Accurate Identification of Pathogenic Mutations Conferring α1-Antitrypsin Deficiency by a Novel Multiplexed Molecular Assay.

BACKGROUND

α1-Antitrypsin deficiency (AATD) is a common, underdiagnosed disease caused by mutations in the polymorphic SERPINA1 gene. AATD often causes COPD, other respiratory ailments, and severe liver disease. AATD underdiagnosis is associated with the lack of a quick, high-precision test for SERPINA1 variants.

RESEARCH QUESTION: Can a rapid and more accurate molecular diagnostic assay be developed that identifies AATD-associated mutations and outperforms current limited methodology?

STUDY DESIGN AND

METHODS

We 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 or buffy coat samples from 177 patients with AATD indication, 176 blood samples from people with presumed normal genotypes in addition to 10 buccal swabs and 10 blood spots (total of 373) were tested to validate the assay. Additionally, 760 whole blood samples from patients with AATD indications were evaluated to identify AATD-associated mutations.

RESULTS

The novel genotyping assay described here accurately detected 23 SERPINA1 single nucleotide polymorphisms (23-SNP AAT assay). Of 177 AATD samples, 96% showed abnormal single nucleotide polymorphisms (SNPs), whereas 9.1% of the 176 presumed normal samples showed abnormal SNPs. The 23-SNP AAT genotypes correlated well with known serum α1-antitrypsin levels. This genotyping assay was more accurate and streamlined than a phenotyping isoelectric focusing assay used to identify AATD variants. For clinical testing, serum α1-antitrypsin protein level determination and the 23-SNP AAT genotyping assay were performed. The 23-SNP AAT assay was successfully implemented using AATD indication patient samples to evaluate the most common SERPINA1 mutations indicative of AATD. The 23-SNP AAT assay has allowed for quick and accurate α1-antitrypsin genotyping of patients.

INTERPRETATION: These findings indicate that we developed a novel, multiplexed genotyping assay that rapidly and accurately identified multiple AATD-associated SERPINA1 SNPs. This assay may be useful to diagnose AATD quickly in patients with pulmonary or hepatic diseases or both of unknown origin.