Mineralization of alpha-1-antitrypsin inclusion bodies in Mmalton alpha-1-antitrypsin deficiency

Abstract

Background: Alpha-1-antitrypsin (AAT) deficiency (AATD) of Z, Mmalton, Siiyama type is associated with liver storage of the mutant proteins and liver disease. The Z variant can be diagnosed on isoelectric focusing (IEF) while Mmalton and Siiyama may be missed or misdiagnosed with this technique. Therefore, molecular analysis is mandatory for their characterization. In particular, that holds true for the Mmalton variant as on IEF profile it resembles the wild M2 subtype.

Methods: This is a retrospective analysis involving review of medical records and of liver biopsy specimens from a series of Mmalton, Z and Siiyama Alpha-1-antitrypsin deficiency patients. The review has been implemented by additional histological stains, electron microscopic observations and 3-D modeling studies of the sites of the mutations.

Results: Z, Mmalton and Siiyama liver specimen contained characteristic intrahepatocytic PAS-D globules. The globules differed in the three variants as only Mmalton cases showed dark basophilic precipitates within the AAT inclusions. The precipitates were visualized in haematoxylin-eosin (H.E.) stained preparations and corresponded to calcium precipitates as demonstrated by von Kossa staining. On immunohistochemistry, ZAAT inclusions were stained by polyclonal as well as monoclonal noncommercial anti-AAT antibody (AZT11), whilst Mmalton and Siiyama inclusion bodies remained negative with the monoclonal anti-Z antibody. 3-D protein analysis allowed to predict more severe misfolding of the Mmalton molecule as compared to Z and Siiyama that could trigger anomalous interaction with endoplasmic reticulum chaperon proteins, namely calcium binding proteins.

Conclusions: Mmalton AAT inclusion bodies contain calcium precipitates inside them that allow the differential diagnosis with Siiyama and ZAAT inclusions in routine histological sections. The study has confirmed the specificity of the monoclonal AZT11 for the Z mutant. Thus, the combination of these two features is crucial for the distinction between the three variants and for predicting the genotype, whose confirmation would definitely require molecular analysis. Our study provides new data on the pathomorphogenesis of Mmalton inclusion bodies whose mineralization could play a central role in disease pathogenesis of Mmalton that is distinct from the Z and Siiyama variants. Calcium is known to be a major effector of cell death either via the increased intracellular concentration or the alteration of homeostasis.

Keywords: Alpha-1-antitrypsin deficiency; Mmalton; calcification.

Fig. 1

(a) Liver tissue section from case 6 Table 1 (Mmalton/Mmalton), with a preserved lobular architecture. Hepatocytes contain eosinophilic inclusions identified as AAT. A few of them mostly located in check-border hepatocytes are centered by dark basophilic material corresponding to calcium precipitates, H.E. 40X, positively stained by von Kossa staining, 100X (Fig. 1a, inset). (b) Liver tissue section from case 1 Table 1 (Mmalton/Mmalton), with fully established cirrhosis, H.E. 40X. The microphotograph shows hepatocytes plenty of cytoplasmic eosinophilic inclusions that were PAS-D positive and immunoreactive with a polyclonal anti-AAT antibody. The largest AAT globule is centered by a dark basophilic material, positively stained by von Kossa staining for calcium

Fig. 2

(a) Electromicrophotogaph from case 6 (Table 1) shows an hepatocyte with dilated cisternae of the ER containing fluffy semi-electron dense material corresponding to the classical appearance of AAT. The largest inclusions contain a large crystalline electrondense material. In addition to the crystalline structures, AAT inclusions contained fine or coarse electrondense granules quite similar to those of other AAT inclusions. This material was also displaying the peaks of calcium on EPMA (10.000 X). (b) Case 7 (a two years old boy with no visible calcium precipitates in H.E. stained preparations). The electronmicrophotograph shows dilated cisternae of ER. At least three AAT inclusions contain sparse electrondense granular precipitates similar to Fig. 2a. A few electrondense lysosomes with lipid inclusions are also seen (8000 X)

Fig. 3

Sites of the Mmalton, Z and Siiyama mutations, and functional regions. (a) Active form of AAT structure (the β-strand region A is colored in blue). (b) Homology model of the latent form of AAT. (c) Superposition of the active AAT and Z variant structures. (d) Detailed view around the sites of Malton and Siiyama mutations highlighting surrounding core domain residues