Fazirsiran for Adults With Alpha-1 Antitrypsin Deficiency Liver Disease: A Phase 2 Placebo Controlled Trial (SEQUOIA).

BACKGROUND & AIMS: Homozygous ZZ alpha-1 antitrypsin (AAT) deficiency produces mutant AAT (Z-AAT) proteins in hepatocytes, leading to progressive liver fibrosis. We evaluated the safety and efficacy of an investigational RNA interference therapeutic, fazirsiran, that degrades Z-AAT messenger RNA, reducing deleterious protein synthesis. METHODS: This ongoing, phase 2 study randomized 40 patients to subcutaneous placebo or fazirsiran 25, 100, or 200 mg. The primary endpoint was percent change in serum Z-AAT concentration from baseline to week 16. Patients with fibrosis on baseline liver biopsy received treatment on day 1, at week 4, and then every 12 weeks and had a second liver biopsy at or after weeks 48, 72, or 96. Patients without fibrosis received 2 doses on day 1 and at week 4. RESULTS: At week 16, least-squares mean percent declines in serum Z-AAT concentration were -61%, -83%, and -94% with fazirsiran 25, 100, and 200 mg, respectively, vs placebo (all P < .0001). Efficacy was sustained through week 52. At postdose liver biopsy, fazirsiran reduced median liver Z-AAT concentration by 93% compared with an increase of 26% with placebo. All fazirsiran-treated patients had histologic reduction from baseline in hepatic globule burden. Portal inflammation improved in 5 of 12 and 0 of 8 patients with a baseline score of >0 in the fazirsiran and placebo groups, respectively. Histologic meta-analysis of histologic data in viral hepatitis score improved by >1 point in 7 of 14 and 3 of 8 patients with fibrosis of >F0 at baseline in the fazirsiran and placebo groups, respectively. No adverse events led to discontinuation, and pulmonary function tests remained stable. CONCLUSIONS: Fazirsiran reduced serum and liver concentrations of Z-AAT in a dose-dependent manner and reduced hepatic globule burden. (ClinicalTrials.gov, Number NCT03945292).

Alpha-defensins inhibit ERK/STAT3 signaling during monocyte-macrophage differentiation and impede macrophage function.

Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder associated with a 5-tenfold decrease in lung levels of alpha-1-antitrypsin (AAT) and an increased risk for obstructive lung disease. α-defensins are cationic broad-spectrum cytotoxic and pro-inflammatory peptides found in the azurophilic granules of neutrophils. The concentration of α-defensins is less than 30 nM in the bronchoalveolar lavage fluid of healthy controls but is up to 6 µM in AATD individuals with significant lung function impairment. Alveolar macrophages are generally classified into pro-inflammatory (M1) or anti-inflammatory (M2) subsets that play distinct roles in the initiation and resolution of inflammation. Therefore, monocyte-macrophage differentiation should be tightly controlled to maintain lung integrity. In this study, we determined the effect of α-defensins on monocyte-macrophage differentiation and identified the molecular mechanism of this effect. The results of this study demonstrate that 2.5 µM of α-defensins inhibit the phosphorylation of ERK1/2 and STAT3 and suppress the expression of M2 macrophage markers, CD163 and CD206. In addition, a scratch assay shows that the high concentration of α-defensins inhibits cell movement by ~ 50%, and the phagocytosis assay using flow cytometry shows that α-defensins significantly reduce the bacterial phagocytosis rate of monocyte-derived macrophages (MDMs). To examine whether exogenous AAT is able to alleviate the inhibitory effect of α-defensins on macrophage function, we incubated MDMs with AAT prior to α-defensin treatment and demonstrate that AAT improves the migratory ability and phagocytic ability of MDMs compared with MDMs incubated only with α-defensins. Taken together, this study suggests that a high concentration of α-defensins inhibits the activation of ERK/STAT3 signaling, negatively regulates the expression of M2 macrophage markers, and impairs innate immune function of macrophages.

Testing Patterns and Disparities for Alpha-1 Antitrypsin Deficiency.

BACKGROUND: Alpha-1 antitrypsin deficiency is an under-recognized genetic cause of chronic lung and liver disease; it remains unclear what the testing frequency and disparities are for alpha-1 antitrypsin deficiency. METHODS: This is a retrospective cohort study of people with newly diagnosed chronic obstructive pulmonary disease and liver disease identified at the University of Florida between January 1, 2012 and December 31, 2021. We performed incidence and prevalence analysis for alpha-1 antitrypsin (AAT) testing and point-biserial correlation analysis for tobacco use and AAT testing. We evaluated characteristics with AAT testing using adjusted multivariable logistic regression. RESULTS: Among 75,810 subjects with newly diagnosed chronic obstructive pulmonary disease and liver disease between 2012 and 2021, 4248 (5.6%) were tested for AAT deficiency. All subjects had an AAT level performed, while 1654 (39%) had phenotype testing. Annual incidence of testing increased for subjects with newly diagnosed chronic obstructive pulmonary disease or liver disease from 2.8% and 5.4%, respectively, in 2012 to 4.1% and 11.3%, respectively, in 2021. Adjusted multivariable regression analysis showed factors favoring AAT testing were White race, and concomitant chronic obstructive pulmonary disease and liver disease. Increasing age, non-White race, current tobacco use, and being a male with chronic obstructive pulmonary disease had lower odds of AAT testing. CONCLUSION: Although slowly improving, testing for AAT deficiency continues to have a low uptake in the clinical setting despite guidelines recommending broader testing. Individuals of White race and those with concomitant chronic obstructive pulmonary disease and liver disease are more likely to be tested, while older subjects, individuals of non-White race, current tobacco use, and men with chronic obstructive pulmonary disease are less favored to be tested.

Multiplexing AAV Serotype-Specific Neutralizing Antibodies in Preclinical Animal Models and Humans.

The accurate assessment of AAV-specific pre-existing humoral immunity due to natural viral infection is critical for the efficient use of clinical gene therapy. The method described in the present study applies equivalent infection conditions to each AAV serotype (AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVAnc80L65). In the current study, we validated the assay by assessing AAV-neutralizing antibody titers in a limited cohort of random human donors and well-established preclinical large animal models, including dogs and non-human primates (NHPs). We achieved a rapid and accurate evaluation of neutralizing titers for each individual subject that can be used for clinical enrollment based on specific AAV serotypes and individualized selection of the most suitable AAV serotype for each specific patient.

Novel SERPINA1 Alleles Identified through a Large Alpha-1 Antitrypsin Deficiency Screening Program and Review of Known Variants.

The SERPINA1 gene encodes the serine protease inhibitor alpha-1 antitrypsin (AAT) and is located on chromosome 14q31-32.3 in a cluster of homologous genes likely formed by exon duplication. AAT has a variety of anti-inflammatory properties. Its clinical relevance is best illustrated by the genetic disease alpha-1 antitrypsin deficiency (AATD) which is associated with an increased risk for chronic obstructive pulmonary disease (COPD) and cirrhosis. While 2 single nucleotide polymorphisms (SNPs) , S and Z, are responsible for more than 95% of all individuals with AATD, there are a number of rare variants associated with deficiency and dysfunction, as well as those associated with normal levels and function. Our laboratory has identified a number of novel AAT alleles that we report in this manuscript. We screened more than 500,000 individuals for AATD alleles through our testing program over the past 20 years. The characterization of these alleles was accomplished by DNA sequencing, measurement of AAT plasma levels and isoelectric focusing at pH 4-5. We report 22 novel AAT alleles discovered through our screening programs, such as Zlittle rock and QOchillicothe, and review the current literature of known AAT genetic variants.

Cigarette smoke exposed airway epithelial cell-derived EVs promote pro-inflammatory macrophage activation in alpha-1 antitrypsin deficiency.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder most commonly secondary to a single mutation in the SERPINA1 gene (PI*Z) that causes misfolding and accumulation of alpha-1 antitrypsin (AAT) in hepatocytes and mononuclear phagocytes which reduces plasma AAT and creates a toxic gain of function. This toxic gain of function promotes a pro-inflammatory phenotype in macrophages that contributes to lung inflammation and early-onset COPD, especially in individuals who smoke cigarettes. The aim of this study is to determine the role of cigarette exposed AATD macrophages and bronchial epithelial cells in AATD-mediated lung inflammation. METHODS: Peripheral blood mononuclear cells from AATD and healthy individuals were differentiated into alveolar-like macrophages and exposed to air or cigarette smoke while in culture. Macrophage endoplasmic reticulum stress was quantified and secreted cytokines were measured using qPCR and cytokine ELISAs. To determine whether there is "cross talk" between epithelial cells and macrophages, macrophages were exposed to extracellular vesicles released by airway epithelial cells exposed to cigarette smoke and their inflammatory response was determined. RESULTS: AATD macrophages spontaneously produce several-fold more pro-inflammatory cytokines as compared to normal macrophages. AATD macrophages have an enhanced inflammatory response when exposed to cigarette smoke-induced extracellular vesicles (EVs) released from airway epithelial cells. Cigarette smoke-induced EVs induce expression of GM-CSF and IL-8 in AATD macrophages but have no effect on normal macrophages. Release of AAT polymers, potent neutrophil chemo attractants, were also increased from AATD macrophages after exposure to cigarette smoke-induced EVs. CONCLUSIONS: The expression of mutated AAT confers an inflammatory phenotype in AATD macrophages which disposes them to an exaggerated inflammatory response to cigarette smoke-induced EVs, and thus could contribute to progressive lung inflammation and damage in AATD individuals.

Alu RNA induces NLRP3 expression through TLR7 activation in α-1-antitrypsin-deficient macrophages.

α-1 antitrypsin (AAT) is a serine protease inhibitor that plays a pivotal role in maintaining lung homeostasis. The most common AAT allele associated with AAT deficiency (AATD) is PiZ. Z-AAT accumulates in cells due to misfolding, causing severe AATD. The major function of AAT is to neutralize neutrophil elastase in the lung. It is generally accepted that loss of antiprotease function is a major cause of COPD in individuals with AATD. However, it is now being recognized that the toxic gain-of-function effect of Z-AAT in macrophage likely contributes to lung disease. In the present study, we determined that TLR7 signaling is activated in Z-MDMs, and the expression level of NLRP3, one of the targets of TLR7 signaling, is significantly higher in Z- compared with M-MDMs. We also determined that the level of endosomal Alu RNA is significantly higher in Z-compared with M-MDMs. Alu RNA is a known endogenous ligand that activates TLR7 signaling. Z-AAT likely induces the expression of Alu elements in MDMs and accelerates monocyte death, leading to the higher level of endosomal Alu RNA in Z-MDMs. Taken together,this study identifies a mechanism responsible for the toxic gain of function of Z-AAT macrophages.

The unfolded protein response to PI*Z alpha-1 antitrypsin in human hepatocellular and murine models.

Alpha-1 antitrypsin (AAT) deficiency (AATD) is an inherited disease caused by mutations in the serpin family A member 1 (SERPINA1, also known as AAT) gene. The most common variant, PI*Z (Glu342Lys), causes accumulation of aberrantly folded AAT in the endoplasmic reticulum (ER) of hepatocytes that is associated with a toxic gain of function, hepatocellular injury, liver fibrosis, and hepatocellular carcinoma. The unfolded protein response (UPR) is a cellular response to improperly folded proteins meant to alleviate ER stress. It has been unclear whether PI*Z AAT elicits liver cell UPR, due in part to limitations of current cellular and animal models. This study investigates whether UPR is activated in a novel human PI*Z AAT cell line and a new PI*Z human AAT (hAAT) mouse model. A PI*Z AAT hepatocyte cell line (Huh7.5Z) was established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of the normal ATT (PI*MM) gene in the Huh7.5 cell line. Additionally, novel full-length genomic DNA PI*Z hAAT and PI*M hAAT transgenic mouse models were established. Using these new models, UPR in Huh7.5Z cells and PI*Z mice were comprehensively determined. Robust activation of UPR was observed in Huh7.5Z cells compared to Huh7.5 cells. Activated caspase cascade and apoptosis markers, increased chaperones, and autophagy markers were also detected in Z hepatocytes. Selective attenuation of UPR signaling branches was observed in PI*Z hAAT mice in which the protein kinase R-like ER kinase and inositol-requiring enzyme1α branches were suppressed while the activating transcription factor 6α branch remained active. This study provides direct evidence that PI*Z AAT triggers canonical UPR and that hepatocytes survive pro-apoptotic UPR by selective suppression of UPR branches. Our data improve understanding of underlying pathological molecular mechanisms of PI*Z AATD liver disease.

Nicotine Exacerbates TAAD Formation Induced by Smooth Muscle-Specific Deletion of the TGF-ß Receptor 2.

Tobacco smoke is an established risk factor for thoracic aortic aneurysms and dissections (TAAD). However, little is known about its underlying mechanisms due to the lack of validated animal models. The present study developed a mouse model that may be utilized to investigate exacerbation of TAAD formation by mimetics of tobacco smoke. TAADs were created via inducible deletion of smooth muscle cell-specific Tgfbr2 receptors. Using this model, the first set of experiments evaluated the efficacy of nicotine salt (34.0 mg/kg/day), nicotine free base (NFB, 5.0 mg 90-day pellets), and cigarette smoke extract (0.1 ml/mouse/day). Compared with their respective control groups, only NFB pellets promoted TAAD dilation (23 ± 3% vs. 12 ± 2%, P = 0.014), and this efficacy was achieved at a cost of >50% acute mortality. Infusion of NFB with osmotic minipumps at extremely high, but nonlethal, doses (15.0 or 45.0 mg/kg/day) failed to accelerate TAAD dilation. Interestingly, costimulation with ß-aminopropionitrile (BAPN) promoted TAAD dilation and aortic rupture at dosages of 3.0 and 45.0 mg/kg/day, respectively, indicating that BAPN sensitizes the response of TAADs to NFB. In subsequent analyses, the detrimental effects of NFB were associated with clustering of macrophages, neutrophils, and T-cells in areas with structural destruction, enhanced matrix metalloproteinase- (MMP-) 2 production, and pathological angiogenesis with attenuated fibrosis in the adventitia. In conclusion, modeling nicotine exacerbation of TAAD formation requires optimization of chemical form, route of delivery, and dosage of the drug as well as the pathologic complexity of TAADs. Under the optimized conditions of the present study, chronic inflammation and adventitial mal-remodeling serve as critical pathways through which NFB exacerbates TAAD formation.

Quantitative measurement of the histological features of alpha-1 antitrypsin deficiency-associated liver disease in biopsy specimens.

BACKGROUND: Pathological mutations in Alpha-1 Antitrypsin (AAT) protein cause retention of toxic polymers in the hepatocyte endoplasmic reticulum. The risk for cirrhosis in AAT deficiency is likely directly related to retention of these polymers within the liver. Polymers are classically identified on liver biopsy as inclusion bodies by periodic acid schiff staining after diastase treatment and immunohistochemistry. However, characterization of the polymer burden within a biopsy sample is limited to a semi-quantitative scale as described by a pathologist. Better methods to quantify polymer are needed to advance our understanding of pathogenesis of disease. Therefore, we developed a method to quantify polymer aggregation from standard histologic specimens. In addition, we sought to understand the relationship of polymer burden and other histologic findings to the presence of liver fibrosis. METHODS: Liver samples from a well-categorized AATD cohort were used to develop histo-morphometric tools to measure protein aggregation. RESULTS: Whole-slide morphometry reliably quantifies aggregates in AATD individuals. Despite very low levels of inclusions present (0-0.41%), accumulation of globules is not linear and is associated with higher fibrosis stages. Immunohistochemistry demonstrates that fibrosis is associated with polymer accumulation and not total AAT. A proportion of patients were found to be "heavy accumulators" with a polymer burden above the upper 25% of normal distribution. Males had significantly more liver inclusions and polymer than females. These measurements also highlight interrelated phenotypes of hepatocellular degeneration and autophagy in AATD liver disease. CONCLUSION: Quantitative inclusion analysis measures AAT accumulation in liver biopsy specimens. Quantification of polymer may identify individuals at risk for progressive disease and candidates for therapeutic interventions. Furthermore, these methods may be useful for evaluating efficacy of drugs targeting accumulation of AAT.

Alpha 1 Antitrypsin-Deficient Macrophages Have Impaired Efferocytosis of Apoptotic Neutrophils.

Alpha 1 antitrypsin deficiency (AATD) is an autosomal co-dominant disorder characterized by a low level of circulating AAT, which significantly reduces protection for the lower airways against proteolytic burden caused by neutrophils. Neutrophils, which are terminally differentiated innate immune cells and play a critical role to clear pathogens, accumulate excessively in the lung of AATD individuals. The neutrophil burden in AATD individuals increases the risk for early-onset destructive lung diseases by producing neutrophil products such as reactive oxygen radicals and various proteases. The level of AAT in AATD individuals is not sufficient to inhibit the activity of neutrophil chemotactic factors such as CXCL-8 and LTB4, which could lead to alveolar neutrophil accumulation in AATD individuals. However, as neutrophils have a short lifespan, and apoptotic neutrophils are rapidly cleared by alveolar macrophages that outnumber the apoptotic neutrophils in the pulmonary alveolus, the increased chemotaxis activity does not fully explain the persistent neutrophil accumulation and the resulting chronic inflammation in AATD individuals. Here, we propose that the ability of alveolar macrophages to clear apoptotic neutrophils is impaired in AATD individuals and it could be the main driver to cause neutrophil accumulation in their lung. This study demonstrates that Z-AAT variant significantly increases the expression of pro-inflammatory cytokines including CXCL-8, CXCL1, LTB4, and TNFα in LPS-treated macrophages. These cytokines play a central role in neutrophil recruitment to the lung and in clearance of apoptotic neutrophils by macrophages. Our result shows that LPS treatment significantly reduces the efferocytosis ability of macrophages with the Z-AAT allele by inducing TNFα expression. We incubated monocyte-derived macrophages (MDMs) with apoptotic neutrophils and found that after 3 h of co-incubation, the expression level of CXCL-8 is reduced in M-MDMs but increased in Z-MDMs. This result shows that the expression of inflammatory cytokines could be increased by impaired efferocytosis. It indicates that the efferocytosis ability of macrophages plays an important role in regulating cytokine expression and resolving inflammation. Findings from this study would help us better understand the multifaceted effect of AAT on regulating neutrophil balance in the lung and the underlying mechanisms.

Neutrophil elastase promotes macrophage cell adhesion and cytokine production through the integrin-Src kinases pathway.

There are a number of respiratory diseases characterized by the presence of excess neutrophil elastase (NE) activity in tissues, including cystic fibrosis and chronic obstructive pulmonary disease (COPD). NE is considered a primary contributor to disease development, but the precise mechanism has yet to be fully determined. We hypothesized that NE alters the function of macrophages (Mɸ) which play a critical role in many physiological processes in healthy lungs. We demonstrate that monocyte-derived Mɸ exposed to NE releases active matrix metalloproteinases (MMPs), increase expression of pro-inflammatory cytokines TNFα, IL-1ß, and IL-8, and reduce capacity to phagocytose bacteria. Changes in Mɸ function following NE treatment were accompanied by increased adhesion and cytoskeleton re-arrangement, indicating the possibility of integrin involvement. To support this observation, we demonstrate that NE induces phosphorylation of kinases from the Src kinase family, a hallmark of integrin signaling activation. Moreover, pretreatment of Mɸ with a specific Src kinase inhibitor, PP2 completely prevents NE-induced pro-inflammatory cytokine production. Taken together these findings indicate that NE participates in lung destruction not only through direct proteolytic degradation of matrix proteins, but also through activation of Mɸ inflammatory and proteolytic functions.

Smoking-related diffuse cystic lung disease.

Smoking tobacco is associated with an array of pulmonary symptoms and diseases. We describe a case of a woman with a spontaneous pneumothorax and diffuse cystic lung disease due to smoking. The presence of diffuse cystic changes in a woman is suggestive of lymphangioleiomyomatosis (LAM); however, her vascular endothelial growth factor-D was normal and surgical lung biopsy and pathology had notable absence of LAM cells and presence of intra-alveolar pigment laden macrophages and intraluminal mucostasis. Smoking-related diffuse cystic lung disease can mimic LAM and is a novel entity with only four other cases reported.

Chemokine signaling axis between endothelial and myeloid cells regulates development of pulmonary hypertension associated with pulmonary fibrosis and hypoxia.

Pulmonary hypertension complicates the care of many patients with chronic lung diseases (defined as Group 3 pulmonary hypertension), yet the mechanisms that mediate the development of pulmonary vascular disease are not clearly defined. Despite being the most prevalent form of pulmonary hypertension, to date there is no approved treatment for patients with disease. Myeloid-derived suppressor cells (MDSCs) and endothelial cells in the lung express the chemokine receptor CXCR2, implicated in the evolution of both neoplastic and pulmonary vascular remodeling. However, precise cellular contribution to lung disease is unknown. Therefore, we used mice with tissue-specific deletion of CXCR2 to investigate the role of this receptor in Group 3 pulmonary hypertension. Deletion of CXCR2 in myeloid cells attenuated the recruitment of polymorphonuclear MDSCs to the lungs, inhibited vascular remodeling, and protected against pulmonary hypertension. Conversely, loss of CXCR2 in endothelial cells resulted in worsened vascular remodeling, associated with increased MDSC migratory capacity attributable to increased ligand availability, consistent with analyzed patient sample data. Taken together, these data suggest that CXCR2 regulates MDSC activation, informing potential therapeutic application of MDSC-targeted treatments.

Targeting the site encoded by SERPINA1*E342K for treating alpha-1 antitrypsin deficiency-associated liver diseases.

Alpha1-antitrypsin (AAT) deficiency predisposes individuals to emphysema and liver diseases such as cirrhosis and hepatocellular carcinoma. The deficiency results from mutations in the SERPIN1A gene encoding AAT molecules that cause hepatotoxic retention within the endoplasmic reticulum. Since the E342K mutation is the basis for destabilization leading to lung and liver pathologies, we used the crystal structure of the mutated AAT as the basis for molecular docking selection of candidate compounds that may bind and stabilize the 342K structural pocket. We identified compounds that inhibited intracellular accumulation of AAT in hepatocytes in vitro. These data suggest that drug binding to a structural site encoded by a mutation associated with AAT deficiency has the potential for clinical utility by modulating conformational transitions.

Analysis of the MILES cohort reveals determinants of disease progression and treatment response in lymphangioleiomyomatosis.

INTRODUCTION: The Multicenter International Lymphangioleiomyomatosis (LAM) Efficacy of Sirolimus (MILES) trial revealed that sirolimus stabilised lung function in patients with moderately severe LAM. The purpose of this study was to further examine the MILES cohort for the effects of racial, demographic, clinical and physiological patient characteristics on disease progression and treatment response in LAM. METHODS: MILES subjects were stratified on the basis of menopausal status (pre-menopausal/post-menopausal), race (Asian/Caucasian), bronchodilator responsiveness (present/absent), initial forced expiratory volume in 1 s (FEV1; 51-70% versus ≤50% predicted) and tuberous sclerosis complex (TSC) association (yes/no). A linear mixed effects model was used to compare slope differences, and nonparametric tests were used to compare medians and proportions between treatment groups in each stratum. RESULTS: In the MILES placebo group, pre-menopausal patients declined 5-fold faster than post-menopausal patients (mean±se FEV1 slope -17±3 versus -3±3 mL·month-1; p=0.003). Upon treatment with sirolimus, both the pre-menopausal (-17±3 versus -1±2 mL·month-1; p<0.0001) and post-menopausal patients (-3±3 versus 6±3 mL·month-1; p=0.04) exhibited a beneficial response in mean±se FEV1 slope compared with the placebo group. Race, LAM subtype, bronchodilator responsiveness or baseline FEV1 did not impact the rate of disease progression in the placebo group or treatment response in the sirolimus group. Menopausal status and race had differential effects on the adverse event profile of sirolimus. Baseline serum vascular endothelial growth factor (VEGF)-D >600 pg·mL-1 identified subgroups of patients who were more likely to decline on placebo and respond to treatment with sirolimus. CONCLUSIONS: In LAM patients, treatment with sirolimus is beneficial regardless of menopausal status, race, bronchodilator responsiveness, baseline FEV1 or TSC association. Serum VEGF-D and menopausal status can help inform therapeutic decisions.

Alpha-1 Antitrypsin Deficiency Liver Disease, Mutational Homogeneity Modulated by Epigenetic Heterogeneity With Links to Obesity.

Alpha-1 antitrypsin deficiency (AATD) liver disease is characterized by marked heterogeneity in presentation and progression, despite a common underlying gene mutation, strongly suggesting the involvement of other genetic and/or epigenetic modifiers. Variation in clinical phenotype has added to the challenge of detection, diagnosis, and testing of new therapies in patients with AATD. We examined the contribution of DNA methylation (5-methylcytosine [5mC]) to AATD liver disease heterogeneity because 5mC responds to environmental and genetic cues and its deregulation is a major driver of liver disease. Using liver biopsies from adults with early-stage AATD and the ZZ genotype, genome-wide 5mC patterns were interrogated. We compared DNA methylation among patients with early AATD, and among patients with normal liver, cirrhosis, and hepatocellular carcinoma derived from multiple etiologic exposures, and linked patient clinical/demographic features. Global analysis revealed significant genomic hypomethylation in AATD liver-impacting genes related to liver cancer, cell cycle, and fibrosis, as well as key regulatory molecules influencing growth, migration, and immune function. Further analysis indicated that 5mC changes are localized, with hypermethylation occurring within a background of genome-wide 5mC loss and with patients with AATD manifesting distinct epigenetic landscapes despite their mutational homogeneity. By integrating clinical data with 5mC landscapes, we observed that CpGs differentially methylated among patients with AATD disease are linked to hallmark clinical features of AATD (e.g., hepatocyte degeneration and polymer accumulation) and further reveal links to well-known sex-specific effects of liver disease progression. Conclusion: Our data reveal molecular epigenetic signatures within this mutationally homogeneous group that point to ways to stratify patients for liver disease risk.

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.