Pathogenic Mutations Conferring α1-Antitrypsin Deficiency Are Identified Accurately by a Novel Multiplexed Molecular Assay

Backgroundα1-Antitrypsin deficiency (AATD) is a common, underdiagnosed disease caused by mutations in the polymorphic SERPINA1 gene. AATD often causes COPD and other respiratory ailments and severe liver disease. AATD underdiagnosis is associated with the lack of a quick, high-precision test for SERPINA1 variants.Research QuestionCan a rapid and more accurate molecular diagnostic assay be developed that identifies AATD-associated mutations and outperforms current limited methodology?Study Design and MethodsWe developed a multiplexed polymerase chain reaction (PCR) assay that that uses mass spectrometry to detect 20 pathogenic SERPINA1 mutations, two normal M allele variants, and an additional variant of unknown significance as an accessible frontline genetic test for AATD. Blood samples from 177 patients with AATD indication, 176 of whom had a presumed normal genotype, and 10 buccal swabs or blood spots were tested to validate the assay. Additionally, 760 whole blood samples from patients with AATD indications were evaluated to identify AATD-associated mutations.ResultsThe novel genotyping assay described here accurately detected 23 SERPINA1 single nucleotide polymorphisms (AAT SNP23). Of 177 AATD samples, 96% showed abnormal SNPs, whereas 9.1% of the 176 presumed normal samples showed abnormal single nucleotide polymorphisms (SNPs). The AAT SNP23 genotypes correlated well with known serum AAT levels. This genotyping assay was more accurate and streamlined than a phenotyping isoelectric focusing assay used to identify AATD variants. For clinical testing, serum AAT protein level determination and the AAT SNP23 genotyping assay were performed. The AAT SNP23 was successfully implemented using AATD indication patient samples to evaluate the most common SERPINA1 mutations indicative of AATD. The AAT SNP23 assay has allowed for quick and accurate AAT genotyping of patients.InterpretationWe developed a novel, multiplexed genotyping assay that rapidly and accurately identified multiple AATD-associated SERPINA1 SNPs. This assay is useful to diagnose AATD quickly in patients with pulmonary or hepatic diseases, or both of unknown origin.