Alpha 1AT Deficiency Research Articles

Characteristics of hepatocellular carcinoma in a murine model of alpha‐1‐antitrypsin deficiency

Individuals with homozygous (ZZ) alpha-1-antitrypsin (alpha1AT) deficiency are at an increased risk for liver damage, cirrhosis and hepatocellular carcinoma (HCC). The transgenic PiZ mouse, expressing the human alpha1AT mutant Z gene, is a valuable model for this disease. We studied PiZ mice in order to identify and characterize mechanisms involved in the development of HCC. Tumor incidence and histology were studied, gene expression levels were surveyed with microarrays, RNA quantified with quantitative real time polymerase chain reaction and protein levels determined with immunoblots and immunohistochemistry. By 16-19 months of age, approximately 69% of the PiZ mice had developed tumors. HCC was present with no evidence of benign adenomas as pre-cancerous lesions. Tumors showed abnormal mitochondria, variable levels of steatosis, globular inclusions of alpha1AT mutant Z protein and metastases. PiZ mice that subsequently developed liver tumors had higher serum levels of alpha1AT mutant Z protein than those that did not develop tumors. Cyclin D1, a cell cycle protein, was upregulated in PiZ livers without tumors compared to Wt. cFOS, a component of AP-1 that may be involved in transforming cells and MCAM, an adhesion molecule likely involved in tumorigenesis and metastases, were elevated in tumors compared with livers without tumors. In the PiZ model, many of the histological characteristics of HCC recapitulated features seen in human HCC, whether from individuals with homozygous ZZ liver disease or from unrelated causes in individuals that were not homozygous ZZ. The accumulation of mutant Z protein altered the regulation of several genes driving proliferation and tumorigenesis.

Genetic Variants of α 1-Antitrypsin

Alpha1-antitrypsin (alpha1-AT) is a 52 kDa sialoglycoprotein. The function of alpha1-antitrypsin is to protect the lower respiratory tract of lungs from proteolytic degradation by neutrophil elastase. Severe genetic deficiency of alpha1-AT is associated with early onset emphysema and liver diseases. alpha1-AT also exhibits anti-inflammatory activities independent of its protease inhibitor function. There are over 90 genetic variants of human alpha1-antitrypsin. These variants occur due to amino acid substitution / deletion which results in charge differences. Based on charge differences these variants have been identified by isoelectric focusing. The two most common deficiency variants are S and Z. The S variant migrates anodal to Z variant. The Z variant migrates most cathodal in isoelectric focusing, hence named Z. In Z variant, the beta-sheet A undergoes expansion, therefore it can easily accept the reactive site loop of a second alpha1-AT molecule and consequently form polymers of alpha1-AT. These polymers of alpha1-AT aggregate in the hepatocytes and show liver and lungs diseases. Contrary to this, the S variant of alpha1-AT is not associated with any significant clinical disease because the conformation of the inhibitor is not altered significantly. The Z related pathologies could be treated by liver transplantation, augmentation therapy, gene therapy, peptide therapy and chemical chaperone therapy. In addition to common deficiency variants, there are several rare deficiency variants of alpha1-AT like Siiyama, Mmalton, Mprocida, Mheerlen, Mmineral springs, Mnichinan, Pduarte, Wbethesda Zaugsberg, and Zbristol. In Siiyama, Mmalton, Mnichinan and Zaugsberg, the beta-sheet A is present in an open state therefore these variants readily undergo polymerization and consequently show aggregation in the hepatocytes. In Mprocida, Mheerlen, Mmineral springs, Pduarte and Wbethesda the conformation is altered significantly therefore these variants become conformationally less stable and thereby undergo intracellular proteolysis. These rare genetic variants show lungs and / or liver disease. There are several null variants of alpha1-AT that are not detected either at the stage of transcription or translation. The examples of some of the null variants are QOcardiff, QOhong kong, QOgranite falls, QObellingham, QOmattawa, QObolton, and QOludwigshafen. The molecular basis of deficiency of these variants also forms the theme of this review.

α1‐Antitrypsin deficiency is not an important cause of childhood liver diseases in a multi‐ethnic Southeast Asian population

We conducted a prospective study to determine the role of alpha1-antitrypsin (alpha1AT) deficiency in the pathogenesis of neonatal cholestasis and other childhood liver diseases in a multi-ethnic Southeast Asian population. Prospective patients with neonatal cholestasis (group 1), other liver diseases (group 2) and children with other medical conditions (group 3) referred to the Paediatric Unit, University of Malaya Medical Centre, Malaysia, from May 2002 to June 2005, were screened for alpha1AT level and phenotype. alpha1AT level below 80 mg/dL was considered as low. Of the 114 patients (group 1, n = 53; group 2, n = 42; group 3, n = 19) screened, seven patients (6% of total; group 1, n = 1; group 2, n = 4; group 3, n = 2) had a alpha1AT level below 80 mg/dL. All had marginally low level (range 57-79 mg/dL), but none had a clinical diagnosis of alpha1AT deficiency. One patient had PiZ- heterozygous phenotype (alpha1AT level 217 mg/dL) while another patient had PiMS heterozygous. alpha1AT deficiency is not an important cause of neonatal cholestasis and childhood liver diseases in Malaysian children. In Malaysian children with neonatal cholestasis or other liver diseases, routine assay for alpha1AT phenotype is not recommended if there is no family history of neonatal cholestasis of uncertain aetiology, or if alpha1AT level is above 80 mg/dL.

Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibrosis in a murine model of cholestasis

Alpha-1 antitrypsin (alpha1-AT) deficiency is the most common genetic cause of liver disease in children. The homozygous alpha1-ATZ mutation (PiZZ) results in significant liver disease in 10% of all affected patients. The alpha1-ATZ mutation also may lead to worse liver injury in the setting of other liver diseases such as cystic fibrosis, nonalcoholic fatty liver disease, and hepatitis C. Although cholestatic injury is common to many forms of liver disease, its effect on the PiZZ phenotype is unknown. To elucidate the interplay of cholestasis and the PiZZ phenotype, we performed bile duct ligation (BDL) on C57BL/6 mice possessing a transgenic alpha1-ATZ mutation and littermate controls. PiZ transgenic mice undergoing BDL developed more liver fibrosis by quantification of Sirius red staining (P = 0.0003) and hydroxyproline (P = 0.007) than wild-type mice after BDL. More activated hepatic stellate cells (HSCs) and apoptotic cells also were observed in the PiZ BDL model. Quantitative real time polymerase chain reaction (PCR) of the endoplasmic reticulum (ER) stress markers CHOP and GRP78 were 4-fold and 2-fold more up-regulated, respectively, in PiZ BDL mice when compared with wild-type BDL mice (P = 0.02, P = 0.02). Increased apoptosis was also noted in PiZ BDL mice by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) and cleaved caspase-3 histological staining. PiZ transgenic mice are more susceptible to liver fibrosis induced by cholestasis from BDL. Cholestasis therefore may lead to increased fibrosis in alpha1-AT deficiency, and the alpha1-ATZ mutation may act as a modifier gene in patients with concurrent cholestatic liver diseases such as cystic fibrosis.

Morphometric examination of native lungs in human lung allograft recipients

The aim of the study was to estimate the degree of lung damage in patients with alpha(1)-antitrypsin (alpha1AT) deficiency, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) at the time of lung transplantation. Using unbiased stereological methods, lung-, bronchial- and vessel-volume, capillary length, and alveolar surface area and densities were estimated in recipient lungs from 21 consecutive patients with pre-transplant diagnoses including COPD (n=7), alpha1AT deficiency (n=6) and CF (n=8). Six unused adult donor lungs served as controls. Information relating to patient demography and pre-transplant lung function was obtained by retrospective chart review. Disease groups differed significantly with respect to demographics and pre-transplant lung function. Total lung volume was similar in all groups. Bronchial volume was significantly larger in CF patients compared to the control group (p<0.0001) and to the other two diagnostic groups: alpha1AT deficiency (p=0.0001) and COPD (p<0.0001). Alveolar surface density and capillary length density were significantly lower in patients with alpha1AT deficiency and COPD compared to controls (p<0.0001, respectively) and to patients with CF (p<0.0002, respectively). There were no correlations between clinical lung function and morphometric measurements. We conclude that unbiased microscopic stereological morphometry is an evolving science with the potential to elucidate pulmonary disease pathogenesis.

Functional results of unilateral lung volume reduction surgery in alpha1-antitrypsin deficient patients

Lung volume reduction surgery (LVRS) has been shown to improve lung function and exercise tolerance in patients with severe emphysema. Some predictors of poor outcome have been described but the role of alpha1-antitrypsin (alpha1-AT) deficiency is still not well known. The aim of this study was to analyze the results of unilateral LVRS in our center according to the alpha1-AT status. The results of LVRS in 17 deficient patients and 35 nondeficient patients were analyzed at 3-6 months and 1 year after surgery. Compared with baseline, a significant improvement of FEV1, partial pressure in arterial blood (PaO2), dyspnea score and walking distance was observed in the two groups at 3-6 months after surgery and the studied parameters remained significantly improved at 1 year in the nondeficient group. By contrast, PaO2 and walking distance returned towards baseline in the deficient group at 1 year whereas improvement of FEV1 and dyspnea score was persistent. Mean values of FEV, at baseline, 3-6 months, and 1 year were 22 +/- 6%, 29 +/- 11%, and 26 +/- 9% and 28 +/- 12%, 38 +/- 17%, and 40 +/- 17% predicted in the deficient group and in the non-deficient group, respectively. In conclusion, the functional benefit is short-lasting in alpha1-AT deficient patients after unilateral LVRS.

Accumulation of Mutant α1-Antitrypsin Z in the Endoplasmic Reticulum Activates Caspases-4 and -12, NFκB, and BAP31 but Not the Unfolded Protein Response

In alpha(1)-antitrypsin (alpha1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein alpha1AT, alpha1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant alpha1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant alpha1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant alpha1AT. The results show that ER retention of polymerogenic mutant alpha1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of alpha1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic alpha1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant alpha1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFkappaB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic alpha1AT mutants.

Results of a case-detection programme for α1-antitrypsin deficiency in COPD patients

Alpha1-antitrypsin (alpha1-AT) deficiency is an underdiagnosed condition in patients with chronic obstructive pulmonary disease (COPD). The present authors have conducted a nationwide case detection programme of alpha1-AT deficiency in unselected patients with COPD using dried blood spots. The first phase analysed samples from 971 patients by determining alpha1-AT concentrations and identifying the deficient Z allele by genotyping using rapid real-time PCR. The second phase analysed 1,166 samples with alpha1-AT concentrations and identified both the S and the Z allele, but only in samples with low alpha1-AT concentrations. A total of eight (0.37%) individuals with the severe deficiency PiZZ were detected. In addition, three patients were identified with the PiSZ genotype in the second phase (0.3%). The global cost of the programme was 41,512, which represents 19.42 per sample and 5,189 per PiZZ detected. A sensitivity analysis demonstrated that performing Z genotype to all samples would have resulted in increased costs of 28 per sample and 7,479.5 per PiZZ case identified. In conclusion, a case detection programme of alpha1-antitrypsin deficiency in patients with chronic obstructive pulmonary disease using dried blood spots is feasible and at a reasonable cost per case detected. Diagnostic yield and costs depend largely on inclusion criteria and the protocol for processing of samples.

The Copenhagen National Lung Transplant Group: Survival after Single Lung, Double Lung, and Heart-Lung Transplantation

To review the 13-year clinical experience of a single center's adult lung transplantation program. From January 1992 to December 2003, 369 lung transplantations were performed on 362 patients. Single lung transplantation was performed in 234 cases, double lung transplantation in 113 cases (comprising en-bloc double lung transplantation in 44 cases and bilateral sequential lung transplantation in 69 cases), heart-lung transplantation in 21 cases, and lobe of lung transplantation in 1 case. Recipient diagnoses included chronic obstructive pulmonary disease (COPD) (n = 175), alpha1 antitrypsin (alpha1AT) deficiency (n = 86), cystic fibrosis (n = 36), pulmonary fibrosis (n = 20), Eisenmenger syndrome and secondary pulmonary hypertension (n = 24), primary pulmonary hypertension (n = 8), sarcoidosis (n = 7), silicosis (n = 4), bronchiectasis (n = 1), and graft-vs-host disease (n = 1). For patients surviving to discharge, the median duration of the intensive care unit stay was 3 days (1-67), and the median duration of the post-operative hospital stay was 37 days (16-144). Mortality for the entire series was 6% at 30 days and 10% at 90 days. The main causes of post-operative inpatient death were primary graft failure (41%), sepsis (29%), cardiac (15%), and hemorrhage (9%). The 1-, 3-, 5-, and 10-year actuarial survival rates for the entire series was 81%, 68%, 63%, and 36%, respectively. There were no significant differences in survival between types of transplant. No significant differences in survival were seen between alpha(1)AT deficiency and COPD patients after stratifying for age. Cox regression analysis demonstrated that age 60 years or older, donor age 50 years or older, and a recipient pre-operative body mass index of 25 or higher were independent predictors of poor survival. This center has 1-, 3-, and 5-year survival rates comparable to other high volume centers. Recipient age, pre-operative body mass index, and donor age significantly influence outcome after lung transplantation.

Alpha-1-Antitrypsin Deficiency: A New Paradigm for Hepatocellular Carcinoma in Genetic Liver Disease *

Liver disease in alpha-1-antitrypsin (alpha1AT) deficiency is caused by a gain-of-toxic function mechanism engendered by the accumulation of a mutant glycoprotein in the endoplasmic reticulum (ER). The extraordinary degree of variation in phenotypical expression of this liver disease is believed to be determined by genetic modifiers and/or environmental factors that influence the intracellular disposal of the mutant glycoprotein or the signal transduction pathways that are activated. Recent investigations suggest that a specific repertoire of signaling pathways are involved, including the autophagic response, mitochondrial- and ER-caspase activation, and nuclear factor kappaB (NFkappaB) activation. Whether activation of these signaling pathways, presumably to protect the cell, inadvertently contributes to liver injury or perhaps protects the cell from one injury and, in so doing, predisposes it to another type of injury, such as hepatocarcinogenesis, is not yet known. Recent studies also suggest that hepatocytes with marked accumulation of alpha1ATZ, globule-containing hepatocytes, engender a cancer-prone state by surviving with intrinsic damage and by chronically stimulating in 'trans' adjacent relatively undamaged hepatocytes that have a selective proliferative advantage. Further, this paradigm may apply to other genetic and infectious liver diseases that are predisposed to hepatocellular carcinoma.

Quantitative isolation of α1AT mutant Z protein polymers from human and mouse livers and the effect of heat

Alpha-1-antitrypsin (alpha1AT) deficiency in its most common form is caused by homozygosity for the alpha1AT mutant Z gene. This gene encodes a mutant Z secretory protein, primarily synthesized in the liver, that assumes an abnormal conformation and accumulates within hepatocytes causing liver cell injury. Studies have shown that mutant alpha1ATZ protein molecules form unique protein polymers. These Z protein polymers have been hypothesized to play a critical role in the pathophysiology of liver injury in this disease, although a lack of quantitative methods to isolate the polymers from whole liver has hampered further analysis. In this study, we demonstrate a quantitative alpha1ATZ polymer isolation technique from whole liver and show that the hepatocellular periodic acid-Schiff-positive globular inclusions that are the histopathological hallmark of this disease are composed almost entirely of the polymerized alpha1ATZ protein. Furthermore, we examine the previously proposed but untested hypothesis that induction of alpha1ATZ polymerization by the heat of physiological fever is part of the mechanism of hepatic alpha1ATZ protein accumulation. The results, however, show that fever-range temperature elevations have no detectable effect on steady-state levels of intrahepatic Z protein polymer in a model in vivo system. In conclusion, methods to separate insoluble protein aggregates from liver can be used for quantitative isolation of alpha1ATZ protein polymers, and the effect of heat from physiological fever may be different in vivo compared with in vitro systems.

Polymerized α1‐antitrypsin is present on lung vascular endothelium. New insights into the biological significance of α1‐antitrypsin polymerization

The damage to lung tissue in chronic obstructive pulmonary disease (COPD) may involve the progressive loss of pulmonary vascular endothelial cells. Endothelial binding of alpha1-antitrypsin (alpha1-AT) derived from plasma has been identified, and alpha1-AT deficiency is a known genetic risk factor associated with alpha1-AT polymerization and COPD development. Therefore, in the present study we aimed to investigate if alpha1-AT is present on the lung vascular endothelium, and if it is in a polymeric form. Postmortem paraffin-embedded tissue specimens from 15 COPD (chronic bronchitis and emphysema) cases with and without Z alpha1-AT (Glu342Lys) deficiency and from 10 cases without signs of COPD were studied. Immunohistochemistry was performed using the streptavidin-biotin method with a monoclonal ATZ11 antibody specific for polymeric alpha1-AT, and polyclonal antibodies against human alpha1-AT and neutrophil elastase. Vascular endothelium showed intense staining for alpha1-AT with the ATZ11 antibody in all cases; however, intensity of staining in patients with alpha1-AT deficiency was greater. No endothelial staining was observed with the anti-elastase antibody. This is the first demonstration that alpha1-AT bound to the vascular endothelium of lungs is in a polymeric form, which also suggests a possible previously unknown role for polymeric alpha1-AT in vivo.

Gene therapy for human α 1-antitrypsin deficiency in an animal model using SV40-Derived vectors

In most genetic diseases, the goal of gene therapy is to deliver a particular transgene; however, sometimes a deleterious gene product must be eliminated. Because of the promise of recombinant simian virus 40 (rSV40) vectors, we tested their ability to deliver a transgene and to target a transcript for destruction by direct administration of the vectors to the liver of an animal model for human alpha1-antitrypsin (alpha1-AT) deficiency. Therapy of human alpha1-AT deficiency requires stable transduction of resting hepatocytes, both to deliver wild-type alpha1-AT and to inhibit production of mutant alpha1-AT. Transgenic mice carrying the mutant human alpha1-AT PiZ allele were treated through an indwelling portal vein catheter with a simian virus 40 (SV40)-derived vector carrying a ribozyme designed to target the human transcript. Treated transgenic mice showed marked decreases of human alpha1-AT messenger RNA and the protein in the liver, and serum levels of human alpha1-AT were decreased to 50% +/- 5% of pretreatment values 3-16 weeks after transduction. Moreover, when normal mice were treated with an SV40-derived vector containing a modified human alpha1-AT complementary DNA engineered to be resistant to cleavage by the alpha1-AT ribozyme, they expressed human alpha1-AT messenger RNA and protein in their livers and serum levels of human alpha1-AT remained >1 microg/mL for 1 year. These results represent the initial steps toward a novel approach to the gene therapy of alpha1-AT deficiency.

Lack of effect of oral 4-phenylbutyrate on serum alpha-1-antitrypsin in patients with alpha-1-antitrypsin deficiency: a preliminary study.

In homozygotes with ZZ genotype alpha-1-antitrypsin (alpha1AT) deficiency, mutant alpha1ATZ protein (alpha1ATZ) accumulates in hepatocytes, rather than being secreted into the blood. Homozygous individuals experience emphysema as a result of reduced levels of circulating alpha1AT in the lung with which to inhibit connective tissue breakdown. Homozygotes may also experience liver disease from the accumulation of alpha1ATZ within hepatocytes, which causes liver damage. A previous study indicated that the compound 4-phenylbutyrate (4-PBA) mediated a significant increase in release of alpha1ATZ from cells in tissue culture and in a mouse model of alpha1AT deficiency. The authors hypothesized that 4-PBA could be used to treat both the liver and lung disease of humans with alpha1AT deficiency. In this preliminary, open label study the authors evaluated the effect of 14 days of oral 4-PBA therapy on alpha1AT blood levels in 10 patients with alpha1AT deficiency. There was no significant increase in alpha1AT blood level associated with 4-PBA administration. Symptomatic and metabolic side effects were significant. 4-PBA did not increase alpha1AT blood levels in humans with alpha1AT deficiency in this preliminary trial.

Protease inhibitor phenotypes and serum alpha-1-antitrypsin levels in patients with COPD: a study from Hong Kong.

Many studies have suggested that an imbalance of protease activation and inhibition might result in COPD with emphysema. Levels of alpha-1-antitrypsin (alpha1-AT), the key protease inhibitor, are genetically determined by alleles that present in many phenotypes/subtypes, some of which are associated with deficiency of the protein. We prospectively evaluated the prevalence of the protease inhibitor (Pi) alleles and phenotypes together with the serum alpha1-AT levels in Chinese patients with COPD. The study population comprised 356 patients with COPD. The male-to-female ratio was 4 : 1 with a mean age of 72.4 years (range 44-93 years). Isoelectric focusing was used for Pi phenotyping/subtyping. The frequencies of Pi alleles and phenotypes were compared with the frequencies in 1085 healthy unrelated Chinese control subjects. The serum alpha1-AT levels were measured by the Cobas Fara assay. PiZ was not detected. No significant difference in distribution of PiM phenotypes/subtypes between patients with COPD and healthy controls was observed, except for M1M3 and M2M3. There was also a significant difference in the proportion of variant S and F alleles between the disease group and the control population. The low prevalence of deficiency Pi phenotypes/subtypes suggests a lack of contribution of alpha1-AT deficiency to the pathogenesis of COPD in Chinese patients. The strategy of launching an alpha1-AT deficiency detection program among COPD patients, based on the recommendation of the World Health Organization, may not be readily applicable in our local setting.

Report of a workshop: quantitative computed tomography scanning in longitudinal studies of emphysema.

It has been reported that quantitative computed tomography (CT) scanning of the lungs showed decreased progression of emphysema in a randomised clinical trial in patients with severe alpha1-antitrypsin (alpha1-AT) deficiency receiving monthly intravenous augmentation therapy with human alpha1-AT. Comparable results were not obtained using rate of decline of forced expiratory volume in one second. Accordingly, the Alpha-1 Foundation convened a workshop to explore the feasibility of using quantitative CT data as a primary outcome variable in trials of drugs for treating alpha1-AT deficiency. This report reviews the following: the principles for the use of modern CT scanners for quantifying emphysema; the methods and data on validation by comparison with measurements of severity of emphysema in inflation-fixed specimens of lungs; and the possibility of decreasing radiation dosage from CT to make it safe and ethically possible to use CT in longitudinal studies. The workshop concluded that it is feasible, safe and ethically possible to use computed tomography in longitudinal studies of emphysema. It recommended that the primary end-point should be a significant shift in the 15th percentile of lung density.

Analyses of hepatocellular proliferation in a mouse model of alpha-1-antitrypsin deficiency.

alpha-1-Antitrypsin (alpha1-AT) deficiency is the most common cause of metabolic pediatric liver disease. Hepatocellular injury is caused by toxicity of the mutant alpha-1-antitrypsin Z (alpha1-ATZ) molecule retained within hepatocytes. In these studies, we used the PiZ transgenic mouse model of alpha1-AT deficiency to examine hepatocellular proliferation in response to chronic liver injury resulting from this metabolic disease. The results showed increased hepatocellular proliferation and caspase 9 activation in male PiZ mice compared with female PiZ and wild-type mice. Hepatic alpha1-AT mRNA and protein expression also were increased in male PiZ mice, suggesting that greater hepatocellular proliferation and caspase activation in males results from increased hepatotoxicity associated with greater intracellular alpha1-ATZ accumulation. Testosterone treatment of female PiZ mice increased alpha1-ATZ expression and hepatocellular proliferation to a level comparable with that in males. In PiZ mice, hepatocytes devoid of intracellular alpha1-AT globules had a proliferative advantage compared with globule-containing hepatocytes. However, this advantage is relative because both globule-containing and globule-devoid hepatocytes exhibited comparable proliferation after partial hepatectomy. In conclusion, these data indicate that intracellular retention of mutant alpha1-ATZ is associated with a regenerative stimulus leading to increased hepatocellular proliferation, that gender-specific signals influence the degree of alpha1-AT expression and associated hepatic injury, and that hepatocytes devoid of alpha1-ATZ have a proliferative advantage over cells that accumulate the mutant protein. This selective proliferation suggests that hepatocellular transplantation may be applicable for treatment of this and other slowly progressive metabolic liver diseases.

Disruption of the Murine .ALPHA.1-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.

Genomic DNA extraction from small amounts of serum to be used for α1-antitrypsin genotype analysis

If laboratory diagnosis of alpha1-antitrypsin (alpha1-AT) deficiency is usually based on its phenotype identification by isoelectric focusing, alpha1-antiprotease inhibitor (Pi)S and PiZ genotypes can also be determined by deoxyribonucleic acid (DNA)-based methods. Recently, several methods have been described for preparing genomic DNA from serum. The aim of the current study was to determine the Pi allele from serum extracted DNA by polymerase chain reaction (PCR) and to compare these results with those obtained with whole blood extracted DNA. Serum alpha1-AT concentration and phenotypic identification were systematically performed in 43 hospitalised patients. Genomic DNA was simultaneously purified from whole blood and from serum. The mutation detection was found using a PCR-mediated site-directed mutagenesis method. Concerning phenotypic identification, 29 patients were MM homozygotes, 11 were heterozygotes for S (MS = 7) or for Z (MZ = 4) and three showed a ZZ phenotype. Genotyping analyses gave identical results with serum and whole blood extracted DNA and all the results were in agreement with the phenotyping results. The authors found that the deoxyribonucleic acid-based test proved to be a reliable tool for alpha1-antitrypsin deficiency diagnosis and appears to be an alternative for the labour intensive alpha1-antitrypsin determination by isoelectric focusing. The authors also concluded that this method yields good quality deoxyribonucleic acid from serum, equal to that extracted from whole blood and is helpful in retrospective studies of multiple genetic markers.

Serum levels of alpha1-antitrypsin predict phenotypic expression of the alpha1-antitrypsin gene.

We conducted a retrospective analysis to determine if both alpha1-antitrypsin serum level and phenotype need be studied when evaluating children for alpha1-AT deficiency. We collected data from patients less than 19 years old who had both serum alpha1-AT level and phenotype determined over a 9-year period (January 1992-December 2000). Eighty-eight patients were identified and 15 had the PiZZ phenotype. The serum alpha1-AT level was below normal (normal 85-215 mg/dl) in all 15 PiZZ patients. Seventy-two of 73 non-PiZZ patients had normal or above normal serum levels. The sensitivity of the serum alpha1-AT level was 100%, and the specificity was 99%. The serum alpha1-AT level had a positive predictive value of 94% and a negative predictive value of 100%. We conclude that serum alpha1-AT levels are highly predictive of the PiZZ phenotype. Determination of the serum alpha1-AT level alone should be the initial test when evaluating for alpha1-AT deficiency.