Variants in autophagy genes MTMR12 and FAM134A are putative modifiers of the hepatic phenotype in α1-antitrypsin deficiency.

BACKGROUND AND AIMS: In the classical form of α1-antitrypsin deficiency, a misfolded variant α1-antitrypsin Z accumulates in the endoplasmic reticulum of liver cells and causes liver cell injury by gain-of-function proteotoxicity in a sub-group of affected homozygotes but relatively little is known about putative modifiers. Here, we carried out genomic sequencing in a uniquely affected family with an index case of liver failure and 2 homozygous siblings with minimal or no liver disease. Their sequences were compared to sequences in well-characterized cohorts of homozygotes with or without liver disease, and then candidate sequence variants were tested for changes in the kinetics of α1-antitrypsin variant Z degradation in iPS-derived hepatocyte-like cells derived from the affected siblings themselves. APPROACH AND RESULTS: Specific variants in autophagy genes MTMR12 and FAM134A could each accelerate the degradation of α1-antitrypsin variant Z in cells from the index patient, but both MTMR12 and FAM134A variants were needed to slow the degradation of α1-antitrypsin variant Z in cells from a protected sib, indicating that inheritance of both variants is needed to mediate the pathogenic effects of hepatic proteotoxicity at the cellular level. Analysis of homozygote cohorts showed that multiple patient-specific variants in proteostasis genes are likely to explain liver disease susceptibility at the population level. CONCLUSIONS: These results validate the concept that genetic variation in autophagy function can determine susceptibility to liver disease in α1-antitrypsin deficiency and provide evidence that polygenic mechanisms and multiple patient-specific variants are likely needed for proteotoxic pathology.

Danish children with ZZ-homozygous alpha-1 antitrypsin deficiency are more affected on liver parameters than children with heterozygosity.

AIM: The longitudinal health status of Danish children with alpha-1 antitrypsin deficiency had never previously been characterised. This study aimed to assess the changes in growth, lung and liver function through childhood in these children. METHODS: Danish children diagnosed between 2005 and 2020 with pathogenic variants in the Serpin family A member 1 gene were included. Retrospective data on growth, lung and liver parameters were obtained from local databases. Anthropometric Z-scores and composite liver scores were computed. Growth and blood results were analysed using robust linear mixed models. RESULTS: The study included 184 children (68 with ZZ-homozygosity, 116 with heterozygosity). The median follow-up time was 7 years [IQR 3.75-9.00] for children with ZZ-homozygosity and 0.5 years [IQR 0.0-2.0] for children with heterozygosity. Both groups had low weight-for-height Z-scores at diagnosis but experienced catch-up growth during the first year of life. In addition, children with ZZ-homozygosity had higher serum concentrations of γ-glutamyl transferase and alanine aminotransferase throughout childhood, when compared with children with heterozygosity. Data proved insufficient to assess lung function properly. CONCLUSION: Children with ZZ-homozygosity were more affected on serum liver parameters throughout childhood when compared with children with heterozygosity. Both groups experienced catch-up growth during the first year of life.

Regulation of PGC1α Downstream of the Insulin Signaling Pathway Plays a Role in the Hepatic Proteotoxicity of Mutant α1-Antitrypsin Deficiency Variant Z.

BACKGROUND & AIMS: Insulin signaling is known to regulate essential proteostasis mechanisms. METHODS: The analyses here examined effects of insulin signaling in the PiZ mouse model of α1-antitrypsin deficiency in which hepatocellular accumulation and proteotoxicity of the misfolded α1-antitrypsin Z variant (ATZ) causes liver fibrosis and cancer. RESULTS: We first studied the effects of breeding PiZ mice to liver-insulin-receptor knockout (LIRKO) mice (with hepatocyte-specific insulin-receptor gene disruption). The results showed decreased hepatic ATZ accumulation and liver fibrosis in PiZ x LIRKO vs PiZ mice, with reversal of those effects when we bred PiZ x LIRKO mice onto a FOXO1-deficient background. Increased intracellular degradation of ATZ mediated by autophagy was identified as the likely mechanism for diminished hepatic proteotoxicity in PiZ x LIRKO mice and the converse was responsible for enhanced toxicity in PiZ x LIRKO x FOXO1-KO animals. Transcriptomic studies showed major effects on oxidative phosphorylation and autophagy genes, and significant induction of peroxisome proliferator-activated-receptor-γ-coactivator-1α (PGC1α) expression in PiZ-LIRKO mice. Because PGC1α plays a key role in oxidative phosphorylation, we further investigated its effects on ATZ proteostasis in our ATZ-expressing mammalian cell model. The results showed PGC1α overexpression or activation enhances autophagic ATZ degradation. CONCLUSIONS: These data implicate suppression of autophagic ATZ degradation by down-regulation of PGC1α as one mechanism by which insulin signaling exacerbates hepatic proteotoxicity in PiZ mice, and identify PGC1α as a novel target for development of new human α1-antitrypsin deficiency liver disease therapies.

Evaluation of cytosine base editing and adenine base editing as a potential treatment for alpha-1 antitrypsin deficiency.

Alpha-1 antitrypsin deficiency (AATD) is a rare autosomal codominant disease caused by mutations within the SERPINA1 gene. The most prevalent variant in patients is PiZ SERPINA1, containing a single G > A transition mutation. PiZ alpha-1 antitrypsin (AAT) is prone to misfolding, leading to the accumulation of toxic aggregates within hepatocytes. In addition, the abnormally low level of AAT secreted into circulation provides insufficient inhibition of neutrophil elastase within the lungs, eventually causing emphysema. Cytosine and adenine base editors enable the programmable conversion of C⋅G to T⋅A and A⋅T to G⋅C base pairs, respectively. In this study, two different base editing approaches were developed: use of a cytosine base editor to install a compensatory mutation (p.Met374Ile) and use of an adenine base editor to mediate the correction of the pathogenic PiZ mutation. After treatment with lipid nanoparticles formulated with base editing reagents, PiZ-transgenic mice exhibited durable editing of SERPINA1 in the liver, increased serum AAT, and improved liver histology. These results indicate that base editing has the potential to address both lung and liver disease in AATD.

Fibrosis-Related Gene Profiling in Liver Biopsies of PiZZ α1-Antitrypsin Children with Different Clinical Courses.

PiZZ (Glu342Lys) α1-antitrypsin deficiency (AATD) is characterized by intrahepatic AAT polymerization and is a risk factor for liver disease development in children. The majority of PiZZ children are disease free, hence this mutation alone is not sufficient to cause the disease. We investigated Z-AAT polymers and the expression of fibrosis-related genes in liver tissues of PiZZ children with different clinical courses. Liver biopsies obtained during 1979-2010 at the Department of Paediatrics, Karolinska University Hospital, Sweden, were subjected to histological re-evaluation, immunohistochemistry and NanoString-based transcriptome profiling using a panel of 760 fibrosis plus 8 bile acid-related genes. Subjects were divided into three groups based on clinical outcomes: NCH (neonatal cholestasis, favourable outcome, n = 5), NCC (neonatal cholestasis, early cirrhosis and liver transplantation, n = 4), and NNCH (no neonatal cholestasis, favourable outcome, n = 5, six biopsies). Hepatocytes containing Z-AAT polymers were abundant in all groups whereas NCC showed higher expression of genes related to liver fibrosis/cirrhosis and lower expression of genes related to lipid, aldehyde/ketone, and bile acid metabolism. Z-AAT accumulation per se cannot explain the clinical outcomes of PiZZ children; however, changes in the expression of specific genes and pathways involved in lipid, fatty acid, and steroid metabolism appear to reflect the degree of liver injury.

Liver Phenotypes of European Adults Heterozygous or Homozygous for Pi∗Z Variant of AAT (Pi∗MZ vs Pi∗ZZ genotype) and Noncarriers.

BACKGROUND & AIMS: Homozygosity for the Pi∗Z variant of the gene that encodes the alpha-1 antitrypsin peptide (AAT), called the Pi∗ZZ genotype, causes a liver and lung disease called alpha-1 antitrypsin deficiency. Heterozygosity (the Pi∗MZ genotype) is a risk factor for cirrhosis in individuals with liver disease. Up to 4% of Europeans have the Pi∗MZ genotype; we compared features of adults with and without Pi∗MZ genotype among persons without preexisting liver disease. METHODS: We analyzed data from the European Alpha-1 Liver Cohort, from 419 adults with the Pi∗MZ genotype, 309 adults with the Pi∗ZZ genotype, and 284 individuals without the variant (noncarriers). All underwent a comprehensive evaluation; liver stiffness measurements (LSMs) were made by transient elastography. Liver biopsies were analyzed to define histologic and biochemical features associated with the Pi∗Z variant. Levels of serum transaminases were retrieved from 444,642 participants, available in the United Kingdom biobank. RESULTS: In the UK biobank database, levels of serum transaminases were increased in subjects with the Pi∗MZ genotype compared with noncarriers. In the Alpha-1 Liver Cohort, adults with Pi∗MZ had lower levels of gamma-glutamyl transferase in serum and lower LSMs than adults with the Pi∗ZZ variant, but these were higher than in noncarriers. Ten percent of subjects with the Pi∗MZ genotype vs 4% of noncarriers had LSMs of 7.1 kPa or more (adjusted odds ratio, 4.8; 95% confidence interval, 2.0-11.8). Obesity and diabetes were the most important factors associated with LSMs ≥7.1 kPa in subjects with the Pi∗MZ genotype. AAT inclusions were detected in liver biopsies of 63% of subjects with the Pi∗MZ genotype, vs 97% of subjects with the Pi∗ZZ genotype, and increased with liver fibrosis stages. Subjects with the Pi∗MZ genotype did not have increased hepatic levels of AAT, whereas levels of insoluble AAT varied among individuals. CONCLUSIONS: Adults with the Pi∗MZ genotype have lower levels of serum transaminases, fewer AAT inclusions in liver, and lower liver stiffness than adults with the Pi∗ZZ genotype, but higher than adults without the Pi∗Z variant. These findings should help determine risk of subjects with the Pi∗MZ genotype and aid in counseling.

Hypertransaminasemia and liver fibrosis associated with haptoglobin retention and anhaptoglobinemia in a paediatric patient.

Cryptogenic elevation of transaminases in childhood can in a few instances be linked to rare hereditary causes. In this paper, a 7-year old girl is reported who was diagnosed with elevated transaminases of unknown origin since infancy. A liver biopsy showed bridging fibrosis, pale eosinophilic intracytoplasmic hepatocellular inclusions and enlarged endoplasmic reticulum cisternae in the hepatocytes. Whole-exome sequencing revealed a homozygous in-frame deletion of 3 base pairs in the haptoglobin gene. The patient is anhaptoglobinemic measured by standard laboratory turbidometry, which was confirmed by Western Blotting and thereby shown to affect both protein chains of haptoglobin. A polyclonal antibody revealed haptoglobin retention in hepatocytes suggesting a defect in haptoglobin secretion. A novel, previously unknown haptoglobin storage disease is suspected to be the reason for the elevated liver enzymes and tissue abnormalities in this patient. The pathophysiology appears to be similar to endoplasmic reticulum storage diseases like alpha-1-antitrypsin-deficiency.

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant.

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

The Mechanism of Mitochondrial Injury in Alpha-1 Antitrypsin Deficiency Mediated Liver Disease.

Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.

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

The maintenance of proteome homeostasis, or proteostasis, is crucial for preserving cellular functions and for cellular adaptation to environmental challenges and changes in physiological conditions. The capacity of cells to maintain proteostasis requires precise control and coordination of protein synthesis, folding, conformational maintenance, and clearance. Thus, protein degradation by the ubiquitin-proteasome system (UPS) or the autophagy-lysosomal system plays an essential role in cellular functions. However, failure of the UPS or the autophagic process can lead to the development of various diseases (aging-associated diseases, cancer), thus both these pathways have become attractive targets in the treatment of protein conformational diseases, such as alpha 1-antitrypsin deficiency (AATD). The Z alpha 1-antitrypsin (Z-AAT) misfolded variant of the serine protease alpha 1-antitrypsin (AAT) is caused by a structural change that predisposes it to protein aggregation and dramatic accumulation in the form of inclusion bodies within liver hepatocytes. This can lead to clinically significant liver disease requiring liver transplantation in childhood or adulthood. Treatment of mice with autophagy enhancers was found to reduce hepatic Z-AAT aggregate levels and protect them from AATD hepatotoxicity. To date, liver transplantation is the only curative therapeutic option for patients with AATD-mediated liver disease. Therefore, the development and discovery of new therapeutic approaches to delay or overcome disease progression is a top priority. Herein, we review AATD-mediated liver disease and the overall process of autophagy. We highlight the role of this system in the regulation of Z-variant degradation and its implication in AATD-medicated liver disease, including some open questions that remain challenges in the field and require further elucidation. Finally, we discuss how manipulation of autophagy could provide multiple routes of therapeutic benefit in AATD-mediated liver disease.

Protease-Antiprotease Imbalance in Bronchiectasis.

Airway inflammation plays a central role in bronchiectasis. Protease-antiprotease balance is crucial in bronchiectasis pathophysiology and increased presence of unopposed proteases activity may contribute to bronchiectasis onset and progression. Proteases' over-reactivity and antiprotease deficiency may have a role in increasing inflammation in bronchiectasis airways and may lead to extracellular matrix degradation and tissue damage. Imbalances in serine proteases and matrix-metallo proteinases (MMPs) have been associated to bronchiectasis. Active neutrophil elastase has been associated with disease severity and poor long-term outcomes in this disease. Moreover, high levels of MMPs have been associated with radiological and disease severity. Finally, severe deficiency of α1-antitrypsin (AAT), as PiSZ and PiZZ (proteinase inhibitor SZ and ZZ) phenotype, have been associated with bronchiectasis development. Several treatments are under study to reduce protease activity in lungs. Molecules to inhibit neutrophil elastase activity have been developed in both oral or inhaled form, along with compounds inhibiting dipeptydil-peptidase 1, enzyme responsible for the activation of serine proteases. Finally, supplementation with AAT is in use for patients with severe deficiency. The identification of different targets of therapy within the protease-antiprotease balance contributes to a precision medicine approach in bronchiectasis and eventually interrupts and disrupts the vicious vortex which characterizes the disease.

Protein Misfolding and Aggregation: The Relatedness between Parkinson's Disease and Hepatic Endoplasmic Reticulum Storage Disorders.

Dysfunction of cellular homeostasis can lead to misfolding of proteins thus acquiring conformations prone to polymerization into pathological aggregates. This process is associated with several disorders, including neurodegenerative diseases, such as Parkinson's disease (PD), and endoplasmic reticulum storage disorders (ERSDs), like alpha-1-antitrypsin deficiency (AATD) and hereditary hypofibrinogenemia with hepatic storage (HHHS). Given the shared pathophysiological mechanisms involved in such conditions, it is necessary to deepen our understanding of the basic principles of misfolding and aggregation akin to these diseases which, although heterogeneous in symptomatology, present similarities that could lead to potential mutual treatments. Here, we review: (i) the pathological bases leading to misfolding and aggregation of proteins involved in PD, AATD, and HHHS: alpha-synuclein, alpha-1-antitrypsin, and fibrinogen, respectively, (ii) the evidence linking each protein aggregation to the stress mechanisms occurring in the endoplasmic reticulum (ER) of each pathology, (iii) a comparison of the mechanisms related to dysfunction of proteostasis and regulation of homeostasis between the diseases (such as the unfolded protein response and/or autophagy), (iv) and clinical perspectives regarding possible common treatments focused on improving the defensive responses to protein aggregation for diseases as different as PD, and ERSDs.

Modulation of calreticulin expression reveals a novel exosome-mediated mechanism of Z variant α1-antitrypsin disposal.

α1-Antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at position 342 in the mature protein, resulting in the Z mutation of the AAT gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes, causing a toxic gain of function. ERdj3 is an ER luminal DnaJ homologue, which, along with calreticulin, directly interacts with misfolded ZAAT. We hypothesize that depletion of each of these chaperones will change the fate of ZAAT polymers. Our study demonstrates that calreticulin modulation reveals a novel ZAAT degradation mechanism mediated by exosomes. Using human PiZZ hepatocytes and K42, a mouse calreticulin-deficient fibroblast cell line, our results show ERdj3 and calreticulin directly interact with ZAAT in PiZZ hepatocytes. Silencing calreticulin induces calcium independent ZAAT-ERdj3 secretion through the exosome pathway. This co-secretion decreases ZAAT aggregates within the ER of hepatocytes. We demonstrate that calreticulin has an inhibitory effect on exosome-mediated ZAAT-ERdj3 secretion. This is a novel ZAAT degradation process that involves a DnaJ homologue chaperone bound to ZAAT. In this context, calreticulin modulation may eliminate the toxic gain of function associated with aggregation of ZAAT in lung and liver, thus providing a potential new therapeutic approach to the treatment of AATD-related liver disease.

Identification of small molecules by screening a mixture-based scaffold compound library for treatment of alpha-1 antitrypsin deficiency.

This study aimed to identify small molecules that have the potential to treat alpha1-antitrypsin deficiency (AATD) by screening compounds available from a mixture-based scaffold library. 93 scaffold libraries (total diversity of >30 million compounds in mixture format) were screened using a cell model of AATD in order to identify samples that could either reduce intracellular aggregation of Z-form AAT protein, increase extracellular secretion of Z-AAT or both. Mixture libraries containing compounds with in vitro activity, for example library 1295, were screened further to identify individual active compounds. The mixture format of the scaffold library allowed for some preliminary structure-activity relationships to be developed and also enabled the rapid selection of a promising scaffold. Utilizing this scaffold, 1295, a collection of individual "control" compounds contained in the 1295 mixture sample were then screened. A sub-library of individual "control" compounds featuring structural diversity at position R1 (1295.R1), was screened and 7 compounds were found to reduce the intracellular accumulation of Z-AAT without affecting cell viability at a concentration of 25ug/ml (about 50 µM). Screening sub-libraries featuring structural diversity at R2 and R3 (1295.R2 and 1295.R3) identified an additional 15 active compounds. Titration experiments identified 3 compounds from the 1295.R2 library that retained activity at 5ug/ml (approx. 10uM). One compound (1295.263) from 1295.R2 decreased intracellular levels of Z-AAT without affecting cell viability and wild-type AAT levels at the concentration of 5ug/ml. Molecular docking of this compound to the Z-AAT crystal structure identified a potential binding site near the C-terminal domain, an identified polymerization site. Our results indicate that screening large mixture-based compound libraries can be used to identify small molecules that may have the potential to treat AATD and other disease.

Alpha-1 antitrypsin deficient individuals have circulating extracellular vesicles with profibrogenic cargo.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD)-mediated liver disease is a toxic "gain-of-function" inflammation in the liver associated with intracellular retention of mutant alpha-1 antitrypsin. The clinical presentation of the disease includes fibrosis, cirrhosis and liver failure. However, the pathogenic mechanism of AATD-mediated liver disease is not well understood. Here, we investigated the role of plasma extracellular vesicles (EVs) in progression of AATD-mediated liver disease. METHODS: EVs were isolated from plasma of AATD individuals with liver disease and healthy controls. Their cytokines and miRNA content were examined by multiplex assay and small RNA sequencing. The bioactivity of EVs was assessed by qPCR, western blot analysis and immunofluorescent experiments using human hepatic stellate cells (HSCs) treated with EVs isolated from control or AATD plasma samples. RESULTS: We have found that AATD individuals have a distinct population of EVs with pathological cytokine and miRNA contents. When HSCs were cultured with AATD plasma derived-EVs, the expression of genes related to the development of fibrosis were significantly amplified compared to those treated with healthy control plasma EVs. CONCLUSION: AATD individuals have a distinct population of EVs with abnormal cytokine and miRNA contents and the capacity to activate HSCs and mediate fibrosis. Better understanding of the components which cause liver inflammation and fibrogenesis, leading to further liver injury, has the potential to lead to the development of new treatments or preventive strategies to prevent AATD-mediated liver disease. Video abstract.

Alpha 1-Antitrypsin Deficiency: A Disorder of Proteostasis-Mediated Protein Folding and Trafficking Pathways.

Human cells express large amounts of different proteins continuously that must fold into well-defined structures that need to remain correctly folded and assemble in order to ensure their cellular and biological functions. The integrity of this protein balance/homeostasis, also named proteostasis, is maintained by the proteostasis network (PN). This integrated biological system, which comprises about 2000 proteins (chaperones, folding enzymes, degradation components), control and coordinate protein synthesis folding and localization, conformational maintenance, and degradation. This network is particularly challenged by mutations such as those found in genetic diseases, because of the inability of an altered peptide sequence to properly engage PN components that trigger misfolding and loss of function. Thus, deletions found in the ΔF508 variant of the Cystic Fibrosis (CF) transmembrane regulator (CFTR) triggering CF or missense mutations found in the Z variant of Alpha 1-Antitrypsin deficiency (AATD), leading to lung and liver diseases, can accelerate misfolding and/or generate aggregates. Conversely to CF variants, for which three correctors are already approved (ivacaftor, lumacaftor/ivacaftor, and most recently tezacaftor/ivacaftor), there are limited therapeutic options for AATD. Therefore, a more detailed understanding of the PN components governing AAT variant biogenesis and their manipulation by pharmacological intervention could delay, or even better, avoid the onset of AATD-related pathologies.

Blockade of Dopamine D2 Receptors as a Novel Approach to Stimulation of Notch1+ Endothelial Progenitor Cells and Angiogenesis in C57BL/6 Mice with Pulmonary Emphysema Induced by Proteases and Deficiency of α1-Antitrypsin.

We studied the effects of spiperone, a selective blocker of dopamine D2 receptors, on the model of pulmonary emphysema provoked by administration of elastase and D-galactosamine hydrochloride to female C57BL/6 mice and characterized by activation of proteases in the lungs and systemic deficiency of its inhibitor α1-antitrypsin. In this model, spiperone prevented the development of inflammatory reaction and reduced the area of emphysematous expanded alveolar tissue. The expression of angiogenic marker CD31 in the lungs increased under these conditions. Regeneration of the damaged microvascular bed under the action of spiperone resulted from recruiting of Notch1+ endothelial progenitor cells (CD45-CD31+CD34+) into the lungs and blockade of the inhibitory effect of dopamine on phosphorylation of VEGF-2 receptors in endothelial cells of different maturity. In addition, spiperone produced a protective effect on hepatocytes and restored the production and secretion of α1-antitrypsin by these cells.

Liver disease related to alpha1-antitrypsin deficiency in French children: The DEFI-ALPHA cohort.

BACKGROUND & AIMS: To identify prognostic factors for liver disease in children with alpha-1 antitrypsin deficiency, irrespective of phenotype, using the DEFI-ALPHA cohort. METHODS: Retrospective, then prospective from 2010, multicentre study including children known to have alpha-1 antitrypsin blood concentration below 0.8 g/L, born in France since 1989. Clinical and biological data were collected. Liver disease was classified as "severe" (portal hypertension, liver failure, liver transplantation or death); "moderate" (persistent abnormal liver biology without portal hypertension); and "mild/none" (normal or almost normal liver biology and native liver). Prognostic factors for severe liver disease were evaluated using a Cox semiparametric model. RESULTS: In January 2017, 153 patients from 19 centres had been included; genotypes were PIZZ in 81.9%, PISZ in 8.1%, other in 10.0%. Mean ± SD follow-up was 4.7 ± 2.1 years. Half of patients had moderate liver disease. Twenty-eight children (18.3%) had severe liver disease (mean age 2.5 years, range: 0-11.6): diagnosis of alpha-1 antitrypsin deficiency was made before two months of age in 65.4%, genotypes were PIZZ in 25 (89.3%), PISZ in 2, PIMlike Z in 1, 15 children underwent liver transplantation, 1 child died at 3 years of age. Neonatal cholestasis was significantly associated with severe liver disease (P = 0.007). CONCLUSION: Alpha-1 antitrypsin-deficient patients presenting with neonatal cholestasis were likely to develop severe liver disease. Some patients with non-homozygous ZZ genotype can develop severe liver disease, such as PISZ and M variants, when associated with predisposing factors. Further genetic studies will help to identify other factors involved in the development of liver complications.

Real-world clinical applicability of pathogenicity predictors assessed on SERPINA1 mutations in alpha-1-antitrypsin deficiency.

The growth of publicly available data informing upon genetic variations, mechanisms of disease, and disease subphenotypes offers great potential for personalized medicine. Computational approaches are likely required to assess a large number of novel genetic variants. However, the integration of genetic, structural, and pathophysiological data still represents a challenge for computational predictions and their clinical use. We addressed these issues for alpha-1-antitrypsin deficiency, a disease mediated by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin. We compiled a comprehensive database of SERPINA1 coding mutations and assigned them apparent pathological relevance based upon available data. "Benign" and "pathogenic" variations were used to assess performance of 31 pathogenicity predictors. Well-performing algorithms clustered the subset of variants known to be severely pathogenic with high scores. Eight new mutations identified in the ExAC database and achieving high scores were selected for characterization in cell models and showed secretory deficiency and polymer formation, supporting the predictive power of our computational approach. The behavior of the pathogenic new variants and consistent outliers were rationalized by considering the protein structural context and residue conservation. These findings highlight the potential of computational methods to provide meaningful predictions of the pathogenic significance of novel mutations and identify areas for further investigation.

Clinical and histologic features of adults with alpha-1 antitrypsin deficiency in a non-cirrhotic cohort.

BACKGROUND & AIMS: Alpha-1 antitrypsin deficiency (AATD) is an uncommonly recognized cause of liver disease in adults, with descriptions of its natural history limited to case series and patient-reported data from disease registries. Liver pathology is limited to selected patients or unavailable. Therefore, we aimed to determine the prevalence and severity of liver fibrosis in an adult AATD population who were not known to have cirrhosis, while defining risk factors for fibrosis and testing non-invasive markers of disease. METHODS: A total of 94 adults with classic genotype 'PI*ZZ' AATD were recruited from North America and prospectively enrolled in the study. Liver aminotransferases and markers of synthetic function, transient elastography, and liver biopsy were performed. RESULTS: The prevalence of clinically significant liver fibrosis (F ≥ 2) was 35.1%. Alanine aminotransferase, aspartate aminotransferase and gamma-glutamyltransferase values were higher in the F ≥ 2 group. Metabolic syndrome was associated with the presence of clinically significant fibrosis (OR 14.2; 95% CI 3.7-55; p <0.001). Additionally, the presence of accumulated abnormal AAT in hepatocytes, portal inflammation, and hepatocellular degeneration were associated with clinically significant fibrosis. The accuracy of transient elastography to detect F ≥ 2 fibrosis was fair, with an AUC of 0.70 (95% CI 0.58-0.82). CONCLUSIONS: Over one-third of asymptomatic and lung affected adults with 'PI*ZZ' AATD have significant underlying liver fibrosis. Liver biopsies demonstrated variable amounts of accumulated Z AAT. The risk of liver fibrosis increases in the presence of metabolic syndrome, accumulation of AAT in hepatocytes, and portal inflammation on baseline biopsy. The results support the hypothesis that liver disease in this genetic condition may be related to a "toxic gain of function" from accumulation of AAT in hepatocytes. LAY SUMMARY: Individuals diagnosed with classic alpha-1 antitrypsin deficiency (ZZ) are at risk of liver injury and scarring, because of the accumulation of abnormal alpha-1 antitrypsin in the liver. A liver biopsy in ZZ individuals can demonstrate the accumulation of alpha-1 antitrypsin within the liver and identify if any associated liver scarring is present. Indviduals with large amounts of alpha-1 antitrypsin on biopsy may be at risk of liver injury and fibrosis. Additional common medical conditions of diabetes, obesity, high cholesterol, and hypertension (known as metabolic syndrome) are associated with a greater degree of liver injury. CLINICAL TRIAL NUMBER: clinicaltrials.gov NCT01810458.