Quantification of circulating alpha-1-antitrypsin polymers associated with different SERPINA1 genotypes.

OBJECTIVES: Alpha-1-antitrypsin deficiency is a genetic disorder caused by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin (AAT), the major serine protease inhibitor in plasma. Reduced AAT levels are associated with elevated risk of developing emphysema mainly due to uncontrolled activity of neutrophil elastase in the lungs. The prevalent Z-AAT mutant and many rare pathogenic AAT variants also predispose to liver disease due to their accumulation as polymeric chains in hepatocytes. Part of these polymers are secreted into the bloodstream and could represent biomarkers of intra-hepatic accumulation. Moreover, being inactive, they further lower lung protection against proteases. Aim of our study is to accurately quantify the percentage of circulating polymers (CP) in a cohort of subjects with different SERPINA1 genotypes. METHODS: CP concentration was measured in plasma or Dried Blood Spot (DBS) by a sensitive sandwich ELISA based on capture by the polymer-specific 2C1 monoclonal antibody. RESULTS: CP were significantly elevated in patients with the prevalent PI*SZ and PI*ZZ genotypes, with considerable intra-genotype variability. Notably, higher percentage of polymers was observed in association with elevated C-reactive protein. CP levels were also increased in carriers of the Mmalton variant, and of Mprocida, I, Plowell and Mherleen in heterozygosity with Z-AAT. CONCLUSIONS: These findings highlight the importance of implementing CP quantification in a clinical laboratory. Indeed, the variable amount of CP in patients with the same genotype may correlate with the variable severity of the associated lung and liver diseases. Moreover, CP can reveal the polymerogenic potential of newly discovered ultrarare AAT variants.

Comprehensive Clinical Diagnostic Pipelines Reveal New Variants in Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency (AATD) is an underdiagnosed disorder associated with mutations in the SERPINA1 gene encoding alpha-1 antitrypsin (AAT). Severe AATD can manifest as pulmonary emphysema and progressive liver disease. Besides the most common pathogenic variants S (E264V) and Z (E342K), many rarer genetic variants of AAT have been found in patients and in the general population. Here we report a panel of new SERPINA1 variants, including 4 null and 16 missense alleles, identified among a cohort of individuals with suspected AATD whose phenotypic follow-up showed inconclusive or atypical results. Because the pathogenic significance of the missense variants was unclear purely on the basis of clinical data, the integration of computational, biochemical, and cellular studies was used to define the associated risk of disease. Established pathogenicity predictors and structural analysis identified a panel of candidate damaging mutations that were characterized by expression in mammalian cell models. Polymer formation, intracellular accumulation, and secretory efficiency were evaluated experimentally. Our results identified two AAT mutants with a Z-like polymerogenic severe deficiency profile (Smilano and Mcampolongo) and three milder variants (Xsarezzo, Pdublin, and Ctiberias). Overall, the experimentally determined behavior of the variants was in agreement with the pathogenicity scores of the REVEL (an ensemble method for predicting the pathogenicity of rare missense variants) predictor, supporting the utility of this bioinformatic tool in the initial assessment of newly identified amino acid substitutions of AAT. Our study, in addition to describing 20 new SERPINA1 variants, provides a model for a multidisciplinary approach to classification of rare AAT variants and their clinical impact on individuals with rare AATD genotypes.

Improving the Laboratory Diagnosis of M-like Variants Related to Alpha1-Antitrypsin Deficiency.

Alpha1-antitrypsin (AAT) is a serine protease inhibitor that is encoded by the highly polymorphic SERPINA1 gene. Mutations in this gene can lead to AAT deficiency (AATD), which is associated with an increased risk of lung and/or liver disease. On the basis of electrophoretic migration, AAT variants are named with capital letters; M (medium) signifies the normal protein. Among pathological variants, the M-like ones represent a heterogeneous group of rare allelic variants that exhibit the same electrophoretic pattern as the M wild-type protein, which makes them difficult to detect with routine methods. In order to avoid their misdiagnosis, the present study defines and validates effective methods for the detection of two pathogenic M-like variants, Mwurzburg and Mwhitstable. Comparison of protein phenotypes using isoelectric focusing of samples that presented the Mwurzburg variant, as revealed by exons 5 sequencing, identified a particular electrophoretic pattern amenable to the Mwurzburg protein. The specific phenotyping pattern was retrospectively validated, thus enabling the detection of 16 patients with Mwurzburg variant among the subjects already tested but not sequenced according to our diagnostic algorithm. The Mwhitstable allele was detected by intron 4 sequencing of SERPINA1 gene. Mwurzburg and Mwhitstable are often misdiagnosed and the introduction of diagnostic improvements can help the clinical management, especially in patients with established lung disease without any other reported risk factors.

Comparison of different algorithms in laboratory diagnosis of alpha1-antitrypsin deficiency.

OBJECTIVES: Alpha1-antitrypsin deficiency (AATD) is an inherited condition that predisposes individuals to an increased risk of developing lung and liver disease. Even though AATD is one of the most widespread inherited diseases in Caucasian populations, only a minority of affected individuals has been detected. Whereas methods have been validated for AATD testing, there is no universally-established algorithm for the detection and diagnosis of the disorder. In order to compare different methods for diagnosing AATD, we carried out a systematic review of the literature on AATD diagnostic algorithms. METHODS: Complete biochemical and molecular analyses of 5,352 samples processed in our laboratory were retrospectively studied using each of the selected algorithms. RESULTS: When applying the diagnostic algorithms to the same samples, the frequency of False Negatives varied from 1.94 to 12.9%, the frequency of True Negatives was 62.91% for each algorithm and the frequency of True Positives ranged from 24.19 to 35.15%. We, therefore, highlighted some differences among Negative Predictive Values, ranging from 0.83 to 0.97. Accordingly, the sensitivity of each algorithm ranged between 0.61 and 0.95. We also postulated 1.108 g/L as optimal AAT cut-off value, in absence of inflammatory status, which points to the possible presence of genetic AATD. CONCLUSIONS: The choice of the diagnostic algorithm has a significant impact on the correct diagnosis of AATD, which is essential for appropriate treatment and medical care. The fairly large number of possible false negative diagnoses revealed by the present paper should also warn clinicians of negative results in patients with clinically-suspected AATD.

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant.

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

Electrophoretic α1-globulin for screening of α1-antitrypsin deficient variants.

Background Available screening procedures for the detection of α1-antitrypsin-deficient (AATD) mutations have suboptimal cost-effectiveness ratios. The aim in this study was to evaluate and compare the viability of a composite approach, primarily based on the α1-globulin fraction, in identifying AAT genetic analysis eligible patients against standard screening procedures, based on clinically compatible profiling and circulating AAT < 1 g/L. Methods A total of 21,094 subjects were screened for AATD and deemed eligible when meeting one of these criteria: α1-globulin ≤2.6%; α1-globulin 2.6%-2.9% and AST: >37 U/L and ALT: > 78 U/L; α1-globulin %: 2.9-4.6% and AST: >37 U/L and ALT: >78 U/L and erythrocyte sedimentation rate (ESR) >34 mm/h and C-reactive protein (CRP) >3 mg/L. Subjects were genotyped for the AAT gene mutation. Detection rates, including those of the rarest variants, were compared with results from standard clinical screenings. Siblings of mutated subjects were included in the study, and their results compared. Results Eighty-two subjects were identified. Among these, 51.2% were found to carry some Pi*M variant versus 15.9% who were clinically screened. The detection rates of the screening, including relatives, were: 50.5% for the proposed algorithm and 18.9% for the clinically-based screening. Pi*M variant prevalence in the screened population was in line with previous studies. Interestingly, 46% of subjects with Pi*M variants had an AAT plasma level above the 1 g/L threshold. Conclusions A composite algorithm primarily based on the α1-globulin fraction could effectively identify carriers of Pi*M gene mutation. This approach, not requiring clinical evaluation or AAT serum determination, seems suitable for clinical and epidemiological purposes.

Molecular diagnosis of alpha1-antitrypsin deficiency: A new method based on Luminex technology.

BACKGROUND: Alpha1-antitrypsin deficiency (AATD) is an under-diagnosed hereditary disorder characterized by reduced serum levels of alpha1-antitrypsin (AAT) and increased risk to develop lung and liver diseases at an early age. AAT is encoded by the highly polymorphic SERPINA1 gene. The most common deficiency alleles are S and Z, but more than 150 rare variants lead to low levels of the protein. To identify these pathological allelic variants, sequencing is required. Since traditional sequencing is expensive and time-consuming, we evaluated the accuracy of A1AT Genotyping Test, a new diagnostic genotyping kit which allows to simultaneously identify and genotype 14 deficiency variants of the SERPINA1 gene based on Luminex technology. METHODS: A total of 418 consecutive samples with AATD suspicion and submitted to the Italian Reference laboratory between January and April 2016 were analyzed both by applying the diagnostic algorithm currently in use, and by applying A1AT Genotyping Test. RESULTS: The assay gave the following results: 101 samples (24.2%) were positive for at least one of the 14 deficiency variants, 316 (75.6%) were negative for all the variants analyzed. The identified mutations showed a 100% correlation with the results obtained with our diagnostic algorithm. Seventeen samples (4%) resulted negative for the assay but sequencing identified other rare pathological variants in SERPINA1 gene. CONCLUSION: The A1AT Genotyping Test assay was highly reliable and robust and allowed shorter diagnostic times. In few cases, it has been necessary to sequence the SERPINA1 gene to identify other rare mutations not included in the kit.

Advances in Identifying Urine/Serum Biomarkers in Alpha-1 Antitrypsin Deficiency for More Personalized Future Treatment Strategies.

Alpha1-antitrypsin deficiency (AATD) is a genetic disorder characterized by reduced serum levels of alpha1-antitrypsin (AAT) and increased risk for developing both early-onset lung emphysema and chronic liver disease. Laboratory diagnosis of AATD is not just a matter of degree, although the AAT serum level is the most important determinant for risk of lung damage. While being a single-gene disease, the clinical phenotype of AATD is heterogeneous. The current standard of care for patients affected by AATD-associated pulmonary emphysema is replacement therapy with weekly i.v. infusions of pooled human purified plasma AAT. Although no treatment for liver disease caused by deposition of abnormal AAT in hepatocytes is available, innovative treatments for this condition are on the horizon. This article aims to provide a critical review of the methodological steps that have marked progress in the detection of indicators described in the literature as being "clinically significant" biomarkers of the disease. The development and routine use of specific biomarkers would help both in identifying which patients and when they are eligible for treatment as well as providing additional parameters for monitoring the disease.

Italian registry of patients with alpha-1 antitrypsin deficiency: general data and quality of life evaluation.

Alpha1-antitrypsin Deficiency (AATD) is a rare hereditary disorder with an estimated prevalence of about 1/5000 individuals in Italy. Deficient patients are at a higher risk of developing lung emphysema and chronic liver disease. The low estimated prevalence of AATD prompted the establishment of a registry with the aim of learning more about the natural history and the quality of care of these patients. The Italian registry for AATD was established in 1996. In this study, genetic and clinical findings of Italian AATD patients are presented. Moreover, we also evaluated the changes in health-related quality of life (HRQoL) in patients with COPD and AAT deficiency over a three-year period, in relation to augmentation therapy. In a period spanning 18 years (1996-2014) a total of 422 adult subjects with severe AATD were enrolled, namely 258 PI*ZZ, 74 PI*SZ, 4 PI*SS and 86 patients with at least one rare deficient allele. The 21.3% frequency for AATD patients with at least one deficient rare variant is the highest so far recorded in national registries of AATD. The registry data allow a detailed characterization of the natural course of the disease and the level of patient care, as well as confirm the usefulness of early AATD detection.

Sanger and Next-Generation Sequencing of AAT.

Sequencing of DNA is normally the final procedure carried out to determine the actual pathogenic variants when the techniques used for genotyping are unable to provide complete identification of both AAT alleles. Gene sequencing of complete SERPINA1 gene by using the Sanger method or next-generation sequencing (NGS) is crucial to enable correct diagnosis in patients with alpha1-antitrypsin deficiency caused by uncommon AAT variants.This protocol explains how to correctly sequence SERPINA1 gene both with Sanger method and NGS.

Comparison among populations with severe and intermediate alpha1-antitrypsin deficiency and chronic obstructive pulmonary disease.

BACKGROUND: Severe alpha1-antitrypsin (AAT) deficiency (AATD) is associated with a high risk of airflow obstruction and emphysema. The risk of lung disease in those with intermediate AAT deficiency is unclear. Our aims were to compare pulmonary function, time of onset of symptoms, and indicators of quality of life among patients with severe AATD (PI*ZZ), patients with intermediate AATD (PI*MZ) from the Italian Registry of AATD with a chronic obstructive pulmonary disease (COPD) cohort of patients without AATD (PI*MM). METHODS: We considered a total of 613 patients: 330 with the PI*ZZ genotype, 183 with the PI*MZ genotype and 100 with the PI*MM genotype. Radiological exams, pulmonary function test, and measurement of quality of life have been performed on all cohorts of patients. RESULTS: The three populations differ significantly in terms of age at COPD/AATD diagnosis (P=0.00001), respiratory function (FEV1, FVC, DLCO P<0.001), quality of life (P=0.0001) and smoking history (P<0.0001). PI*ZZ genotype had 24.9 times a higher likelihood of developing airflow obstruction. The MZ genotype is not associated with a significant early risk of airflow obstruction. CONCLUSIONS: The comparison of populations with PI*ZZ, MZ and MM genotypes allows to delineate the role of alpha1-antitrypsin deficiency on respiratory function and on the impact on quality of life, in relation to other risk factors. These results highlight the crucial role of primary and secondary prevention on smoking habits in PI*MZ subjects and the importance of an early diagnosis.

Clinical phenotypes of Italian and Spanish patients with α1-antitrypsin deficiency.

With the aim of providing better clinical characterisation of patients with α1-antitrypsin deficiency (AATD), we analysed the data of adult patients with severe AATD enrolled in the Spanish and Italian national registries. We assessed 745 subjects, 416 of whom were enrolled in the Spanish registry and 329 in the Italian registry. 57.2% were male and 64.9% were smokers or former smokers with a mean±sd age of 49.9±13.8 years. Most (81.2%) were index cases, mainly having the PI*ZZ genotype (73.4%), and the mean±sd diagnostic delay was 9.0±12.1 years. Patients with chronic bronchitis were younger, had better preserved lung function and lower tobacco consumption. Overlap patients (chronic obstructive pulmonary disease with asthma) were mainly females, more frequently never-smokers and received respiratory medications more often. 48% of emphysema, 27.5% of chronic bronchitis and 44.8% of overlap subjects were receiving augmentation therapy. Compared with PI*ZZ patients (n=547), the PI*SZ (n=124) subjects were older at diagnosis and had more preserved lung function, despite a higher mean smoking consumption. Early diagnosis of AATD is still an unmet need. Augmentation therapy is administered to similar proportions of patients with different clinical phenotypes. PI*ZZ patients in both registries had more severe respiratory disease than those with PI*SZ, despite lower smoking levels.

Serum levels and genotype distribution of α1-antitrypsin in the general population.

RATIONALE: α1-Antitrypsin (AAT) deficiency is one of the commonest rare respiratory disorders worldwide. Diagnosis, assessment of risk for developing chronic obstructive pulmonary disease (COPD), and management of replacement therapy require the availability of precise and updated ranges for protein serum levels. OBJECTIVE: This paper aims to provide ranges of serum AAT according to the main genotype classes in the general population. METHODS: The authors correlated mean AAT serum levels with the main SERPINA1 variants (M1Ala/M1Val (rs6647), M3 (rs1303), M2/M4 (rs709932), S (rs17580) and Z (rs28929474)) in 6057 individuals enrolled in the Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) cohort. RESULTS: The following ranges (5th-95th percentile) of AAT were found in the serum (g/litre): 1.050-1.640 for PI*MM, 0.880-1.369 for PI*MS, 0.730-1.060 for PI*SS, 0.660-0.997 for PI*MZ and 0.490-0.660 for PI*SZ. There was very little overlap in AAT serum levels between genotype classes generally not believed to confer an enhanced health risk (MM and MS) and those associated with an intermediate AAT deficiency and a potentially mildly enhanced health risk (SS, MZ). CONCLUSION: This work resulted in three important findings: technically updated and narrower serum ranges for AAT according to PI genotype; a suggestion for a population-based 'protective threshold' of AAT serum level, used in decision-making for replacement therapy; and more precise ranges framing the intermediate AAT deficiency area, a potential target for future primary prevention.

SERPINA1 gene variants in individuals from the general population with reduced alpha1-antitrypsin concentrations.

BACKGROUND: Individuals with severe deficiency in serum alpha(1)-antitrypsin (AAT) concentrations are at high risk for developing chronic obstructive pulmonary disease (COPD), whereas those carrying the PI*MZ genotype are at slightly increased risk. Testing appropriate subgroups of the population for AAT deficiency (AATD) is therefore an important aspect of COPD prevention and timely treatment. We decided to perform an exhaustive investigation of SERPINA1 gene variants in individuals from the general population with a moderately reduced serum AAT concentration, because such information is currently unavailable. METHODS: We determined the Z and S alleles of 1399 individuals enrolled in the Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) with serum AAT concentrations < or = 1.13 g/L and submitted 423 of these samples for complete exon 2-->5 sequencing. RESULTS: We found that 900 of 1399 samples (64%), carried the normal PI*MM genotype, whereas 499 samples (36%) carried at least 1 SERPINA1 deficiency variant. In the subpopulations in which AAT concentrations ranged from > 1.03 to < or = 1.13 and from > 0.93 to < or = 1.03 g/L, individuals with the PI*MM genotype represented the majority (86.5% and 53.8%, respectively). The PI*MS genotype was predominant (54.9%) in the AAT range of 0.83 to 0.93 g/L, whereas PI*MZ represented 76.4% in the AAT range of > 0.73 to < or = 0.83 g/L. CONCLUSIONS: This analysis provided a detailed molecular definition of intermediate AATD, which would be helpful in the diagnostic setting.

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 prevent secretion of α1-antitrypsin (α1-AT) and cause predisposition to pulmonary and liver diseases. The heterogeneity of clinical manifestations in AATD is related to the complexity of biological function of α1-AT. 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. Lung damage progression in AATD can also be related to body mass index, exacerbation rate, sex, environmental exposure and specific mutations of SERPINA1. Recent randomised controlled trials, together with previous observational work, have provided compelling evidence for the importance of early detection and intervention in order to enable patients to receive appropriate treatment and preserve functional lung tissue.

The pathological Trento variant of alpha-1-antitrypsin (E75V) shows nonclassical behaviour during polymerization.

Severe alpha-1-antitrypsin deficiency (AATD) is most frequently associated with the alpha-1-antitrypsin (AAT) Z variant (E342K). ZZ homozygotes exhibit accumulation of AAT as polymers in the endoplasmic reticulum of hepatocytes. This protein deposition can lead to liver disease, with the resulting low circulating levels of AAT predisposing to early-onset emphysema due to dysregulation of elastinolytic activity in the lungs. An increasing number of rare AAT alleles have been identified in patients with severe AATD, typically in combination with the Z allele. Here we report a new mutation (E75V) in a patient with severe plasma deficiency, which we designate Trento. In contrast to the Z mutant, Trento AAT was secreted efficiently when expressed in cellular models but showed compromised conformational stability. Polyacrylamide gel electrophoresis (PAGE) and ELISA-based analyses of the secreted protein revealed the presence of oligomeric species with electrophoretic and immunorecognition profiles different from those of Z and S (E264V) AAT polymers, including reduced recognition by conformational monoclonal antibodies 2C1 and 4B12. This altered recognition was not due to direct effects on the epitope of the 2C1 monoclonal antibody which we localized between helices E and F. Structural analyses indicate the likely basis for polymer formation is the loss of a highly conserved stabilizing interaction between helix C and the posthelix I loop. These results highlight this region as important for maintaining native state stability and, when compromised, results in the formation of pathological polymers that are different from those produced by Z and S AAT.

Case-finding for alpha1-antitrypsin deficiency in Kazakh patients with COPD.

BACKGROUND: Alpha-1-antitrypsin deficiency (AATD) is an under-diagnosed condition in patients with chronic obstructive pulmonary disease (COPD). The aim of this study was to screen for AATD in Kazakh patients with COPD using dried blood spot specimens. METHODS: The alpha1-antitrypsin (AAT) concentration was determined by nephelometry, PCR was used to detect PiS and PiZ alleles; and isoelectric focusing was used to confirm questionable genotype results and detect rare AAT variants. RESULTS: To this aim, 187 Kazakh subjects with COPD were recruited. Blood samples were collected as dried blood spot. Genotyping of 187 samples revealed 3 (1.6%) PI*MZ and 1 (0.53%) PI*MS, Phenotyping identified also two sample (1.1%) with phenotype PiMI. Allelic frequencies of pathological mutations Z, S and I resulted 0.8%, 0.3%, 0.5%, respectively, in COPD Kazakh population. CONCLUSION: This study proved that AATD is present in the Kazakh population. These results support the general concept of targeted screening for AAT deficiency in countries like Kazakhstan, with a large population of COPD patients and low awareness among care-givers about this genetic condition.

NOD2/CARD15 gene polymorphisms in idiopathic pulmonary fibrosis.

BACKGROUND: Micro-organisms, behaving in a non-infectious fashion, may be among the exogenous factor(s) believed to trigger idiopathic pulmonary fibrosis (IPF). One possible strategy to identify an individual's susceptibility to such microbial triggers, which are likely to be ubiquitous, is to investigate the molecular processes involved in their recognition. NOD2/CARD15 is a specific pattern recognition receptor protein, whose genetic variants have been previously associated with susceptibility to Crohn's disease. AIM: The aim of this work was to determine the frequencies of the three major NOD2/CARD 15 gene mutations (R702W, G908R and 1007fsinC) in a series of 76 subjects affected by IPF, and to compare them with those found in three groups of controls: a group with sarcoidosis (a disorder in which an involvement of the NOD2/CARD15 gene has already been investigated and rejected in different ethnic groups; 67 subjects) and two groups of healthy subjects (218 and 208 subjects, respectively), matched for gender, age, and ethnicity. RESULTS: We found no differences in frequencies of NOD2/CARD15 gene polymorphisms among the four groups investigated. CONCLUSION: We conclude that the NOD2/CARD15 gene is not likely to be involved in susceptibility to IPF in Italians.

How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

The Z deficiency in α1-antitrypsin (A1ATD) is an under-recognized condition. Alpha1-antitrypsin (A1AT) is the main protein in the α1-globulin fraction of serum protein electrophoresis (SPE); however, evaluation of the α1-globulin protein fraction has received very little attention. Serum Z-type A1AT manifests in polymeric forms, but their interference with quantitative immunoassays has not been reported. Here, 214 894 samples were evaluated by SPE at the G. Fracastoro Hospital of Verona, Italy. Patients with an A1AT level ≤ 0.92 g/L were recalled to complete A1ATD diagnosis. In parallel, to qualitatively and quantitatively characterize A1AT, sera samples from 10 PiZZ and 10 PiMM subjects obtained at the National Institute of Tuberculosis and Lung Diseases in Warsaw, Poland, were subjected to non-denaturing 7.5% PAGE and 7.5% SDS-PAGE followed by Western blot. Moreover, purified A1AT was heated at 60°C and analyzed by a non-denaturing PAGE and 4-15% gradient SDS-PAGE followed by Western blot as well as by isolelectrofocusing and nephelometry. A total of 966 samples manifested percentages ≤ 2.8 or a double band in the alpha1-zone. According to the nephelometry data, 23 samples were classified as severe (A1AT ≤ 0.49 g/L) and 462 as intermediate (A1AT >0.49≤ 1.0 g/L) A1ATD. Twenty subjects agreed to complete the diagnosis and an additional 21 subjects agreed to family screening. We detected 9 cases with severe and 26 with intermediate A1ATD. Parallel experiments revealed that polymerization of M-type A1AT, when measured by nephelometry or isolelectrofocusing, yields inaccurate results, leading to the erroneous impression that it was Z type and not M-type A1AT. We illustrate the need for confirmation of Z A1AT values by "state of the art" method. Clinicians should consider a more in-depth investigation of A1ATD in patients when they exhibit serum polymers and low α1-globulin protein levels by SPE.