Stefania Ottaviani

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 α1-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.

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

With the aim of providing better clinical characterisation of patients with &agr;1-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.

Screening for Alpha 1 antitrypsin deficiency in Tunisian subjects with obstructive lung disease: a feasibility report.

BackgroundAATD is one of the most common inherited disorders in the World. However, it is generally accepted that AATD in North African populations is not a risk factor for lung and/or liver disease, based on a number of small studies. We therefore planned a screening study for detection of AATD in patients with OLD in a cohort of patients from Kairouan in central Tunisia. Methods: One hundred twenty patients with OLD (asthma, emphysema, COPD) were enrolled in the screening programme. Laboratory diagnosis for AATD was performed according to current diagnostic standards.ResultsWe found that 6/120 OLD patients carried an AAT deficient allele, 1 PI*MZ, 1 PI*MPlowel, 3 PI*MMmalton, 1 PI*MMwurzburg.Conclusionthis pilot study demonstrated that alleles related to deficiency of AAT are not absent in the Tunisian population, and that rare AATD variants prevailed over commonest PI*Z variant. These results would support a larger scale screening for AATD in Tunisia.

SERPINA1 PiZ and PiS Heterozygotes and Lung Function Decline in the SAPALDIA Cohort

Background Severe alpha1-antitrypsin (AAT) deficiency is a strong risk factor for COPD. But the impact of gene variants resulting in mild or intermediate AAT deficiency on the longitudinal course of respiratory health remains controversial. There is indication from experimental studies that pro-inflammatory agents like cigarette smoke can interact with these variants and thus increase the risk of adverse respiratory health effects. Therefore, we tested the effect of the presence of a protease inhibitor (Pi) S or Z allele (PiMS and PiMZ) on the change in lung function in different inflammation-exposed subgroups of a large, population-based cohort study. Methodology and Principal Findings The SAPALDIA population includes over 4600 subjects from whom SERPINA1 genotypes for S and Z alleles, spirometry and respiratory symptoms at baseline and after 11 years follow-up, as well as proxies for inflammatory conditions, such as detailed smoking history, obesity and high sensitivity C-reactive protein (hs-CRP), were available. All analyses were performed by applying multivariate regression models. There was no overall unfavourable effect of PiMS or PiMZ genotype on lung function change. We found indication that PiZ heterozygosity interacted with inflammatory stimuli leading to an accelerated decline in measures in use as indices for assessing mild airway obstruction. Obese individuals with genotype PiMM had an average annual decline in the forced mid expiratory flow (ΔFEF25-75%) of 58.4 ml whereas in obese individuals with PiMZ it amounted to 92.2 ml (p = 0.03). Corresponding numbers for persistent smokers differed even more strongly (66.8 ml (PiMM) vs. 108.2 ml (PiMZ), p = 0.005). Equivalent, but less strong associations were observed for the change in the FEV1/FVC ratio. Conclusions We suggest that, in addition to the well established impact of the rare PiZZ genotype, one Z allele may be sufficient to accelerate lung function decline in population subgroups characterized by elevated levels of low grade inflammation.

C reactive protein and alpha1-antitrypsin: relationship between levels and gene variants

The first step in laboratory diagnosis of alpha1-antitrypsin deficiency (AATD) is the determination of alpha1-antitrypsin (AAT) serum levels; these levels in turn are influenced by the inflammatory status. C reactive protein (CRP) has been proposed as a marker of systemic inflammation. Single nucleotide polymorphisms (SNPs) in the CRP gene have been associated with differences in baseline CRP levels. The purpose of this study was to investigate the relationship between CRP and AAT in the AATD diagnostic setting and to verify whether variations in the CRP gene could influence CRP. We determined AAT and CRP levels in 362 consecutive dried blood spot (DBS) samples submitted for AATD diagnosis and genotyped 3 CRP gene SNPs (rs1205, rs3093077, and rs3091244) associated with variations in serum CRP concentrations. To this aim, we developed a method to measure CRP in a DBS with a good correlation with CRP measurement in serum (r2=0.9927). We showed then that systemic inflammatory status parallels increased levels of AAT (80% of subjects with intermediate AATD and a CRP>0.8 mg/dL had an AAT level above the cut-off of 113 mg/dL) and that this increase might mask the presence of AATD variants. No association was detected between CRP levels and the 3 CRP gene polymorphisms. Simultaneous determination of CRP and AAT is useful in the correct diagnosis of heterozygotes carrying intermediate AATD genotypes; their genetic influence on the CRP level is negligible.

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.

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.

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

BackgroundAlpha-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.MethodsThe 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.ResultsTo 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.ConclusionThis 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.

Inherited alpha1-antitrypsin deficiency: is the level the key message?

Fifty-one years ago, two scientists from the Malmö University, Carl Bertil Laurell and Sten Eriksson, first reported five individuals affected by inherited alpha1-antitrypsin ( 1-AT) deficiency [1]. Of particular interest, this original nucleus of described patients, already included the spectrum of clinical phenotypic variability associated with this genetic disorder: absence of any respiratory disease, juvenile or late pulmonary emphysema, disseminated bronchiectasis. Since then, knowledge on the related epidemiology, pathophysiology, pathogenesis, clinical course, and treatment has dramatically improved, as evidenced by the authoritative published review articles [2,3]. Still, there are a number of open questions awaiting on answer. In particular, we currently have relatively solid information on the worldwide epidemiology of 1-AT; this information has led to the consensus that 1-AT is likely the most common rare respiratory disorder [4]. Nevertheless, the diagnostic rate for 1-AT is still dramatically low, with no more than 5—10% of expected individuals actually being recognized. This is true in all developed countries, irrespective of the local prevalence of the disorder and the quality of the diagnostic programs in place. Therefore, it has never been more evident that ‘‘. . . inherited 1-AT is not a rare condition, but a condition rarely diagnosed’’ [5]. We are thus heartened by the fact that the opening article in a thematic series of the Revue de Maladies Respiratoires was devoted to the laboratory diagnosis for inherited 1-AT deficiency. In this paper, Balduyck et al. provide an exhaustive review of the methodologies used to perform a ( s p p