Update on Research, Diagnosis and Management of Alpha1‐Antitrypsin Deficiency

Abstract

Formed in response to a World Health Organization recommendation, the Alpha One International Registry (AIR) is a multinational research program focused on alpha1‐antitrypsin (AAT) deficiency. Each of the nearly 20 participating countries maintains a National Registry of patients with AAT deficiency and contributes to an international database in Malmö, Sweden, that is designed to increase understanding of AAT deficiency as well as safeguard patient confidentiality. AIR members are engaged in active and wide‐ranging investigations to improve the diagnosis, monitoring and therapy of the disease. The AIR membership meets biennially to exchange views and research findings. The third biennial meeting was held in Barcelona, Spain, June 11–13, 2003. A wide range of AAT deficiency‐related topics were addressed, encompassing molecular and cellular pathophysiologic mechanisms, clinical epidemiology, diagnostic advances, current and investigational therapeutic approaches, and progress in registry development. Valuable cross‐fertilization of concepts and scientific observations was apparent between AAT deficiency research and other fields of biomedicine. The proceedings of the meeting are summarized in this report.

• Trypsin inhibitors
• Emphysema
• Phenotype
• Liver diseases
• Registries

Introduction

Affecting one in 1,600 to 5,000 individuals, alpha1‐antitrypsin (AAT) deficiency is one of the most common hereditary disorders in Caucasians. AAT deficiency is characterized by low serum levels of AAT, a predisposition to early‐onset emphysema, and less commonly, liver disease, including neonatal jaundice and adult cirrhosis and hepatoma. In order to overcome the significant challenges that exist when investigating uncommon disorders, in recent years a concerted effort has been made to bring together all relevant information on research into the underlying pathophysiology and the clinical management of AAT deficiency, as illustrated by the establishment of a substantial number of registries for the disorder. Although much remains to be elucidated, progress continues to be made. Moreover, it has become clear that advances in AAT deficiency research can contribute to the understanding of other diseases and that approaches developed for other medical areas are now being applied to AAT deficiency.

The Alpha One International Registry (AIR), formed in 1998, is a multinational research project with the involvement of nearly 20 countries. AIR has committed to organizing biennial scientific meetings to update the diverse areas of research and clinical development relating to AAT deficiency [[1]]. The 3rd AIR meeting was held in Barcelona, Spain in June 2003, involving 25 speakers and covering a wide‐ranging array of issues, from basic research to clinical findings. In a review of recent developments with regard to clinical management of AAT deficiency, Dr. Robert A. Sandhaus concluded that very little has changed in the management of lung and liver disease of AAT deficiency in the past two years. However, progress is steadily being made in unraveling the pathophysiology of both lung and liver and identifying risk factors for disease in AAT deficiency, and a number of new approaches to therapy are on the horizon. The presentations are summarized in this report.

The Laurell Lecture

In recognition of the 40th anniversary of the 1963 seminal work on AAT deficiency by Dr. Carl‐Bertil Laurell, Head of the Department of Clinical Chemistry in Malmö, Sweden [[2]], the AIR Laurell Lecture was established, and Dr. Robin W. Carrell was chosen to present the first such lecture. Dr. Laurell's original observation has led to progress in medicine extending far beyond AAT deficiency. Dr. Carrell noted that an observation in a clinical laboratory in Sweden 40 years ago has opened up new fields in biology and medicine, and has done so long before the significance was recognized by science as a whole. Numerous avenues of research and clinical progress can be traced to Dr. Laurell's astute observation of the absence of the alpha1‐band among the electrophoretic abnormalities of lung patients. Dr. Carrell summarized Dr. Laurell's contribution with a quote by Goethe: “When the king finds gold, the subjects all begin to dig the soil.”

Not only is AAT the archetype of the serpin supergene family but it also has been the prototype for an entire category of disorders—the conformational diseases [[3]]. The serine proteinase inhibitors, or serpins, are a superfamily of proteins that are found in organisms from plants to viruses to animals. In humans, the family includes in addition to AAT, proteins as diverse as α‐antichymotrypsin, C1 inhibitor, antithrombin, and plasminogen activator inhibitor‐1, which have key regulatory functions in the inflammatory, complement, coagulation, and fibrinolytic cascades [[4]]. The serpins all possess an exposed mobile reactive center loop consisting of 14 amino acid residues that acts like a mouse trap for serine proteinases and enables the serpins to inhibit the serine proteases irreversibly (Fig. 1). The design of this reactive loop makes serpins very effective antiproteinases, but also leaves them susceptible to conformational transitions that can cause disease.

Figure 1. Tertiary structure of AAT.

In individuals with the PiZ (proteinase inhibitor) phenotype, a single nucleotide mutation causes the AAT molecules to accumulate in the endoplasmic reticulum (ER) of hepatocytes rather than be secreted. In the ER, there is a rapid sequential interaction between the reactive center loop of one AAT molecule and the main β‐sheet of a second. The consequence in PiZ individuals is the formation of long polymers of AAT, with each molecule linked by its reactive loop to the sheet of the next [[5]]. Characteristic inclusion bodies in the liver, which are readily recognized on periodic acid‐Schiff staining, develop over time. The accumulation of aberrant forms of individual proteins rather than a failure to produce the protein is the common theme in conformational diseases. Conformational diseases such as the common dementias, Alzheimer's disease, prion diseases, and the serpinopathies are all caused by structural rearrangements within a protein that transform it into a pathological species. Recently in a study of familial neurodegenerative disease, inclusion‐body formations containing neuroserpin, a brain‐specific serpin, were found sufficient to cause neurodegeneration [[6]]. The onset and severity of the disease was found to be associated with the rate and magnitude of the neuronal protein aggregation.

Efforts are underway to develop small peptides that can selectively inhibit polymerization of the Z allele of the AAT protein [[7]], and the early results have been promising. Dr. Carrell expressed confidence that in due course there will be a cure or treatment for this condition, but it could take a decade.

Lung Disease

The hallmark of AAT deficiency is the development of early‐onset severe lower zone emphysema in individuals with the PiZZ genotype. The unchecked action of neutrophil elastase on lung parenchyma leads to alveolar destruction.

AAT is the major defense in the lung against neutrophil elastase. Severe AAT deficiency is the only defined genetic risk factor for chronic obstructive pulmonary disease (COPD), and individuals with susceptible genotypes are at risk for severe, early‐onset COPD. Cigarette smoking markedly increases the risk and rate of development of emphysema in patients with severe AAT deficiency.

Dr. Robert A. Stockley reviewed the occurrence of exacerbations in AAT deficiency. In an attempt to establish a widely accepted definition, a COPD working group recently defined exacerbations as “a sustained worsening of the patient's condition, from the stable state and beyond normal day‐to‐day variations, that is acute in onset and necessitates a change in regular medication in a patient with underlying COPD” [[8]]. The criteria for COPD exacerbation proposed by Anthonisen et al. [[9]] are still widely accepted: breathlessness, sputum volume and sputum purulence. Increased numbers of exacerbations have been found to be related to a faster decline in lung function in individuals with AAT deficiency [[10]]. Exacerbations have been associated with bacterial and viral infections, pollution, airway irritability/obstruction, and increased temperature, and there is a considerable challenge faced in delineating the contribution of each of these factors.

A classic study by Hill et al. [[11]] demonstrated that individuals with AAT deficiency have increased elastase activity at both the start and resolution of the exacerbation when compared with other COPD patients. Moreover, this study showed that both at the start and resolution of an exacerbation, the lung airways in AAT deficiency individuals are more neutrophilic, as evidenced by increased myeloperoxidase, sputum leukotriene B4 (LTB4) and sputum interleukin‐8 (IL‐8) levels. Increased exacerbation frequency has also been reported in those AAT deficiency individuals with chronic sputum expectoration [[12]]. Exacerbations last approximately twice as long in AAT deficiency patients as in other COPD patients who have an equivalent degree of lung function impairment. Exacerbations do not occur in 40–50% of AAT deficiency individuals each year, but those AAT deficiency individuals who do experience exacerbations have 2.4 on average. Although this predictor is not clear‐cut, lower forced expiratory volume in 1 sec (FEV₁) appears to be associated with increased risk of exacerbations. One study has indicated that patients on augmentation therapy have fewer exacerbations (i.e., the number of infections per year) compared with those not on augmentation therapy [[13]].

High resolution computerized tomography (CT) scanning is the most sensitive non‐invasive method to diagnose emphysema. According to Dr. Saher Shaker, quantitative CT has shown good correlation with the pathological extent of emphysema, lung function tests, especially diffusion capacity, and to a lesser extent measures of airway obstruction like FEV₁ [[14]]. Quantitative CT is reproducible and more sensitive than lung function testing in monitoring the progression of emphysema and also correlates with the patient's health status. However, a major confounder in the use of quantitative CT is the depth of inspiration. The lung density almost doubles from full inspiration to full expiration. CT lung density should be adjusted for the level of inspiration, and from 10 to 75 percentile density, double logarithmic transformation and a coefficient of − 1.3 should be applied.

Another indicator of lung dysfunction is expiratory flow limitation (EFL), which is the maximal expiratory flow achieved during tidal breathing and signifies intrathoracic airflow obstruction. EFL promotes dynamic pulmonary hyperinflation and can adversely affect hemodynamics as well as contribute to dyspnea [[15]]. Dr. Luciano Corda reported that EFL was observed frequently in AAT deficiency patients and that they had a significantly greater probability of exhibiting EFL when supine (70%) than seated (30%).

Role of Phenotype

The gene locus specifying the AAT protein is polymorphic, and more than 70 variants have now been identified, according to Dr. Sabina Janciauskiene. However, the M, S and Z alleles are most prevalent. The PiM allele, the predominant allele, results in normal AAT levels, as shown in Fig. 2 [[16]]. The PiS allele accounts for 2–3% of the alleles and is associated with mildly reduced AAT levels, whereas the PiZ allele accounts for only 1% of alleles but is associated with severely reduced AAT levels (16% of normal) and the characteristic emphysema of AAT deficiency.

Figure 2. Serum AAT levels in various Pi phenotypes. Based on the data of Brantly and Wittes [[16]].

The risk of developing COPD in heterozygotes is still not clearly delineated and has been a subject of controversy. Dr. Morten Dahl reviewed the risk of lung disease in the heterozygote PiMZ phenotype. A longitudinal study spanning up to 18 years among 9,187 participants in the population‐based Copenhagen City Heart Study found that the 451 PiMZ heterozygotes had 31% lower levels of AAT than PiMM genotypes. Their rate of FEV₁ decrease was 19% greater than the average values in persons with the PiMM genotype, and they had a 30% increase in risk for airway obstruction, a 50% increase in risk for COPD and a 50% increase in risk of hospitalization or death from COPD [[17]][[18]]. Based on an as yet unpublished meta‐analysis of 17 studies, the odds ratio of developing COPD is 2.3 in PiMZ individuals and 13 in PiZZ individuals. In the meta‐analysis it was observed that the type of study design affected the outcome, with the odds ratio of COPD in PiMZ individuals being 1.3 in population‐based studies and 3.1 in case‐control studies.

While the risk of lung dysfunction is much lower in PiMZ than PiZZ individuals, the PiMZ genotype is present in the population in a much higher frequency than the PiZZ genotype. Consequently, the actual number of PiMZ patients with COPD could be similar to that of PiZZ patients with COPD. Evidence suggests that other genetic and environmental factors contribute to the development of COPD in PiMZ individuals and that in certain subsets PiMZ may be associated with a relatively large risk for developing COPD. On the other hand, data from the Copenhagen City Heart Study suggest that the PiMZ phenotype may be associated with reduced risk of ischemic cerebrovascular and ischemic heart disease, and in that regard the PiMZ phenotype could prove beneficial.

The prevalence of the PiSZ genotype in most European countries is 1 in 1000, according to Dr. Niels Seersholm. The genotype is more prevalent in Spain compared with other European countries [[19]]. The prevalence in the USA is estimated to be 1 in 1270. However, few individuals of this genotype are in the various registries, and therefore the natural history of lung disease has not been extensively studied. In the Copenhagen City Heart Study [[18]], airway obstruction was increased in PiSZ individuals (40%) compared with PiMM (15%). A Swedish study of 94 PiSZ individuals revealed that only a small fraction of persons with the PiSZ phenotype are at increased risk of developing pulmonary emphysema, and such individuals are affected at an older age than persons with the PiZ phenotype [[20]]. A cohort study of 50 nonsmokers demonstrated that PiSZ may confer little or no added risk of lung disease [[21]]. PiSZ smokers may carry a small additional risk. Life expectancy in PiSZ individuals appears to be normal.

Lung Transplants

As reported by Dr. Sandhaus, a recent analysis of the United Network for Organ Sharing (UNOS) lung transplant database showed that, with regard to mortality at 3, 5 and 10 years, AAT deficiency individuals receiving double lung transplants had better outcomes than any other group, including the cystic fibrosis patients. However, analysis of results from the National Emphysema Treatment Trial (NETT) indicated that patients with emphysema and lower lobe predominance, as is characteristic of AAT deficiency patients, did not benefit from lung reduction surgery [[22]]. The NETT report did not stratify results according to disease etiology such as AAT deficiency, however.

Pathophysiology of Lung Disease

The pathogenesis of emphysema is still incompletely understood, and it is increasingly clear that complex, interacting pathways are involved in its initiation and progression and the failure of repair processes [[23]]. While the protease/antiprotease hypothesis is still “alive and well”, animal models are elucidating how other cellular components and biochemical pathways can also contribute to the lung damage seen in emphysema, according to Dr. Robert M. Senior. Emphysema can be modeled in many ways, and mice have become the predominant species for these studies. Three categories of experimental design are currently being pursued: 1) inducing emphysema in normal mice by challenge with exogenous agents; 2) studying airspace enlargement in naturally occurring genetic mutant mouse strains, such as blotchy and pallid strains; and 3) creating targeted mutagenesis (“knockout”) or transgenic mice. Transgenic and gene‐related mouse models of emphysema are dominating research currently. Among the advantages are that many genes have been cloned, techniques for genetic manipulation of mice are well established, and large numbers of mice can be generated quickly. However, it is still not clear how accurately these models reflect human biology and pathology. There are now many examples linking proteases and emphysema, and different proteases appear to be involved in different models. In addition to neutrophil elastase, other potential enzymes implicated are macrophage elastase, interstitial collagenase, and cysteine proteases.

Two recent papers have addressed the role of macrophage matrix metaloproteinase‐12 (MMP‐12)—also known as macrophage elastase—in inflammation and subsequent emphysema [[24]][[25]]. Mice lacking α V beta 6 integrin have high levels of lung MMP‐12 and develop emphysema. Transforming growth factor (TGF‐b1) is activated by binding to alpha V β 6 integrin, and active TGF‐β down‐regulates the expression of MMP‐12 in macrophages. The hypothesis has been advanced that emphysema may occur in the α V β 6‐deficient mice due to overexpression of MMP‐12 following a failure to activate TGF‐β [[24]][[26]]. In another mouse model of cigarette smoke‐induced emphysema, MMP‐12 was found to mediate smoke‐induced inflammation by releasing tissue necrosis factor‐alpha (TNF‐α) from macrophages, with subsequent endothelial activation, neutrophil influx, and proteolytic matrix breakdown caused by neutrophil‐derived proteases [[25]]. Research in this field is very active with recent models revealing complex pathways. These models are opening new directions for the study of human emphysema.

Dr. Jean‐Michel Sallenave further elaborated on the role of cytokines in the pathogenesis of COPD [[27]]. Exacerbations have been linked to increased expression of pro‐inflammatory cytokines, but the relative importance of these changes remains to be determined. Although there have been significant advances in understanding the pathophysiology of COPD, the role of inflammation in the pathogenesis of the condition is just beginning to be elucidated. Available evidence suggests that cytokines such as interleukin‐1 (IL‐1), TNF‐α, and interferon‐g (IFN‐γ) can initiate emphysema and that macrophages, neutrophils, and CD8 T cells also play a role. Cytokines such as IL‐8, the chemokine RANTES and interleukin‐13 derived from neutrophils, eosinophils and CD8 T cells, appear to be involved in COPD exacerbations. Even less explored is the role of cytokines and growth factors, such as vascular endothelial growth factor (VEGF), in lung hemostasis and repair. While there is not yet a clear picture of the varied roles of cytokines in emphysema, a growing number of models have demonstrated the proof of principle that cytokines do have a function in inducing emphysema, at least as assessed by animal models [[25]][[28]][[29]].

Dr. Joan Albert Barberà reported on yet another cell type—the endothelial cell—that appears to play a role in the progression of COPD. Increased pulmonary artery pressure is a predictor of poor clinical outcome in COPD patients, and pulmonary vascular abnormalities start at early stages of the disease [[30]][[31]]. Endothelial dysfunction can lead to pulmonary hypertension. Smooth muscle cell proliferation, as well as elastin and collagen deposition, in the thickened intimas of pulmonary arteries in moderate COPD patients and smokers has been reported [[32]]. Cigarette smoke products may initiate vascular changes by a direct effect on endothelium and/or an inflammatory mechanism.

Liver Disease

PiZZ AAT deficiency is the most common genetic/metabolic cause of end stage liver disease in children, accounting for approximately 10% of pediatric liver transplantation in Europe/USA, according to Dr. Dino Hadzic. Severe liver disease has two peaks with decompensation either within the first 2 years of life or in puberty. The most common presentation is neonatal hepatitis, and liver deposits of AAT have been demonstrated as early as 17–20 weeks of gestation [[33]]. Physiological perinatal prevalence of hydrophobic bile acids may add to prolonged neonatal jaundice, but the majority of children who will not need liver transplantation clear their jaundice by 6 months of age. Chronic liver disease will develop in 78% of PiZZ infants with neonatal hepatitis syndrome, and 28% will either require a transplant or die.

In contrast to PiZZ individuals, liver disease in PiSS and PiSZ individuals is much less well documented [[34]][[35]][[36]]. The PiS allele is more common than PiZ in some parts of Europe, such as the Iberian peninsula where the prevalence may be as high as 28%. It has been reported that PiZZ patients with acute liver decompensation may exhibit a PiSZ‐like pattern on electrophoretic phenotyping, and this could lead to an erroneous exaggeration of the actual incidence of childhood liver disease associated with PiSZ [[37]]. A study conducted at King's College Hospital in London included 9 PiSS and 20 PiSZ individuals. The PiSS individuals had a variety of liver problems, including biliary atresia, glycogen storage disease type III, urea cycle defect and steato‐hepatitis (Mauriac syndrome). However, after a mean follow‐up of 62 months in individuals with PiSS and 24 months in those with PiSZ, there was no clinical evidence of liver disease, except in relation to associated pathologies. Thus, possession of PiSS and PiSZ phenotype per se does not induce liver disease in childhood. Liver involvement in PiS individuals may be subclinical, and prospective community‐based studies in areas with high prevalence of the S allele are needed. Three studies that investigated the effect of rare Pi phenotypes on liver function have also recently been published [[38]][[39]][[40]].

A number of presentations at the meeting underscored the need to explore further the pathophysiological processes taking place in the liver so that directed therapies can be developed. To date, there are no specific therapies for liver disease associated with AAT deficiency. The mutant PiZ protein has a single amino acid substitution that prevents it from folding properly. The AAT PiZ protein polymerizes and is retained in the ER rather than secreted into blood and body fluids. While the assumption has been that the retention of this aggregated PiZ glycoprotein elicits liver injury, the possibility exists that the accumulation of Z polymer per se does not cause liver disease, according to Dr. Richard Sifers. Dr. Jeffrey H. Teckman stressed that the mechanisms of liver injury, including the role of mutant AAT in triggering injury, are still poorly understood. For instance, the PiSaar mutation results in prolonged intracellular retention even though the proteins do not have polymeric properties [[41]]. The mapping of the flux of AAT PiZ through the cell from synthesis, chaperone binding, polymerization, retention and degradation and even to the small amount that is secreted is key to understanding liver pathophysiology.

The glycoprotein ER‐associated degradation (GERAD) system that has been found to be a major component of the eukaryotic cell's “biosynthetic quality control” system [[42]]. GERAD is one of the numerous checkpoints in eukaryotic cells that maintain fidelity of genetic information. On their passage through the cell, secretory proteins such as AAT are first transported by a molecular chaperone through the ER where they undergo conformational maturation. Recent studies have shown that an asparagine‐linked oligosaccharide of ATT serves a vital role in ER transport, processing and proper folding. The chaperone protein calnexin can only bind to ATT after the removal of two glucose molecules from the oligosaccharide. Binding of calnexin results in the shedding of another glucose and the folding of the AAT molecule. The PiZ molecule cannot be folded properly and therefore is marked for degradation. The oligosaccharide chain has also been found to play an essential role in the signal for degradation. The removal of one mannose plus a non‐native protein structure are both required for degradation.

Degradation of the misfolded or polymerized AAT molecules occurs primarily in the proteosome [[43]]. However, nonproteosomal autophagy may also be involved. In autophagy, cytosol and intracellular organelles, such as ER, are first sequestered from the rest of the cytoplasm, allowing them to be degraded subsequently within lysosomes. The concentration of the PiZ protein may determine whether it is degraded by proteosomes or lysosomes. Mitochondrial autophagy may play a role in the mechanism of liver cell injury in AAT deficiency [[44]], but mitochondrial injury is only one part of the story.

A mouse model study has demonstrated an altered response to stress, such as fasting, in the AAT deficiency liver, including an incapacity for further increase in activated autophagic response, a developmental state‐specific rise in AAT‐containing globules, and higher mortality [[45]]. The liver of AAT deficiency individuals could be very susceptible to perturbations and insults, and will suffer more injury than a normal liver for any given insult. Recent research in the laboratory of Dr. Teckman has suggested that many common medications may be injurious to the liver of AAT deficiency individuals.

Diagnosis and Rare Phenotypes of AAT Deficiency

Dr. Edward J. Campbell stressed that in all countries in which AAT deficiency is prevalent, the majority of individuals with the disease have not yet been detected. In Sweden and Denmark, approximately 15–20% of all deficient individuals have been detected. However, in the remainder of the world, that percentage is much lower. Of the more than 80,000 AAT deficiency individuals estimated to be in the USA, only 4,000 to 5,000 (5–6%) have been diagnosed.

World Health Organization recommendations were issued in 1997 that all patients with COPD, all adults and adolescents with asthma and all individuals with a family history of AAT deficiency, as well as neonates, children and adults with unexplained liver disease, should be screened, and that those with abnormal results on screening should undergo phenotyping [[46]]. Three recommended categories of testing were immunoassay for serum levels of AAT, phenotyping based on isoelectrophoretic focusing of the AAT protein and genotyping using polymerase chain reaction (PCR) to analyze DNA. At least two independent tests are required to establish a diagnosis of AAT deficiency, and all approaches have their limitations.

The prevalence of genotypes other than PiZZ is approximately 6–10%, i.e., 17 of 182 (9.3%) US patients and 6 of 103 (5.8%) British patients. Diagnostic testing is complicated by the presence of Null alleles, which result in a total lack of circulating protein. Prior to the availability of DNA‐based testing, Null alleles could be identified with certainty only through familial patterns of inheritance. In PCR‐based genotyping, primers for both the PiZ mutation and the normal sequence allow “counting” of the PiZ alleles present. Individuals with an allele having normal sequence at residue 342, but only Z protein identified by phenotyping have a Null allele. Unlike the PiZ mutation which results from a single specific mutation, Null alleles can result from a number of mutations, including ones that result in T splicing abnormalities, deletion of AAT coding exons and premature stop codons. In an analysis of 25 AAT deficiency individuals with genotypes other than PiZZ and 29 individuals with suspected PiNull alleles, a total of 24 different deficient alleles, including 17 novel ones, were identified. The presence of Null alleles at relatively high frequency, and the variety of Null alleles represented, pose severe challenges for the use of genotyping alone for diagnosis of AAT deficiency.

In Northern Italy, a unique screening program has tracked rare AAT phenotypes and liver‐test abnormalities during infancy [[38]]. The authors concluded that only a neonatal screening based on phenotyping can detect these rare carriers early in life.

The ATZ11 monoclonal antibody has been used for immunological detection of PiZ‐positive hepatocytes [[47]] and detects a conformation‐dependent neoepitope on both polymerized AAT and native AAT‐elastase complex. Dr. Janciauskiene reported that this antibody detects AAT‐elastase complexes in the endothelium layer, and this finding requires further investigation. Moreover, AAT deficiency is related to high levels of circulating AAT‐polymers, and the detection of circulating and tissue‐bound AAT polymers might prove valuable for various clinical applications. For instance, ATZ11‐based enzyme‐linked immunosorbent plasma assays allow sensitive and specific detection of Z carriers [[48]][[49]]. Tissue‐bound AAT‐polymers can serve as endothelial cell markers [[50]].

With the sequencing of the human genome complete, researchers are now focusing on creating a map of human genetic variation based on common haplotype patterns. Dr. Sally Plummer reported on the construction of a complete single nucleotide polymorphism (SNP) map of the AAT gene. Using this approach, 16 polymorphisms have been identified in AAT to date. There appears to be one major haplotype with many other rare haplotypes. The goal is to identify informative haplotype tag SNPs that will allow a reduction in the number of loci that need to be included in future association studies.

Registries and Foundation Update

Dr. Charlie Strange reviewed findings and ongoing activities from two US‐based registries: the Alpha‐1 Research Registry [[51]] and the National Heart Lung and Blood Institute (NHLBI) Registry [[52]][[53]]. The Alpha‐1 Research Registry, which was initiated in 1997, has grown to 2280 individuals, and 274 new PiZZ individuals were enrolled in 2002. Several projects of the Alpha‐1 Research Registry are in progress such as the Family Linkage initiative, in which families are encouraged to register as a “linked” family, and the Alpha Coded Testing (ACT). In ACT, a mailed fingerstick test approach identified 709 PiMM individuals, 613 PiMZ, 97 PiMS, 53 PiZZ, 39 PiSZ and 8 PiSS.

The NHLBI‐sponsored Registry for AAT deficiency in North America enrolled 1,129 subjects from 1988 to 1992, and follow‐up continued through 1996. A recent analysis of data from the NHLBI Registry was performed to identify risk factors for decrease in FEV₁. The study provided evidence that age, male sex, bronchial hyper‐responsiveness, baseline serum AAT concentrations and smoking were among the risk factors. Dust exposure and chronic bronchitis phenotype were new risk factors for decrease in FEV₁ identified by the univariate analysis.

Dr. Claes‐Goran Löfdahl provided an update on AIR. This registry now consists of 1,296 individuals, primarily PiZZ phenotypes. The only other phenotype with substantial representation is PiSZ, comprising 7–8% of the registry. Ex‐smokers compose 60% of the registry, never smokers 31% and current smokers 9%. The predominant FEV₁ (% predicted) in never smokers was 100%, whereas it was 20–40% in ex‐smokers and current smokers. In the ex‐smokers, the number of cigarette pack years was significantly correlated with decrease in lung function. However, even in never smokers, a decrease in lung function with increasing age can be seen, and by the age of 60, the mean value of FEV₁ is in the lower range of normal. The decline in lung function from smoking was significantly less in PiSZ individuals compared with PiZZ individuals. When categorized according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines, 35% of the PiZZ individuals were in Stage 0, i.e., at risk but with lung function still normal [[54]]. The most rapid decline in lung function, as tracked by FEV₁, was in those PiZZ individuals in GOLD Stage 3, i.e., severe COPD. The registry contains patients covering the spectrum of severity and may be well suited for interventional studies.

Dr. Tomas Sveger provided an update on a Swedish birth cohort with AAT deficiency that had been followed for almost 30 years [[55]]. Neonatal screening for AAT deficiency undertaken in Sweden between 1972 and 1974 identified 129 infants with severe AAT deficiency (phenotype PiZ). This cohort has been followed prospectively, and participation is currently approximately 65% with an additional 15% still answering questionnaires [[35]][[56]][[57]]. The participants are nearing 30 years of age and continue to be in excellent health. Their FEV₁ values are within normal ranges and have not changed since about 18 years of age. None has clinical symptoms of liver disease, although a few have marginally increased liver enzymes occasionally. The question of whether emphysema and late onset liver disease can be prevented in those individuals who have neonatal detection of AAT deficiency and are given preventative advice remains unanswered.

The new Alpha‐1 Disease Management and Prevention (ADMAP) program from AlphaNet is about to be launched in the USA. This disease management program is a new approach, and grew out of the realization that the majority of those 2,700 AAT deficiency patients being treated in the USA are seen by a physicians who has only one AAT deficiency patient. Therefore, educational efforts directed at patients and families are probably the most effective way to increase knowledge about the disorder and bring the most current understanding of the disorder to the attention of healthcare professionals. The program is designed to foster collaborative self‐management, and will include nurse training and testing.

Augmentation Therapy for Lung Disease

Dr. James K. Stoller reviewed seven salient studies on clinical efficacy of augmentation therapy [[13]][[14]][[53]][[58]][[59]][[60]][[61]]. These studies ranged from observational studies to cohort studies (with either historical or concurrent controls) to one randomized controlled trial. The randomized trial of 56 ex‐smokers with AAT deficiency receiving monthly therapy found that augmentation conferred overall no significant benefit in slowing FEV₁ decline, but the analysis of lung density measured by CT showed a trend to slower decline [[14]].

The NHLBI Registry study was a nonrandomized comparison of 927 patients either receiving or not receiving AAT replacement. The rate of FEV₁ decline was significantly less in those receiving augmentation therapy (66 ± 5 vs. 93 ± 11 mL/year; p = 0.03) only in the subgroup with FEV₁ 35–49% predicted [[53]]. In this study subjects receiving augmentation therapy also had a significantly decreased mortality risk. In another large nonrandomized cohort study comparing 198 German patients receiving weekly augmentation infusions with 97 untreated Danish patients [[58]], the mean annual decline in FEV₁ was significantly less in treated patients only in the subgroup with FEV₁ 31–65% predicted (62 vs. 83 mL, p = 0.04). Both these studies are concordant in showing that augmentation therapy recipients experience a slower rate of decline in FEV₁, especially in those individuals with a moderate degree of airflow obstruction.

In a longitudinal follow‐up of AAT deficiency patients before and during augmentation therapy the largest benefit was observed in those with better preserved lung function [[60]]. This is in contrast to the findings of other observational studies which showed the largest benefits in moderate degrees of airflow obstruction. A novel Web‐based survey of individuals with the PiZ phenotype found that augmentation therapy appeared to be associated with a marked reduction in the frequency and severity of lung infections [[13]]. Two or more infections per year occurred in 65% of respondents before augmentation therapy but in only 18% after therapy began, whereas 55% of those not receiving augmentation therapy experienced two or more infections per year. In a study on the effect of augmentation therapy on bronchial inflammation, short‐term therapy increased lung fluid AAT concentrations to protective levels and was associated with a fall in LTB4, which is thought to be central to the airway inflammation in AAT deficiency [[61]]. In contrast to the benefit from augmentation therapy in the other studies, eight weeks of augmentation therapy in 12 AAT deficiency individuals did not appreciably reduce the rate of elastin degradation, as measured by the rate of urinary excretion of desmosine [[59]]. Dr. Stoller concluded that more studies are needed and that the results of observational studies need to be interpreted with caution.

Prolastin® (Bayer AG, Leverkusen, Germany), which has been available since 1987 and is plasma‐derived, has been the only augmentation therapy available. Two new plasma‐derived augmentation products are currently being introduced in the USA: Aralast™ (Baxter BioScience, Westlake Village, California, USA) and Zemaira™ (Aventis Behring, Marburg, Germany). Also in development are augmentation products, both plasma‐derived and recombinant, that can be inhaled.

Dr. Jorge Jorquera reported that a new product developed by Instituto Grifols, S.A (Barcelona, Spain) and expected to be introduced in 2003, was stable for at least 15 days at both 5°C and 30°C when the contents of several vials of therapy were reconstituted in polypropylene bags equipped with a sterility filter. Because of sterility concerns, the recommended shelf‐life of currently available augmentation products is only hours. A system with an extended shelf‐life could prove of great utility in home care settings.

Potential New Therapeutic Approaches

Thus far the mainstay of therapy has been administration of exogenous AAT in order to augment circulating levels. A variety of additional approaches in early stages of development are also being pursued. Among these are restoring endogenous capacity for AAT synthesis and secretion through gene therapy. A potential cellular therapy involves the transplantation of immortalized hepatocytes. An alternative cellular therapy would entail use of embryonal stem cells to generate replacement hepatocytes. Other investigational approaches include synthetic elastase inhibitors that might reduce the need for AAT, hyaluronic acid that might prevent elastase‐mediated damage, retinoids that might reverse pathologic changes occurring due to AAT deficiency and chaperone molecules that might minimize mislocalization and misfolding of mutant AAT.

A Phase I AAT gene therapy clinical trial is approved and scheduled to start at the University of Florida College of Medicine, according to Dr. Terry Spencer. The trial will involve the intramuscular (IM) injection of the AAT gene using a recombinant adeno‐associated virus (rAAV) vector. This safety study will be an open‐label, single‐dose escalation study with 4 cohorts of 3 subjects each. rAAV‐2 is a non‐pathogenic parvovirus that is a common inhabitant of the upper respiratory tract. Gene therapy, which is a direct extension of protein replacement strategy, has the potential for single‐dose treatment and may be able to provide stable plasma levels.

Skeletal muscle has been shown in animal studies to be particularly efficient for expressing DNA encoded by the AAV vector [[62]]. AAT secretion following IM gene therapy was sustained for up to a year and a half in a mouse model, and there is persistence of rAAV‐hAAT DNA in skeletal muscle for up to 18 months. Further studies are currently being done with another vector, AAV‐1, aimed at increasing the levels of AAT expression. Efficient transgene expression and increased AAT serum levels have also been successfully demonstrated in a non‐human primate model [[63]]. This model was additionally used to demonstrate that the risks of immune reaction and germline transmission after intramuscular injection of rAAV‐bAAT are relatively low within the range of vector doses studied. Efficient, sustained expression of AAT transgene has been demonstrated in mice, rabbits and baboons [[64]]. Other gene therapy approaches for AAT deficiency are also being explored [[65]][[66]][[67]].

An approach in the preclinical investigation phase [[68]] described by Dr. Mark Zern is the immortalization of hepatocytes through the transduction of hepatocytes with the catalytic component of human telomerase, the telomerase reverse transcriptase (TERT). Telomeres are located on the tip of chromosomes. Each time a cell divides the telomeres shorten, and eventually the cell dies. Telomerase lengthens telomeres, allowing the cell to divide indefinitely. The number of cells used in therapy could be tailored to the patient. Individuals with primary lung disease may merely require the transplantation of an adequate number of cells to provide the AAT protein. Those with liver and lung disease will require enough cells to provide for adequate liver function as well as enough AAT protein for their lungs. Immortalized hepatocytes could potentially be used for direct liver transplantation or in a bioartificial liver. In preclinical studies, human fetal hepatocytes transduced with TERT have been in continuous culture for one and a half years, compared to controls which went into senescence at approximately 100 days. The immortalized cells were not found to be tumorigenic in mouse studies.

Another approach proposed by Dr. Juan Domínguez‐Bendala is the use of embryonic stem cells to create replacement hepatocytes. This is a promising new area, but without relevant publications thus far. The development of this approach will be hindered unless a suitable animal model of AAT deficiency can be developed. In the first report of its kind, Dr. Zern described the preliminary success of his research group, under the leadership of Dr. Hitoshi Shirahasi, in achieving the differentiation of human embryonic stem cells along a hepatocyte lineage.

Among other therapeutic approaches discussed by Dr. Sandhaus are synthetic elastase inhibitors, hyaluronic acid, retinoids and chaperone proteins. An endogenous polysaccharide in normal lung, hyaluronic acid is depleted in emphysema. In hamsters intratracheal administration of hyaluronic acid blocked the marked decrease in airspace enlargement after elastase instillment [[69]]. The protective effect of hyaluronic acid may reflect its ability to bind to lung elastic fibers, thus preventing elastase‐mediated breakdown. Retinoids are important in regulating lung growth and development, and in a rat model of emphysema all‐trans‐retinoic acid reversed the loss of lung elastic recoil and the reductions in numbers of alveoli and volume‐corrected alveolar surface area [[70]]. Protein chaperones might potentially reverse the cellular mislocalization or misfolding of mutant AAT. One chemical chaperone, 4‐phenylbutyric acid, increased secretion of functionally active AAT PiZ in a model cell culture system [[71]]. The diverse and numerous presentations at the meeting brought into focus the extensive efforts being made on many fronts to improve the understanding, diagnosis and treatment of AAT deficiency.

Appendix

Meeting Chairman

• Robert A. Stockley (UK, Chairman of AIR)

• Scientific Coordinators

• Marc Miravitlles (Spain)

• Niels Seersholm (Denmark)

• Scientific Committee

• Robert A. Stockley (UK)

• Marc Miravitlles (Spain)

• Maurizio Luisetti (Italy)

• Bruce C. Trapnell (USA)

• Niels Seersholm (Denmark)

• David A. Lomas (UK)

• Dino Hadzic (UK)

• Invited Speakers and Chairs

• Gill Ainsle (Cape Town, South Africa)

• Bruno Balbi (Pavia, Italy)

• Joan A. Barberà (Barcelona, Spain)

• Mark L. Brantly (Gainesville, Florida, USA)

• Edward Campbell (Salt Lake City, Utah, USA)

• Robin Carrell (Cambridge, UK)

• Morten Dahl (Copenhagen, Denmark)

• Asger Dirksen (Copenhagen, Denmark)

• Juan Domínguez‐Bendala (Miami, Florida, USA)

• Sarah Everett (Boston, Massachusetts, USA)

• Shane Fitch (Cádiz, Spain)

• Dino Hadzic (London, UK)

• John Humphries (Research Triangle Park, North Carolina, USA)

• Sabina Janciauskiene (Copenhagen, Denmark)

• Claes‐Goran Löfdhal (Lund, Sweden)

• David A. Lomas (Cambridge, UK)

• Maurizio Luisetti (Pavia, Italy)

• Marc Miravitlles (Barcelona, Spain)

• Eeva Piitulainen (Malmö, Sweden)

• Sally Plummer (Nottingham, UK)

• Erich Russi (Zurich, Switzerland)

• Jean‐Michel Sallenave (Edinburgh, Scotland)

• Sandy Sandhaus (Denver, Colorado, USA)

• Niels Seersholm (Copenhagen, Denmark)

• Bob Senior (St. Louis, Missouri, USA)

• Saher Shaker (Copenhagen, Denmark)

• Richard Sifers (Houston, Texas, USA)

• Edwin K. Silverman (Boston, Massachusetts, USA)

• Gordon L. Snider (Boston, Massachusetts, USA)

• Terry Spencer (Gainesville, Florida, USA)

• Robert A. Stockley (Birmingham, UK)

• Jan Stolk (Leiden, Netherlands)

• James K. Stoller (Cleveland, Ohio, USA)

• Charlie Strange (Charleston, South Carolina, USA)

• Tomas Sveger (Malmö, Sweden)

• Jeffrey H. Teckman (St. Louis, Missouri, USA)

• Bruce C. Trapnell (Cincinnati, Ohio, USA)

• Gerard M. Turino (New York, New York, USA)

• Claus Vogelmeier (Marburg, Germany)