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Background: Alpha-1 antitrypsin deficiency (AATD) is characterized by reduced serum levels of the AAT protein and predisposes to liver and lung disease. The characterization at structural level of novel pathogenic SERPINA1 mutants coding for circulating AAT could provide novel insights into the mechanisms of AAT misfolding. The present study aimed to provide a practical framework for the identification and analysis of new AAT mutations, combining structural simulations and clinical data., Methods: We analysed a total of five mutations (four not previously described) in a total of six subjects presenting moderate to severe AATD: Gly95Alafs*18, Val210Glu, Asn247Ser, Pi*S + Asp341His and Pi*S + Leu383Phe + Lys394Ile. Clinical data, genotyping and phenotyping assays, structural mapping, and conformational characterization through molecular dynamic (MD) simulations were developed and combined., Results: Newly discovered AAT missense variants were localized both on the interaction surface and the hydrophobic core of the protein. Distribution of mutations across the structure revealed Val210Glu at the solvent exposed s4C strand and close to the "Gate" region. Asn247Ser was located on the accessible surface, which is important for glycan attachment. On the other hand, Asp341His, Leu383Phe were mapped close to the "breach" and "shutter" regions. MD analysis revealed the reshaping of local interactions around the investigated substitutions that have varying effects on AAT conformational flexibility, hydrophobic packing, and electronic surface properties. The most severe structural changes were observed in the double- and triple-mutant (Pi*S + Asp341His and Pi*S + Leu383Phe + Lys394Ile) molecular models. The two carriers presented impaired lung function., Conclusions: The results characterize five variants, four of them previously unknown, of the SERPINA1 gene, which define new alleles contributing to the deficiency of AAT. Rare variants might be more frequent than expected, and therefore, in discordant cases, standardized screening of the S and Z alleles needs complementation with gene sequencing and structural approaches. The utility of computational modelling for providing supporting evidence of the pathogenicity of rare single nucleotide variations is discussed., (© 2022. The Author(s).)

Background: Alpha-1 antitrypsin deficiency (AATD) is considered one of the most common genetic diseases and is characterised by the misfolding and polymerisation of the alpha-1 antitrypsin (AAT) protein within hepatocytes. The relevance of circulating polymers (CP) of AAT in the pathogenesis of lung and liver disease is not completely understood. Therefore, the main objective of our study was to determine whether there is an association between the levels of CP of AAT and the severity of lung and liver disease., Method: This was a cross-sectional study in patients with different phenotypes of AATD and controls. To quantify CP, a sandwich ELISA was performed using the 2C1 monoclonal antibody against AAT polymers. Sociodemographic data, clinical characteristics, and liver and lung parameters were collected., Results: A cohort of 70 patients was recruited: 32 Pi*ZZ (11 on augmentation therapy); 29 Z-heterozygous; 9 with other genotypes. CP were compared with a control group of 47 individuals (35 Pi*MM and 12 Pi*MS). ZZ patients had the highest concentrations of CP (p < 0.001) followed by Z heterozygous. The control group and patients with Pi*SS and Pi*SI had the lowest CP concentrations. Pi*ZZ also had higher levels of liver stiffness measurements (LSM) than the remaining AATD patients. Among patients with one or two Z alleles, two patients with lung and liver impairment showed the highest concentrations of CP (47.5 µg/mL), followed by those with only liver abnormality (n = 6, CP = 34 µg/mL), only lung (n = 18, CP = 26.5 µg/mL) and no abnormalities (n = 23, CP = 14.3 µg/mL). Differences were highly significant (p = 0.004)., Conclusions: Non-augmented Pi*ZZ and Z-patients with impaired lung function and increased liver stiffness presented higher levels of CP than other clinical phenotypes. Therefore, CP may help to identify patients more at risk of developing lung and liver disease and may provide some insight into the mechanisms of disease., (© 2021. The Author(s).)

Background: α1-Antitrypsin deficiency (AATD) is associated with a high risk of developing lung and liver disease. Despite being one of the most common hereditary disorders worldwide, AATD remains under-diagnosed and prolonged delays in diagnosis are usual. The aim of this study was to validate the use of buccal swab samples and serum circulating DNA for the complete laboratory study of AATD., Methods: Sixteen buccal swab samples from previously characterized AATD patients were analyzed using an allele-specific genotyping assay and sequencing method. In addition, 19 patients were characterized by quantification, phenotyping and genotyping using only serum samples., Results: The 16 buccal swab samples were correctly characterized by genotyping. Definitive results were obtained in the 19 serum samples analyzed by quantification, phenotyping and genotyping, thereby performing the complete AATD diagnostic algorithm., Conclusions: Buccal swab samples may be useful to expand AATD screening programs and family studies. Genotyping using DNA from serum samples permits the application of the complete diagnostic algorithm without delay. These two methods will be useful for obtaining more in depth knowledge of the real prevalence of patients with AATD.

Background and Objectives: Alpha-1-antitrypsin deficiency (AATD) is associated with a high risk for the development of early-onset emphysema and liver disease. A large majority of subjects with severe AATD carry the ZZ genotype, which can be easily detected. Another rare pathologic variant, the Mmalton allele, causes a deficiency similar to that of the Z variant, but it is not easily recognizable and its detection seems to be underestimated. Therefore, we have included a rapid allele-specific genotyping assay for the detection of the Mmalton variant in the diagnostic algorithm of AATD used in our laboratory. The objective of this study was to test the usefulness of this new algorithm for Mmalton detection., Materials and Methods: We performed a retrospective revision of all AATD determinations carried out in our laboratory over 2 years using the new diagnostic algorithm. Samples with a phenotype showing one or two M alleles and AAT levels discordant with that phenotype were analyzed using the Mmalton allele-specific genotyping assay., Results: We detected 49 samples with discordant AAT levels; 44 had the MM and five the MS phenotype. In nine of these samples, a single rare Mmalton variant was detected. During the study period, two family screenings were performed and four additional Mmalton variants were identified., Conclusion: The incorporation of the Mmalton allele-specific genotyping assay in the diagnostic algorithm of AATD resulted in a faster and cheaper method to detect this allele and avoided a significant delay in diagnosis when a sequencing assay was required. This methodology can be adapted to other rare variants. Standardized algorithms are required to obtain conclusive data of the real incidence of rare AAT alleles in each region., Competing Interests: The authors report no conflicts of interest in this work.

Background: α1-Antitrypsin deficiency (AATD) is an autosomal codominant disorder associated with a high risk of developing lung and liver disease. The most common deficient alleles are known as Z and S. However, another deficient variant, called Mmalton, which causes a deficiency similar to variant Z, is considered to be the second cause of severe AATD in Spain. Nevertheless, the Mmalton allele is not recognizable by usual diagnostic techniques and therefore, its real prevalence is underestimated. We describe a rapid real-time PCR and melting curves assay designed for the detection of Mmalton AATD., Methods: We tested the applicability of this new technique for the identification of the Mmalton allele in AATD screening using whole blood, dried blood spot (DBS) and serum samples. Mmalton heterozygote and homozygote samples and samples without this allele were included in the study., Results: This new assay is able to detect homozygous and heterozygous genotypes in the same reaction and in a single step, giving matching results with those obtained by SERPINA1 gene sequencing., Conclusions: This technology is optimal for working with small amounts of DNA, such as in DBS and even with residual DNA present in serum samples, allowing improvement in routine algorithms of AATD diagnosis or large-scale screening. This method will be useful for obtaining more in depth knowledge of the real incidence of the Mmalton variant.

The effect of hereditary alpha-1 antitrypsin (AAT) deficiency can manifest clinically in the form of chronic obstructive pulmonary disease (COPD). AAT deficiency (AATD) is defined as a serum concentration lower than 35% of the expected mean value or 50 mg/dl (determined by nephelometry). It is associated in over 95% of cases with Pi*ZZ genotypes, and much less frequently with other genotypes resulting from combinations of Z, S, rare and null alleles. A systematic qualitative review was made of 107 articles, focusing mainly on an active search for AATD in COPD patients and intravenous (iv) treatment with AAT. On the basis of this review, the consultant committee of the Spanish Registry of Patients with AATD recommends that all COPD patients be screened for AATD with the determination of AAT serum concentrations, and when these are low, the evaluation must be completed with phenotyping and, on occasions, genotyping. Patients with severe AATD COPD should receive the pharmacological and non-pharmacological treatment recommended in the COPD guidelines. There is enough evidence from large observational studies and randomized placebo-controlled clinical trials to show that the administration of iv AAT reduces mortality and slows the progression of emphysema, hence its indication in selected cases that meet the inclusion criteria stipulated in international guidelines. The administration of periodic infusions of AAT is the only specific treatment for delaying the progression of emphysema associated with AATD., (Copyright © 2014 SEPAR. Published by Elsevier Espana. All rights reserved.)

Hereditary alpha-1-antitrypsin (α1-AT) deficiency predisposes to pulmonary emphysema. The objective of this study is to demonstrate the limitations of some laboratory methods used in the study of the deficiency, and which may produce errors in interpretation and detection of uncommon alleles. Two clinical cases are described: the index patient, who had pulmonary emphysema with α1-AT levels less than 12 mg/dL, was erroneously classified as a homozygote of the normal allelic variant PI MM using a rapid genotype method; the mother of the patient, asymptomatic, with low levels (60 mg/dL), was also classified as PI MM. The gene sequencing classified the index patient as a carrier of the PI Clayton null allele and PI Mmalton deficient. The mother was a PI Clayton/PI heterozygote carrier. These results highlight the difficulties in diagnosing the deficiency, as the well as the need to reach a consensus on methods for this study., (Copyright © 2010 SEPAR. Published by Elsevier Espana. All rights reserved.)

Objective: Alpha1-antitrypsin (AAT) deficiency is a codominant autosomal genetic disorder that predisposes a patient to chronic obstructive pulmonary disease and emphysema. Specific treatment is systemic, consisting of intravenous infusion of AAT. The protocol currently recommended by the Spanish Registry is infusion of 180 mg/kg every 21 days. The objective of this study was to assess the pharmacokinetic behavior of AAT and estimate the level of protection, defined as the percentage of time that the AAT plasma concentration was above the assumed protective threshold of 50 mg/dL with the usual protocol and with other alternative ones., Material and Methods: Plasma concentrations at 4 times were analyzed for 9 patients to profile the pharmacokinetics of AAT. The data were fitted to a single compartment open model with the WinNonlin software package. The duration of protection was estimated by simulating the evolution of AAT plasma activity over time according to the model constructed based on data recorded in the study., Results: Five men and 4 women (mean weight, 69 kg; range, 59-84 kg) were given a mean AAT dose of 12.06 g (range, 11-15 g). The mean (SD) volume infused was 516.67 (88.17) mL. The half-life of AAT was 8.7 days and the volume of distribution was 127.6 mL/kg. The currently recommended treatment protocol (180 mg/kg every 21 days) gave a level of protection of 67% (considering 60 mg/dL to be protective threshold) or 76% (for a threshold of 50 mg/dL). Protection values for the alternative protocol of 120 mg/kg every 14 days were 82% and 100%, respectively. For the alternative protocol of 60 mg/kg every 7 days, protection was 100% for both thresholds., Conclusions: Profiling the pharmacokinetic behavior of AAT has enabled the coverage time to be assessed for several treatment protocols. The regimen of 120 mg/kg every 14 days had the most appropriate profile.

Alpha1-antitrypsin (alpha1-AT) deficiency is an underdiagnosed condition in patients with chronic obstructive pulmonary disease (COPD). The present authors have conducted a nationwide case detection programme of alpha1-AT deficiency in unselected patients with COPD using dried blood spots. The first phase analysed samples from 971 patients by determining alpha1-AT concentrations and identifying the deficient Z allele by genotyping using rapid real-time PCR. The second phase analysed 1,166 samples with alpha1-AT concentrations and identified both the S and the Z allele, but only in samples with low alpha1-AT concentrations. A total of eight (0.37%) individuals with the severe deficiency PiZZ were detected. In addition, three patients were identified with the PiSZ genotype in the second phase (0.3%). The global cost of the programme was 41,512, which represents 19.42 per sample and 5,189 per PiZZ detected. A sensitivity analysis demonstrated that performing Z genotype to all samples would have resulted in increased costs of 28 per sample and 7,479.5 per PiZZ case identified. In conclusion, a case detection programme of alpha1-antitrypsin deficiency in patients with chronic obstructive pulmonary disease using dried blood spots is feasible and at a reasonable cost per case detected. Diagnostic yield and costs depend largely on inclusion criteria and the protocol for processing of samples.

Background: Population studies indicate that alpha-1-antitrypsin (AAT) deficiency is an under diagnosed disease. Although alpha-1 serum protein is widely known to accompany AAT deficiency, the diagnostic utility of measuring the alpha-1 band to screen for this condition has not been assessed in the literature., Subjects and Method: Electropherograms with alpha-1 band widths under the reference values were collected over a period of 3 months. The Pi phenotype of AAT was identified for these sera by isoelectric point determination. The phenotypes were compared to those obtained for the population of the same geographic area (n = 440). The alpha-1 band reference values were obtained from 73 healthy individuals with no Pi phenotype deficiency. Moreover, the alpha-1 band was also measured for a group of 17 PiZZ deficient patients., Results: We analyzed 7,305 electropherograms. One hundred four individuals (1.4%) without hypoproteinemia had alpha-1 readings below reference (set at 2.3%). The phenotypes in this group were 25 PiMM (24%), 52 PiMS (54%), 13 PiMZ (12.5%) and 5 PiSS 5 (5%). The odds ratios (CI 95%) in comparison with the normal population were, respectively, 0.10 (0.16-0.06); 4.58 (2.97-7.04); 4.35 (2.09-9.04) and 5.51 (1.66-18.16) (p < 10-5 in all cases except PiSS, which was p < 0.05). The levels for PiZZ patients were 1.4% +/- 0.3% (range 1.0%-2.1%)., Conclusions: Three times fewer subjects with a normal PiMM phenotypes are found among individuals with low alpha-1 band serum protein levels, and many more of such individuals are carriers of Z allele heterozygotes. Alpha-1 band readings in patients with AAT deficiency (PiZZ phenotype) have alpha-1 values below reference. Measuring alpha-1 protein is an easy technique, within the expertise of any laboratory, and may be very useful for screening for AAT deficiency in patients with chronic respiratory diseases.

Background: Severe alpha-1-antitrypsin (AAT) deficiency is caused by homozygous inheritance of gene Z, and is associated with a high risk of developing pulmonary emphysema. Determination of frequencies of different genes associated with the deficiency (especially S and Z) gives a clue to estimate the number of individuals homozygous PiZZ, carrying a high risk for pulmonary disease, in any given population., Patients and Methods: Pi phenotypes of 440 healthy individuals were determined by means of isoelectrofocusing in polyacrylamide gel. Seric values of AAT were determined by immunonephelometry. Mean age of participants was 30 years (range 18-49 yrs.). Results are compared with other published series., Results: Distribution of phenotypes was: PiMM 333 individuals (75%), PiMS 84 (19%), PiMZ 14 (3%), PiSS 4 (0.9%), PiM 3 (0.6%), PiMF 1 (0.2%), PiMP 1 (0.2%). The corresponding gene frequencies were Pi*M 87%, Pi*S 10.4%, and Pi*Z 1.5%. Normal values of AAT (phenotype PiMM) established in our laboratory were 116-232 mg/dl (21-41 micromol/I) (mean +/- 2 SD). According to Hardy-Weinberger equation, expected frequency of PiZZ individuals in our area would be 225 per million., Conclusions: The frequency of Z gen individuals observed in our study is one of the highest in the Iberian Peninsula, but lower than the frequency in northern Europe. According to these results, AAT deficiency (PiZZ) is not a rare condition in contrast with the small number of patients diagnosed. The gen frequency of the S variant is higher than that of the rest of Europe, and similar to others found in some Spanish populations.