Molecular characterization of new variants associated with alpha-1-antitrypsin deficiency (AATD)

Alpha-1 antitrypsin deficiency (AATD) is an autosomal recessive disorder caused by mutations in the SERPINA1 gene, which encodes the 52-kD plasma glycoprotein alpha-1 antitrypsin (AAT), the main in-hibitor of serine proteases released by neutrophils at inflammatory sites. Missense mutations of SERPINA1 often result in conformationally altered AAT variants with a variable tendency to form intracellular poly-mers in hepatocytes, predisposing to liver disease and the consequent secretory deficiency. In addition to the most common pathogenic variant Z (Glu342Lys), which accumulates as ordered aggregates in the en-doplasmic reticulum (ER) of hepatocytes, many other rare genetic variants have been identified in both patients and the general population by genetic screening. We have investigated a panel of 17 new AAT variants found in Italian patients in order to define their pathogenic risk and to identify new functionally crucial regions of the AAT molecule. We first used bioinformatics tools to predict the functional effects of the missense mutations and to select the likely pathogenic ones to be characterized in mammalian cell models. Polymers formation was evaluated by the tendency of the variants to accumulate as intracellular insoluble aggregates and by immunofluorescence staining, while secretion deficiency was analyzed by quantifying AAT in the culture media. Our results identified two severe AAT mutants with a Z-like profile and three milder variants. Another AAT variant revealed an atypical polymeric pattern that is currently being investigated. The overall behavior of the variants was in agreement with the pathogenicity scores of the REVEL predictor, supporting the utility of this bioinformatic tool in the initial assessment of newly identified amino acid substitutions of AAT. However, molecular and cellular characterization is required to better define the mutant-specific pathogenetic mechanisms in AATD patients with rare genotypes.