Herbert L. Bonkovsky

A variant upstream of IFNL3 ( IL28B ) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus

Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA sequencing in primary human hepatocytes activated with synthetic double-stranded RNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a new transiently induced region that harbors a dinucleotide variant ss469415590 (TT or ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590[ΔG] is a frameshift variant that creates a novel gene, designated IFNL4, encoding the interferon-λ4 protein (IFNL4), which is moderately similar to IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians. Transient overexpression of IFNL4 in a hepatoma cell line induced STAT1 and STAT2 phosphorylation and the expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.

Causes, Clinical Features, and Outcomes From a Prospective Study of Drug-Induced Liver Injury in the United States

BACKGROUND & AIMS Idiosyncratic drug-induced liver injury (DILI) is among the most common causes of acute liver failure in the United States, accounting for approximately 13% of cases. A prospective study was begun in 2003 to recruit patients with suspected DILI and create a repository of biological samples for analysis. This report summarizes the causes, clinical features, and outcomes from the first 300 patients enrolled. METHODS Patients with suspected DILI were enrolled based on predefined criteria and followed up for at least 6 months. Patients with acetaminophen liver injury were excluded. RESULTS DILI was caused by a single prescription medication in 73% of the cases, by dietary supplements in 9%, and by multiple agents in 18%. More than 100 different agents were associated with DILI; antimicrobials (45.5%) and central nervous system agents (15%) were the most common. Causality was considered to be definite in 32%, highly likely in 41%, probable in 14%, possible in 10%, and unlikely in 3%. Acute hepatitis C virus (HCV) infection was the final diagnosis in 4 of 9 unlikely cases. Six months after enrollment, 14% of patients had persistent laboratory abnormalities and 8% had died; the cause of death was liver related in 44%. CONCLUSIONS DILI is caused by a wide array of medications, herbal supplements, and dietary supplements. Antibiotics are the single largest class of agents that cause DILI. Acute HCV infection should be excluded in patients with suspected DILI by HCV RNA testing. The overall 6-month mortality was 8%, but the majority of deaths were not liver related.

Incidence of Hepatocellular Carcinoma and Associated Risk Factors in Hepatitis C-Related Advanced Liver Disease

BACKGROUND & AIMS Although the incidence of hepatocellular carcinoma (HCC) is increasing in the United States, data from large prospective studies are limited. We evaluated the Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis (HALT-C) cohort for the incidence of HCC and associated risk factors. METHODS Hepatitis C virus-positive patients with bridging fibrosis or cirrhosis who did not respond to peginterferon and ribavirin were randomized to groups that were given maintenance peginterferon for 3.5 years or no treatment. HCC incidence was determined by Kaplan-Meier analysis, and baseline factors associated with HCC were analyzed by Cox regression. RESULTS 1,005 patients (mean age, 50.2 years; 71% male; 72% white race) were studied; 59% had bridging fibrosis, and 41% had cirrhosis. During a median follow-up of 4.6 years (maximum, 6.7 years), HCC developed in 48 patients (4.8%). The cumulative 5-year HCC incidence was similar for peginterferon-treated patients and controls, 5.4% vs 5.0%, respectively (P= .78), and was higher among patients with cirrhosis than those with bridging fibrosis, 7.0% vs 4.1%, respectively (P= .08). HCC developed in 8 (17%) patients whose serial biopsy specimens showed only fibrosis. A multivariate analysis model comprising older age, black race, lower platelet count, higher alkaline phosphatase, esophageal varices, and smoking was developed to predict the risk of HCC. CONCLUSIONS We found that maintenance peginterferon did not reduce the incidence of HCC in the HALT-C cohort. Baseline clinical and laboratory features predicted risk for HCC. Additional studies are required to confirm our finding of HCC in patients with chronic hepatitis C and bridging fibrosis.

Outcome of sustained virological responders with histologically advanced chronic hepatitis C

Retrospective studies suggest that subjects with chronic hepatitis C and advanced fibrosis who achieve a sustained virological response (SVR) have a lower risk of hepatic decompensation and hepatocellular carcinoma (HCC). In this prospective analysis, we compared the rate of death from any cause or liver transplantation, and of liver‐related morbidity and mortality, after antiviral therapy among patients who achieved SVR, virologic nonresponders (NR), and those with initial viral clearance but subsequent breakthrough or relapse (BT/R) in the HALT‐C (Hepatitis C Antiviral Long‐Term Treatment Against Cirrhosis) Trial. Laboratory and/or clinical outcome data were available for 140 of the 180 patients who achieved SVR. Patients with nonresponse (NR; n = 309) or who experienced breakthrough or relapse (BT/R; n = 77) were evaluated every 3 months for 3.5 years and then every 6 months thereafter. Outcomes included death, liver‐related death, liver transplantation, decompensated liver disease, and HCC. Median follow‐up for the SVR, BT/R, and NR groups of patients was 86, 85, and 79 months, respectively. At 7.5 years, the adjusted cumulative rate of death/liver transplantation and of liver‐related morbidity/mortality in the SVR group (2.2% and 2.7%, respectively) was significantly lower than that of the NR group (21.3% and 27.2%, P < 0.001 for both) but not the BT/R group (4.4% and 8.7%). The adjusted hazard ratio (HR) for time to death/liver transplantation (HR = 0.17, 95% confidence interval [CI] = 0.06‐0.46) or development of liver‐related morbidity/mortality (HR = 0.15, 95% CI = 0.06‐0.38) or HCC (HR = 0.19, 95% CI = 0.04‐0.80) was significant for SVR compared to NR. Laboratory tests related to liver disease severity improved following SVR. Conclusion: Patients with advanced chronic hepatitis C who achieved SVR had a marked reduction in death/liver transplantation, and in liver‐related morbidity/mortality, although they remain at risk for HCC. (HEPATOLOGY 2010;)

Recommendations for the Diagnosis and Treatment of the Acute Porphyrias

Key Summary Points Early Diagnosis of Acute Porphyria Consider in all adults with unexplained symptoms seen in acute porphyrias (Table 2); certain clinical features are suggestive: women of reproductive age; abdominal pain; muscle weakness; hyponatremia; and dark or reddish urine. Establish diagnosis promptly by testing for increased porphobilinogen in a single-void urine (we recommend the Trace PBG Kit [Thermo Trace/DMA, Arlington, Texas]). If porphobilinogen is increased, begin treatment immediately. To establish the type of acute porphyria, save the same urine sample for measurement of ALA, porphobilinogen, and porphyrin levels, and measure plasma porphyrin levels, fecal porphyrin levels, and erythrocyte porphobilinogen deaminase levels (Table 5 and Figure). Treatment of the Acute Attack Hospitalize patient for control of acute symptoms and withdraw all unsafe medications (see Table 3) and other possible precipitating factors. Provide nutritional support and symptomatic and supportive treatment; consider seizure precautions, especially if patient is hyponatremic; use medications that are known to be safe in the acute porphyrias; and use intravenous fluids to correct dehydration and electrolyte imbalances, narcotic analgesics for pain, phenothiazine for nausea or vomiting, and -adrenergic blockers for hypertension and symptomatic tachycardia. Begin hemin (3 to 4 mg/kg daily for at least 4 days) as soon as possible. Intravenous glucose alone (10%, at least 300 g daily) may resolve mild attacks (mild pain, no paresis, or hyponatremia) or can be given while awaiting delivery of hemin. Monitor patient closely: Check vital capacity (if impaired, place patient in intensive care) and neurologic status, including muscle strength (especially proximal); check serum electrolytes, creatinine, and magnesium levels at least daily; and watch for bladder distention. Follow-up Educate patient and family about the disease, its inheritance, precipitating factors, and important preventive measures. Encourage patients to wear medical alert bracelets and keep records of diagnostic studies and recommended therapy. Treat chronic manifestations (such as pain and depression) and disability. Provide access to genetic testing for patient and family members. The acute porphyrias are well-defined genetic disorders of heme biosynthesis characterized by acute life-threatening attacks of nonspecific neurologic symptoms (1). Although the specific enzyme and gene defects have been identified, diagnosis and treatment of these 4 disorders still present formidable challenges because their symptoms and signs mimic other, more common conditions. Delaying diagnosis and treatment of acute porphyric attacks can be fatal or can cause long-term or permanent neurologic damage. Updated, consistent recommendations for timely diagnosis and treatment of these disorders have been lacking, despite the existence of rapid, sensitive, and specific biochemical tests (2) and the availability of an effective therapy, which was first described more than 30 years ago (3) and was approved by the U.S. Food and Drug Administration (FDA) more than 20 years ago. Formation of an Expert Panel and Basis of Recommendations Concerns about misdiagnosis, delayed diagnosis, and inappropriate therapy prompted the American Porphyria Foundation to assemble a panel of experts on the acute porphyrias who were selected on the basis of their clinical and research experience and their contributions to the medical literature. The panel, which represents specialties including internal medicine, pediatrics, genetics, gastroenterology, hepatology, and hematology, was charged with formulating updated recommendations for diagnosing and treating the acute porphyrias. With support from the American Porphyria Foundation, the panel members convened for a day-long meeting to formulate clinical recommendations. Two members, assisted by a medical writer funded by the Foundation, prepared a draft manuscript based on the panels discussion and recommendations. All panel members participated in the review and revision of the manuscript and agreed to the final version. Recommendations are based on the clinical experience of the authors and their review of the literature. Because the acute porphyrias are rare, most of the literature consists of reviews, small series, and case studies. A detailed MEDLINE search on treatment of acute attacks, for example, revealed 71 papers (55 in English and 16 with English abstracts) published between 1966 and October 2004. Of these, 41 were single-case reports, 13 were case series of 10 or fewer patients, and 17 (11 in English) were studies with more than 10 patients (4-20). Altogether, 53 papers discuss more than 1000 patients who received hemin therapy with or without initial treatment with glucose. The American Porphyria Foundation partially funded this review. This nonprofit organization provides information and support to patients with porphyria and their physicians. It receives funding from private sources in addition to a nonrestricted grant from Ovation Pharmaceuticals, the manufacturer of hemin for injection (Panhematin), the only FDA-approved hemin therapy for the acute porphyrias. The Foundation and Ovation Pharmaceuticals had no role in the literature review, the formulation of recommendations, or the drafting and revising of the manuscript. Overview of the Acute Porphyrias Acute Porphyrias Are Inborn Errors of Heme Biosynthesis Each of the acute porphyrias results from the deficient activity of a distinct enzyme in the heme biosynthetic pathway (1). Porphyrias are classified as hepatic or erythroid, depending on whether most of the heme biosynthetic intermediates arise from, and accumulate in, the liver or in developing erythrocytes. They are also classified clinically as acute or cutaneous on the basis of their major clinical manifestations. Of the 5 hepatic porphyrias, 4 characteristically present with acute attacks of neurologic manifestationshence the designation acute porphyrias, a term that does not fully describe the clinical features, which can be prolonged and chronic. Table 1 shows the genetic and enzymatic features of the 4 acute hepatic porphyrias (21): acute intermittent porphyria, hereditary coproporphyria, variegate porphyria, and the very rare 5-aminolevulinic acid (ALA)dehydratase porphyria. The combined prevalence of these diseases is approximately 5 cases per 100000 persons (1). Numerous mutations have been identified for each disorder. The major manifestations of the acute porphyrias are neurologic, including neuropathic abdominal pain, peripheral neuropathy, and mental disturbances (Table 2) (1, 4, 22-25). These develop during adult life, are more common in women than in men, and are treated by methods to restore heme homeostasis. Variegate porphyria and, much less commonly, hereditary coproporphyria can also cause chronic, blistering lesions on sun-exposed skin that are identical to those in porphyria cutanea tarda, a much more common condition. Photocutaneous lesions may occur without neuropathic manifestations. Table 1. Characteristics of the 4 Acute Porphyrias Table 2. Common Presenting Symptoms and Signs of Acute Porphyria In addition to their highly variable neurologic signs and symptoms, the acute porphyrias are distinct from other porphyrias because of their common overproduction of the porphyrin precursors ALA (an amino acid), and porphobilinogen (a pyrrole). This striking biochemical feature is important for laboratory diagnosis and has implications for pathogenesis of the neurologic manifestations. While porphyrins (tetrapyrroles) are also increased, their measurement is of little value for initial diagnosis because they are also increased (in urine, feces, erythrocytes, or plasma) in other porphyrias and many other medical conditions. Pathogenesis of Acute Attacks The enzyme deficiency in each disorder is partial (approximately 50% of normal in the 3 most common acute porphyrias), and the remaining enzyme activity is usually sufficient for heme homeostasis. Because ALA dehydratase activity normally greatly exceeds that of the other enzymes in the pathway, a more severe deficiency of this enzyme (5% of normal) is required to cause manifestations of ALA-dehydratase porphyria. These enzymatic defects predispose the affected persons to the effects of precipitating factors, including many drugs (for example, barbiturates, anticonvulsants, rifampin, and progestins), endogenous steroid hormones (especially progesterone), fasting, dieting, smoking, and stress from illness, all of which can increase the demand for hepatic heme and induce synthesis of ALA synthase, the first enzyme in the heme biosynthetic pathway. Because hepatic ALA synthase is rate-controlling, production of heme pathway intermediates increases to the point at which the inherited partial enzyme deficiency becomes limiting, and intermediates accumulate in the liver. Porphobilinogen and ALA levels are increased in all patients with acute symptoms of these disorders and in some who are asymptomatic. The cause of hepatic overproduction of porphyrin precursors in the acute porphyrias is better understood than are the mechanisms for neurologic damage. Presumably, symptoms result primarily from the porphyrin precursors themselves rather than a deficiency of heme in nerve tissue (26, 27). Chronic symptoms and signs may reflect previous, unresolved neurologic damage. In the very rare cases of homozygous acute intermittent porphyria (26), variegate porphyria (28), and hereditary coproporphyria (29), severe neurologic manifestations begin in childhood. An allogeneic liver transplantation in a woman with heterozygous acute intermittent porphyria normalized her urinary ALA and porphobilinogen levels in 24 hours and completely eliminated her recurrent neurologic attacks, which supports the hepatic overproduction of porphyrin precursors in causing the neurologic symptoms (27). Si

Non-alcoholic steatohepatitis and iron: increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis.

BACKGROUND/AIMS Non-alcoholic steatohepatitis (NASH) is increasingly recognized, and its pathogenesis is believed to involve increased oxidative stress. Elevated levels of serum ferritin and positive liver iron stains are often observed in patients with NASH, and the pathogenesis of liver injury due to iron is also thought to involve oxidative stress. The aim of this study was to determine whether there is an association of NASH and mutations in the HFE gene associated with hereditary hemochromatosis (HHC). METHODS Clinical, laboratory, and histopathological data on all 57 subjects with a final diagnosis of NASH seen between August 1990 and August 1997 at our Liver Center were analyzed. Thirty-six Caucasian subjects (23 men) with NASH underwent mutational analyses of HFE gene mutations performed. The prevalence of HFE gene mutations was compared to that in 348 Caucasian normal controls. Data were analyzed by both parameteric and non-parametric methods with similar results. RESULTS One subject (2.8%) with NASH was homozygous for the C282Y mutation and six (16.7%) were heterozygous, compared with 0%, and 11.2%, respectively, of controls. Two (5.6%) subjects with NASH were homozygous for the H63D mutation and 16 (44.4%) were heterozygous, whereas 2.9% and 26.4%, respectively, of controls had these genotypes. The prevalence of heterozygosity (61.1%) for either mutation was significantly higher in subjects with NASH than in controls (38%) (p = 0.008), and the prevalence of homozygosity or heterozygosity combined in NASH subjects (69.4%) was significantly higher than for controls (40.5%, p = 0.001). Sex (63-67% male) and age at diagnosis of NASH did not differ between those with or without HFE mutations, but men with NASH were significantly more likely than women to have the H63D mutation (15/23 vs. 3/13, p<0.05) Levels of serum ferritin, iron, transferrin saturation levels, and the degree of hepatic iron staining were significantly higher (p<0.05) in subjects with NASH who carried an HFE mutation than in those without. Differences in hepatic iron concentrations or hepatic iron indices between NASH subjects with and without HFE mutations were not significant. Those with C282Y mutations had significantly more hepatic fibrosis than those without (p<0.05). Those with HFE mutations had significantly higher levels of serum ALT (90+/-11 [mean +/- SE]) than those without (55+/-6; p = 0.02). CONCLUSION The prevalences of the HFE gene mutations associated with hereditary hemochromatosis are increased among North American subjects with NASH.

Des-γ-Carboxy Prothrombin and α-Fetoprotein as Biomarkers for the Early Detection of Hepatocellular Carcinoma

BACKGROUND & AIMS The outcome of patients with hepatocellular carcinoma (HCC) remains poor because of late diagnosis. The aim of this study was to compare the accuracy of alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) in the early diagnosis of HCC. METHODS Among 1031 patients randomized in the Hepatitis C Antiviral Long-term Treatment Against Cirrhosis (HALT-C) Trial, a nested case-control study of 39 HCC cases (24 early stage) and 77 matched controls was conducted to compare the performance of AFP and DCP. Testing was performed on sera from 12 months prior (month -12) to the time of HCC diagnosis (month 0). RESULTS The sensitivity and specificity of DCP at month 0 was 74% and 86%, respectively, at a cutoff of 40 mAU/mL and 43% and 100%, respectively, at a cutoff of 150 mAU/mL. The sensitivity and specificity of AFP at month 0 was 61% and 81% at a cutoff of 20 ng/mL and 22% and 100% at a cutoff of 200 ng/mL. At month -12, the sensitivity and specificity at the low cutoff was 43% and 94%, respectively, for DCP and 47% and 75%, respectively, for AFP. Combining both markers increased the sensitivity to 91% at month 0 and 73% at month 12, but the specificity decreased to 74% and 71%, respectively. Diagnosis of early HCC was triggered by surveillance ultrasound in 14, doubling of AFP in 5, and combination of tests in 5 patients. CONCLUSIONS Biomarkers are needed to complement ultrasound in the detection of early HCC, but neither DCP nor AFP is optimal.

Predicting cirrhosis in patients with hepatitis C based on standard laboratory tests: Results of the HALT-C cohort†

Knowledge of the presence of cirrhosis is important for the management of patients with chronic hepatitis C (CHC). Most models for predicting cirrhosis were derived from small numbers of patients and included subjective variables or laboratory tests that are not readily available. The aim of this study was to develop a predictive model of cirrhosis in patients with CHC based on standard laboratory tests. Data from 1,141 CHC patients including 429 with cirrhosis were analyzed. All biopsies were read by a panel of pathologists (blinded to clinical features), and fibrosis stage was determined by consensus. The cohort was divided into a training set (n = 783) and a validation set (n = 358). Variables that were significantly different between patients with and without cirrhosis in univariate analysis were entered into logistic regression models, and the performance of each model was compared. The area under the receiver‐operating characteristic curve of the final model comprising platelet count, AST/ALT ratio, and INR in the training and validation sets was 0.78 and 0.81, respectively. A cutoff of less than 0.2 to exclude cirrhosis would misclassify only 7.8% of patients with cirrhosis, while a cutoff of greater than 0.5 to confirm cirrhosis would misclassify 14.8% of patients without cirrhosis. The model performed equally well in fragmented and nonfragmented biopsies and in biopsies of varying lengths. Use of this model might obviate the requirement for a liver biopsy in 50% of patients with CHC. In conclusion, a model based on standard laboratory test results can be used to predict histological cirrhosis with a high degree of accuracy in 50% of patients with CHC. (HEPATOLOGY 2005.)

Mechanism of Sodium Arsenite-mediated Induction of Heme Oxygenase-1 in Hepatoma Cells ROLE OF MITOGEN-ACTIVATED PROTEIN KINASES

Heme oxygenase-1 is an inducible enzyme that catalyzes heme degradation and has been proposed to play a role in protecting cells against oxidative stress-related injury. We investigated the induction of heme oxygenase-1 by the tumor promoter arsenite in a chicken hepatoma cell line, LMH. We identified a heme oxygenase-1 promoter-driven luciferase reporter construct that was highly and reproducibly expressed in response to sodium arsenite treatment. This construct was used to investigate the role of mitogen-activated protein (MAP) kinases in arsenite-mediated heme oxygenase-1 gene expression. In LMH cells, sodium arsenite, cadmium, and heat shock, but not heme, induced activity of the MAP kinases extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. To examine whether these MAP kinases were involved in mediating heme oxygenase-1 gene expression, we utilized constitutively activated and dominant negative components of the ERK, JNK, and p38 MAP kinase signaling pathways. Involvement of an AP-1 site in arsenite induction of heme oxygenase-1 gene expression was studied. We conclude that the MAP kinases ERK and p38 are involved in the induction of heme oxygenase-1, and that at least one AP-1 element (located −1576 base pairs upstream of the transcription start site) is involved in this response.

Drug-Induced Liver Injury Network (DILIN) Prospective Study : Rationale, Design and Conduct

AbstractBackground: Drug-induced liver injury (DILI) is an uncommon adverse drug reaction of increasing importance to the medical community, pharmaceutical industry, regulatory agencies and the general public. Objectives: The Drug-Induced Liver Injury Network (DILIN) was established to advance understanding and research into DILI by initiating a prospective registry of patients with bona fide DILI for future studies of host clinical, genetic, environmental and immunological risk factors. The DILIN was also charged with developing standardized nomenclature, terminology and causality assessment instruments. Methods: Five clinical sites, a data coordinating centre and senior scientists from the National Institute of Diabetes and Digestive and Kidney Diseases initiated the DILIN prospective study in September 2004. Eligible patients are required to meet minimal laboratory or histological criteria within 6 months of DILI onset and have other competing causes of liver injury excluded. Patients in the general community setting with pre-existing HIV, hepatitis B virus or hepatitis C virus infections and/or abnormal baseline liver biochemistries are eligible for enrolment. In addition, subjects with liver injury due to herbal products are eligible to participate. Control patients without DILI are also to be recruited in the future. Results: All referred subjects undergo an extensive review of available laboratory, pathology and imaging studies. Subjects who meet pre-defined eligibility criteria at the 6-month study visit are followed for 2 years to better define the natural history of chronic DILI. Causality assessment is determined by a panel of three hepatologists who independently assign a causality score ranging from 1 (definite) to 5 (unlikely) as well as a severity score ranging from 1 (mild) to 5 (fatal). During the first 3 years, 367 subjects were enrolled into the DILIN prospective study. Conclusion: DILIN is a multicentre research network charged with improving our understanding of the aetiologies, risk factors and outcomes of DILI in the US. The network is meeting the targeted enrolment of ten patients per month and is developing a repository of clinical data and biological samples for future studies of DILI pathogenesis and outcome.