Rapid analysis of alpha1-antitrypsin PiZ genotype by a real-time PCR approach.
alpha1-Antitrypsin (AAT) deficiency is a common inherited cause of emphysema and cirrhotic liver disease. Current laboratory diagnosis of Pi (proteinase inhibitor) status by protein analysis depends on the availability of blood samples and has a limited accuracy. Single-strand conformational polymorphism (SSCP) analysis and direct DNA sequencing can be performed from blood cells or from tissue samples, but it is a time-consuming procedure not suitable for screening purposes. We used a Light-Cycler assisted PCR approach to identify the PiZ mutation and to determine hetero- and homozygous carrier status from whole blood and from paraffin-embedded archival tissue specimens. The results were compared to those obtained by standard PCR amplification followed by SSCP and direct DNA sequencing. Light-Cycler assisted PCR identified heterozygous PiZ mutations in 16 samples, a homozygous PiZ status in three cases, and wild-type PiM in five control samples. In all cases the results were confirmed by SSCP and direct DNA sequencing. Light-Cycler assisted PCR has a high detection rate for the PiZ mutation. It can be performed from blood or from fixed archival tissues, requires only small amounts of DNA, and allows a rapid diagnosis on a high output level.
Ortiz-Pallardó
M E
ME
Medizinische Universitäts-Poliklinik Bonn, Germany.
Zhou
H
H
Fischer
H P
HP
Neuhaus
T
T
Sachinidis
A
A
Vetter
H
H
Brüning
T
T
Ko
Y
Y
D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000737:chemistry; D005838:Genotype; D006801:Humans; D008099:Liver / Q000473:pathology; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA; D000515:alpha 1-Antitrypsin / Q000235:genetics*; D019896:alpha 1-Antitrypsin Deficiency / Q000235:genetics*
D017931:DNA Primers; C043392:SERPINA1 protein, human; D000515:alpha 1-Antitrypsin; D004247:DNA