Detection of alpha-1-antitrypsin PiZ individuals by SSCP and DNA sequencing in formalin-fixed and paraffin-embedded tissue: a comparison with immunohistochemical analysis

Background/Aim: The role of alpha-l-antitrypsin (AAT) deficiency in the dev elopment of cirrhosis and carcinoma of the liver can be investigated from t he analysis of archival biopsy specimens, Immunohistochemistry can visualiz e the storage of defective protease inhibitor (Pi) variant Z, but does not allow differentiation between homozygous and heterozygous patients, The aim of the study was to establish a method for the detection of the PiZ mutati on on the gene level. Methods: Liver biopsy and autopsy samples in which AAT deficiency was detec ted immunohistochemically by a monoclonal PiZ-antibody were analyzed by sin gle-strand conformational polymorphism (SSCP) to reliably determine hetero- and homozygote carrier state in the absence of blood samples and to confir m the histological diagnosis. The accuracy of SSCP was verified by direct D NA sequencing. Results: Tissue slices (>0.8 cm(2)) from 29 consecutive cases with immunohi stochemically detected PiZ depositions and from ten PiZ-negative control ca ses were provided for extraction and amplification of DNA, In comparison to wild-type sequence of AAT exon V, all 29 cases showed band shifts on SSCP analysis, with a heterozygous pattern in 28 patients and a homozygous patte rn in one patient, DNA sequence analysis revealed the same single-base muta tion at position 342 of AAT exon V. Conclusions: SSCP analysis proved a sensitive and specific technique for th e detection of the PiZ mutation at the gene level, which allowed unequivoca l differentiation between heterozygous and homozygous PiZ status from paraf fin-embedded archival tissue specimens, Besides a use in diagnostic patholo gy, this technique could be valuable for prenatal diagnosis and population screening purposes.