α-1-Antitrypsin (A1AT) deficiency is the most common genetic cause of liver disease in children and the most common genetic disease leading to liver transplantation in children (1). A1AT deficiency-associated liver disease is caused by homozygosity for the abnormal Z allele, resulting in misfolding of the protein and accumulation of the abnormal protein in the endoplasmic reticulum of the hepatocyte. This so-called gain of negative function in the liver triggers a variety of cellular responses that lead to variable degrees of liver damage (1).
Although the risk of liver disease in individuals with A1AT deficiency is well described, the role of A1AT heterozygosity as an a priori cause, or predisposing factor, for chronic liver disease is controversial. The possible role of A1AT heterozygosity as a modifier of other forms of adult-onset liver disease has been suggested with increasing frequency in recent years. Early observations of select adult populations with end-stage liver disease revealed a higher prevalence of A1AT heterozygosity among patients who underwent liver transplantation and a greater number of A1AT heterozygotes with cryptogenic cirrhosis as compared with other causes of end-stage liver disease (2,3). More recent studies have identified a higher-than-expected prevalence of A1AT heterozygosity among patients with hepatitis B infection and among patients who received a liver transplant for hepatitis C or nonalcoholic fatty liver disease (4–6). As a potential genetic modifier, the presence of A1AT variant alleles may explain some of the phenotypic variability seen in these disorders and allow more accurate prediction of disease outcome.
With respect to pediatric liver disease, the role of A1AT as a modifier gene has not been examined. We hypothesized that non-M A1AT allele variants are more common in children with chronic liver disease than in the general population.
PATIENTS AND METHODS
After approval by the Cincinnati Children's Hospital Medical Center (CCHMC) Institutional Review Board, A1AT phenotypes were retrieved on all of the children evaluated at CCHMC for chronic liver disease from January 1997 to January 2004. Patients were identified and phenotypes were collected by a combination of chart review, query of the laboratory database and query of our prospectively maintained Liver Transplant Database.
Before 1999, phenotyping was performed in the CCHMC Research Foundation using polyacrylamide gel isoelectric focusing, as previously described (7). Since 1999, A1AT phenotyping has been performed by Quest Diagnostics Nichols Institute, San Juan Capistrano, CA. Patients were phenotyped using the Pi classification system, which assigns a letter to A1AT variants according to position of migration of the protein band in the gel, using alphabetic order from low to high isoelectric point. Phenotyping in this fashion represents one level of identifying genetic variation in the A1AT gene. The alleles reported using this method are M, S, Z and Other. For those patients who received a liver transplant, A1AT phenotypes were obtained pretransplant.
Patients with chronic liver disease for whom an A1AT phenotype was not available were excluded from analysis. Patients with homozygous A1AT deficiency (Pi ZZ) were also excluded from analysis. Additional clinical data were collected on patients who were evaluated for liver transplantation. These data included sex, race, age at transplant and age at death.
For all patients with biliary atresia, age at portoenterostomy and presence of congenital anomalies were recorded. A patient was classified as having “congenital” biliary atresia based on the presence of ≥1 major anomaly (polysplenia, intestinal malrotation, congenital heart disease, dextrocardia). Rapid progression of disease in this population was defined as loss of native liver (death or transplant) before 24 months of age.
The distribution of A1AT alleles among the entire Cincinnati Liver Disease population was compared with data from the largest American database, published by de Serres (8) in 2002, using the χ2 test. Multiple subsets of the entire Cincinnati Liver Disease population were also compared with each other and with de Serres's data using χ2analysis and Fisher exact test. Demographic variables and outcomes were compared using Student t test and χ2 analysis. P < 0.05 was considered statistically significant.
A1AT phenotypes were available on 264 children evaluated at CCHMC for chronic liver disease. Of these, 23 had A1AT deficiency (ZZ phenotype) and were excluded from analysis. The A1AT phenotypes of the remaining 241 patients were as follows: 205 MM, 17 MS, 10 MZ, 1 SZ, 2 M/Other, 1 FS and 5 FM. The distribution of A1AT alleles was significantly different in the entire CCHMC Liver Disease population as compared with published population-based data (P < 0.001) (8). The CCHMC Liver Disease population had a reduced frequency of the M allele and a 4-fold increase in the frequencies of the Z and Other alleles compared with published data (Table 1).
In an effort to determine whether this observed difference was caused by a subset of patients within the entire CCHMC Liver Disease population, the population was divided into “biliary atresia” (n = 67) and “other liver disease” (n = 174). In each of these groups, the distribution of A1AT allele frequencies remained statistically different from the published reference database (Table 1).
To investigate whether non-M alleles were differentially associated with a more severe course of disease, we divided the CCHMC Liver Disease population into transplant (n = 117) and nontransplant (n = 124) subsets on the basis of whether the patient had been evaluated for liver transplantation listing. A1AT allele frequencies for both of these subsets were also statistically different from published data, with an increased frequency of S, Z and Other alleles in the transplant group and an increased frequency of Z alleles in the nontransplant group (Table 1).
The 117 patients listed for liver transplantation were examined for the impact of A1AT heterozygosity on outcome, as defined by pretransplant death, age at listing and age at first transplant. With use of these measures, no impact of A1AT heterozygosity on outcome was identified in the transplant population as a whole (Table 2). Within the group of children evaluated for transplantation, however, the frequency of non-M alleles was higher in children with biliary atresia with rapid progression of disease as defined by the loss of native liver by death or transplantation before 24 months of age (Table 3; Fig. 1). The groups did not differ in age at portoenterostomy or proportion of patients with the congenital form of biliary atresia (Table 3). A similar association between A1AT heterozygosity and outcome was not observed in children with other liver diseases.
In this retrospective analysis of children with chronic liver disease seen at a single US center, the distribution of A1AT alleles was significantly different in our liver disease cohort as compared with reference epidemiological data (8). This difference arose primarily from increased frequencies of the abnormal Z and Other alleles. The differences in allele frequencies persisted when multiple subsets of the liver disease population were compared with published data.
Previous attempts to identify an association between A1AT heterozygosity and chronic liver disease in adult patients have focused primarily on the Z allele and have reported conflicting results (2–5,9–12). Most have reported a positive association between MZ heterozygosity and the presence or progression of various forms of chronic liver disease common in the adult population, including alcoholic cirrhosis, viral hepatitis, nonalcoholic fatty liver disease and cryptogenic cirrhosis. An increased frequency of the Z allele in 29 pediatric patients with cryptogenic cirrhosis as compared with 100 normal controls was previously reported (13). Our findings corroborate this observation. We found a significantly increased frequency of the Z allele in a heterogeneous population representing many forms of chronic pediatric liver disease (except A1AT deficiency), regardless of the severity of disease. Furthermore, our findings expand this association to include other phenotypic variants of the A1AT protein (eg, non-M, non-Z alleles).
Among the non-M, non-Z alleles identified in our population, the most commonly reported was the F allele. This allelic variant of the A1AT protein results from a single nucleotide polymorphism (C > T) leading to an amino acid substitution (arginine to cysteine) at position 223 (14). Although the F protein is expressed in serum at low normal levels, it exhibits dysfunctional inhibitory activity against human neutrophil elastase in vitro and has been associated with chronic obstructive pulmonary disease (15–17). The effect of the F allele in the liver has not been explored.
Our findings also implicate A1AT heterozygosity as a potential marker for disease severity in children with biliary atresia referred for liver transplant evaluation. In these patients, the presence of a non-M allele was associated with earlier age at transplant listing and rapid progression of disease. This could not be explained by a systematic difference in the age of the patients at the time of Kasai operation or by the presence of the congenital form of biliary atresia. In fact, none of the patients with A1AT heterozygosity had the congenital form of biliary atresia.
These observations are particularly intriguing in view of the fact that despite a relatively uniform age of onset of symptoms, the clinical course of patients with biliary atresia is variable, with a significant subset of patients requiring liver transplantation by 2 y of age (18,19). Although the cessation of biliary flow after portoenterostomy is strongly associated with poor long-term outcome with the native liver, the biological factors contributing to the progression of liver disease are largely unknown. In this context, non-M alleles emerge as one of these factors.
The potential contribution of non-M A1AT alleles to progression of liver disease is in keeping with the concept of synergistic heterozygosity, in which the inheritance of concurrent partial defects in >1 biological pathway or in multiple steps of a single pathway alters the phenotypic expression and outcome of a disease state (20). This concept has already proved important in several forms of chronic liver disease. For example, mutations in the gene responsible for the Alagille syndrome (jagged 1) have been found in infants with biliary atresia and are associated with the need for liver transplantation before 5 y of age (21). Similarly, a polymorphism in the gene for glutathione-S-transferase P1 is associated with an 8-fold increase in the risk of liver disease in pediatric patients with cystic fibrosis (22). Common heterozygous mutations in HFE, the gene responsible for hereditary hemochromatosis, are associated with increased risk of fibrogenesis and disease progression in adults with chronic hepatitis C infection (22,23). With regard to A1AT in particular, heterozygosity has been implicated as a risk factor for severe cystic fibrosis liver disease as well as for pulmonary asbestosis (24,25).
One of the limitations of this study is the potential for population stratification, that is, the possibility that the differences in allele frequencies between our chronic liver disease population and the published data may be caused by systematic differences in ancestry rather than association of A1AT heterozygosity with chronic liver disease. Lack of availability of detailed ethnic data on both populations makes it impossible to rule out population stratification as a potential confounding factor. In addition, as a retrospective study, inclusion of patients was limited to those for whom an A1AT phenotype was available. This leads to a potential for selection bias on several levels because a phenotype may not have been collected as a component of clinical care or may have been unrecoverable from the medical record. A true evaluation of the impact of A1AT heterozygosity on pediatric liver disease requires across-the-board screening of the entire disease population. This will be facilitated by efforts such as the Biliary Atresia Research Consortium (http://www.barcnetwork.org) and the Cholestatic Liver Disease Consortium (http://rarediseasesnetwork.epi.usf.edu/clic/index.htm).
It is important to note that in the present study, evaluation of outcome in patients with biliary atresia was limited to those who had been referred for liver transplantation evaluation. As such, our sample selected for patients with a poor outcome. Furthermore, inclusion of other variables likely to affect progression of disease (eg, use of steroids, episodes of cholangitis) is equally important but was beyond the scope of this investigation.
In spite of these limitations, our study provides new information on the potential role of the A1AT gene as a modifier of pediatric liver disease in general and a marker of rapid progression of disease in children with biliary atresia in particular. Our finding that “Other” alleles are more frequent in children with liver disease as compared with the reference population implies that the pathogenic mechanisms by which A1AT variants may influence the disease phenotype are not solely dependent on the intracellular accumulation of misfolded A1AT protein. It is possible that the variant allele(s) may influence the antiprotease properties of A1AT in the extracellular environment, as has been shown with the F variant, or modulate the activated phenotype of inflammatory cells (15,26–30). The loss of these broader biological effects of A1AT, including an antiviral effect and antiapoptotic effect, has been implicated as a risk factor for HIV infection and as an additional contributing factor to pulmonary emphysema (31–33). Formal examination of these possibilities will require a comprehensive gene-sequencing analysis to define the molecular basis of the “Other” Pi phenotypes, followed by elucidation of their functional significance with regard to both antiproteolytic and immunomodulating activity. The discovery of prevalent genetic polymorphisms in subjects with “Other” alleles will potentiate future studies to directly examine the functional consequences of less common A1AT variants, and in doing so may provide unique insight into the role of this protein in the hepatic microenvironment.
The authors acknowledge the support of the American Association for the Study of Liver Disease/Schering Plough Advanced Hepatology Fellowship Award (to K.M.C.) and the American Liver Foundation/Alpha One Foundation Innovative Hepatology Seed Grant (to J.A.B.).
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