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Characterization of a novel DNA polymorphism in the human CYP21 gene and application for DNA diagnosis of congenital adrenal hyperplasia
Author(s) -
Lee YongHo,
Park EunSook,
Kang ShinHye,
Kim Hogeun,
Lee JinYong,
Lee JinSung
Publication year - 2000
Publication title -
clinical endocrinology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.055
H-Index - 147
eISSN - 1365-2265
pISSN - 0300-0664
DOI - 10.1046/j.1365-2265.2000.01090.x
Subject(s) - restriction fragment length polymorphism , biology , genetics , microbiology and biotechnology , haplotype , exon , restriction site , allele , restriction enzyme , polymerase chain reaction , gene
OBJECTIVES Congenital adrenal hyperplasia (CAH) is a common endocrine disorder and CYP21 (21‐OH, EC 1.14.99.10) deficiency is the most common cause of the disease. The presence of a pseudogene and a wide range of mutation types often makes DNA diagnosis difficult. Analysis of mutant alleles from patients with CAH identified a new DNA polymorphism in exon 10. To test the usefulness of this polymorphism, linkage analysis was performed using three RFLP's at both end of the CYP21 gene in patients and controls. DESIGN AND PATIENTS Genomic DNA was extracted from 21 unrelated patients and 39 unaffected individuals. Haplotyping analysis was performed for three RFLPs, Msp I and Fnu4H I in intron 2 and a new Sma I RFLP in exon 10. All three polymorphic sites were characterized by DNA sequencing and usefulness of these RFLPs for DNA diagnosis was tested in patients' families. MEASUREMENT CAH patients were diagnosed by clinical symptoms and biochemical tests. Allele frequencies and heterozygosities were studied for three RFLP's in patients and controls using polymerase chain reaction (PCR). RESULTS A new Sma I RFLP showed a sequence difference as G or A at the nucleotide position 2694 in exon 10. Sequences at the Msp I and Fnu 4HI polymorphic sites were T or C at the nucleotide position 395 and 453, respectively. All of these RFLPs showed biallelic DNA polymorphisms and codominant segregation in family analysis. Heterozygosities were 0.31 for Msp I, 0.48 for Fnu 4HI and 0.44 for Sma I in normal individuals. There was no linkage disequilibrium for three RFLPs between patients and controls. CONCLUSIONS The Sma I RFLP can be useful for linkage analysis and DNA diagnosis in conjunction with RFLPs in intron 2 in CAH families because the polymorphic site is within the active gene at the 3′ end. DNA sequencing results revealed that these RFLPs were created by gene conversions as with other mutations in the CYP21 gene.