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Purpose: In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu)., Methods: Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics., Results: All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production., Conclusion: Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.

Purpose: Hardikar syndrome (MIM 612726) is a rare multiple congenital anomaly syndrome characterized by facial clefting, pigmentary retinopathy, biliary anomalies, and intestinal malrotation, but with preserved cognition. Only four patients have been reported previously, and none had a molecular diagnosis. Our objective was to identify the genetic basis of Hardikar syndrome (HS) and expand the phenotypic spectrum of this disorder., Methods: We performed exome sequencing on two previously reported and five unpublished female patients with a clinical diagnosis of HS. X-chromosome inactivation (XCI) studies were also performed., Results: We report clinical features of HS with previously undescribed phenotypes, including a fatal unprovoked intracranial hemorrhage at age 21. We additionally report the discovery of de novo pathogenic nonsense and frameshift variants in MED12 in these seven individuals and evidence of extremely skewed XCI in all patients with informative testing., Conclusion: Pathogenic missense variants in the X-chromosome gene MED12 have previously been associated with Opitz-Kaveggia syndrome, Lujan syndrome, Ohdo syndrome, and nonsyndromic intellectual disability, primarily in males. We propose a fifth, female-specific phenotype for MED12, and suggest that nonsense and frameshift loss-of-function MED12 variants in females cause HS. This expands the MED12-associated phenotype in females beyond intellectual disability.

Purpose: Galactokinase (GALK1) deficiency is a rare hereditary galactose metabolism disorder. Beyond cataract, the phenotypic spectrum is questionable. Data from affected patients included in the Galactosemias Network registry were collected to better characterize the phenotype., Methods: Observational study collecting medical data of 53 not previously reported GALK1 deficient patients from 17 centers in 11 countries from December 2014 to April 2020., Results: Neonatal or childhood cataract was reported in 15 and 4 patients respectively. The occurrence of neonatal hypoglycemia and infection were comparable with the general population, whereas bleeding diathesis (8.1% versus 2.17-5.9%) and encephalopathy (3.9% versus 0.3%) were reported more often. Elevated transaminases were seen in 25.5%. Cognitive delay was reported in 5 patients. Urinary galactitol was elevated in all patients at diagnosis; five showed unexpected Gal-1-P increase. Most patients showed enzyme activities ≤1%. Eleven different genotypes were described, including six unpublished variants. The majority was homozygous for NM_000154.1:c.82C>A (p.Pro28Thr). Thirty-five patients were diagnosed following newborn screening, which was clearly beneficial., Conclusion: The phenotype of GALK1 deficiency may include neonatal elevation of transaminases, bleeding diathesis, and encephalopathy in addition to cataract. Potential complications beyond the neonatal period are not systematically surveyed and a better delineation is needed.

Niemann-Pick type C (NPC)1 disease is a rare genetic condition in which the function of the lysosomal cholesterol transporter NPC1 protein is impaired. Consequently, sphingolipids and cholesterol accumulate in lysosomes of all tissues, triggering a cascade of pathological events that culminate in severe systemic and neurological symptoms. Lysosomal cholesterol accumulation is also a key factor in the development of atherosclerosis and NASH. In these two metabolic diseases, the administration of plant stanol esters has been shown to ameliorate cellular cholesterol accumulation and inflammation. Given the overlap of pathological mechanisms among atherosclerosis, NASH, and NPC1 disease, we sought to investigate whether dietary supplementation with plant stanol esters improves the peripheral features of NPC1 disease. To this end, we used an NPC1 murine model featuring a Npc1 -null allele ( Npc1 nih ), creating a dysfunctional NPC1 protein. Npc1 nih mice were fed a 2% or 6% plant stanol ester-enriched diet over the course of 5 weeks. During this period, hepatic and blood lipid and inflammatory profiles were assessed. Npc1 nih mice fed the plant stanol-enriched diet exhibited lower hepatic cholesterol accumulation, damage, and inflammation than regular chow-fed Npc1 nih mice. Moreover, plant stanol consumption shifted circulating T-cells and monocytes in particular toward an anti-inflammatory profile. Overall, these effects were stronger following dietary supplementation with 6% stanols, suggesting a dose-dependent effect. The findings of our study highlight the potential use of plant stanols as an affordable complementary means to ameliorate disorders in hepatic and blood lipid metabolism and reduce inflammation in NPC1 disease., (Copyright © 2020 Magro dos Reis et al.)

Purpose: PMM2-CDG is the most common congenital disorder of glycosylation (CDG), which presents with either a neurologic or multisystem phenotype. Little is known about the longitudinal evolution., Methods: We performed data analysis on PMM2-CDG patients' clinical features according to the Nijmegen CDG severity score and laboratory data. Seventy-five patients (28 males) were followed up from 11.0 ± 6.91 years for an average of 7.4 ± 4.5 years., Results: On a group level, there was no significant evolution in overall clinical severity. There was some improvement in mobility and communication, liver and endocrine function, and strabismus and eye movements. Educational achievement and thyroid function worsened in some patients. Overall, the current clinical function, the system-specific involvement, and the current clinical assessment remained unchanged. On follow-up there was improvement of biochemical variables with (near) normalization of activated partial thromboplastin time (aPTT), factor XI, protein C, antithrombin, thyroid stimulating hormone, and liver transaminases., Conclusion: PMM2-CDG patients show a spontaneous biochemical improvement and stable clinical course based on the Nijmegen CDG severity score. This information is crucial for the definition of endpoints in clinical trials.

Background: Autosomal recessive polycystic kidney disease (ARPKD) with congenital hepatic fibrosis (CHF) causes portal hypertension and its complications. A transjugular intrahepatic portosystemic shunt (TIPSS) could serve as a symptomatic treatment for portal hypertension-related symptoms in these children., Aims: To study the effect of TIPSS on portal hypertension, liver and kidney function and the long term complications., Materials and Methods: We report on 5 children with CHF treated with a TIPSS to manage severe portal hypertension related symptoms., Results: Mean follow-up time was 7 years and 2 months. At the end of follow-up there was a reduction of spleen size (p = 0.715) and hypersplenism with a rise in platelet count (p = 0.465). Esophageal varices and ascites disappeared in all patients. Liver and kidney function remained stable. In two patients endotipsitis was suspected and two patients developed an in-stent stenosis. There was no sign of encephalopathy in our patients., Conclusion: TIPSS using ePTFE-covered stent is a feasible and effective alternative for surgical portosystemic shunting in children with CHF, also on the long term. It can postpone the need of a liver transplantation but close monitoring remains important for early diagnosis of endotipsitis or stent dysfunction related to stenosis., (Copyright © 2018 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Introduction: Cobalamin C (cblC) deficiency is the most common inborn error of vitamin B 12 metabolism. Renal failure attributed to thrombotic microangiopathy (TMA) has occasionally been described in the late-onset presentation of cblC deficiency, but kidney lesions associated with cblC deficiency remain poorly defined. This study aims to describe the characteristics of kidney disease in cblC deficiency, and to provide a comparative histological analysis with cblC-independent renal TMA., Methods: We performed a multicenter retrospective study including 7 patients with cblC deficiency and 16 matched controls with cblC-independent TMA. The patients included were aged 6 to 26 years at the time of the first manifestations. All patients presented with acute renal failure, proteinuria, and hemolysis; 5 patients required dialysis., Results: The histological study revealed arteriolar and glomerular TMA in all patients. After comparison with the cblC-independent TMA control group, a vacuolated aspect of the glomerular basement membrane and the intensity of glomerular capillary wall IgM deposits were more present in cblC deficiency patients than in controls. Six patients were treated with hydroxycobalamin. All of them improved, with disappearance of hemolysis, and 3 of the 4 patients requiring renal replacement therapy were weaned off dialysis., Conclusion: This study provides a precise description of kidney pathology in cblC deficiency. Due to major therapeutic implications, we suggest that patients with renal TMA be screened for cblC deficiency regardless of age, particularly when the kidney biopsy provides evidence of long-lasting TMA, including a vacuolated aspect of the glomerular basement membrane and glomerular capillary wall IgM deposition.

Objective: Heterozygous familial hypercholesterolemia (HeFH) is an autosomal dominant disorder leading to premature atherosclerosis. Guidelines recommend initiating statins early to reduce low-density lipoprotein cholesterol (LDL-C). Studies have evaluated rosuvastatin in children aged ≥10 years, but its efficacy and safety in younger children is unknown., Methods: Children with HeFH and fasting LDL-C >4.92 mmol/L (190 mg/dL) or >4.10 mmol/L (>158 mg/dL) with other cardiovascular risk factors received rosuvastatin 5 mg daily. Based on LDL-C targets (<2.85 mmol/L [<110 mg/dL]), rosuvastatin could be uptitrated to 10 mg (aged 6-9 years) or 20 mg (aged 10-17 years). Treatment lasted 2 years. Changes in lipid values, growth, sexual maturation, and adverse events (AEs) were assessed., Results: The intention-to-treat analysis included 197 patients. At 24 months, LDL-C was reduced by 43, 45, and 35% vs baseline in patients aged 6-9, 10-13, and 14-17 years, respectively (P < .001 for all groups). Most AEs were mild. Intermittent myalgia was reported in 11 (6%) patients and did not lead to discontinuation of rosuvastatin treatment. Serious AEs were reported by 9 (5%) patients, all considered unrelated to treatment by the investigators. No clinically important changes in hepatic biochemistry were reported. Rosuvastatin treatment did not appear to adversely affect height, weight, or sexual maturation., Conclusions: In HeFH patients aged 6-17 years, rosuvastatin 5-20 mg over 2 years significantly reduced LDL-C compared with baseline. Treatment was well tolerated, with no adverse effects on growth or sexual maturation., (Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Background: Statin therapy is recommended for children with familial hypercholesterolemia (FH), but most children do not reach treatment targets., Objective: Here we present the design and results at baseline of the ongoing CHARON study, to evaluate the safety and efficacy of rosuvastatin., Methods: This study comprises an international 2-year open label, titration-to-goal study in 198 children with heterozygous FH aged 6 to 18 years, with rosuvastatin in a maximum dose of 10 mg (<10 years of age) or 20 mg (older children). In addition, 64 unaffected siblings were enrolled as controls. The primary efficacy outcome is the change from baseline in low-density lipoprotein cholesterol, and the secondary outcome is the change in carotid intima-media thickness (c-IMT) in patients with FH compared with their siblings. The primary safety outcomes are growth and sexual maturation; secondary outcomes are the change in other lipoprotein levels and the incidence of adverse events, discontinuation rates, and abnormal laboratory values., Results: At baseline, mean age of patients with FH was 12.1 ± 3.3 years, 44% were boys, and mean low-density lipoprotein cholesterol levels were 6.1 ± 1.3 mmol/L (235.9 ± 48.7 mg/dL). Mean c-IMT was 0.399 mm (95% CI, 0.392-0.406 mm) in children with FH versus 0.377 (95% CI, 0.366-0.388 mm) in unaffected siblings (P = .001)., Conclusions: At baseline, as expected according to on previous observations, children with FH proved to have a greater c-IMT than their healthy siblings. These differences had already occurred at a very young age, which emphasizes the importance of considering early statin initiation in this high-risk population., (Copyright © 2013 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Background: Cystic fibrosis (CF) can be a devastating disease. Disorders in essential fatty acid state are increasingly reported and various supplementation trials have been performed in an attempt to improve outcomes. However, the mechanisms leading to these disturbances remain elusive. We wanted to investigate the role of the diseased CF lung on fatty acid profiles., Methods: We compared fatty acid profiles in patients with CF after lung transplantation (n=11) to age-matched healthy controls and homozygous F508del patients (n=22 each)., Results: Compared to healthy controls, in patients with CF, there are decreased levels of docosahexaenoic, linoleic and arachidonic acid and increased levels of mead acid. In patients that underwent a lung transplantation, levels of docosahexaenoic, linoleic and arachidonic acid were normal. Mead acid did not decrease significantly., Conclusions: The diseased CFTR deficient lung is a major determinant in the disturbed fatty acid profile in CF., (Copyright © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Pruritus is a disabling complication of cholestatic liver disorders. Its management remains challenging. Ultraviolet B (UVB) phototherapy has been successfully used to treat pruritus in other indications., Methods: This is an observational case series. The study population consists of 13 patients (10 females, mean age 52 years) with pruritus due to different cholestatic liver disorders: PBC (n=4), PSC (n=2), drug-induced (n=3) and persistent cholestasis after liver transplantation (LT) (n=4). Serum alkaline phosphatase levels were: 686 ± 363 μ/L and serum bile acids levels: 147 ± 15 μmol/L. In all patients, conventional medical treatment had failed to control pruritus. Perception of pruritus was recorded by the visual analogue scale (VAS)., Results: The mean follow-up was 3 years. Ten patients (77%) had more than 60% reduction in perceived pruritus of which 4 had more than an 80% reduction. Median [25-75% percentiles] VAS score before and after treatment decreased from 8.0 [8.0-10] to 2.0 [1.5-2.1] (p<0.001). The mean number of irradiations required to obtain this effect was 26 ± 17 (average duration of phototherapy: 8 weeks). No significant changes in cholestatic serum markers were observed. Four patients (30%) needed an additional phototherapy course because of recurrent pruritus and in all of them again a marked improvement of pruritus was observed. The therapy was well tolerated, except in two patients who developed, during retreatment, pronounced erythema in one case and paresthesia in the other case., Conclusions: UVB phototherapy appears to be a promising and well tolerated treatment also for cholestasis-associated pruritus., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The development of hepatocellular adenomas and - more rarely - carcinoma in the liver of patients with Glycogen Storage Disease type Ia (GSDIa) is a well-known complication of the disease. The pathophysiology of adenoma and carcinoma development in these patients is, however, hitherto largely unknown and is thought to be related to the metabolic control of the patient and/or the type of mutations in the G6PC gene. We report here on a very illustrative case of adenoma and carcinoma formation in a previously undiagnosed 42 year old male GSDIa patient (enzymatically and genetically proven). He had two episodes of mild hypoglycaemia in childhood, never required formal treatment, showed normal growth, and only mild lactate increases after prolonged starvation. He was a long-distance runner for most of his adult life, without the need for more than normal carbohydrate intake before/during exertion. To gain a better view on the type of adenoma formed in this patient, molecular studies were performed. We show here that in this patient with mild GSDIa without recurrent hypoglycaemic episodes, adenoma and carcinoma formation still occurred and that malignant transformation of adenoma here is associated with CTNNB1 mutations and a typical mRNA profile of a beta-catenin activated lesion., (Copyright 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Cystic fibrosis-associated liver disease (CFLD) is the second cause of mortality in CF. The prevalence is estimated to be 26-45%, but sensitive diagnostic tools are lacking. We investigated whether non-invasive liver elastography (Fibroscan) could serve as a screening tool., Methods: Fibroscan measurements were performed in 66 CF patients. Age-specific cutoff values were determined in a control population (n=59). The measurements were compared to clinical data, bi-yearly biochemistry and ultrasound., Results: Fibroscan was easy to perform in this patient population. There were 14 patients (21%) with abnormal liver stiffness measurements. Liver stiffness was significantly increased in patients with clinical CFLD (11.2 kPa versus 5.1 kPa), biochemical CFLD (7.4 kPa versus 5.4 kPa) or ultrasonographical CFLD (8.2 versus 4.3 kPa) (p<0.02 for all)., Conclusions: Fibroscan is an objective measure and is easy to perform in CF patients, even in children and could provide a valuable tool to detect, and quantify CFLD.

Hepatitis virus replication in the liver is often accompanied by inflammation resulting in the formation of reactive oxygen species (ROS) and nitric oxide (NO) and these may induce cell death. We investigated whether the expression of HBx or HCV core protein in HepG2 cells has an influence on the sensitivity of these cells for oxidative radicals. Our previous study, using the inducible HBV model of HepAD38, revealed that oxidative-stress-related genes are upregulated by virus replication. In the present study, we examined the intracellular pro-oxidant status with dichlorofluorescein (DCF) in HepG2 cell lines transfected with HBx, HbsAg and HCV core. Baseline intracellular oxidative levels were not different in the cell lines expressing viral proteins as compared to control. However, when these cells were exposed to H(2)O(2), the viral protein expressing cells, especially those expressing HBx, showed a reduced level of ROS. This suggests that HBx and HCV core transfected cells can convert H(2)O(2) to less reactive compounds at a higher rate than the control cells. When HBx or HCV core expressing cells were exposed to peroxynitrite (a highly reactive product formed under physiological conditions through interaction of superoxide (O(2)(-)) with NO) these cells were less sensitive to induction of cell death. In addition, these cell lines were less prone to cell death when exposed to H(2)O(2) directly. In conclusion, HBx and HCV core expression in HepG2 cells leads to a survival benefit under oxidative stress which in vivo can be induced during inflammation.

Background/aims: Hepatic stellate cells (HSC) have been hypothesised to derive from the neural crest, based on their expression of multiple neural/neuroendocrine features and their contacts with autonomic nerve endings., Methods: We studied the emergence of HSC in the liver during embryonic development in a transgenic mouse line expressing yellow fluorescent protein (YFP) in all neural crest cells and their derivatives. Cellular YFP expression in these mice was compared with desmin expression between embryonic day (E) 11.5 and adulthood., Results: YFP was abundantly expressed in neural crest cells delaminating from the neural tube and in all known neural crest-derived structures and cell populations. In particular, YFP expressing cells perfectly mimicked the spatial and temporal pattern of enteric nervous system development from neural crest cells migrating from the postotic region. Cells within the adrenal medulla were also YFP positive. Analysis of the liver showed that desmin-expressing, stellate-shaped, perisinusoidally located HSC were evident from E11.5 onwards. However, no detectable YFP expression was seen in the developing liver or in HSC, from E11.5 until adulthood., Conclusions: These findings suggest HSC do not descend from the neural crest, and therefore may derive from the septum transversum mesenchyme, from endoderm or from the mesothelial liver capsule.

In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver.

Background/aims: Hepatic stellate cells (HSC) are commonly considered the precursor population of septal myofibroblasts (MF) in cirrhosis. We studied the distribution and expression profile of mesenchymal (myo)fibroblast-like populations in fibrotic and cirrhotic liver, in an attempt to elucidate their possible interrelationships., Methods: Fibrotic/cirrhotic livers (from 22 human explants and from two rat models: carbon tetrachloride intoxication, bile duct-ligation) were studied by means of immunohistochemistry (single and double immunostaining) with antibodies raised against desmin, alpha-smooth muscle actin (alpha SMA), glial fibrillary acidic protein (GFAP), neural-cell adhesion molecule (N-CAM), synaptophysin, neurotrophins, neurotrophin receptors and alpha B-crystallin (ABCRYS)., Results: Septal MF showed the same expression profile as portal MF, in human and rat, being alpha SMA/ABCRYS/brain-derived nerve growth factor/GFAP-expression, with additional N-CAM- and desmin-expression in rat portal/septal MF. Perisinusoidally located HSC stained with all tested markers, MF at the septal/parenchymal interface showed an expression profile, intermediate between the profiles of HSC and portal/septal MF., Conclusions: In advanced fibrosis and in cirrhosis, regardless of cause or species, three distinct mesenchymal (myo)fibroblast-like liver cell subpopulations can be discerned: portal/septal MF, interface MF and perisinusoidally located HSC. The fact that septal MF share more characteristics with portal MF than with HSC might suggest descent.