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, M_wurzburg and M_whitstable. Comparison of protein phenotypes using isoelectric focusing of samples that presented the M_wurzburg variant, as revealed by exons 5 sequencing, identified a particular electrophoretic pattern amenable to the M_wurzburg protein. The specific phenotyping pattern was retrospectively validated, thus enabling the detection of 16 patients with M_wurzburg variant among the subjects already tested but not sequenced according to our diagnostic algorithm. The M_whitstable allele was detected by intron 4 sequencing of SERPINA1 gene. M_wurzburg and M_whitstable 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.

Keywords: Mwhitstable; Mwurzburg; SERPINA1 gene; alpha1-antitrypsin; laboratory diagnosis; rare pathogenic variants.

Figure 1

IEF pattern of M-like variants obtained by Sebia Hydrasys^® System on DBS samples. M2, M4, M6, M7, and M8 represent the different M isoforms [11,12]. (Arrows show the specific pattern of M_wurzburg).

Figure 2

(A) Electropherogram of M_wurzuburg variant in homozygosis; (B) Electropherogram of M_wurzuburg variant in heterozygosis; (C) Electropherogram of M_whitstable variant in heterozygosis.

Figure 2

(A) Electropherogram of M_wurzuburg variant in homozygosis; (B) Electropherogram of M_wurzuburg variant in heterozygosis; (C) Electropherogram of M_whitstable variant in heterozygosis.

Figure 3

Frequencies of genotypes carrying the M_wurzburg allele analysed in the Centre for Diagnosis of Inherited Alpha-1 Antitrypsin Deficiency in Pavia in the last 16 years.