Parenteral nutrition emulsion inhibits CYP3A4 in an iPSC derived liver organoids testing platform.

OBJECTIVES: Parenteral nutrition (PN) is used for patients of varying ages with intestinal failure to supplement calories. Premature newborns with low birth weight are at a high risk for developing PN associated liver disease (PNALD) including steatosis, cholestasis, and gallbladder sludge/stones. To optimize nutrition regimens, models are required to predict PNALD. METHODS: We have exploited induced pluripotent stem cell derived liver organoids to provide a testing platform for PNALD. Liver organoids mimic the developing liver and contain the different hepatic cell types. The organoids have an early postnatal maturity making them a suitable model for premature newborns. To mimic PN treatment we used medium supplemented with either clinoleic (80% olive oil/20% soybean oil) or intralipid (100% soybean oil) for 7 days. RESULTS: Homogenous HNF4a staining was found in all organoids and PN treatments caused accumulation of lipids in hepatocytes. Organoids exhibited a dose dependent decrease in CYP3A4 activity and expression of hepatocyte functional genes. The lipid emulsions did not affect overall organoid viability and glucose levels had no contributory effect to the observed results. CONCLUSIONS: Liver organoids could be utilized as a potential screening platform for the development of new, less hepatotoxic PN solutions. Both lipid treatments caused hepatic lipid accumulation, a significant decrease in CYP3A4 activity and a decrease in the RNA levels of both CYP3A4 and CYP1A2 in a dose dependent manner. The presence of high glucose had no additive effect, while Clinoleic at high dose, caused significant upregulation of interleukin 6 and TLR4 expression.

Hunting for the elusive target antigen in gestational alloimmune liver disease (GALD).

The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in Escherichia coli using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial-temporal regulation of histone arginine methylation in neurodevelopment.

BACKGROUND: Oxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency. RESULTS: We report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. CONCLUSIONS: This study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

Scalable production of tissue-like vascularized liver organoids from human PSCs.

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

Aagenaes syndrome/lymphedema cholestasis syndrome 1 is caused by a founder variant in the 5'-untranslated region of UNC45A.

BACKGROUND & AIMS: Lymphedema cholestasis syndrome 1 or Aagenaes syndrome is a condition characterized by neonatal cholestasis, lymphedema, and giant cell hepatitis. The genetic background of this autosomal recessive disease was unknown up to now. METHODS: A total of 26 patients with Aagenaes syndrome and 17 parents were investigated with whole-genome sequencing and/or Sanger sequencing. PCR and western blot analyses were used to assess levels of mRNA and protein, respectively. CRISPR/Cas9 was used to generate the variant in HEK293T cells. Light microscopy, transmission electron microscopy and immunohistochemistry for biliary transport proteins were performed in liver biopsies. RESULTS: One specific variant (c.-98G>T) in the 5'-untranslated region of Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome. Nineteen were homozygous for the c.-98G>T variant and seven were compound heterozygous for the variant in the 5'-untranslated region and an exonic loss-of-function variant in UNC45A. Patients with Aagenaes syndrome exhibited lower expression of UNC45A mRNA and protein than controls, and this was reproduced in a CRISPR/Cas9-created cell model. Liver biopsies from the neonatal period demonstrated cholestasis, paucity of bile ducts and pronounced formation of multinucleated giant cells. Immunohistochemistry revealed mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2). CONCLUSIONS: c.-98G>T in the 5'-untranslated region of UNC45A is the causative genetic variant in Aagenaes syndrome. IMPACT AND IMPLICATIONS: The genetic background of Aagenaes syndrome, a disease presenting with cholestasis and lymphedema in childhood, was unknown until now. A variant in the 5'-untranslated region of the Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome, providing evidence of the genetic background of the disease. Identification of the genetic background provides a tool for diagnosis of patients with Aagenaes syndrome before lymphedema is evident.

Mycophenolate mofetil use is associated with reduced incidence of food allergy in liver transplanted children.

OBJECTIVES: The incidence of food allergy in children following liver transplantation is high and the pathogenesis is still not known. We aimed to identify risk factors for development of food allergies in liver transplant children. METHODS: 107 children and adolescents who underwent liver transplantation from 1999 to 2019 were included. Data were retrospectively collected from medical records included total and specific IgE, eosinophil cationic protein and eosinophil count 12 months after transplantation and at yearly follow up (median follow-up). RESULTS: 24/107 (22%) patients reported clinical food reactions. Median time from transplantation to debut of food allergy was 1.6 (IQR 0.6-3.3) years. Mycophenolate mofetil (MMF) was discontinued in 24/78 patients (31%) due to side effects. Children treated with MMF in addition to tacrolimus one year after transplantation reported less food allergy (12.5% vs. 37.8%, p=0.003) and sensitization to food allergens one year after transplantation (8.9% vs. 17.8%, p=0.02) than those not receiving MMF. Tacrolimus trough levels did not differ between the patients treated with MMF and those who were not. Treatment with MMF two years after transplantation was associated with less food allergy (p=0.001) and food sensitization (p=0.002), also when adjusted for age at transplantation (p=0.006 and p=0.03, respectively) or for use of basilixmab (p=0.015 and p=0.018, respectively). Basiliximab was also associated with less food allergy. CONCLUSIONS: Use of MMF one and two years after transplantation was associated with less food allergy and sensitization against food allergens. The effect of MMF was not due to reduced trough levels of of tacrolimus. An infographic is available for this article at: https://links.lww.com/MPG/C821.

Impaired Neurocognitive Performance in Children after Liver Transplantation.

OBJECTIVES: To assess longitudinal neurocognitive development after liver transplantation and evaluate factors associated with neurocognitive performance. STUDY DESIGN: Data from neurocognitive testing of 65 children (aged <18 years) who underwent liver transplantation at Oslo University Hospital between 1995 and 2018 were collected from the testing program after transplantation. The parent-reported version of the Behavior Rating Inventory of Executive Function was used to assess executive function. RESULTS: A total of 104 neurocognitive tests were conducted on 65 patients. At the first test, conducted at a median of 4.1 years (IQR, 1.5-5.3 years) after transplantation and at a median age of 6.7 years (IQR, 5.4-10.5 years), the mean full-scale IQ (FSIQ) was 91.7 ± 14, and the mean verbal comprehension index was 92.0 ± 14.5. In the 30 patients tested more than once, there was no significant difference in FSIQ between the first test at a median age of 5.8 years (IQR, 5.2-8.5 years) and the last test at a median age of 10.8 years (IQR, 9.8-12.9 years) (87.4 ± 12.9 vs 88.5 ± 13.2; P = .58). Compared with the patients who underwent transplantation a age >1 year (n = 35), those who did so at age <1 year (n = 30) had a lower FSIQ (87.1 ± 12.6 vs 96.6 ± 13.8; P = .005) and lower verbal comprehension index (87.3 ± 13.8 vs 95.4 ± 13.0; P = .020). Age at transplantation (P = .005; adjusted for cholestasis: P = .038) and transfusion of >80 mL/kg (P = .004; adjusted for age at transplantation: P = .046) were associated with FSIQ. CONCLUSIONS: Young age at transplantation and large blood transfusions during transplantation are risk factors for poor neurocognitive performance later in life. Children who undergo transplantation before 1 year of age have significantly lower neurocognitive performance compared with those who do so later in childhood. Cognitive performance did not improve over time after transplantation.

Spectrum of Fontan-associated liver disease assessed by MRI and US in young adolescents.

PURPOSE: Patients with Fontan circulation are at risk of developing hepatic fibrosis/cirrhosis. The mechanisms and disease development are unclear and early secondary liver cancer is a concern. This study will describe hepatic imaging findings in a national cohort of adolescents with Fontan circulation. METHODS: The patients prospectively underwent abdominal contrast enhanced magnetic resonance imaging (MRI) including diffusion-weighted imaging. Images were assessed for criteria of fibrosis/cirrhosis including characterization of hepatic nodules. These nodules were in addition, assessed by ultrasonography (US). Nodules ≥ 1 cm were investigated and monitored to evaluate malignant transformation. Clinical and hepatic serological data were recorded. RESULTS: Forty-six patients, median age of 16.5 years (15.4-17.9 years) were enrolled. All patients underwent US examination and MRI was performed in 35/46 patients. On MRI, 60% had hepatomegaly and 37% had signs of fibrosis/cirrhosis. Seven patients had together 13 nodules ≥ 1 cm in diameter. Only 4/13 (17%) where seen on US. Nodules had variable MRI signal characteristics including hepatobiliary contrast enhancement and two nodules revealed portal venous phase 'wash-out' on the first examination. No further imaging signs of malignancy were revealed during the follow-up period of median 24.4 (7-42) months. CONCLUSION: The majority of adolescents with Fontan circulation had imaging findings of fibrosis/cirrhosis of varying severity. US had low detection rate of hepatic nodules compared to MRI. The imaging work-up before transition to adult cardiology care did not reveal findings suggestive of malignancy. However, the high prevalence of Fontan-associated liver disease calls for surveillance strategies even in childhood.

MicroRNA in dried blood spots from patients with Aagenaes syndrome and evaluation of pre-analytical and analytical factors.

BACKGROUND: Circulatory miRNAs are promising biomarkers. The feasibility of using miRNA from dried blood spots (DBS) was investigated using newborn screening cards from patients with cholestasis-lymphedema syndrome (Aagenaes syndrome) and controls. METHODS: Total amount of miRNA and specific miRNAs from DBS were analyzed. miRNA was also obtained from newborn screening cards in patients with cholestasis-lymphedema syndrome/Aagenaes syndrome and in healthy newborns. RESULTS: No differences in miRNA concentrations were found between multispotted samples and samples with one single drop of blood and between central and peripheral punches. Ten repeated freeze-thaw cycles did not significantly change miRNA levels from controls. miR-299 (1.73-fold change, p = 0.034) and miR-365 (1.46-fold change, p = 0.011) were upregulated and miR-30c (0.72-fold change, p = 0.0037), miR-652 (0.85-fold change, p = 0.025), and miR-744 (0.72-fold change, p = 0.0069) were downregulated in patients with Aagenaes syndrome at birth compared to controls. CONCLUSIONS: miRNAs were not affected by multispotting or punch location and were stable throughout repeated freeze-thaw cycles. miRNA in dried blood spots could be used to detect differential expression of miRNA in newborns with Aagenaes syndrome and healthy controls in newborn screening cards. Dried blood spots may be a useful source to explore circulating miRNA as biomarkers. IMPACT: Circulating miRNAs can be useful biomarkers. miRNAs from dried blood spots were not affected by multispotting or punch location and were stable throughout repeated freeze-thaw cycles. Discrimination between patients and controls are allowed even with few individuals. Early after birth, patients with cholestasis-lymphedema syndrome exhibit miRNA profiles associated with liver fibrosis. This study demonstrated that newborn screening cards may be a useful source for studying miRNA as the technical variability is smaller than biological variation.

Partial Biliary Diversion May Promote Long-Term Relief of Pruritus and Native Liver Survival in Children with Cholestatic Liver Diseases.

INTRODUCTION: Rare cholestatic liver diseases may cause debilitating pruritus in children. Partial biliary diversion (PBD) may relieve pruritus and postpone liver transplantation which is the only other alternative when conservative treatment fails. The aim was to report long-term outcome after PBD in a population of 26 million people during a 25-year period. MATERIALS AND METHODS: This is an international, multicenter retrospective study reviewing medical journals. Complications were graded according to the Clavien-Dindo classification system. RESULTS: Thirty-three patients, 14 males, underwent PBD at a median of 1.5 (0.3-13) years at four Nordic pediatric surgical centers. Progressive familial intrahepatic cholestasis was the most common underlying condition. Initially, all patients got external diversion, either cholecystojejunostomy (25 patients) or button placed in the gallbladder or a jejunal conduit. Early complications occurred in 14 (42%) patients, of which 3 were Clavien-Dindo grade 3. Long-term stoma-related complications were common (55%). Twenty secondary surgeries were performed due to stoma problems such as prolapse, stricture, and bleeding, or conversion to another form of PBD. Thirteen children have undergone liver transplantation, and two are listed for transplantation due to inefficient effect of PBD on pruritus. Serum levels of bile acids in the first week after PBD construction were significantly lower in patients with good relief of pruritus than in those with poor effect (13 [2-192] vs. 148 [5-383] µmol/L; p = 0.02). CONCLUSION: PBD may ensure long-term satisfactory effect on intolerable pruritus and native liver survival in children with cholestatic liver disease. However, stoma-related problems and reoperations are common.

Hepatic magnetic resonance T1-mapping and extracellular volume fraction compared to shear-wave elastography in pediatric Fontan-associated liver disease.

BACKGROUND: Children with Fontan circulation are at risk of developing hepatic fibrosis/cirrhosis. Reliable noninvasive monitoring techniques are lacking or under development. OBJECTIVE: To investigate surrogate indicators of hepatic fibrosis in adolescents with Fontan circulation by evaluating hepatic magnetic resonance (MR) T1 mapping and extracellular volume fraction measurements compared to US shear-wave elastography. MATERIALS AND METHODS: We analyzed hepatic native T1 times and extracellular volume fractions with modified Look-Locker inversion recovery. Liver stiffness was analyzed with shear-wave elastography. We compared results between 45 pediatric patients ages 16.7±0.6 years with Fontan circulation and 15 healthy controls ages 19.2±1.2 years. Measurements were correlated to clinical and hemodynamic data from cardiac catheterization. RESULTS: MR mapping was successful in 35/45 patients, revealing higher hepatic T1 times (774±44 ms) than in controls (632±52 ms; P<0.001) and higher extracellular volume fractions (47.4±5.0%) than in controls (34.6±3.8%; P<0.001). Liver stiffness was 1.91±0.13 m/s in patients vs. 1.20±0.10 m/s in controls (P<0.001). Native T1 times correlated with central venous pressures (r=0.5, P=0.007). Native T1 was not correlated with elastography in patients (r=0.2, P=0.1) or controls (r = -0.3, P=0.3). Extracellular volume fraction was correlated with elastography in patients (r=0.5, P=0.005) but not in controls (r=0.2, P=0.6). CONCLUSION: Increased hepatic MR relaxometry and shear-wave elastography values in adolescents with Fontan circulation suggested the presence of hepatic fibrosis or congestion. Central venous pressure was related to T1 times. Changes were detected differently with MR relaxometry and elastography; thus, these techniques should not be used interchangeably in monitoring hepatic fibrosis.

Cytokine profile in children with food allergy following liver transplantation.

BACKGROUND: LTX in children is associated with increased risk of food allergy, and the mechanisms underlying this are unknown. We wanted to study whether plasma cytokine profile differed in liver transplanted children, with and without food allergy, and whether it differed from untransplanted children with CLD. METHODS: Plasma cytokines, total and specific IgE in nine patients with food allergy were compared with 13 patients without food allergy following LTX, and also with seven untransplanted patients with CLD. RESULTS: No difference was found in the cytokine profile between liver transplanted patients with and without food allergy. Transplanted patients with food allergy having received a prescription of epinephrine had a significantly higher total IgE (2033 [234-2831] vs 10 [5-41] IU/L, P = .002) and MIP-1b (52 [37-96] vs 36 [32-39], P = .035) compared with transplanted patients without food allergy. Two patients with severe food allergy responded favorably to conversion from tacrolimus-based immunosuppression to MMF and corticosteroids with reduction in clinical symptoms, total IgE, specific IgE, IL-1ra, IL-4, RANTES, PDGF, MIP-1a, and TNFα. The transplantation group had higher levels of IL-1b, IL-5, IL-7, IL-13, GCSF, IFNγ, and MIP-1a compared with the CLD group. CONCLUSIONS: No overall difference was found in plasma cytokine profile between patients with and without food allergy. Patients with severe food allergy had significant elevation of MIP-1b. Discontinuation of tacrolimus reduced total and specific IgE and changed plasma cytokine profile. The plasma cytokine profile in liver transplanted children was different compared with children with CLD.

Growth Differentiation Factor 15 in Children with Chronic Kidney Disease and after Renal Transplantation.

Growth differentiation factor 15 (GDF-15) is strongly associated with cardiovascular disease (CVD). The aim of our study was to evaluate plasma and urinary levels of GDF-15 after pediatric renal transplantation (Rtx) and in children with chronic kidney disease (CKD) and its associations to cardiovascular risk factors. In this cross-sectional study, GDF-15 was measured in plasma and urine from 53 children with a renal transplant and 83 children with CKD and related to cardiovascular risk factors (hypertension, obesity, and cholesterol) and kidney function. Forty healthy children served as a control group. Plasma levels of GDF-15 (median and range) for a Tx (transplantation) cohort, CKD cohort, and healthy controls were, respectively, 865 ng/L (463-3039 ng/L), 508 ng/L (183-3279 ng/L), and 390 ng/L (306-657 ng/L). The CKD and Tx cohorts both had significantly higher GDF-15 levels than the control group (p < 0.001). Univariate associations between GDF-15 and hyperuricemia (p < 0.001), elevated triglycerides (p = 0.028), low HDL (p = 0.038), and obesity (p = 0.028) were found. However, mGFR (p < 0.001) and hemoglobin (p < 0.001) were the only significant predictors of GDF-15 in an adjusted analysis. Urinary GDF-15/creatinine ratios were 448 ng/mmol (74-5013 ng/mmol) and 540 ng/mmol (5-14960 ng/mmol) in the Tx cohort and CKD cohort, respectively. In the CKD cohort, it was weakly correlated to mGFR (r = -0.343, p = 0.002). Plasma levels of GDF-15 are elevated in children with CKD and after Rtx. The levels were not associated with traditional cardiovascular risk factors but strongly associated with renal function.

Measured GFR by Utilizing Population Pharmacokinetic Methods to Determine Iohexol Clearance.

INTRODUCTION: There is an increasing demand for accurately measured glomerular filtration rate (GFR). Iohexol serum clearance has become a new gold standard, but it is challenging when GFR is low and 24-hour sampling is required for accurate results. The primary aim of this study was to develop an iohexol pharmacokinetic population model for accurate determination of individual GFR using limited sampling for up to 5 hours also when renal function is <40 ml/min. METHODS: A nonparametric iohexol population pharmacokinetic model was developed with rich data from 176 patients. In a validation cohort of 43 patients, a model-determined GFR (iohexol clearance) using different limited sampling strategies for up to 5 hours was compared with the strategy currently used in routine care, a log-linear 2-point method. In all, 1526 iohexol concentrations were used, from patients ranging in age from 1 to 82 years and GFR from 14 to 149 ml/min. RESULTS: The clinical 2-point method showed insufficient agreement compared with reference values; 15% of GFR values had an error of greater than ±10% even when sampling for 24 hours when estimating GFR <40 ml/min per 1.73 m2 (standard procedure). Restricted sampling the first 5 hours with the population model required 4 samples to determine GFR accurately. This strategy showed excellent agreement with the reference; <3% of GFR values had an error greater than ±10 %. CONCLUSION: Using an iohexol population pharmacokinetic model allows for accurate determination of GFR within 5 hours when applying 4 optimally timed samples, even in patients with GFR <40 ml/min.

Progressive loss of bone mass in children with Fontan circulation.

OBJECTIVE: We investigated bone mineral density (BMD) at different ages after the Fontan completion, and we evaluated the relationship between BMD, vitamin D levels, and pertinent patient variables. METHODS: A cross-sectional sample of 64 patients was examined with dual-energy X-ray absorptiometry (DXA) scans to determine BMD. Of these patients, 24 were also examined with BoneXpert software to determine bone mass density (BMX), expressed as the bone health index (BHI). Blood samples from all patients were analyzed. Patients were divided into three different age groups; A: 4-9 years old (n = 22), B: 10-15 years old (n = 21), and C: 16-18 years old (n = 21). RESULTS: Overall, BMD z scores were (mean ± SD): -1.0 ± 1.3 for the lumbar spine and -0.2 ± 1.2 for the total body. Groups B and C had significantly lower z score values compared to group A. Of patients in group C, 35% had z score values ≤-2 SD of the mean of the healthy population. There was no difference related to systemic ventricular anatomy (left or right); however, patients with lateral tunnels had lower BMD than patients with extra cardiac conduits. Overall, the BHI z score was (mean ± SD): -1.2 ± 0.9, but low BMX did not correlate with low BMD. The 25-hydroxy vitamin D level was 58 ± 30 nmol/L. Vitamin D levels decreased with age: in group C, 33.3% of patients exhibited vitamin D deficiencies. Vitamin D levels were not correlated with bone mineral densities. CONCLUSION: BMD levels decreased with age in patients with Fontan circulation. Different bone components were involved. Vitamin D levels also decreased with age, but they were not consistently associated with bone mineral densities. The single factor most predictive of low BMD was a lateral tunnel Fontan, compared to an extra cardiac Fontan.