Single-cell multiomics guided mechanistic understanding of Fontan-associated liver disease.

The Fontan operation is the current standard of care for single-ventricle congenital heart disease. Individuals with a Fontan circulation (FC) exhibit central venous hypertension and face life-threatening complications of hepatic fibrosis, known as Fontan-associated liver disease (FALD). The fundamental biology and mechanisms of FALD are little understood. Here, we generated a transcriptomic and epigenomic atlas of human FALD at single-cell resolution using multiomic snRNA-ATAC-seq. We found profound cell type-specific transcriptomic and epigenomic changes in FC livers. Central hepatocytes (cHep) exhibited the most substantial changes, featuring profound metabolic reprogramming. These cHep changes preceded substantial activation of hepatic stellate cells and liver fibrosis, suggesting cHep as a potential first "responder" in the pathogenesis of FALD. We also identified a network of ligand-receptor pairs that transmit signals from cHep to hepatic stellate cells, which may promote their activation and liver fibrosis. We further experimentally demonstrated that activins A and B promote fibrotic activation in vitro and identified mechanisms of activin A's transcriptional activation in FALD. Together, our single-cell transcriptomic and epigenomic atlas revealed mechanistic insights into the pathogenesis of FALD and may aid identification of potential therapeutic targets.

Event-free survival of maralixibat-treated patients with Alagille syndrome compared to a real-world cohort from GALA.

BACKGROUND AND AIMS: Alagille syndrome (ALGS) is characterized by chronic cholestasis with associated pruritus and extrahepatic anomalies. Maralixibat, an ileal bile acid transporter inhibitor, is an approved pharmacologic therapy for cholestatic pruritus in ALGS. Since long-term placebo-controlled studies are not feasible or ethical in children with rare diseases, a novel approach was taken comparing 6-year outcomes from maralixibat trials with an aligned and harmonized natural history cohort from the G lobal AL agille A lliance (GALA) study. APPROACH AND RESULTS: Maralixibat trials comprise 84 patients with ALGS with up to 6 years of treatment. GALA contains retrospective data from 1438 participants. GALA was filtered to align with key maralixibat eligibility criteria, yielding 469 participants. Serum bile acids could not be included in the GALA filtering criteria as these are not routinely performed in clinical practice. Index time was determined through maximum likelihood estimation in an effort to align the disease severity between the two cohorts with the initiation of maralixibat. Event-free survival, defined as the time to first event of manifestations of portal hypertension (variceal bleeding, ascites requiring therapy), surgical biliary diversion, liver transplant, or death, was analyzed by Cox proportional hazards methods. Sensitivity analyses and adjustments for covariates were applied. Age, total bilirubin, gamma-glutamyl transferase, and alanine aminotransferase were balanced between groups with no statistical differences. Event-free survival in the maralixibat cohort was significantly better than the GALA cohort (HR, 0.305; 95% CI, 0.189-0.491; p <0.0001). Multiple sensitivity and subgroup analyses (including serum bile acid availability) showed similar findings. CONCLUSIONS: This study demonstrates a novel application of a robust statistical method to evaluate outcomes in long-term intervention studies where placebo comparisons are not feasible, providing wide application for rare diseases. This comparison with real-world natural history data suggests that maralixibat improves event-free survival in patients with ALGS.

Immunosuppression-Free Life after Pediatric Liver Transplant: A Case-Control Study from the Society of Pediatric Liver Transplant (SPLIT) Registry.

OBJECTIVE: To compare long-term outcomes of pediatric liver transplant (LT) recipients off immunosuppression (IS) with matched controls on IS using data from the Society of Pediatric Liver Transplant (SPLIT) registry. STUDY DESIGN: This was a retrospective case-control study. SPLIT participants <18 years of age, ≥4 years after isolated LT, and off IS for ≥1 year (cases) were age- and sex-matched 1:2 to patients with the same primary diagnosis and post-LT follow-up duration (controls). Primary outcomes included retransplantation, allograft rejection, IS comorbidities, and prevalence of SPLIT-derived composite ideal outcome (c-IO) achieved at the end of the follow-up period. Differences were compared using multiple linear regression for continuous outcomes and logistic regression for dichotomous data. RESULTS: The study cohort was composed of 33 cases (42.4% male, 60.6% biliary atresia, median age at LT of 0.7 [P25, P75, 0.5, 1.6] years, median IS withdrawal time of 9 [P25, P75, 6, 12] years after LT) and 66 age- and sex-matched controls. No cases required retransplantation. Cases and controls had similar growth parameters, laboratory values, calculated glomerular filtration rates, rates of post-transplant lymphoproliferative disease, graft rejection, and attainment of c-IO. CONCLUSIONS: No differences in allograft rejection rates, IS complications, or c-IO prevalence were seen between SPLIT patients off IS and age- and sex-matched controls remaining on IS. Discontinuation of IS most commonly occurred in the context of rigorously designed IS withdrawal trials. The available sample size was small, affecting generalizability to the broader pediatric LT population.

Liver Biopsy Leads to Serendipitous Diagnosis of Glycogen Storage Disease Type IX in a Patient With Fontan-Associated Liver Disease.

Fontan-associated liver disease (FALD) is a form of congestive hepatopathy resulting from Fontan palliation procedures in patients with single ventricle physiology. Although there is variation between pediatric centers, the surveillance for FALD may include liver biopsies for assessment of degree of fibrosis. Our report describes a 7-year-old girl with hypoplastic left heart syndrome who underwent Fontan palliation at age 2, and presented with disproportionate hepatomegaly, elevated liver enzymes, and increased stiffness on liver elastography. Liver biopsy showed diffuse hepatocellular cytoplasmic glycogenation, leading to the diagnosis of glycogen storage disease IX. This case demonstrates the importance of investigating unexpected physical exam findings and the potential for serendipitous benefit of liver biopsy in FALD.

Safety and Immunogenicity of Live Viral Vaccines in a Multicenter Cohort of Pediatric Transplant Recipients.

Importance: Live vaccines (measles-mumps-rubella [MMR] and varicella-zoster virus [VZV]) have not been recommended after solid organ transplant due to concern for inciting vaccine strain infection in an immunocompromised host. However, the rates of measles, mumps, and varicella are rising nationally and internationally, leaving susceptible immunocompromised children at risk for life-threating conditions. Objective: To determine the safety and immunogenicity of live vaccines in pediatric liver and kidney transplant recipients. Design, Setting, and Participants: This cohort study included select pediatric liver and kidney transplant recipients who had not completed their primary MMR and VZV vaccine series and/or who displayed nonprotective serum antibody levels at enrollment between January 1, 2002, and February 28, 2023. Eligibility for live vaccine was determined by individual US pediatric solid organ transplant center protocols. Exposures: Exposure was defined as receipt of a posttransplant live vaccine. Transplant recipients received 1 to 3 doses of MMR vaccine and/or 1 to 3 doses of VZV vaccine. Main Outcome and Measure: Safety data were collected following each vaccination, and antibody levels were obtained at 0 to 3 months and 1 year following vaccination. Comparisons were performed using Mann-Whitney U test, and factors associated with development of postvaccination protective antibodies were explored using univariate analysis. Results: The cohort included 281 children (270 [96%] liver, 9 [3%] kidney, 2 [1%] liver-kidney recipients) from 18 centers. The median time from transplant to enrollment was 6.3 years (IQR, 3.4-11.1 years). The median age at first posttransplant vaccine was 8.9 years (IQR, 4.7-13.8 years). A total of 202 of 275 (73%) children were receiving low-level monotherapy immunosuppression at the time of vaccination. The majority of children developed protective antibodies following vaccination (107 of 149 [72%] varicella, 130 of 152 [86%] measles, 100 of 120 [83%] mumps, and 124 of 125 [99%] rubella). One year post vaccination, the majority of children who initially mounted protective antibodies maintained this protection (34 of 44 [77%] varicella, 45 of 49 [92%] measles, 35 of 42 [83%] mumps, 51 of 54 [94%] rubella). Five children developed clinical varicella, all of which resolved within 1 week. There were no cases of measles or rubella and no episodes of graft rejection within 1 month of vaccination. There was no association between antibody response and immunosuppression level at the time of vaccination. Conclusions and Relevance: The findings suggest that live vaccinations may be safe and immunogenic after solid organ transplant in select pediatric recipients and can offer protection against circulating measles, mumps, and varicella.

Status 1B designation does not adequately prioritize children with acute-on-chronic liver failure for liver transplantation.

Acute-on-chronic liver failure (ACLF) is an acute decompensation of chronic liver disease leading to multiorgan failure and mortality. The objective of this study was to evaluate characteristics and outcomes of children with ACLF who are at the highest priority for liver transplantation (LT) on the United Network for Organ Sharing (UNOS) database-listed as status 1B. The characteristics and outcomes of 478 children with ACLF listed as status 1B on the UNOS LT waiting list from 2007-2019 were compared with children with similar or higher priority listing for transplant: 929 with acute liver failure (ALF) listed as status 1A and 808 with metabolic diseases and malignancies listed as status 1B (termed "non-ACLF"). Children with ACLF had comparable rates of cumulative organ failures compared with ALF (45% vs. 44%; p > 0.99) listings, but higher than non-ACLF (45% vs. 1%; p < 0.001). ACLF had the lowest LT rate (79%, 84%, 95%; p < 0.001), highest pre-LT mortality (20%, 11%, 1%; p < 0.001), and longest waitlist time (57, 3, 56 days; p < 0.001), and none recovered without LT (0%, 4%, 1%; p < 0.001). In survival analyses, ACLF was associated with an increased adjusted hazard ratio (HR) for post-LT mortality (HR, 1.50 vs. ALF [95% confidence interval, CI, 1.02-2.19; p = 0.04] and HR, 1.64 vs. non-ACLF [95% CI, 1.15-2.34; p = 0.01]). ACLF has the least favorable waitlist and post-LT outcomes of all patients who are status 1A/1B. Increased prioritization on the LT waiting list may offer children with ACLF an opportunity for enhanced outcomes.

Inborn error of metabolism patients after liver transplantation: Outcomes of 35 patients over 27 years in one pediatric quaternary hospital.

Liver transplantation (LT) has been used for many years as a therapeutic option for certain inborn errors of metabolism (IEMs). Here we present one institution's 27 years of experience with LT in IEMs. Our objective is to assess the outcomes of IEM patients who have undergone LT, which we hypothesize to be generally successful for prevention of metabolic decompensation. A retrospective chart review was performed on patients with urea cycle defects, organic acidemias, and amino acidopathies who underwent LT at the Children's Hospital of Philadelphia. Thirty-five patients with the following conditions have undergone LT: tyrosinemia (8), methylmalonic acidemia (7), maple syrup urine disease (6), citrullinemia (6), ornithine transcarbamylase deficiency (4), propionic acidemia (2), and argininosuccinate lyase deficiency (2). Average age at transplantation was 3.6 years. Three patients are now deceased. One patient suffered a metabolic stroke posttransplant. No episodes of metabolic decompensation have been noted. Thirty-five patients received LT with generally favorable outcome. None sustained metabolic decompensation posttransplant. As has been reported previously, LT does not ameliorate pre-existing developmental differences or risk to other organ systems. Further research is needed to aid in standardization of care and follow-up, as most patients no longer follow with a geneticist.

Live virus vaccination following pediatric liver transplantation: Outcomes from two academic children's hospitals.

Pediatric liver transplant (LT) recipients are often transplanted at a young age, precluding them from receiving live virus vaccinations (LVV) such as varicella (VZV) vaccine and measles, mumps and rubella. This places them at profound risk for vaccine preventable illness. We sought to detail safety of vaccination. This was a retrospective cohort study of pediatric LT recipients at two children's hospitals. Among 204 LT recipients included in the study, 97 received at least one LVV after LT. Six patients who did not receive LVV after transplant had evidence of vaccine-preventable infection following vaccination (one disseminated VZV disease, five VZV-related rash), while one patient who received LVV after transplant developed a diffuse VZV-related rash. Rejection rates were the same between those that did and did not receive a live virus vaccine post-transplant. There were no serious adverse events caused by vaccination post-transplant. LVV following LT was safe at our two institutions, although there exist limitations in our study due to its retrospective study design. Larger scale studies should be performed to evaluate the effectiveness of LVV in relation to immunosuppression.

Center Variability in Acute Rejection and Biliary Complications After Pediatric Liver Transplantation.

Transplant center performance and practice variation for pediatric post-liver transplantation (LT) outcomes other than survival are understudied. This was a retrospective cohort study of pediatric LT recipients who received transplants between January 1, 2006, and May 31, 2017, using United Network for Organ Sharing (UNOS) data that were merged with the Pediatric Health Information System database. Center effects for the acute rejection rate at 1 year after LT (AR1) using UNOS coding and the biliary complication rate at 1 year after LT (BC1) using inpatient billing claims data were estimated by center-specific rescaled odds ratios that accounted for potential differences in recipient and donor characteristics. There were 2216 pediatric LT recipients at 24 freestanding children's hospitals in the United States during the study period. The median unadjusted center rate of AR1 was 36.92% (interquartile range [IQR], 22.36%-44.52%), whereas that of BC1 was 32.29% (IQR, 26.14%-40.44%). Accounting for recipient case mix and donor factors, 5/24 centers performed better than expected with regard to AR1, whereas 3/24 centers performed worse than expected. There was less heterogeneity across the center effects for BC1 than for AR1. There was no relationship observed between the center effects for AR1 or BC1 and center volume. Beyond recipient and allograft factors, differences in transplant center management are an important driver of center AR1 performance, and less so of BC1 performance. Further research is needed to identify the sources of variability so as to implement the most effective solutions to broadly enhance outcomes for pediatric LT recipients.

Live Vaccines in Pediatric Liver Transplant Recipients: "To Give or Not to Give".

Content available: Author Audio Recording.

Multiple organ involvement and ICU considerations for the care of acute liver failure (ALF) and acute on chronic liver failure (ACLF) in children.

Liver disease results in approximately 15,000 pediatric hospitalizations per year in the United States and is a significant burden to child health. Major etiologies of liver failure and indications for transplantation in children include: biliary atresia, metabolic/genetic conditions, toxins, infections, tumors, and immune-mediated liver/biliary injury. Children requiring transplantation are placed on the United Network of Organ Sharing waitlist including those with acute liver failure (ALF) and acute on chronic liver failure (ACLF). ALF is a clinical syndrome in which a previously healthy child develops rapid-onset hepatic dysfunction, and becomes critically ill with multiple organ dysfunction within days. ACLF, by contrast, is generally described as an acute decompensation of pre-existing chronic liver disease (CLD) brought on by a precipitating event, with higher risk of mortality. Children with ALF and ACLF receive multidisciplinary care in pediatric intensive care units (ICUs) due to multiple organ system involvement and high risk of decompensation in these patients. The care of these patients requires a holistic approach that addresses the complex interplay between hepatic and extra-hepatic organ systems. This review will define and describe ALF and ACLF in the pediatric population, and outline the effects of ALF and ACLF on individual organ systems with diagnostic and management considerations in the ICU while awaiting liver transplantation.

Growth in Children with a Fontan Circulation.

OBJECTIVE: To evaluate growth in a population of patients with Fontan circulation. STUDY DESIGN: We performed a cross-sectional evaluation of patients followed in our multidisciplinary Fontan clinic from January 2011 through August 2015. We reviewed the historical data, anthropometry, clinical, and laboratory studies and performed bivariate and multivariate analysis of factors associated with height z score. RESULTS: Patients (n = 210) were included in the study at median age 11.07 years (8.3, 14.73 years) (43% female); 138 (65%) had a dominant right systemic ventricle and 92 (44%) hypoplastic left heart syndrome. Median age at completion of Fontan circulation was 31 months (7.6, 135.8 months). Median height z score was -0.58 (-1.75, 0.26). Twenty-five (12%) had current or past history of protein-losing enteropathy (PLE). Median height z score for those with current or past history of PLE was -2.1 (-2.46, 1.24). Multivariate analysis revealed positive associations between height z score and body mass index z score, time since Fontan, mid-parental height, dominant systemic ventricle type, and serum alkaline phosphatase. Height correlated negatively with known genetic syndrome, PLE, use of stimulant or oral steroid medication. CONCLUSIONS: Children with Fontan circulation have mild deficits in height, with greater deficits in those with PLE. Height z score improves with time postsurgery. Improving weight, leading to improved body mass index, may be a modifiable factor that improves growth in those who are underweight. Biochemical markers may be helpful screening tests for high-risk groups in whom to intensify interventions.

Early detection of SARS-CoV-2 and other infections in solid organ transplant recipients and household members using wearable devices.

The increasing global prevalence of SARS-CoV-2 and the resulting COVID-19 disease pandemic pose significant concerns for clinical management of solid organ transplant recipients (SOTR). Wearable devices that can measure physiologic changes in biometrics including heart rate, heart rate variability, body temperature, respiratory, activity (such as steps taken per day) and sleep patterns, and blood oxygen saturation show utility for the early detection of infection before clinical presentation of symptoms. Recent algorithms developed using preliminary wearable datasets show that SARS-CoV-2 is detectable before clinical symptoms in >80% of adults. Early detection of SARS-CoV-2, influenza, and other pathogens in SOTR, and their household members, could facilitate early interventions such as self-isolation and early clinical management of relevant infection(s). Ongoing studies testing the utility of wearable devices such as smartwatches for early detection of SARS-CoV-2 and other infections in the general population are reviewed here, along with the practical challenges to implementing these processes at scale in pediatric and adult SOTR, and their household members. The resources and logistics, including transplant-specific analyses pipelines to account for confounders such as polypharmacy and comorbidities, required in studies of pediatric and adult SOTR for the robust early detection of SARS-CoV-2, and other infections are also reviewed.

Hepatic Involvement in Aicardi-Goutières Syndrome.

Aicardi-Goutières syndrome (AGS) is a monogenic type-I interferonopathy that results in neurologic injury. The systemic impact of sustained interferon activation is less well characterized. Liver inflammation is known to be associated with the neonatal form of AGS, but the incidence of AGS-related hepatitis across lifespan is unknown.We compared natural history data including liver enzyme levels with markers of inflammation, (liver-specific autoantibodies and interferon signaling gene expression[ISG] scores). Liver enzymes were classified as normal or elevated by the fold increase over the upper limit of normal (ULN). The highest increases were designated as hepatitis, defined as aspartate-aminotransferase or alanine-aminotransferase threefold ULN, or gamma-glutamyl transferase 2.5-fold ULN. A larger cohort was used to further characterize the longitudinal incidence of liver abnormalities and the association with age and genotype.Across the AGS cohort (n = 102), elevated liver enzymes were identified in 76 individuals (74.5%) with abnormalities at a level consistent with hepatitis in 29 individuals (28.4%). SAMHD1 mutations were less likely to be associated with hepatitis (log-rank test; p = 0.011). Hepatitis was associated with early-onset disease and microcephaly (log-rank test; microcephaly p = 0.0401, age onset p = 0.0355). While most subjects (n = 20/33) were found to have liver-specific autoantibodies, there was no association between the presence of autoantibodies or ISG scores with hepatitis-level enzyme elevations.In conclusion, all genotypes of AGS are associated with transient elevations of liver enzymes and the presence of liver-associated autoantibodies. This adds to our growing understanding of the systemic pathology AGS.

Efficacy and Safety of Immunosuppression Withdrawal in Pediatric Liver Transplant Recipients: Moving Toward Personalized Management.

BACKGROUND AND AIMS: Tolerance is transplantation's holy grail, as it denotes allograft health without immunosuppression and its toxicities. Our aim was to determine, among stable long-term pediatric liver transplant recipients, the efficacy and safety of immunosuppression withdrawal to identify operational tolerance. APPROACH AND RESULTS: We conducted a multicenter, single-arm trial of immunosuppression withdrawal over 36-48 weeks. Liver tests were monitored biweekly (year 1), monthly (year 2), and bimonthly (years 3-4). For-cause biopsies were done at investigators' discretion but mandated when alanine aminotransferase or gamma glutamyltransferase exceeded 100 U/L. All subjects underwent final liver biopsy at trial end. The primary efficacy endpoint was operational tolerance, defined by strict biochemical and histological criteria 1 year after stopping immunosuppression. Among 88 subjects (median age 11 years; 39 boys; 57 deceased donor grafts), 33 (37.5%; 95% confidence interval [CI] 27.4%, 48.5%) were operationally tolerant, 16 were nontolerant by histology (met biochemical but failed histological criteria), and 39 were nontolerant by rejection. Rejection, predicted by subtle liver inflammation in trial entry biopsies, typically (n = 32) occurred at ≤32% of the trial-entry immunosuppression dose and was treated with corticosteroids (n = 32) and/or tacrolimus (n = 38) with resolution (liver tests within 1.5 times the baseline) for all but 1 subject. No death, graft loss, or chronic, severe, or refractory rejection occurred. Neither fibrosis stage nor the expression level of a rejection gene set increased over 4 years for either tolerant or nontolerant subjects. CONCLUSIONS: Immunosuppression withdrawal showed that 37.5% of selected pediatric liver-transplant recipients were operationally tolerant. Allograft histology did not deteriorate for either tolerant or nontolerant subjects. The timing and reversibility of failed withdrawal justifies future trials exploring the efficacy, safety, and potential benefits of immunosuppression minimization.

End-Organ Function and Exercise Performance in Patients With Fontan Circulation: What Characterizes the High Performers?

Background The physiologic hallmarks of the Fontan circulation-chronically elevated central venous pressures and low cardiac output-have significant effects not only on cardiovascular status but also impact other organ systems. Exercise capacity is limited in many and declines with age, accelerating in adolescence, but with wide variability. We explore the relationship between exercise performance and end-organ function in outpatient subjects with a Fontan circulation. Methods and Results This is a cross-sectional analysis of subject end-organ characterization from our outpatient Fontan circulation clinic with peak oxygen consumption (peak Vo2) at cardiopulmonary exercise testing as the primary outcome. We perform linear regression to assess associations between clinical characteristics and peak Vo2 as well as the magnitude of the association of clinical characteristics with peak Vo2. Of 265 subjects age 12.8 (9.5-16.4) years, there is a negative correlation between age and peak Vo2 (-0.49, P<0.001). Of those undergoing ramp cycle exercise testing, 34% perform above 80% predicted peak Vo2. Variables positively associated with peak Vo2 and their effect size include vitamin D sufficiency (+3.00, P=0.020) and absolute lymphocyte count (+0.23, P=0.005). Status as overweight/obese (-3.91, P=0.003) and hemoglobin (-0.77, P=0.003) are negatively associated. Neither ventricular morphology, timing of Fontan palliation, nor Fontan circulation type affect peak Vo2. Conclusions Higher peak Vo2 in those with a Fontan circulation is associated with younger age, vitamin D sufficiency, absence of overweight/obese, lower hemoglobin, and a healthier hepatic profile. Whether exercise training or other initiatives can modify organ characteristics in those with a Fontan circulation is worthy of exploration.

Biliary Excretion Noted on Hepatobiliary Iminodiacetic Acid Scan Does Not Exclude Diagnosis of Biliary Atresia.

A hepatobiliary iminodiacetic acid (HIDA) scan is frequently used in an attempt to exclude biliary atresia in infants who are cholestatic. We present 6 cases of confirmed biliary atresia in infants who had biliary patency reported on HIDA scan. We demonstrate that misinterpreted HIDA scans led to delayed diagnosis and surgical intervention for biliary atresia.

Alagille syndrome mutation update: Comprehensive overview of JAG1 and NOTCH2 mutation frequencies and insight into missense variant classification.

Alagille syndrome is an autosomal dominant disease with a known molecular etiology of dysfunctional Notch signaling caused primarily by pathogenic variants in JAGGED1 (JAG1), but also by variants in NOTCH2. The majority of JAG1 variants result in loss of function, however disease has also been attributed to lesser understood missense variants. Conversely, the majority of NOTCH2 variants are missense, though fewer of these variants have been described. In addition, there is a small group of patients with a clear clinical phenotype in the absence of a pathogenic variant. Here, we catalog our single-center study, which includes 401 probands and 111 affected family members amassed over a 27-year period, to provide updated mutation frequencies in JAG1 and NOTCH2 as well as functional validation of nine missense variants. Combining our cohort of 86 novel JAG1 and three novel NOTCH2 variants with previously published data (totaling 713 variants), we present the most comprehensive pathogenic variant overview for Alagille syndrome. Using this data set, we developed new guidance to help with the classification of JAG1 missense variants. Finally, we report clinically consistent cases for which a molecular etiology has not been identified and discuss the potential for next generation sequencing methodologies in novel variant discovery.