Serum Alpha 1AT Research Articles

Deletion/frameshift mutation in the alpha 1-antitrypsin null allele, PI*QObolton.

The most common deficiency allele of the protease inhibitor (PI) alpha 1-antitrypsin (alpha 1AT) is PI*Z. Other rare deficiency alleles of alpha 1AT are of two types: those producing low but detectable amounts of alpha 1AT (less than 20% of normal serum concentrations), and null alleles producing less than 1% of normal alpha 1AT and therefore not detectable by routine quantitative methods. We have previously used DNA polymorphisms and family data to determine heterozygosity in an individual producing low levels of serum alpha 1AT (12% of normal) of PI type Mmalton. By DNA analysis we observed the typical haplotype associated with PI*Mmalton and a unique null haplotype associated with the allele PI*QObolton. The QObolton allele produces no detectable serum alpha 1AT. We have cloned and sequenced the QObolton allele from a phage genomic library. Deletion of a single cytosine residue near the active site of alpha 1 AT in exon V results in a frameshift causing an in-frame stop codon downstream of the deletion. This stop codon leads to premature termination of protein translation at amino acid 373, resulting in a truncated protein. The truncated protein is predicted to have an altered carboxy terminus (amino acids 363-372) and will lack structurally important amino acids.

Alpha 1-antitrypsin deficiency caused by the alpha 1-antitrypsin Nullmattawa gene. An insertion mutation rendering the alpha 1-antitrypsin gene incapable of producing alpha 1-antitrypsin.

alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder associated with reduced serum alpha 1AT levels and the development of pulmonary emphysema. An alpha 1AT gene is defined as "Null" when no alpha 1AT in serum is attributed to that alpha 1AT gene. Although all alpha 1AT Null genes have identical phenotypic consequences (i.e. no detectable alpha 1AT in the serum), different genotypic mechanisms can cause the Null state. This study defines the molecular basis for the alpha 1AT gene Nullmattawa, identified and cloned from genomic DNA of an individual with the Null-Null phenotype and emphysema resulting from the heterozygous inheritance of the Nullmattawa and Nullbellingham genes. Sequencing of exons Ic-V and all exon-intron junctions of the Nullmattawa gene demonstrated it was identical to the common normal M1(Val213) alpha 1AT gene except for the insertion of a single nucleotide within the coding region of exon V, causing a 3' frameshift with generation of a premature stop signal. Family analysis using oligonucleotide probes specific for the Nullmattawa sequence demonstrated the gene was inherited in an autosomal fashion. Examination of blood monocytes demonstrated that a normal-sized, 1.8-kb alpha 1AT mRNA transcript is associated with the Nullmattawa gene and in vitro translation of mRNA with the Nullmattawa mutation showed it translated at a normal rate but produced a truncated alpha 1AT protein. Additionally, retroviral transfer of the alpha 1AT Nullmattawa cDNA to murine fibroblasts demonstrated no detectable intracellular or secreted alpha 1AT, despite the presence of alpha 1AT Nullmattawa mRNA transcripts. These findings are consistent with the concept that the molecular pathophysiology of Nullmattawa is likely manifested at a posttranslational level. The identification of the Nullmattawa gene supports the concept that Null alpha 1AT alleles represent a heterogenous group in which very different mechanisms cause the identical phenotypic state.

Characterization of the gene and protein of the alpha 1-antitrypsin "deficiency" allele Mprocida.

The "deficiency" group of alpha 1-antitrypsin (alpha 1AT) alleles is characterized by alpha 1AT genes that code for alpha 1AT present in serum but in amounts insufficient to protect the lower respiratory tract from progressive destruction by its burden of neutrophil elastase. Mprocida, a rare alpha 1AT allele associated with alpha 1AT serum levels less than 10 mg/dl (normal 150-350 mg/dl), codes for an alpha 1AT molecule that focuses on immobilized pH gradient isoelectric gels slightly cathodal to the common normal M1 (Val213) protein. On a per molecule basis, Mprocida has a mildly reduced function as an inhibitor, with an association rate constant for human neutrophil elastase of 7.0 +/- 0.1 x 10(6) M-1 s-1 (normal M1 (Val213) 9.3 +/- 0.8 x 10(6), p less than 0.01). The Mprocida molecule behaves normally in vivo with a half-life similar to normal M1 alpha 1AT molecules. Restriction endonuclease mapping demonstrates that the cloned Mprocida gene was grossly intact. Sequencing of all the exons, exon-intron junctions, and the major promoter region demonstrated Mprocida to be identical to the M1 (Val213) gene except for a single base substitution in exon II coding for amino acid 41 of the mature protein (M1 (Val213) Leu41 CTG----Mprocida Pro41 CCG). Usefully, the coding sequence of the alpha 1AT residues 40-41 is recognized by the restriction endonuclease PvuII so that using a probe corresponding to this region of exon II, the Mprocida mutation can be rapidly identified by Southern analysis. Evaluation of the crystallographic structure of alpha 1AT suggests the Leu41 to Pro41 mutation may disrupt alpha-helix A in the region of Pro21-Ser45, suggesting the possibility that the alpha 1AT Mprocida molecule is unstable and degraded intracellularly prior to secretion.

Biochemical Efficacy and Safety of Monthly Augmentation Therapy for α1-Antitrypsin Deficiency

The hereditary disorder alpha 1-antitrypsin (alpha 1AT) deficiency results in the development of emphysema due to a diminished anti-neutrophil elastase screen of the lower respiratory tract. Specific therapy for this disorder is available in the form of weekly intravenous infusions of human plasma alpha 1AT, which effectively reconstitute the anti-elastase screen of the lung in these individuals. In an attempt to reduce the frequency of therapy we evaluated the ability of monthly infusions of alpha 1AT to provide equivalent lower respiratory tract protection against neutrophil elastase. Intravenous infusion of 250 mg/kg of alpha 1AT at 28-day intervals to nine individuals with alpha 1AT deficiency and emphysema was carried out for 12 months. Serum alpha 1AT levels exceeded the protective threshold for an average of 25 days after each dose of alpha 1AT was administered. Furthermore, the postinfusion level of alpha 1AT in the nadir lung epithelial lining fluid was fivefold greater than the preinfusion level, and the anti-neutrophil elastase capacity of the nadir epithelial lining fluid also was elevated significantly, nearly threefold above the preinfusion level. These results indicate that monthly administration of human alpha 1AT is fully capable of adequately augmenting serum and lung alpha 1AT levels and anti-elastase capacity and is therefore a rational alternative to weekly therapy.

Characterization of the M1(Ala213) type of alpha 1-antitrypsin, a newly recognized, common "normal" alpha 1-antitrypsin haplotype.

alpha 1-Antitrypsin (alpha 1AT) is a highly pleomorphic 52-kDa serum glycoprotein that functions as the major inhibitor of neutrophil elastase. Of these, the most common normal alpha 1AT haplotypes identified by isoelectric focusing (IEF) of serum are those of the M family, including M1, M2, and M3. In the course of studying the alpha 1AT type Z gene, we identified a restriction endonuclease BstEII polymorphism in the M1 gene that predicted the existence of a previously unidentified, but relatively common, haplotype of M, referred to as M1(Ala213) [Nukiwa, T., Satoh, K., Brantly, M. L., Ogushi, F., Fells, G. A., Courtney, M., & Crystal, R. G. (1986) J. Biol. Chem. 261, 15989-15994]. In this study we have cloned both alpha 1AT genes from an individual heterozygous for the M1(Ala213) and M1(Val213) haplotypes. Sequencing of the coding exons of both demonstrated that they are identical except for the Ala-Val difference at residue 213. The codominant transmission of the M1(Ala213) gene was demonstrated in a family study. Evaluation of 39 genomic samples of Caucasians with the IEF haplotype M1 demonstrated haplotype frequencies of 68% for M1(Val213) and 32% for M1(Ala213). alpha 1AT serum levels of individuals inheriting the M1(Ala213) gene in a homozygous fashion were in the same range as those for homozygous M1(Val213) as was the rate of association of the M1(Ala213) protein with neutrophil elastase.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of tamoxifen as a therapy to augment alpha-1-antitrypsin concentrations in Z homozygous alpha-1-antitrypsin-deficient subjects.

Tamoxifen, an agent that binds to intracytoplasmic estrogen receptors, was evaluated as a possible means of increasing alpha-1-antitrypsin (alpha 1AT) synthesis and/or secretion and thus alpha 1AT serum levels in subjects with the homozygous form of alpha 1AT deficiency. Administration of tamoxifen (10 mg twice daily) to 30 Z homozygotes for a 30-day period was not associated with adverse reactions. However, although serum alpha 1AT levels increased significantly (p less than 0.03), the increase was minor (average pretreatment levels, 32 +/- 1 mg/dl; levels at 30 days of therapy, 35 +/- 1 mg/dl) and far below the "threshold" level of 80 mg/dl considered "protective" against an increased risk for emphysema. Thus, while the concept that increasing alpha 1AT synthesis and/or secretion is a rational goal for treating the Z homozygous form of alpha 1AT deficiency, tamoxifen will not be useful in this regard.

Identification of a second mutation in the protein-coding sequence of the Z type alpha 1-antitrypsin gene.

This study reports the entire nucleotide sequence of the protein coding region sequence of the alpha 1-antitrypsin (alpha 1AT) Z gene, a common form of the alpha 1AT gene associated with serum alpha 1AT deficiency. In addition to Glu342 to Lys342 mutation in exon V which has been previously identified by peptide analysis, another point mutation (GTG to GCG in exon III) in the gene sequence predicts a second amino acid substitution (Val213 to Ala213) in the Z protein. This Val213 to Ala213 mutation was confirmed to be a general finding in Z type alpha 1AT gene by evaluating genomic DNA from 40 Z haplotypes using synthetic oligonucleotide gene probes directed toward the mutated exon III sequences in the Z gene. Furthermore, the exon III Val213 to Ala213 mutation eliminates a BstEII restriction endonuclease site in the alpha 1AT Z gene, allowing rapid identification of this Val213 to Ala213 substitution at the genomic DNA level. Surprisingly, when genomic DNA samples from individuals thought to be homozygous for the M1 gene (the most common alpha 1AT normal haplotype) were evaluated with BstEII, 23% of the M1 haplotypes were BstEII site negative, thus identifying a new form of M1 (i.e. M1(Ala213], likely identical to M1 but with an isoelectric focusing "silent" amino acid substitution (Val213 to Ala213). Although the relative importance of the newly identified exon III Val213 to Ala213 mutation to the pathogenesis of the abnormalities associated with the Z gene is not known, it is likely that M1(Ala213) gene represents a common "normal" polymorphism of the alpha 1AT gene that served as an evolutionary intermediate between the M1(Val213) and Z genes.

Biosynthesis, processing, and secretion of M and Z variant human alpha 1-antitrypsin.

The Z genetic variant of human alpha 1-antitrypsin (alpha 1AT) is associated with decreased serum alpha 1AT levels, hepatic inclusion bodies, and an increased risk of lung and liver disease. We studied the biosynthesis, processing, and secretion of normal and Z variant alpha 1AT in cell-free translation systems, reconstituted in vitro processing systems, and in the Xenopus oocyte secretory system. Human liver mRNA was prepared from normal subjects (PiMM) and from individuals homozygous for alpha 1AT deficiency (PiZZ). Cell-free translation resulted in the synthesis of 49,000-Da preproteins with a 23-amino acid signal sequence. The genetic variants were synthesized at comparable levels and could be distinguished on the basis of charge. The majority of the amino acids in the ZZ signal peptide were identified and found to be the same as those comprising the MM signal sequence. These proteins were co-translationally processed with similar efficiency by dog pancreas microsomes, producing 52,000-Da glycoproteins which were completely translocated across the endoplasmic reticulum membrane. When the human liver RNA preparations were injected into Xenopus oocytes, both of the alpha 1AT variants were synthesized intracellularly and alpha 1AT was detected in the medium of all oocytes injected with MM RNA. However, the Z variant accumulated within the microsomal vesicles of the cell and was undetectable or present at decreased levels in the medium. We conclude that the single amino acid substitution in the Z variant of alpha 1AT does not affect its synthesis or co-translational processing but that it strongly affects its transport from the rough endoplasmic reticulum through the secretory pathway.

A new allele of alpha 1-antitrypsin: PI NADELAIDE.

The allele PI NADELAIDE (PI NADE) was named in accord with nomenclature guidelines and specifies a new co-dominant variant of alpha 1AT. Discovery was achieved by IEF and the isoelectric point of NADE is between N and NHAM. Familial inheritance of PI NADE was demonstrated and both PI M2NADE and PI M3NADE phenotypes were observed. The mobility of PI NADE is identical to PI M by both starch and agarose electrophoresis. PI NADE apparently confers normal alpha 1AT serum concentrations and is probably unrelated to disease.

Alpha-1-antitrypsin in umbilical cord serum: Pi phenotypes and relationships with idiopathic respiratory distress syndrome

The concentrations and phenotypes of serum alpha-1-antitrypsin (alpha1AT) were determined in 650 newborn infants. The distribution of these 650 subjects among the various Pi phenotypes confirms the higher frequency reported for the PiS allele in Latin populations. Serum alpha1AT levels vary between one phenotype and the other. Besides, at birth, infants weighing more than 2,500 g have alpha1AT levels significantly higher (P less than 0.001) than infants weighing less than 2,500 g; this difference in serum alpha1AT concentrations is due to the low alpha1AT levels found in preterm infants. The significantly lower alpha1AT concentrations found in preterms is associated with a higher risk of developing IRDS and with a mean birth weight under 2,000 g. Infants who develop IRDS frequently have lower alpha1AT levels than those who do not develop the syndrome, independently from body weight. On the basis of serum alpha1AT quantitation, newborn infants may be separated into two groups, characterized respectively by concentrations above or below 150 mg%. From our data, it appears that if the group with an alpha1AT concentration lower than 150 mg% is phenotyped, it is possible to differentiate infants with a high risk of fatal IRDS from individuals with a "pathological" phenotype.

Alpha1-Antitrypsin: 1. Evidence for two genes causing low concentrations in serum; 2. Association of heterozygosity and Chronic obstructive lung disease

Alpha1-antitrypsin (alpha1-at) is one of several proteinase inhibitors in human serum. It is a glycoprotein with molecular weight of approximately 50,000. The normal concentration in serum is 213±32 mg/100 ml. The corresponding trypsin inhibiting capacity is between 1.0 and 1.3 mg of trypsin inhibited by one ml of serum. During recent years, different phenotypes of alpha1-at have been identified and a genetic model with several codominant alleles at one locus has been developed. Two phenotypes can be distinguished quantitatively and electrophoretically while others can be recognized by electrophoresis only. I consider here only the phenotypes with lower than normal alpha1-at concentrations in serum. The method most commonly used to determine the total trypsin inhibiting capacity of serum is an inhibition assay with H,α-benzoyl-arginine-p-nitroanilide as substrate for trypsin. A convenient method for a specific determination of alpha1-at is the radial immunodiffusion employing an antiserum against alpha1-at. For the electrophoretic distinction of the different phenotypes starch gel electrophoresis at an acidic pH (4.95) in combination with a second electrophoresis at an alkaline pH (8.65) in agarose gel that contains an antiserum at alpha1-at is a sensitive method. With this technique, the alpha1-at phenotypes of heterozygotes and homozygotes for the two genes, Pin and Pz (nomenclature of Fagerhol), that cause lower concentrations of serum alpha1-at, can be recognized as a characteristic pattern of bands. The protein that is determined by the gene Piz moves on electrophoresis more slowly towards the anode than that determined by Pi”, but both are moving slower than the normal protein controlled by the most common gene PiM. In heterozygotes, both patterns of alpha1-at can be recognized. The resulting phenotypes are called: M/M, S/S, Z/Z, M/S, M/Z, etc. The alpha1-at concentrations for these phenotypes are in mg/100 ml of serum: M/M; 213±32, S/S; 122±10, Z/Z; 25±6, M/S; 159±39, M/Z; 125±46. These concentrations are only found under normal physiologic conditions. In the presence of an inflammation, during pregnancy, and due to certain steroid hormones, the alpha1-at concentration of normal individuals can rise to levels 100 percent above the starting value, and heterozygotes can show values in the normal range of 200 mg percent. The electrophoretic characteristics are, however, independent of concentration and for that reason the electrophoretic determination of the alpha1-at phenotype is more reliable than the quantitative one, particularly in patients with inflammation.