Ann Lab Med.  2015 Sep;35(5):535-539. 10.3343/alm.2015.35.5.535.

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

• Hong G
• Park HD
• Choi R
• Jin DK
• Kim JH
• Ki CS
• Lee SY
• Song J
• Kim JW

• KMID: 2369771
• DOI: http://doi.org/10.3343/alm.2015.35.5.535

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.

Keyword

CYP21A2; Pseudogene; Restriction fragment length polymorphism; Multiple ligation-dependent probe amplification; Korea

MeSH Terms

Adrenal Hyperplasia, Congenital*
Alleles
Gene Conversion
Genotype
Haplotypes
Humans
Incidence
Korea
Polymerase Chain Reaction*
Polymorphism, Restriction Fragment Length*
Pseudogenes
Spectrum Analysis

Figure

• 

  Fig. 1 Chromatogram of a novel mutation, c.492delA (p. Glu164Aspfs*24), in patient 1 and the sibling of patient 1.

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