Comprehensive Mutation Analysis of the CYP21A2 Gene
Open accessAbstract
Congenital adrenal hyperplasia, due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of adrenal steroidogenesis caused by mutations in the CYP21A2 gene. Direct comparison of established and novel methodologies of CYP21A2 genetic analysis in a large cohort representing a wide range of genotypes has not been previously reported. We genotyped a cohort of 129 unrelated patients with 21-OHD, along with 145 available parents, using Southern blot (SB) analysis, multiplex ligation-dependent probe amplification (MLPA), PCR-based restriction fragment length polymorphism (RFLP) analysis, multiplex minisequencing and conversion-specific PCR, duplication-specific amplification, and DNA sequencing. CYP21A2 genotyping identified four duplicated CYP21A2 genes (1.53%) and 79 chimeric CYP21A1P/CYP21A2 genes (30.15%). Parental SB data were essential for determining the CYP21 haplotype in three cases, whereas PCR-based RFLP analysis was necessary for MLPA results to be accurately interpreted in the majority of cases. The comparison of different methods in detecting deletion and duplication showed that MLPA with PCR-based RFLP was comparable with SB analysis, with parental data of 100% sensitivity and specificity. DNA sequencing was required for the identification of 16 (6.1%) rare point mutations and determination of clinically significant chimera junction sites. MLPA with PCR-based RFLP analysis is an excellent substitute for SB analysis in detecting CYP21A2 deletion and duplication and a combination of MLPA, PCR-based RFLP, duplication-specific amplification, and DNA sequencing is a convenient and comprehensive strategy for mutation analysis of the CYP21A2 gene in patients with 21-OHD.
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All subjects had comprehensive genetic records, including a complete CAH and CAH-X genotype by a CLIA-accredited laboratory.
“All subjects had comprehensive genetic records, including a complete CAH and CAH-X genotype by a CLIA-accredited laboratory (PreventionGenetics, LLC, Marshfield, WI), as well as copy numbers of TNXB exons 35, 40, C4A, C4B genes determined as previously described [10,32,33].”
From Pseudogene TNXA Variants May Interfere with the Genetic Testing of CAH-X (2023) · cites this paper as [32]
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Comprehensive analysis of congenital adrenal hyperplasia using long-read sequencing
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Pseudogene TNXA Variants May Interfere with the Genetic Testing of CAH-X
Lao, Q., Zhou, K., Parker, M., Faucz, F. R., & Merke, D. P. (2023) · Genes · doi:10.3390/genes14020265 · Cited by 2 · see citing works
“5 kb long-range PCR with frequent heterogeneous insert–deletions across the locus [32].”
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“RFLP analysis, among the earliest molecular tools for CYP21A2 testing, is inexpensive and technically simple, yet it detects only variants that create or abolish restriction sites and offers low resolution for complex alleles (54).”
- Authors
- Xu, Z., Chen, W., Merke, D. P., & McDonnell, N. B.
- Affiliations
- National Institute on Aging (US)
- National Institutes of Health (US)
- Corresponding author
- McDonnell, Nazli B.
- ORCID
- Merke, Deborah P.
- Venue
- The Journal of Molecular Diagnostics
- Publisher
- Elsevier BV
- Published
- 2013-11
- Volume
- 15
- Issue
- 6
- Pages
- 745-753
- ISSN
- 1525-1578 (ISSN-L)
- PMC
- PMC5803549
- OpenAlex
- W21482950
- Cited by
- 62
- References
- 13
- Citations by year
- 2026: 7 · 2025: 5 · 2024: 8 · 2023: 4 · 2022: 5 · 2021: 10 · 2020: 3 · 2019: 5 · 2018: 4 · 2017: 2 · 2016: 3 · 2015: 3 · 2014: 2 · 2013: 1
- FWCI
- 1.34
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Molecular Diagnostics (publishedVersion, cc-by-nc-nd)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- American Society for Investigative Pathology
- National Institutes of Health
- U.S. Public Health Service
- National Institute on Aging
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sexual Differentiation and Disorders
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Keywords
- CYP21A2, congenital adrenal hyperplasia, 21-hydroxylase deficiency, mutation analysis, multiplex ligation-dependent probe amplification, Southern blot, haplotype inference, point mutations
- MeSH terms
- Adrenal Hyperplasia, Congenital, DNA Mutational Analysis, Humans, Polymorphism, Restriction Fragment Length, Steroid 21-Hydroxylase, Polymerase Chain Reaction, Gene Dosage, Molecular Diagnostic Techniques, Observational Studies as Topic
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