Definition
Alpha-1 antitrypsin deficiency is a genetic diseas affecting the lung and liver and it is defined by a reduced concentration of alpha-1 antitrypsin (AAT) in the serum and/or by the identification of a defective genotype. Although AAT is mainly produced in the liver, its main function is to protect the lung against proteolytic damage from neutrophil elastase. The AATD is caused by various mutations in the SERPINA1 gene that determine polymerization and accumulation of AAT protein within hepatocytes. The accumulation and the consequent reduction in the serum levels of AAT cause liver and lung disease, and the latter occurring mainly as early emphysema.
Figure 1. Schematic model of pathophysiology of Alpha-1 Antitrypsin Deficiency
Genetics and biology
Alpha-1 antitrypsin deficiency is an autosomal recessive disease. The SERPINA1 gene (gene size 12.2 kb), which encodes the alpha-1 antitrypsin protein, is located on chromosome 14 (14q31-32.3). The gene is highly pleomorphic, and until today more than 100 allelic variants have been identified. The variants can broadly be classified according to their effects on levels of serum alpha-1 antitrypsin protein. The M alleles (M1 to M6) are defined like “normal variants” because they are associated with normal serum AAT protein levels. Disease manifestation is associated with null variants or genotypes, resulting in impaired gene expression or protein synthesis. The characteristics of the common variants are summarised in Table 1.
The majority of individuals with lung or liver disease are homozygous for the alleles Z or S (ZZ and SS phenotype), or heterozygous (MS, MZ, or SZ phenotype), all of which result in diminished serum AAT levels.
Pathophysiology
The most common variant protein of AAT to cause severe clinical disease is the Z variant, characterized by the mutation Glu342Lys, that results in abnormalities in the tertiary structure of the protein. The S variant of AAT, however, is characterized by a single amino acid mutation in position 264 in the protein where a glutamine is replaced by a valine aminoacid: the homozygous S variant of alpha-1-antitrypsin results in an instable protein that is easily degraded outside the hepatocyte and affects the half-life of the S variant.
The Z variant, however, have consequences at several levels, including gene deletion, degradation of unstable mRNA transcripts, aggregation of AAT in the rough endoplasmatic reticulum (RER), degradation of the AAT protein before translocation to the Golgi complex, and release AAT. Protein synthesis in the RER of hepatocytes is delayed, so that approximately 85% of synthesized molecules polymerize into large conglomerates: these polymers cannot be processed further and accumulate in the RER (Figure 2).
Figure 2. a) Folding and active state of Alpha-1 antitrypsin. b) Molecular mechanism of polymerisation determined by misfolding of Alpha-1 antitrypsin protein in AATD
Only a few nonpolymerized molecules are released into the blood. In individuals with the ZZ genotype, the antiproteolytic activity of AAT against the most important substrate, neutrophil elastase, is approximately 5 times less than normal AAT levels.
In hepatocytes, continuous accumulation of AAT molecules may result in cell injury and later in cell death. Patients with AATD have a highly increased risk of liver fibrosis and cirrhosis. The amount of accumulated polymerized proteins correlates with the stage of the cirrhosis.
In the lung tissue, the balance between proteases and antiproteases is alterated: the majority of the released proteases remain active and slowly destroy lung matrix components, alveolar structures, and blood vessels: the progressive destruction results in chronic obstructive bronchitis and lung emphysema (Figure 3).
Figure 3. Model of the molecular and cellular basis of emphysema in AATD
Epidemiology
Alpha1-antitrypsin deficiency (AATD) is a diseas that affects about one in 2000-5000 individuals. Women and men are affected in equal numbers. Prevalence estimates for typical deficiency genotypes of the disease are presented in Table 2.
In all countries, however, the number of clinically identified patients is less than the anticipated prevalence based on allele frequencies: it is estimated that from 10% to 35% of individuals with homozygous ZZ genotypes do not exhibit clinical symptoms. The reasons underlying the lack of a direct relationship between the genotype and the phenotype are not understood.
Clinical manifestation
The AATD is a metabolic disorder that predisposes the affected individual to chronic pulmonary disease, in addition to chronic liver disease, cirrhosis, and hepatocellular carcinoma. Clinical manifestations are always present in patients with complete absence of serum alpha-1 antitrypsin (null variants). The majority of patients with ZZ or SZ genotypes, and some others with the SS genotype, have pulmonary or hepatic symptoms. Severe lung and liver disease are rarely observed in the same person. Heterozygous individuals, with both a normal and a variant allele (MZ or MS), rarely develop clinical symptoms. In most patients with symptomatic AATD, the dominant manifestation is lung disease: the symptoms appear earlier and may proceed faster if additional risk factors are present, like smoke or air pollutants. The mean life expectancy of homozygous patients (ZZ and SS variant) is from 48 to 52 years for smokers and from 60 to 68 years for nonsmokers. Severe pulmonary impairment, manifesting as COPD (chronic obstructive pulmonary disease) and panacinar lung emphysema, may be expected if the AAT serum concentration is below the protective threshold of 35% of the normal mean value (≤0.8 g/L or ≤11 μmol/L). Pulmonary symptoms (cough, sputum expectoration, and exertional dyspnea) may occur by the age of 30 to 40 years.
Hepatic symptoms manifest initially as hepatitis, followed later by fibrosis or cirrhosis. The underlying cause may be the intrahepatic accumulation of polymerized alpha-1 antitrypsin molecules. Subsequent intrahepatic cholestasis may lead to diminished resorption of lipids and lipophilic vitamins. The incidence of primary liver cell carcinoma is higher than in other hepatic diseases.
Diagnosis
The complete laboratory diagnosis of AATD is based on combination of quantitative and qualitative methods. The main method of diagnosis is the detection of low serum levels of AAT as well as phenotypic confirmation. Because of AAT is an acute-phase protein, its synthesis may be up-regulated during all states of inflammation. It is therefore recommended that C-reactive protein and AAT levels are determined simultaneously, and that AAT concentration results are rejected if C-reactive protein levels are abnormal. Because of the variability in the sensitivity of serum electrophoresis, estimation of the AAT concentration using this method is less reliable than direct measurement of the serum concentrations. The measurement of plasma/serum AAT concentration is always the initial test performed in clinically suspected individuals. Nevertheless, only the AAT phenotype or genotype identification allows the full medical verification of the diagnosis.
Among the various techniques of either AAT variant phenotyping or genotyping accepted by reference medical centers worldwide,
- the isoelectric focusing
- real-time-PCR
- restriction fragment-length polymorphism PCR (RFLP-PCR)
represent the best diagnostic tests for screening ATTD. Usually the search for the two major mutations (Z and S alleles) is carried out by PCR amplification, from genomic DNA, and direct sequencing of exon 3 (variant S) and 5 (variant Z) of the AAT gene.
The main indications for AATD screening are newborns with prolonged jaundice or nonspecific signs of liver disease, patients with chronic pulmunary disease (COPD, bronchial asthma, bronchiectasis, immotilecilia syndrome, frequently recurring airway infections) or with chronic liver disease. Genetic counselling for affected families is recommended, as well as tests to identify relatives at risk. Prenatal diagnosis is possible, but may not predict the future severity of the disease.
Treatment
At this time, there is no cure for AATD, but there are treatments that can improve symptoms. The principal medical treatment is similar to the usual treatment for patients with COPD and consists of chronic application of the long-acting beta-2 agonists, formoterol or salmeterol, combined with the long-acting anticholinergic tiotropium. Lung transplantation may be an option in more severe clinical cases. Staying away from cigarette smoke is crucial. In addition to this standard treatment, specific therapy consisting of infusion of purified alpha-1 antitrypsin is currently available.
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Chemical structure and synthesis
Alpha-1 antitrypsin is a 52-kDa protein composed of 394 aminoacids. It belongs to the group of Serpins and it is predominantly produced in the liver and released into the blood. It is synthesized within the endoplasmic reticulum (RER) of hepatocytes, where it undergoes multiple complex foldings and insertions of carbohydrate side (the AAT includes 3 glycosylated side chains coupled to asparagine). The molecule has a globular tertiary structure, and its reactive site loop, that contains the neutrophil elastase binding site to inhibit enzymes, is on a surface protrusion (Figure 4).
Figure 4. Three-dimensional model of Alpha-1 antitrypsin
Here is where the most important amino acid is present, a methionine in position 358, an aminoacid susceptible to conversion to methionine sulfoxide by oxidants from cigarette smoke, rendering it much less potent as an inhibitor of neutrophil elastase.
Protein Aminoacids Percentage
Physiologic function
The normal daily production of AAT is approximately 34 mg/kg, and the physiologic serum concentration for adults ranges from 1.5 to 3.0 g/L (20-52 μmol/L). Under normal conditions, AAT is constitutively produced and, like an acute phase protein, it is up-regulated during inflammation, infection, cancer and pregnancy. The most important physiologic function of AAT occours in pulmonary tissue. The large surface of the lung is continuously exposed to a high burden of airborne pathogens, resulting in frequent cellular immune responses: during the resultant process of phagocytosis, proteases and oxidants are released into the adjacent lung tissue. The most important protease is neutrophil elastase, which has a high potential to destroy lung matrix components: the AAT inactives this proteolytic enzymes, therefore protecting the lung tissue (Figure 5).
Figure 5. Action of Alpha-1 antitrypsin in the lung tissue
In addition to its antiprotease activity, AAT seems to have an important role in the regulation of inflammatory processes in the lung, with anti-inflammatory action: it may inhibit immune responses, stimulate tissue repair and matrix production, and have antibacterial activities.
Acquired Alpha-1 Antitrypsin deficiency
- HNF1 reduced activity is a transcriptional activator of many hepatic genes including
- albumin
- alpha1-antitrypsin
- alpha- and beta-fibrinogen
