent Probe Amplification Assay. Ann Lab Med. 2015;35(5):535-9. PubMed PMID: 26206692.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - CYP21A2 mutation analysis in Korean patients with congenital adrenal hyperplasia using complementary methods: sequencing after long-range PCR and restriction fragment length polymorphism analysis with multiple ligation-dependent probe amplification assay. AU - Hong,Geehay, AU - Park,Hyung Doo, AU - Choi,Rihwa, AU - Jin,Dong Kyu, AU - Kim,Jae Hyeon, AU - Ki,Chang Seok, AU - Lee,Soo Youn, AU - Song,Junghan, AU - Kim,Jong Won, PY - 2014/08/14/received PY - 2015/01/16/revised PY - 2015/06/17/accepted PY - 2015/7/25/entrez PY - 2015/7/25/pubmed PY - 2016/12/15/medline KW - CYP21A2 KW - Korea KW - Multiple ligation-dependent probe amplification KW - Pseudogene KW - Restriction fragment length polymorphism SP - 535 EP - 9 JF - Annals of laboratory medicine JO - Ann Lab Med VL - 35 IS - 5 N2 - CYP21A2 mutation analysis of congenital adrenal hyperplasia (CAH) is challenging because of the genomic presence of a homologous CYP21A2 pseudogene and the significant incidence of pseudogene conversion and large deletions. The objective of this study was to accurately analyze the CYP21A2 genotype in Korean CAH patients using a combination of complementary methods. Long-range PCR and restriction fragment length polymorphism analyses were performed to confirm valid amplification of CYP21A2 and to detect large gene conversions and deletions before direct sequencing. Multiple ligation-dependent probe amplification (MLPA) analysis was conducted concurrently in 14 CAH-suspected patients and six family members of three patients. We identified 27 CYP21A2 mutant alleles in 14 CAH-suspected patients. The c.293-13A>G (or c.293-13C>G) was the most common mutation, and p.Ile173Asn was the second, identified in 25% and 17.9% of alleles, respectively. A novel frame-shift mutation of c.492delA (p.Glu 164Aspfs*24) was detected. Large deletions were detected by MLPA in 10.7% of the alleles. Mutation studies of the six familial members for three of the patients aided in the identification of haplotypes. In summary, we successfully identified CYP21A2 mutations using both long-range PCR and sequencing and dosage analyses. Our data correspond relatively well with the previously reported mutation spectrum analysis. SN - 2234-3814 UR - https://www.unboundmedicine.com/medline/citation/26206692/CYP21A2_mutation_analysis_in_Korean_patients_with_congenital_adrenal_hyperplasia_using_complementary_methods:_sequencing_after_long_range_PCR_and_restriction_fragment_length_polymorphism_analysis_with_multiple_ligation_dependent_probe_amplification_assay_ L2 - http://www.annlabmed.org/journal/viewJournal.html?year=2015&vol=35&page=535 DB - PRIME DP - Unbound Medicine ER -