The ten years following this discovery were marked with events of a lifetime for alpha1-antitrypsindeficiency (AATD) in Sweden. In his captivating review , Robin Carrell told how the original description of Laurell & Eriksson promoted an extraordinary, lively and productive environment in Sweden, and in Malmö in particular. Kjell Ohlsson, Jan Olof Jeppson, Magne Fagerhöl and Diane Cox, the latter arriving in Malmö from Norway and Canada, respectively, focused their work on the explanation of the complex electrophoretic heterogeneity of AAT, eventually contributing to the development of Pi nomenclature for AAT variants [6-8]. In the meantime, Christeer Larson provided evidence for the interaction of smoking with AATD , thus contributing to the current oxidation stress/proteinase imbalance hypothesis of the pathogenesis of emphysema, and Tomas Sveger performed the Swedish newborn national screening for
Introduction: COPD has complex etiologies involving both genetic and environmental determinants. Among genetic determinants, the most recognized is a severe PiZZ (Glu342Lys) inherited alpha1-antitrypsindeficiency (AATD). Nonetheless, AATD patients present a heterogeneous clinical evolution, which has not been completely explained by sociodemographic or clinical factors. Here we performed the gene expression profiling of blood cells collected from mild and severe COPD patients with PiZZ AATD. Our aim was to identify differences in messenger RNA (mRNA) and microRNA (miRNA) expressions that may be associated with disease severity.
In our study of 300 COPD patients 20 were identified as having AAT deficiency (group1), 160 with intermediate level of AAT (group2) and 120 having normal concentration (group3). Among the deficient subjects 25% had PiMZ phenotypes followed by PiZZ, SZ and SS with percentage of 10%, 50% and 15%, respectively. People who have two deleterious AAT alleles such as Z, S are at risk for diseases related to AAT deficiency as a results of the lower level of circulating AAT protease inhibitor [5, 6]. The mean of AAT concentration in this group was found to be 0.5 g/l which explained the reduction in pulmonary functions in this group. The obtained results were in agreement with those reported that the threshold of pulmonary damage is less than or equal to 60 mg/dl of AAT concentration [20-22].
An important fraction of COPD cases harbor a major genetic determinant, inherited ZZ (Glu342Lys) α1- antitrypsindeficiency (AATD). AAT is an acute phase protein, main inhibitor of neutrophil elastase and a modulator of host inflammatory responses . It is well established that the Z variant of AAT forms polymers and is retained in the endoplasmic reticulum (ER) of the hepatocytes  which leads to markedly impede (up to 90%) levels of the circulating AAT protein. The lack of AAT predisposes a person to develop early onset, rapidly progressive COPD where emphysema is a major compo- nent  whereas the accumulation of abnormally folded AAT protein increases the risk to develop chronic liver disease. Cirrhosis in ZZ AATD individuals may become clinically apparent at any age, with the peak incidence typically occurring in elderly never-smokers who have survived without developing severe emphysema .
Estimation of alpha1-antitrypsin enzyme was done using serum samples of patients who were clinically diagnosed with (COPD), smokers who have a smoking history of at least 10 pack years and normal healthy individuals. The quantification of AAT enzyme was done using two methods, Immune Nephlometry (BN Prospec, Siemens, Erlangen, Germany) and sandwich enzyme linked immune sorbent assay (IRE 96 Reader, SFRI, France). The results of both methods were compared. Immune nephlometric readings were higher than that of ELISA (Figure 1). Both techniques are immune complex reactions, ELISA seemed to be more sensitive.
We investigated the molecular behaviour of the novel AATD- associated variants by expressing them in liver-derived cells (Hepa1.6) and performing pulse-chase experiments (Figure 2). As controls we included M and Z AAT, as well as Mwurzburg (Pro369Ser), a previously described AAT deficiency variant whose amino acid substitution occurs next to that of Etaurisano . As expected, the newly synthesized M AAT was present at the end of the pulse period in the NP40-soluble cell fraction as a single band of about 50 kD, which corresponds to the high mannose intermediate form (Figure 2A, arrowhead). During the chase period, the protein is converted to a fully glycosylated mature protein (approx. 54 kD) that is rapidly secreted into the medium (Figure 2A, arrow). Only a small amount of M AAT was transiently found as immature polypeptide in the NP40- insoluble cell fraction. On the other hand, the newly synthesized Z protein was mainly retained in the cells as immature and significantly accumulated in the NP40-insoluble fractions, confirming its tendency to form large insoluble complexes (Figure 2A). The novel variants showed a reduction of protein secretion and were found in the NP40-insoluble fraction, suggesting that they formed large insoluble intracellular com- plexes similarly to Z AAT, although to different extents. The AAT distribution at different time points in the intracellular soluble and insoluble fractions, as well as in the cell media, was quantified by densitometry and expressed as percentages of the total AAT amount found at time 0 of chase (Figure 2B). At 240 min of chase, when about 78% of the wild-type M is found in the culture medium, the different mutants show reduced secretion (Z 16%, Mwurzburg 27%, Etaurisano 52%, Mpisa 22% and Yorzinuovi 11%). At the same time point, when the wild-type M is undetectable in the NP40-insoluble fraction, all the mutants show an accumulation of detergent insoluble complexes (Z 21%, Mwurzburg 13%, Etaurisano 13%, Mpisa 13% and Yorzinuovi 43%).
In this study, we extended the SERPINA1 gene in- vestigation to variants not detectable by PI*S and PI*Z genotyping. With this strategy, we found two samples with the rare deficiency variant I (frequency 1.1%). The I mutation occurs at the residue, arginine 39, which is involved in the formation of an ionic bond with glutamic acid 264 . Although no subjects with severe AATD deficiency were detected in this study, we found six out of 187 (5%) subjects positive for so called “ intermediate genetic AAT deficiency ” [mean (SD) AAT level: 0.85 g/L (0.17)]. These findings are consistent with the hypothesis that intermediate Table 3 Demographic and clinical data of patients with AAT deficiency
Dr Abboud has participated in two advisory board meetings for Talecris Biotherapeutics. He has also given three presentations on alpha1-antitrypsindeficiency sponsored by Talecris to Asthma & COPD Educators in Vancouver, to Respiratory Therapists in Edmonton, and to Respira- tory Physicians at the University of Alberta in Edmonton in 2008. Lastly, as of 2008, he has been participating as a local principal investigator in Vancouver in a multicenter clinical trial evaluating weekly IV infusions of a purified human alpha1-antitrypsin preparation in treating emphysema due to severe antitrypsindeficiency, sponsored by CSL Behring Biotherapies for Life.
Reports published from the prospective follow-up study of ZZ and SZ individuals up to age 26 years, focusing on clinical health, lung and liver function tests and plasma markers of the protease/protease inhibitor balance, have shown that ZZ and SZ subjects had significantly higher plasma concentrations of α 2-MG, ACT and antithrombin III  at age 8 and 18 compared with MM control sub- jects. Recent findings at age 26 for ZZ and SZ subjects with normal lung function and only marginal deviations in liver test results showed significantly higher plasma SLPI levels, but not α 2-MG, compared to age matched healthy MM subjects . In contrast, at age 31, we find in this study that ZZ and SZ individuals have no significant dif- ference in plasma SLPI and ACT levels compared to MM controls. The higher plasma α2-MG, SLPI and ACT levels reported in AAT deficiency subjects at younger ages were previously attributed to an unidentified compensatory
inhibitory activity. It might represent a physiologic mech- anism of regulating α 1 -AT activity, especially in acute con- ditions (for example during defense against infections agents) , in which an excess of α 1 -AT would interfere with the physiologic role of proteinases. α 1 -AT is indeed a highly specialised proteinase inhibitor , but the pres- ence in nature of several, robust mechanisms of α 1 -AT downregulation (i.e. inherited deficiency, susceptibility to oxidative stress and proteolysis, polymerization) would imply the occurrence of intrinsic risks related to the over- expression of a nearly perfect and immortal inhibitor. Therefore, the formation of supramolecular complexes SP-A/ α 1 -AT might be a sort of reserve mechanism, taking place in case of need.
Alpha-1 antitrypsin (AAT) deficiency is a hereditary dis- order first reported in the early 1960s when emphysema was described in patients with low plasma levels of AAT protein . The condition is associated with substantially increased risk for the development of pulmonary emphy- sema by the third or fourth decades of life and is also associated with risks for development of hepatic disease , cutaneous panniculitis , bronchiectasis , vasculi- tis , Wegener ’ s granulomatosis , and lung cancer . AAT deficiency is characterised by misfolding of the AAT protein and belongs to a class of genetic diseases termed conformational disorders .
The combination of alpha-1 antitrypsin (AAT) deficiency, ANCA-vasculitis, and aortic aneurysm has been rarely described in literature. We report an eventually fatal case in a 70-year-old patient who initially presented with giant cell arteritis and ANCA associated glomerulonephritis. Several years later, he presented with aortic dissection due to large vessel vasculitis, raising the suspicion of AAT deficiency, as two first-line relatives had chronic obstructive pulmonary disease, while they never smoked. This diagnosis was confirmed by AAT electrophoresis and immunohistochemistry on a temporal artery biopsy. Considering AAT deficiency in these cases might lead to a more timely diagnosis.
The SERPINA1 gene is highly pleiomorphic with over 100 alleles identified to date . Mutations which confer an increased risk of developing pulmonary emphysema and/or liver disease are those in which deficiency alleles are combined in homozygous or heterozygous states, yielding AAT serum levels below a putative protective threshold of 11 μ M. The most common variants asso- ciated with disease are the Z (Glu342Lys) and S (Glu264- Val) mutations, caused by a single amino acid replacement of glutamic acid at positions 342 and 264 of the polypep- tide, respectively . The class of SERPINA1 variants termed “ null ” mutations lead to a complete absence of AAT production and while extremely rare, confer a parti- cularly high risk of emphysema .
allele is associated with severe AATD with serum levels ranging between ⩽ 29 and 52 mg·dL −1 . Controversial results have been published revealing chronic obstructive pulmonary disease (COPD) risk among protease inhibitor (Pi)MZ heterozygotes. A normal protease/antiprotease balance exists in healthy (MM) individuals, in which high levels of MAAT surround the resting neutrophil . During exposure to increasing levels of interleukin (IL)-8 the cell moves down a concentration gradient of AAT and up a gradient of IL-8, thus leading to neutrophil migration to the area of inflammation . Nonsmoking MZ individuals have intermediate levels of AAT and increased sputum IL-8 levels and neutrophil counts. In AATD individuals homozygous for the Z allele, low levels of circulating ZAAT surround the neutrophil and the described AAT gradient is grossly disrupted, resulting in increased chemotactic responsiveness of neutrophils with an overwhelmed anti-protease defence that contributes to the development of COPD. In smoking MZ individuals, reactive oxygen species in cigarette smoke inactivate AAT, resulting in a protease/antiprotease imbalance with increased amounts of neutrophil elastase. Polymerisation of ZAAT protein and increased amounts of IL-8 intensify neutrophil influx into the MZ lung, which could facilitate the development of COPD [2, 8]. In fact, ZAAT polymers found within lungs can also fuel inflammation in the AAT deficiency . Since ZAAT polymers are chemotactic for neutrophils, co-localisation can trigger the release of myeloperoxidase and upregulation of neutrophil adhesion molecules . It has been found that ZAAT polymers co-localise with neutrophils in the alveoli of patients with AAT deficiency .
deficiency of the chemotactic factor inactivator. When normal human serum and a 1 -AT- deficient human sera are chemotactically activated by incubation with immune precipitates, substantially more chemotactic activity is generated in a 1 -AT-deficient serum. These data indicate that in a 1 -AT-deficient serum there is an imbalance in the generation and control of chemotactic factors. It is suggested that the theory regarding development of pulmonary emphysema in patients lacking the a 1 -antitrypsin in their serum should be modified to take into account a deficiency of the chemotactic factor inactivator.
gressive familial intrahepatic cholestasis (1, 2, 3, and 4), citrin deficiency, bile acid synthetic defects, alpha-1-antitrypsindeficiency, Wolman disease, neonatal ichthyosis-sclerosing cho- langitis syndrome, cerebrotendinous xantho- matosis, mitochondrial DNA Depletion syndro- me, hereditary tyrosinemia type I, galactose- mia, fructose intolerance, cystic fibrosis, and ARC syndrome were ruled out by the next gen- eration sequencing (NGS) of a panel of 41 genes known to cause genetic cholestasis disorders in children.