Update on α₁-antitrypsin deficiency

α₁-Antitrypsin deficiency (AATD) is an inherited metabolic disorder in which mutations in the coding sequence of the SERPINA1 gene, also known as the proteinase inhibitor (Pi) system, prevent secretion of α₁-antitrypsin (α₁-AT) and cause predisposition to pulmonary and liver diseases. In particular, AATD has been associated with the development of chronic obstructive pulmonary disease (COPD), characterised by permanent destruction of the alveolar distal unit of the terminal bronchioles (emphysema) and increased risk for infectious exacerbations [1].

Severe AATD is inherited as an autosomal recessive disorder with codominant expression, as each allele contributes 50% of the total circulating enzyme inhibitor. Severely reduced serum α₁-AT levels occur from the inheritance of two Pi-deficient alleles at the SERPINA1 gene on chromosome 14 (14q32.1), with most cases resulting from homozygous inheritance of the Z allele (p.E366K; giving the genotype known as PiZZ), although more than 100 pathological variants have been so far identified.

Any individuals heterozygous for a pathological variant should not be simply labelled as “healthy carriers”, since the risk of lung diseases in this category can vary largely, according to the gene mutation and environmental exposure.

Pathogenesis of AATD in the lungs

α₁-AT is a 52-kDa glycoprotein mainly synthesised and secreted by hepatocytes into the bloodstream. Nevertheless, lung tissue is the principal target of α₁-AT, since the protein is a serine-proteinase inhibitor and it is crucial in maintaining protease–antiprotease homeostasis in the lungs.

The principal pathophysiological pathway is associated with neutrophil recruitment and the release of serine proteinases, especially neutrophil elastase, which causes collateral tissue damage due to inadequate α₁-AT protection. The role of inhibition that α₁-AT carries out towards neutrophil elastase is well known (figure 1a) [2]. The imbalance of the protease/antiprotease activity in favour of the neutrophil serine proteases can result in a self-perpetuating cycle of inflammation and respiratory tissue damage. Moreover, α₁-AT also inhibits two other serine proteinases, namely cathepsin G and proteinase 3, which are produced by neutrophils and cause lung damage. New findings have emerged on the role of α₁-AT in inhibiting a broader range of proteases, such as metalloproteases and cysteine-aspartic proteases [3]. Furthermore, α₁-AT may have other anti-inflammatory and immunomodulatory effects, including reduction of Toll-like receptor expression, reduction of neutrophil adherence to the endothelium, and reduction of selected proinflammatory cytokines in the lungs [4, 5]. In this picture, it is pretty clear what impact a deficiency or lack of α₁-AT could have on lung tissue protection (figure 1b).

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Figure 1

Roles and functions of α₁-AT in lungs of a) individuals with normal levels of protein, b) patients with deficient or null mutations, and c) patients with deficient and polymerogenic mutations of the SERPINA1 gene. HNE: human neutrophil elastase; ER: endoplasmic reticulum.

Cigarette smoking is an additional risk factor, which accelerates the development of lung pathologies in individuals with AATD, as supported by the pallid mice model [6]. Oxidative modifications of α₁-AT are induced by components of cigarette smoke, as well as by oxidants and enzymes (e.g. myeloperoxidase) released by cells at sites of inflammation. Although oxidative modifications do not abolish the anti-inflammatory effects of α₁-AT [3], the oxidation of the P1 methionine (methionine 358 or methionine 351) to methionine sulfoxide significantly reduces the ability of α₁-AT to inhibit neutrophil elastase released by neutrophils during inflammatory processes in the lungs [7]. The oxidation of methionine in α₁-AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the effective anti-elastase protection in the lungs, but also converts α₁-AT into a proinflammatory mediator; the oxidised α₁-AT, which is generated in the airways, interacts directly with epithelial cells to release chemokines that attract macrophages into the airways [8].

Recent studies have demonstrated that, even in α₁-AT non-deficient individuals, cigarette smoking disables the endothelial pro-survival effect of α₁-AT, which may contribute to chronic lung damage in susceptible individuals [9]. However, the lack of function of α₁-AT is not the only mechanism by which this protein contributes to lung impairment. The enhanced tendency of Z-type α₁-AT to combine in oligomeric assemblies is well documented in hepatocytes and it has been recently demonstrated in bronchial epithelial cells [10]. Polymeric forms of Z-type α₁-AT are less active as elastase inhibitors, and may also possess proinflammatory properties (figure 1c) [11]. Polymers secreted from hepatocytes can contribute to circulating polymers [12], which have also been found in lung lavage. Extracellular polymers are chemotactic and stimulatory for human neutrophils and may contribute to inflammatory neutrophil infiltration in the lungs [13]. Moreover, the observation that cigarette smoke accelerates polymerisation of Z-type α₁-AT by oxidative modifications [14] has linked two of the major prevailing hypotheses in COPD, namely oxidants and proteinases in Z-type α₁-AT-related emphysema, and added the new idea that the polymers in the lung could promote lung inflammation.

Similarly to what occurs in “usual” COPD, an important adaptive immune inflammation, comprising B, CD4⁺ and CD8⁺ lymphocytes and lymphoid follicles, is a prominent feature in AATD [15]. Baraldo et al. [15] showed that lymphoid follicles in AATD and usual COPD were markedly increased when compared with control groups, and their number correlated negatively with the ratio of forced expiratory volume in 1 s (FEV₁) to forced vital capacity (FVC). These results suggest that the extent of inflammation in AATD is similar to that found in severe usual COPD; thus, the inflammatory mechanisms involving the adaptive immune system, known to be important in usual COPD, seem also to be at play in AATD.

Another interesting role of α₁-AT polymers implies an increased tendency of Z-type α₁-AT towards hydrophobic interactions. In fact, α₁-AT polymers have a better propensity to bind fatty acids and upregulate the expression of angiopoietin-like protein 4 (Angptl4), thus suggesting a novel role for α₁-AT in lipid homeostasis and immune regulation [3].

Different α₁-AT variants have a different prognosis

There is considerable heterogeneity in clinical presentation among AATD patients, since this disorder predisposes to lung and liver disease, but it might also manifest with granulomatosis with polyangiitis and panniculitis. Various studies demonstrate that lower levels of α₁-AT are associated with a risk of HIV type 1 infection [16], type II diabetes mellitus [17], spontaneous abortions [18] and pre-eclampsia [19].

The clinical pulmonary manifestations are rather various, although the “classical” clinical phenotype still remains the basal panacinar emphysema; nevertheless, some patients display other radiological patterns, such as centrilobular emphysema and bronchiectasis. Signs and symptoms of pulmonary involvement with AATD closely resemble those of other patients with COPD. However, a very recent study on 500 severe AATD subjects has provided evidence that nearly 46% of all participants were diagnosed with either asthma or allergic disease [20], and a higher incidence of the main AATD alleles has recently been detected in pulmonary Langerhans cell histiocytosis [21].

When attempting to explain the heterogeneity of clinical manifestations, the complexity of biological function of α₁-AT is not negligible. Interactions of α₁-AT with other molecules may lead to degradation, complex formation, oxidation, self-assembly or other modifications. Genetic mutations together with cigarette smoke, which is known to induce post-translational modifications such as oxidation or polymerisation, may alter α₁-AT bidirectional intracellular traffic in endothelial cells and thus determine its functional bioavailability in certain lung compartments [22].

It is known that the stepwise reduction in plasma α₁-AT is associated with greater risks of spirometry-defined airway obstruction and COPD, and there is a clear association between α₁-AT concentration in plasma and common genotypes of SERPINA1 [23]. Pathological α₁-AT variants are either “null”, with no detectable levels of α₁-AT in serum and generally due to a premature stop codon, or “deficient”, where a point mutation causes different grades of retention in the hepatocytes and, consequently, reduced levels of α₁-AT in plasma. The most common severely deficient variant is Z-type α₁-AT (p.E366K), but several other deficient variants, often referred to as “rare”, have been identified over the last decades. The alleles bearing missense mutations result in conformationally altered proteins that have variable degrees of degradation/accumulation in the endoplasmic reticulum and different degrees of plasma deficiency. Nonrespiratory clinical manifestations, mainly associated with intracellular accumulation of α₁-AT polymers, are listed in table 1.

The progression of lung damage in severe AATD

Individuals with severe AATD have, as a consequence of pathological SERPINA1 alleles inherited from both parents, α₁-AT plasma concentrations below the canonical protective threshold of 11 μM, corresponding to 50 mg⋅dL⁻¹ or 0.5 g⋅L⁻¹ [23]. Most of our knowledge about the natural history of severe AATD (PiZZ) has come from the follow-up data from a Swedish birth cohort [24]. The purpose of the Swedish neonatal screening was to determine the frequency of liver disease during the neonatal period in AATD; therefore, no details about lung diseases were reported in the first publication [24]. Anyway, lung clinical manifestations in AATD during infancy are still reported as case reports. Although respiratory symptoms do not usually appear during childhood, recent studies conducted in a cohort of children and teenagers diagnosed with severe or intermediate AATD have suggested that oxidative stress is a feature of severe AATD at an early stage, as indicated by lower total glutathione and reduced glutathione levels, decreased catalase activity and increased glutathione peroxidase activity [25], as well as reduced telomere length [26], in children and teenagers with severe AATD compared to controls.

The examination of the Swedish cohort at about 18 years of age reported essentially normal lung function [27], but high prevalence of asthma symptoms especially among ever-smokers was reported at 22 years of age [28]. At 35 years of age, the PiZZ ever-smokers had significantly lower transfer coefficient of the lung for carbon monoxide (K_CO) values and 15th percentile density than the control subjects [29]. The prevalence of recurrent wheezing was significantly higher among the PiZZ ever-smokers than the PiZZ never-smokers, which could indicate early symptoms of COPD [30].

The UK Antitrypsin Deficiency Assessment and Programme for Treatment (ADAPT) programme demonstrated in a group of 101 PiZ individuals, mainly between the fourth and sixth decades of life, that FEV₁ decline was greatest in those with moderately severe disease, and this showed associations with bronchodilator reversibility, body mass index and male sex and exacerbation rate [31]. K_CO decline, conversely, was greatest in severe disease [31]. In the same cohort, decline of lung function was predicted by outdoor pollution, in particular exposure to ozone and particles with a 50% cut-off aerodynamic diameter of 10 μm (PM₁₀) [32]. An observational study in patients with severe AATD enrolled in the Spanish and Italian national registries showed that, among the three principal clinical phenotypes (emphysema, chronic bronchitis and asthma), patients with chronic bronchitis were younger, had more preserved lung function and lower tobacco consumption, whereas patients with asthma–COPD overlap were more frequently never-smokers and female [33]. The role of smoking is crucial in the natural history of lung damage progression in severe AATD individuals, even if it also partly explains the heterogeneity in lung disease. Cigarette smoking was the greatest predictor of impairment in FEV₁ and diffusing capacity of the lung for carbon monoxide (D_LCO) in the PiZZ cohorts (139 individuals) identified from the Irish National AATD Registry and (surprisingly) passive smoke exposure in childhood resulted in a greater total pack-year smoking history [34]. In severe AATD the impact of smoking is greater at the beginning of the habit, as demonstrated by the steeper decline in lung function with the first 20 pack-years of smoking compared with consequent consumption for PiZZ and PiSZ patients [33]. Later on, intensive (ex-) smokers had diminished differences in quality of life and exacerbation frequency between PiZZ and PiSZ individuals, as recently demonstrated by data from the German registry for individuals with AATD [35].

The PiZZ subjects identified by the neonatal Swedish programme had a significantly shorter survival time at the age of 35 years than the controls of the Swedish general population, yet the never-smoking PiZZ individuals had a similar life expectancy to the control never-smokers. Mortality due to respiratory disease was markedly increased in PiZZ smoker Swedish subjects compared with the age- and sex-matched Swedish population [30]. Lower body mass index has also been linked with greater progression of lung disease and mortality in PiZ patients [36].

The lung damage progression associated with intermediate AATD

According to the recently published European Respiratory Society statement on AATD, “Never-smoking PiMZ subjects do not have an increased risk for COPD” and “Smoking PiMZ and PiSZ subjects have an increased risk of COPD compared to smoking PiMM subjects” [37]. Data about lung damage progression in intermediate AATD, principally due to the presence in heterozygosity of Z or S alleles, are difficult to obtain because of scant screening programmes in the general population. Data from the Copenhagen City Heart Study and the SAPALDIA (Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults) epidemiological studies have partially filled this gap. Thanks to the population-based Danish cohort with 21-year follow-up data between the fourth and sixth decades of life and triple measurements of lung function, Dahl et al. [38] reported that the average annual decrease in FEV₁ was 19% greater in persons with the PiMZ genotype and 169% greater in persons with the PiSZ genotype, in comparison with a 21-mL average annual decrease in persons with the PiMM genotype. Studies based on the SAPALDIA cohort indicated that the PiMZ genotype was associated with an accelerated average annual decline in forced expiratory flow at 25–75% of FVC (FEF_25–75%), in smoking and obesity subgroups from the general population, in comparison to the PiMM genotype [39]. Moreover, a statistically significant interaction (p<0.0001) was observed between the PiMZ genotype and high levels of exposure to vapours, gas, dusts and fumes (VGDF) on annual change in FEF_25–75% [40]. A similar interaction of statistical significance (p=0.03) was observed between the PiMZ genotype and high-level VGDF exposure on annual change in FEV₁/FVC [40]. More recently, a robust family-based study to determine the risk of COPD in PiMZ individuals indicated that PiMZ heterozygotes are at an increased risk for impaired lung function and COPD, and cigarette smoke exposure exerts a significant modifier effect [41].

A comparison of lung function decline among PiMZ and PiZZ subjects has not yet been examined, although this matter does deserve attention.

The paradigm of null mutations

The identification of the exact molecular mechanisms underlying AATD would definitely help in the definition of different diagnostic risk levels for the liver and lung disease displayed by varying AATD genotypes. For example, the identification of alleles associated with a high degree of α₁-AT polymerisation, such as Z or M_malton, would indicate the need for a more extensive liver evaluation, whereas null alleles do not indicate a risk for liver pathology. Regarding a possible relationship between the severity of lung disease and different α₁-AT genotypes, most of the data are focused on the Z mutation. Progression and severity of lung diseases are little considered in cohorts of AATD patients with rare pathological variants, mainly because their rareness. The only exceptions are null mutations, where interest is raised by the total absence of α₁-AT. As a consequence of the fact that serum levels of α₁-AT are correlated with the severity of the pulmonary phenotype, subjects with null mutations in homozygote fashion should be considered a subgroup at particularly high risk of emphysema within AATD. Among the lung function measurements, FEV₁ as a percentage of predicted and K_CO as a percentage of predicted were statistically lower in subjects who were null/null, in comparison to PiZZ [42]. Moreover, evidence of the recurrence of lung symptoms (dyspnoea, cough) and lung diseases (emphysema, asthma, chronic bronchitis) was reported in M/null subjects aged >45 years, irrespective of their smoking habit [43].

The simplification of the pathogenetic mechanisms of null mutations, since no polymer effect needs to be considered, is interesting in terms of lung damage progression. Null/null patients have a total absence of α₁-AT in vivo; therefore, their clinical follow-up would allow a comprehensive investigation of the role of α₁-AT as a serine-proteinase inhibitor.

Augmentation therapy and lung damage progression: a meta-analysis

The current standard of care for patients affected by AATD-associated pulmonary emphysema is replacement therapy by weekly intravenous infusion of pooled human plasma purified α₁-AT. By the dose usually recommended (60 mg per kg bodyweight, weekly), the plasma level of α₁-AT is kept over the protective threshold (>50 mg⋅dL⁻¹) for the 7 days preceding the next infusion.

The appropriateness and utility of this therapy has been a topic of intense debate. There are some meta-analyses and reviews about it, and the recent European Respiratory Society statement on AATD dedicated to this matter an exhaustive analysis based on standard systematic review [37], which showed that intravenous augmentation therapy reduces the progression of emphysema as assessed by computed tomography (CT) densitometry. The reduction of the rate of lung density decline with augmentation therapy compared with placebo and the reduction of lung function decline in observational studies are reported in table 2. Because of expected between-study heterogeneity, a random effects model was employed for meta-analysis; pooled differences in density and FEV₁ slopes were estimated using the META program (https://mathgen.stats.ox.ac.uk/genetics_software/meta/meta.html). Figure 2 presents the individual study and pooled density and FEV₁ slopes and slope differences. Our meta-analysis failed to show a significant effect on FEV₁ decline (standardised mean difference (SMD) −0.132 mL⋅year⁻¹, 95% CI −0.406 to 0.113 mL⋅year⁻¹; p=0.269), while it showed a positive effect on CT densitometry, with a density SMD of −0.275 g⋅L⁻¹⋅year⁻¹ (95% CI −0.499 to −0.0506 g⋅L⁻¹⋅year⁻¹; p=0.016) between active treatment and placebo. This confirms the results of a previous Cochrane systematic review [51]; however, statistical methods and conclusions were different. We state that the protective effect of augmentation therapy is primarily attributable to the blunted decline of lung density, because CT lung density reflects emphysema lung destruction (and thus disease severity) better than FEV₁ does. In contrast, changes in FEV₁ are less sensitive, so that several hundred patients would need to be randomised to capture a significant effect in a few years of follow-up. Although many of the observational studies showed a benefit of treatment on the rate of FEV₁ decline, the potential for bias is greater than in a randomised controlled trial, and the data should be interpreted with caution.

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Figure 2

Forest plot of studies included in the meta-analysis of a) CT scan density and b) FEV₁ data.