Alpha 1AT Deficiency Research Articles

Alpha 1-antitrypsin. Hope on the horizon for emphysema sufferers?

Alpha 1-Antitrypsin (alpha 1AT) deficiency is the most common genetic cause of liver disease in children and emphysema in adults. Therapy for pulmonary disease attributable to alpha 1AT deficiency includes alpha 1AT augmentation therapy along with supportive measures. The alpha 1AT preparation that is currently used for therapy is derived from fractionated plasma. The results of clinical trials suggest that augmentation therapy with alpha 1AT slows the progression of emphysema and causes few adverse events. Patients with plasma levels of alpha 1AT that are < 11 mumol/L and who have airway obstruction should be considered for augmentation therapy. Novel approaches include the administration of aerosolised alpha 1AT, recombinant alpha 1AT, gene therapy and synthetic elastase inhibitors.

Alpha-1-Antitrypsin Deficiency

Homozygous PIZZ alpha 1-antitrypsin deficiency, which has an incident of 1 in 1600 to 1 in 2000 live births, is the most common genetic cause of liver disease in children. It is also associated with chronic liver disease and hepatocellular carcinoma in adults. It is a well-known cause of pulmonary emphysema. Although emphysema is due to uninhibited proteolytic destruction of the connective tissue backbone of the lung, liver disease is thought to result from the toxic effects of the mutant alpha 1AT molecule retained within the endoplasmic reticulum of liver cells. Screening studies done by Sveger in Sweden have shown that only 10 to 15% of the PIZZ population develop clinically significant liver disease over the first 20 years of life. Recent studies have suggested that a subgroup of PIZZ individuals are predisposed to liver injury because of an inefficient degradation of mutant alpha 1ATZ within the endoplasmic reticulum. Altered migration of the abnormal alpha 1ATZ molecule in isoelectric focussing gels is the basis of the diagnosis of alpha 1AT deficiency. Treatment of alpha 1AT deficiency-associated liver disease is mostly supportive. Liver replacement therapy has been used successfully for severe liver injury. An increasing number of patients with severe emphysema have undergone lung transplantation.

Pharmacokinetic Study of α1-Antitrypsin Infusion in α1-Antitrypsin Deficiency

To ascertain how long 120 mg/kg alpha1-antitrypsin concentrate (alpha1-AT-C), administered I.V. every 2 weeks, can maintain alpha1-antitrypsin (alpha1-AT) serum levels above 70 to 80 mg/dL. Secondary objectives were to summarize the nature, severity, and relationship of a plasma-derived alpha1-AT-C infusion to any side effects. This was an open-label uncontrolled pharmacokinetic study. Alpha1-AT-C was administered I.V. every 2 weeks for 10 infusions in 23 patients with PIZ alpha1-AT deficiency. Serum alpha1-AT levels and neutralizing elastase activity were measured preinfusion, postinfusion, and at nadir. During two infusion periods, daily serum alpha1-AT and neutralizing elastase activities were measured on the seventh to 14th days. Five patients received BAL assays for alpha1-AT and neutralizing elastase activity. Adverse events were recorded in a patient diary and by a nurse at each infusion visit. The 120-mg/kg dose of alpha1-AT-C could not maintain nadir serum protective levels above 70 or 80 mg/dL for the entire 14-day dosing interval in most patients. None of the patients had alpha1-AT levels above 80 mg/dL for all 14 days. The serum alpha1-AT and neutralizing elastase levels correlated suggesting functional activity. The BAL alpha1-AT and neutralizing elastase activities were low and did not correlate with serum levels. Alpha1-AT-C at 120 mg/kg administered every 2 weeks did not maintain nadir serum alpha1-AT levels above 70 to 80 mg/dL for a 14-day dosing interval. Higher doses every 2 weeks or decreased interval between infusions may be required.

Molecular basis, clinical consequences and diagnosis of alpha-1 antitrypsin deficiency.

(1) Deficiency of alpha AT is one of the most common hereditary diseases affecting Caucasians in Europe. The alpha 1AT protein is extremely pleomorphic, and around 90 variants due to mutations have been recognized. The prime functions of alpha 1AT is to inhibit neutrophil elastase, and a proportion of individuals who are deficient in alpha 1AT develop emphysema. The most common deficiency variant (Z) is also associated with liver disease. The main site of alpha 1AT synthesis is in the liver. Not all deficient individuals are affected by lung or liver disease, however, so that other factors (genetic and environmental) are clearly important. (2) Investigation of alpha 1AT status is essential in any child or adult presenting with chronic liver disease. The genetic cause cannot be identified clinically or by any other laboratory investigation. The diagnosis carries important prognostic consequences and is important for other family members. Patients with emphysema should have their Pi type determined, especially if they are under the age of 50, have never smoked or there is a suggestive family history. Asymptomatic individuals who are homozygous type Z should be referred to a chest physician for a clinical and radiological assessment together with lung function tests. (3) Several laboratory tests are available to detect alpha 1AT deficiency, and the choice of test(s) will depend on circumstances. Quantitation of the serum protein is simple and cheap. Because alpha 1AT is an acute phase protein, however, quantitation used in isolation may give false negative results which are clearly unacceptable, particularly in association with paediatric liver disease. Phenotyping by isoelectric focusing requires some experience in distinguishing SZ and ZZ phenotypes, and phenotyping should ideally be used in conjunction with quantitation because heterozygous null phenotypes may appear identical to homozygous normal phenotypes. (4) Prenatal diagnosis is usually performed by DNA analysis of CVS samples obtained at 11-13 weeks. Because of the risk that CVS samples might be contaminated by maternal tissue, assays which are less likely to detect minor contaminants are preferable. At present, use of DNA tests is confined to prenatal diagnosis, but the availability of simple tests and the possibility of unequivocal identification of S and Z alleles means that these tests are likely to find greater use in the near future.

Alpha1-antitrypsin gene mutation hot spot associated with the formation of a retained and degraded null variant [corrected; erratum to be published

Null alpha1-antitrypsin (alpha1AT) alleles represent the end of a continuum of variants associated with profound alpha1AT deficiency and an increased risk of emphysema. This study characterizes the molecular basis of QOclayton, a new example of an alpha1AT null allele arising from a mutational hot spot in the alpha1AT gene. The QOclayton allele is identical to the normal M1(V213) alpha1AT allele except for an insertion of a cytosine. This insertion occurs in the alpha1AT sequence which normally has seven cytosines corresponding to amino acid residues 360 to 362. The QOclayton mutation is located in the same reiterated DNA sequence as the alpha1AT QObolton deletion mutation and the insertion mutation allele QOsaarbruecken. The QOclayton cytosine insertion causes a 3' frameshift and results in the formation of a termination codon at residue 376, the same consequence as the alpha1AT QOmattawa mutation (L353 T-insertion with a 3' frameshift). To determine the molecular mechanisms responsible for the absence of alpha1AT associated with the QOclayton gene, an in vitro model of QOclayton was established using Chinese hamster ovary cells (CHO) transfected with the QOclayton gene. These cells were evaluated for alpha1AT mRNA expression, protein synthesis and secretion. Although the QOclayton gene expresses a similar amount of alpha1AT mRNA as compared with the normal alpha1AT gene, no QOclayton protein is secreted. Protein trafficking and double-label immunofluorescence demonstrate that the QOclayton protein is retained in the rough endoplasmic reticulum or pre-Golgi compartment and is degraded (t1/2 = 6.5 h). Since QOmattawa, QObolton, and QOsaarbruecken have similar termination sites in the alpha1AT mRNA, they may share a similar intracellular fate.

Degradation of a Mutant Secretory Protein, α1-Antitrypsin Z, in the Endoplasmic Reticulum Requires Proteasome Activity

Degradation of proteins that are retained in the quality control apparatus of the endoplasmic reticulum (ER) has been attributed to a third proteolytic system, distinct from the lysosomal and the cytoplasmic ubiquitin-dependent proteosomal proteolytic pathways. However, several recent studies have shown that ER degradation of a mutant membrane protein, CFTRdeltaF508, is at least in part mediated from the cytoplasmic side by the 26 S proteasome. In this study, we examined the possibility that ER degradation of mutant secretory protein alpha1-antitrypsin (alpha1-AT) Z, the mutant protein associated with infantile liver disease and adult-onset emphysema of alpha1-AT deficiency, is mediated by the proteasome. The results show that a specific proteasome inhibitor, lactacystin, inhibits ER degradation of alpha1-ATZ in transfected human fibroblast cell lines and in a cell-free microsomal translocation system. Although it is relatively easy to conceptualize how a transmembrane protein like CFTRDeltaF508 might be accessible on the cytoplasmic aspect of the ER membrane for ubiquitination and degradation by the proteasome, it is more difficult to conceptualize how this might occur for a luminal polypeptide. The results show that, once within the lumen of the ER, alpha1-ATZ interacts with the transmembrane molecular chaperone calnexin and specifically induces the polyubiquitination of calnexin. The results, therefore, provide evidence that the proteasome, from its cytoplasmic localization, induces the degradation of the luminal alpha1-ATZ molecule by first attacking the cytoplasmic tail of calnexin molecules that are associated with alpha1-ATZ.

C-antineutrophil cytoplasmic antibody positivity in vasculitis patients is associated with the Z allele of alpha-1-antitrypsin, and P-antineutrophil cytoplasmic antibody positivity with the S allele

Antineutrophil cytoplasmic antibodies (ANCA) in vasculitis have either cANCA or pANCA patterns as defined by immunofluorescence. The target autoantigen of cANCA is usually proteinase 3 (PR3), whereas that of pANCA is usually myeloperoxidase (MPO). Alpha-1-antitrypsin (alpha 1AT) is the major physiological inhibitor of PR3, while MPO is an inhibitor of alpha 1AT. To determine whether there was an association between ANCA positive vasculitis, ANCA pattern, and alpha 1AT deficiency alleles, we studied alpha 1AT phenotypes of 99 cANCA and 99 pANCA positive vasculitis patients by isoelectric focusing and immunoblotting, and compared them with 2310 controls from the same geographical area. C-ANCA patients showed an increased frequency of the Z allele (0.055 versus 0.018 in controls), conferring a relative risk of 3. They showed no increase in frequency of the S allele. P-ANCA patients showed an increased frequency of the S allele (0.091 versus 0.046 in controls) conferring a relative risk of 2. The frequency of the Z allele also appeared to be increased (0.030 versus 0.018 in controls), but this was not statistically significant. These findings demonstrate an association between ANCA-positive vasculitis and deficiency phenotypes of alpha 1AT, and suggest a role for alpha 1AT in the development of systemic vasculitis.

Polarity of secretion of alpha 1-antitrypsin by human respiratory epithelial cells after adenoviral transfer of a human alpha 1-antitrypsin cDNA.

alpha 1-Antitrypsin (alpha AT) deficiency, a hereditary cause of progressive emphysema, can potentially be treated by transfer of a functional human alpha 1AT gene to the respiratory epithelium. For such an approach to be successful, alpha 1AT must be provided to both the interstitium and the epithelial surface--that is, the alpha 1AT directed by the transferred gene must be secreted to both the apical and basolateral surfaces of the epithelial cells. To evaluate this concept, a recombinant, replication-deficient adenoviral vector (Ad-alpha 1AT) containing a human alpha 1AT cDNA driven by an adenovirus major late promoter was used to infect Bet-1A human respiratory epithelial cells. The infected cells expressed Ad-alpha 1AT-directed mRNA transcripts and synthesized and secreted functional human alpha 1AT as shown by [35S]methionine labeling and immunoprecipitation of a 52-kD glycosylated human alpha 1AT molecule capable of interacting with neutrophil elastase, its natural substrate. Bet-1A cells grown on microporous polycarbonate membranes formed tight junctions (resistance > 150 omega x cm2). After infection with Ad-alpha 1AT, [35S]methionine labeling and enzyme-linked immunoassay demonstrated that alpha 1AT was secreted into both the apical and basolateral compartments, with an average apical to basolateral ratio of 1.9 +/- 0.2. Thus, human respiratory epithelial cells infected with a recombinant adenoviral vector containing a human alpha 1AT cDNA secrete alpha 1AT across both the apical and basolateral cell membranes, suggesting that the respiratory epithelium could serve as a target for in vivo gene therapy of alpha 1AT deficiency.

Abdominal aortic aneurysms and alpha1‐antitrypsin

The incidence of abdominal aortic aneurysms (AAAs) has been reported to be increased in heterozygous (PiMZ) and homozygous (PiZZ) alpha 1-antitrypsin (alpha 1 AT) deficiency. We report a further case of AAA in a PiSZ alpha 1AT-deficient female with primary biliary cirrhosis (PBC). Moreover, to assess the risk of AAA in patients with PiZZ alpha 1AT deficiency, we conducted a retrospective study based on 30 consecutive autopsied adult cases of severe alpha 1AT deficiency identified during the period 1963-1993 in the city of Malmö, Sweden, during which autopsies were performed in 49,548, or 58.4%, of all residents in the city who died. From the homozygote frequency in the general population, 50 of these would be expected to have alpha 1AT deficiency. The disease had been diagnosed in 35, and autopsies had been performed in 30 adults. Each autopsied case was matched with four controls selected from the same autopsy register, and the Mantel-Haenszel odds ratio (ORmh) was calculated. We found one case of AAA amongst the 30 PiZZ individuals autopsied, and one case amongst 120 autopsied controls (ORmh = 2.65, 95% confidence limits, 0.47-15.0; P = 0.273). We found no significant evidence of the relationship between AAA and the PiZZ polymorphism, although previously reported by others. At least in severe alpha 1AT deficiency, any coexistence of the PiZZ phenotype and AAA would seem to be fortuitous.

Purification of alpha-1-antitrypsin monomer by preparative electrophoresis.

Alfa-1-antitrypsin (alpha 1AT) was purified by pseudoligand chromatography and preparative electrophoresis from the serum of a patient with alpha 1AT deficiency. The combination of the two techniques yielded a high grade batch of alpha 1AT monomer and this was successfully used to purify the protein from the serum of PiMIM1, PiMIM2, and PiZZ phenotype subjects. This procedure should facilitate structural studies of alpha 1AT variants susceptible to intracellular accumulation.

Family study of alpha 1-antitrypsin deficiency: effects of cigarette smoking, measured genotype, and their interaction on pulmonary function and biochemical traits.

To gain insight into the variable expression of lung disease in alpha 1-antitrypsin (alpha 1AT) deficiency, five quantitative variables including forced expiratory volume at 1 sec (FEV1), forced expiratory flow rate between 25 and 75% of forced vital capacity (FEF25-75), total serum alpha 1AT, oxidized serum alpha 1AT, and total serum immunoglobulin E (IgE) were measured in alpha 1AT deficient individuals and their families. The effect of a known, measured genotype (the Pi type) was estimated for each quantitative trait; the influence of mode of case ascertainment on the measured genotype effect was also assessed. These analyses showed that total alpha 1AT levels are strongly influenced by Pi type; IgE levels are unaffected by Pi type; and FEV1, FEF25-75, and oxidized alpha 1AT are moderately influenced by Pi type. The effect of genotype-by-environment interaction between Pi type and pack-years of cigarette smoking on the five quantitative phenotypes was studied using an analysis of covariance. Significant Pi x pack-years interaction was evident for FEV1, but this effect is confounded in this data set with the Pi x age interaction. Probands who were ascertained because they had chronic obstructive pulmonary disease (COPD) do not demonstrate the significant Pi x pack-years interaction effect of the Pi x pack-years subjects ascertained for other reasons demonstrate. The effect of the Pi x pack-years interaction on FEV1 was no longer significant on a transformed scale, (FEVf12,) thus providing an additive scale for future data analysis. The increased sensitivity of Pi MZ individuals in our sample to cigarette smoking reduced the Pi x packs-years interaction effect on FEF25-75 to borderline significance. This investigation has provided an opportunity to incorporate both measured genotype and genotype-by-environment interaction analyses into the study of the variable expression of lung disease in Pi Z individuals.

Temperature gradient gel electrophoresis: rapid detection of alpha-1-antitrypsin deficiency carriers.

The homozygous state of the alpha-1-antitrypsin (alpha 1AT) deficiency variant Z is associated with severe liver damage in early childhood and progressive lung emphysema in adulthood. A single base transition (G to A in codon 342) in exon V is causing the severe disease. The Glu342 to Lys342 mutation can be detected conventionally by isoelectric focusing (IEF) or on the DNA level by the newly developed method of temperature gradient gel electrophoresis (TGGE). It is the aim of this study to describe the TGGE technique, to compare the results with conventional IEF, and to discuss its efficiency for different diagnostic applications.

Alpha 1-antitrypsin deficiency, complement activation, and chronic liver disease.

Activation of the complement system, the main humoral mediator of inflammation, is restrained by the action of enzyme inhibitors including alpha 1-antitrypsin. Deficiency leads to chronic liver disease in about one in five children with this genetic defect. Complement activation was investigated in 34 children with alpha 1 AT deficiency (12 with minimal, 10 with moderate, and 12 with severe liver disease) and in 38 sex and age matched normal children by measuring the complement parent molecules C3, C4, the C3d fragment and by calculating the C3d:C3 ratio. C3 and C4 were lower in children with severe liver disease compared with controls, indicating impairment of hepatic protein synthesis or complement consumption. The C3d activation fragment was higher in all the patient groups when compared with controls while the C3d:C3 ratio, a measure of activation independent of the concentrations of the parent molecule, was higher in patients than in controls and increased with the degree of disease severity. These results suggest that complement may have a role in the pathogenesis of the chronic liver disease associated with alpha 1AT deficiency.

Neutrophil accumulation in the lung in alpha 1-antitrypsin deficiency. Spontaneous release of leukotriene B4 by alveolar macrophages.

The emphysema of alpha 1-antitrypsin (alpha 1AT) deficiency is conceptualized to result from insufficient alpha 1AT allowing neutrophil elastase to destroy lung parenchyma. In addition to the deficiency of alpha 1AT in these individuals resulting from mutations in the alpha 1AT gene, it is recognized that, for unknown reasons, there are also increased numbers of neutrophils in their lungs compared with normal individuals. With the knowledge that alveolar macrophages have surface receptors for neutrophil elastase, we hypothesized that the neutrophil accumulation in the lower respiratory tract in alpha 1AT deficiency may result, in part, from release of neutrophil chemotactic activity by alveolar macrophages as they bind uninhibited neutrophil elastase. Consistent with this hypothesis, alpha 1AT-deficient alveolar macrophages spontaneously released nearly threefold more neutrophil chemotactic activity than normal alveolar macrophages. Analysis of alpha 1AT-deficient macrophage supernates by reverse-phase HPLC, molecular sieve chromatography, radioimmunoassay, and absorption with anti-LTB4 antibody revealed that the majority of the chemotactic activity was leukotriene B4 (LTB4), a mediator absent from normal macrophage supernates. Consistent with this hypothesis, incubation of normal macrophages with human neutrophil elastase resulted in the release of the same neutrophil chemotactic mediator. Furthermore, purified human alpha 1AT was able to prevent the neutrophil elastase from stimulating the macrophages to release the chemotactic factor. Together, these findings suggest that the absence of a normal antineutrophil elastase screen in the lower respiratory tract permits free neutrophil elastase to bind to alveolar macrophages, resulting in the release of LTB4, a process which attracts neutrophils to the alveoli of alpha 1AT deficient individuals, thus accelerating the lung destruction that characterizes this disorder.

Use of a Highly Purified α1-Antitrypsin Standard to Establish Ranges for the Common Normal and Deficient α1-Antitrypsin Phenotypes

Diagnosis of the hereditary disorder alpha 1-antitrypsin (alpha 1AT) deficiency is critically dependent on quantification of serum levels of alpha 1AT, a 52-kDa antiprotease that serves to protect the lung from destruction by neutrophil elastase. Although the measurement of serum alpha 1AT levels is not difficult, there is no international standard for alpha 1AT, and investigators in the field recognize that widely used commercially available standards vary by as much as 50 percent. To establish accurate ranges for the common normal and deficient alpha 1AT phenotypes, the present study uses a purified alpha 1AT standard to quantify the alpha 1AT serum levels of 443 individuals with common normal and deficient alpha 1AT phenotypes, including MM, ZZ, SS, MZ, MS, and SZ. Based on the observed values, a statistical model was developed to generate predicted frequency distributions of alpha 1AT serum levels for each of these phenotypes. Based on these studies, the ranges (5th to 95th percentile) for alpha 1AT serum levels of the common phenotypes are: MM, 20 to 53 mumol/L; SS, 20 to 48 mumol/L; ZZ, 3.4 to 7.0 mumol/L; MZ, 15 to 42 mumol/L; MS, 18 to 52 mumol/L; and SZ, 10 to 23 mumol/L. This alpha 1AT standard and these ranges are being used for the National alpha 1-Antitrypsin Deficiency Registry organized under the auspices of the National Heart, Lung, and Blood Institute.

α1‐antitrypsin deficiency and liver disease

Alpha 1-antitrypsin (alpha 1AT) deficiency, one of the most common lethal hereditary disorders among Caucasians, is associated with emphysema in adults, while in children it is associated with liver disease. Produced in the liver and released into the plasma, alpha 1AT serves as the body's major inhibitor of neutrophil elastase, a powerful proteolytic enzyme capable of degrading extracellular structural proteins. The pathogenesis of the liver disease associated with alpha 1AT deficiency is not as well understood, but is clearly linked to specific mutations in coding exons of the alpha 1AT gene, and the resulting accumulation of alpha 1AT within hepatocytes. At present, therapy for the liver disease associated with alpha 1AT deficiency is symptomatic, with liver transplantation as a last resort. New strategies are being developed to suppress the accumulation of alpha 1AT by transferring the normal gene into the liver.

Siiyama (serine 53 (TCC) to phenylalanine 53 (TTC)). A new alpha 1-antitrypsin-deficient variant with mutation on a predicted conserved residue of the serpin backbone

alpha 1-antitrypsin (alpha 1AT), a plasma serine protease inhibitor, increases the risk of precocious pulmonary emphysema in individuals when deficient. Although more than 25 years have passed since a deficiency in the serum level of alpha 1AT was reported, it is only recently that the consequence of the amino acid replacement which leads to the deficient state has been discussed in terms of the crystallographic structure of alpha 1AT and the amino acid residues conserved in the superfamily to which it belongs. Our case involved a 38-year-old Japanese male with alpha 1AT deficiency which was analyzed and identified as a new deficient variant. The serum alpha 1AT of the proband migrated to the S position of the reference serum which is more cathodal than M1, the predominant normal variant, when isoelectric focusing (pH 4.2-4.9) is performed by a combination of Western blotting and crossed immunoelectrophoresis. The new deficient variant is designated as Siiyama after his birthplace. Although liver biopsy specimen showed no apparent pathological findings, PAS-positive with diastase-resistant inclusion bodies and immunoreactive aggregates were detected in several hepatocytes. In addition, similar alpha 1AT mRNA transcript levels were observed in peripheral blood leukocytes from the proband and healthy subjects by Northern analysis. All the coding exons (exon Ic, II, III, IV, and V) of the alpha 1AT gene of the proband and his family were amplified by polymerase chain reaction and followed by direct sequencing. A single missense mutation, Ser53 (TCC) to Phe53 (TTC was identified in exon II of the proband's alpha 1AT gene. All his family examined were heterozygous at this base. Ser53 is one of the most conserved residues as predicted by Huber and Carrell (Huber, R., and Carrell, R. W. (1989) Biochemistry 28, 8951-8966) and is thought to contribute to the organization of the internal core element of the alpha 1AT molecule. The mutational matrix number of Ser to Phe substitution is -3, indicating that this change is evolutionally rare. In this regard, a possible explanation for the deficient state in alpha 1AT Siiyama is that the change from an uncharged polar to a nonpolar amino acid imposed on the conserved serpin backbone exerts severe effects on the integrity of the molecule, and hence alters the intracellular processing of alpha 1AT.

Adenovirus-Mediated Transfer of a Recombinant α1-Antitrypsin Gene to the Lung Epithelium in Vivo

The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin (alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.

A null deficiency allele of alpha 1-antitrypsin, QOludwigshafen, with altered tertiary structure.

The most common deficiency allele of the plasma protease inhibitor alpha 1-antitrypsin (alpha 1AT) is PI*Z. Some rare deficiency alleles of alpha 1AT produce low but detectable amounts of plasma alpha 1AT (1-20% of normal), which can be differentiated by isoelectric focusing. Others, designated null (QO) alleles, produce no alpha 1AT detectable by routine quantitative methods. We have previously described a method using DNA polymorphisms, haplotypes, and polyacrylamide isoelectric focusing gels, to differentiate various deficiency alleles. Based on haplotypes, we previously identified, in eight patients, five different null alleles, four of which had been previously sequenced. We have now analyzed all 12 null alleles in these eight patients, using allele-specific oligonucleotide probes, and have identified six different null alleles. We have cloned and sequenced one of these, PI*QOludwigshafen, which has a base substitution in exon II, replacing isoleucine 92 in the normal sequence with an asparagine. This substitution of a polar for a nonpolar amino acid occurs in one of the alpha-helices and is predicted to disrupt the tertiary structure. A total of 13 different alpha 1AT deficiency alleles, 6 of them null alleles, have been sequenced to date.

A Family Study of the Variability of Pulmonary Function in α1-Antitrypsin Deficiency: Quantitative Phenotypes

A group of 52 alpha 1-antitrypsin-deficient individuals of phenotype Pi Z and 117 of their relatives underwent a protocol including pulmonary function testing, completion of a questionnaire, and blood donation. Our population permitted a minimum frequency estimate (7 x 10(-4)) for Pi null alleles. Five quantitative phenotypes were measured, including FEV1, FEF25-75, total serum alpha 1AT, oxidized serum alpha 1AT, and total serum IgE. We found that (1) total alpha 1AT levels were higher in Pi Z subjects with lung function impairment (FEV1 less than or equal to 65% of predicted) than in Pi Z subjects who were not impaired; (2) Pi Z subjects with lung function impairment had elevated serum levels of oxidized alpha 1AT; and (3) IgE levels were relatively elevated in first-degree Pi MZ relatives of impaired Pi Z subjects. Moreover, FEV1 tended to be relatively reduced in heterozygous parents of impaired Pi Z subjects, suggesting that a subset of Pi MZ individuals are at risk for development of lung disease because of familial factors. These results represent an initial step toward the development of intermediate phenotypes that will be predictive of a severe course in alpha 1AT deficiency; they suggest that, in addition to cigarette smoking, atopic predisposition and undetermined familial factors may be important codeterminants of lung disease progression.