Homozygous inheritance of the Z mutation (exon V, Glu342GAG----Lys342AAG), the most common cause of alpha-1-antitrypsin (alpha 1AT) deficiency, is associated with a high risk for emphysema and liver disease. This study presents a rapid and accurate approach to definitive genotypic diagnosis of the Z homozygous state using a combination of polymerase chain reaction amplification of exon V of the alpha 1AT gene and ribonuclease cleavage of an exon V-specific antisense RNA probe. Taking advantage of the concept that ribonuclease A will cleave at points of mismatch of RNA-DNA hybrids, a 0.79 kb antisense RNA probe was designed with complementarity to the sense strand of exon V of the alpha 1AT gene (the site of the Z mutation) along with small regions of the 5' and 3' flanking sequences. After amplification of exon V of the alpha 1AT gene from genomic DNA by the polymerase chain reaction, the amplified DNA was analyzed by hybridization to a 32P-labeled exon V antisense RNA probe followed by digestion with RNase A. Any substitution mutations resulting in DNA-RNA mismatch were detected by evaluation with polyacrylamide gel electrophoresis under denaturing conditions followed by autoradiography (expected fragment lengths: 0.33 kb when the exon V probe hybridized to the normal amplified genomic DNA, 0.25 and 0.08 kb fragments when the exon V probe hybridized to the amplified genomic DNA with the Z mutation). Double-blinded evaluation of genomic DNA of 36 individuals (phenotypes MM n = 14, MZ n = 5, ZZ n = 16, ZNull n = 1; included among the "M" alleles were representatives of all the major normal M alleles) demonstrated definitive diagnosis of the Z mutation with absolute specificity for all 36 specimens, i.e., ZZ homozygotes, MZ heterozygotes, and normals were all detected accurately. This approach should be useful not only for screening for the Z mutation of the alpha 1AT gene, but by this type of analysis, mutational alterations of the alpha 1AT gene can be screened for without prior knowledge of the sequence changes and without complex cloning and sequencing methods.

Original languageEnglish
Pages (from-to)329-334
Number of pages6
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Issue number4
StatePublished - Oct 1989


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