Introduction and Objective

Alpha-1 antitrypsin deficiency (A1ATD) is an under-diagnosed autosomal co-dominant hereditary disorder for which there is treatment. A1AT corresponds to a protease inhibitor encoded by the SERPINA1 gene. More than 300 mutations have been identified in this gene. Some of them cause a deficiency of the protein. Genetic testing is a powerful tool that helps in the diagnosis of the disorder. Next Generation Sequencing (NGS) allows the identification of all pathogenic variants in SERPINA1. This work aimed to validate in Progenika an assay to accurately sequence SERPINA1 exons and adjacent intronic regions.

Methods

We designed an NGS SERPINA1 sequencing assay using Illumina MiSeq platform. A total of 582 DNA samples from Saliva, Dried Blood Spots and Cell Lines were analyzed. The validation included Precision, Sensitivity, Specificity, Accuracy and Sample Plexing studies. More than 99.8% of the A1AT allele variant combinations were represented, including SNVs and small InDels in heterozygosis and homozygosis. Data analysis was performed through CLC Genomics Workbench using a dedicated pipeline.

Results and Conclusion

Agreement values of 100% were obtained in the four verification studies using Sanger sequencing as reference. The input DNA concentration range was defined as 0.05 ng/?L ? 200 ng/?L. Also, absolute genotype agreement was obtained for the sample plexing study with Progenika A1AT Genotyping Test based on Luminex xMAP technology. A sample plexing up to 384 was verified. This developed and validated A1AT NGS assay constitutes a reliable and powerful tool for the diagnostic of A1AT deficiency.