title: Molecular diagnosis of 21-hydroxylase deficiency: detection of four mutations on a single gel.
abstract: Previous studies of the molecular basis of 21-hydroxylase deficiency have shown four common gene conversion mutations in exons 7 and 8. Current molecular diagnostic protocols use allele-specific oligonucleotide hybridization (ASOH) to individually detect each of these mutations and the corresponding normal alleles. This method is costly, labor intensive, and may not provide quantitative results. To expedite molecular diagnosis in families with 21-hydroxylase deficiency, we have designed and implemented single-strand conformational polymorphism (SSCP) analysis. We applied SSCP analysis to 12 families in whom mutations in exons 7 or 8 had been previously identified by ASOH. Using a single polymerase chain reaction (PCR) amplification, unique conformers can be assigned to three mutations: V281L, Q318X, and R356W. The fourth mutation, T insertion at nucleotide 1761, was detected by heteroduplex analysis of the same PCR product. Thus, we were able to identify all four mutations using a single PCR product on a single gel.
authors.affiliation: Department of Pediatrics, University of Pittsburgh, Children's Hospital of Pittsburgh, Pennsylvania 15213.
authors.forename: S F
authors.initials: SF
authors.lastname: Siegel
authors.forename: E P
authors.initials: EP
authors.lastname: Hoffman
authors.forename: M
authors.initials: M
authors.lastname: Trucco
mesh_terms: D000293:Adolescent; D000312:Adrenal Hyperplasia, Congenital / Q000235:genetics*; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010375:Pedigree; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics
chemical_list: D009692:Nucleic Acid Heteroduplexes