α1-Antitrypsin phenotypes and associated serum protein concentrations in a large clinical population.

BACKGROUND: α1-Antitrypsin (AAT) deficiency variants reduce the concentration of serum AAT protease inhibitor and can lead to the development of pulmonary and hepatic disease. Relative frequencies of rare AAT variant phenotypes (non-M, Z, and S) and associated serum concentrations in the clinical population have not been thoroughly described. METHODS: Protein phenotypes were determined by isoelectric focusing electrophoresis for 72,229 consecutive samples. Phenotype frequencies, median serum concentrations, and central 95% concentration intervals were determined for observed phenotypes. Concurrent AAT phenotype and concentration data were used to evaluate the efficacy of using serum AAT concentration alone to detect AAT deficiency. RESULTS: Age, race, and sex had only slight effects on the median 95% serum protein concentration intervals of the 58,087 PiMM (wild type) phenotype specimens. Positive predictive values were calculated for the detection of potential deficiency phenotypes at different serum cutoff concentrations, aiding potential screening effort design. For example, the PiZZ deficiency phenotype (n = 814) could be detected at 99.5% sensitivity and 96.5% specificity using a cutoff of ≤ 85 mg/dL. However, at-risk specimens with two putative deleterious variants (Z, S, I, F, P, T, and Null variants) were detected with only 85.9% sensitivity at this cutoff (n = 1,661). Rare phenotype variants were observed in 2.5% of samples. CONCLUSIONS: This analysis provides novel information on serum AAT concentrations associated with different AAT phenotypes and provides insight into the severity of depression of AAT concentration in the presence of rare deficiency variants. Additionally, it allows for evaluation of efficacy of testing algorithms incorporating AAT serum concentration determination.

[Genetic variants of alpha-1 antitrypsin: classification and clinical implications]. / Genetyczne warianty alfa-1 antytrypsyny ­ klasyfikacjai znaczenie kliniczne

Inherited alpha-1 antitrypsin deficiency is listed among the three most common genetic disorders in Caucasians. It considerably increases the risk of progressive obstructive lung diseases, mostly chronic obstructive pulmonary disease, as well as chronic liver disorders, hepatitis, cirrhosis, and cancer. It is estimated that more than 5.5% of the Polish population carries one of the most common deficiency phenotypes, which might be relatively easily detected due to low alpha-1 antitrypsin serum concentration. However, as well as being quantitative, alpha-1 antitrypsin deficiency might also be qualitative. These dysfunctional alpha-1 antitrypsin variants are characterized by scarce antiproteolytic activity and quite often by fully effective protein production resulting in normal serum levels. Consequently, dysfunctional variant identification is possible only by means of pheno- or genotyping. This review presents clinically useful characteristics of main genetic alpha-1 antitrypsin variants.

[Proteins of the inter-alpha trypsin inhibitor (ITI) family. A major role in the biology of the extracellular matrix]. / Les protéines de la famille de l'ITI (inter-alpha trypsine inhibiteur). Un rôle majeur dans la biologie de la matrice extra-cellulaire.

A glycoprotein with a high inhibitory activity against trypsin was isolated in 1961 from human plasma and named inter-alpha trypsin inhibitor (ITI). Since then, several other proteins that share antigenic and structural similarities with ITI have been identified and classified as members of the ITI protein family. These glycoproteins built up from different combinations of four polypeptides HC1, HC2, HC3 and bikunin are encoded by four genes H1, H2, H3, L on three chromosomes. Bikunin has two proteinase inhibitor domains and belongs to the Kunitz-type protease inhibitor family; it displays an inhibitory activity against trypsin, leukocyte elastase and plasmin. The heavy chains do not have any protease inhibitory properties but have the capacity to interact in vitro and in vivo with hyaluronic acid. This binding promotes the stability of the extra-cellular matrix. Consequently, the ITI protein family is suspected of playing a key role in the extra-cellular matrix biology. Isolation of free heavy chains in bronchial secretions and the recent emphasis about the bikunin role in tumoral invasion should enhance the interest about ITI protein family in the pathophysiology of chronic bronchopulmonary diseases or lung cancer progression.

Subtyping of group-specific component and protease inhibitor by rapid isoelectric focusing on PhastSystem.

A rapid method on PhastSystem was used to investigate the distribution of group-specific component (GC) and protease inhibitor (PI) subtypes and their gene frequencies from 190 unrelated healthy donors of the Han population in Beijing. Laboratory-made gels (pH 4.5-5.4 and pH 4.2-4.9) were used for analysis of GC and PI, respectively. Sample loading was 1.5 microliters. The separation and visualization time was 0.5 h in each. Gene frequencies were as follows: GC*1F = 0.4891, GC*1S = 0.2432, GC*2 = 0.2678; rare GC variants were discovered in seven cases. The results for PI were: PI*M1 = 0.7542, PI*M2 = 0.1808, PM*M3 = 0.0650. Good agreement between the observed and expected values in both GC and PI subtyping (for GC, sigma chi 2 = 1.4043, 0.7 < P < 0.8; for PI, sigma chi 2 = 1.1233, 0.7 < P < 0.8) was obtained.

Correlation of alpha-1-antitrypsin (PI) polymorphism between blood and semen.

The correlation between isoprotein types of alpha-1-antitrypsin (PI) in blood and semen samples from the same individual was determined in 48 Japanese men by the combined technique of isoelectric focusing with immobilized pH gradients and immunoblotting. Five common PI types (M1, M1M2, M1M3, M2 and M2M3) were detected in the blood plasma samples. However, PI-specific bands in semen migrated more cathodally than those in plasma into a pI region of approximately 5.05, and about 17% of the semen samples could not be phenotyped: the rest were easily phenotyped and their PI types were found to correlate with the type found in the corresponding blood samples. PI typing could therefore provide an additional discriminant characteristic for the forensic examination of individualization from semen samples.

[Respiratory involvement in ankylosing spondylarthritis: relations to alpha-1-antitrypsin phenotypes and tobacco consumption]. / Atteinte respiratoire de la spondylarthrite ankylosante: ses rapports avec les phénotypes de l'alpha-1-antitrypsine et la consommation de tabac.

The distribution of alpha-1-antitrypsin phenotypes was similar in 555 controls and 98 patients with ankylosing spondylitis: the MM phenotype (including "main" MM subtypes, i.e., M2M2 and M3M3, and "secondary" MM subtypes) was found in 86% of subjects and "rare" phenotypes combining M, F, S, and Z in 14%. Six per cent of the controls and none of the ankylosing spondylitis patients had the M4M4 phenotype (p < 0.01). Respiratory function tests were performed in 49 patients with axial ankylosing spondylitis and 30 controls matched on sex, age, body mass index, smoking status, nonsteroidal antiinflammatory drug use and distribution of "main" and "secondary" phenotypes (no subjects in this study had "rare" phenotypes); the significant reduction in chest expansion seen in the ankylosing spondylitis group (5.6 +/- 2.7 cm versus 8.7 +/- 1.2; p < 0.001) was correlated with total capacity (p < 0.04) and vital capacity (p < 0.001). Restrictive ventilatory dysfunction was seen in four ankylosing spondylitis patients versus no controls (p < 0.02). Proximal airway obstruction, pulmonary distension and decreases in the diffusing capacity for carbon monoxide were seen in similar proportions of ankylosing spondylitis patients and controls. In the ankylosing spondylitis group, evidence of pulmonary distension included increases in mean residual functional capacity and mean residual volume (105.6 +/- 21.2% versus 94.8 +/- 17.4, p < 0.03, and 100.3 +/- 22.8% versus 88.6 +/- 17.9, p < 0.04, respectively) and bullous emphysema in the lung bases in two patients (versus no controls). In the small subgroup of ankylosing spondylitis patients with lung distension or a decreased diffusing capacity for carbon monoxide, smokers and nonsmokers were evenly balanced but subjects with "secondary" phenotypes outnumbered those with "main" phenotypes (p < 0.02); in contrast, our data suggested that smoking may play the central role in the proximal airway obstruction. Our findings suggest that in addition to previously established causes of pulmonary involvement in ankylosing spondylitis a "secondary" MM phenotype (i.e., neither M2M2 nor M3M3) may be a risk factor for lung distension and impaired diffusing capacity for carbon monoxide.

[Clinical usefulness of phenotyping of alpha 1-antitrypsin]. / Utilité clinique du phénotypage de l'alpha 1-antitrypsine.

Analysis by isoelectric focusing of serum alpha 1-antitrypsin phenotype of 167 consecutive cases from an occupational health outpatient clinic, from university hospital departments and from private practitioners showed an excessive presence of rare gene alleles S, Z, I and anodal variants compared to their frequencies in blood donors from Lausanne or in the general Swiss population. It seems that analysis has been well grounded in most cases and helps to establish diagnosis in many respiratory diseases, in unexplained liver cirrhosis and even in aortic rupture.

Improved method for identifying alpha 1-antitrypsin Pi M subtypes by isoelectric focusing in agarose.

We describe an improved method for the classification of alpha 1-antitrypsin variants by isoelectric focusing in agarose. Identification of the three Pi M subtypes can now be made by using a narrow-range carrier ampholyte (pH 4.2-4.9) and pretreating serum with dithioerythritol-iodoacetic acid to enhance band resolution. Phenotype results for two groups of Pi M homo- and heterozygotes are compared to illustrate the improved accuracy of the new method.

[Subtyping of Pi in human bloodstains by isoelectric focusing].

Subtyping of Pi (alpha 1-AT) in 90 human bloodstains stored under different conditions was carried out by using ULPAGIF. The detectable time of Pi subtypes of all bloodstains kept at room temperature (12-20 degrees C), 4 degrees C and-15 degrees C was 10 days, 4 weeks and 4 weeks respectively. Beyond this time limit, the detectable rate decreased gradually. The minimal detectable quantity, of fresh bloodstains was 4 microliters. The factors influencing the detection of Pi subtypes in bloodstains were discussed.

Human alpha-1-antitrypsin subtyping by hybrid isoelectric focusing in miniaturized polyacrylamide gel.

The polymorphism of alpha-1-antitrypsin (PI) has been studied by hybrid isoelectric focusing in miniaturized immobilized pH gradient gels, with an interelectrode distance of 55 mm, in two narrow ranges of pH 4.35-4.75 and 4.35-4.55), following rehydration with pH 4.2-4.9 carrier ampholytes. The use of the separator N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) in combination with carrier ampholytes for gel rehydration has been shown to enhance PI band sharpness. The influence of different additives (sucrose, sorbitol and glycerol) on the PI band pattern has also been evaluated. Glycerol has been shown to be responsible for the change in the relative mobility of the M3 band. The analysis of the minor M-7 isoprotein zone by hybrid isoelectric focusing followed by silver staining has permitted a more reliable classification of PIM subtypes. A population study carried out with 164 unrelated individuals living in Spain is also presented.

[Alpha 1-antitrypsin polymorphism]. / Polimorfizm alfa1-antytrypsyny.

Discovery, nomenclature and some genetical and molecular aspect of alpha 1-antitrypsin polymorphism are discussed. Particular attention has been paid to the deficiency variants and their role in human pathology.

Review of isoelectric focusing for Gc, PGM1, Tf, and Pi subtypes: population distributions.

Isoelectric focusing (IEF) as a method for differentiating macromolecules with minor differences in isoelectric points has demonstrated an increase in the degree of genetic polymorphisms of the blood. Studies over the last 5 to 6 years have shown that genetic marker systems such as transferrin (TF), phosphoglucomutase (PGM1), the vitamin D-binding globulin (GC), and A1 antitrypsin (PI) are a great deal more polymorphic than observed using conventional electrophoresis. Additional genetic variants have been detected or further defined in such systems as esterase D (ESD) and hemoglobin (HB) to name a few. The increased heterozygosity levels of these genetic marker systems identified by IEF have added to their value in forensic medicine and resulted in further resolution of racial and population affinities. IEF should prove to be a valuable anthropological tool for measuring population structure and genetic distances.

Genetic typing of alpha1-antitrypsin by immunofixation electrophoresis, identification of subtypes of Pi M.

Prolonged electrophoresis in alkaline agarose gels, followed by immunofixation, is a valuable addition to acid starch gel electrophoresis and isoelectric focusing for the genetic phenotyping of alpha1-antitrypsin. This technique is helpful in clarifying certain variants and in ascertaining types in serum and amniotic fluid samples with secondary changes. In addition, heterogeneity may be detected within the variants found at pH 4.95, analogous to hemoglobin polymorphism. Two "new" variants, PiMLamb and PiMBaldwin, have been detected by a combination of immunofixation electrophoresis and acid starch gel electrophoresis.