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Alpha-1 antitrypsin deficiency: A re-surfacing adult liver disorder

  • Malin Fromme
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Health Care Provider of the European Reference Network on Rare Liver Disorders (ERN RARE LIVER), Aachen, Germany
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  • Carolin V. Schneider
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Health Care Provider of the European Reference Network on Rare Liver Disorders (ERN RARE LIVER), Aachen, Germany
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  • Christian Trautwein
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Health Care Provider of the European Reference Network on Rare Liver Disorders (ERN RARE LIVER), Aachen, Germany
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  • Nicola Brunetti-Pierri
    Affiliations
    Telethon Institute of Genetics and Medicine, Pozzuoli, 80078 Naples, Italy

    Department of Translational Medicine, Federico II University of Naples, Naples, Italy
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  • Pavel Strnad
    Correspondence
    Corresponding author. Address: Coordinating Center for Alpha-1 Antitrypsin Deficiency-related Liver Disease of the European Reference Network (ERN) “Rare Liver” and the European Association for the Study of the Liver (EASL) Registry Group “Alpha1-Liver” University Hospital Aachen, Pauwelsstr. 30, 52074 Aachen, Germany; Tel.: +49 241 80-35324.
    Affiliations
    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Health Care Provider of the European Reference Network on Rare Liver Disorders (ERN RARE LIVER), Aachen, Germany
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Published:November 27, 2021DOI:https://doi.org/10.1016/j.jhep.2021.11.022

      Summary

      Alpha-1 antitrypsin deficiency (AATD) arises from mutations in the SERPINA1 gene encoding alpha-1 antitrypsin (AAT) that lead to AAT retention in the endoplasmic reticulum of hepatocytes, causing proteotoxic liver injury and loss-of-function lung disease. The homozygous Pi∗Z mutation (Pi∗ZZ genotype) is responsible for the majority of severe AATD cases and can precipitate both paediatric and adult liver diseases, while the heterozygous Pi∗Z mutation (Pi∗MZ genotype) is an established genetic modifier of liver disease. We review genotype-related hepatic phenotypes/disease predispositions. We also describe the mechanisms and factors promoting the development of liver disease, as well as approaches to evaluate the extent of liver fibrosis. Finally, we discuss emerging diagnostic and therapeutic approaches for the clinical management of this often neglected disorder.

      Keywords

      Background

      Alpha-1 antitrypsin deficiency (AATD) is one of the most common potentially life-threatening genetic disorders. It predisposes patients to lung and liver damage, and both organs constitute the most prevalent causes of AATD-related mortality.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      AATD arises from mutation-based misfolding of the anti-protease alpha-1 antitrypsin (AAT), which is primarily expressed in hepatocytes and then secreted into the bloodstream to protect the lungs from proteolytic degradation by neutrophil elastase. Mutations in SERPINA1 lead to enhanced protein degradation and/or aggregation in the endoplasmic reticulum (ER) of hepatocytes, thereby causing proteotoxic liver stress and damage (Fig. 1). Proteolytic lung damage results from decreased systemic AAT levels and the resulting insufficient inhibition of proteases (Fig. 1).
      • Janciauskiene S.
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      • Greulich T.
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      The multifaceted effects of alpha1-antitrypsin on neutrophil functions.
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      • Welte T.
      The discovery of alpha1-antitrypsin and its role in health and disease.
      Clinically, AATD manifests as early-onset pan-lobular lung emphysema and/or chronic obstructive pulmonary disease whose development might be slowed down by AAT augmentation therapy.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      ,
      • Greene C.M.
      • Marciniak S.J.
      • Teckman J.
      • Ferrarotti I.
      • Brantly M.L.
      • Lomas D.A.
      • et al.
      alpha1-Antitrypsin deficiency.
      Lung damage represents the leading cause of death in patients with severe AATD, and lung destruction progresses more rapidly in smokers.
      • Tanash H.A.
      • Ekström M.
      • Rönmark E.
      • Lindberg A.
      • Piitulainen E.
      Survival in individuals with severe alpha 1-antitrypsin deficiency (PiZZ) in comparison to a general population with known smoking habits.
      Given the decreased secretion of AAT into the bloodstream in individuals with AATD, measurement of serum levels of AAT constitutes a cost-effective screening assay, and the diagnosis is further established by genetic analysis, which might be complemented with AAT protein phenotyping.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      ,
      • Strange C.
      • Moseley M.A.
      • Jones Y.
      • Schwarz L.
      • Xie L.
      • Brantly M.L.
      Genetic testing of minors for alpha1-antitrypsin deficiency.
      • Alpha-1 antitrypsin (AAT) is a secreted protein produced primarily in hepatocytes.
      Figure thumbnail gr1
      Fig. 1Pathophysiology, clinical manifestations, and treatment approaches for AAT deficiency.
      AAT deficiency results from mutations in the gene encoding AAT (named SERPINA1) that lead to increased degradation and/or retention of AAT in the liver. The misfolding and polymerisation of AAT confers proteotoxic stress that promotes the development of cirrhosis and liver tumours. The lack of AAT in the systemic circulation results in an insufficient inhibition of proteases (loss-of-function phenotype) that leads to proteolytic lung injury and pan-lobular emphysema. Histologically, the retention of AAT can be visualised as inclusion bodies in PAS-D staining. The characteristic homozygous Pi∗Z mutation leads to an ∼85% decrease in AAT secretion, and monomeric/polymeric Z-AAT is degraded by ERAD and autophagy. The resulting ER stress and/or environmental triggers stimulate AAT production, thereby causing a vicious cycle. Therapeutic approaches for AATD-related liver disease include siRNA, decreasing AAT production and secretion, and autophagy enhancers diminishing protein accumulation. Finally, polymerisation inhibitors may facilitate both secretion and degradation. AAT, alpha-1 antitrypsin; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; PAS-D, periodic acid-Schiff-diastase; siRNA, small-interfering RNA.
      More than 100 variants of SERPINA1, the gene encoding AAT, have been described. They are grouped based on the migration of mutant AAT in the electric field.
      • Strnad P.
      • Nuraldeen R.
      • Guldiken N.
      • Hartmann D.
      • Mahajan V.
      • Denk H.
      • et al.
      Broad spectrum of hepatocyte inclusions in humans, animals, and experimental models.
      ,
      • Ferrarotti I.
      • Carroll T.P.
      • Ottaviani S.
      • Fra A.M.
      • O'Brien G.
      • Molloy K.
      • et al.
      Identification and characterisation of eight novel SERPINA1 Null mutations.
      For example, Pi∗M indicates the medium (i.e., normal) velocity of the wild-type allele. Pi∗Z (rs28929474) and Pi∗S (rs17580) are the most clinically relevant variants and display very slow or slow movement, respectively (Table 1).
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      ,
      • Fagerhol M.K.
      • Laurell C.B.
      The Pi system-inherited variants of serum alpha 1-antitrypsin.
      The rare Pi∗F isoform exhibits fast migration, while the Pi∗Q0 alleles constitute a heterogeneous group of variants that yield no detectable protein in serum (Table 1).
      • Lee J.H.
      • Brantly M.
      Molecular mechanisms of alpha1-antitrypsin null alleles.
      • Talamo R.C.
      • Langley C.E.
      • Reed C.E.
      • Makino S.
      1 -Antitrypsin deficiency: a variant with no detectable 1 -antitrypsin.
      • Sinden N.J.
      • Koura F.
      • Stockley R.A.
      The significance of the F variant of alpha-1-antitrypsin and unique case report of a PiFF homozygote.
      Finally, some variants, such as Pi∗Mmalton or Pi∗Mprocida, produce functionally deficient AAT with migration similar to the wild-type isoform (Table 1).
      • Lomas D.A.
      • Irving J.A.
      • Gooptu B.
      Serinopathies.
      Table 1Overview of the most prominent alpha-1 antitrypsin deficiency alleles with their clinical characteristics.
      rs numberDeficiency allelesSERPINA1 variantCharacteristic genotypes and corresponding clinical features
      rs28929474Zp.Glu342LysStrong (Pi∗ZZ)/mild (Pi∗MZ): predisposition to liver disease and lung emphysema
      rs17580Sp.Glu264ValPi∗SZ: moderate predisposition to liver disease and lung emphysema

      Pi∗SS: only minimal risk
      Fp.Arg223CysLung emphysema in compound heterozygotes
      rs28931570Ip.Arg39CysNo clear phenotype
      rs121912713Pittsburghp.Met358ArgBleeding disorder via inhibition of thrombin and factor XI
      Trentop.Glu75ValLung emphysema in coinheritance with Pi∗Z
      rs775982338Mmaltonp.Phe52del (M2 variant)Liver disease and lung emphysema in homozygotes
      rs28931568Mmineral springsp.Gly67GluLung emphysema in homozygotes
      rs28931569Mprocidap.Leu41ProLung emphysema in homozygotes
      rs199422211Q0bellinghamp.Lys217∗Lung emphysema in homozygotes and compound heterozygotes
      Q0boltonp.Δ1 bpPro362Lung emphysema in homozygotes and compound heterozygotes
      rs267606950Q0granitefallsp.Δ1 bpTyr160Lung emphysema in coinheritance with Pi∗Z
      rs1057519610Q0hongkongp.Δ2 bpLeu318Lung emphysema in homozygotes and compound heterozygotes
      AAT, alpha-1 antitrypsin; Pi∗MZ, AAT genotype with heterozygosity for the Pi∗Z variant; Pi∗SS, AAT genotype with homozygosity for the Pi∗S variant; Pi∗SZ, AAT genotype with compound heterozygosity for the Pi∗Z and Pi∗S variants; Pi∗ZZ, AAT genotype with homozygosity for the Pi∗Z variant; rs, reference SNP ID number. All deficiency alleles are displayed with their rs number, mutation, and clinical features.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      ,
      • Sinden N.J.
      • Koura F.
      • Stockley R.A.
      The significance of the F variant of alpha-1-antitrypsin and unique case report of a PiFF homozygote.
      ,
      • Lomas D.A.
      • Irving J.A.
      • Gooptu B.
      Serinopathies.
      Classic severe AATD (genotype Pi∗ZZ) is caused by the homozygous Pi∗Z variant and affects approximately 1:2,000 individuals of European descent (Fig. 2).
      • Teckman J.H.
      Liver disease in alpha-1 antitrypsin deficiency: current understanding and future therapy.
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      • Blanco I.
      • Bueno P.
      • Diego I.
      • Pérez-Holanda S.
      • Casas-Maldonado F.
      • Esquinas C.
      • et al.
      Alpha-1 antitrypsin Pi∗Z gene frequency and Pi∗ZZ genotype numbers worldwide: an update.
      It is characterised by markedly decreased concentrations of serum AAT that confer a strong predisposition to lung disease (Table 2).
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      In contrast, the heterozygous genotype Pi∗MZ can be found in 1:30 Caucasians and results in normal/slightly decreased AAT serum concentrations (Table 2, Fig. 2). It predisposes individuals to lung emphysema when additional risk factors, such as smoking, are present.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      The compound heterozygous genotype Pi∗SZ arises from the simultaneous presence of both the Pi∗Z and the Pi∗S variants in trans. It affects 1:500 Caucasians, displays intermediate AAT serum concentrations, and moderately increases susceptibility to lung disease (Table 2, Fig. 2).
      • Blanco I.
      • Bueno P.
      • Diego I.
      • Perez-Holanda S.
      • Lara B.
      • Casas-Maldonado F.
      • et al.
      Alpha-1 antitrypsin Pi∗SZ genotype: estimated prevalence and number of SZ subjects worldwide.
      ,
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      The Pi∗SS genotype (i.e., homozygous Pi∗S allele) is approximately as common as Pi∗SZ but does not seem to constitute a clinically relevant risk factor for the development of liver disease.
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      Figure thumbnail gr2
      Fig. 2AATD genotypes and factors promoting the development of AATD-related liver disease.
      (A) The figure summarises the frequency of the most relevant AATD genotypes in Caucasians, their odds of developing cirrhosis, and their share in adult liver transplantations performed in Europe. Pi∗ZZ and Pi∗MZ refer to a homozygous and heterozygous presence of the Pi∗Z mutation, whereas Pi∗SZ denotes compound heterozygosity for both the Pi∗S and Pi∗Z mutations. Notably, unlike Pi∗ZZ, Pi∗MZ contributes to rather than causes cirrhosis. (B) The established non-genetic risk factors associated with the occurrence of advanced liver disease in individuals with AATD are shown. Pi∗MZ, AAT genotype with heterozygosity for the Pi∗Z variant; Pi∗SS, AAT genotype with homozygosity for the Pi∗S variant; Pi∗SZ, AAT genotype with compound heterozygosity for the Pi∗Z and Pi∗S variants; Pi∗ZZ, AAT genotype with homozygosity for the Pi∗Z variant.
      Table 2Range of alpha-1 antitrypsin serum levels and proportion of patients with Pi∗ZZ, Pi∗SZ, and Pi∗MZ who have elevated liver enzymes.
      SERPINA1 genotypeRange of serum AAT (mg/dl)AST ≥ULN (%)ALT ≥ULN (%)GGT ≥ULN (%)
      Pi∗ZZ20-45151122
      Pi∗SZ75-1205918
      Pi∗MZ90-2105717
      AAT, alpha-1 antitrypsin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase; Pi∗MZ, AAT genotype with heterozygosity for the Pi∗Z variant; Pi∗SS, AAT genotype with homozygosity for the Pi∗S variant; Pi∗SZ, AAT genotype with compound heterozygosity for the Pi∗Z and Pi∗S variants; Pi∗ZZ, AAT genotype with homozygosity for the Pi∗Z variant; ULN, upper limit of normal (sex-specific). Liver enzyme levels are based on data obtained from the UK Biobank in participants without an obvious liver comorbidity.
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      Range of AAT serum levels is in mg/dl.
      American Thoracic Society/European Respiratory Society statement: standards for the diagnosis and management of individuals with alpha-1 antitrypsin deficiency.

      Mechanisms of AATD-related liver injury

      The severe homozygous Pi∗Z variant leads to an ∼85% decrease in the amount of secreted protein and an accumulation of the Z-AAT protein at the production site (i.e., the ER).
      • Lomas D.A.
      • Evans D.L.
      • Finch J.T.
      • Carrell R.W.
      The mechanism of Z alpha 1-antitrypsin accumulation in the liver.
      ,
      • Teckman J.H.
      • Blomenkamp K.S.
      Pathophysiology of alpha-1 antitrypsin deficiency liver disease.
      Seventy percent of the protein is degraded, and 15% of the protein forms insoluble polymers. While ER-associated degradation (ERAD) removes monomeric Z-AAT,
      • Christianson J.C.
      • Shaler T.A.
      • Tyler R.E.
      • Kopito R.R.
      OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD.
      autophagy is the primary degradation machinery for Z-AAT polymers (Fig. 1).
      • Teckman J.H.
      • Perlmutter D.H.
      Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response.
      Polymerised AAT is rather indistinct in routine H&E sections but yields characteristic purple, roundish inclusions after periodic acid-Schiff-diastase (PAS-D) staining (Fig. 3).
      • Strnad P.
      • Nuraldeen R.
      • Guldiken N.
      • Hartmann D.
      • Mahajan V.
      • Denk H.
      • et al.
      Broad spectrum of hepatocyte inclusions in humans, animals, and experimental models.
      Immunohistochemistry with an anti-Pi∗Z antibody constitutes the most sensitive method to visualise Pi∗Z accumulation (Fig. 3).
      • AATD presents with liver disease due to proteotoxicity of the mutant AAT and lung emphysema due to a loss-of-function mechanism.
      • Inherited variants of the SERPINA1 gene encoding AAT may impair AAT secretion and give rise to alpha-1 antitrypsin deficiency (AATD).
      Figure thumbnail gr3
      Fig. 3Histological presentation of Pi∗ZZ-associated liver disease.
      Liver sections from a 61-year-old man with the Pi∗ZZ genotype and fibrosis stage 4 following (A) H&E staining, (B) PAS-D staining, and (C) immunohistochemistry using an antibody specific for the Pi∗Z variant of SERPINA1.
      • Fu D.A.
      • Campbell-Thompson M.
      Immunohistochemistry staining for human alpha-1 antitrypsin.
      PAS-D, periodic acid-Schiff-diastase; Pi∗ZZ, AAT genotype with homozygosity for the Pi∗Z variant.
      Several tools have been employed to delineate the consequences of Pi∗Z-related liver injury. Among them, mice transgenic for the human Pi∗Z variant (termed Pi∗Z mice) develop chronic proteotoxic liver injury with characteristic PAS-D-positive globules but do not display a lung phenotype due to the unaffected synthesis of the wild-type murine AAT. Notably, mice with a quintuple deletion of endogenous AAT alleles (in contrast to humans, mice have multiple genes encoding AAT) have recently become available and may constitute the more relevant genetic model for lung disease but not for liver disease.
      • Borel F.
      • Sun H.
      • Zieger M.
      • Cox A.
      • Cardozo B.
      • Li W.
      • et al.
      Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema.
      Regardless, Pi∗Z mice have yielded several important insights into AATD-related liver disease. For example, enhancement of autophagic degradation, either via gene transfer of the master regulator TFEB or via autophagy-enhancing drugs (such as carbamazepine or rapamycin), diminished Z-AAT accumulation and ameliorated liver injury (Fig. 1).
      • Pastore N.
      • Ballabio A.
      • Brunetti-Pierri N.
      Autophagy master regulator TFEB induces clearance of toxic SERPINA1/α-1-antitrypsin polymers.
      • Hidvegi T.
      • Ewing M.
      • Hale P.
      • Dippold C.
      • Beckett C.
      • Kemp C.
      • et al.
      An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis.
      • Kaushal S.
      • Annamali M.
      • Blomenkamp K.
      • Rudnick D.
      • Halloran D.
      • Brunt E.M.
      • et al.
      Rapamycin reduces intrahepatic alpha-1-antitrypsin mutant Z protein polymers and liver injury in a mouse model.
      The mouse model was also instrumental in determining the extent of ER-related stress. Although no activation of the unfolded stress response was detected in cells expressing Z-AAT, an ER overload response was observed.
      • Lawless M.W.
      • Greene C.M.
      • Mulgrew A.
      • Taggart C.C.
      • O'Neill S.J.
      • McElvaney N.G.
      Activation of endoplasmic reticulum-specific stress responses associated with the conformational disease Z alpha 1-antitrypsin deficiency.
      Among the ER stress-related pathways, NF-kB activation constitutes a protective response promoting protein degradation.
      • Mukherjee A.
      • Hidvegi T.
      • Araya P.
      • Ewing M.
      • Stolz D.B.
      • Perlmutter D.H.
      NFκB mitigates the pathological effects of misfolded α1-antitrypsin by activating autophagy and an integrated program of proteostasis mechanisms.
      On the other hand, JNK phosphorylation and increased expression of the proapoptotic protein CHOP (also known as DDIT3) accelerated cell death and the development of liver injury via the transcriptional upregulation of SERPINA1, resulting in an overload of proteotoxic Z-AAT.
      • Pastore N.
      • Attanasio S.
      • Granese B.
      • Castello R.
      • Teckman J.
      • Wilson A.A.
      • et al.
      Activation of the c-Jun N-terminal kinase pathway aggravates proteotoxicity of hepatic mutant Z alpha1-antitrypsin.
      ,
      • Attanasio S.
      • Ferriero R.
      • Gernoux G.
      • De Cegli R.
      • Carissimo A.
      • Nusco E.
      • et al.
      CHOP and c-JUN up-regulate the mutant Z α(1)-antitrypsin, exacerbating its aggregation and liver proteotoxicity.
      JNK activation also activated FOXO3 (forkhead box O3) and upregulated microRNA-34b/c expression. Notably, microRNA-34b/c reduced profibrotic signalling of platelet-derived growth factor, and its deletion in Pi∗Z mice resulted in the early development of liver fibrosis.
      • Piccolo P.
      • Ferriero R.
      • Barbato A.
      • Attanasio S.
      • Monti M.
      • Perna C.
      • et al.
      Up-regulation of miR-34b/c by JNK and FOXO3 protects from liver fibrosis.
      Moreover, mitochondrial injury is another well-established consequence of Z-AAT accumulation observed both in Pi∗Z mice and in human liver specimens.
      • Perlmutter D.H.
      Liver injury in alpha1-antitrypsin deficiency: an aggregated protein induces mitochondrial injury.
      Finally, the mouse model was instrumental in investigating the role of cofactors in the modulation of liver disease. In that respect, overexpression of hepatitis B surface protein, as seen in individuals with chronic hepatitis B infection, was found to aggravate liver injury, fibrosis, and the frequency of hepatocellular carcinoma in Pi∗Z mice,
      • Kuscuoglu D.
      • Bewersdorf L.
      • Wenzel K.
      • Gross A.
      • Kobazi Ensari G.
      • Luo Y.
      • et al.
      Dual proteotoxic stress accelerates liver injury via activation of p62-Nrf2.
      while the mild iron accumulation seen in homeostatic iron regulator (Hfe) gene knockout animals did not have a significant effect.
      • Guldiken N.
      • Hamesch K.
      • Schuller S.M.
      • Aly M.
      • Lindhauer C.
      • Schneider C.V.
      • et al.
      Mild iron overload as seen in individuals homozygous for the alpha-1 antitrypsin Pi∗Z variant does not promote liver fibrogenesis in HFE knockout mice.
      Notably, administration of the non-steroidal anti-inflammatory drug indomethacin increased liver damage via activation of the IL-6-STAT-3 pathway in Pi∗Z mice.
      • Rudnick D.A.
      • Shikapwashya O.
      • Blomenkamp K.
      • Teckman J.H.
      Indomethacin increases liver damage in a murine model of liver injury from alpha-1-antitrypsin deficiency.
      Finally, Pi∗Z mice are used to evaluate the efficacy of new treatment strategies. For example, the administration of antisense oligonucleotides or RNA interference effectively decreased hepatic Pi∗Z accumulation and ameliorated/reversed the development of the associated disease in this mouse model.
      • Guo S.
      • Booten S.L.
      • Aghajan M.
      • Hung G.
      • Zhao C.
      • Blomenkamp K.
      • et al.
      Antisense oligonucleotide treatment ameliorates alpha-1 antitrypsin-related liver disease in mice.
      In addition to transgenic animals, several cellular models are available. Patient-derived induced pluripotent stem cells have attracted significant interest, since they can be differentiated into hepatocyte-like cells and might reflect interindividual variability in the expression of liver disease.
      • Yusa K.
      • Rashid S.T.
      • Strick-Marchand H.
      • Varela I.
      • Liu P.Q.
      • Paschon D.E.
      • et al.
      Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.
      • Hannan N.R.
      • Segeritz C.P.
      • Touboul T.
      • Vallier L.
      Production of hepatocyte-like cells from human pluripotent stem cells.
      • Tafaleng E.N.
      • Chakraborty S.
      • Han B.
      • Hale P.
      • Wu W.
      • Soto-Gutierrez A.
      • et al.
      Induced pluripotent stem cells model personalized variations in liver disease resulting from α1-antitrypsin deficiency.
      Activation of the unfolded protein response and inflammatory networks were the primary alterations seen in the Pi∗ZZ-derived cell lines.
      • Segeritz C.P.
      • Rashid S.T.
      • de Brito M.C.
      • Serra M.P.
      • Ordonez A.
      • Morell C.M.
      • et al.
      hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α(1)-antitrypsin deficiency.
      Although limited by the availability of human tissues, the generation of liver organoids from Lgr5+ stem cells constitutes another attractive approach.
      • Huch M.
      • Gehart H.
      • van Boxtel R.
      • Hamer K.
      • Blokzijl F.
      • Verstegen M.M.
      • et al.
      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      The first published data are encouraging and point toward the decreased production of hepatocyte-specific genes in Pi∗ZZ organoids.
      • Gómez-Mariano G.
      • Matamala N.
      • Martínez S.
      • Justo I.
      • Marcacuzco A.
      • Jimenez C.
      • et al.
      Liver organoids reproduce alpha-1 antitrypsin deficiency-related liver disease.
      This is well in line with transgenic animal data showing downregulated HNF4α (hepatocyte nuclear factor-4 alpha)-related signalling which resulted in the loss of liver zonation.
      • Piccolo P.
      • Annunziata P.
      • Soria L.R.
      • Attanasio S.
      • Barbato A.
      • Castello R.
      • et al.
      Down-regulation of hepatocyte nuclear factor-4α and defective zonation in livers expressing mutant Z α1-antitrypsin.
      Finally, several cell lines overexpressing Pi∗Z or other SERPINA1 variants have been developed. The advantage of cell lines is that they are relatively easy to handle. However, they are not well suited to study processes that occur in terminally differentiated hepatocytes. On the other hand, they are useful for dissecting basic molecular mechanisms. For example, they were instrumental in the identification of a potentially novel degradative process based on the delivery of polymerised Z-AAT directly to the lysosome.
      • Fregno I.
      • Fasana E.
      • Bergmann T.J.
      • Raimondi A.
      • Loi M.
      • Soldà T.
      • et al.
      ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport.
      Moreover, they yielded important insights into the handling of mutated AAT by ERAD. This is facilitated by ER mannosidase I, which removes the protein from the calnexin-calreticulin refolding machinery.
      • Wu Y.
      • Swulius M.T.
      • Moremen K.W.
      • Sifers R.N.
      Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation.
      ,
      • Sun A.H.
      • Collette J.R.
      • Sifers R.N.
      The cytoplasmic tail of human mannosidase Man1b1 contributes to catalysis-independent quality control of misfolded alpha1-antitrypsin.
      In subsequent steps, misfolded AAT is stabilised by interactions with chaperones and lectins, such as OS9, ER lectin 1, or GRP94 (or HSP90B2P), and translocates to the cytosol to ultimately be degraded by the proteasome.
      • Christianson J.C.
      • Shaler T.A.
      • Tyler R.E.
      • Kopito R.R.
      OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD.
      ,
      • Greenblatt E.J.
      • Olzmann J.A.
      • Kopito R.R.
      Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum.
      However, these data, as well as other data generated in cell lines, await validation in animal models or human samples. Meanwhile, several human observations need to be further dissected in experimental models. These include an alteration in lipid metabolism, with more pronounced liver steatosis and decreased levels of serum triglycerides, as well as identification of extracellular vesicles with profibrogenic cargo in sera from individuals with Pi∗ZZ.
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      ,
      • Khodayari N.
      • Oshins R.
      • Holliday L.S.
      • Clark V.
      • Xiao Q.
      • Marek G.
      • et al.
      Alpha-1 antitrypsin deficient individuals have circulating extracellular vesicles with profibrogenic cargo.

      Paediatric liver disease in AATD

      Although this review is focused on liver disease in adults, it needs to be highlighted that AATD also causes paediatric liver injury, typically in the form of neonatal cholestasis.
      • Ruiz M.
      • Lacaille F.
      • Berthiller J.
      • Joly P.
      • Dumortier J.
      • Aumar M.
      • et al.
      Liver disease related to alpha1-antitrypsin deficiency in French children: the DEFI-ALPHA cohort.
      The strongest evidence stems from the Swedish neonatal screening programme that identified 120 newborns with Pi∗ZZ out of 200,000 newborns, while a variety of other reports relied on data from tertiary centres.
      • Sveger T.
      Liver disease in alpha1-antitrypsin deficiency detected by screening of 200,000 infants.
      • Sveger T.
      • Eriksson S.
      The liver in adolescents with alpha 1-antitrypsin deficiency.
      • Mostafavi B.
      • Piitulainen E.
      • Tanash H.A.
      Survival in the Swedish cohort with alpha-1-antitrypsin deficiency, up to the age of 43-45 years.
      In the population-based cohort, 12% of Pi∗ZZ neonates displayed prolonged jaundice, and 8% of these neonates had severe liver disease.
      • Sveger T.
      Liver disease in alpha1-antitrypsin deficiency detected by screening of 200,000 infants.
      Although biochemical abnormalities were seen in >50% of the Pi∗ZZ neonates identified by newborn screening, the values often normalised during follow-up.
      • Sveger T.
      • Eriksson S.
      The liver in adolescents with alpha 1-antitrypsin deficiency.
      As a result, only 2-3% of Pi∗ZZ children develop end-stage liver disease that may require liver transplantation, and at age 18, only 12% of them display elevated alanine aminotransferase (ALT) or gamma-glutamyltransferase (GGT).
      • Ruiz M.
      • Lacaille F.
      • Berthiller J.
      • Joly P.
      • Dumortier J.
      • Aumar M.
      • et al.
      Liver disease related to alpha1-antitrypsin deficiency in French children: the DEFI-ALPHA cohort.
      ,
      • Sveger T.
      • Eriksson S.
      The liver in adolescents with alpha 1-antitrypsin deficiency.
      ,
      • Fromme M.
      • Oliverius M.
      • Strnad P.
      DEFI-ALFA: the French key to the alpha1 mystery?.
      Interestingly, increased CHOP, which upregulates SERPINA1 transcription, was detected in diseased livers of children but not adults with Pi∗ZZ, suggesting that CHOP plays an important role in hepatic disease by increasing the burden of proteotoxic Z-AAT, particularly in the paediatric population.
      • Attanasio S.
      • Ferriero R.
      • Gernoux G.
      • De Cegli R.
      • Carissimo A.
      • Nusco E.
      • et al.
      CHOP and c-JUN up-regulate the mutant Z α(1)-antitrypsin, exacerbating its aggregation and liver proteotoxicity.
      Notably, a clinically significant paediatric liver disease in less severe genotypes, such as Pi∗MZ or Pi∗SZ, is exceedingly rare and might be at least in part caused by additional comorbidities, such as cystic fibrosis.
      • Ruiz M.
      • Lacaille F.
      • Berthiller J.
      • Joly P.
      • Dumortier J.
      • Aumar M.
      • et al.
      Liver disease related to alpha1-antitrypsin deficiency in French children: the DEFI-ALPHA cohort.
      • While >100 SERPINA1 variants have been reported, the heterozygous and homozygous Pi*Z variants (Pi*MZ/Pi*ZZ) are the most clinically relevant genotypes.

      Novel insights into adult AATD-associated liver disease

      Recent data on AATD-related adult liver disease largely come from 3 independent sources. First, the United Kingdom Biobank (UKB) constitutes a population-based cohort study comprising approximately 500,000 individuals with available AATD genotyping and clinical data. The recruited volunteers gave informed consent for data linkage to medical reports, which allowed the researchers to collect the available ICD codes. After excluding individuals with pathological alcohol consumption and viral hepatitis, the cohort was left with >17,000 individuals with Pi∗MZ, >800 with Pi∗SZ, and ∼140 with Pi∗ZZ.
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      Although the lack of a detailed assessment of liver fibrosis is a limitation of this cohort, the population-based recruitment of participants independently of their AATD genotype constitutes a major advantage, given that most individuals with AATD remain undiagnosed their whole life.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      Second, the EASL AATD consortium recruited >400 individuals with Pi∗MZ, ∼240 with Pi∗SZ, and nearly 600 study participants with Pi∗ZZ from 14 European countries, the US, and Australia. All individuals underwent standard serological testing and non-invasive assessment of liver fibrosis by transient elastography. While this cohort possesses only a limited number of liver biopsies,
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      ,
      • Strnad P.
      • Buch S.
      • Hamesch K.
      • Fischer J.
      • Rosendahl J.
      • Schmelz R.
      • et al.
      Heterozygous carriage of the alpha1-antitrypsin Pi∗Z variant increases the risk to develop liver cirrhosis.
      ,
      • Schneider C.V.
      • Hamesch K.
      • Gross A.
      • Mandorfer M.
      • Moeller L.S.
      • Pereira V.
      • et al.
      Liver phenotypes of European adults heterozygous or homozygous for Pi∗Z variant of AAT (Pi∗MZ vs Pi∗ZZ genotype) and non-carriers.
      it includes systematic biobanking of blood samples, thus making it a valuable resource for ancillary studies. The third cohort from North America included 94 individuals with Pi∗ZZ evaluated by routine laboratory tests, non-invasive assessment of liver fibrosis in most participants, and liver biopsies.
      • Clark V.C.
      • Marek G.
      • Liu C.
      • Collinsworth A.
      • Shuster J.
      • Kurtz T.
      • et al.
      Clinical and histologic features of adults with alpha-1 antitrypsin deficiency in a non-cirrhotic cohort.

      Liver enzymes in AATD – as seen on a population basis

      The UKB constitutes the largest resource for the assessment of routine liver function tests. Mean ALT values were significantly higher in all analysed AATD genotypes compared to those in non-carriers, but differences between the genotypes were modest. On the other hand, mean aspartate aminotransferase (AST) concentrations were clearly the highest in individuals with Pi∗ZZ, but they were also elevated in those with Pi∗MZ and Pi∗SZ (Table 2). GGT concentrations were comparable in non-carriers and individuals with Pi∗MZ, Pi∗SS, and Pi∗SZ. Although participants with Pi∗ZZ more often presented with elevated serum GGT levels compared to non-carriers, the differences were only borderline significant (Table 2).
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      Notably, serum GGT elevation may reflect metabolic alterations or the predominant involvement of the periportal area in AATD liver disease.
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      ,
      • Clark V.C.
      • Marek G.
      • Liu C.
      • Collinsworth A.
      • Shuster J.
      • Kurtz T.
      • et al.
      Clinical and histologic features of adults with alpha-1 antitrypsin deficiency in a non-cirrhotic cohort.
      Compared to the UKB, elevated liver function tests were found to be more frequent in the participants of the EASL AATD consortium, likely because UKB was a cohort of healthy individuals.
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      Regardless, the available studies clearly demonstrate that only a minority of individuals with AATD display liver enzymes outside the normal range. Elevated serum liver enzyme concentrations are uncommon, even in individuals with Pi∗ZZ, the most severe common AATD genotype. Consequently, elevated liver enzymes in patients with AATD should always trigger a thorough diagnostic workup.
      • Pi*ZZ causes neonatal hepatitis and cirrhosis in adults.

      ICD-10 diagnoses of liver fibrosis/cirrhosis and primary malignant liver neoplasms in AATD

      Data linkage to UKB participants' medical reports enabled the evaluation of the prevalence of liver-related ICD-10 codes. It demonstrated a strong predisposition of individuals with Pi∗ZZ to liver fibrosis/cirrhosis and primary malignant neoplasms of the liver.
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      The risk of liver fibrosis/cirrhosis in those with Pi∗ZZ was 20 times higher than that in non-carriers (Fig. 2), which is well in line with liver stiffness measurements reported by the EASL AATD consortium, as well as with the increased need for liver transplantation in individuals with AATD.
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      ,
      • Adam R.
      • Karam V.
      • Delvart V.
      • O'Grady J.
      • Mirza D.
      • Klempnauer J.
      • et al.
      Evolution of indications and results of liver transplantation in Europe. A report from the European Liver Transplant Registry (ELTR).
      In contrast, only very limited data on the susceptibility of individuals with Pi∗ZZ to liver tumours are available. While the UKB analysis indicated an odds ratio >40, further studies are needed to clarify liver cancer risk in individuals with Pi∗ZZ.
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      Individuals with Pi∗SZ were at a 3-fold higher risk of liver fibrosis/cirrhosis, while the risk of liver cancer was 7-fold higher (Fig. 2). Individuals with Pi∗MZ displayed a slightly increased odds ratio of 1.7 for liver fibrosis and a similar risk for liver-related mortality (Fig. 2),
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      ,
      • Schneider C.V.
      • Hamesch K.
      • Strnad P.
      Response to: the Pi∗MZ allele in alpha-1 antitrypsin increases liver-related outcomes in a population-based study.
      whereas their risk of liver cancer was not significantly increased. Notably, the risk of liver fibrosis/cirrhosis and/or a liver tumour in Pi∗SZ carriers was comparable to that in individuals homozygous for the patatin-like phospholipase domain-containing protein 3 (PNPLA3: rs738409) and transmembrane 6 superfamily member 2 (TM6SF2: rs5854926) single nucleotide polymorphisms, while the risks in individuals with Pi∗ZZ clearly exceeded the risks reported for these well-established disease modifiers.
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      Given the high risk of liver cancer development, we advocate regular hepatic ultrasound follow-ups for those with Pi∗ZZ and those with Pi∗SZ and significant liver fibrosis. However, larger prospective studies are needed to justify this approach.

      Evaluation of liver fibrosis and pathology in individuals with AATD

      Although liver biopsy remains the gold standard for the evaluation of liver fibrosis, pathology data on patients with AATD are limited.
      • Tapper E.B.
      • Lok A.S.
      Use of liver imaging and biopsy in clinical practice.
      Clark et al. examined 94 adult Pi∗ZZ biopsies, and the data revealed a prevalence of clinically significant liver fibrosis (i.e., fibrosis grade F ≥2) of 35.1% in individuals with Pi∗ZZ.
      • Clark V.C.
      • Marek G.
      • Liu C.
      • Collinsworth A.
      • Shuster J.
      • Kurtz T.
      • et al.
      Clinical and histologic features of adults with alpha-1 antitrypsin deficiency in a non-cirrhotic cohort.
      When established clinical routine PAS-D staining was used, characteristic roundish AAT inclusions were seen in 95% of participants with Pi∗ZZ (Fig. 3). Notably, they became more abundant at advanced liver fibrosis stages.
      • Schneider C.V.
      • Hamesch K.
      • Gross A.
      • Mandorfer M.
      • Moeller L.S.
      • Pereira V.
      • et al.
      Liver phenotypes of European adults heterozygous or homozygous for Pi∗Z variant of AAT (Pi∗MZ vs Pi∗ZZ genotype) and non-carriers.
      Similar findings were made by the EASL AATD consortium, which also showed that immunohistochemical staining with the Pi∗Z-specific antibody had higher sensitivity than PAS-D staining (Fig. 3). Only 40% of individuals with Pi∗MZ harbour Z-AAT inclusions by PAS-D vs. 63% by immunohistochemistry with an antibody specific for polymeric Z-AAT.
      • Schneider C.V.
      • Hamesch K.
      • Gross A.
      • Mandorfer M.
      • Moeller L.S.
      • Pereira V.
      • et al.
      Liver phenotypes of European adults heterozygous or homozygous for Pi∗Z variant of AAT (Pi∗MZ vs Pi∗ZZ genotype) and non-carriers.
      ,
      • Fu D.A.
      • Campbell-Thompson M.
      Immunohistochemistry staining for human alpha-1 antitrypsin.
      These data demonstrate that the presence/absence of inclusion bodies by staining cannot be used to diagnose/rule out AATD. While the average number of AAT inclusions was much lower in Pi∗MZ livers than in Pi∗ZZ livers, the abundance of AAT inclusions in those with the same genotype increased with fibrosis stage. This suggests that individuals with advanced liver fibrosis might have an impaired ability to degrade misfolded Z-AAT, which may lead to a vicious cycle of protein accumulation and liver injury.
      Liver stiffness measurement by transient elastography was the most precise method for the recognition of advanced liver fibrosis (i.e., fibrosis grade F ≥3).
      • Clark V.C.
      • Marek G.
      • Liu C.
      • Collinsworth A.
      • Shuster J.
      • Kurtz T.
      • et al.
      Clinical and histologic features of adults with alpha-1 antitrypsin deficiency in a non-cirrhotic cohort.
      ,
      • Kumpers J.
      • Fromme M.
      • Schneider C.V.
      • Trautwein C.
      • Denk H.
      • Hamesch K.
      • et al.
      Assessment of liver phenotype in adults with severe alpha-1 antitrypsin deficiency (Pi∗ZZ genotype).
      In contrast, there is no optimal tool for the detection of intermediate liver fibrosis, and multiple approaches (serum GGT, serum ALT, liver stiffness measurement by transient elastography, and AST-to-platelet ratio index [APRI], which is a serum-based fibrosis predictor) yielded only modest results.
      • Clark V.C.
      • Marek G.
      • Liu C.
      • Collinsworth A.
      • Shuster J.
      • Kurtz T.
      • et al.
      Clinical and histologic features of adults with alpha-1 antitrypsin deficiency in a non-cirrhotic cohort.
      Although there are multiple other methods to non-invasively investigate liver fibrosis,
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      their sensitivity and specificity remain to be validated against liver biopsies. As with other liver diseases, magnetic resonance elastography might provide an accurate assessment, but current evidence is very limited.
      • Kim R.G.
      • Nguyen P.
      • Bettencourt R.
      • Dulai P.S.
      • Haufe W.
      • Hooker J.
      • et al.
      Magnetic resonance elastography identifies fibrosis in adults with alpha-1 antitrypsin deficiency liver disease: a prospective study.

      Phenotype of Pi∗ZZ and Pi∗SZ individuals

      Both the EASL AATD consortium and the biopsy study by Clark et al. demonstrated a high liver fibrosis burden in individuals with Pi∗ZZ and identified male sex, age ≥50 years, presence of metabolic syndrome, and increased liver enzyme levels as risk factors for clinically significant liver fibrosis (i.e., fibrosis stage ≥2 on an F0-F4 scale) (Fig. 2).
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      ,
      • Clark V.C.
      • Marek G.
      • Liu C.
      • Collinsworth A.
      • Shuster J.
      • Kurtz T.
      • et al.
      Clinical and histologic features of adults with alpha-1 antitrypsin deficiency in a non-cirrhotic cohort.
      Notably, individuals with Pi∗ZZ might be susceptible to the development of liver steatosis, and an impaired ability to secrete lipids might be responsible for this defect.
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      On the other hand, no correlation between the extent of lung and liver fibrosis was observed.
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      Importantly, significant or even advanced liver fibrosis was sometimes seen even in individuals with relatively low liver stiffness measurements. These data need to be considered when counselling patients, and based on that evidence, we advocate a low threshold for performing liver biopsy, particularly in young individuals with repeatedly elevated liver enzyme levels.
      Compared to individuals with Pi∗ZZ, those with Pi∗SZ seem to display a substantially lower liver fibrosis burden,
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.
      which is consistent with their moderate risk of AATD-related lung disease.
      • Lara B.
      • Miravitlles M.
      Spanish registry of patients with alpha-1 antitrypsin deficiency; comparison of the characteristics of PISZ and PIZZ individuals.
      However, factors promoting the development of liver fibrosis are similar between individuals with Pi∗ZZ and Pi∗SZ (i.e., male sex, age ≥50 years, obesity, and the presence of diabetes mellitus) (Fig. 2).
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.

      Heterozygous carriage of the Pi∗Z variant (Pi∗MZ genotype) as a disease modifier

      Unlike the Pi∗ZZ genotype, Pi∗MZ status is a risk factor rather than a disease-causing agent, and “second hits” are necessary to induce disease.
      • Schneider C.V.
      • Hamesch K.
      • Gross A.
      • Mandorfer M.
      • Moeller L.S.
      • Pereira V.
      • et al.
      Liver phenotypes of European adults heterozygous or homozygous for Pi∗Z variant of AAT (Pi∗MZ vs Pi∗ZZ genotype) and non-carriers.
      In line with that theory, individuals with Pi∗MZ in the UKB displayed only a 1.7-fold increase in liver-related mortality.
      • Schneider C.V.
      • Hamesch K.
      • Strnad P.
      Response to: the Pi∗MZ allele in alpha-1 antitrypsin increases liver-related outcomes in a population-based study.
      However, the simultaneous presence of other liver disorders or risk factors substantially increased their liver-related risks. This has been particularly clearly demonstrated in individuals with cystic fibrosis and alcoholic/non-alcoholic fatty liver disease (ALD/NAFLD), in whom heterozygous Pi∗Z carriage markedly increased the risk of cirrhosis.
      • Strnad P.
      • Buch S.
      • Hamesch K.
      • Fischer J.
      • Rosendahl J.
      • Schmelz R.
      • et al.
      Heterozygous carriage of the alpha1-antitrypsin Pi∗Z variant increases the risk to develop liver cirrhosis.
      ,
      • Bartlett J.R.
      • Friedman K.J.
      • Ling S.C.
      • Pace R.G.
      • Bell S.C.
      • Bourke B.
      • et al.
      Genetic modifiers of liver disease in cystic fibrosis.
      A large genome-wide association study supported this finding by showing that the Pi∗Z variant was associated with an odds ratio for ALD-/NAFLD-related cirrhosis that surpasses the risk conferred by other established genetic modifiers of liver disease, such as PNPLA3 p. Ile148Met (rs738409), hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13):T (rs72613567), and TM6SF2 p. Glu167Lys (rs5854926).
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • Xin Y.
      • Schurmann C.
      • Stevis P.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.
      The fibrosis-promoting effect of Pi∗Z in cystic fibrosis might be related to the predisposition to gallstone formation of the Pi∗Z variant, because cystic fibrosis causes cholestatic liver disease.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      The other risk factors seem to be shared with other Pi∗ZZ and Pi∗SZ individuals (i.e., obesity and diabetes mellitus were the strongest modifiers, and age ≥50 years was a weak risk factor) (Fig. 2).
      • Schneider C.V.
      • Hamesch K.
      • Gross A.
      • Mandorfer M.
      • Moeller L.S.
      • Pereira V.
      • et al.
      Liver phenotypes of European adults heterozygous or homozygous for Pi∗Z variant of AAT (Pi∗MZ vs Pi∗ZZ genotype) and non-carriers.
      These data are not surprising, since obesity is known to amplify the profibrogenic effect of genetic variants.
      • Stender S.
      • Kozlitina J.
      • Nordestgaard B.G.
      • Tybjærg-Hansen A.
      • Hobbs H.H.
      • Cohen J.C.
      Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci.
      ,
      • Williams R.
      • Aspinall R.
      • Bellis M.
      • Camps-Walsh G.
      • Cramp M.
      • Dhawan A.
      • et al.
      Addressing liver disease in the UK: a blueprint for attaining excellence in health care and reducing premature mortality from lifestyle issues of excess consumption of alcohol, obesity, and viral hepatitis.
      In AATD, both obesity and diabetes may aggravate the occurrence of ER stress and are associated with increased oxidative stress and lipolysis.
      • Petersen M.C.
      • Shulman G.I.
      Mechanisms of insulin action and insulin resistance.
      ,
      • Lebeaupin C.
      • Vallée D.
      • Hazari Y.
      • Hetz C.
      • Chevet E.
      • Bailly-Maitre B.
      Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease.
      The greater risk in older individuals is also not surprising, since they are exposed to the inherited condition for a longer time. Moreover, aging may impair the efficiency of misfolded protein degradation.
      • Schneider C.V.
      • Hamesch K.
      • Gross A.
      • Mandorfer M.
      • Moeller L.S.
      • Pereira V.
      • et al.
      Liver phenotypes of European adults heterozygous or homozygous for Pi∗Z variant of AAT (Pi∗MZ vs Pi∗ZZ genotype) and non-carriers.
      ,
      • Leidal A.M.
      • Levine B.
      • Debnath J.
      Autophagy and the cell biology of age-related disease.
      While it was clearly shown to predispose individuals to ALD-/NAFLD-related fibrosis, the contribution of the Pi∗MZ status to other liver disorders, such as haemochromatosis or viral hepatitis, remains unclear (Table 3).
      • Pi*MZ predisposes individuals with cystic fibrosis and alcohol-/metabolic-associated fatty liver disease to the development of advanced liver disease.
      Table 3Current evidence for the Pi∗MZ genotype as a modifier of liver disease.
      EvidenceOdds ratioReferences
      Overall population++Increased liver-related mortality (HR ∼1.7), higher odds for diabetic/obese individualsSchneider et al., 2020
      • Schneider C.V.
      • Hamesch K.
      • Gross A.
      • Mandorfer M.
      • Moeller L.S.
      • Pereira V.
      • et al.
      Liver phenotypes of European adults heterozygous or homozygous for Pi∗Z variant of AAT (Pi∗MZ vs Pi∗ZZ genotype) and non-carriers.


      Luukkonen et al., 2021
      • Luukkonen P.K.
      • Salomaa V.
      • Åberg F.
      The Pi∗MZ allele in alpha-1 antitrypsin increases liver-related outcomes in a population-based study.


      Schneider et al., 2021
      • Schneider C.V.
      • Hamesch K.
      • Strnad P.
      Response to: the Pi∗MZ allele in alpha-1 antitrypsin increases liver-related outcomes in a population-based study.


      Fromme et al., 2021
      • Fromme M.
      • Schneider C.V.
      • Pereira V.
      • Hamesch K.
      • Pons M.
      • Reichert M.C.
      • et al.
      Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.


      Hakim et al., 2021
      • Hakim A.
      • Moll M.
      • Qiao D.
      • Liu J.
      • Lasky-Su J.A.
      • Silverman E.K.
      • et al.
      Heterozygosity of the alpha 1-antitrypsin Pi∗Z allele and risk of liver disease.
      NAFLD-related cirrhosis++OR ∼2-7Regev et al., 2006
      • Regev A.
      • Guaqueta C.
      • Molina E.G.
      • Conrad A.
      • Mishra V.
      • Brantly M.L.
      • et al.
      Does the heterozygous state of alpha-1 antitrypsin deficiency have a role in chronic liver diseases? Interim results of a large case-control study.


      Cacciottolo et al., 2014
      • Cacciottolo T.M.
      • Gelson W.T.
      • Maguire G.
      • Davies S.E.
      • Griffiths W.J.
      Pi∗Z heterozygous alpha-1 antitrypsin states accelerate parenchymal but not biliary cirrhosis.


      Abul-Husn et al., 2018
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • Xin Y.
      • Schurmann C.
      • Stevis P.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.


      Strnad et al., 2019
      • Strnad P.
      • Buch S.
      • Hamesch K.
      • Fischer J.
      • Rosendahl J.
      • Schmelz R.
      • et al.
      Heterozygous carriage of the alpha1-antitrypsin Pi∗Z variant increases the risk to develop liver cirrhosis.
      ALD-related cirrhosis++OR ∼2-6Goltz et al., 2014
      • Goltz D.
      • Hittetiya K.
      • Vossing L.M.
      • Kirfel J.
      • Spengler U.
      • Fischer H.P.
      Alpha(1)-antitrypsin PiMZ heterozygosity has an independent aggravating effect on liver fibrosis in alcoholic liver disease.


      Cacciottolo et al., 2014
      • Cacciottolo T.M.
      • Gelson W.T.
      • Maguire G.
      • Davies S.E.
      • Griffiths W.J.
      Pi∗Z heterozygous alpha-1 antitrypsin states accelerate parenchymal but not biliary cirrhosis.


      Abul-Husn et al., 2018
      • Abul-Husn N.S.
      • Cheng X.
      • Li A.H.
      • Xin Y.
      • Schurmann C.
      • Stevis P.
      • et al.
      A protein-truncating HSD17B13 variant and protection from chronic liver disease.


      Strnad et al., 2019
      • Strnad P.
      • Buch S.
      • Hamesch K.
      • Fischer J.
      • Rosendahl J.
      • Schmelz R.
      • et al.
      Heterozygous carriage of the alpha1-antitrypsin Pi∗Z variant increases the risk to develop liver cirrhosis.
      Chronic hepatitis B-associated advanced liver fibrosis+Smaller studies, OR ∼10 (1 study)Propst et al., 1992
      • Propst T.
      • Propst A.
      • Dietze O.
      • Judmaier G.
      • Braunsteiner H.
      • Vogel W.
      High prevalence of viral infection in adults with homozygous and heterozygous alpha 1-antitrypsin deficiency and chronic liver disease.


      Hashemi et al., 2005
      • Hashemi M.
      • Alavian S.M.
      • Ghavami S.
      • de Serres F.J.
      • Salehi M.
      • Doroudi T.
      • et al.
      High prevalence of alpha 1 antitrypsin phenotypes in viral hepatitis B infected patients in Iran.


      Kuscuoglu et al., 2021
      • Kuscuoglu D.
      • Bewersdorf L.
      • Wenzel K.
      • Gross A.
      • Kobazi Ensari G.
      • Luo Y.
      • et al.
      Dual proteotoxic stress accelerates liver injury via activation of p62-Nrf2.
      Chronic hepatitis C-associated advanced liver fibrosis+/-Several studies, OR ∼4 in one of themPropst et al., 1992
      • Propst T.
      • Propst A.
      • Dietze O.
      • Judmaier G.
      • Braunsteiner H.
      • Vogel W.
      High prevalence of viral infection in adults with homozygous and heterozygous alpha 1-antitrypsin deficiency and chronic liver disease.


      Eigenbrodt et al., 1997
      • Eigenbrodt M.L.
      • McCashland T.M.
      • Dy R.M.
      • Clark J.
      • Galati J.
      Heterozygous alpha 1-antitrypsin phenotypes in patients with end stage liver disease.


      Serfaty et al., 1997
      • Serfaty L.
      • Chazouillères O.
      • Poujol-Robert A.
      • Morand-Joubert L.
      • Dubois C.
      • Chrétien Y.
      • et al.
      Risk factors for cirrhosis in patients with chronic hepatitis C virus infection: results of a case-control study.


      Regev et al., 2006
      • Regev A.
      • Guaqueta C.
      • Molina E.G.
      • Conrad A.
      • Mishra V.
      • Brantly M.L.
      • et al.
      Does the heterozygous state of alpha-1 antitrypsin deficiency have a role in chronic liver diseases? Interim results of a large case-control study.
      Haemochromatosis-associated advanced liver fibrosis+/-Multiple studies, conflicting resultsRabinovitz et al., 1992
      • Rabinovitz M.
      • Gavaler J.S.
      • Kelly R.H.
      • Van Thiel D.H.
      Association between heterozygous alpha 1-antitrypsin deficiency and genetic hemochromatosis.


      Kaserbacher et al., 1993
      • Kaserbacher R.
      • Propst T.
      • Propst A.
      • Graziadei I.
      • Judmaier G.
      • Vogel W.
      Association between heterozygous alpha 1-antitrypsin deficiency and genetic hemochromatosis.


      Elzouki et al., 1995
      • Elzouki A.N.
      • Hultcrantz R.
      • Stål P.
      • Befrits R.
      • Eriksson S.
      Increased PiZ gene frequency for alpha 1 antitrypsin in patients with genetic haemochromatosis.


      Fargion et al., 1996
      • Fargion S.
      • Bissoli F.
      • Fracanzani A.L.
      • Suigo E.
      • Sergi C.
      • Taioli E.
      • et al.
      No association between genetic hemochromatosis and alpha1-antitrypsin deficiency.


      Schaefer et al., 2015
      • Schaefer B.
      • Haschka D.
      • Finkenstedt A.
      • Petersen B.S.
      • Theurl I.
      • Henninger B.
      • et al.
      Impaired hepcidin expression in alpha-1-antitrypsin deficiency associated with iron overload and progressive liver disease.


      Guldiken et al., 2019
      • Guldiken N.
      • Hamesch K.
      • Schuller S.M.
      • Aly M.
      • Lindhauer C.
      • Schneider C.V.
      • et al.
      Mild iron overload as seen in individuals homozygous for the alpha-1 antitrypsin Pi∗Z variant does not promote liver fibrogenesis in HFE knockout mice.
      Cystic fibrosis-associated advanced liver disease++OR ∼5Bartlett et al., 2009
      • Bartlett J.R.
      • Friedman K.J.
      • Ling S.C.
      • Pace R.G.
      • Bell S.C.
      • Bourke B.
      • et al.
      Genetic modifiers of liver disease in cystic fibrosis.


      Boëlle et al., 2019
      • Boëlle P.Y.
      • Debray D.
      • Guillot L.
      • Corvol H.
      SERPINA1 Z allele is associated with cystic fibrosis liver disease.
      Liver transplantation+Up to 10% of liver transplant recipients display Pi∗MZ genotypeCarey et al., 2013
      • Carey E.J.
      • Iyer V.N.
      • Nelson D.R.
      • Nguyen J.H.
      • Krowka M.J.
      Outcomes for recipients of liver transplantation for alpha-1-antitrypsin deficiency-related cirrhosis.


      Schaefer et al., 2018
      • Schaefer B.
      • Mandorfer M.
      • Viveiros A.
      • Finkenstedt A.
      • Ferenci P.
      • Schneeberger S.
      • et al.
      Heterozygosity for the alpha-1-antitrypsin Z allele in cirrhosis is associated with more advanced disease.
      AATD, alpha-1 antitrypsin deficiency; ALD, alcohol-related liver disease; HR, hazard ratio; NAFLD, non-alcoholic fatty liver disease; OR, odds ratio; Pi∗MZ, AAT genotype with heterozygosity for the Pi∗Z variant. +/-, conflicting data; +, weak positive evidence; ++, robust positive evidence.

      Therapeutic options for AATD-related liver disease

      For the treatment of AATD-related lung disease, intravenous augmentation therapy with plasma-purified AAT was approved by the Food and Drug Association in 1987 as the first disease-specific therapy for AATD.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      The RAPID trial, the first randomised, placebo-controlled trial of AAT augmentation therapy, later showed a significant reduction in the annual rate of lung density loss as a surrogate for lung emphysema by AAT augmentation. However, it did not show a significant effect on quality of life, forced expiratory volume values, or exacerbations of chronic obstructive pulmonary disease.
      • Chapman K.R.
      • Burdon J.G.
      • Piitulainen E.
      • Sandhaus R.A.
      • Seersholm N.
      • Stocks J.M.
      • et al.
      Intravenous augmentation treatment and lung density in severe α1 antitrypsin deficiency (RAPID): a randomised, double-blind, placebo-controlled trial.
      ,
      • McElvaney N.G.
      • Burdon J.
      • Holmes M.
      • Glanville A.
      • Wark P.A.
      • Thompson P.J.
      • et al.
      Long-term efficacy and safety of α1 proteinase inhibitor treatment for emphysema caused by severe α1 antitrypsin deficiency: an open-label extension trial (RAPID-OLE).
      • Weekly intravenous augmentation therapy with AAT has clinical efficacy in Pi∗ZZ individuals with lung involvement, and its immunomodulatory effects might also be effective in other disorders.
      While AAT augmentation therapy constitutes a disease-specific treatment of AATD-related lung disease, liver transplantation represents the only available relief for severe AATD-related liver disease.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      ,
      • Chapman K.R.
      • Burdon J.G.
      • Piitulainen E.
      • Sandhaus R.A.
      • Seersholm N.
      • Stocks J.M.
      • et al.
      Intravenous augmentation treatment and lung density in severe α1 antitrypsin deficiency (RAPID): a randomised, double-blind, placebo-controlled trial.
      ,
      • McElvaney N.G.
      • Burdon J.
      • Holmes M.
      • Glanville A.
      • Wark P.A.
      • Thompson P.J.
      • et al.
      Long-term efficacy and safety of α1 proteinase inhibitor treatment for emphysema caused by severe α1 antitrypsin deficiency: an open-label extension trial (RAPID-OLE).
      Although data are limited, the existing evidence demonstrates excellent survival and rapid normalisation of serum AAT concentrations in both adults and children following liver transplantation.
      • Guillaud O.
      • Jacquemin E.
      • Couchonnal E.
      • Vanlemmens C.
      • Francoz C.
      • Chouik Y.
      • et al.
      Long term results of liver transplantation for alpha-1 antitrypsin deficiency.
      • Carey E.J.
      • Iyer V.N.
      • Nelson D.R.
      • Nguyen J.H.
      • Krowka M.J.
      Outcomes for recipients of liver transplantation for alpha-1-antitrypsin deficiency-related cirrhosis.
      • Townsend S.A.
      • Edgar R.G.
      • Ellis P.R.
      • Kantas D.
      • Newsome P.N.
      • Turner A.M.
      Systematic review: the natural history of alpha-1 antitrypsin deficiency, and associated liver disease.
      Notably, since individuals with AATD who have cirrhosis may rapidly decompensate,
      • Schaefer B.
      • Mandorfer M.
      • Viveiros A.
      • Finkenstedt A.
      • Ferenci P.
      • Schneeberger S.
      • et al.
      Heterozygosity for the alpha-1-antitrypsin Z allele in cirrhosis is associated with more advanced disease.
      evaluation for liver transplantation should be considered in the early stages. In line with the genotype-associated susceptibilities described above, <10% of individuals with Pi∗ZZ who underwent liver transplantation had an additional liver disorder, compared to 40% and 90% of transplanted patients with Pi∗SZ and Pi∗MZ, respectively.
      • Carey E.J.
      • Iyer V.N.
      • Nelson D.R.
      • Nguyen J.H.
      • Krowka M.J.
      Outcomes for recipients of liver transplantation for alpha-1-antitrypsin deficiency-related cirrhosis.
      Although the implantation of a liver without AATD is thought to “cure” the disease, a decline in lung function was seen in some liver transplanted individuals.
      • Carey E.J.
      • Iyer V.N.
      • Nelson D.R.
      • Nguyen J.H.
      • Krowka M.J.
      Outcomes for recipients of liver transplantation for alpha-1-antitrypsin deficiency-related cirrhosis.
      Nevertheless, the risk of lung-related death seems to be low in carefully selected individuals.
      • Carey E.J.
      • Iyer V.N.
      • Nelson D.R.
      • Nguyen J.H.
      • Krowka M.J.
      Outcomes for recipients of liver transplantation for alpha-1-antitrypsin deficiency-related cirrhosis.
      Several approaches addressing AATD-related liver disease have yielded promising results in preclinical studies, and some of them have already been translated into clinical trials (Fig. 1).
      • Turner A.M.
      • Stolk J.
      • Bals R.
      • Lickliter J.D.
      • Hamilton J.
      • Christianson D.R.
      • et al.
      Hepatic-targeted RNA interference provides robust and persistent knockdown of alpha-1 antitrypsin levels in ZZ patients.
      In an important proof-of-concept study, Burrows et al. demonstrated the ability of chemical chaperones to increase the secretion of mutant AAT.
      • Burrows J.A.
      • Willis L.K.
      • Perlmutter D.H.
      Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: a potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency.
      Similarly, small peptides or intrabodies have been shown to block AAT polymerisation.
      • Mahadeva R.
      • Dafforn T.R.
      • Carrell R.W.
      • Lomas D.A.
      6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis.
      • Chang Y.P.
      • Mahadeva R.
      • Chang W.S.
      • Shukla A.
      • Dafforn T.R.
      • Chu Y.H.
      Identification of a 4-mer peptide inhibitor that effectively blocks the polymerization of pathogenic Z alpha1-antitrypsin.
      • Ordóñez A.
      • Pérez J.
      • Tan L.
      • Dickens J.A.
      • Motamedi-Shad N.
      • Irving J.A.
      • et al.
      A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.
      Recently, Lomas et al. performed an extensive high-throughput screen and identified a small molecule that corrected AAT misfolding and increased secretion, both in vitro and in vivo.
      • Lomas D.A.
      • Irving J.A.
      • Arico-Muendel C.
      • Belyanskaya S.
      • Brewster A.
      • Brown M.
      • et al.
      Development of a small molecule that corrects misfolding and increases secretion of Z α(1) -antitrypsin.
      These efforts are supported by recent advances in our understanding of the Z-AAT polymerisation process.
      • Faull S.V.
      • Elliston E.L.K.
      • Gooptu B.
      • Jagger A.M.
      • Aldobiyan I.
      • Redzej A.
      • et al.
      The structural basis for Z α(1)-antitrypsin polymerization in the liver.
      For clinical studies, small-interfering RNAs (siRNAs) are currently leading the way. They inhibit the production of mutated proteins, thereby alleviating proteotoxic stress. The employed siRNA is conjugated with N-acetylgalactosamine residues that mediate its hepatocyte-specific uptake via the asialoglycoprotein receptor.
      • Remih K.
      • Amzou S.
      • Strnad P.
      Alpha1-antitrypsin deficiency: new therapies on the horizon.
      Recently, the first data documenting the potential efficacy of this approach has become available. In the ARO-AAT2002 open-label trial (NCT03946449), 4 patients with Pi∗ZZ underwent 3 subcutaneous injections with ARO-AAT with a biopsy after 24 weeks, while 5 patients received 5 injections and were biopsied at week 48. siRNA treatment resulted in marked reductions in both serum and hepatic Z-AAT levels.
      • Strnad P.
      • Mandorfer M.
      • Choudhury G.
      • Griffiths W.
      • Turner A.M.
      • Trautwein C.
      • et al.
      ARO-AAT reduces serum and intra-hepatic Z-AAT protein in PiZZ alpha-1 antitrypsin deficient patients with liver disease leading to improvements in clinically relevant liver biomarkers.
      Moreover, treated patients presented with decreased serum ALT and GGT concentrations.
      • Strnad P.
      • Mandorfer M.
      • Choudhury G.
      • Griffiths W.
      • Turner A.M.
      • Trautwein C.
      • et al.
      ARO-AAT reduces serum and intra-hepatic Z-AAT protein in PiZZ alpha-1 antitrypsin deficient patients with liver disease leading to improvements in clinically relevant liver biomarkers.
      Most importantly, 6 out of the 9 individuals displayed an improvement in liver fibrosis, including 2 individuals with cirrhosis at baseline. Two additional siRNA trials are either currently recruiting or are expected to be recruiting in the near future (NCT03945292, NCT04174118) and will even include individuals with Pi∗ZZ and pre-existing cirrhosis.
      • While no treatment is currently available for AATD-related liver disease, siRNA-based approaches have yielded encouraging results in early phase II clinical trials.
      Another pathway for decreasing the hepatic burden of AAT is autophagy, which degrades protein polymers that are too large to be processed via the proteasome.
      • Perlmutter D.H.
      Autophagic disposal of the aggregation-prone protein that causes liver inflammation and carcinogenesis in alpha-1-antitrypsin deficiency.
      In human studies, autophagy-inducing drugs, such as carbamazepine, are being used. A randomised and placebo-controlled trial assessing the safety and efficacy of a 52-week treatment with carbamazepine in individuals with PiMZ/Pi∗ZZ and cirrhosis is currently in phase II (NCT01379469), with the first results expected by the end of this year.
      An alternative approach relying on a folding corrector named VX-864 has recently been investigated in a phase II clinical trial in individuals with Pi∗ZZ (NCT04474197). While it significantly increased serum AAT concentrations, further development of the compound was discontinued since its efficacy was deemed insufficient to confer a clinical benefit. While the way ahead is still long, this strategy should be pursued further since it may potentially alleviate both lung and liver disease. Other orally administered folding correctors, namely ZF874, have recently been investigated in a phase I, double-blinded, randomised, placebo-controlled clinical trial with healthy volunteers and individuals with Pi∗MZ (NCT04443192).
      • Zhou A.
      • Stein P.E.
      • Huntington J.A.
      • Sivasothy P.
      • Lomas D.A.
      • Carrell R.W.
      How small peptides block and reverse serpin polymerisation.
      ,
      • Yamasaki M.
      • Sendall T.J.
      • Pearce M.C.
      • Whisstock J.C.
      • Huntington J.A.
      Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer.
      As the number of treatment strategies is growing, personalised approaches need to be developed for individuals with different genotypes and stages of liver disease. Moreover, genetic assessment should be expanded to include sequencing of modifier genes to identify individuals at risk of severe liver disease who will benefit the most from early therapeutic interventions.
      While the current siRNA approach seems to be the most straightforward option for individuals with Pi∗ZZ who have a high load of intrahepatic Z-AAT and advanced liver fibrosis, its long-term lung safety needs to be evaluated. As a result, concomitant AAT augmentation therapy may need to be considered to address lung disease. Additionally, Pi∗Z-specific silencing represents an attractive option for individuals with Pi∗MZ/Pi∗SZ and liver disease. Alternatively, folding correctors or substances stimulating protein degradation that do not diminish AAT serum levels may prove beneficial for these cases and for individuals with lower fibrosis stages.

      Therapeutic effects of AAT

      AAT possesses versatile immunomodulatory and cytoprotective functions. For example, AAT overexpression was able to extend the lifespan of Drosophila.
      • Strnad P.
      • McElvaney N.G.
      • Lomas D.A.
      Alpha1-Antitrypsin deficiency.
      ,
      • Yuan Y.
      • DiCiaccio B.
      • Li Y.
      • Elshikha A.S.
      • Titov D.
      • Brenner B.
      • et al.
      Anti-inflammaging effects of human alpha-1 antitrypsin.
      In line with this, AAT supplementation was beneficial in experimental models of graft-vs.-host disease and might even be helpful in humans with this condition (Table 4). It has been successfully investigated in experimental lung, kidney, and islet transplantation models (Table 4), while its efficacy in liver grafts warrants systematic investigation. Moreover, AAT supplementation exerts a protective function in several models of acute liver injury and in experimental alcohol-related liver disease.
      • Jedicke N.
      • Struever N.
      • Aggrawal N.
      • Welte T.
      • Manns M.P.
      • Malek N.P.
      • et al.
      α-1-antitrypsin inhibits acute liver failure in mice.
      ,
      • Grander C.
      • Schaefer B.
      • Schwärzler J.
      • Grabherr F.
      • de Graaf D.M.
      • Enrich B.
      • et al.
      Alpha-1 antitrypsin governs alcohol-related liver disease in mice and humans.
      Finally, AAT inhibits TMPRSS2 protease, which enables the entry of SARS-CoV-2 into cells, and the therapeutic effect of AAT for this condition is currently being assessed in several clinical trials (Table 4).
      • Wettstein L.
      • Weil T.
      • Conzelmann C.
      • Müller J.A.
      • Groß R.
      • Hirschenberger M.
      • et al.
      Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection.
      ,
      • Ritzmann F.
      • Chitirala P.
      • Krüger N.
      • Hoffmann M.
      • Zuo W.
      • Lammert F.
      • et al.
      Therapeutic application of alpha-1 antitrypsin in COVID-19.
      Table 4Overview of the studies on alpha-1 antitrypsin supplementation.
      DiseaseStudy
      Experimental
      Acute liver failureJedicke et al., 2014
      • Jedicke N.
      • Struever N.
      • Aggrawal N.
      • Welte T.
      • Manns M.P.
      • Malek N.P.
      • et al.
      α-1-antitrypsin inhibits acute liver failure in mice.
      Alcohol-related liver diseaseGrander et al., 2021
      • Grander C.
      • Schaefer B.
      • Schwärzler J.
      • Grabherr F.
      • de Graaf D.M.
      • Enrich B.
      • et al.
      Alpha-1 antitrypsin governs alcohol-related liver disease in mice and humans.
      Graft-vs.-host diseaseTawara et al., 2012
      • Tawara I.
      • Sun Y.
      • Lewis E.C.
      • Toubai T.
      • Evers R.
      • Nieves E.
      • et al.
      Alpha-1-antitrypsin monotherapy reduces graft-versus-host disease after experimental allogeneic bone marrow transplantation.


      Marcondes et al., 2014
      • Marcondes A.M.
      • Karoopongse E.
      • Lesnikova M.
      • Margineantu D.
      • Welte T.
      • Dinarello C.A.
      • et al.
      α-1-Antitrypsin (AAT)-modified donor cells suppress GVHD but enhance the GVL effect: a role for mitochondrial bioenergetics.


      Geiger et al., 2019
      • Geiger S.
      • Ozay E.I.
      • Geumann U.
      • Hereth M.K.
      • Magnusson T.
      • Shanthalingam S.
      • et al.
      Alpha-1 antitrypsin-expressing mesenchymal stromal cells confer a long-term survival benefit in a mouse model of lethal GvHD.
      Kidney transplantationDaemen et al., 2000
      • Daemen M.A.
      • Heemskerk V.H.
      • van't Veer C.
      • Denecker G.
      • Wolfs T.G.
      • Vandenabeele P.
      • et al.
      Functional protection by acute phase proteins alpha(1)-acid glycoprotein and alpha(1)-antitrypsin against ischemia/reperfusion injury by preventing apoptosis and inflammation.
      Islet transplantationLewis et al., 2005; 2008
      • Lewis E.C.
      • Shapiro L.
      • Bowers O.J.
      • Dinarello C.A.
      Alpha1-antitrypsin monotherapy prolongs islet allograft survival in mice.
      ,
      • Lewis E.C.
      • Mizrahi M.
      • Toledano M.
      • Defelice N.
      • Wright J.L.
      • Churg A.
      • et al.
      alpha1-Antitrypsin monotherapy induces immune tolerance during islet allograft transplantation in mice.


      Koulmanda et al., 2008; 2012
      • Koulmanda M.
      • Bhasin M.
      • Hoffman L.
      • Fan Z.
      • Qipo A.
      • Shi H.
      • et al.
      Curative and β cell regenerative effects of α1-antitrypsin treatment in autoimmune diabetic NOD mice.
      ,
      • Koulmanda M.
      • Bhasin M.
      • Fan Z.
      • Hanidziar D.
      • Goel N.
      • Putheti P.
      • et al.
      Alpha 1-antitrypsin reduces inflammation and enhances mouse pancreatic islet transplant survival.


      Abecassis et al., 2014
      • Abecassis A.
      • Schuster R.
      • Shahaf G.
      • Ozeri E.
      • Green R.
      • Ochayon D.E.
      • et al.
      α1-antitrypsin increases interleukin-1 receptor antagonist production during pancreatic islet graft transplantation.
      Lung inflammationJonigk et al., 2013
      • Jonigk D.
      • Al-Omari M.
      • Maegel L.
      • Müller M.
      • Izykowski N.
      • Hong J.
      • et al.
      Anti-inflammatory and immunomodulatory properties of α1-antitrypsin without inhibition of elastase.
      Lung transplantationGao et al., 2014
      • Gao W.
      • Zhao J.
      • Kim H.
      • Xu S.
      • Chen M.
      • Bai X.
      • et al.
      α1-Antitrypsin inhibits ischemia reperfusion-induced lung injury by reducing inflammatory response and cell death.


      Iskender et al., 2014
      • Iskender I.
      • Sakamoto J.
      • Nakajima D.
      • Lin H.
      • Chen M.
      • Kim H.
      • et al.
      Human α1-antitrypsin improves early post-transplant lung function: pre-clinical studies in a pig lung transplant model.


      Lin et al., 2018
      • Lin H.
      • Chen M.
      • Tian F.
      • Tikkanen J.
      • Ding L.
      • Andrew Cheung H.Y.
      • et al.
      α(1)-Anti-trypsin improves function of porcine donor lungs during ex-vivo lung perfusion.


      Götzfried et al., 2018
      • Götzfried J.
      • Smirnova N.F.
      • Morrone C.
      • Korkmaz B.
      • Yildirim A.
      • Eickelberg O.
      • et al.
      Preservation with α(1)-antitrypsin improves primary graft function of murine lung transplants.
      COVID-19Wettstein et al., 2021
      • Wettstein L.
      • Weil T.
      • Conzelmann C.
      • Müller J.A.
      • Groß R.
      • Hirschenberger M.
      • et al.
      Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection.
      Clinical
      Graft-vs.-host diseaseMagenau et al., 2018
      • Magenau J.M.
      • Goldstein S.C.
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      • et al.
      α(1)-Antitrypsin infusion for treatment of steroid-resistant acute graft-versus-host disease.
      Type 1 diabetesBrener et al., 2018
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      Conclusions

      Recent studies in preclinical models and patients have greatly improved our understanding of AATD-related liver disease. Depending on the genotype, AATD may either cause liver disease or be a disease modifier. Despite the gain in knowledge, the vast majority of individuals with AATD, even those with a severe Pi∗ZZ genotype, remain undiagnosed and, in the case of liver disease, are mislabelled as having alcohol-related liver disease or idiopathic cirrhosis. Moreover, several important questions remain unanswered (Table 5). Given that multiple therapeutic products are already or will soon be under clinical investigation, we need to improve the testing and diagnosis of AATD. We also need genetic screening to systematically identify individuals with AATD who will develop liver disease, particularly when additional environmental risk factors are present. A minimisation of coexistent risk factors is warranted for individuals with AATD genotypes conferring an increased risk of liver fibrosis (i.e., Pi∗MZ, Pi∗SZ, and Pi∗ZZ). Inclusion in clinical trials should be attempted for those with significant liver fibrosis to stop/attenuate the development of liver scarring. Finally, longitudinal studies are needed to better evaluate the rate of progression and to more precisely calculate liver cancer risk. A systematic comparison between paediatric and adult cases, as well as long-term, standardised follow-up of affected children, is also warranted (Table 5).
      Table 5Key research questions for individuals with alpha-1 antitrypsin deficiency.
      Pi∗MZIs the modifier role liver disease aetiology-specific?

      Is the contribution of AATD-related liver disease significant enough to warrant specific therapeutic intervention?
      Pi∗ZZAre mechanisms underlying paediatric and adult liver disease identical?

      Are there any genetic and/or environmental modifiers? Can individuals at risk of severe liver disease be identified by specific tests?

      How to measure disease activity/identify rapid progressors?

      What are the most effective and clinically relevant endpoints for evaluating the efficacy of investigational products?
      AATD, alpha-1 antitrypsin deficiency; Pi∗MZ, AAT genotype with heterozygosity for the Pi∗Z variant; Pi∗ZZ, AAT genotype with homozygosity for the Pi∗Z variant.

      Abbreviations

      AAT, alpha-1 antitrypsin; AATD, alpha-1 antitrypsin deficiency; LSM, liver stiffness measurement; PAS-D, periodic acid-Schiff-diastase; Pi∗M, normal AAT allele; Pi∗S, mutant SERPINA1 allele variant termed ‘S’; Pi∗Z, mutant SERPINA1 allele variant termed ‘Z’; Pi∗MZ, AAT genotype with heterozygosity for the Pi∗Z variant; Pi∗SZ, AAT genotype with compound heterozygosity for the Pi∗Z and Pi∗S variants; Pi∗ZZ, AAT genotype with homozygosity for the Pi∗Z variant; PNPLA3, patatin-like phospholipase domain-containing protein 3; SERPINA1, serpin family A member 1 (AAT gene); siRNA, small-interfering RNA; TM6SF2, transmembrane 6 superfamily member 2; UKB, United Kingdom Biobank.

      Financial support

      This work was supported by the EASL registry grant on alpha-1 antitrypsin-related liver disease; the Deutsche Forschungsgemeinschaft (DFG) consortium SFB/TRR57 “Liver fibrosis” (both to P.S. and C.T.); the DFG grant STR1095/6-1 (to P.S.); and unrestricted research grants from CSL Behring , Vertex Pharmaceuticals and Arrowhead Pharmaceuticals (to P.S.).

      Authors’ contributions

      Drafting of the manuscript: M.F. and P.S. Critical revision of the manuscript for important intellectual content: M.F., C.V.S., C.T., N.B.P., and P.S. Figures and tables: M.F. Obtained funding: C.T. and P.S. All authors approved the final version of this manuscript. All authors take responsibility for the integrity of the data and the accuracy of the data analysis.

      Conflict of interest

      All authors declare no support from any organization other than the abovementioned organizations for the submitted work, no financial relationships with any organizations that might have an interest in the submitted work in the previous 5 years, and no other associations or activities that could appear to have influenced the submitted work. Hence, all authors declare themselves independent of funders concerning this manuscript.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

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