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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
JAMA. Author manuscript; available in PMC Jul 15, 2013.
Published in final edited form as:
PMCID: PMC3711243
NIHMSID: NIHMS254899
GENETIC MODIFIERS OF LIVER DISEASE IN CYSTIC FIBROSIS
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, Carlo Castellani, MD, Marco Cipolli, MD, Carla Colombo, MD, John L. Colombo, MD, Dominique Debray, MD, Adriana Fernandez, MD, Florence Lacaille, MD, Milan Macek, Jr., MD, DSc, Marion Rowland, MB, PhD, Francesco Salvatore, MD, Christopher J. Taylor, MD, Claire Wainwright, MD, Michael Wilschanski, MBBS, Dana Zemková, CSc, 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,* and Michael R. Knowles, MD*, for the Gene Modifier Study Group
Cystic Fibrosis/Pulmonary Research and Treatment Center (Drs Bartlett and Knowles, Ms Pace, and Mr Hannah) and Department of Genetics (Dr Lange and Mr Wang), School of Medicine, and Department of Biostatistics, School of Public Health (Drs Zou, Wright, and Lange, and Mr Wang), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; 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, 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, Nebraska (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 (Ms 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).
Corresponding Author: Michael R. Knowles, MD, Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, 7019 Thurston-Bowles Bldg., CB# 7248, Chapel Hill, NC 27599 (knowles/at/med.unc.edu)
*These authors jointly directed the project.
Context
A subset (~3–5%) of patients with cystic fibrosis (CF) develops severe liver disease (CFLD) with portal hypertension.
Objective
To assess whether any of 9 polymorphisms in 5 candidate genes (SERPINA1, ACE, GSTP1, MBL2, and TGFB1) are associated with severe liver disease in CF patients.
Design, Setting, and Participants
A 2-stage design was used in this case–control study. CFLD subjects were enrolled from 63 U.S., 32 Canadian, and 18 CF centers outside of North America, with the University of North Carolina at Chapel Hill (UNC) as the coordinating site. In the initial study, we studied 124 CFLD patients (enrolled 1/1999–12/2004) and 843 CF controls (patients without CFLD) by genotyping 9 polymorphisms in 5 genes previously implicated 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 CFLD patients (enrolled 1/2005–2/2007) and 1088 CF controls.
Main Outcome Measures
We compared differences in distribution of genotypes in CF patients with severe liver disease versus CF patients without CFLD.
Results
The initial study showed CFLD to be associated with the SERPINA1 (also known as α1-antiprotease and α1-antitrypsin) Z allele (P value=3.3×10−6; odds ratio (OR) 4.72, 95% confidence interval (CI) 2.31–9.61), and with transforming growth factor β-1 (TGFB1) codon 10 CC genotype (P=2.8×10−3; OR 1.53, CI 1.16–2.03). In the replication study, CFLD was associated with the SERPINA1 Z allele (P=1.4×10−3; OR 3.42, CI 1.54–7.59), 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 (P=1.5×10−8; OR 5.04, CI 2.88–8.83).
Conclusion
The SERPINA1 Z allele is a risk factor for liver disease in CF. Patients who carry the Z allele are at greater odds (OR ~5) to develop severe liver disease with portal hypertension.
Cystic fibrosis (CF) is a recessive monogenic disorder characterized by multi-organ involvement and clinical heterogeneity that is incompletely explained by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene.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 CF liver 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 CF patients 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 severe liver disease in CF with portal hypertension (CFLD), we studied 9 functional variants in 5 genes previously studied in CF liver disease, including α1-antitrypsin (also known as α1-antiprotease, α1AP, SERPINA1)18, angiotensin-converting enzyme (ACE)19, glutathione S-transferase (GSTP1)20, mannose-binding lectin 2 (MBL2)21 and transforming growth factor β1 (TGFB1).19 Our initial study compared polymorphic genotypes in these candidate modifier genes in CF subjects with CFLD and “control” CF patients (without CFLD, who were at least 15 years of age). We tested our initial findings in a second study in different populations of CF patients with and without CFLD.
Patients - Initial (First) Study
Of the 158 CF patients evaluated for severe liver disease (CFLD; portal hypertension; enrolled 1/1999–12/2004), there were 128 patients who fulfilled criteria from 22 CF Centers in 10 countries (Australia, 8; Canada, 17; Czech Republic, 17; Germany, 3; Italy, 28; Netherlands, 1; Scotland, 2; Slovakia, 4; Turkey, 4; and U.S., 44). For patients without 2 defined mutations in CFTR, we tested further, using a panel of 70 mutations (Tm Bioscience/Luminex CFTR mutation detection assay). After genotyping was complete, >95% of CFLD patients with 2 defined mutations in CFTR had 2 pancreatic insufficient (PI) mutations (eTable 1). The 843 control patients (CF patients without CLFD) were enrolled from the U.S. (759 patients from 42 Centers) and Canada (84 patients from 32 Centers). The majority of the CF controls were ascertained from the GMS Lung Study population (DF508 homozygotes; 92.6%)5, Most of the other controls had biallelic PI mutations (see online supplement, Methods). These controls without CFLD were ≥ 15 years of age (1 SD above the mean age of diagnosis of CFLD), in order to exclude younger patients who might have occult liver disease.
Patients - Replication (Second) Study
Of the 191 CF patients evaluated for CFLD (portal hypertension; enrolled 1/2005–2/2007), there were 139 CF patients who fulfilled criteria from 35 CF Centers in 10 countries (Argentina, 5; Australia, 5; Canada, 24; Chile, 1; France, 9; Ireland, 8; Israel, 7; Italy, 14; United Kingdom, 4; and U.S., 62). The percentage of PI CFTR genotypes in CFLD subjects was similar to those in the initial study (eTable 1). The 1088 control patients (CF patients ≥ 15 years without CFLD) were ascertained from five countries (Canada, 391 patients from 32 Centers; Czech Republic, 30 patients; Ireland, 6 patients; Italy, 71 patients; and U.S., 590 patients from 54 Centers). The majority of the CF controls had 2 PI mutations (93.5%; mostly DF508/DF508 62.8%) (online supplement, Methods).
Enrollment Criteria
All patients had a diagnosis of CF, confirmed by sweat test and/or CFTR genotyping. CFLD was defined as cirrhosis in patients ≥ 2 years of age, confirmed by imaging (ultrasound, CT, MRI) showing hepatic parenchymal abnormalities of portal hypertension (esophageal varices, portal-systemic collaterals, splenomegaly) in the absence of another cause for liver disease. Data were independently reviewed by 2 hepatologists (P.R.D. and S.C.L.) 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 ethnic background, which were subject-defined.
We excluded 30 (19%) and 52 (27%) subjects 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 a liver transplant (26 in initial study; 21 in replication study), source documents were obtained from dates prior to transplant. 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 and written informed consent was obtained.
Exclusion from Analysis Based on Age of 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 (± 5.4) years (eFigure 1).15,2225 The diagnosis of CFLD was first established after the age of 30 years in 7 subjects (ages of 32, 33, 35, 40, 43, 44, and 47 years), which is ≥ 4 SD above the mean of the normal distribution. Therefore, these patients were excluded from the genetic analyses (4 from the initial study, and 3 from the replication study).
Data Collection
Patients received a unique identifier code, and data was 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 transplant), liver biopsy, endoscopy and colonoscopy.
DNA Extraction and Genotyping
DNA was extracted from peripheral blood leukocytes using standard protocols.26 Genetic polymorphisms were determined by direct sequencing, by microsphere-based genotyping using Illumina BeadArray™ technology (San Diego, CA) and by site-directed mutagenesis (online supplement, Methods).
Histochemistry/Histopathology
Immunohistochemistry with polyclonal rabbit anti-α-1 antitrypsin antibody and monoclonal mouse anti-CD68 (clone KP1) antibody (Dako Canada, Ontario, Canada), was performed on the Benchmark XT™ auto-immunostainer (Ventana Medical Systems, Tucson, AZ) 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 α-1 antitrypsin, heat epitope retrieved for CD68, peroxidase, and endogenous biotin blocked using Ventana proprietary reagents. Sections were hematoxylin counterstained for nuclear detail (eFigure 2).
Statistical Analysis
Genotype distributions were tested for consistency with expected Hardy-Weinberg equilibrium (HWE) proportions for cases and controls in the initial, replication and combined studies, using all subjects and then restricted to Caucasian subjects, using the PLINK software package (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 and unadjusted P values are reported, along with P values that were significant (P<0.05) after Bonferroni (adjusted for 9 tests) correction (appended to Tables). Analyses were performed using all samples, and only Caucasian samples.
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 (online supplement, Methods). Varying levels of covariate adjustment in the logistic regression models were made for ethnicity (as a 5-level categorical variable for all samples), gender, CFTR genotype and TGFB1 codon 10 genotype. Tests of interactions were performed to assess whether the odds of CFLD differed between males and females by SERPINA1 genotype. Odds ratios (and corresponding 95% confidence intervals) 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, using all samples and then using Caucasian subjects only.
Analysis of variance models were used to assess whether gender, SERPINA1 Z allele and CFTR genotype were associated with age of diagnosis of CFLD in the combined sample. Data were analyzed on all CFLD cases and Caucasian CFLD cases with a reported age at diagnosis with covariate adjustment for self-reported ancestry.
To estimate population attributable risk, we used a modified form of Levin’s classic formula for population attributable fraction by replacing relative risk estimates with odd ratios, and using the proportion of control subjects carrying the Z allele as an estimate of the probability of exposure2931 (online supplement, Methods). While this estimate is not exact, given our case–control sampling design (over-sampled older CF patients without CFLD), this estimate should provide a reasonable approximation, due to the modest frequency of CFLD in CF patients (~5%).
Clinical Features - Initial Study
Characteristics of the initial group of 124 CF patients with severe liver disease and 843 CF patients without CFLD are shown in Table 1. The CFLD group was younger at enrollment, had more males, and slightly fewer Caucasians. The CFTR mutations in CFLD patients were representative of PI mutations in North American and European CF patients (Table 1, eTable 1)1. The prevalence of meconium ileus at birth in CFLD patients (18.2%) is comparable to the control (no CFLD) group, and typical for the general CF population with pancreatic insufficient CFTR mutations.1
Table 1
Table 1
Initial study: Characteristics of cystic fibrosis patients with (CFLD) and without (CF no LD) severe liver disease
Abnormalities in biochemical tests of the liver (AST, ALT and GGT) were not predictive of CFLD and showed no correlation with markers of hepatocellular synthetic dysfunction, such as international normalized ratio (INR) and serum albumin (Table 2). Preoperative assessment of data available from a subset of patients (n=22) who underwent liver transplant (n=43) showed a similar distribution of abnormal total bilirubin and, albumin values as the non-transplanted patients (Table 2, footnote “b” and “c”).
Table 2
Table 2
Summary of clinical lab values for CF patients with 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 (P=3.3×10−6; OR 4.72, CI 2.31–9.61) andTGFB1 codon 10 (P=2.8×10−3; OR 1.53, CI 1.16–2.03). The SERPINA1 Z allele displayed association for all CFLD patients, but was more prominent in females (eTable 2). Similar results were seen when the analysis was restricted to Caucasians (data not shown). It is noteworthy that small effects for the non-significant polymorphisms would not be detected with sufficient power by this study (see online supplement Methods – Power Analysis; eTable 3). The genotypes and minor allele frequencies for genetic variants in patients without CFLD were similar to those previously reported.57,1821
Table 3
Table 3
Initial study: Prevalence of polymorphic genotypes according to CF patients with (CFLD) and without (no CFLD) severe liver disease
Clinical Features - Replication Study
Based on the associations for the SERPINA1 Z allele and TGFB1 codon 10 (Table 3), we enrolled additional CF 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 (eTable 1), prevalence of meconium ileus (23.8%) and liver function abnormalities (Table 2, Table 4).
Table 4
Table 4
Replication study: Characteristics of cystic fibrosis patients with (CFLD) and without (CF no LD) severe liver disease
Cochran-Armitage Trend Test of Association – Replication Study
The association was replicated for the SERPINA1 Z allele (P=1.4×10−3; OR 3.42, CI 1.54–7.59); Table 5), but the association was more prominent in males (eTable4), in contrast to the initial study (eTable 2). Similar results were seen when analyses were restricted to Caucasians (data not shown). The association of the TGFB1 codon 10 variant was not replicated for all patients (Table 5) or for males or females when analyzed separately (data not shown).
Table 5
Table 5
Replication study: Prevalence of polymorphic genotypes according to CF patients with (CFLD) and without (no CFLD) severe liver disease
Cochran-Armitage Trend Test of Association – 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 (P=9.9×10−9; OR 4.17, CI 2.46–7.05 eTable 4); similar evidence for association was observed in analyses restricted to Caucasian subjects in the initial plus replication populations(data not shown ).
Hardy-Weinberg Equilibrium
All polymorphisms had genotype distributions consistent with Hardy-Weinberg equilibrium (P > 0.01) 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 – Initial plus Replication Study
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, gender and CFTR genotype. Results remained consistent when using all subjects or Caucasian subjects only, with respect to both statistical significance estimates (P=1.5×10−8 or P=6.3×10−8, respectively) and odds ratio estimates (OR 5.04, CI 2.88–8.83 for all patients vs. OR 4.87, CI 2.75–8.64 for Caucasians only). In addition, we saw no evidence for interactions between gender and the SERPINA1 Z allele in all subjects or only Caucasians. Similar results were obtained by logistic regression adjusting only for ethnicity in the complete sample, and models that additionally adjusted for the TGFB1 codon 10 genotype (eTable 5).
Population Attributable Risk
We combined the initial and replication groups, and the population attributable risk for the Z allele was estimated to be 6.7% (Caucasians only, 6.6%) (see online supplement, Methods). 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 of Diagnosis of CFLD
The mean and median age of recognition (diagnosis) of portal hypertension in all CFLD patients was ~10–11 years (eFigure 1) and 90% of patients had CFLD diagnosed before 20 years of age. Males had an earlier age of diagnosis of CFLD than females for all subjects (males=8.5 yrs, females=10.5 years; P=0.007), and for self-reported Caucasians (males=9.7 years, females=11.5 years; P=0.027). 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 CFLD subject carrying a single copy of the SERPINA1 Z allele accumulated SERPINA1 protein within hepatocytes adjoining the fibrosed portal tracts, but SERPINA1 protein is not seen in hepatocytes of a CFLD patient without the Z allele (eFigure 2).
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 that are relevant to the development of severe liver disease in CF, i.e., 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 disease1821, 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 CF patients with portal hypertension (“cases”), reflecting hepato-biliary cirrhosis, and key source documents were reviewed independently by 2 experts to confirm the CFLD phenotype. Second, for CF patients without CFLD (“controls”), we studied only patients who were ≥ 15 years of age, in order to exclude younger patients with predisposition to develop CFLD. Third, we enrolled a large number of CF patients with and without CFLD in order to improve statistical power. For the initial study, ~50% of the CFLD patients were from outside North America and 93% were self-described as Caucasian, and all control CF patients were from North America. For the replication (second) study, a slightly greater percentage of CFLD patients were from North America (63% versus 50%), and there were some control CF patients (10%) 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 non-significant polymorphisms in other genes would not be detected with sufficient power by this study. The association of the SERPINA1 Z allele with CFLD contrasts to a previous “negative” study, which 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 to most genetic association studies (odds ratio ~5) when gender, ethnicity and CFTR genotype were included as covariates. The strength of the association of the SERPINA1 Z allele with CFLD varied by gender for the initial versus the replication studies, but the overall odds were not statistically different for females and males when all subjects 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 our CF patients without CFLD (controls) is similar to that reported for > 85,000 individuals genotyped in pertinent regions of the world (1.20%).32,33 (eFigure 3 and eTable 4).
The mechanism of the SERPINA1 Z allele as an adverse modifier of liver disease in CF patients likely reflects the dual stimulation of hepatic stellate cells by inflammatory mediators from both CFTR-deficient cholangiocytes and hepatocytes containing the misfolded SERPINA1 protein, i.e., these inflammatory stimuli induce hepatic stellate cells to migrate and proliferate in the bile duct regions in a pro-fibrogenic manner.1013,16,3438 Bile duct ligation with resultant cholestasis induces more activated stellate cells and fibrosis in the liver of homozygous transgenic PiZ versus 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 non-alcoholic fatty liver disease.37,38,40
By studying a large number of CF patients 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 diagnosed earlier, in males; 2) specific CFTR mutations do not correlate with CFLD, but CFTR mutations with residual function (pancreatic sufficient mutations) are uncommon in subjects with CFLD; 3) hepatic synthetic function is preserved for long durations in most CFLD patients; 4) thrombocytopenia due to hypersplenism is common in subjects 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 (portal hypertension) develops in pediatric patients by age 10–12 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 CF adults, despite progressive increase in longevity; more than 90% of our CFLD patients were diagnosed by age 20, with a mean (and median) age of diagnosis of 10–11 years. We were not able to confirm any association of meconium ileus with CFLD, as previously reported in some, but not all, studies;1517,2225,41 the prevalence of meconium ileus in our patients with CFLD (~20%) was similar to that reported for pancreatic exocrine insufficient patients.1
In summary, we studied 2 large populations of CF patients with and without liver disease and portal hypertension in order 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 CF patients are carriers), but the odds ratio 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 CF patients 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, to screen for genetic polymorphisms. The availability of this first marker, SERPINA1 Z allele, for the development of severe liver disease in CF illustrates the possibility of identifying risk factors in CF patients early in life, conceptually as a secondary component of neonatal screening after the diagnosis of CF is confirmed.
Supplementary Material
Supp Data
Acknowledgments
Funding/Support:
This study was supported by grants from the Cystic Fibrosis Foundation (SILVER0Z00, KNOWLE00A0, DRUMM04P0, DRUMM0A00); the National Institutes of Health (NIH R01GM074175, DK066368, GCRC RR00046, CTSA UL1RR025747); the Prince Charles Hospital Foundation; the OWHC/CIHR Fellowship; VZFNM00064203 and NS9488/3 from the Czech Ministry of Health; 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 Canadian Cystic Fibrosis Foundation.
Role of the Sponsor: The funding organizations had no role in the design and conduct of the study; in the collection, management, analysis, and the interpretation of the data; or in the preparation, review or approval of the manuscript.
Participating Groups
United States of America
Alabama: University of Alabama at Birmingham, Birmingham -- J.P. Clancy; Pulmonary Associates, Mobile -- L.J. Sindel.
Alaska: Providence Alaska Medical Center, Anchorage -- D.M. Roberts and V. Roberts.
Arizona: Phoenix Children’s Hospital, Phoenix -- P.J. Radford and N. Argel; University of Arizona, Tucson -- W.J. Morgan and J.L. Douthit.
Arkansas: University of Arkansas Medical Sciences, Little Rock -- D.E. Schellhase, P. Anderson and A. Taggart.
California: University of California, Davis School of Medicine, Davis -- B. Morrissey; Children’s Hospital of LA, Los Angeles -- A.C.G. Platzker, M.S. Woo, L. Fukushima and E. Hsu; Northern California Kaiser Permanente Medical Care Program, Oakland -- G.F. Shay; Bay Area Pediatric Pulmonary, Oakland -- K.A. Hardy; Lucile Packard Children's Hospital, Stanford University Medical Center, Palo Alto -- R.B. Moss and C.E. Dunn; University of California-San Diego, Children’s Hospital, San Diego -- M.S. Pian; Naval Medical Center, San Diego -- H.A. Wojtczak and L. Burns; California Pacific Medical Center, San Francisco -- N.R. Henig; University of California San Francisco, San Francisco -- D.W. Nielson; Ventura County Medical Center, Ventura -- C. Landon and A. Thompson.
Colorado: Denver Children’s Hospital, Denver -- F.J. Accurso; University of Colorado Adult Cystic Fibrosis Program, Denver -- J.A. Nick and M. Jones.
Connecticut: Connecticut Children's Medical Center, Hartford -- C. Lapin and V.M. Drapeau; Cystic Fibrosis Care Center, Yale University, New Haven -- M.E. Egan.
Delaware: Nemours/Alfred I. Dupont Hospital for Children, Wilmington -- R. Padman.
Washington, D.C.
Children’s National Medical Center, Washington -- G.B. Winnie and C. George.
Florida: University of Florida, Gainesville -- E.L. Olson; Batchelor Children's Institute, University of Miami, Miami -- M.J. Light; Nemours Children’s Clinic - Orlando, Orlando -- D.E. Geller; All Children's Hospital, St. Petersburg -- M. Gondor and J. Flanary.
Georgia: Emory University CF Center, Atlanta -- A.A. Stecenko; Medical College of Georgia, Augusta -- M.F. Guill.
Illinois: Northwestern University Feinberg School of Medicine, Chicago -- S.A. McColley; Children’s Memorial Hospital, Chicago -- S.A. McColley and E.M. Potter; Loyola University Medical Center, Maywood -- Y. Chung and M. Garvey.
Indiana: Riley Hospital for Children, Indianapolis -- M.S. Howenstine; Indiana University School of Medicine - Adult Cystic Fibrosis Center, Indianapolis -- A. Sannuti and J. Yeley.
Iowa: Blank Children's Hospital, Des Moines -- D.G. Sloven; University of Iowa, Iowa City -- R.C. Ahrens and M. Teresi.
Kansas: PriVia, The Research Centers of Via-Christi, Wichita -- C.M. Riva.
Louisiana: Tulane University School of Medicine, New Orleans -- S. Davis, B. Quiniones-Ellis and C. Gabor.
Maine: Eastern Maine Medical Center, Bangor -- T.F. Lever and R. Welch; Maine Pediatric Specialty Group, Portland -- A. Cairns and M. Corrigan.
Maryland: Johns Hopkins University School of Medicine, Baltimore -- P.L. Zeitlin and L. Brass.
Massachusetts: Massachusetts General Hospital, Boston -- H. Dorkin; Children’s Hospital, Boston -- H. Levy and I. Huntington; University of Massachusetts Memorial Health Care, Worcester -- B.P. O'Sullivan.
Michigan: University of Michigan Health System, Ann Arbor -- R.H. Simon, S.Z. Nasr, C.N. Lumeng and M.E. Ball; Children’s Hospital of Michigan – Wayne State, Detroit -- D.S. Toder; Michigan State University CF Center, East Lansing -- R.E. Honicky; Spectrum Health Hospitals, Grand Rapids -- S. Fitch and L. Contreras.
Minnesota: University of Minnesota, Minneapolis -- W.E. Regelmann, J.R. Phillips; Children's Hospitals and Clinics of Minnesota, Minneapolis -- J. McNamara and M. Johnson.
Mississippi: University of Mississippi Medical Center, Jackson -- F.E. Ruiz and K.G. Adcock.
Missouri: University of Missouri, Columbia -- P. Konig; Children's Mercy Hospital, Kansas City -- P. Black and J.D. Weigel; Saint Louis University, St. Louis -- B.E. Noyes and V.L. Kociela; Washington University School of Medicine, St. Louis -- T. Ferkol, Jr. and M. Boyle.
Nebraska: University of Nebraska Medical Center, Omaha -- J.L. Colombo.
Nevada: University of Nevada, Cystic Fibrosis Center, Las Vegas -- T. Brascia.
New Hampshire: Dartmouth Hitchcock Medical Center, Lebanon -- H.W. Parker.
New Jersey: Monmouth Medical Center, Long Branch -- R.L. Zanni; Atlantic Health/Morristown Memorial Hospital, Morristown -- S.B. Fiel and P. Lomas.
New Mexico: University of New Mexico, Albuquerque -- J. Taylor-Cousar.
New York: The Women and Children's Hospital of Buffalo, Buffalo -- D. Borowitz; Schneider Children's Hospital, New Hyde Park -- J.K. DeCelie-Germana; Long Island Jewish Medical Center, New Hyde Park -- R. Cohen and M. Gannon; Columbia University, New York -- E.A. DiMango; Columbia University Medical Center, New York -- A.A. Mencin, S.J. Lobritto and M. Benitez; St. Vincent’s Hospital, New York -- P.A. Walker, M.N. Berdella and E. Langfelder-Schwind; University of Rochester, Rochester -- C.L. Ren and A.K. Rovitelli; Upstate Medical University, SUNY, Syracuse -- R.D. Anbar and D.M. Lindner; Samaritan Medical Center, Watertown -- R.G. Perciaccante; Maria Fareri Children's Hospital at Westchester Medical Center, Westchester -- A.J. Dozor.
North Carolina: University of North Carolina at Chapel Hill, Chapel Hill -- M.R. Knowles and M.W. Leigh; Duke University, Durham -- J. Taylor-Cousar, J.A. Voynow and K.J. Auten; Wake Forest University Baptist Medical Center, Winston-Salem -- M.S. Schechter.
Ohio: Akron Children's Hospital, Akron -- G.J. Omlor and D.A. Ouellette; Cincinnati Children’s Hospital, Cincinnati -- C.L. Karp; University of Cincinnati Medical Center, Cincinnati -- P.M. Joseph; Rainbow Babies and Children's Hospital, CWRU, Cleveland -- M.W. Konstan; Nationwide Children's Hospital, Columbus -- K.S. McCoy; Children’s Medical Center, Dayton -- F. Royce and S. Bartosik; Toledo Children’s Hospital, Toledo -- P.A. Vauthy and M.L. Vauthy.
Oklahoma: Tulsa Cystic Fibrosis Center, Tulsa -- J.C. Kramer and S. Hensel.
Pennsylvania: Geisinger Health System, Danville -- C.R. Perez; Penn State Children's Hospital and Penn State University College of Medicine, Hershey -- N.J. Thomas and J.C. Hess; Penn Presbyterian Medical Center, Philadelphia -- D.S. Holsclaw; Children's Hospital of Philadelphia-University of Pennsylvania, Philadelphia -- T.F. Scanlin, R. Rubenstein, C. Murray and M. Skotleski; Drexel University College of Medicine, Philadelphia -- S.B. Fiel, W.P. Sexauer, A. Ko and J. Hillman; University of Pittsburgh, Pittsburgh -- D.M. Orenstein.
Rhode Island: Rhode Island Hospital, Providence -- M.S. Schechter.
South Carolina: Medical University of South Carolina, Charleston -- P.A. Flume; University of South Carolina School of Medicine, Columbia -- D. Brown.
Tennessee: University of Tennessee Health Science Center, Memphis -- R. Schoumacher and B. Culbreath; Vanderbilt University Medical Center, Nashville -- P.E. Moore and B. Slovis.
Texas: Cook Children's Health Care System, Fort Worth -- N. Dambro and J. Garbarz; Texas Children’s Hospital - Baylor, Houston -- P.W. Hiatt; Wilford Hall Medical Center, San Antonio -- K.N. Olivier; University of Texas Health Science Center, Tyler -- R. Amaro and L. Macleod.
Utah: University of Utah Pulmonary Division, Salt Lake City -- T.G. Liou.
Virginia: University of Virginia Medical Center, Charlottesville -- D.K. Froh; Children's Hospital of The King's Daughters, Norfolk -- C.E. Epstein; Virginia Commonwealth University, Richmond -- J. Schmidt, G. Elliot, R. Williams, M. Anderson and J. Gadd.
Washington: Seattle Children’s Hospital/University of Washington, Seattle -- R.L. Gibson, S. McNamara and K. Worrell; University of Washington School of Medicine, Seattle -- S.M. Moskowitz; Deaconess Medical Center, Spokane -- M. McCarthy, C. Llewellyn and S. Wicks.
West Virginia: West Virginia University, Morgantown -- K.S. Moffett and L.S. Baer.
Wisconsin: University of Wisconsin, Adult CF Center, Madison -- G.A. do Pico and L.M. Makholm; University of Wisconsin, CF-Pediatric Pulmonary Center, Madison -- M.J. Rock and S.R. Osmond; Medical College of Wisconsin, Milwaukee -- J. Biller; Children's Hospital of Wisconsin, Milwaukee -- T. Miller.
Argentina
Cystic Fibrosis Center, La Plata -- A. Fernandez and F. Renteria.
Australia
Prince Charles Hospital, Chermside, QLD -- S.C. Bell; Herston Royal Children’s Hospital, Brisbane, QLD -- C. Wainwright and P. Lewindon; The Children's Hospital - Westmead, Sydney, NSW -- H. Selvadurai and K. Gaskin.
Belgium
Universitair Ziekenhuis Gent, Gent -- S. Van Biervliet.
Canada
Alberta Children’s Hospital, Calgary, Alberta -- M. Montgomery; Adult Cystic Fibrosis Clinic, University of Calgary Medical Clinic of the Foothills Medical Center, Calgary, Alberta -- H.R. Rabin and J. Leong; University of Alberta Hospitals - Paediatric, Edmonton, Alberta -- P. Zuberbuhler; University of Alberta Hospitals - Adult Clinic, Edmonton, Alberta -- N.E. Brown, Adult & Pediatric Clinics -- J. Tabak; BC Children’s Hospital, Vancouver, British Columbia -- A.G.F. Davidson; St. Paul’s Adult CF Clinic, Vancouver, British Columbia -- E.M. Nakielna; Victoria General Hospital, Victoria, British Columbia -- B. Habbick; Royal Jubilee Hospital - Adult, Victoria, British Columbia -- I. Waters and S. Wiltse; Health Sciences Centre, Winnipeg, Manitoba -- W. Kepron; Children’s Hospital of Winnipeg - Paediatric, Winnipeg, Manitoba -- H. Pasterkamp; Saint John Regional Hospital, Clinic 2, St. John, New Brunswick -- D.N. Garey and G. Bishop; Janeway Child Health and Rehabilitation Centre, St. John’s and Health Science Centre, St. John’s, Newfoundland -- M. Noseworthy; Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia -- R.T. Michael, A.M. Dale and F.A. Gosse; IWK Health Centre, Halifax, Nova Scotia -- W. Robinson; Hospital for Sick Children, Toronto -- P.R. Durie and M. Corey; Hamilton Health Science Corp. - Adults, Hamilton, Ontario -- A. Freitag; Hamilton Health Science Corp. - Paediatric, Hamilton, Ontario -- L. Pedder; Hôtel Dieu Hospital - Paediatric, Kingston, Ontario -- R. Van Wylick; Hotel Dieu Hospital - Adult, Kingston, Ontario -- M.D. Lougheed and L. Kodiattu; Kitchener-Waterloo Health Center of Grand River Hospital - Adult, Kitchener, Ontario -- M. Jackson; Kitchener-Waterloo Health Center of Grand River Hospital - Paediatric, Kitchener, Ontario -- K. Malhotra; London Health Sciences - Children's Hospital of Western Ontario, London, Ontario -- B. Lyttle; London Health Sciences - Adult, London, Ontario -- N.A.M. Paterson; Ottawa General Hospital, Ottawa, Ontario -- S. Aaron; Children’s Hospital of Eastern Ontario, Ottawa, Ontario -- M. Boland, T. Kovesi and A. Smith; Sudbury Regional Hospital, Laurentian Site, Sudbury, Ontario -- V.J. Kumar and S. Zinger; St. Michael’s Hospital, Toronto, Ontario -- E. Tullis; Complexe Hospitalier de la Sagamie Hôpital de Chicoutimi, Chicoutimi, Quebec -- F. Simard; Sherbrooke - Centre Universitaire de Sante de L’Estrie, Fleurimont, Quebec -- L. Rivard and A. Cantin; Centre Hospitalier Regional de L’Outaouais, Hull, Quebec -- G. Cote; Montreal Children’s Hospital of McGill University, Montreal, Quebec -- L.C. Lands; Hôpital Sainte-Justine, Montreal, Quebec -- J.E. Marcotte; Montreal Chest Institute, McGill University Health Centre, Montreal, Quebec -- E. Matouk; Centre Hospitalier de l'Université de Montréal-Hôtel-Dieu, Montreal, Quebec -- Y. Berthiaume, A. Jeanneret and M. Van Spall; Centre Hospitalier de l'Universite Laval, Québec, Quebec -- G. Rivard; Centre Hospitalier Regional de Rimouski, Rimouski, Quebec -- J. Boucher; Centre Hospitalier Rouyn-Noranda, Rouyn-Noranda, Quebec -- N. Petit; Regina General Hospital, Regina, Saskatchewan -- B. Holmes; Royal University Hospital, Saskatoon, Saskatchewan -- D. Cotton and K. Ramlall.
Chile
Facultad de Medicina, Clinica Alemana-Universidad del Desarrollo, Santiago -- G. Repetto.
Czech Republic
CF Center, Charles University, Prague -- M. Macek, Jr., V. Vavrova, J. Bartosova and L. Fila.
France
Centre Hospitalier Universitaire de Bicêtre, Le Kremlin-Bicêtre Cedex -- D. Debray; Hopital Necker-Enfants Malades, Paris -- F. Lacaille; CF Center, University Hospital Robert Debré, Paris -- A. Munck.
Germany
Medizinische Hochschule Hannover, Hannover -- B. Tümmler.
Ireland
Our Lady’s Children’s Hospital Crumlin, Dublin -- M. Rowland and B. Bourke; Our Lady’s Children’s Hospital, Crumlin, Dublin -- G. Canny; St. Vincent’s University Hospital, Elm Park, Dublin -- C. Gallagher.
Israel
Carmel Hospital, Haifa -- J. Rivlin; Shaare Zedek Medical Center, Jerusalem -- E. Picard; Schneider Children's Hospital, Petach Tikvah -- H. Blau; Hadassah University Organization, Jerusalem -- M. Wilschanski, C. Springer and E. Kerem; Safra Children's Hospital, Tel Hashomer -- Y. Yahav and Y. Bujanover.
Italy
Cystic Fibrosis Center, Pediatric Clinics University of Genova, G. Gaslini Children Hospital, Genoa -- R. Casciaro; Pediatrics, University of Milan, Milan -- C. Colombo; University of Naples Federico II, Naples -- G. Castaldo, F. Salvatore and V. Raia; Centro Fibrosi Cistica, Verona -- M. Cipolli and C. Castellani.
Netherlands
Erasmus University Rotterdam, Rotterdam -- M. Sinaasappel and D. Dooijes.
Scotland
Royal Hospital for Sick Children, Glasgow -- S.C. Ling.
Slovakia
Institute of Molecular Physiology & Genetics, Bratislava -- H. Kayserova.
Turkey
Pediatric Pulmonology, Hacettepe University, Ankara -- U. Ozcelik, N. Kiper and D. Dogru.
United Kingdom
University of Sheffield, Sheffield -- C.J. Taylor and J. McGaw.
Footnotes
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, Wright, Lange, Durie, Knowles.
Acquisition of data: Bartlett, Friedman, 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, Silverman, Drumm, Durie, Knowles.
Analysis and interpretation of data: Bartlett, Friedman, Ling, Pace, Corey, Zielenski, Wang, Zou, Silverman, Wright, Lange, Durie, Knowles.
Drafting of the manuscript: Bartlett, Friedman, Wright, Lange, Durie, Knowles.
Critical revision of the manuscript for important intellectual content: Bartlett, Friedman, Ling, Pace, Bell, Bourke, Castaldo, Cipolli, C. Colombo, Debray, Lacaille, Macek, Rowland, Salvatore, Wainwright, Wilschanski, Corey, Zielenski, Dorfman, Drumm, Wright, Lange, Durie, Knowles.
Statistical Analysis: Wang, Zou, Wright, Lange.
Obtained funding: Bartlett, Friedman, Bell, Bourke, Castaldo, Castellani, Cipolli, C. Colombo, Macek, Rowland, Salvatore, Taylor, Wainwright, Corey, Zielenski, Dorfman, Zou, Silverman, Drumm, Wright, Lange, Durie, Knowles.
Administrative, technical, or material support: Bartlett, Friedman, Pace, Hannah, Phillips, Silverman.
Study supervision: Bartlett, Friedman, Ling, Pace, Hannah, Durie, Knowles.
Financial Disclosures: No financial conflicts were disclosed.
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; and Sarah Norris*, BS), the Hospital for Sick Children in Toronto (Mary Christofi*, BSc; Jennifer Breaton*, RN, BN, MHSc (C); and Nicole Anderson*, HonsBSc, CCRP), Case Western Reserve University (Colette Bucur*, CNP), and Hadassah University Hospital (Netta Malka*, RN, BSN and Limor Cohen*, RN, BSN); to the University of North Carolina Center for Bioinformatics (Airong Xu*, MD, MSIS; David Fargo*, PhD; and Hemant Kelkar, PhD, director) and Department of Pathology and Laboratory Medicine (Zhaoqing Zhou*, PhD, FACMG) for genotyping support; and Wanda O’Neal, PhD, director of the 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. In conclusion, we express our gratitude to all the patients and their families for making this study possible.
*(Received salary compensation for their contributions)
Additional Information: eTables 1 through 5, eFigures 1 through 3, and supplemental information are available at http://www.jama.com.
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