J Korean Soc Endocrinol.  1994 Sep;9(3):219-227.

Molecular Genetic Studies on the Human CYP21A2 Gene (1)

• Park BK
• Woo HO
• Woo HW

Abstract

Congenital adrenal hyperplasia, especially due to steroid-12-hydroxylase(P450c21) deficiency, is one of the most common autosomal recessive inborn errors at adrenal steroidogenesis in Korean. Molecular genetic analysis has demonstrated that there are two steroid 21-hydroxylase genes, CYP21A1P and CYP21A2. The CYP21A2 gene encodes P450c21, whereas the CYP21A1P gene is a pseudogene. Since there is 98 percent homology between the CYP21A1P and CYP21A2 gene in nucleotide sequences, it has hampered the characterization of molecular defects in the CYP21A2 gene.In this study, efforts have been made to selectively PCR amplify the CYP21A2 gene and test feasibility of DNA microextraction from Guthrie card for prospective use of molecular screening. This study was also aimed at investigating deletion mutations in P450c21 deficient patients, as well as allele frequencies and average heterozygosity of exon 1 A/C polymorphism in Korean newborns. Genomic DNAs were obtained from Guthrie cards of 50 Korean newborns by microextraction method and these DNAs were analyzed by PCR-allele specific oligonucleotide(ASO) hybridization. First part of the CYP21A2 gene has been successfully amplified and digested by restriction enzyme using Taq I or Kpn I, subsequently run on 1.5% agarose gel to confirm its specificity. The anterior 1141 bp PCR product was utilized to examine the frequency and average heterozygosity of exon 1 A/C polymorphism in 100 alleles by ASO dot blot hybridization. Amplified genomic DNAs from four P450c21 deficient patients out of three families were screened by PCR to see if any one has complete deletion of the CYP21A2 gene.The results were as follows;1) The average 1230ng of genomic DNA was obtained form single semi-circled Guthrie card of 1/2 inch diameter by microextraction method, which has been successfully used for DNA analysis.2) The PCR amplified anterior 1141 bp product from the CYP21A2 gene was digested by Kpn I, generating 309 bp, 832 bp fragments, not by Taq I, indicating its specificity.3) The frequencies of exon 1 nucleotide 138 A/C polymorphism in Korean population were 0.81, 0.91 respectively, and average heterozygosity was 0.31.4) None of four P450c21 deficient patients turned out to carry complete deletion of the CYP21A2 gene based on selective PCR amplification of the CYP21A2 gene.In conclusion, dried blood spots from Guthrie card can be sued for DNA analysis because of easy sample collection, bandling, shipment, and DNA extraction feasibility. The selective PCR amplification of the CYP 21A2 gene will pave the way for molecular characterization in P450c21 deficient patients. The exon 1 A/C polymorphism can by efficiently used for molecular diagnosis of P450c21 deficiency in informative families, though it has a drawback of handling radioactive material.

MeSH Terms

Adrenal Hyperplasia, Congenital
Alleles
Base Sequence
Diagnosis
DNA
Exons
Gene Frequency
Humans*
Infant, Newborn
Mass Screening
Methods
Molecular Biology*
Polymerase Chain Reaction
Prospective Studies
Pseudogenes
Sensitivity and Specificity
Sepharose
Sequence Deletion
Steroid 21-Hydroxylase
DNA
Sepharose
Steroid 21-Hydroxylase