Increased Frequency of Heterozygous Alpha-1-Antitrypsin Deficiency in Liver Explants From Nonalcoholic Steatohepatitis Patients.

Cirrhotic explanted livers occasionally have unexpected periodic acid-Schiff-diastase (PASD)-positive globules within the hepatocyte cytoplasm. It is often unclear whether this finding is a nonspecific consequence of cirrhosis or is indicative of an underlying alpha-1-antitrypsin deficiency (A1ATD) contributing to the cirrhosis. In this study, explanted livers were retrospectively evaluated for histopathology (including PASD status with confirmatory alpha-1-antitrypsin [A1AT] immunohistochemistry [IHC]), and chart review provided etiology of liver failure and general clinical parameters. Real-time polymerase chain reaction was used to detect A1AT genotype (SERPINA1 S and Z alleles) by melting curve analysis on liver explant tissue from selected cases. Of 196 explanted livers, 21 (11%) had PASD+ globules, which were significantly enriched in patients with a clinical diagnosis of nonalcoholic steatohepatitis (NASH; 47%) compared with other causes (P < 0.001). IHC confirmed all PASD+ globules were A1AT+, with 20 of 21 cases demonstrating diffuse A1AT staining. In an expanded NASH cohort, 42% (14/33) of explants had PASD+ globules, 92% of which were homozygous (n = 1) or heterozygous (n = 11) for the SERPINA1 Z allele, corresponding to nearly 40% of all NASH patients. Overall, the Z allele was present in 10% of all tested liver explants, with 85% of PASD+ cases genotyping homozygous (n = 2) or heterozygous (n = 20), which is far in excess of the estimated 2% in the general population. These results indicate PASD+ A1AT globules (with confirmatory genotyping showing at least 1 Z allele) are commonly observed in NASH, suggesting a synergistic relationship toward liver fibrosis. In addition, the high frequency of SERPINA1 Z alleles in liver transplantation patients supports the utility of pretransplant genotyping.

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

BACKGROUND: α-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 A1AT 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 °C (SD = 0.30) for mutant S alleles, 48.6 °C (SD = 0.28) for non-S alleles, 61.2 °C (SD = 0.34) for mutant Z alleles, and 54.7 °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.