High-Speed Detection of the Two Common α1-Antitrypsin Deficiency Alleles Pi*Z and Pi*S by Real-Time Fluorescence PCR and Melting Curves

摘要

Protease inhibitor 1 (α1-antitrypsin; AT) is the main serum inhibitor of proteolytic enzymes. In AT deficiency, enzymes such as neutrophil elastase can damage the lung tissues, leading to pulmonary emphysema. More than 90 different alleles have been identified to date for the protease inhibitor 1 gene (PI) . The three most important variants are type M (90% of population), type S (PiS) , and type Z (PiZ) , of which types S and Z are two of the more common abnormal variants. Homozygotes of type Z have a considerable reduction in the serum AT concentration and may develop pulmonary emphysema or hepatic cirrhosis. SZ-heterozygotes are less severely affected (1)(2)(3). The allele frequencies of the most common alleles, PiS and PiZ , are 0.02–0.04 or 0.01–0.02, respectively, with 0.2% of the general population being homozygous for either the PiZ or PiS allele. Typically, different phenotypes have been characterized by isoelectric focusing.PCR-based technologies are now being used widely for the identification of the mutations underlying the PiZ allele (Glu342Lys, GAG to AAG) and the PiS allele (Glu264Val, GAA to GTA), thus simplifying mutation detection (4). Although these methods give rise to unequivocal results, they are time-consuming and require optimization of the PCR reaction to eliminate nonspecific PCR products that would disturb the genetic analysis. Recently, a new detection methodology based on hybridization of amplicon-specific oligonucleotides with adjacent fluorophores capable of fluorescence resonance energy transfer was introduced and was used in a new high-speed thermal cycler (LightCyclerTM; Roche Diagnostics) that uses glass capillaries and hot air for heating (5). This technology enables the real-time detection of the specific PCR product followed by detection of the mutation by identification of the melting behavior of one of the two hybridization oligonucleotides (6). To this end, one …