alpha1-antitrypsin: common subtypes of Pi M.

More than 20 different alleles are so far known at the Pi locus, corresponding to a total variant phenotype frequency of about 10% in most western Europeans. The common phenotype Pi M constitutes the remaining major group. Now it has been possible to identify three subtypes M1, M1M2 and M2, corresponding to the gene products of two common alleles PiM1 and PiM2, segregating as autosomal codominant alleles. Preliminary gene frequencies are reported for eight populations, the PiM2 frequency varying from 0.20 in Maris (USSR) to 0.02 in Bantus (Kenya).     

Alpha-1-antitrypsin (PI) subtypes in Russians and Poles.

alpha-1-antitrypsin (PI) subtypes were studied in Poles and Russians. The frequencies of the PI alleles were similar in the two populations, with the exception of the Z allele, whose frequency was significantly lower in Poles. The M3 allele frequency, which is highly heterogeneous in European populations, has medium frequencies in Poles and Russians.     

Disruption of the murine alpha1-antitrypsin/PI2 gene.

Alpha-1-antitrypsin (alpha1-AT) is a member of the serine protease inhibitor family regulating numerous proteolytic processes. The genetic disorder, alpha1-AT deficiency, is well known as a cause of hereditary pulmonary emphysema and liver cirrhosis. To create an animal model of human alpha1-AT deficiency, we disrupted the major murine isoform PI2, which is similar to human alpha1-AT and is one of 7 alpha1-AT isoforms found in the mouse. The ability of the serum to inhibit the activities of human leukocyte elastase (HLE) and human chymotrypsin (CYT) was significantly lower in heterozygous mice (alpha1-AT/PI2 -/+) than wild-type (alpha1-AT/PI2 +/+) mice (73.2% vs. 100% for HLE and 67.8% vs.100% for CYT, respectively; P<0.05). The distribution of genotypes among F(2) progeny was not in accordance with Mendelian distribution (P<0.01), as the percentages of wild-type, heterozygotes and homozygotes were 47.8%, 37.3% and 14.9%, respectively. Thus, it is likely that impairment of the protease inhibitor had a critical effect on fetus development. The alpha1-AT/PI2 deficient mouse will be a useful animal model for elucidating the function of alpha1-AT in fetal development, studying the mechanisms of chronic inflammatory disease and evaluating therapeutic candidates for the treatment of inflammatory disease.     

Molecular abnormality of PI S variant of human alpha1-antitrypsin.

Alpha1-antitrypsin variant protein was purified to homogeneity from a PI S-S subject with a mild deficiency of plasma trypsin inhibiting capacity. Molecular weight, specific trypsin inhibitory activity, and composition of amino acids and carbohydrates were similar to the proteins purified from Pi M-M individuals with normal alpha1-antitrypsin activity. The structural difference between the normal and the variant alpha1-antitrypsin was elucidated by peptide mapping of their tryptic digests. An amino acid substitution of glutamic acid in the normal protein to valine in the variant protein was found. The result is consistent with the previously reported amino acid substitution in Pi S-Christchurch.     

Sequence data of the rare deficient alpha 1-antitrypsin variant PI Zaugsburg.

Weidinger et al. recognized a rare deficient PI-variant, named PI Zaugsburg (PI Zaug), by using isoelectric focusing with a narrow pH gradient. The serum alpha 1-antitrypsin (alpha 1AT) level determined quantitatively in an individual carrying the phenotype PI M1Zaug revealed a value of 50%-60% of the normal range. The frequency of the deficient PI*Zaug allele is still unknown. Haplotyping the Zaug-affected chromosome, we found a pattern different from the common PI*Z allele described by Cox et al. Therefore, we directly sequenced the coding exons of both genes (M1 and Zaug) after PCR amplification. Zaug sequence data analysis showed the presence of the common PI*Z allele-specific mutation (M1 Glu342 GAG to Z Lys342 AAG) surprisingly occurring in an M2 ancestral gene. This is not consistent with the heretofore common finding, by Nukiwa et al. and others, that this mutation is derived from an M1 (Ala213) background gene. No further mutations were found in the PI Zaug gene.     

PI LBEI and PI JHOU: two new alpha-1-antitrypsin alleles

Summary Two new variants of alpha-1-antitrypsin were found in a Chinese population by isoelectric focusing in polyacrylamide gel. They have been named Lbeijing (PI^*LBEI) and Jhouyao (PI^*JHOU) respectively. With the samples reduced and alkylated, both the variants were anodal to M1 and cathodal to L. Omitting the alkylation step, however, the relative mobilities of L and Jhouyao were altered. Under this condition, a characteristic of Jhouyao was revealed by isoelectric focusing in immobilized pH gradient 4.4–4.9, i.e., while its band 6 became anodal to L, band 4 was still cathodal to L.     

Hormonal regulation of serum alpha 1-antitrypsin and hepatic alpha 1-antitrypsin mRNA in rats

We evaluated the effects of pituitary dependent hormones on alpha 1-antitrypsin in male rats. Hepatic alpha 1-antitrypsin mRNA was measured by in vitro translation and by specific hybridization with a mouse cDNA alpha 1-antitrypsin probe. Hypophysectomy caused a 50-75% decrease in serum elastase inhibitory capacity (measuring functional alpha 1-antitrypsin) and hepatic alpha 1-antitrypsin mRNA content. In hypophysectomized animals, no increase in elastase inhibitory capacity or alpha 1-antitrypsin mRNA levels by translation was found when met-human growth hormone alone or corticosterone, dihydrotestosterone and thyroxine were given together. Growth hormone increased alpha 1-antitrypsin mRNA by hybridization to a small extent. Addition of growth hormone to the combination of corticosterone, dihydrotestosterone, and thyroxine increased serum elastase inhibitory capacity and alpha 1-antitrypsin mRNA. We conclude that growth hormone acts synergistically with the other pituitary dependent hormones to regulate serum and hepatic mRNA levels of alpha 1-antitrypsin.     

Polymorphism of alpha1-antitrypsin in Pamir populations. Reproductive compensation--possible mechanism for maintaining genetic diversity gor PI genes in human populations

The distribution of the phenotype and gene frequencies of alpha 1-antitrypsin among Pamir's aborigines localized at high altitudes was studied. The Kirghizes of Murgab studied include mainly the mongoloid component in their composition. Populations of the Khuf river valley in West Pamir anthropologically belong to South caucasoids. The following frequencies of PI genes have been registered in Kirghizes (N = 102): M1 = 0.6961, M2 = 0.2108, M3 = 0.0539, Z = 0.0245, I = 0.0049, S = 0.0049, N = 0.0049; in the Khuf population (N = 122): M1 = 0.7910, M2 = 0.0943, M3 = 0.0984, Z = 0.0082, I = 0.0041, S = 0.0041; in the Pastkhuff population (N = 38): M1 = 0.7237, M2 = 0.1579, M3 = 0.1053, Z = 0.0132. A parallele biodemographic investigation in the Murgab population showed that couples, with one of the partners carrying the rare variant of PI demonstrated statistically significant increase in successful outcomes of pregnancies. The same cohort has displayed lower infant mortality rates, the absence of miscarried fetus and stillborn babies. Our results point to the possible existence of a mechanism of reproductive compensation serving to uphold the genetic diversity of PI genes.     

Alpha-1-antitrypsin (PI) polymorphism in France, with special regard to the PI*Z allele.

Alpha-1-antitrypsin (PI) phenotypes were studied in a sample of more than 5,000 individuals from cities throughout France. Special interest was paid to the PI*Z allele whose average frequency, based on the present work plus results from the literature, was 0.0130. This figure was used to estimate the number of PI Z homozygotes in France. In accordance with previous studies, the frequency of the PI*S allele was found to increase towards the southern parts of France.     

Alpha 1-antitrypsin Wbethesda: molecular basis of an unusual alpha 1-antitrypsin deficiency variant

Molecular analysis of alpha 1-antitrypsin (alpha 1AT) Wbethesda revealed that it differs from the normal M1 (Ala213) allele by a single base mutation causing an amino acid substitution Ala336 GCT----Thr ACT. Evaluation of alpha 1AT biosynthesis directed by the Wbethesda allele showed that although Wbethesda alpha 1AT mRNA was translated normally in vitro, transfection of the Wbethesda cDNA into COS-I cells was associated with human alpha 1AT secretion of 50% that of cells transfected with a normal alpha 1AT cDNA. The pattern of alpha 1AT biosynthesis was not intracellular accumulation as observed with the common Z alpha 1AT deficiency allele, but reduced intracellular alpha 1AT, suggesting intracellular degradation of the newly synthesized Wbethesda molecule. Together these observations suggest that in heterozygous combination with a Z or Null alpha 1AT allele, the Wbethesda variant causes "alpha 1AT deficiency", thus classifying it as an alpha 1AT "at risk" allele for emphysema.     

Improved resolution of PI (alpha 1-antitrypsin) phenotypes by a large-scale immobilized pH gradient

PI variants and PI M subtypes were examined by isoelectric focusing in a preformed immobilized pH gradient. Conditions were found that permit reliable classification of PI variants and of most PI M subtypes: the pH gradient ranges from 4.3 to 4.8 on a polyacrylamide gel with a length of 20 cm and a separation distance of approximately 16-17 cm (delta pH = 0.025 U/cm). The PI phenotypes observed are presented.     

Alpha 1-antitrypsin Null(isola di procida): an alpha 1-antitrypsin deficiency allele caused by deletion of all alpha 1-antitrypsin coding exons.

alpha 1-Antitrypsin (alpha 1AT) deficiency, a common hereditary disorder responsible for emphysema in Caucasians of northern European descent, is caused by single base substitutions, deletions, or additions in the seven exons (IA-IC and II-V), of the 12.2-kb alpha 1AT gene located on chromosome 14 at q31-32.3. Of the five known representatives of the "null" group of alpha 1AT-deficiency alleles (alpha 1AT genes incapable of producing alpha 1AT protein detectable in serum) evaluated at the gene level, all result from mutations causing the formation of stop codons in coding exons of the alpha 1AT gene. The present study identifies an alpha 1AT allele (referred to as "Null(isola di procida")) caused by complete deletion of the alpha 1AT coding exons. The Null(isola di procida) allele was identified in an individual with heterozygous inheritance of M(procida) (an allele associated with alpha 1AT deficiency) and a null allele. Although results of karyotypic analysis were normal, quantification of the copies of alpha 1AT genes in this individual revealed that the index case had only half the normal copies of alpha 1AT genes. Cloning and mapping of the Null(isola di procida) gene demonstrated a deletion of a 17-kb fragment that included exons II-V of the alpha 1AT structural gene. As a consequence of the deletion, the normal noncoding exons (IA-IC) were followed by exons II-V of the downstream alpha 1AT-like gene. Sequence analysis of the deletion demonstrated a 7-bp repeat sequence (GAGGACA) both 5' to the deletion and at the 3' end of the deletion, a 4-bp palindromic sequence (ACAG vs. CTGT) bracketing the deletion, and a novel inserted 4-bp sequence (CCTG) at the breakpoint, suggesting that the mechanism of the deletion may have been "slipped mispairing."     

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

Genetic polymorphism of alpha 1-antitrypsin in green monkeys studied by isoelectric focusing and family analysis

24 variants of alpha 1-antitrypsin (alpha 1-AT) were recognized in sera of 120 wild and capture-born African green monkeys by isoelectrofocusing in Ampholine PAG-plates (pH 4-6.5) and western blotting with antihuman alpha 1-AT serum. All variants had much more cathodal position than human alpha 1-AT and revealed very high microheterogeneity which was slightly different from the observed in human alpha 1-AT. The alpha 1-AT banding pattern allowed to postulate existence of 10 codominant alleles in the Pi locus of African green monkeys. The reality of 8 alleles was proved by family analysis which included 45 monkey birth cases. Two other alleles were absent in the parents available. Thus, alpha 1-AT is the most polymorphic among the known serum proteins of African green monkeys. The latter can be useful for molecular systematics of these primates.     

Genetic polymorphism of the alpha 1-antitrypsin system in the native population of the Kazakh SSR

Phenotypes of alpha 1-antitrypsin of 218 natives from three ethno-historical regions of the Kazakh SSR were detected by isoelectric focusing in ultrathin layer polyacrylamide gel. Gene frequencies of the PiM1, PiM2, PiM3 subtypes in the summary extract were as follows: 0.8477, 0.1372 and 0.0106, respectively; the total gene frequency of two rare variants (PiN and PiZ) was 0.0046. The observed distribution of Pi subtypes shows good agreement with the Hardi-Weinberg equation. The analysis of the interpopulation intraethnic variability of the alpha 1-antitrypsin phenotype and allele frequencies in Kazakhs revealed clear local diversity. The frequency of PiM1 in the natives from North-Central ethno-historical region was reliably lower and that of PiM2 higher than in populations from South-East and West regions. The extracts analysed did not differ in the PiM3 incidences. The results of these studies were compared with the literature data for alpha 1-antitrypsin polymorphism in populations of the Euro-Asia. It is shown that PiM1 and PiM2 frequencies in the Kazakhs differ from the corresponding mean values both in mongoloid and europeoid groups. At the same time, they do not correspond to the intermediate frequency estimations, which could be expected from the fact of mixed origin of the Kazakh people and their border-line geographical position between Europe and Asia. Possible reason for such discrepancy is discussed.