Improving the Laboratory Diagnosis of M-like Variants Related to Alpha1-Antitrypsin Deficiency

Abstract

Alpha1-antitrypsin (AAT) is a serine protease inhibitor that is encoded by the highly polymorphic SERPINA1 gene. Mutations in this gene can lead to AAT deficiency (AATD), which is associated with an increased risk of lung and/or liver disease. On the basis of electrophoretic migration, AAT variants are named with capital letters; M (medium) signifies the normal protein. Among pathological variants, the M-like ones represent a heterogeneous group of rare allelic variants that exhibit the same electrophoretic pattern as the M wild-type protein, which makes them difficult to detect with routine methods. In order to avoid their misdiagnosis, the present study defines and validates effective methods for the detection of two pathogenic M-like variants, Mwurzburg and Mwhitstable. Comparison of protein phenotypes using isoelectric focusing of samples that presented the Mwurzburg variant, as revealed by exons 5 sequencing, identified a particular electrophoretic pattern amenable to the Mwurzburg protein. The specific phenotyping pattern was retrospectively validated, thus enabling the detection of 16 patients with Mwurzburg variant among the subjects already tested but not sequenced according to our diagnostic algorithm. The Mwhitstable allele was detected by intron 4 sequencing of SERPINA1 gene. Mwurzburg and Mwhitstable are often misdiagnosed and the introduction of diagnostic improvements can help the clinical management, especially in patients with established lung disease without any other reported risk factors.

Comprehensive Clinical Diagnostic Pipelines Reveal New Variants in Alpha-1 Antitrypsin Deficiency

“…Many rare variants are not easily diagnosed with routine laboratory techniques, thus contributing to misclassification and misdiagnosis. For example, phenotyping by IEF usually does not allow the precise identification of M-like AAT variants, such as M malton , M procida (40) as they are easily confused with M (normal) protein. Furthermore, Null (Q0) variants in heterozygosity are invisible to IEF and can only be indirectly observed through their effects on circulating levels.…”

“…Most rare alleles can only be detected by molecular biology techniques, such as SERPINA1 gene sequencing (1,41). Sometimes, depending on the complexity of the case, sequencing of exons might not be sufficiently informative and more extensive analysis by whole gene sequencing is required to detect rare exonic deletions, alterations in promoters or deep intronic regions not covered by standard sequencing (9,40). It has been recently demonstrated that the choice of diagnostic algorithm can have a significant impact on the accurate diagnosis of AATD, which is essential for appropriate medical care and treatment (42).…”