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Original Contribution |

Genetic Modifiers of Liver Disease in Cystic Fibrosis FREE

Jaclyn R. Bartlett, PhD; Kenneth J. Friedman, PhD; Simon C. Ling, MB, ChB; Rhonda G. Pace, BS; Scott C. Bell, MD; Billy Bourke, MD; Giuseppe Castaldo, MD, PhD; Carlo Castellani, MD; Marco Cipolli, MD; Carla Colombo, MD; John L. Colombo, MD; Dominique Debray, MD; Adriana Fernandez, MD; Florence Lacaille, MD; Milan Macek, MD, DSc; Marion Rowland, MB, PhD; Francesco Salvatore, MD, PhD; Christopher J. Taylor, MD; Claire Wainwright, MD; Michael Wilschanski, MBBS; Dana Zemková, PhD; William B. Hannah, BS; M. James Phillips, MD; Mary Corey, PhD; Julian Zielenski, PhD; Ruslan Dorfman, PhD; Yunfei Wang, MS; Fei Zou, PhD; Lawrence M. Silverman, PhD; Mitchell L. Drumm, PhD; Fred A. Wright, PhD; Ethan M. Lange, PhD; Peter R. Durie, MD; Michael R. Knowles, MD; for the Gene Modifier Study Group
[+] Author Affiliations

Author Affiliations: Cystic Fibrosis/Pulmonary Research and Treatment Center (Drs Bartlett and Knowles, Ms Pace, and Mr Hannah) and Department of Genetics (Mr Wang and Dr Lange), School of Medicine, and Department of Biostatistics, School of Public Health (Mr Wang and Drs Zou, Wright, and Lange), University of North Carolina at Chapel Hill; Center for Molecular Biology and Pathology, Laboratory Corporation of America, Research Triangle Park, North Carolina (Dr Friedman); Division of Gastroenterology, Hepatology and Nutrition (Drs Ling and Durie), Pediatric Laboratory Medicine (Dr Phillips), Child Health Evaluative Sciences (Dr Corey), Genetics and Genome Biology (Drs Zielenski and Dorfman), and Physiology and Experimental Medicine (Dr Durie), The Hospital for Sick Children, and the University of Toronto (Drs Ling, Corey, and Durie); Toronto, Ontario, Canada; Adult Cystic Fibrosis Centre, Prince Charles Hospital, Chermside, Queensland, Australia (Dr Bell); Children's Research Centre, Our Lady's Children's Hospital, Crumlin (Drs Bourke and Rowland) and University College Dublin School of Medicine and Medical Science (Dr Rowland), Dublin, Ireland; CEINGE-Advanced Biotechnologies and Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Naples, Italy (Drs Castaldo and Salvatore); Cystic Fibrosis Centre, Azienda Ospedaliera di Verona, Verona, Italy (Drs Castellani and Cipolli); CF Center, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, University of Milan, Milan, Italy (Dr C. Colombo); Department of Pediatrics, Pulmonary Section, University of Nebraska Medical Center, Omaha (Dr J. Colombo); Service d'Hépatologie Pédiatrique, Centre Hospitalier Universitaire de Bicêtre, Le Kremlin-Bicêtre Cedex, France (Dr Debray); Cystic Fibrosis Center, La Plata Children's Hospital, La Plata, Buenos Aires, Argentina (Dr Fernandez); the Pediatric Hepato-Gastroenterology-Nutrition Unit, Necker-Enfants Malades Hospital, Paris, France (Dr Lacaille); Departments of Biology and Medical Genetics (Dr Macek) and Pediatrics (Dr Zemková), Charles University and University Hospital Motol, Prague, Czech Republic; Department of Paediatric Gastroenterology, Academic Unit of Child Health, University of Sheffield, Sheffield, United Kingdom (Dr Taylor); Queensland Children's Respiratory Centre, Royal Children's Hospital, Brisbane, Queensland, Australia (Dr Wainwright); Department of Pediatric Gastroenterology, Hadassah Medical Organization, Jerusalem, Israel (Dr Wilschanski); Department of Pathology, University of Virginia, Charlottesville, Virginia (Dr Silverman); and Departments of Pediatrics and Genetics, Case Western Reserve University, Cleveland, Ohio (Dr Drumm).


JAMA. 2009;302(10):1076-1083. doi:10.1001/jama.2009.1295.
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Context A subset (≈ 3%-5%) of patients with cystic fibrosis (CF) develops severe liver disease with portal hypertension.

Objective To assess whether any of 9 polymorphisms in 5 candidate genes (α1-antitrypsin or α1-antiprotease [SERPINA1], angiotensin-converting enzyme [ACE], glutathione S-transferase [GSTP1], mannose-binding lectin 2 [MBL2], and transforming growth factor β1 [TGFB1]) are associated with severe liver disease in patients with CF.

Design, Setting, and Participants Two-stage case-control study enrolling patients with CF and severe liver disease with portal hypertension (CFLD) from 63 CF centers in the United States as well as 32 in Canada and 18 outside of North America, with the University of North Carolina at Chapel Hill as the coordinating site. In the initial study, 124 patients with CFLD (enrolled January 1999-December 2004) and 843 control patients without CFLD were studied by genotyping 9 polymorphisms in 5 genes previously studied as modifiers of liver disease in CF. In the second stage, the SERPINA1 Z allele and TGFB1 codon 10 genotype were tested in an additional 136 patients with CFLD (enrolled January 2005-February 2007) and 1088 with no CFLD.

Main Outcome Measures Differences in distribution of genotypes in patients with CFLD vs patients without CFLD.

Results The initial study showed CFLD to be associated with the SERPINA1 Z allele (odds ratio [OR], 4.72; 95% confidence interval [CI], 2.31-9.61; P = 3.3 × 10−6) and with TGFB1 codon 10 CC genotype (OR, 1.53; 95% CI, 1.16-2.03; P = 2.8 × 10−3). In the replication study, CFLD was associated with the SERPINA1 Z allele (OR, 3.42; 95% CI, 1.54-7.59; P = 1.4 × 10−3) but not with TGFB1 codon 10. A combined analysis of the initial and replication studies by logistic regression showed CFLD to be associated with SERPINA1 Z allele (OR, 5.04; 95% CI, 2.88-8.83; P = 1.5 × 10−8).

Conclusions The SERPINA1 Z allele is a risk factor for liver disease in CF. Patients who carry the Z allele are at greater risk (OR, ≈ 5) of developing severe liver disease with portal hypertension.

Cystic fibrosis (CF) is a recessive monogenic disorder characterized by multiorgan involvement and clinical heterogeneity that is incompletely explained by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene (OMIM 602421).1 Patients with CF, including those homozygous for DF508, exhibit a range of lung disease severity, and genetic variability in non-CFTR genes contributes to risk for severity of pulmonary disease.27

Intrinsic abnormalities in the liver of persons with CF reflect loss of CFTR (Cl channel) function on the apical membrane of cholangiocytes.8,9 This dysfunction is predicted to result in defective (sluggish) bile flow and is associated with a cholangiocyte-induced inflammatory response with activation and proliferation of hepatic stellate cells, which results in cholangitis and fibrosis in focal portal tracts.1013 However, only a small fraction (≈ 3%-5%) of patients with CF develops severe liver disease characterized by cirrhosis with portal hypertension (CFLD)1; thus, non-CFTR genetic variability may contribute to risk for severe liver disease.1417

To determine the association between non-CFTR genetic polymorphisms and CFLD, we studied 9 functional variants in 5 genes previously studied in CF liver disease, including α1-antitrypsin (also known as α1-antiprotease [SERPINA1, OMIM 107400]),18 angiotensin-converting enzyme (ACE [OMIM 106180]),19 glutathione S-transferase (GSTP1 [OMIM 134660]),20 mannose-binding lectin 2 (MBL2 [OMIM 154545]),21 and transforming growth factor β1 (TGFB1 [OMIM 190180]).19 Our initial study compared polymorphic genotypes in these candidate modifier genes in persons with CFLD and in control patients without CFLD aged at least 15 years. We tested our initial findings in a second study in different populations of patients with and without CFLD.

Patients

Initial Study. Of the 158 patients with CF evaluated for CFLD (enrolled January 1999-December 2004), 128 fulfilled criteria from 22 CF centers in 10 countries (Australia [8], Canada [17], Czech Republic [17], Germany [3], Italy [28], the Netherlands [1], Scotland [2], Slovakia [4], Turkey [4], and United States [44]). For patients without 2 defined mutations in CFTR, we performed further testing using a panel of 70 mutations (CFTR mutation detection assay; Tm Bioscience/Luminex, Austin, Texas). After genotyping was complete, more than 95% of patients with CFLD and with 2 defined mutations in CFTR had 2 pancreatic insufficient mutations. The 843 control patients without CFLD were enrolled from the United States (759 from 42 centers) and Canada (84 from 32 centers). The majority of the control patients were ascertained from the GMS Lung Study population (DF508 homozygotes; 92.6%).5 Most of the other controls had biallelic pancreatic insufficient mutations. These controls without CFLD were 15 years or older (1 SD above the mean age of diagnosis of CFLD), to exclude younger patients who might have occult liver disease.

Replication Study. Of the 191 patients with CF evaluated for CFLD (enrolled January 2005-February 2007), 139 fulfilled criteria from 35 CF centers in 10 countries (Argentina [5], Australia [5], Canada [24], Chile [1], England [4], France [9], Ireland [8], Israel [7], Italy [14], and United States [62]). The percentage of pancreatic insufficient CFTR genotypes in patients with CFLD was similar to those in the initial study. The 1088 control patients (≥15 years and without CFLD) were ascertained from 5 countries (Canada [391 from 32 centers], Czech Republic [30 patients], Ireland [6 patients], Italy [71 patients], and United States [590 from 54 centers]). The majority of the controls had 2 pancreatic insufficient mutations (93.5%; mostly DF508/DF508 [62.8%]).

Enrollment Criteria

All patients had a diagnosis of CF, confirmed by sweat test, CFTR genotyping, or both. CFLD was defined as cirrhosis in patients 2 years or older, confirmed by imaging (ultrasound, computed tomography, magnetic resonance imaging) showing hepatic parenchymal abnormalities and portal hypertension (esophageal varices, portal-systemic collaterals, splenomegaly) in the absence of another cause for liver disease. Data were independently reviewed by 2 hepatologists (S.C.L., P.R.D.) with experience in CFLD to ensure inclusion and exclusion criteria were met, using case report forms, radiology and endoscopy reports, and clinical notes. When there was no consensus, the reviewers requested additional information to clarify the diagnosis of CFLD. No patient was excluded because of race or ethnicity, defined by patient self-report.

We excluded 30 (19%) and 52 (27%) patients originally submitted for the initial and replication studies, respectively, with a presumed diagnosis of CFLD, because they had milder liver disease without portal hypertension or inadequate documentation. For the 47 patients with confirmed CFLD who had undergone a liver transplant (26 in initial study; 21 in replication study), source documents were obtained from dates prior to transplantation. Exclusion criteria for the CFLD group included portal vein thrombosis or other causes of liver disease (alcohol abuse, biliary atresia, clinically significant viral hepatitis, use of parenteral nutrition, and Wilson disease). The study was approved by the institutional review boards of all participating institutions; all participants (or their parent) provided written informed consent.

Exclusion From Analysis Based on Age at Diagnosis of CFLD

In common with previous reports, we found the mean age of diagnosis of CFLD (first documentation of portal hypertension) to be 10.6 (SD, 5.4) years.15,2225 The diagnosis of CFLD was first established after age 30 years in 7 patients (aged 32, 33, 35, 40, 43, 44, and 47 years), which is 4 or more SDs above the mean of the normal distribution. Therefore, these patients were excluded from the genetic analyses (4 from the initial study, 3 from the replication study).

Data Collection and Laboratory Methods

Patients received a unique identifier code, and data were stored in a secure database in the UNC Bioinformatics Center. Clinical data on standard case report forms included self-reported race/ethnicity, pancreatic exocrine status, medical history, physical examination, laboratory blood work values, and abdominal radiology reports. In addition, we reviewed the following procedure reports if available: liver explant pathology (from liver transplantation), liver biopsy, endoscopy, and colonoscopy.

DNA was extracted from peripheral blood leukocytes using standard protocols.26 Genetic polymorphisms were determined by direct sequencing, microsphere-based genotyping using Illumina BeadArray technology (Illumina Inc, San Diego, California), and site-directed mutagenesis.

Immunohistochemistry with polyclonal rabbit anti–α1-antitrypsin antibody and monoclonal mouse anti-CD68 (clone KP1) antibody (Dako Canada Inc, Mississauga, Ontario, Canada), was performed on the Benchmark XT autoimmunostainer (Ventana Medical Systems, Tucson, Arizona) at dilutions of 1:3000 and 1:5000, respectively. Immunodetection was performed using the Ventana i-VIEW DAB, LSAB kit. Tissue sections were dewaxed, enzyme pretreated for α1-antitrypsin, heat epitope retrieved for CD68, peroxidase, and endogenous biotin blocked using Ventana proprietary reagents. Sections were hematoxylin-eosin counterstained for nuclear detail.

Statistical Analysis

Genotype distributions were tested for consistency with expected Hardy-Weinberg equilibrium proportions for case and control patients in the initial, replication, and combined studies, using all patients and then restricted to white patients, using PLINK version 1.03 (http://pngu.mgh.harvard.edu/~purcell/plink/).27

For the initial study, the association between polymorphisms and CFLD was assessed using Cochran-Armitage trend tests.28 All tests were 2-sided; unadjusted P values are reported, along with P values that were significant (P < .05) after Bonferroni correction adjusting for 9 tests. Analyses were performed using all patients and then restricted to white patients.

For the replication study, the association between 2 polymorphisms from the initial study (SERPINA1 Z allele and TGFB1 codon 10) and CFLD was assessed using Cochran-Armitage trend tests. Initial and replication samples were subsequently combined and analyzed for the SERPINA1 Z allele using Cochran-Armitage trend tests and logistic regression models. Varying levels of covariate adjustment in the logistic regression models were made for ethnicity (as a 5-level categorical variable for all samples), sex, CFTR genotype, and TGFB1 codon 10 genotype. Tests of interactions were performed to assess whether the odds of CFLD differed between male and female patients by SERPINA1 genotype. Odds ratios (ORs), corresponding 95% confidence intervals (CIs), and uncorrected P values are reported. Bonferroni correction was applied to assess overall statistical significance in the replication and combined analyses (adjusting for 2 tests in the replication and 9 tests in the combined sample). Analyses were performed separately, first using all samples and then using those from white patients only.

Analysis of variance models were used to assess whether sex, SERPINA1 Z allele, and CFTR genotype were associated with age of diagnosis of CFLD in the combined sample. Data were analyzed on all CFLD case patients with covariate adjustment for self-reported ancestry, and on white CFLD case patients with a reported age at diagnosis.

To estimate population attributable risk, we used a modified form of the classic Levin formula for population attributable fraction by replacing relative risk estimates with ORs and using the proportion of control patients carrying the Z allele as an estimate of the probability of exposure.2931

While this estimate is not exact, given our case-control sampling design (oversampled older patients without CFLD), this estimate should provide a reasonable approximation, owing to the modest frequency of CFLD in patients with CF (≈ 5%).

Clinical Features

Initial Study. Characteristics of the initial group of 124 patients with CFLD and 843 patients without CFLD are shown in Table 1. The CFLD group was younger at enrollment, had more male patients, and had slightly fewer white patients. The CFTR mutations in patients with CFLD were representative of pancreatic insufficient mutations in North American and European patients with CF (Table 1).1 The prevalence of meconium ileus at birth in patients with CFLD (18.2%) is comparable to that in the control group and typical for the general CF population with pancreatic insufficient CFTR mutations.1

Table Graphic Jump LocationTable 1. Initial Study: Characteristics of Patients With Cystic Fibrosis With or Without Severe Liver Disease

Abnormalities in biochemical tests of the liver (aspartate transaminase, alanine transaminase, and γ-glutamyl transferase) were not predictive of CFLD and also were not markers of hepatocellular synthetic dysfunction, such as international normalized ratio and levels of serum albumin (Table 2). Preoperative assessment of data available from a subset of patients (n = 22) who underwent liver transplantation (n = 43) showed a distribution of abnormal total bilirubin and albumin values similar to that of the nontransplanted patients (Table 2).

Table Graphic Jump LocationTable 2. Summary of Clinical Laboratory Values for Patients With Cystic Fibrosis and Severe Liver Disease

Replication Study. Based on the associations for the SERPINA1 Z allele and TGFB1 codon 10 (Table 3), we enrolled additional patients with and without CFLD to test for replication (Table 4). The characteristics of the replication patients were similar to those in the initial study (Table 1), including the distribution of specific CFTR mutations, prevalence of meconium ileus (23.8%), and liver function abnormalities (Table 2 and Table 4).

Table Graphic Jump LocationTable 3. Initial Study: Prevalence of Polymorphic Genotypes in Patients With Cystic Fibrosis With or Without Severe Liver Disease
Table Graphic Jump LocationTable 4. Replication Study: Characteristics of Patients With Cystic Fibrosis With or Without Severe Liver Disease
Cochran-Armitage Trend Test of Association

Initial Study. In the analysis of previously studied gene modifiers of liver disease in CF (Table 3), association was seen only for the SERPINA1 Z allele (OR, 4.72; 95% CI, 2.31-9.61; P = 3.3 × 10−6) and TGFB1 codon 10 (OR, 1.53; 95% CI, 1.16-2.03; P = 2.8 × 10−3). The SERPINA1 Z allele displayed association for all patients with CFLD but was more prominent in female patients. Similar results were seen when the analysis was restricted to white patients (data not shown). It is noteworthy that small effects for the nonsignificant polymorphisms would not be detected with sufficient power by this study. The genotypes and minor allele frequencies for genetic variants in patients without CFLD were similar to those previously reported.57,1821

Replication Study. The association was replicated for the SERPINA1 Z allele (OR, 3.42; 95% CI, 1.54-7.59; P = 1.4 × 10−3) (Table 5), but the association was more prominent in male patients, in contrast to the initial study. Similar results were seen when analyses were restricted to white patients (data not shown). The association of the TGFB1 codon 10 variant was not replicated for all patients (Table 5) or for male or female patients when analyzed separately (data not shown).

Table Graphic Jump LocationTable 5. Replication Study: Prevalence of Polymorphic Genotypes in Patients With Cystic Fibrosis With or Without Severe Liver Disease

Initial Plus Replication Study. When the initial and replication populations were combined for analysis using Cochran-Armitage trend tests, the SERPINA1 Z allele displayed very robust association with CFLD (OR, 4.17; 95% CI, 2.46-7.05; P = 9.9 × 10−9); similar evidence for association was observed in analyses restricted to white patients in the initial plus replication populations (data not shown).

Hardy-Weinberg Equilibrium

All polymorphisms had genotype distributions consistent with Hardy-Weinberg equilibrium (P > .01) in the initial, replication, and combined samples, irrespective of how samples were partitioned according to ethnicity and CFLD status.

Logistic Regression for the Z Allele

We combined the initial and replication groups and performed logistic regression for the SERPINA1 Z allele to estimate the odds of CFLD, adjusting for the covariates of ethnicity, sex, and CFTR genotype. Results remained consistent when using all patients or white patients only, with respect to statistical significance estimates (P = 1.5 × 10−8 or P = 6.3 × 10−8, respectively) as well as OR estimates (OR, 5.04; 95% CI, 2.88-8.83 for all patients vs OR, 4.87; 95% CI, 2.75-8.64 for white patients only). In addition, we saw no evidence for interactions between sex and the SERPINA1 Z allele in all patients or only white patients. Similar results were obtained by logistic regression adjusting only for ethnicity in the complete sample and in models that additionally adjusted for the TGFB1 codon 10 genotype.

Population Attributable Risk

We combined the initial and replication groups and estimated the population attributable risk for the Z allele to be 6.7% (white patients only, 6.6%). A similar result was obtained using another method of estimating the probability of exposure, namely the average Z allele frequency of patients from North America, Europe, and Australia (data not shown).

Age at Diagnosis of CFLD

The mean and median age of recognition (diagnosis) of portal hypertension in all patients with CFLD was approximately 10 to 11 years, and 90% of patients had CFLD diagnosed before age 20 years. Male patients had an earlier age of diagnosis of CFLD than female patients for all patients (males, 8.5 years; females, 10.5 years; P = .007), and for white patients (males, 9.7 years; females, 11.5 years; P = .03). Age at diagnosis of CFLD was not associated with the presence of the SERPINA1 Z allele, CFTR genotype, or self-reported ancestry.

Liver Histopathology

A patient with CFLD carrying a single copy of the SERPINA1 Z allele accumulated SERPINA1 protein within hepatocytes adjoining the fibrosed portal tracts, but SERPINA1 protein was not seen in hepatocytes of a patient with CFLD and without the Z allele.

Previous studies have suggested that genetic polymorphisms may act as modifiers of liver disease in cystic fibrosis, but these studies were small and phenotyping did not address the development of severe (biliary) cirrhosis associated with portal hypertension.1821 To increase the likelihood of identifying genetic modifiers relevant to the development of severe liver disease in CF, ie, cirrhosis with portal hypertension, we performed 2 sequential studies in different groups of patients. The initial study involved 5 candidate genes that had previously been studied as modifiers of CF liver disease,1821 and the replication study tested for confirmation of SERPINA1 Z allele and TGFB1 codon 10 variant as modifiers of severe liver disease in CF.

This study had 3 key design features. First, we used rigorous criteria to identify patients with CF and portal hypertension (cases), reflecting hepatobiliary cirrhosis, and key source documents were reviewed independently by 2 experts to confirm the CFLD phenotype. Second, for patients without CFLD (controls), we studied only those 15 years or older, to exclude younger patients with predisposition to develop CFLD. Third, we enrolled a large number of patients with and without CFLD to improve statistical power. For the initial study, approximately 50% of the case patients were from outside North America, and 93% were self-described as white; all control patients were from North America. For the replication (second) study, a slightly greater percentage of case patients were from North America (63% vs 50%), and some control patients (10%) were from outside North America.

Genetic analyses of the initial cohort showed that a single copy of the SERPINA1 Z allele and each additional copy of the TGFB1 codon 10 C allele were associated with significantly increased odds of CFLD. In the replication study, the SERPINA1 Z allele was confirmed as a modifier of liver disease in CF, whereas the TGFB1 codon 10 variant was not confirmed. It is noteworthy that small effects of the nonsignificant polymorphisms in other genes would not be detected with sufficient power by this study. The association of the SERPINA1 Z allele with CFLD contrasts with results from a previous “negative” study that used less stringent phenotypic markers of CF liver disease, such as liver function tests, which do not correlate with severity of CFLD (portal hypertension).18

When the initial and replication study populations were combined for joint analysis by multivariable logistic regression, the magnitude of the effect of the SERPINA1 Z allele was large compared with most genetic association studies (OR, ≈ 5) when sex, ethnicity, and CFTR genotype were included as covariates. The strength of the association of the SERPINA1 Z allele with CFLD varied by sex for the initial vs the replication studies, but the overall odds were not statistically different for female and male patients when all patients were analyzed. Population stratification is unlikely to account for the results for the SERPINA1 Z allele; the prevalence of the Z allele (1.14%) in patients without CFLD (controls) in our study is similar to that reported for more than 85 000 individuals genotyped in pertinent regions of the world (1.20%).32,33

The mechanism of the SERPINA1 Z allele as an adverse modifier of liver disease in patients with CF likely reflects the dual stimulation of hepatic stellate cells by inflammatory mediators from CFTR-deficient cholangiocytes as well as hepatocytes containing the misfolded SERPINA1 protein, ie, these inflammatory stimuli induce hepatic stellate cells to migrate and proliferate in the bile duct regions in a profibrogenic manner.1013,16,3438 Bile duct ligation with resultant cholestasis induces more activated stellate cells and fibrosis in the liver of homozygous transgenic PiZ vs wild-type mice, which is compatible with this proposed mechanism of the Z allele as an adverse modifier in CF.39 Further studies are necessary to better define the pathogenesis of the Z allele in CFLD.

The Z allele variant causes misfolding of the SERPINA1 protein, which results in an accumulation of protein in hepatocytes. The most prevalent CFTR mutation, DF508, is also a misfolding mutation, expressed predominantly in cholangiocytes in the liver.813,3438 However, it is unlikely that folding mutations in CFTR and SERPINA1 induce an amplified, adverse effect on the proteosomal degradation pathway, because these 2 genes are predominantly expressed in 2 different cell types in the liver.8,9,3438 Furthermore, heterozygosity for the Z allele is associated with the risk and progression of a variety of liver diseases, including cryptogenic cirrhosis, biliary atresia, viral hepatitis, alcoholic cirrhosis, and nonalcoholic fatty liver disease.37,38,40

By studying a large number of patients with CF and with well-defined severe liver disease and portal hypertension, we confirmed and refuted some previous observations and discovered new information about the clinical features of these patients. We confirmed that (1) CFLD is more common and is diagnosed earlier in male individuals; (2) specific CFTR mutations do not correlate with CFLD, but CFTR mutations with residual function (pancreatic sufficient mutations) are uncommon in individuals with CFLD; (3) hepatic synthetic function is preserved for long durations in most patients with CFLD; (4) thrombocytopenia due to hypersplenism is common in individuals with portal hypertension due to CFLD; (5) liver biochemical tests are poorly predictive of severe liver disease and portal hypertension in CF; and (6) severe liver disease with portal hypertension develops in pediatric patients by age 10 to 12 years.1417,2225 In addition, we made a striking observation about the age distribution of diagnosis of severe liver disease, whereby the prevalence of severe liver disease does not increase in adults with CF, despite progressive increase in longevity; more than 90% of the patients with CFLD in our study were diagnosed by age 20 years, with a mean (and median) age of diagnosis of 10 to 11 years. We were not able to confirm any association of meconium ileus with CFLD, as has been reported in some,15,16,2225 but not all,17,41 studies; the prevalence of meconium ileus in patients with CFLD in our study (≈21%) was similar to that reported for patients with pancreatic exocrine insufficiency.1

In summary, we studied 2 large populations of patients with CF with and without liver disease and portal hypertension to test genes previously studied as modifiers of liver disease. Of these candidate genes, only the SERPINA1 Z allele was significantly associated with CFLD and portal hypertension. This polymorphism is relatively uncommon in CF (≈ 2.2% of patients with CF are carriers), but the OR for association with severe liver disease is relatively high (≈ 5) for the contribution of a genetic modifier to a mendelian disorder. Moreover, the estimated population attributable risk among patients with CF is 6.7%. From a clinical perspective, a rare variant with large penetrance (such as the Z allele) may be more useful than a common variant with low penetrance in screening for genetic polymorphisms. The identification of the SERPINA1 Z allele as the first marker for the development of severe liver disease in CF illustrates the possibility of identifying CF risk factors early in life, conceptually as a secondary component of neonatal screening after the diagnosis of CF is confirmed.

Corresponding Author: Michael R. Knowles, MD, Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, 7019 Thurston-Bowles Bldg, CB#7248, Chapel Hill, NC 27599 (knowles@med.unc.edu).

Author Contributions: Dr Knowles had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Bartlett, Friedman, Ling, Pace, Zou, Silverman, Lange, Durie, Knowles.

Acquisition of data: Bartlett, Friedman, Pace, Bell, Bourke, Castaldo, Castellani, Cipolli, C. Colombo, J. Colombo, Debray, Fernandez, Lacaille, Macek, Rowland, Salvatore, Taylor, Wainwright, Wilschanski, Zemkova, Hannah, Corey, Zielenski, Dorfman, Drumm, Durie, Knowles.

Analysis and interpretation of data: Bartlett, Friedman, Ling, Pace, Salvatore, Hannah, Phillips, Wang, Zou, Wright, Lange, Durie, Knowles.

Drafting of the manuscript: Bartlett, Friedman, Pace, Zou, Lange, Knowles.

Critical revision of the manuscript for important intellectual content: Bartlett, Ling, Pace, Bell, Bourke, Castaldo, Castellani, Cipolli, C. Colombo, J. Colombo, Debray, Fernandez, Lacaille, Macek, Rowland, Salvatore, Taylor, Wainwright, Wilschanski, Zemkova, Hannah, Phillips, Corey, Zielenski, Dorfman, Wang, Silverman, Drumm, Wright, Lange, Durie, Knowles.

Statistical analysis: Bartlett, Wang, Zou, Wright, Lange, Knowles.

Obtained funding: Friedman, Bell, Rowland, Zielenski, Drumm, Durie, Knowles.

Administrative, technical, or material support: Bartlett, Friedman, Pace, Bell, Bourke, Castaldo, Castellani, Cipolli, C. Colombo, J. Colombo, Debray, Lacaille, Rowland, Salvatore, Taylor, Wainwright, Wilschanski, Hannah, Phillips, Corey, Zielenski, Dorfman, Zou, Wright, Durie, Knowles.

Study supervision: Bartlett, Pace, Lange, Knowles.

Drs Durie and Knowles jointly directed the project.

Financial Disclosures: None reported.

Funding/Support: This study was supported by grants from the Cystic Fibrosis Foundation (SILVER0Z00, KNOWLE00A0, DRUMM04P0, DRUMM0A00); the National Institutes of Health (NIH R01GM074175, NIH/NIDDK DK066368, CTRC RR00046, CTSA UL1RR025747); the Prince Charles Hospital Foundation; the OWHC/CIHR Fellowship; the Czech Ministry of Health (VZFNM00064203, NS9488/3); MIUR, Rome, Italy, and Regione Campania, Italy; a Fellowship in Rare Diseases from The Health Research Board Grant (RFRD–05-07); Genome Canada through the Ontario Genomics Institute (2004-OGI-3-05); the Lloyd Carr-Harris Foundation; and the Canadian Cystic Fibrosis Foundation.

Role of the Sponsor: The funding organizations had no role in the design and conduct of the study; the collection, management, analysis, and the interpretation of the data; or the preparation, review, or approval of the manuscript.

The Gene Modifier Study Group:Alabama: J.P. Clancy, L.J. Sindel; Alaska: D.M. Roberts, V. Roberts; Arizona: P.J. Radford, N. Argel, W.J. Morgan, J.L. Douthit; Arkansas: D.E. Schellhase, P. Anderson, A. Taggart; California: B. Morrissey, A.C.G. Platzker, M.S. Woo, L. Fukushima, E. Hsu, G.F. Shay, K.A. Hardy, R.B. Moss, C. E. Dunn, M.S. Pian, H.A. Wojtczak, L. Burns, N.R. Henig, D.W. Nielson, C. Landon, A. Thompson; Colorado: F.J. Accurso, J.A. Nick, M. Jones; Connecticut: C. Lapin, V.M. Drapeau, M.E. Egan; Delaware: R. Padman; Washington, DC: G.B. Winnie, C. George; Florida: E.L. Olson, M.J. Light, D.E. Geller, M. Gondor, J. Flanary; Georgia: A.A. Stecenko, M.F. Guill; Illinois: S.A. McColley, E.M. Potter, Y. Chung, M. Garvey; Indiana: M.S. Howenstine, A. Sannuti, J. Yeley; Iowa: D.G. Sloven, R.C. Ahrens, M. Teresi; Kansas: C.M. Riva; Louisiana: S. Davis, B. Quiniones-Ellis, C. Gabor; Maine: T.F. Lever, R. Welch, A. Cairns, M. Corrigan; Maryland: P.L. Zeitlin, L. Brass; Massachusetts: H. Dorkin, H. Levy, I. Huntington, B.P. O’Sullivan; Michigan: R.H. Simon, S.Z. Nasr, C.N. Lumeng, M.E. Ball, D.S. Toder, R.E. Honicky, S. Fitch, L. Contreras; Minnesota: W.E. Regelmann, J.R. Phillips, J. McNamara, M. Johnson; Mississippi: F.E. Ruiz, K.G. Adcock; Missouri: P. Konig, P. Black, J.D. Weigel, B.E. Noyes, V.L. Kociela, T. Ferkol Jr, M. Boyle; Nebraska: J.L. Colombo; Nevada: T. Brascia; New Hampshire: H.W. Parker; New Jersey: R.L. Zanni, S.B. Fiel, P. Lomas; New Mexico: J. Taylor-Cousar; New York: D. Borowitz, J.K. DeCelie-Germana, R. Cohen, M. Gannon, E.A. DiMango, A.A. Mencin, S.J. Lobritto, M. Benitez, P.A. Walker, M.N. Berdella, E. Langfelder-Schwind, C.L. Ren, A.K. Rovitelli, R.D. Anbar, D.M. Lindner, R.G. Perciaccante, A.J. Dozor; North Carolina: M.R. Knowles, M.W. Leigh, J. Taylor-Cousar, J.A. Voynow, K.J. Auten, M.S. Schechter; Ohio: G.J. Omlor, D.A. Ouellette, C.L. Karp, P.M. Joseph, M.W. Konstan, K.S. McCoy, F. Royce, S. Bartosik, P.A. Vauthy, M.L. Vauthy; Oklahoma: J.C. Kramer, S. Hensel; Pennsylvania: C.R. Perez, N.J. Thomas, J.C. Hess, D.S. Holsclaw, T.F. Scanlin, R. Rubenstein, C. Murray, M. Skotleski, S.B. Fiel, W.P. Sexauer, A. Ko, J. Hillman, D.M. Orenstein; Rhode Island: M.S. Schechter; South Carolina: P.A. Flume, D. Brown; Tennessee: R. Schoumacher, B. Culbreath, P.E. Moore, B. Slovis; Texas: N. Dambro, J. Garbarz, P.W. Hiatt, K.N. Olivier, R. Amaro, L. Macleod; Utah: T.G. Liou; Virginia: D.K. Froh, C.E. Epstein, J. Schmidt, G. Elliot, R. Williams, M. Anderson, J. Gadd; Washington: R.L. Gibson, S. McNamara, K. Worrell, S.M. Moskowitz, M. McCarthy, C. Llewellyn, S. Wicks; West Virginia: K.S. Moffett, L.S. Baer; Wisconsin: G.A. do Pico, L.M. Makholm, M.J. Rock, S.R. Osmond, J. Biller, T. Miller; Argentina: A. Fernandez, F. Renteria; Australia: S.C. Bell, C. Wainwright, P. Lewindon, H. Selvadurai, K. Gaskin; Belgium: S. Van Biervliet; Canada: M. Montgomery, H. R. Rabin, J. Leong, P. Zuberbuhler, N.E. Brown, J. Tabak, A.G.F. Davidson, E.M. Nakielna, B. Habbick, I. Waters, S. Wiltse, W. Kepron, H. Pasterkamp, D.N. Garey, G. Bishop, M. Noseworthy, R.T. Michael, A.M. Dale, F.A. Gosse, W. Robinson, P.R. Durie, M. Corey, A. Freitag, L. Pedder, R. Van Wylick, M.D. Lougheed, L. Kodiattu, M. Jackson, K. Malhotra, B. Lyttle, N.A.M. Paterson, S. Aaron, M. Boland, T. Kovesi, A. Smith, V.J. Kumar, S. Zinger, E. Tullis, F. Simard, L. Rivard, A. Cantin, G. Cote, L.C. Lands, J.E. Marcotte, E. Matouk, Y. Berthiaume, A. Jeanneret, M. Van Spall, G. Rivard, J. Boucher, N. Petit, B. Holmes, D. Cotton, K. Ramlall; Chile: G. Repetto; Czech Republic: M. Macek Jr, V. Vavrova, J. Bartosova, L. Fila; England: C.J. Taylor, J. McGaw; France: D. Debray, F. Lacaille, A. Munck; Germany: B. Tümmler; Ireland: M. Rowland, B. Bourke, G. Canny, C. Gallagher; Israel: J. Rivlin, E. Picard, H. Blau, M. Wilschanski, C. Springer, E. Kerem, Y. Yahav, Y. Bujanover; Italy: R. Casciaro, C. Colombo, G. Castaldo, F. Salvatore, V. Raia, M. Cipolli, C. Castellani; the Netherlands: M. Sinaasappel, D. Dooijes; Scotland: S.C. Ling; Slovakia: H. Kayserova; Turkey: U. Ozcelik, N. Kiper, D. Dogru. The full list, including institutions, appears here.

Additional Contributions: We are indebted to the research coordinators from the University of North Carolina at Chapel Hill (Allison Handler, RN, BSN, MS, CCRC*; Lori Jee, MSN, RN, FNP-C*; Sally Wood, BS*; Sonya Adams, BS*; Leia Charnin, BA*; Sarah Norris, BS*); the Hospital for Sick Children, Toronto, Ontario, Canada (Mary Christofi, BSc*; Jennifer Breaton, RN, BN, MHSc (C)*; Nicole Anderson, HonsBSc, CCRP*); Case Western Reserve University (Colette Bucur, CNP*); and Hadassah University Hospital (Netta Malka, RN, BSN*; Limor Cohen, RN, BSN*). We are also indebeted to the University of North Carolina Center for Bioinformatics (Airong Xu, MD, MSIS*; David Fargo, PhD*; and Hemant Kelkar, PhD) and Department of Pathology and Laboratory Medicine (Zhaoqing Zhou, PhD*) for genotyping support; and Wanda O’Neal, PhD, Molecular Biology Core Laboratory for the Cystic Fibrosis/Pulmonary Research and Treatment Center at the University of North Carolina at Chapel Hill, for useful discussion. We are extremely grateful to Beth Godwin, BA,* for administrative support and to Sarah Norris, BS,* for editorial assistance. We express our gratitude to all the patients and their families for making this study possible. *Indicates individuals who received salary compensation for their contributions.

Welsh MJ, Ramsey BW, Accurso FJ, Cutting GR. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York, NY: McGraw-Hill; 2001:5121-5188
Mekus F, Ballmann M, Bronsveld I, Bijman J, Veeze H, Tummler B. Categories of deltaF508 homozygous cystic fibrosis twin and sibling pairs with distinct phenotypic characteristics.  Twin Res. 2000;3(4):277-293
PubMed
Sontag MK, Accurso FJ. Gene modifiers in pediatrics: application to cystic fibrosis.  Adv Pediatr. 2004;51:5-36
PubMed
Vanscoy LL, Blackman SM, Collaco JM,  et al.  Heritability of lung disease severity in cystic fibrosis.  Am J Respir Crit Care Med. 2007;175(10):1036-1043
PubMed   |  Link to Article
Drumm ML, Konstan MW, Schluchter MD,  et al; Gene Modifier Study Group.  Genetic modifiers of lung disease in cystic fibrosis.  N Engl J Med. 2005;353(14):1443-1453
PubMed   |  Link to Article
Bremer LA, Blackman SM, Vanscoy LL,  et al.  Interaction between a novel TGFB1 haplotype and CFTR genotype is associated with improved lung function in cystic fibrosis.  Hum Mol Genet. 2008;17(14):2228-2237
PubMed   |  Link to Article
Dorfman R, Sandford A, Taylor C,  et al.  Complex two-gene modulation of lung disease severity in children with cystic fibrosis.  J Clin Invest. 2008;118(3):1040-1049
PubMed
Cohn JA, Strong TV, Picciotto MR, Nairn AC, Collins FS, Fitz JG. Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells.  Gastroenterology. 1993;105(6):1857-1864
PubMed
Kinnman N, Lindblad A, Housset C,  et al.  Expression of cystic fibrosis transmembrane conductance regulator in liver tissue from patients with cystic fibrosis.  Hepatology. 2000;32(2):334-340
PubMed   |  Link to Article
Colombo C. Liver disease in cystic fibrosis.  Curr Opin Pulm Med. 2007;13(6):529-536
PubMed   |  Link to Article
Feranchak AP, Sokol RJ. Cholangiocyte biology and cystic fibrosis liver disease.  Semin Liver Dis. 2001;21(4):471-488
PubMed   |  Link to Article
Schuppan D, Afdhal NH. Liver cirrhosis.  Lancet. 2008;371(9615):838-851
PubMed   |  Link to Article
Tsukada S, Parsons CJ, Rippe RA. Mechanisms of liver fibrosis.  Clin Chim Acta. 2006;364(1-2):33-60
PubMed   |  Link to Article
Castaldo G, Fuccio A, Salvatore D,  et al.  Liver expression in cystic fibrosis could be modulated by genetic factors different from the cystic fibrosis transmembrane regulator genotype.  Am J Med Genet. 2001;98(4):294-297
PubMed   |  Link to Article
Colombo C, Apostolo MG, Ferrari M,  et al.  Analysis of risk factors for the development of liver disease associated with cystic fibrosis.  J Pediatr. 1994;124(3):393-399
PubMed   |  Link to Article
Feranchak AP. Hepatobiliary complications of cystic fibrosis.  Curr Gastroenterol Rep. 2004;6(3):231-239
PubMed   |  Link to Article
Wilschanski M, Rivlin J, Cohen S,  et al.  Clinical and genetic risk factors for cystic fibrosis-related liver disease.  Pediatrics. 1999;103(1):52-57
PubMed   |  Link to Article
Frangolias DD, Ruan J, Wilcox PJ,  et al.  Alpha 1-antitrypsin deficiency alleles in cystic fibrosis lung disease.  Am J Respir Cell Mol Biol. 2003;29(3, pt 1):390-396
PubMed   |  Link to Article
Arkwright PD, Pravica V, Geraghty PJ,  et al.  End-organ dysfunction in cystic fibrosis: association with angiotensin I converting enzyme and cytokine gene polymorphisms.  Am J Respir Crit Care Med. 2003;167(3):384-389
PubMed   |  Link to Article
Henrion-Caude A, Flamant C, Roussey M,  et al.  Liver disease in pediatric patients with cystic fibrosis is associated with glutathione S-transferase P1 polymorphism.  Hepatology. 2002;36(4, pt 1):913-917
PubMed
Gabolde M, Hubert D, Guilloud-Bataille M, Lenaerts C, Feingold J, Besmond C. The mannose binding lectin gene influences the severity of chronic liver disease in cystic fibrosis.  J Med Genet. 2001;38(5):310-311
PubMed   |  Link to Article
Colombo C, Battezzati PM, Crosignani A,  et al.  Liver disease in cystic fibrosis: a prospective study on incidence, risk factors, and outcome.  Hepatology. 2002;36(6):1374-1382
PubMed
Corbett K, Kelleher S, Rowland M,  et al.  Cystic fibrosis-associated liver disease: a population-based study.  J Pediatr. 2004;145(3):327-332
PubMed   |  Link to Article
Debray D, Lykavieris P, Gauthier F,  et al.  Outcome of cystic fibrosis-associated liver cirrhosis: management of portal hypertension.  J Hepatol. 1999;31(1):77-83
PubMed   |  Link to Article
Lamireau T, Monnereau S, Martin S, Marcotte JE, Winnock M, Alvarez F. Epidemiology of liver disease in cystic fibrosis: a longitudinal study.  J Hepatol. 2004;41(6):920-925
PubMed   |  Link to Article
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989
Purcell S, Neale B, Todd-Brown K,  et al.  PLINK: a tool set for whole-genome association and population-based linkage analyses.  Am J Hum Genet. 2007;81(3):559-575
PubMed   |  Link to Article
Armitage P. Tests for linear trends in proportions and frequencies.  Biometrics. 1955;11(3):375-386
Link to Article
Levin ML. The occurrence of lung cancer in man.  Acta Unio Int Contra Cancrum. 1953;9(3):531-541
PubMed
Yang Q, Khoury MJ, Friedman JM, Flanders WD. On the use of population attributable fraction to determine sample size for case-control studies of gene-environment interaction.  Epidemiology. 2003;14(2):161-167
PubMed
Levin ML, Bertell RRE. simple estimation of population attributable risk from case-control studies.  Am J Epidemiol. 1978;108(1):78-79
PubMed
de Serres FJ, Blanco I, Fernandez-Bustillo E. PI S and PI Z alpha-1 antitrypsin deficiency worldwide: a review of existing genetic epidemiological data.  Monaldi Arch Chest Dis. 2007;67(4):184-208
PubMed
de Serres FJ, Blanco I, Fernandez-Bustillo E. Genetic epidemiology of alpha-1 antitrypsin deficiency in North America and Australia/New Zealand: Australia, Canada, New Zealand and the United States of America.  Clin Genet. 2003;64(5):382-397
PubMed   |  Link to Article
Eigenbrodt ML, McCashland TM, Dy RM, Clark J, Galati J. Heterozygous alpha 1-antitrypsin phenotypes in patients with end stage liver disease.  Am J Gastroenterol. 1997;92(4):602-607
PubMed
Fischer HP, Ortiz-Pallardo ME, Ko Y, Esch C, Zhou H. Chronic liver disease in heterozygous alpha1-antitrypsin deficiency PiZ.  J Hepatol. 2000;33(6):883-892
PubMed   |  Link to Article
Graziadei IW, Joseph JJ, Wiesner RH, Therneau TM, Batts KP, Porayko MK. Increased risk of chronic liver failure in adults with heterozygous alpha1-antitrypsin deficiency.  Hepatology. 1998;28(4):1058-1063
PubMed   |  Link to Article
Lawless MW, Greene CM, Mulgrew A, Taggart CC, O’Neill SJ, McElvaney NG. Activation of endoplasmic reticulum-specific stress responses associated with the conformational disease Z alpha 1-antitrypsin deficiency.  J Immunol. 2004;172(9):5722-5726
PubMed
Perlmutter DH. Pathogenesis of chronic liver injury and hepatocellular carcinoma in alpha-1-antitrypsin deficiency.  Pediatr Res. 2006;60(2):233-238
PubMed   |  Link to Article
Mencin A, Seki E, Osawa Y,  et al.  Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibrosis in a murine model of cholestasis.  Hepatology. 2007;46(5):1443-1452
PubMed   |  Link to Article
Regev A, Guaqueta C, Molina EG,  et al.  Does the heterozygous state of alpha-1 antitrypsin deficiency have a role in chronic liver diseases? interim results of a large case-control study.  J Pediatr Gastroenterol Nutr. 2006;43:(suppl 1)  S30-S35
PubMed   |  Link to Article
Slieker MG, Deckers-Kocken JM, Uiterwaal CS, van der Ent CK, Houwen RH. Risk factors for the development of cystic fibrosis related liver disease.  Hepatology. 2003;38(3):775-776
PubMed   |  Link to Article

Figures

Tables

Table Graphic Jump LocationTable 1. Initial Study: Characteristics of Patients With Cystic Fibrosis With or Without Severe Liver Disease
Table Graphic Jump LocationTable 2. Summary of Clinical Laboratory Values for Patients With Cystic Fibrosis and Severe Liver Disease
Table Graphic Jump LocationTable 3. Initial Study: Prevalence of Polymorphic Genotypes in Patients With Cystic Fibrosis With or Without Severe Liver Disease
Table Graphic Jump LocationTable 4. Replication Study: Characteristics of Patients With Cystic Fibrosis With or Without Severe Liver Disease
Table Graphic Jump LocationTable 5. Replication Study: Prevalence of Polymorphic Genotypes in Patients With Cystic Fibrosis With or Without Severe Liver Disease

References

Welsh MJ, Ramsey BW, Accurso FJ, Cutting GR. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York, NY: McGraw-Hill; 2001:5121-5188
Mekus F, Ballmann M, Bronsveld I, Bijman J, Veeze H, Tummler B. Categories of deltaF508 homozygous cystic fibrosis twin and sibling pairs with distinct phenotypic characteristics.  Twin Res. 2000;3(4):277-293
PubMed
Sontag MK, Accurso FJ. Gene modifiers in pediatrics: application to cystic fibrosis.  Adv Pediatr. 2004;51:5-36
PubMed
Vanscoy LL, Blackman SM, Collaco JM,  et al.  Heritability of lung disease severity in cystic fibrosis.  Am J Respir Crit Care Med. 2007;175(10):1036-1043
PubMed   |  Link to Article
Drumm ML, Konstan MW, Schluchter MD,  et al; Gene Modifier Study Group.  Genetic modifiers of lung disease in cystic fibrosis.  N Engl J Med. 2005;353(14):1443-1453
PubMed   |  Link to Article
Bremer LA, Blackman SM, Vanscoy LL,  et al.  Interaction between a novel TGFB1 haplotype and CFTR genotype is associated with improved lung function in cystic fibrosis.  Hum Mol Genet. 2008;17(14):2228-2237
PubMed   |  Link to Article
Dorfman R, Sandford A, Taylor C,  et al.  Complex two-gene modulation of lung disease severity in children with cystic fibrosis.  J Clin Invest. 2008;118(3):1040-1049
PubMed
Cohn JA, Strong TV, Picciotto MR, Nairn AC, Collins FS, Fitz JG. Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells.  Gastroenterology. 1993;105(6):1857-1864
PubMed
Kinnman N, Lindblad A, Housset C,  et al.  Expression of cystic fibrosis transmembrane conductance regulator in liver tissue from patients with cystic fibrosis.  Hepatology. 2000;32(2):334-340
PubMed   |  Link to Article
Colombo C. Liver disease in cystic fibrosis.  Curr Opin Pulm Med. 2007;13(6):529-536
PubMed   |  Link to Article
Feranchak AP, Sokol RJ. Cholangiocyte biology and cystic fibrosis liver disease.  Semin Liver Dis. 2001;21(4):471-488
PubMed   |  Link to Article
Schuppan D, Afdhal NH. Liver cirrhosis.  Lancet. 2008;371(9615):838-851
PubMed   |  Link to Article
Tsukada S, Parsons CJ, Rippe RA. Mechanisms of liver fibrosis.  Clin Chim Acta. 2006;364(1-2):33-60
PubMed   |  Link to Article
Castaldo G, Fuccio A, Salvatore D,  et al.  Liver expression in cystic fibrosis could be modulated by genetic factors different from the cystic fibrosis transmembrane regulator genotype.  Am J Med Genet. 2001;98(4):294-297
PubMed   |  Link to Article
Colombo C, Apostolo MG, Ferrari M,  et al.  Analysis of risk factors for the development of liver disease associated with cystic fibrosis.  J Pediatr. 1994;124(3):393-399
PubMed   |  Link to Article
Feranchak AP. Hepatobiliary complications of cystic fibrosis.  Curr Gastroenterol Rep. 2004;6(3):231-239
PubMed   |  Link to Article
Wilschanski M, Rivlin J, Cohen S,  et al.  Clinical and genetic risk factors for cystic fibrosis-related liver disease.  Pediatrics. 1999;103(1):52-57
PubMed   |  Link to Article
Frangolias DD, Ruan J, Wilcox PJ,  et al.  Alpha 1-antitrypsin deficiency alleles in cystic fibrosis lung disease.  Am J Respir Cell Mol Biol. 2003;29(3, pt 1):390-396
PubMed   |  Link to Article
Arkwright PD, Pravica V, Geraghty PJ,  et al.  End-organ dysfunction in cystic fibrosis: association with angiotensin I converting enzyme and cytokine gene polymorphisms.  Am J Respir Crit Care Med. 2003;167(3):384-389
PubMed   |  Link to Article
Henrion-Caude A, Flamant C, Roussey M,  et al.  Liver disease in pediatric patients with cystic fibrosis is associated with glutathione S-transferase P1 polymorphism.  Hepatology. 2002;36(4, pt 1):913-917
PubMed
Gabolde M, Hubert D, Guilloud-Bataille M, Lenaerts C, Feingold J, Besmond C. The mannose binding lectin gene influences the severity of chronic liver disease in cystic fibrosis.  J Med Genet. 2001;38(5):310-311
PubMed   |  Link to Article
Colombo C, Battezzati PM, Crosignani A,  et al.  Liver disease in cystic fibrosis: a prospective study on incidence, risk factors, and outcome.  Hepatology. 2002;36(6):1374-1382
PubMed
Corbett K, Kelleher S, Rowland M,  et al.  Cystic fibrosis-associated liver disease: a population-based study.  J Pediatr. 2004;145(3):327-332
PubMed   |  Link to Article
Debray D, Lykavieris P, Gauthier F,  et al.  Outcome of cystic fibrosis-associated liver cirrhosis: management of portal hypertension.  J Hepatol. 1999;31(1):77-83
PubMed   |  Link to Article
Lamireau T, Monnereau S, Martin S, Marcotte JE, Winnock M, Alvarez F. Epidemiology of liver disease in cystic fibrosis: a longitudinal study.  J Hepatol. 2004;41(6):920-925
PubMed   |  Link to Article
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989
Purcell S, Neale B, Todd-Brown K,  et al.  PLINK: a tool set for whole-genome association and population-based linkage analyses.  Am J Hum Genet. 2007;81(3):559-575
PubMed   |  Link to Article
Armitage P. Tests for linear trends in proportions and frequencies.  Biometrics. 1955;11(3):375-386
Link to Article
Levin ML. The occurrence of lung cancer in man.  Acta Unio Int Contra Cancrum. 1953;9(3):531-541
PubMed
Yang Q, Khoury MJ, Friedman JM, Flanders WD. On the use of population attributable fraction to determine sample size for case-control studies of gene-environment interaction.  Epidemiology. 2003;14(2):161-167
PubMed
Levin ML, Bertell RRE. simple estimation of population attributable risk from case-control studies.  Am J Epidemiol. 1978;108(1):78-79
PubMed
de Serres FJ, Blanco I, Fernandez-Bustillo E. PI S and PI Z alpha-1 antitrypsin deficiency worldwide: a review of existing genetic epidemiological data.  Monaldi Arch Chest Dis. 2007;67(4):184-208
PubMed
de Serres FJ, Blanco I, Fernandez-Bustillo E. Genetic epidemiology of alpha-1 antitrypsin deficiency in North America and Australia/New Zealand: Australia, Canada, New Zealand and the United States of America.  Clin Genet. 2003;64(5):382-397
PubMed   |  Link to Article
Eigenbrodt ML, McCashland TM, Dy RM, Clark J, Galati J. Heterozygous alpha 1-antitrypsin phenotypes in patients with end stage liver disease.  Am J Gastroenterol. 1997;92(4):602-607
PubMed
Fischer HP, Ortiz-Pallardo ME, Ko Y, Esch C, Zhou H. Chronic liver disease in heterozygous alpha1-antitrypsin deficiency PiZ.  J Hepatol. 2000;33(6):883-892
PubMed   |  Link to Article
Graziadei IW, Joseph JJ, Wiesner RH, Therneau TM, Batts KP, Porayko MK. Increased risk of chronic liver failure in adults with heterozygous alpha1-antitrypsin deficiency.  Hepatology. 1998;28(4):1058-1063
PubMed   |  Link to Article
Lawless MW, Greene CM, Mulgrew A, Taggart CC, O’Neill SJ, McElvaney NG. Activation of endoplasmic reticulum-specific stress responses associated with the conformational disease Z alpha 1-antitrypsin deficiency.  J Immunol. 2004;172(9):5722-5726
PubMed
Perlmutter DH. Pathogenesis of chronic liver injury and hepatocellular carcinoma in alpha-1-antitrypsin deficiency.  Pediatr Res. 2006;60(2):233-238
PubMed   |  Link to Article
Mencin A, Seki E, Osawa Y,  et al.  Alpha-1 antitrypsin Z protein (PiZ) increases hepatic fibrosis in a murine model of cholestasis.  Hepatology. 2007;46(5):1443-1452
PubMed   |  Link to Article
Regev A, Guaqueta C, Molina EG,  et al.  Does the heterozygous state of alpha-1 antitrypsin deficiency have a role in chronic liver diseases? interim results of a large case-control study.  J Pediatr Gastroenterol Nutr. 2006;43:(suppl 1)  S30-S35
PubMed   |  Link to Article
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