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Comparing the Southern blot method and polymerase chain reaction product analysis for chimeric RCCX detection in CYP21A2 deficiency.

Abstract
The 3.2-kb TaqI-produced fragment of the CYP21A1P pseudogene and the 3.7-kb TaqI-produced fragment of the functional CYP21A2 gene exist on chromosome 6p21.3. We used the polymerase chain reaction (PCR) product and Southern blot method with TaqI endonuclease digestion to identify a chimeric RCCX module in two unrelated patients with congenital adrenal hyperplasia (CAH). After TaqI cleavage, the PCR product analysis revealed that patient 1 with the chimeric CYP21A1P/CYP21A2 gene in one allele and IVS2-12A/C>G in combination with the 707-714del mutation in the other allele produced a configuration of 3.2- and 2.4-kb fragments. Patient 2, who carried IVS2-12A/C>G in combination with the 707-714del mutation in one allele and the chimeric TNXA/TNXB gene in the other allele, presented with 3.2- and 2.3-kb fragments. However, Southern blot analysis showed that patients 1 and 2 produced 3.2-, 2.4-, and 2.5-kb fragments. We conclude that the chimeric CYP21A1P/CYP21A2 gene, IVS2-12A/C>G in combination with the 707-714del mutation, and the chimeric TNXA/TNXB gene cannot be distinguished by the Southern blot method. Conversely, the chimeric TNXA/TNXB gene was identified in the PCR product analysis due to the appearance of the 2.37-kb fragment, which indicates the occurrence of the chimeric TNXA/TNXB formation extending to the boundary of TNXA in the RCCX region.
AuthorsHsien-Hsiung Lee, Yann-Jinn Lee, Mei-Chyn Chao
JournalAnalytical biochemistry (Anal Biochem) Vol. 399 Issue 2 Pg. 293-8 (Apr 15 2010) ISSN: 1096-0309 [Electronic] United States
PMID19961824 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright 2009 Elsevier Inc. All rights reserved.
Chemical References
  • TNXA protein, human
  • Tenascin
  • tenascin X
  • CYP21A2 protein, human
  • Steroid 21-Hydroxylase
Topics
  • Adrenal Hyperplasia, Congenital (genetics)
  • Alleles
  • Blotting, Southern (methods)
  • Humans
  • Polymerase Chain Reaction (methods)
  • Steroid 21-Hydroxylase (genetics, metabolism)
  • Tenascin (genetics, metabolism)

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