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-1-antitrypsin (AAT) deficiency in the development of cirrhosis and carcinoma of the liver can be investigated from the analysis of archival biopsy specimens. Immunohistochemistry can visualize the storage of defective protease inhibitor (Pi) variant Z, but does not allow differentation between homozygous and heterozygous patients. The aim of the study was to establish a method for the detection of the PiZ mutation on the gene level. Methods: Liver biopsy and autopsy samples in which AAT deficiency was detected immunohistochemically by a monoclonal PiZ-antibody were analyzed by single-strand conformational polymorphism (SSCP) to reliably determine hetero- and homozygote carrier state in the absence of blood samples and to confirm the histological diagnosis. The accuracy of SSCP was verified by direct DNA sequencing. Results: Tissue slices (>0.8 cm2) from 29 consecutive cases with immunohistochemically detected PiZ depositions and from ten PiZ-negative control cases 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 pattern in one patient. DNA sequence analysis revealed the same single-base mutation at position 342 of AAT exon V. Conclusions: SSCP analysis proved a sensitive and specific technique for the detection of the PiZ mutation at the gene level, which allowed unequivocal differentiation between heterozygous and homozygous PiZ status from paraffin-embedded archival tissue specimens. Besides a use in diagnostic pathology, this technique could be valuable for prenatal diagnosis and population screening purposes.