Magnetic Resonance Liver Iron Concentration Can Guide Venesection Decision-Making in Hyperferritinemia.

BACKGROUND: The clinical benefit of venesection in suspected iron overload can be unclear and serum ferritin may overestimate the degree of iron overload. AIMS: To help inform practice, we examined magnetic resonance liver iron concentration (MRLIC) in a cohort investigated for haemochromatosis. METHODS: One hundred and six subjects with suspected haemochromatosis underwent HFE genotyping and MRLIC with time-matched serum ferritin and transferrin saturation values. For those treated with venesection, volume of blood removed was calculated as a measure of iron overload. RESULTS: Forty-seven C282Y homozygotes had median ferritin 937 µg/l and MRLIC 4.83 mg/g; MRLIC was significantly higher vs non-homozygotes for any given ferritin concentration. No significant difference in MRLIC was observed between homozygotes with and without additional risk factors for hyperferritinemia. Thirty-three compound heterozygotes (C282Y/H63D) had median ferritin 767 µg/l and MRLIC 2.58 mg/g; ferritin < 750 µg/l showed 100% specificity for lack of significant iron overload (< 3.2 mg/g). 79% of C282Y/H63D had additional risk factors-mean MRLIC was significantly lower in this sub-group (2.4 mg/g vs 3.23 mg/g). 26 C282Y heterozygous or wild-type had median ferritin 1226 µg/l and MRLIC 2.13 mg/g; 69% with additional risk factors had significantly higher ferritin concentrations (with comparable MRLIC) and ferritin < 1000 µg/l showed 100% specificity for lack of significant iron overload. In 31 patients (26 homozygotes, 5 C282Y/H63D) venesected to ferritin < 100 µg/l, MRLIC and total venesection volume correlated strongly (r = 0.749), unlike MRLIC and serum ferritin. CONCLUSION: MRLIC is an accurate marker of iron overload in haemochromatosis. We propose serum ferritin thresholds in non-homozygotes which, if validated, could tailor cost-effective use of MRLIC in venesection decision-making.

Pharmacogenomics of drug-induced liver injury (DILI): Molecular biology to clinical applications.

A 21-year old woman was admitted to hospital with a two-week history of painless jaundice, fatigue and anorexia having previously been fit and well. One month prior to presentation, the patient had taken a five-day course of amoxicillin-clavulanic acid for an infected skin cyst. Otherwise, she was only on the oral contraceptive pill and reported minimal alcohol intake. On examination, she was deeply jaundiced, but alert and oriented with no asterixis. She had no stigmata of chronic liver disease, but hepatomegaly extending 3 cm from below the right subcostal margin was evident. Investigations showed: white cell count 13.4 × 109/L (normal 3.6-9.3), haemoglobin 11.8 g/dl (normal 11-15), platelet count 356 × 109/L (normal 170-420), sodium 138 mmol/L (normal 134-144), potassium 3.5 mmol/L (normal 3.5-5.0), creatinine 32 µmol/L (normal 40-75), albumin 30 g/L (normal 35-48), alanine aminotransferase 707 IU/L (normal 15-54), alkaline phosphatase 151 IU/L (normal 30-130), bilirubin 384 µmol/L (normal 7-31) and prothrombin time 27.2 s (normal 11.7-14). Screening for hepatitis A, B, C, E, Epstein-Barr virus, cytomegalovirus and autoimmune hepatitis was negative. Tests for anti-smooth muscle, antinuclear, and anti-liver-kidney microsomal-1 antibodies were negative; immunoglobulin levels and ceruloplasmin levels were normal. Liver ultrasonography demonstrated a liver of normal contour with no biliary dilatation, a normal spleen size and patent vessels. Liver biopsy revealed severe portal interface hepatitis with lobular inflammation and scant plasma cells. Her clinical condition deteriorated in the following days with prothrombin time and bilirubin rising to 56.6 s and 470 µmol/L, respectively. At follow-up after 11 days, her alanine aminotransferase level was 1,931 IU/L. She developed grade 2 hepatic encephalopathy 14 days after presentation, and was listed for a super-urgent liver transplant. Human leucocyte antigen (HLA) typing was performed as a part of preparatory investigations and showed the patient carried the HLA haplotype HLA-DRB1∗15:02-DQB1∗06:01. Following orthotopic transplantation of a deceased donor graft her explant histology revealed severe ongoing hepatitis with multi-acinar necrosis (Fig. 1A and B). This case raised a number of important questions about the diagnosis of drug-induced liver injury and tools available for clinicians to make the best decisions for patient care: In this Grand Rounds article, we will explore these questions, describing the pathophysiology, diagnostic and prognostic biomarkers, and clinical management of drug-induced liver injury. We will also discuss ongoing areas of uncertainty.

Liver transplantation in adults with liver disease due to common variable immunodeficiency leads to early recurrent disease and poor outcome.

Common variable immunodeficiency (CVID) is the most common form of primary immunodeficiency characterized by antibody deficiency, recurrent bacterial infections, and autoimmunity. Advanced chronic liver disease occurs in a subset of patients with CVID and manifests with various histological features, such as nodular regenerative hyperplasia, inflammation, fibrosis, and cholangiopathy. We present a case series characterizing the outcomes in adult patients transplanted for primary CVID-related liver disease. We discuss the unique transplantation challenges faced in this primary immunodeficiency group including susceptibility to infections and early disease recurrence. There is a statistically significant decrease in 3-year and 5-year survival after liver transplantation in those with CVID-related liver disease (55% at 3 and 5 years) compared with all-comers (89% at 3 years, 81% at 5 years), prompting a need for discussion of suitability of transplantation in this group of patients as well as methods for reducing posttransplantation risk such as scrupulous search for infectious agents and reduction of immunosuppression. Liver Transplantation 24 171-181 2018 AASLD.

Impact of D181V and A69T on the function of ferroportin as an iron export pump and hepcidin receptor.

Mutations in the only known mammalian iron exporter ferroportin cause a rare iron overload disorder termed ferroportin disease. Two distinct clinical phenotypes are caused by different disease mechanisms: mutations in ferroportin either cause loss of iron export function or gain of function due to resistance to hepcidin, the peptide hormone that normally downregulates ferroportin. The aim of the present study was to examine the disease mechanisms of the thus far unclassified A69T and D181V ferroportin mutations. We overexpressed wild-type and mutant ferroportin fused to green fluorescent protein in human embryonic kidney cells and used a (59)Fe-assay, intracellular ferritin concentrations, confocal microscopy and flow cytometry to study iron export function, subcellular localization and the responsiveness to hepcidin. While the A69T ferroportin mutation seems not to affect the iron export function it causes dose-dependent hepcidin resistance. We further found that D181V mutated ferroportin is iron export defective and hepcidin resistant, similar to the loss of function mutations A77D and C367X. This indicates that intact iron export might be necessary for hepcidin-induced downregulation of ferroportin. This hypothesis was investigated by studying the hepcidin response under modulation of iron availability. Incubation of wild-type ferroportin overexpressing cells with holo-transferrin increases the hepcidin effect whereas chelating extracellular ferrous iron causes hepcidin resistance. In this study we present data that postulates to classify the D181V ferroportin mutation as loss of function and the A69T mutation as dose-dependent hepcidin resistant and outline a possible causal link between iron export function and the hepcidin effect.

Prevalence and risk factors for liver involvement in individuals with PiZZ-related lung disease.

RATIONALE: α1-Antitrypsin deficiency is one of the most common heritable human diseases, predisposing to liver and lung injury. Significant heterogeneity in phenotypic expression is well documented, but less is known of the prevalence, severity, and correlates of chronic liver disease among individuals presenting with lung disease. OBJECTIVES: To determine the frequency of and risk factors for severe liver fibrosis and cirrhosis among individuals with PiZZ-related lung disease. METHODS: A well-characterized cohort of 57 PiZZ adults attending a tertiary referral respiratory clinic was screened prospectively for clinical, laboratory, radiologic, and (when appropriate) histologic evidence of chronic liver disease. MEASUREMENTS AND MAIN RESULTS: Thirty-six (63.2%) of 57 had a history or clinical findings suggestive of liver disease; or had one or more abnormalities of liver function, or liver ultrasound, and 24 of these underwent liver biopsy. Ten (17.5%) had evidence of severe fibrosis or cirrhosis and were more likely to have higher body mass index (P = 0.04), alanine transaminase (P = 0.0001), alkaline phosphatase (P = 0.0009), prothrombin time (P = 0.0005), and maximal vital capacity (VCmax) (P = 0.04); lower platelet count (P = 0.007); abnormal liver echogenicity (P < 0.001); and splenomegaly (P = 0.001) at ultrasound. Screening with liver ultrasound provided a sensitivity and negative predictive value for severe fibrosis or cirrhosis of 100%, as were the specificity and positive predictive value for platelet count less than or equal to 174,000 per mm(3) and splenomegaly. Among individuals undergoing liver biopsy, fibrosis stage correlated with increasing VCmax (P = 0.02) and % predicted VCmax (P = 0.05), and decreasing residual volume/total lung capacity (TLC) (P = 0.02) and % predicted residual volume/TLC (P = 0.05). CONCLUSIONS: Significant chronic liver disease is common in PiZZ individuals with lung disease and can be screened effectively by a combination of conventional tests of liver function, platelet count, and liver ultrasound.

Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans.

Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. Here, we identify, confirm, and replicate the biomarker performance characteristics of candidate proteins in patients with DILI at onset (DO; n = 133) and follow-up (n = 120), acute non-DILI at onset (NDO; n = 63) and follow-up (n = 42), and healthy volunteers (HV; n = 104). Area under the receiver operating characteristic curve (AUC) for cytoplasmic aconitate hydratase, argininosuccinate synthase, carbamoylphosphate synthase, fumarylacetoacetase, fructose-1,6-bisphosphatase 1 (FBP1) across cohorts achieved near complete separation (range: 0.94-0.99) of DO and HV. In addition, we show that FBP1, alone or in combination with glutathione S-transferase A1 and leukocyte cell-derived chemotaxin 2, could potentially assist in clinical diagnosis by distinguishing NDO from DO (AUC range: 0.65-0.78), but further technical and clinical validation of these candidate biomarkers is needed.

Identification of mutations in SLC40A1 that affect ferroportin function and phenotype of human ferroportin iron overload.

BACKGROUND & AIMS: Patients with ferroportin iron overload due to loss-of-function mutations in SLC40A1 have macrophage iron overload, hyperferritinemia, and normal transferrin saturation. In contrast, hepatocellular iron storage, hyperferritinemia, and increased saturation of transferrin are a distinct clinical presentation of ferroportin iron overload that results from SLC40A1 mutations that confer resistance of ferroportin to hepcidin-mediated inactivation. METHODS: SLC40A1 was sequenced in patients from 2 independent pedigrees affected by hepatic iron overload unrelated to HFE. Functions of the ferroportin variants were tested in vitro. RESULTS: A patient heterozygous for the variant p.W158C in SLC40A1 presented with macrophage iron overload, hyperferritinemia, and normal transferrin saturation. A patient with hepatocellular iron storage, hyperferritinemia, and increased transferrin saturation was heterozygous for p.H507R. Expression of the p.W158C form of ferroportin in 293T cells resulted in defective trafficking to the plasma membrane and reduced iron export activity; the iron export activity of cells that expressed the p.H507R form of ferroportin did not differ from cells that expressed ferroportin without this mutation. The p.H507R of ferroportin localizes normally to the plasma membrane but is resistant to hepcidin-mediated inactivation. Addition of a synthetic peptide derived from ferroportin without these mutations (amino acids 500-518) decreased the inhibitory activity of hepcidin in cells, whereas a peptide from the same region, with p.H507R, had no effect on hepcidin activity. CONCLUSIONS: The variant p.W158C in SLC40A1 impairs intracellular trafficking of ferroportin, resulting in reduced iron export. The variant p.H507R does not bind hepcidin in vitro and results in apparent hepcidin resistance.

A comparison of MR elastography and 31P MR spectroscopy with histological staging of liver fibrosis.

OBJECTIVES: Conventional imaging techniques are insensitive to liver fibrosis. This study assesses the diagnostic accuracy of MR elastography (MRE) stiffness values and the ratio of phosphomonoesters (PME)/phosphodiesters (PDE) measured using (31)P spectroscopy against histological fibrosis staging. METHODS: The local research ethics committee approved this prospective, blinded study. A total of 77 consecutive patients (55 male, aged 49 ± 11.5 years) with a clinical suspicion of liver fibrosis underwent an MR examination with a liver biopsy later the same day. Patients underwent MRE and (31)P spectroscopy on a 1.5 T whole body system. The liver biopsies were staged using an Ishak score for chronic hepatitis or a modified NAS fibrosis score for fatty liver disease. RESULTS: MRE increased with and was positively associated with fibrosis stage (Spearman's rank = 0.622, P < 0.001). PME/PDE was not associated with fibrosis stage (Spearman's rank = -0.041, p = 0.741). Area under receiver operating curves for MRE stiffness values were high (range 0.75-0.97). The diagnostic utility of PME/PDE was no better than chance (range 0.44-0.58). CONCLUSIONS: MRE-estimated liver stiffness increases with fibrosis stage and is able to dichotomise fibrosis stage groupings. We did not find a relationship between (31)P MR spectroscopy and fibrosis stage. KEY POINTS: Magnetic resonance elastography (MRE) and MR spectroscopy can both assess the liver. MRE is superior to ( 31 ) P MR spectroscopy in staging hepatic fibrosis. MRE is able to dichotomise liver fibrosis stage groupings. Gradient-echo MRE may be problematic in genetic haemochromatosis.

First report of post-transplant autoimmune hepatitis recurrence following SARS-CoV-2 mRNA vaccination.

Whilst vaccination for the SARS-CoV-2 virus has been successful in reducing the severity and burden of the COVID-19 pandemic, there have been recent reports of mRNA vaccines triggering autoimmune hepatitis in the native liver. There have been no descriptions thus far of recurrent 'autoimmune hepatitis' after liver transplantation in the context of SARS-CoV-2 vaccination. We describe a patient transplanted for autoimmune hepatitis who was stable for many years until they had immune-mediated flares coinciding with Pfizer-BioNTech mRNA vaccination. Intravenous steroid treatment was required to suppress histologically evident interface hepatitis. We firmly believe that mRNA vaccination was responsible for this 'recurrence' and that clinicians should be vigilant for this reaction in patients transplanted for autoimmune hepatitis.

Synonymous mutation in adenosine triphosphatase copper-transporting beta causes enhanced exon skipping in Wilson disease.

Wilson disease (WD) is caused by biallelic pathogenic variants in adenosine triphosphatase copper-transporting beta (ATP7B); however, genetic testing identifies only one or no pathogenic ATP7B variant in a number of patients with WD. Synonymous single-nucleotide sequence variants have been recognized as pathogenic in individual families. The aim of the present study was to evaluate the prevalence and disease mechanism of the synonymous variant c.2292C>T (p.Phe764=) in WD. A cohort of 280 patients with WD heterozygous for a single ATP7B variant was investigated for the presence of c.2292C>T (p.Phe764=). In this cohort of otherwise genetically unexplained WD, the allele frequency of c.2292C>T (p.Phe764=) was 2.5% (14 of 560) compared to 7.1 × 10-6 in the general population (2 of 280,964 in the Genome Aggregation Database; p < 10-5 ; Fisher exact test). In an independent United Kingdom (UK) cohort, 2 patients with WD homozygous for p.Phe764= were identified. RNA analysis of ATP7B transcripts from patients homozygous or heterozygous for c.2292C>T and control fibroblasts showed that this variant caused high expression of an ATP7B transcript variant lacking exon 8. Conclusion: The synonymous ATP7B variant c.2292C>T (p.Phe764=) causes abnormal messenger RNA processing of ATP7B transcripts and is associated with WD in compound heterozygotes and homozygotes.

Clinical presentation and molecular pathophysiology of autosomal dominant hemochromatosis caused by a novel ferroportin mutation.

UNLABELLED: Mutations in the SLC40A1 gene, which encodes ferroportin, are associated with autosomal dominant hemochromatosis. Ferroportin is inhibited directly by hepcidin, a key iron-regulatory peptide, and functional consequences of SLC40A1 mutations account for observed phenotypic differences in patients with ferroportin disease. We describe a large pedigree with a novel SLC40A1 mutation and, through in vitro analysis, elucidate the associated molecular mechanism of iron overload. The entire coding sequence of the SLC40A1 gene was sequenced in a pedigree, presenting with autosomal dominant hyperferritinemia. The functional effects of a novel SLC40A1 mutation were studied by overexpression of wild-type and mutant ferroportin fusion proteins in human embryonic kidney cells. Iron export was studied in these cells using (59)Fe transport assays; subcellular localization of ferroportin was examined by way of confocal microscopy. A novel SLC40A1 mutation p.R489K segregated with iron overload in a family with clinical and histopathological signs of macrophage-type ferroportin disease. Human embryonic kidney cells overexpressing p.R489K ferroportin showed decreased iron export capacity when compared with wild-type ferroportin overexpressing cells. Subcellular localization studies demonstrated that p.R489K ferroportin was retained abnormally within an intracellular compartment. CONCLUSION: We report a novel pathological SLC40A1 variant associated with abnormal cell surface expression of ferroportin due to intracellular retention of the mutant protein. These findings predict macrophage-type ferroportin disease, the phenotype observed in this kindred. Study of the molecular cell biology of ferroportin and its mutants is key to understanding the pathogenesis of this increasingly recognized form of hemochromatosis, which responds poorly to conventional therapy.

Juvenile haemochromatosis.

Juvenile haemochromatosis is a severe inherited iron-loading disorder that can present in children and adolescents. Typical manifestations include heart failure, endocrine failure (including diabetes and hypogonadism), cirrhosis, and arthropathy. Compared with HFE haemochromatosis, juvenile haemochromatosis affects female and male individuals similarly, presents at a younger age, and causes multiple organ dysfunction; the principle of iron loading into tissues from the gut is shared by both forms, but the process is far more rapid in juvenile haemochromatosis. Juvenile haemochromatosis is initially recognised by extreme increases of serum ferritin and transferrin saturation, which is supported by an MRI showing iron deposition in the heart and liver. MRI software techniques allow quantification of iron in these organs, and can therefore be used to monitor progress. Juvenile haemochromatosis is autosomal recessive and is generally associated with mutations in HJV (type 2A) or HAMP (type 2B). Mutations in TFR2 cause an intermediate severity phenotype (type 3), but this phenotype can cross over into the juvenile haemochromatosis spectrum so it might need to be additionally considered during diagnosis. Treatment needs to be administered without delay, in the form of aggressive iron chelation, and a multidisciplinary approach is essential. Because iron is removed, organ function is restored, which could obviate the need for cardiac or liver transplantation. Substantial restoration of health can ensue, but patients require life-long monitoring. Family screening is an important component of the management of juvenile haemochromatosis. Genetic advances which underpin the haemochromatosis types also clarify the role of iron metabolism in health and disease, particularly the role of hepcidin in regulating iron homoeostasis. Therefore, juvenile haemochromatosis is an important condition to understand; it can present insidiously in children and adolescents, and awareness of the diagnosis is needed to inform early recognition and treatment.

First report of liver transplantation in Blau syndrome: The challenges faced in this rare granulomatous liver disease.

Blau syndrome is a rare autoinflammatory granulomatous disease caused by variants in the NOD2 gene, classically presenting in childhood. Hepatic manifestations are recognized including cholestasis and granulomatous liver disease. We describe a novel NOD2 gene variant c.1471A > C, p.(Met491Leu) in an adult who developed cirrhotic complications despite selective immunotherapy, including recurrent esophageal bleeding and spontaneous bacterial peritonitis which resulted in liver transplantation. He required a second liver transplant as his first graft failed due to ischemic cholangiopathy. Disease recurrence has been observed (hitherto unreported). Of 84 patients with Blau syndrome treated with antibody therapy, five hepatic cases responded to anti-TNF therapy, with promising results if instigated before decompensation occurs. We report the first case of liver transplantation for Blau syndrome in an adult with a novel NOD2 variant. Blau related liver disease can reoccur post transplantation and is an important consideration for any future graft. LAY SUMMARY: Blau syndrome is a rare immune disease which presents in childhood. We describe the first liver transplant for this condition following development of progressive liver disease in adulthood. The patient had a newly described variant in the Blau gene (NOD2). We discuss the effectiveness of antibody therapy currently being used to control the disease, and the role of liver transplantation in Blau syndrome.

Variants in PCSK7, PNPLA3 and TM6SF2 are risk factors for the development of cirrhosis in hereditary haemochromatosis.

BACKGROUND: Cirrhosis develops in <10% of individuals homozygous for the C282Y variant in the homeostatic iron regulator (HFE) gene. Carriage of PCSK7:rs236918 is associated with an increased risk of cirrhosis in this population. AIM: To determine if genetic variants significantly associated with the risk of alcohol- and NAFLD-related cirrhosis also modulate the cirrhosis risk in C282Y homozygotes. METHODS: Variants in PCSK7, PNPLA3, TM6SF2, MBOAT7 and HSD17B13 were genotyped in 1319 C282Y homozygotes, from six European countries, of whom 171 (13.0%) had cirrhosis. Genotypic and allelic associations with the risk for developing cirrhosis were assessed, adjusting for age and sex. Fixed effects meta-analyses of the adjusted summary data for each country were performed. Post hoc association testing was undertaken in the 131 (76.6%) cases and 299 (26.0%) controls with available liver histology. RESULTS: Significant associations were observed between PCSK7:rs236918 (OR = 1.52 [95% CI 1.06-2.19]; P = 0.022; I2  = 0%); PNPLA3:rs738409 (OR = 1.60 [95% CI 1.22-2.11]; P = 7.37 × 10-4 ; I2  = 45.5%) and TM6SF2:rs58542926 (OR = 1.94 [95% CI 1.28-2.95]; P = 1.86 × 10-3 ; I2  = 0%) and the cirrhosis risk in C282Y homozygotes. These findings remained significant in the subpopulation with available liver histology. The population-attributable fractions were 5.6% for PCSK7:rs236918, 13.8% for PNPLA3:rs738409, 6.5% for TM6SF2:rs58542926 and 24.0% for carriage of all three variants combined. CONCLUSIONS: The risk of cirrhosis associated with carriage of PCSK7:rs236918 was confirmed in this much larger population of C282Y homozygotes. In addition, PNPLA3:rs738409 and TM6SF2:rs58542926 were established as significant additional risk factors. More detailed genetic testing of C282Y homozygotes would allow risk stratification and help guide future management.

Ferroportin disease: a systematic meta-analysis of clinical and molecular findings.

BACKGROUND & AIMS: Classical ferroportin disease is characterized by hyperferritinemia, normal transferrin saturation, and iron overload in macrophages. A non-classical form is characterized by additional hepatocellular iron deposits and a high transferrin saturation. Both forms demonstrate autosomal dominant transmission and are associated with ferroportin gene (SLC40A1) mutations. SLC40A1 encodes a cellular iron exporter expressed in macrophages, enterocytes, and hepatocytes. The aim of the analysis is to determine the penetrance of SLC40A1 mutations and to evaluate in silico tools to predict the functional impairment of ferroportin mutations as an alternative to in vitro studies. METHODS: We conducted a systematic review of the literature and meta-analysis of the biochemical presentation, genetics, and pathology of ferroportin disease. RESULTS: Of the 176 individuals reported with SLC40A1 mutations, 80 were classified as classical phenotype with hyperferritinemia and normal transferrin saturation. The non-classical phenotype with hyperferritinemia and elevated transferrin saturation was present in 53 patients. The remaining patients had normal serum ferritin or the data were reported incompletely. Despite an increased hepatic iron concentration in all biopsied patients, significant fibrosis or cirrhosis was present in only 11%. Hyperferritinemia was present in 86% of individuals with ferroportin mutations. Bio-informatic analysis of ferroportin mutations showed that the PolyPhen score has a sensitivity of 99% and a specificity of 67% for the discrimination between ferroportin mutations and polymorphisms. CONCLUSIONS: In contrast to HFE hemochromatosis, ferroportin disease has a high penetrance, is genetically heterogeneous and is rarely associated with fibrosis. Non-classical ferroportin disease is associated with a higher risk of fibrosis and a more severe overload of hepatic iron.