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.

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.

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.

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.

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.

A retrospective study assessing fully covered metal stents as first-line management for malignant biliary strictures.

OBJECTIVES: Fully covered self-expanding metal stents (FCSEMS) constitute the first type of metal stent that can easily be removed endoscopically and/or intraoperatively, which may be advantageous in the management of distal malignant biliary strictures (DMBS). To assess the efficacy of FCSEMS as first-line treatment for DMBS, we compared patency, survival and complication rates between FCSEMS, uncovered self-expanding metal stents (USEMS) and plastic stents (PS). METHODS: This was a multicentre retrospective study of 315 consecutive patients with DMBS, who underwent endoscopic retrograde cholangiopancreatography and stenting (FCSEMS, USEMS or PS) at two hospitals between 1 January 2007 and 31 December 2013. Stent patency and patient survival were compared using the Kaplan-Meier method; complication rates were compared using Fisher's exact test; and Cox regression analysis was used to screen for confounding factors. RESULTS: FCSEMS were associated with prolonged stent patency (median=145 days) compared with USEMS (median=110 days, P<0.003) and PS (median=34 days, P<0.001). Biliary sepsis rates were lower for FCSEMS compared with PS (4.7 vs. 17.8%, P=0.02), whereas pancreatitis rates were higher for FCSEMS compared with USEMS (7.8 vs. 1.0%, P=0.04), but not PS (2.6%, P=NS). CONCLUSION: The use of FCSEMS as first-line management for DMBS is associated with longer patency and reduced complication rates compared with the use of PS. However, the higher rate of pancreatitis compared with USEMS requires further evaluation in a large randomized controlled trial.

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.

Pi*Z heterozygous alpha-1 antitrypsin states accelerate parenchymal but not biliary cirrhosis.

OBJECTIVE: The degree to which heterozygous forms of alpha-1 antitrypsin (A1AT), principally MZ, causes liver disease is uncertain. If heterozygosity is a relevant cofactor, over-representation in patients with end-stage liver disease would be predicted. We therefore assessed the prevalence and disease-related distribution of A1AT heterozygosity in the largest cohort to date for this purpose. METHODS: We retrospectively analysed 1036 patients assessed for liver transplantation at our unit between 2003 and 2010. A1AT heterozygotes were identified on the basis of isoelectric focusing and/or histology, showing A1AT globule deposition consistent with an abnormal phenotype. RESULTS: Z-allele frequency was the highest in patients with nonalcoholic steatohepatitis (NASH) cirrhosis (20.3%), followed by patients with 'other parenchymal' diseases (11.9%), alcohol-related liver disease (9.9%), autoimmune disease (8.6%), hepatitis C (6.1%), hepatitis B (3.0%) and biliary disease (1.9%). Compared with the heterozygote frequency in the general European population of 9.0%, the heterozygote frequency was significantly higher among patients with NASH cirrhosis (P≤0.0001) and lower in the biliary subgroup (P=0.004). The prevalence of MZ heterozygosity was significantly increased in cirrhosis because of both alcohol (9.9%) and NASH (17.3%) compared with the general European population (2.8%; P<0.0001). CONCLUSION: Accumulation of misfolded A1AT aggregates appears to accelerate progression, in which the hepatocyte is the key injured cell. Heterozygous A1AT states worsen prognosis, particularly in NASH and alcohol-related cirrhosis, and should be identified at presentation. In cases in which genetic screening is not readily available, a low threshold for isoelectric focusing and routine specific histochemical staining of liver biopsy specimens are warranted to identify these patients.

Orthotopic liver transplantation in an adult with cholesterol ester storage disease.

Cholesterol ester storage disease (CESD) is a rare autosomal recessive lipid storage disorder associated with mutations of the gene encoding lysosomal acid lipase, manifestations of which include chronic liver disease and early atherosclerosis. Although normally presenting in childhood, severity is variable and the condition can occasionally remain undetected until middle age. Typical presentation is with asymptomatic hepatosplenomegaly and hyperlipidaemia, though the condition is probably underdiagnosed. Treatment is supportive and may include attention to cardiovascular risk factors. Phase I/II trials of enzyme replacement therapy are ongoing, but this approach remains experimental. We present the case of a 42-year-old woman diagnosed with CESD in childhood who ran an indolent course until re-presentation with cirrhotic hydrothorax. She underwent orthotopic liver transplantation but required re-transplantation for hepatic artery thrombosis. She remains well with excellent graft function 2 years later. Although atherosclerosis was apparent at assessment, and may have contributed to hepatic artery thrombosis, partial correction of the metabolic defect and restoration of liver function by transplantation together with ongoing medical therapy should permit reasonable survival over the longer term from both a liver and a vascular perspective. This is the first reported case of orthotopic liver transplantation for CESD in an adult, which was the only available option to improve survival. The case highlights the importance of monitoring patients with CESD through adulthood and suggests that liver replacement at a later stage may yet be indicated and remain of benefit.