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

Abstract

BackgroundAlpha-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.MethodsThis 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.ResultsZ, 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.ConclusionsMmalton 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.

Known Mutations at the Cause of Alpha-1 Antitrypsin Deficiency an Updated Overview of SERPINA1 Variation Spectrum

“…These modifications were hypothesized to upregulate ER chaperones fostering cell necrosis in a distinct molecular process from one of the Z and S Iiyama alleles. 80 The haplotype analysis of M Malton and M Palermo alleles disclosed their independent origin, indicating that the p. Phe52del mutation occurred at least twice. 2 In addition, such data were used to estimate their time to the most common recent ancestor, which in both instances is close to 5000 years old.…”

“…These modifications were hypothesized to upregulate ER chaperones fostering cell necrosis in a distinct molecular process from one of the Z and S Iiyama alleles. 80 …”

The Discovery of Endoplasmic Reticulum Storage Disease. The Connection between an H&E Slide and the Brain

“…No AAT was detected in the SER or Golgi apparatus, thereby indicating a defective translocation from the RER along the secretory pathway. The crystal structure (3D modelling) of the Z protein could explain the mechanism of aggregation as due to a unique molecular interaction between the reactive center loop of one molecule and the gap in the A-sheet of another [8,39]. In 1975, Jeppsson et al [40] were able to extract and purify the ZAAT from a homozygous cirrhotic liver and to raise the monoclonal ATZ11 antibody, capable of specifically interacting with Pi Z AAT [41] and detecting ZAAT molecules in the polymerized form [42].…”

“…There is an additional variant of AAT that is stored within hepatocytes and that equally predisposes to liver cirrhosis, the Siiyama, whose mutation, Ser53Phe, has been localized at exon III [48]. By using the above histopathological methodology, the three variants can be distinguished under the light microscope: all of them present the same H&E appearance and the same PASD positivity and immunoreactivity with polyclonal AAT antibodies, but Mmalton and Siiyama do not react with the monoclonal anti-Z-AAT antibody and only Mmalton AAT inclusions present calcium precipitates [8]. 3D protein analysis has allowed us to predict more severe misfolding of the Mmalton molecule as compared to Z and Siiyama, which could trigger anomalous interaction with endoplasmic reticulum chaperons, namely, calcium binding proteins [8].…”

The Autophagy Pathway: A Critical Route in the Disposal of Alpha 1-Antitrypsin Aggregates That Holds Many Mysteries

“…We previously mentioned that the hallmark for the detection of AATD in liver cells is the presence of PAS-positive and diastase-resistant globules, also named IBs. These structures represent dilated ER due to aggregated mutant protein retention [ 50 , 51 ]. However, these IBs are distinct compartments from the ER, even though they do stain positive for ER components (ribosomes and luminal ER protein, such as protein disulfide isomerase) and negative for lysosomal markers (such as LC3 and LAMP1).…”