Real-Time PCR to Detect alpha-1 Antitrypsin S and Z Alleles in Formalin-Fixed Paraffin-Embedded Tissue

Background: alpha-1 Antitrypsin (A1AT) deficiency is an autosomal recessive genetic disease with incomplete penetrance that can cause pulmonary and liver disease. Multiple methods are available to determine A1AT genotype using peripheral blood specimens, but none are validated to detect A1AT alleles in formalin-fixed paraffin-embedded (FFPE) tissue. Methods: A real-time PCR assay was validated to detect the SERPINA1 S and Z alleles (NM_000295.4: c.863A>T, p.E288V and c.1096G>A, p.E366K, respectively) in FFPE liver tissue using allele-specific dual hybridization probes and melting curve analysis. Validation experiments were performed on genomic DNA samples (n = 11) with A1 AT genotypes previously determined by orthogonal methods. Results: The S and Z allele assays accurately genotyped all FFPE validation specimens that had a threshold cycle <32. Validation samples produced mean melting temperatures of 55.4 degrees C (SD = 0.30) for mutant S alleles, 48.6 degrees C (SD = 0.28) for non-S alleles, 61.2 degrees C (SD = 0.34) for mutant Z alleles, and 54.7 degrees C (SD = 0.19) for non-Z alleles. Samples failing to meet quality control parameters were infrequent. Conclusions: Poor PCR amplification because of low nucleic acid concentration in small biopsy specimens and time-dependent degradation in specimens stored for extended periods were the most common reasons for assay failure. The ability to determine A1AT genotype from archived surgical pathology specimens can facilitate research on the role of A1AT globules in liver disease.