Abstract
BACKGROUND
The molecular mechanisms underlying Fontan-associated liver disease (FALD) remain largely unknown.
OBJECTIVES
This study aimed to assess intrahepatic transcriptomic differences among patients with FALD according to the degree of liver fibrosis and clinical outcomes.
METHODS
This retrospective cohort study included adults with the Fontan circulation. Baseline clinical, laboratory, imaging, and hemodynamic data as well as a composite clinical outcome (CCO) were extracted from medical records. Patients were classified into early or advanced fibrosis. RNA was isolated from formalin-fixed paraffin-embedded liver biopsy samples; RNA libraries were constructed with the use of an rRNA depletion method and sequenced on an Illumina Novaseq 6000. Differential gene expression and gene ontology analyses were performed with the use of DESeq2 and Metascape.
RESULTS
A total of 106 patients (48% male, median age 31 years [IQR: 11.3 years]) were included. Those with advanced fibrosis had higher B-type natriuretic peptide levels and Fontan, mean pulmonary artery, and capillary wedge pressures. The CCO was present in 23 patients (22%) and was not predicted by advanced liver fibrosis, right ventricular morphology, presence of aortopulmonary collaterals, or Fontan pressures on multivariable analysis. Samples with advanced fibrosis had 228 upregulated genes compared with early fibrosis. Samples with the CCO had 894 upregulated genes compared with those without the CCO. A total of 136 upregulated genes were identified in both comparisons and were enriched in cellular response to cytokine stimulus or oxidative stress, VEGFA-VEGFR2 signaling pathway, TGF-β signaling pathway, and vasculature development.
CONCLUSIONS
Patients with FALD and advanced fibrosis or the CCO exhibited upregulated genes related to inflammation, congestion, and angiogenesis.
Keywords: Fontan-associated liver disease, Fontan outcomes, liver fibrosis, single ventricle, transcriptomics
Patients with a functional single ventricle represent a population with highly complex anatomy, physiology, and management challenges. Since its first description in 1971, the Fontan operation has been performed for palliation of single ventricle physiology.1 Several evolutionary modifications have been made to improve long-term outcomes, but short- and long-term complications, including Fontan-associated liver disease (FALD),2 continue to occur after the Fontan palliation. Preliminary studies have demonstrated that hepatic fibrosis develops early after the Fontan palliation3 and will be present in virtually all patients,2–4 depending mostly on duration of Fontan physiology rather than hemodynamics.4,5 Decompensated cirrhosis will be apparent in only some patients, and a minority will develop hepatocellular carcinoma.5 The pathophysiology of FALD includes chronic passive congestion due to elevated systemic venous pressures,6,7 as well as chronic low cardiac output. This physiology results in decreased oxygen delivery to centrilobular cells, zone 3 hepatocyte atrophy, sinusoidal fibrosis, eventual bridging fibrosis, and finally cardiac cirrhosis.8,9
With >70,000 post-Fontan patients worldwide now reaching adulthood,2 there is a need to better characterize the molecular pathways underlying FALD. We assessed intrahepatic gene expression profiles in adults with the Fontan circulation compared with donor control subjects to test the hypotheses that: 1) despite clinical heterogeneity, patients with the Fontan circulation and advanced fibrosis exhibit a distinctive gene transcriptome that contrasts with that of early fibrosis and controls; 2) these differences involve enriched pathways related to angiogenesis; and 3) molecular phenotyping can identify Fontan subgroups that exhibit distinct clinical features and prognosis.
METHODS
FONTAN STUDY POPULATION.
The Fontan patient group consisted of adults (≥18 years old) who had at least 2 visits at the Ahmanson/UCLA Adult Congenital Heart Disease Center from January 2005 to December 2021 and had tissue available from at least 1 liver biopsy. The study was approved by the UCLA Institutional Review Board. Baseline clinical, laboratory, imaging, and hemodynamic information was extracted from medical records, and the results closest to the date of liver biopsy within a 1-year period (ie, within 6 months before or after the liver biopsy) were recorded. These variables included age, sex, height, weight, body mass index (BMI), race/ethnicity, cardiac anatomy, genetic syndromes, age at Fontan palliation, cardiac medications (beta-blockers, antiplatelets, anticoagulants, loop diuretics, renin-angiotensin-aldosterone system inhibitors, phosphodiesterase-5 inhibitors, endothelin receptor antagonists, antiarrhythmics), diabetes, hypertension, obesity (BMI >30 kg/m2), hepatitis C, alcohol use, arrhythmias, NYHA functional class, single ventricle systolic function and atrioventricular valve regurgitation (according to echocardiography or cardiac magnetic resonance imaging [MRI]), creatinine, platelet count, hemoglobin, aspartate transaminase, alanine transaminase, gamma-glutamyltransferase), international normalized ratio, alpha-fetoprotein, total bilirubin, B-type natriuretic peptide (BNP), liver ultrasound, MRI and computed tomographic characteristics, exercise-induced desaturation, peak VO2, VE/VCO2, high exercise capacity (peak VO2 >80% of the predicted value for the general nonaffected population of equivalent age, sex, and body size),10 low exercise capacity (peak VO2 <50% of the predicted value for the general nonaffected population of equivalent age, sex, and body size),10 Fontan pressures, pulmonary artery pressures, pulmonary capillary wedge pressures, single ventricle end-diastolic pressure, Qs, pulmonary vascular resistance (PVR), PVR index (PVRi), systemic vascular resistance (SVR), SVR index (SVRi), Fontan pathway obstruction (angiographic evidence of stenosis along the Fontan pathway with at least 1 mm Hg gradient),10 diastolic dysfunction (single ventricle end-diastolic pressure or pulmonary capillary wedge pressure ≥12 mm Hg at baseline or ≥15 mm Hg after volume or contrast load),10 elevated PVR (PVRi ≥2 Wu·m2),10 and aortopulmonary and venous-venous collaterals (assessed by means of invasive angiography).
LIVER TISSUE PROCUREMENT AND PROCESSING.
The Ahmanson/UCLA Adult Congenital Heart Disease Center routinely performs cardiac catheterization along with invasive angiography and liver biopsy every 10 years as part of the clinical protocol applied to patients with the Fontan circulation. In addition, liver biopsy is obtained as part of the clinical multidisciplinary evaluation during heart or combined heart and liver transplant evaluation. Archived formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with the Fontan circulation who have had liver biopsies was processed by the Translational Pathology Core Laboratory. Slides were independently interpreted by 2 liver pathologists, and classification into early (less than bridging fibrosis) or advanced (bridging fibrosis or cirrhosis) fibrosis was determined by consensus. Normal liver tissue and post—liver transplantation biopsy tissue were used as control samples.
RNA PREPARATION AND SEQUENCING.
Five to six 10-μmol/L sections from each FFPE tissue block were used for RNA extraction. Total RNA was isolated from the liver sections with the use of the RNeasy FFPE kit (Qiagen cat. no. 73504). RNA samples with DV200 ≥30% were processed for library construction with the use of rRNA depletion methods (Roche KAPA RNA HyperPrep Kit w/Ribo Erase, KK8561). Sequencing was performed with the use of the Illumina Novaseq 6000 platform (2 × 100 bp) to the depth of 28 to 110 million reads per library at the UCLA Technology Center for Genomics and Bioinformatics. The FASTQ raw reads were mapped with the use of Spliced Transcripts Alignment to a Reference (STAR; version 2.7.10a)11 to the human reference genome GRCh38 with default parameters. The counts for each gene were generated with the use of the –quantMode GeneCounts function along the STAR alignment.
DIFFERENTIAL GENE EXPRESSION ANALYSES.
Transcripts were first quality filtered to exclude the genes with a mean read count <5 in each sample. Transcripts without gene annotation also were excluded with the use of the R package biomaRt. Data normalization and differential expression analyses were carried out with the use of the R package DESeq2 version 1.38.1.11 After estimation of size factors and dispersion, the likelihood ratio test with negative binomial generalized linear model was used for fitting the data across different groups (control and early and advanced Fontan fibrosis, reporting differentially expressed genes [DEGs] taking into account control transcriptome expression). Sequencing depth normalized gene counts were obtained from DESeq-DataSet. DEGs were defined by the 5% false discovery rate (Benjamini-Hochberg method) threshold and 2-fold change for significance. To minimize possible false discoveries, DESeq function was performed again following permutations on the variables of fibrosis or composite clinical outcome. The genes identified in the differential test after permutations were filtered out.
GENE ONTOLOGY AND GENE-DISEASE ASSOCIATION ANALYSES.
Gene ontology and gene-disease association (DisGeNET) enrichment analyses were performed with the use of Metascape platform.12 The DisGeNET platform integrates information of human gene-disease associations from various repositories.13,14
STATISTICAL ANALYSIS OF CLINICAL VARIABLES.
Continuous and categoric data were reported as median (IQR) and n (%), respectively. Between-group differences in the baseline variables described above were examined with the use of Kruskal-Wallis or Pearson chi-square (or Fisher exact) tests as appropriate. For continuous covariates, we performed Dwass, Steel, Critchlow-Fligner post hoc multiple comparison analysis to test for 2-way group differences. A composite clinical outcome reflecting end-organ dysfunction outside the heart since the date of the Fontan was developed and included decompensated cirrhosis (ascites requiring paracentesis, esophageal variceal bleeding, or hepatic encephalopathy), hepatocellular carcinoma, need for liver transplantation, protein-losing enteropathy, chronic kidney disease stage 4 or higher, or death. A Kaplan-Meier curve stratified by early vs advanced fibrosis and the log-rank test were used to compare time to event relative to the date of the Fontan between the 2 groups (the group without fibrosis was excluded from the analysis). A multivariable Cox proportional hazards model was built using covariates that were considered clinically meaningful. The proportional hazards assumption was tested for this model and it was fulfilled. These analyses were performed with the use of SAS 9.4 (SAS Institute).
DATA AVAILABILITY.
RNA sequencing raw data and normalized gene counts can be requested from the corresponding author.
RESULTS
BASELINE CHARACTERISTICS ACCORDING TO DEGREE OF FIBROSIS.
Of 130 adults with the Fontan circulation who had 152 liver biopsies (20 patients had 2 liver biopsies and 2 had 3 liver biopsies), 106 patients with 112 adequate RNA quality samples were included. Fourteen samples were used as control (5 from normal livers and 9 from post-transplantation biopsies). Fifty-five (51.8%) were women, 71 (66.9%) were White, and 29 (27.4%) were Hispanic. Fifteen patients (14.2%) had no fibrosis, 50 (47.1%) had early fibrosis, and 41 (38.7%) had advanced fibrosis (Supplemental Figure 1). Baseline characteristics are detailed in Table 1. Although aortopulmonary collaterals were identified in all groups, those without fibrosis were more likely to have aortopulmonary collaterals (46.7% without fibrosis, 10.0% with early fibrosis, 26.8% with advanced fibrosis; P = 0.0332). Higher SVRi was seen in the group without fibrosis (25.7 WU·m2 [IQR: 9.2 Wu·m2] without fibrosis, 19.5 WU·m2 [IQR: 11.3 WU·m2] with early fibrosis, 18.1 WU·m2 [IQR: 9.4 WU·m2] with advanced fibrosis; P = 0.014). Those with advanced fibrosis were more likely to be on phosphodiesterase-5 inhibitors (6.7% without fibrosis, 38.0% with early fibrosis, 43.9% with advanced fibrosis; P = 0.033), have diastolic dysfunction (40.0% without fibrosis, 48.0% with early fibrosis, 70.7% with advanced fibrosis; P = 0.017) and higher BNP (44 pg/mL [IQR: 46 pg/mL] without fibrosis, 47 pg/mL [IQR: 91 pg/mL] with early fibrosis, 95 pg/mL [IQR: 130 pg/mL] with advanced fibrosis; P = 0.016). Similarly, they also had a higher VE/VCO2 slope (27.1 [IQR: 6] without fibrosis; 27.7 [IQR: 5.8] with early fibrosis; 30.9 [IQR: 6.4] with advanced fibrosis; P = 0.009) and Fontan (14.0 mm Hg [IQR: 5.0 mm Hg] without fibrosis; 14.5 mm Hg [IQR: 3.0 mm Hg] with early fibrosis; 17.0 mm Hg [IQR: 6.0 mm Hg] with advanced fibrosis; P < 0.001), mean pulmonary artery (13.5 mm Hg [IQR: 5.0 mm Hg] without fibrosis; 14.0 mm Hg [IQR: 3.0 mm Hg] with early fibrosis; 17.0 mm Hg [IQR: 5.0 mm Hg] with advanced fibrosis; P < 0.001), and pulmonary capillary wedge (10.0 mm Hg [IQR: 4.0 mm Hg] without fibrosis; 10.0 mm Hg [IQR: 4.0 mm Hg] with early fibrosis; 12.0 mm Hg [IQR: 5.0 mm Hg] with advanced fibrosis; P = 0.005) pressures. Post hoc analysis comparing those without fibrosis vs those with advanced fibrosis showed significant differences in BNP (P = 0.017), Fontan (P < 0.001), mean pulmonary artery (P = 0.001), and pulmonary capillary wedge (P = 0.019) pressures, as well as SVR (0.029) and SVRi (P = 0.005). When comparing those with early fibrosis vs advanced fibrosis, significant differences were found in Fontan (P < 0.001), mean pulmonary artery (P < 0.001) and pulmonary capillary wedge (P = 0.016) pressures.
TABLE 1.
Baseline Characteristics of Adults With the Fontan Circulation According to the Degree of Liver Fibrosis
| No Fibrosis (n = 15) | Early Fibrosis (n = 50) | Advanced Fibrosis (n = 41) | P Valuea | |
|---|---|---|---|---|
|
| ||||
| Left systemic ventricle morphology | 8 (53.3) | 35 (70.0) | 20 (48.8) | 0.078 |
| Age at Fontan, y | 4.0 [6.0] | 5.0 [7.0] | 4.0 [5.0] | 0.722 |
| Male | 10 (66.7) | 24 (48.0) | 17 (41.5) | 0.247 |
| Age at liver biopsy, y | 27.8 [6.4] | 32.9 [10.8] | 29.3 [11.4] | 0.129 |
| Liver biopsy route | 0.467 | |||
| Transjugular | 14 (93.3) | 41 (82.0) | 28 (68.3) | |
| Percutaneous | 1 (6.7) | 9 (18.0) | 10 (24.4) | |
| Liver explant | 0 (0.0) | 0 (0.0) | 2 (5.0) | |
| Type of Fontan | 0.363 | |||
| Atriopulmonary | 3 (20.0) | 19 (38.0) | 12 (29.3) | |
| Bjork | 0 (0.0) | 1 (2.0) | 1 (2.4) | |
| Extracardiac | 4 (26.7) | 5 (10.0) | 11 (26.8) | |
| Lateral tunnel (including intra-atrial) | 8 (53.3) | 23 (46.0) | 14 (34.2) | |
| Other | 0 (0.0) | 2 (4.0) | 3 (7.3) | |
| Fenestration | 5/13 (38.5) | 12/43 (27.9) | 16/37 (43.2) | 0.350 |
| Fenestration closure | 3/5 (60.0) | 7/11 (63.6) | 12/14 (85.7) | 0.353 |
| Fontan conversion | 2 (13.3) | 10 (20.0) | 15 (36.6) | 0.099 |
| Heterotaxy | 1 (6.7) | 5 (10.0) | 7 (17.1) | 0.459 |
| Diabetes | 0 (0.0) | 2 (4.0) | 4 (9.8) | 0.428 |
| Hypertension | 1 (6.7) | 3 (6.0) | 5 (12.2) | 0.712 |
| Hepatitis C | 0 (0.0) | 2 (4.0) | 3 (7.3) | 0.693 |
| Obesity | 1 (6.7) | 8 (16.0) | 6 (14.6) | 0.752 |
| Body mass index, kg/m2 | 22.7 [5.7] | 23.8 [7.6] | 24.2 [7.3] | 0.957 |
| Hemidiaphragm paresis | 0 (0.0) | 1 (2.0) | 2 (4.9) | 0.735 |
| Smoking | 1 (6.7) | 3 (6.0) | 3 (7.3) | 1.000 |
| Alcohol | 8 (53.3) | 18 (36.0) | 13 (31.7) | 0.327 |
| Drugsb | 4 (26.7) | 8 (16.0) | 7 (17.1) | 0.630 |
| Previous thromboembolism | 1 (6.7) | 14 (28.0) | 10 (24.4) | 0.218 |
| Protein-losing enteropathy | 0 (0.0) | 4 (8.0) | 5 (12.2) | 0.402 |
| Arrhythmiac | 6 (40.0) | 34 (68.0) | 25 (61.0) | 0.148 |
| Pacemaker | 4 (26.7) | 19 (38.0) | 20 (48.8) | 0.286 |
| Defibrillator | 1 (6.7) | 1 (2.0) | 3 (7.3) | 0.352 |
| NYHA functional class | 0.064 | |||
| I | 12 (80.0) | 26 (52.0) | 15 (36.6) | |
| II | 3 (20.0) | 17 (34.0) | 21 (51.2) | |
| III | 0 (0.0) | 5 (10.0) | 3 (7.3) | |
| IV | 0 (0.0) | 0 (0.0) | 2 (4.9) | |
| Beta-blockers | 8 (53.3) | 24 (48.0) | 18 (43.9) | 0.811 |
| PDE-5 inhibitors | 1 (6.7) | 19 (38.0) | 18 (43.9) | 0.033e |
| ERAs | 1 (6.7) | 2 (4.0) | 2 (4.9) | 0.841 |
| Antiplatelets | 9 (60.0) | 25 (50.0) | 23 (56.1) | 0.737 |
| Anticoagulants | 4 (26.7) | 27 (54.0) | 19 (46.3) | 0.176 |
| Loop diuretics | 5 (33.3) | 28 (56.0) | 25 (61.0) | 0.178 |
| ACE inhibitors | 9 (60.0) | 23 (46.0) | 19 (46.3) | 0.610 |
| ARBs | 1 (6.7) | 6 (12.0) | 2 (4.9) | 0.516 |
| Aldosterone antagonists | 3 (20.0) | 17 (34.0) | 21 (51.2) | 0.068 |
| Digoxin | 3 (20.0) | 9 (18.0) | 10 (24.4) | 0.754 |
| Other antiarrhythmics | 2 (13.3) | 16 (32.0) | 8 (19.5) | 0.214 |
| Creatinine, mg/dL | 0.8 [0.2] | 0.8 [0.2] | 0.9 [0.4] | 0.223 |
| Platelets, × 109/L | 170.0 [53.0] | 158.0 [82.0] | 134.5 [60.0] | 0.106 |
| Hemoglobin, g/dL | 15.5 [4.0] | 15.1 [2.9] | 15.1 [2.7] | 0.692 |
| Albumin, g/dL | 4.7 [0.6] | 4.7 [0.7] | 4.4 [1.0] | 0.057 |
| INRd | 1.2 [0.0] | 1.2 [0.3] | 1.3 [0.4] | 0.230 |
| AFP, ng/mL | 3.0 [8.0] | 2.0 [1.0] | 3.0 [2.0] | 0.489 |
| BNP, pg/mL | 44.0 [46.0] | 47.0 [91.0] | 95.0 [130.0] | 0.016e |
| Systemic ventricle function | 0.181 | |||
| Normal or mildly reduced | 12 (80.0) | 45 (90.0) | 31 (75.6) | |
| Moderately or severely reduced | 3 (20.0) | 5 (10.0) | 10 (24.4) | |
| AVV regurgitation | 0.161 | |||
| None, trace or mild | 13 (86.7) | 36 (72.0) | 25 (61.0) | |
| Moderate or severe | 2 (13.3) | 14 (28.0) | 16 (39.0) | |
| Liver ultrasound characteristics | 14 (15.9) | 38 (43.2) | 36 (40.9) | |
| Nodular contour | 5 (35.7) | 24 (63.2.0) | 24 (66.7) | 0.118 |
| Splenomegaly | 4 (28.6) | 11 (29.0) | 13 (36.1) | 0.772 |
| Abdominal collateral vessels | 1 (7.1) | 0 (0.0) | 1 (2.8) | 0.155 |
| Ascites | 0 (0.0) | 7 (18.4) | 8 (22.2) | 0.165 |
| Elevated liver stiffness | 4 (28.6) | 14 (36.8) | 14 (38.9) | 0.790 |
| Liver mass | 0 (0.0) | 3 (7.9) | 1 (2.8) | 0.514 |
| Liver MRI characteristics | 5 (20.0) | 14 (56.0) | 6 (24.0) | |
| Nodular contour | 5 (100.0) | 9 (64.3) | 4 (66.7) | 0.405 |
| Splenomegaly | 1 (20.0) | 4 (28.6) | 3 (50.0) | 0.611 |
| Ascites | 0 (0.0) | 2 (14.3) | 2 (33.3) | 0.468 |
| Abdominal collateral vessels | 0 (0.0) | 1 (7.1) | 1 (16.7) | 0.697 |
| Elevated stiffness | 0 (0.0) | 0 (0.0) | 2 (33.3) | 0.083 |
| Liver mass | 0 (0.0) | 0 (0.0) | 2 (33.3) | 0.083 |
| Liver CT characteristics | 6(11.1) | 26 (48.1) | 22 (40.8) | |
| Nodular contour | 4 (66.7) | 21 (80.8) | 20 (90.9) | 0.275 |
| Splenomegaly | 4 (66.7) | 8 (30.8) | 15 (68.2) | 0.032 |
| Abdominal collateral vessels | 2 (33.3) | 6 (23.1) | 10 (45.5) | 0.270 |
| Ascites | 3 (50.0) | 11 (42.3) | 11 (50.0) | 0.926 |
| Liver mass | 0 (0.0) | 3 (11.5) | 2 (9.1) | 1.000 |
| Cardiopulmonary stress testing characteristics | 13 (15.1) | 40 (46.5) | 33 (38.4) | |
| Baseline O2Sat, % | 93.0 [3.0] | 92.5 [6.0] | 92.0 [6.0] | 0.655 |
| Peak O2Sat, % | 92.0 [5.0] | 89.0 [8.0] | 90.0 [7.0] | 0.616 |
| Exercise-induced desaturation (peak O2 – baseline O2), % | -2.0 [2.0] | -3.0 [5.0] | -2.0 [3.0] | 0.576 |
| Peak VO2, % of predicted | 61.0 [17.0] | 59.0 [22.0] | 55.5 [26.0] | 0.645 |
| VE/VCO2 slope | 27.1 [6.0] | 27.7 [5.8] | 30.9 [6.4] | 0.009e |
| High exercise capacity | 2 (13.3) | 6 (12.0) | 7 (17.1) | n/a |
| Low exercise capacity | 2 (13.3) | 9 (18.0) | 13 (31.7) | n/a |
| Cardiac catheterization characteristics | 15 (14.9) | 50 (49.5) | 36 (35.6) | |
| Fontan pressure, mm Hg | 14.0 [5.0] | 14.5 [3.0] | 17.0 [6.0] | <0.001e |
| Mean PAP, mm Hg | 13.5 [5.0] | 14.0 [3.0] | 17.0 [5.0] | <0.001e |
| Pulmonary capillary wedge pressure, mm Hg | 10.0 [4.0] | 10.0 [4.0] | 12.0 [5.0] | 0.005e |
| Single ventricle end-diastolic pressure, mm Hg | 10.0 [5.0] | 10.0 [5.5] | 10.0 [3.0] | 0.609 |
| Qs, L/min | 4.3 [1.4] | 4.5 [1.9] | 5.0 [2.6] | 0.273 |
| Qs index, L/min/m2 | 2.5 [0.7] | 2.7 [1.1] | 2.7 [1.6] | 0.153 |
| PVR, WU | 0.9 [0.4] | 0.9 [0.7] | 1.1 [0.8] | 0.485 |
| PVRi, WU·m2 | 1.7 [0.8] | 1.6 [1.3] | 2.0 [1.5] | 0.674 |
| SVR, WU | 13.0 [4.3] | 11.3 [7.3] | 10.2 [5.6] | 0.067 |
| SVRi, WU·m2 | 25.7 [9.2] | 19.5 [11.3] | 18.1 [9.4] | 0.014e |
| Fontan pathway obstruction | 0 (0.0) | 7 (14.0) | 8 (19.5) | 0.115 |
| Diastolic dysfunction | 6 (40.0) | 24 (48.0) | 29 (70.7) | 0.017e |
| Elevated PVR | 4 (26.7) | 18 (36.0) | 17 (41.5) | 0.290 |
| Aortopulmonary collaterals | 7 (46.7) | 5 (10.0) | 11 (26.8) | 0.006e |
| Venous-venous collaterals | 8 (53.3) | 26 (52.0) | 18 (43.9) | 0.698 |
Values are n (%), n/N (%), or median [IQR].
P values from chi-square test for independence (or Fisher exact test when appropriate) for categoric variables and Kruskal-Wallis test for continuous variables. P values do not include missing values on any given variable. Frequencies were calculated among those with available data and not among total number in each column.
Including marijuana, cocaine, 3,4-methylenedioxy-methamphetamine, lysergic acid diethylamide, and others.
Defined as an atrial or ventricular rate of >100 beats/min lasting for >30 seconds, or <30 seconds if associated with hemodynamic instability, or of any duration if arrhythmia therapy is used. This definition excluded sinus tachycardia.
Regardless of the use of coumadin. Those using warfarin were instructed to hold this medication 3 days before cardiac catheterization.
P < 0.05.
ACE = angiotensin-converting enzyme; AFP = alpha-fetoprotein; ARB = angiotensin receptor blocker; AVV = atrioventricular valve; BNP = B-type natriuretic peptide; CT = computed tomography; ERA = endothelin receptor antagonist; INR = international normalized ratio; MRI = magnetic resonance imaging; PDE-5 = phosphodiesterase-5; PVR = pulmonary vascular resistance; Qs = systemic blood flow; SVR = systemic vascular resistance.
COMPOSITE CLINICAL OUTCOME AND ASSOCIATED FACTORS.
Outcomes among patients with the Fontan circulation according to degree of liver fibrosis during the follow-up period of 29.5 years (IQR: 9.1 years) are presented in Table 2. Those with advanced liver fibrosis on biopsy were more likely to experience the composite clinical outcome (0 [0%] without fibrosis, 8 [16.0%] with early fibrosis, and 15 [36.6%] with advanced fibrosis; P = 0.005). None of the individual components of the composite clinical outcome was significantly associated with advanced fibrosis. Survival analysis showed that those with advanced fibrosis were more likely to have the composite clinical outcome compared with those with early fibrosis (P = 0.016) (Figure 1). In a multivariable Cox proportional hazards model (Supplemental Table 1) controlling for right ventricular morphology, aortopulmonary collaterals, and Fontan pressures, there was no difference in the risk of the composite clinical outcome between advanced or early fibrosis (HR: 1.32; 95% CI: 0.44–3.93; P = 0.621). In fact, there were no differences in the risk of the composite clinical outcome based on right ventricular morphology (HR: 1.47; 95% CI: 0.59–3.69; P = 0.672), presence of aortopulmonary collaterals (HR: 2.03; 95% CI: 0.69–5.96; P = 0.198), or Fontan pressures (HR: 1.08; 95% CI: 0.94–1.25; P = 0.258).
TABLE 2.
Outcomes Among Patients With the Fontan Circulation According to Liver Biopsy Results
| No Fibrosis | Early Fibrosis | Advanced Fibrosis | P Value | |
|---|---|---|---|---|
|
| ||||
| Thromboembolism | 1 (6.7) | 1 (2.0) | 1 (2.4) | 0.527 |
| Arrhythmias | 4 (26.7) | 14 (28.0) | 7 (17.0) | 0.215 |
| Composite clinical outcomea | 0 (0.0) | 8 (16.0) | 15 (36.6) | 0.005b |
| Decompensated cirrhosis | 0 (0.0) | 4 (8.0) | 7 (17.1) | 0.138 |
| Ascites requiring paracentesis | 0 (0.0) | 3 (6.0) | 5 (12.2) | 0.402 |
| Esophageal variceal bleeding | 0 (0.0) | 1 (2.0) | 2 (4.9) | 0.740 |
| Hepatic encephalopathy | 0 (0.0) | 0 (0.0) | 0 (0.0) | n/a |
| Hepatocellular carcinoma | 0 (0.0) | 2 (4.0) | 1 (2.4) | 1.000 |
| Chronic kidney disease | 0 (0.0) | 1 (2.0) | 1 (2.4) | 0.115 |
| Fontan circulatory failure | 1 (6.7) | 11 (22.0) | 15 (36.6) | 0.056 |
| Liver transplantation | 0 (0.0) | 5 (10.0) | 8 (19.5) | 0.109 |
| Heart transplantation | 1 (6.7) | 9 (18.0) | 13 (31.7) | 0.090 |
| Death | 0 (0.0) | 3 (6.0) | 3 (7.3) | 0.858 |
Values are n (%).
Composite clinical outcome of decompensated cirrhosis (ascites requiring paracentesis, esophageal variceal bleeding, hepatic encephalopathy), hepatocellular carcinoma, need for liver transplantation, protein-losing enteropathy, chronic kidney disease stage 4 or higher, or death.
P<0.05.
FIGURE 1. Survival Analysis.
Kaplan-Meier curve comparing time to the composite clinical outcome, relative to the date of Fontan palliation, between patients with early vs advanced fibrosis.
FALD HAS DISTINCT mRNA EXPRESSION PROFILES COMPARED WITH CONTROL SAMPLES AND ACCORDING TO DEGREE OF FIBROSIS AND COMPOSITE CLINICAL OUTCOME.
Liver samples from patients with early Fontan fibrosis had 67 DEGs (30 upregulated) compared with normal liver control samples and 50 DEGs (17 upregulated) compared with post-transplantation biopsies. Liver samples from patients with advanced fibrosis had 231 DEGs (228 upregulated) compared with those with early fibrosis (Figure 2A). Liver samples from patients with the composite clinical outcome had 906 DEGs (894 upregulated) compared with those without it (Figure 2B). A total of 136 DEGs were identified in both comparisons, and they were enriched in various cellular responses and signaling pathways, including response to wounding, extracellular matrix organization, regulation of cell adhesion, interleukin-18, mitogen-activated protein kinase, and transforming growth factor (TGF)-β signaling pathways, vasculature development, and angiogenesis (Figure 3A). Significant correlations of these 136 DEGs were found with various diseases in the DisGeNET database, including idiopathic pulmonary arterial hypertension, lung diseases, cardiac fibrosis, vascular diseases, and endothelial dysfunction (Figure 3B). The distribution of specific DEGs involved in FALD pathophysiology according to the degree of fibrosis (including those associated with liver fibrosis from other etiologies) is shown in Figure 4A, those associated with tissue fibrosis in other organs are shown in Figure 4B, and association with the presence or absence of the composite clinical outcome is shown in Figure 5.
FIGURE 2. DEGs in Advanced Fibrosis and the Composite Clinical Outcome.
(A) Volcano plot of 231 differentially expressed genes (DEGs) (228 upregulated) in patients with FALD and advanced fibrosis vs early fibrosis. Highlighted genes were among the top upregulated genes in advanced fibrosis. (B) Volcano plot of 906 DEGs (894 upregulated) in patients with Fontan-associated liver disease (FALD) and the composite clinical outcome vs those without it. Highlighted genes were among the top upregulated genes in FALD patients with the composite clinical outcomes. The likelihood ratio test with negative binomial generalized linear model was used for fitting and DEGs analysis with the use of R package DESeq2. DEG cutoffs: Log2 fold change (FC) ≥1 and adjusted P value (Padj) <0.05 with the use of the Benjamini-Hochberg method. NS = not significant; Sig = significant.
FIGURE 3. Upregulated Gene Enrichment Analyses in Advanced Fibrosis and the Composite Clinical Outcome.
(A) Representative terms of gene ontology and pathway enrichment analysis using 136 upregulated genes both in advanced liver fibrosis and in patients with the composite clinical outcomes. (B) Top associated human diseases of gene disease association analysis (DisGeNet) using these 136 upregulated genes.
FIGURE 4. Selected DEGs in FALD According to the Degree of Fibrosis.
(A) Genes associated with liver fibrosis from etiologies other than FALD. (B) Representative top upregulated genes associated with tissue fibrosis in other organs. Expression of each gene was plotted with the use of violin in combination with box plots among patients with FALD according to the degree of liver fibrosis. Expression levels were plotted using normalized gene counts in log10 scale. Adjusted P value calculated with the use of the Benjamini-Hochberg method for multiple comparison testing. ns = not significant; other abbreviations as in Figure 2.
FIGURE 5. Selected DEGs in FALD According to the Composite Clinical Outcomes.
(A) Genes associated with liver fibrosis from etiologies other than FALD. (B) Representative top upregulated genes associated with tissue fibrosis in other organs. Combined violin and box plots of gene expression among FALD patients according to the composite clinical outcome. The likelihood ratio test with negative binomial generalized linear model was used for DEG analysis. Expression levels were plotted using normalized gene counts in log10 scale. Adjusted P value calculated with the use of the Benjamini-Hochberg method for multiple comparison testing. Abbreviations as in Figure 2.
DISCUSSION
In this first broad analysis of gene expression in liver tissue from a retrospectively identified cohort of patients with FALD, we reveal several important findings. First, patients with FALD and early fibrosis have a distinct transcriptome compared with control samples. Second, patients with FALD and advanced fibrosis have a distinct transcriptome vs those with early fibrosis. Third, patients with FALD and advanced fibrosis are more likely to experience the composite clinical outcome, but this association did not reach significance in a multivariable model. Fourth, patients with FALD and the composite clinical outcome have a distinct transcriptome compared with those without the composite clinical outcome. And fifth, we identified overlapping DEGs between those with advanced fibrosis as well as the composite clinical outcome, and identified pathways related to proinflammatory responses and increased oxidative stress (HSPB1, IRF1, SOCS3, and NFKB2), impaired vascular endothelial function (HSPA1A, HSPA1B, ND5, ND6, NR4A2, SOD3, and KLF2), enriched angiogenesis and vasculature development (RHOB, COL1A1, NR4A1, NOTCH3, and SERPINE1), TGF-β signaling pathways, etc (Central Illustration). These findings are important given that an inflammatory infiltrate on liver biopsies was not found, and we excluded samples with active hepatocellular carcinoma. These results expand our insights into FALD pathophysiology and show potential candidate genes that could serve as biomarkers of adverse outcomes.
CENTRAL ILLUSTRATION. Intrahepatic Transcriptomics, Advanced Fibrosis, and Clinical Outcomes in Fontan-Associated Liver Disease.
In this study we identified differentially expressed genes (DEGs) related to inflammation, congestion, and angiogenesis among those with advanced vs early liver fibrosis as well as those who experienced the composite clinical outcome (CCO) vs those who did not.
Out of all the overlapping DEGs identified in our study, COL1A1, RASD1, CHI3L1, MTRNR2, MUC5B, SLCO4A1, SOD3, and WNK2 have been previously described in transcriptomic profiling of obesity-related nonalcoholic steatohepatitis.15 Our findings demonstrate that angiogenesis may be particularly important in FALD. This complex, dynamic, and growth factor–dependent process leading to the formation of new blood vessels from preexisting ones is strongly associated with scar formation and sinusoidal remodeling in chronic liver diseases.10 Several genes are involved in this process, including VEGF and ANGPT2.16 High serum angiopoietin-2 levels (protein expressed from ANGPT2) have been associated with liver cirrhosis and hepatocellular carcinoma among samples from nonalcoholic steatohepatitis,17 and several studies are evaluating this as a potential therapeutic target.18–20 In a cohort study involving patients with the Fontan circulation, angiopoietin-2 levels were found to be significantly higher in patients with active or recent arrhythmias.21 Although we did not find ANGPT2 as one of our overlapping genes, we identified the VEGFA-VEGFR2 signaling pathway, which is the major pathway that activates angiogenesis.
STUDY STRENGTHS AND LIMITATIONS.
This study has several strengths, including a highly diverse and phenotypically characterized cohort that had longitudinal care at a large adult congenital heart disease referral center with expertise in combined heart and liver transplantation, as well as achieving >80% high-quality RNA extraction from FFPE liver biopsies. Moreover, the patients had undergone cardiac catheterization in a standard fashion, limiting the heterogeneity of hemodynamic measurements.
Intrahepatic gene expression profiles are largely driven by the effect of parenchymal cells, and this represents a limitation when analyzing bulk transcriptomics. Several recent discoveries in the molecular biology of liver cirrhosis (from noncardiac causes) have highlighted the important role that nonparenchymal liver cells (immune, endothelial, and mesenchymal cells) play in its development. Gene expression profiles of nonparenchymal cells are underrepresented in the whole tissue liver RNA-seq data. Other methods, such as single-cell RNA sequencing or digital spatial profiling,22 may be helpful in identifying the gatekeepers of advanced liver fibrosis in FALD. Furthermore, the assessment of aortopulmonary collaterals was not systematically performed in all patients unless the patients presented for organ transplantation, and this might influence our univariate as well as multivariable Cox proportional hazards results. A third limitation is the small cohort size, which could have influenced the precision of our estimates. Fourth, ascites can be a consequence of elevated central venous pressure alone and may not imply inherent liver failure per se. And fifth, we did not perform comparisons between FALD and other causes of liver fibrosis to confirm etiology-specific findings.
CONCLUSIONS
Patients with FALD exhibit transcriptomic differences according to the degree of fibrosis and the presence of the composite clinical outcome. These genes are involved in pathways related to inflammation, congestion, and angiogenesis.
Supplementary Material
PERSPECTIVES.
COMPETENCY IN MEDICAL KNOWLEDGE:
Intrahepatic transcriptomics identifies differentially expressed genes related to inflammation, congestion, and angiogenesis in the pathophysiology of Fontan-associated liver fibrosis.
TRANSLATIONAL OUTLOOK:
Confirmation of gatekeeping pathways involved in Fontan-associated liver disease could improve risk stratification when selecting patients for heart vs combined heart and liver transplantation and facilitate the development of strategies for prevention of advanced hepatic fibrosis.
FUNDING SUPPORT AND AUTHOR DISCLOSURES
Dr Bravo-Jaimes was supported by the Adult Congenital Heart Association Research Grant 2021. Dr Klomhaus was supported by the National Institutes of Health (NIH)/National Center for Advancing Translational Science UCLA CTSI grant no. UL1TR001881. Dr Böstrom was supported by NIH/National Heart, Lung, and Blood Institute grant nos. HL81397 and HL154548. Dr Aboulhosn was supported by the Streisand/American Heart Association Endowed Chair in Cardiology. Dr Kaldas was supported by the Kelly Lee Tarantello Endowed Chair in Integrative Liver Transplantation. The authors have reported that they have no relationships relevant to the contents of this paper to disclose.
ABBREVIATIONS AND ACRONYMS
- BNP
B-type natriuretic peptide
- DEG
differentially expressed gene
- FALD
Fontan-associated liver disease
- FFPE
formalin-fixed paraffin-embedded
- PVR
pulmonary vascular resistance
- SVR
systemic vascular resistance
- VE/VCO2 slope
ventilatory efficiency
Footnotes
The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’ institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit the Author Center.
APPENDIX For a supplemental table and figure, please see the online version of this paper.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
RNA sequencing raw data and normalized gene counts can be requested from the corresponding author.