The new england journal of medicine

Review Article

Dan L. Longo, M.D., Editor

Alpha1-Antitrypsin Deficiency Pavel Strnad, M.D., Noel G. McElvaney, D.Sc., and David A. Lomas, Sc.D.​​

lpha1-antitrypsin (AAT) deficiency is one of the most common From the Department of Internal Med­ genetic diseases. Most persons carry two copies of the wild-type M allele icine III, University Hospital RWTH of SERPINA1, which encodes AAT, and have normal circulating levels of the ­(Rheinisch–Westfälisch Technische Hoch­ A schule) Aachen, Aachen, Germany (P.S.); protein. Ninety-five percent of severe cases of AAT deficiency result from the homo- the Irish Centre for Genetic Lung Dis­ zygous substitution of a single amino acid, Glu342Lys (the Z allele), which is present ease, Royal College of Surgeons in Ire­ in 1 in 25 persons of European descent (1 in 2000 persons of European descent land, Beaumont Hospital, Dublin (N.G.M.); and UCL Respiratory, Division of Medi­ are homozygotes). Mild AAT deficiency typically results from a different amino cine, Rayne Institute, University College acid replacement, Glu264Val (the S allele), which is found in 1 in 4 persons in the London, London (D.A.L.). Address re­ Iberian peninsula. However, many other alleles have been described that have vari- print requests to Dr. Lomas at UCL Re­ spiratory, Rayne Institute, University Col­ able effects, such as a lack of protein production (null alleles), production of mis- lege London, London WC1E 6JF, United folded protein, or no effect on the level or function of circulating AAT (Table 1). Kingdom, or at ­d​.­lomas@​­ucl​.­ac​.­uk. AAT is synthesized in the liver and secreted into the circulation, where its pri- N Engl J Med 2020;382:1443-55. mary role is to protect lung tissue against attack by the enzyme neutrophil elas- DOI: 10.1056/NEJMra1910234 tase. Point mutations can lead to retention of AAT in the liver, causing liver disease Copyright © 2020 Massachusetts Medical Society. through a toxic “gain of function,” whereas the lack of an important circulating proteinase inhibitor predisposes homozygotes with severe deficiency to early-onset emphysema (“loss of function”). The high frequency of genetic variants suggests that AAT mutations confer a selective advantage, perhaps by amplifying the in- flammatory response to invasive respiratory and gastrointestinal infection.2 We review the current understanding of the pathophysiology of AAT deficiency and discuss how this knowledge has led to new therapeutic strategies.

Pathophysiology and Clinical Features of Liver Disease

AAT is synthesized within hepatocytes, intestinal and pulmonary alveolar cells, neutrophils, macrophages, and the cornea. The liver produces approximately 34 mg of AAT per kilogram of body weight per day, leading to a plasma level of 0.9 to 1.75 mg per milliliter, with a half-life of 3 to 5 days. AAT levels can rise by 100% in persons with a normal proteinase inhibitor (PI) genotype (MM) during the acute-phase response, but the increase is markedly attenuated in persons with severe deficiency alleles. AAT is synthesized within the endoplasmic reticulum and secreted through the Golgi apparatus. Severe deficiency alleles — such as the com- mon Z (Glu342Lys) allele and the rare Siiyama (Ser53Phe), Mmalton (∆Phe52), and King’s (His334Asp) alleles — do not affect synthesis but cause approximately 70% of the mutant AAT to be degraded in the endoplasmic reticulum within hepatocytes,3 15% to be secreted, and 15% to form ordered polymers.4,5 These polymers are partially degraded by autophagy6,7 and a small amount is secreted,8,9 but a propor- tion persists within the endoplasmic reticulum as inclusions that are positive on periodic acid–Schiff staining and resistant to diastase; such inclusions are the

n engl j med 382;15 nejm.org April 9, 2020 1443 The new england journal of medicine Case report Case Case report Case report Case Case report Case Case report Case Case report Case Epidemiology gotes seen sporadically in the United Kingdom and Kingdom United the in sporadically seen Canada Japan in the United States United the in quency: 1 in 5 persons in southern Europe, 1 Europe, southern in persons 5 in 1 quency: per ­ among 23 in 1 States, United the in 30 in in 1 Australia, in descent European of sons New in descent European of those among 26 Africa, Asia, in nonexistent or rare Zealand; Australians Aboriginal and Disease reported only in compound heterozy ­ compound in only reported Disease Most common rare deficiency allele in Sardinia; in allele deficiency rare common Most Rare, but most common deficiency allele in allele deficiency common most but Rare, Caribbean person Afro - Caribbean an in described Unusual; Most common deficiency variant; carrier fre ­ carrier variant; deficiency common Most Clinical Features emphysema in homozygote or com ­ or homozygote in emphysema heterozygote Z pound may confer a predisposition to em ­ to predisposition a confer may physema ease and emphysema in homozygotes in emphysema and ease zygotes but less severe than in ZZ; cirrhosis ZZ; in than severe less but heterozygotes SZ in reported Neonatal jaundice; presumed high risk of risk high presumed jaundice; Neonatal No clear disease association disease clear No known Not Coinheritance with the Z deficiency allele deficiency Z the with Coinheritance established association with liver dis ­ liver with association Well - established Liver disease and emphysema in homo ­ in emphysema and disease Liver Emphysema in homozygotes in Emphysema Emphysema seen in SZ heterozygotes SZ in seen Emphysema Compound heterozygote with Z with heterozygote Compound Fatal bleeding disorder bleeding Fatal High risk of emphysema in homozygotes in emphysema of risk High Molecular Basis of Disease endoplasmic reticulum; delayed reticulum; endoplasmic secretion polymers; reduced serum protein serum reduced polymers; deficient) functionally but neutrophil elastase; slow formation slow elastase; neutrophil polymers of ization; low serum level serum ization; low ization; low serum level serum low ization; thesis but no polymerization; low polymerization; no but thesis level serum tion; can form heteropolymers with heteropolymers form can tion; AAT Z intracellular degradation; reduced degradation; intracellular circulating of activity catalytic protein Rapid polymerization in hepatocyte in polymerization Rapid Protein misfolding; can form hetero ­ form can misfolding; Protein secretion normal (i.e., mutant II Type Reduced association rate constant with constant rate association Reduced Intracellular degradation and polymer ­ and degradation Intracellular Intracellular degradation and polymer ­ and degradation Intracellular Abnormal posttranslational biosyn ­ posttranslational Abnormal Protein misfolding and reduced secre ­ reduced and Protein misfolding Polymer formation Function altered to an antithrombin an to altered Function Unstable protein structure leading to leading structure protein Unstable -Antitrypsin (AAT) Deficiency.* (AAT) -Antitrypsin 1

rs17580 Mutation Gly67Glu Arg39Cys Ser53Phe Leu41Pro Glu264Val Gly349Arg Arg223Cys Lys154Asn Met358Arg rs28931570 rs55819880 rs28931568 rs28931569 Hisp334Asp rs121912713 rs775982338 and rs Number‡ Δ52Phe (M2 variant) (M2 Δ52Phe malton mineral springs procida iiyama M I Iners King’s Deficiency alleles Deficiency F M Variant† M S S Queen’s Pittsburgh Key Alleles Associated with Alpha with Associated Alleles Key 1. Table

1444 n engl j med 382;15 nejm.org April 9, 2020 Alpha1-Antitrypsin Deficiency Case report Case Case report Case Case report Case report Case Epidemiology rier frequency: 1 in 27 persons in northern in 27 persons in 1 frequency: rier 75 in 1 States, United the in 83 in 1 Europe, 1 Australia, in descent European persons of New in descent European of persons 46 in Korea, China, Japan, in seen not Zealand; Africa and western northern or Malaysia, Most common severe deficiency variant; car ­ variant; deficiency severe common Most Case reports (persons of Chinese descent) Chinese of (persons reports Case Clinical Features ciation with liver disease and emphy ­ and disease liver with ciation more be may heterozygotes MZ sema; and obstruction airflow to susceptible disease liver chronic with Z with and compound heterozygotes compound and and compound heterozygotes compound and pound heterozygote pound heterozygotes compound and established asso ­ well - established homozygotes, In Emphysema in compound heterozygote compound in Emphysema High risk of emphysema in homozygotes in emphysema of risk High High risk of emphysema in homozygotes in emphysema of risk High Severe emphysema reported in Z com ­ Z in reported emphysema Severe homozygotes in emphysema of risk High Molecular Basis of Disease ization; low serum level serum low ization; dation and no secreted protein secreted no and dation gation (no polymerization), degra ­ polymerization), (no gation protein secreted no and dation, Intracellular degradation and polymer ­ and degradation Intracellular Nonclassical polymer formation polymer Nonclassical No detectable AAT mRNA AAT detectable No Truncated protein; intracellular degra ­ intracellular protein; Truncated No detectable AAT mRNA AAT detectable No aggre ­ intracellular protein; Truncated , and King’s variants; slower polymer formation of S, I, Queen’s AAT; a change in the inhibitory activity Pittsburgh AAT ; secre ­ malton , M

AAT deficiency alleles with different functional effects are listed. The delta symbol denotes deletion, and mRNA messenger RNA . 1 iiyama Glu75Val Mutation Glu342Lys rs28929474 rs199422211 and rs Number‡ Lys217 stop codon stop Lys217 codon at 373 at codon codon 334 at codon Δ1bpPro362, causing stop causing Δ1bpPro362, stop causing Δ1bpTyr160, rs267606950 stop causing Δ2bpLeu318, rs1057519610 bellingham bolton granitefalls hongkong Z alleles (QO) Null QO Trento QO QO QO Variant† The information is from Lomas et al. tion of an inactive protein, Iners AAT; and some the many null variants that result in no secreted protein. The rs number is the reference SNP cluster identification number. The single or initial letter in each name reflects the position of migration variant on isoelectric - focusing gels (e.g., A indicates fastest migration, M moderate and Z the slowest migration); rest of name represents site first description (in case Iners, is un known). Deficiency results from a range effects on pro ­ tein: rapid polymerization of the Z, S *  ‡  † 

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“Gain of function” Neonatal hepatitis or cirrhosis

Proteotoxic stress

↓Circulating AAT

PAS-D–positive inclusions

Misfolded AAT Polymerization

↓AAT function

“Loss of function” Emphysema Macrophage Neutrophil • Cysteinyl TNF cathepsins Up-regulation TNF- receptor Proteolytic • MMP 1 or 2 lung damage Activation NE NE NE NE • Lactoferrin • MMP BLT1 Neutrophil • hCAP-18 elastase LTB CXCR1 4 Inhibition NE by AAT Lactoferrin ROS Autoantibody AIRWAY TLR TACE EGFR MUCUS

EPITHELIUM Panlobular Interleukin-8 emphysema

CXCR1 BLOOD VESSEL

histologic hallmark of the disease (Fig. 1). deficiency1 (Table 1). The rate at which most Milder deficiency alleles, such as the S (Glu- AAT mutants form polymers correlates directly 264Val), I (Arg39Cys), and Queen’s (Lys154Asn) with alterations in the protein structure, as indi- alleles, also form polymers of AAT but at a much cated by altered thermal stability.10 slower rate, in keeping with only a mild plasma The key longitudinal, population-based co-

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with PI ZZ AAT deficiency, and a large European Figure 1 (facing page). Pathophysiology of Alpha1-­ Antitrypsin (AAT) Deficiency. analysis that relied on a noninvasive assessment Misfolded AAT forms ordered polymers that accumu­ showed clinically significant liver fibrosis in 20 to late as hepatocyte inclusions, which are positive on 36% of such persons.15,16 Risk factors for ad- ­periodic acid–Schiff staining with diastase digestion vanced fibrosis include male sex, the metabolic (PAS-D). This misfolded protein causes proteotoxic syndrome, and obesity. The exact effect of alco- stress and a gain-of-function liver disease. A deficiency of AAT results in an excess of neutrophil elastase (NE), hol consumption in persons with the PI ZZ geno- which in turn induces mucin production and secretion type remains to be determined, but the available and increases expression of other proteases and inflam­ evidence suggests that it promotes disease pro- matory cytokines. AAT is not just an antiprotease but gression. Handling of the Z variant of AAT is also a potent antiinflammatory agent, regulating neu­ delayed in hepatocytes derived from induced trophil chemotaxis, degranulation, autoimmunity, and apoptosis through interactions with interleukin-8, leuko­ pluripotent stem cells from persons who have 17 triene B4 (LTB4), and tumor necrosis factor α (TNF-α). liver disease associated with AAT deficiency. AAT is inactivated by oxidation, proteolytic cleavage, Whether this finding, along with DNA methyla- and polymerization. Thus, with a deficiency of AAT, tion marks,18 can be used to identify persons at neutrophils are increased and protease activity is un­ risk for the development of liver disease is not opposed. Structural damage and susceptibility to infec­ tion occur, leading to tissue damage and emphysema. known. BLT1 denotes LTB4 receptor 1, CXCR1 C-X-C motif che­ mokine receptor 1, EGFR epidermal growth factor re­ ceptor, hCAP-18 human cathelicidin antimicrobial pep­ Pathophysiology and Clinical tide 18, MMP matrix metalloproteinase, ROS reactive Features of Lung Disease oxygen species, TACE TNF-α converting enzyme, and TLR toll-like receptor. AAT was first described as a serine protease in- hibitor, and it is the loss of this activity in the lung that has informed our understanding of the hort study of AAT deficiency followed 127 per- pathophysiology of the associated lung disease sons with the PI ZZ genotype and 54 with the PI (Fig. 1). AAT inhibits neutrophil elastase, which, SZ genotype from birth to 45 years of age.11,12 A when unopposed, can cleave many of the struc- total of 73% of infants with the PI ZZ genotype tural proteins of the lung as well as innate im- for AAT deficiency had an elevated serum ala- mune proteins. Airspace enlargement ensues nine aminotransferase level in the first 12 months when elastase is instilled into the lung in animal of life, but the level was abnormal in only 15% models, and neutrophil elastase–knockout mice by 12 years of age. The serum bilirubin level was are protected from emphysema that is induced by elevated in 11% of such infants in the first few exposure to cigarette smoke.19 In the classic months of life but was normal by 6 months of loss-of-function concept, the Z form of AAT fails age. Cholestatic jaundice developed in 10% of to reach the lung in sufficient quantities and infants with the PI ZZ genotype, and clinical takes longer to inhibit neutrophil elastase.20 features of liver disease without jaundice devel- Oxidants in cigarette smoke can further incapaci- oped in 6%. The clinical symptoms usually re- tate Z AAT by oxidizing its active-site methio- solved by the second year of life, but 15% of nines and increasing polymerization,21 both of persons with cholestatic jaundice had progres- which render it unable to inhibit neutrophil sion to juvenile cirrhosis. The risk of death from elastase. This illustrates the pivotal role of liver disease among children with the PI ZZ smoking in AAT deficiency, which contributes to genotype was 2 to 3%, but none of the survivors earlier and more severe lung disease. had clinical symptoms of liver disease at 12 Other lung proteases, such as proteinase-3, years of age.13 Persons in the cohort study who metalloproteases, cysteine proteases, and bacte- had the PI ZZ genotype and had a history of to- rial proteases, have proteolytic activity that is bacco smoking had hyperinflation and emphy- similar to the activity of neutrophil elastase and sema at 37 to 39 years of age, whereas age- may also play a role.22 Some, such as cysteinyl matched adults with the PI MM genotype who proteases and metalloproteases, are induced and had never smoked did not have evidence of hy- activated by neutrophil elastase, and inhibition perinflation and emphysema.14 of neutrophil elastase decreases their activity in A cross-sectional biopsy study showed clini- vivo. AAT also binds a variety of proteins and cally significant liver fibrosis in 35% of adults fatty acids,23 and loss or moderation of these

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functions as a result of AAT deficiency may in- reported in persons with the PI ZZ genotype. crease inflammation. In persons with AAT defi- Among them, neutrophilic panniculitis is char- ciency, AAT is inactivated in the lung not just by acterized by painful subcutaneous nodules and oxidation but also by proteolytic cleavage and the presence of neutrophil infiltrates in the sub- polymerization.24 Moreover, polymerized Z AAT cutis; it occurs in less than 1% of persons with has a distinct proinflammatory effect, acting as the PI ZZ genotype. Disorders that are overrep- a potent neutrophil chemoattractant.24 Thus, in resented in persons with AAT deficiency include persons with AAT deficiency, the neutrophil antineutrophil cytoplasmic antibody (ANCA)– number is increased, protease activity is unop- associated vasculitis, chronic kidney disease, posed, and susceptibility to infection and struc- diabetes,30 and metabolic alterations, with de- tural damage develop (Fig. 1). creased levels of serum triglycerides and very-low- The clinical manifestations of lung disease density lipoproteins.16 associated with AAT deficiency are mainly indis- tinguishable from those of nonhereditary em- Risk of Disease among physema. This is partly why severe AAT defi- Heterozygotes ciency remains undiagnosed in approximately 90% of cases, with an interval of 5 to 7 years The PI MZ genotype (i.e., heterozygous Z car- from the onset of symptoms to diagnosis. The riage) is a common form of AAT deficiency, oc- classic clinical description of lung disease asso- curring in up to 4% of the population with Euro- ciated with AAT deficiency is of early-onset ob- pean ancestry.31 These persons are susceptible to structive lung disease in persons with moderate multiple disorders (Table 2), partly due to lower cigarette consumption and panacinar emphyse- levels of circulating AAT but also because the ma affecting mainly the lower lobes. Rigid ad- secreted Z AAT is less effective at inhibiting herence to these indicators to prompt testing neutrophil elastase. has led to underdiagnosis, late diagnosis, and Although population-based studies have failed misdiagnosis of AAT deficiency. Up to 37% of to show an association between PI MZ status people with severe AAT deficiency have predomi- and COPD or have shown only a weak associa- nantly upper-lobe involvement,25 with bronchiec- tion with emphysema,34 studies enriched for per- tasis as a common radiologic manifestation. sons at risk have shown that PI MZ carriers have Even when this diagnostic algorithm is used for a clear predisposition to COPD, at least among young people with chronic obstructive pulmo- smokers.35,36 These data suggest that PI MZ car- nary disease (COPD), AAT deficiency is often di- riers have a low absolute risk for the develop- agnosed late, at a point when the lung disease ment of a clinically relevant lung disease but that has become irreversible.26,27 In the National Heart, the risk increases significantly with additional Lung, and Blood Institute registry for severe genetic and environmental factors. This risk pat- AAT deficiency, the average age at diagnosis was tern is similar to that described for pediatric 46 years, but the mean forced expiratory volume liver disease, which is rarely seen in PI MZ car- 40,41 in 1 second (FEV1) and diffusing capacity of the riers without coexisting conditions. The ab- lung for carbon monoxide (DLco) were 47% and solute risk of liver disease among adult PI MZ 50% of the predicted value, respectively.28 Early carriers is unknown, but the risk is increased by diagnosis is important in helping people make the presence of additional coexisting conditions, lifestyle changes, reducing occupational risk, and such as nonalcoholic fatty liver disease, alcohol providing access to new therapies, whereas a misuse, and cystic fibrosis.37-39 Indeed, in a re- delayed diagnosis is associated with poor func- cent genomewide association study, the Z allele tional status and COPD-related outcomes.29 was associated with the highest odds ratio for the development of cirrhosis associated with alco- 38 AAT Deficiency as a Systemic holic or nonalcoholic liver disease. Disorder PI MZ carriers have an increased incidence of gallstone disease34 and an increased susceptibil- Although lung disease and liver disease are the ity to immune disorders such as ANCA-associated most prominent disorders associated with AAT vasculitis.32 Particularly high odds were reported deficiency, several other conditions have been for disorders associated with myeloperoxidase-

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Table 2. Overview of Clinical Conditions Associated with AAT Deficiency.*

Disease Odds Ratio (95% CI) Study

PI MZ PI ZZ ANCA-associated vasculitis 2.9 (2.2–3.9)† ND Merkel et al.,32 Rahmattulla et al.33 Gallstone disease 1.3 (1.3–1.4) 1.3 (0.7–2.5) Ferkingstad et al.34 Emphysema (population-based studies) 1.4 (1.2–1.7) 28 (18–44) Ferkingstad et al.34 COPD (population-based studies)‡ 1–3 4.8 (3.0–7.9) Ferkingstad et al.,34 Foreman et al.35 COPD (case–control studies)‡ 3–10 ND Molloy et al.36 CFLD 5.0 (2.9–8.8) ND Bartlett et al.37 NAFLD cirrhosis 3–7 ND Abul-Husn et al.38 Alcoholic liver cirrhosis 3.4–6 ND Strnad et al.39 Advanced liver fibrosis (general population) ND 9–20 Hamesch et al.16

* ANCA denotes antineutrophil cytoplasmic antibody, CFLD cystic fibrosis–associated liver disease with portal hyperten­ sion, CI confidence interval, COPD chronic obstructive pulmonary disease, NAFLD nonalcoholic fatty liver disease, ND not determined, PI MZ proteinase inhibitor genotype MZ, and PI ZZ proteinase inhibitor genotype ZZ. † Higher odds ratios have been reported for vasculitis associated with proteinase 3–reactive ANCA with cytoplasmic staining (c-ANCA) and vasculitis associated with myeloperoxidase-reactive ANCA with perinuclear staining (p-ANCA). ‡ Higher odds ratios were reported for current and former smokers. reactive ANCA with perinuclear staining (p-ANCA) also to test specific patient groups for AAT defi- and those associated with proteinase 3–reactive ciency. All persons with COPD, liver disease, ANCA with cytoplasmic staining (c-ANCA). ANCA- poorly responsive asthma, c-ANCA vasculitis (in associated vasculitis can occur with other AAT >90% of cases, the antibody is specific for pro- variants such as the S allele33 and may represent teinase 3), panniculitis, or bronchiectasis, in a loss-of-function phenotype, since proteinase 3 addition to first-degree relatives of people with is a target protease for AAT. Although the mech- AAT deficiency, should be tested.26,27 anisms leading to lung or liver disease in PI MZ The first step in testing is measurement of carriers are probably analogous to those identi- the AAT level in serum. This measurement should fied in PI ZZ carriers, the factors leading to the be accompanied by an assessment of C-reactive increased incidence of gallstones in PI MZ carriers protein, since AAT is an acute-phase reactant remain to be determined. The compound hetero- that increases during infection or inflammation. zygous PI SZ genotype is more common than PI A serum level higher than or equal to 1.1 g per ZZ and is characterized by AAT serum levels that liter in the presence of a normal C-reactive pro- are intermediate between those associated with tein level can be taken as evidence of normal the PI MZ and PI ZZ genotypes. In keeping with AAT status.43 If the serum AAT level is less than this observation, lung disease is less likely to 1.1 g per liter, or if there is a strong clinical develop in PI SZ carriers than in PI ZZ homozy- concern, then the clinician should request either gotes,42 but as with MZ carriers, smoking sig- phenotyping or genotyping in a specialist labo- nificantly increases the risk of COPD. Children ratory. In inconclusive cases, gene sequencing who are PI SZ carriers rarely have clinically rele- should be performed. Patients should be referred vant liver disease.40,41 Liver disease in adult PI SZ to a center specializing in AAT deficiency for heterozygotes has been reported in small studies follow-up.27 Some guidelines suggest simultane- and remains to be systematically assessed. ous testing of AAT levels and genotyping.44 Smoking cessation is central for all forms of Diagnosis AAT deficiency. People with severe AAT defi- ciency (the PI ZZ, ZNull, or NullNull genotype) Lung Disease should be monitored with spirometry, DLco, the AAT deficiency is underdiagnosed, and it is im- 6-minute walk test, and health-related quality- portant not merely to consider the condition but of-life questionnaires.27 The frequency of moni-

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toring depends on the degree of impairment, but range × 100) ÷ the platelet count, and the Fibro- monitoring every 6 months for the first few years sis-4 (FIB-4) score, calculated as the patient’s years is helpful for establishing a baseline and identi- of age × AST × ALT−0.5 ÷ the platelet count (in which fying signs of rapid decline, with a change to ALT denotes alanine aminotransferase).15 For once-a-year evaluations thereafter. The role of both equations, platelet count is measured at 109 computed tomographic (CT) assessment of lung per liter, and aminotransferase in units per liter. density in monitoring remains uncertain because The γ-glutamyltransferase level performs better of a lack of correlation with other surrogate as a noninvasive marker than the APRI, the FIB-4 markers that are used more routinely in clinical score, or aminotransferase measurements. The trials, such as lung function or quality of life.45 use of these biomarkers in liver disease has been Follow-up for patients with the PI MZ geno- reviewed recently,49 but cutoff values remain to type is more contentious. MZ and SZ heterozy- be defined for liver disease associated with AAT gotes who do not smoke have no increased risk deficiency. Yearly liver ultrasound screening has of lung disease, but those who do smoke have a been proposed, with scans every 6 months to significantly increased risk, as compared with screen for hepatocellular carcinoma in patients relatives who are PI MM carriers.34,36 Patients with cirrhosis, portal hypertension, or persis- with established COPD should be followed ac- tently abnormal liver-function tests. cording to standard protocols, but it is unclear whether they require closer follow-up, since it is Treatment uncertain whether the decline in lung function is accelerated after smoking cessation, as com- Treatment for the lung disease associated with pared with lung function in PI MM carriers with AAT deficiency is the same as treatment for COPD. More rare AAT variants, such as the F COPD. The only licensed disease-specific ther­

(Arg223Cys), I (Arg39Cys), and Mmalton (Δ52Phe) apy for AAT deficiency is intravenous augmenta- alleles, are reported to be associated with in- tion therapy with plasma-purified AAT. This creased susceptibility to COPD only when inher- therapy was approved by the Food and Drug ited with a Z or null allele. Null homozygotes Administration in 1987 for lung disease associ- lack circulating AAT and have more severe lung ated with AAT deficiency on the basis of bio- disease than PI ZZ or PI SZ carriers but do not chemical efficacy and pharmacokinetics, without have an increased risk of liver disease.27 Even proof of clinical efficacy. Randomized, con- with definitive identification of an AAT defi- trolled trials have focused on decreased loss of ciency genotype, substantial variation is noted in lung density as the primary efficacy outcome. the phenotypic presentation of the disease. The The largest of these studies strongly suggested a most important differentiator is cigarette smok- decreased loss of lung density with augmenta- ing, but occupational exposures, such as expo- tion therapy in people with AAT deficiency but

sure to kerosene or dust, can also play a role in no effects on other measures, such as FEV1, the phenotypic presentation.46 A number of poly- quality of life, or exacerbation of COPD.45,50 morphisms have been identified in lung and The liver disease associated with the accumu- liver disease associated with AAT deficiency,47,48 lation of mutant AAT within hepatocytes is ex- but their importance has yet to be fully defined. acerbated by secondary factors that also affect hepatic function, such as fat and alcohol. It is Liver Disease recommended that persons with AAT deficiency All PI ZZ carriers should be monitored for liver and normal liver function maintain a normal disease at a center that specializes in AAT defi- body-mass index and consume alcohol within ciency. Given its invasive nature, liver biopsy is recommended limits. Persons with advanced not acceptable for follow-up of asymptomatic liver disease associated with AAT deficiency patients. Transient elastography is useful to rule should abstain from alcohol. No therapy is cur- out advanced fibrosis (stage F3 or F4) but is less rently approved for liver disease associated with effective at lower levels of fibrosis, for which it AAT deficiency other than transplantation for performs similarly to the aspartate aminotrans- persons with advanced disease. ferase (AST)–to-platelet ratio index (APRI), cal- An understanding of the molecular and struc- culated as (AST ÷ the upper limit of the normal tural basis for the disease has underpinned new

1450 n engl j med 382;15 nejm.org April 9, 2020 Alpha1-Antitrypsin Deficiency approaches that may come to fruition in the next far been unsuccessful.60 All these approaches 5 to 10 years (Fig. 2). Some of these approaches require detailed evaluation before they can be are at the preclinical stage of investigation, and introduced into clinical practice. others are in early-phase clinical trials. The under- lying genetic defect may be corrected by means Future Directions for Research of CRISPR (clustered regularly interspaced short palindromic repeats), stem-cell technology,52 or The antiinflammatory and tissue-protective prop- replacement hepatocytes,53 and the production of erties of AAT are underscored by several studies mutant protein may be “switched off” by means suggesting its usefulness in transplantation med- of gene silencing.54 The polymerization interme- icine (Fig. 3). AAT can ameliorate experimental- diates may be stabilized with small molecules55 ly induced kidney and lung ischemia–reperfusion or intrabodies (antibodies expressed inside a cell injury61,62 and augment the function of murine to alter its function),56 intracellular polymers and porcine lung transplants.63 Treatment with may be cleared by stimulating autophagy with AAT has been shown to have an antiinflamma- the use of sirolimus and carbamazepine,57,58 and tory effect on insulin-sensitive tissues and was secretion may be enhanced by manipulating pro- beneficial in experimental islet-transplantation teostasis networks.59 Attempts to read through models, reducing islet-cell loss and inducing im- premature stop codons of null variants have so mune tolerance.64 These data prompted phase 1

M

Z

DNA Protein precursor M P

Linkage between reactive center Linkage between loop and C-terminal triple- strand motif Reduce production of the Use intra-ER chaperones to β-sheet A Stimulate the autophagy pathway mutant protein with siRNA, promote folding or Block the formation of polymers by to clear formed polymers (e.g., correct the genetic defect with degradation (e.g., treatment stabilizing the intermediate in the treatment with carbamazepine, gene or cell therapy with small molecules or folding pathway or the monomer sirolimus, or overexpression of the proteostasis modulators) (e.g., treatment with small molecules autophagy regulator transcription or intrabodies) factor EB)

Figure 2. Strategies for Treating AAT Deficiency. Mature wild­type AAT (M) folds through an intermediate (M*) and is then secreted through the Golgi apparatus. The Z mutation allows the intermediate to form polymers, which accumulate within the cell. Current evidence supports two pathological β­strand linkages: be­ tween the reactive center loop and β­sheet A (left)4 and through a C­terminal triple­strand motif (right).51 Strategies for treating AAT de­ ficiency address different steps in this pathway: reducing production of the mutant protein with small interfering RNA (siRNA) and cor­ recting the genetic defect with gene or cell therapy, using chaperones within the endoplasmic reticulum (ER) to promote folding or degradation (e.g., treatment with small molecules or proteostasis modulators), blocking the formation of polymers by stabilizing the in­ termediate in the folding pathway or the monomer (e.g., treatment with small molecules or intrabodies), and stimulating the autophagy pathway to clear formed polymers (P) (e.g., treatment with carbamazepine, sirolimus, or overexpression of the autophagy regulator tran­ scription factor EB).

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tration of AAT increased the ratio of regulatory T cells to effector T cells, thereby mimicking the GVHD Type 1 diabetes findings in experimental models. However, fur- ↑Treg cells ↓Inflammation ther trials are needed to assess the efficacy of ↓Inflammation ↑Immunotolerance AAT in humans. ↑Survival ↑Survival End Points for Phase 2 Trials The first approval for AAT augmentation therapy AAT was based purely on biochemical efficacy and pharmacokinetics.69 Early studies of a new drug involve single ascending doses, followed by mul- tiple ascending doses, an approach that addresses Experimental safety and tolerability but may also reveal a sig- Clinical trials nal for efficacy and the dose at which this can Lung Kidney be achieved. The results of large phase 3 studies suggest that it is unlikely that shorter phase 2 studies will show robust changes in lung density Transplantation co on CT scans, FEV1, DL , pulmonary exacerba- 50 ↓Oxidative stress tions, or quality of life. As a minimum, these ↓Reperfusion injury phase 2 studies should generate data similar to ↑Survival the original study reported by Wewers et al.,69 which showed increased levels of AAT in blood and lung epithelial-lining fluid, above a protec- Figure 3. AAT in Immunology and Transplantation. tive threshold, along with an increased capacity AAT can ameliorate experimentally induced kidney and lung ischemia– for neutrophil elastase inhibition in lung epithe- reperfusion injury and augment the function of murine and porcine lung lial-lining fluid. The observed downstream anti- transplants. Treatment with AAT has an antiinflammatory effect on insulin­ inflammatory effects of inhibiting neutrophil sensitive tissues and may have a role in the treatment of type 1 diabetes. It elastase, such as normalizing neutrophil chemo- also induces immune tolerance and increases regulatory T (Treg) cells and may therefore be effective in the management of graft­versus­host disease taxis and degranulation and protecting innate (GVHD). immune proteins from proteolytic inactivation, are also desirable (Fig. 1). Surrogate markers of efficacy might provide early indications of treat- ment response. These markers include desmosine and 2 clinical trials that showed the safety and and isodesmosine, unique cross-linkers of ma- side-effects profile of AAT administration in ture elastin fibers that can be measured in plas- children with type 1 diabetes.65 ma, bronchoalveolar-lavage (BAL) fluid, or urine,70 Treatment with AAT induces immune toler- and Aα-Val360, a specific marker of neutrophil ance and increases regulatory T cells.66 This elastase activity in plasma.70 process is of particular relevance in graft-versus- These markers may also be effective for evalu- host disease (GVHD), a potentially lethal conse- ating the effects of non–AAT neutrophil elastase quence of allogeneic hematopoietic stem-cell inhibitors, which can increase elastase neutral- transplantation. In keeping with the data from izing capacity in blood and epithelial-lining islet transplantation, AAT administration de- fluid, while also showing downstream anti–neu- creased mortality and reduced proinflammatory trophil elastase effects similar to those of AAT. cytokine levels in three different mouse models However, some of the antiinflammatory effects of GVHD.67 Moreover, it enhanced the recovery of AAT augmentation therapy may reflect a of regulatory T cells and decreased the number of wider antiprotease and antiinflammatory profile alloreactive effector T cells. These findings un- not related solely to inhibition of neutrophil elas- derpinned a multicenter clinical study showing tase (Fig. 1). Recent studies have shown an in- the safety of intravenous AAT in patients with flammatory imprint in plasma and BAL fluid glucocorticoid-refractory GVHD.68 The adminis- from persons with AAT deficiency, which is

1452 n engl j med 382;15 nejm.org April 9, 2020 Alpha1-Antitrypsin Deficiency ameliorated by augmentation therapy.70 The anti- small population of persons with well-character- inflammatory effects of therapeutic candidates ized disease, many of whom are already receiv- can be assessed by measuring reductions in in- ing augmentation therapy and would be loath to flammatory markers such as proinflammatory stop such therapy if they were assigned to a cytokines (tumor necrosis factor α, interleu- placebo group.54 kin-17, granulocyte–macrophage colony-stimulat- ing factor, macrophage inflammatory protein 1, AAT Deficiency as a Paradigm and macrophage migration inhibitory factor) and of Conformational Diseases macrophage, lymphocyte, eosinophil, or mast- cell activating cytokines in BAL fluid. A problem AAT is the archetypal member of the serine pro- with biomarkers in lung disease associated with teinase inhibitor, or serpin, superfamily. The AAT deficiency is the requirement for BAL, since process of mutation-driven polymerization in the procedure for obtaining the samples is dif- other members of the family has been shown to ficult to standardize across sites. Attempts to form a group of diseases, the serpinopathies.74 use sputum biomarkers have met with variable Mutants of neuroserpin polymerize in the brain, success.71 AAT can be detected in exhaled-breath causing familial encephalopathy with neuroser- condensates,72 and the response of volatile or- pin inclusion bodies, an autosomal dominant ganic compounds to AAT augmentation has dementia.75 Mutants of antithrombin, C1 inhibi- been analyzed with the use of composite nano- tor, and alpha1-antichymotrypsin are retained as sensor arrays.73 However, these methods remain polymers within hepatocytes, causing a circulat- to be tested in large-scale clinical trials, and ing deficiency associated with thrombosis, angio- newer plasma-based assays of inflammation are edema, and emphysema, respectively.74 The aber- anticipated. rant β-strand linkage that underlies AAT deficiency The biomarkers of choice in early-stage stud- and the serpinopathies has similarities to the ies of liver disease associated with AAT defi- linkages formed in amyloidosis and by the pro- ciency depend on the intervention being as- teins underlying prion disease and Parkinson’s, sessed. If the intervention is aimed at promoting Huntington’s, and Alzheimer’s diseases. Indeed, secretion of Z AAT from the liver, it would be the approaches being developed to treat these expected to increase circulating AAT levels and conditions are similar: the use of small mole- anti–neutrophil elastase capacity in plasma and cules to block the aberrant protein linkages, BAL fluid, decrease desmosine and isodesmosine monoclonal antibodies to stabilize intermediates, in these compartments, and have antiinflamma- and more recently, small interfering RNA ap- tory effects. The liver can be imaged with tran- proaches to silence protein expression. Thus, sient elastography15,16 or magnetic resonance AAT deficiency and the serpinopathies provide a elastography, and the findings correlate well useful model for the protein linkages, the effect with the stage of fibrosis and may change even of mutations, the propagation of polymeric struc- over the short term.49 The role of blood liver- tures, and therapeutic strategies for other con- function tests in phase 2 studies is uncertain, formational diseases. although γ-glutamyl transpeptidase shows some Dr. Strnad reports receiving grant support and lecture fees promise as a noninvasive marker of fibrosis.15 from Grifols and CSL Behring, grant support and advisory board Silencing-RNA approaches would decrease sys- fees from Arrowhead Pharmaceuticals, advisory board fees from Dicerna Pharmaceuticals, and lecture fees from Alnylam Phar- temic and lung AAT levels and antiprotease maceuticals; Dr. McElvaney, receiving advisory board fees from protection; unless accompanied by concomitant Vertex and CSL Behring, grant support and fees for serving as an intravenous augmentation therapy, such ap- adjudicator from Grifols, and grant support from Novo Nordisk; and Dr. Lomas, receiving fees for serving on an award jury for proaches might require systemic and BAL evalu- Grifols, grant support from GlaxoSmithKline, consulting fees ations to make sure that there was no increase and license income from Biomarin, and holding pending pat- in lung inflammation.54 A number of early-phase ent 1906708.1 on small molecules to prevent the polymeriza- tion of antitrypsin, licensed by UCL Business to Biomarin. No studies of AAT deficiency–associated liver dis- other potential conflict of interest relevant to this article was ease evaluate liver biopsy specimens for polymer reported. burden and fibrosis as biomarkers of response, Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. but these invasive tests are a potential barrier to We thank Carolin Victoria Schneider and Sile Kelly for help recruitment. Another obstacle is the relatively with earlier versions of the figures.

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