Mutations In ABCB11 Research Articles

A homozygous ABCB4 mutation causing an LPAC syndrome evolves into cholangiocarcinoma

Low phospholipid-associated cholelithiasis (LPAC) is characterized by the association of ABCB4 mutations and low biliary phospholipid concentration with symptomatic and recurring cholelithiasis. In the present study, we reported a case of a 63-year-old woman, who presented a biliary pain beginning at the age of 30, recurrent after cholecystectomy, along with “comet-tail shadows” revealed by ultrasonography thus, fulfilling the diagnosis of LPAC. This disease evolved into a cholangiocarcinoma. To understand the molecular basis of this phenotype, we performed the ABCB4 gene sequencing, followed by in silico analysis and Q-RT-PCR assay. The results displayed a homozygous missense sequence variation (c.140G > A, p.Arg47Gln), predicted as pathogenic according to MutPred. Accordingly, this gave rise to a decreased hepatic ABCB4 mRNA level and structural alterations of the mutated protein. Eventually, we reported, here, the first description of an ABCB4 missense mutation (p.Arg47Gln) at homozygous state in a Tunisian LPAC syndrome. An elucidation of its functional consequences was performed. Besides, this case suggests that the delayed diagnosis of LPAC syndrome and the lack of UDCA treatment may contribute in the development of complications, such as cholangiocarcinoma.

Progressive Familial Intrahepatic Cholestasis in Korea: A Clinicopathological Study of Five Patients.

BackgroundProgressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomal recessive liver diseases that present as neonatal cholestasis. Little is known of this disease in Korea.MethodsThe records of five patients histologically diagnosed with PFIC, one with PFIC1 and four with PFIC2, by liver biopsy or transplant were reviewed, and ATP8B1 and ABCB11 mutation status was analyzed by direct DNA sequencing. Clinicopathological characteristics were correlated with genetic mutations.ResultsThe first symptom in all patients was jaundice. Histologically, lobular cholestasis with bile plugs was the main finding in all patients, whereas diffuse or periportal cholestasis was identified only in patients with PFIC2. Giant cells and ballooning of hepatocytes were observed in three and three patients with PFIC2, respectively, but not in the patient with PFIC1. Immunostaining showed total loss of bile salt export pump in two patients with PFIC2 and focal loss in two. Lobular and portal based fibrosis were more advanced in PFIC2 than in PFIC1. ATP8B1 and ABCB11 mutations were identified in one PFIC1 and two PFIC2 patients, respectively. One PFIC1 and three PFIC2 patients underwent liver transplantation (LT). At age 7 months, one PFIC2 patient was diagnosed with concurrent hepatocellular carcinoma and infantile hemangioma in an explanted liver. The patient with PFIC1 developed steatohepatitis after LT. One patient showed recurrence of PFIC2 after 10 years and underwent LT.ConclusionsPFIC is not rare in patients with neonatal cholestasis of unknown origin. Proper clinicopathologic correlation and genetic testing can enable early detection and management.

From the Editor’s desk…: June 2019

From the Editor’s desk…: June 2019

Clinical utility of genomic analysis in adults with idiopathic liver disease

Adult patients suffering from liver disease of unknown cause represent an understudied and underserved population. The use of whole-exome sequencing (WES) for the assessment of a broader spectrum of non-oncological diseases, among adults, remains poorly studied. We assessed the utility of WES in the diagnosis and management of adults with unexplained liver disease despite comprehensive evaluation by a hepatologist and with no history of alcohol overuse. We performed WES and deep phenotyping of 19 unrelated adult patients with idiopathic liver disease recruited at a tertiary academic health care center in the US. Analysis of the exome in 19 cases identified 4 monogenic disorders in 5 unrelated adults. Patient 1 suffered for 18 years from devastating complications of undiagnosed type 3 familial partial lipodystrophy due to a deleterious heterozygous variant in PPARG. Molecular diagnosis enabled initiation of leptin replacement therapy with subsequent normalization of liver aminotransferases, amelioration of dyslipidemia, and decreases in daily insulin requirements. Patients 2 and 3 were diagnosed with MDR3 deficiency due to recessive mutations in ABCB4. Patient 4 with a prior diagnosis of non-alcoholic steatohepatitis was found to harbor a mitochondrial disorder due to a homozygous pathogenic variant in NDUFB3; this finding enabled initiation of disease preventive measures including supplementation with antioxidants. Patient 5 is a lean patient with hepatic steatosis of unknown etiology who was found to have a damaging heterozygous variant in APOB. Genomic analysis yielded an actionable diagnosis in a substantial number (∼25%) of selected adult patients with chronic liver disease of unknown etiology. This study supports the use of WES in the evaluation and management of adults with idiopathic liver disease in clinical practice. We performed whole-exome sequencing in 19 adult patients with unexplained liver disease after an unrevealing conventional work-up performed by a hepatologist. In 5 cases, genomic analysis led to a diagnosis and informed treatment and management of the disease. Therefore, we suggest using whole-exome sequencing in the evaluation and management of adults with unexplained liver disease.

Molecular Mechanisms for Protection of Hepatocytes against Bile Salt Cytotoxicity.

Biliary lipids consist mainly of bile salts, phospholipids and cholesterol, which form mixed micelles and vesicles. Bile salts play various physiological roles but have damaging effects on cell membranes due to their detergent properties. The cytotoxicity of bile salts on hepatocytes leads to liver injuries and is largely determined by the bile salt species, the concentrations of bile salts, phospholipids and cholesterol, and the lipid composition of cell membranes. In bile, monomers and simple micelles of bile salts coexist with mixed micelles and vesicles in dynamic equilibrium, and contribute to the cytotoxicity on hepatocytes. The ATP-binding cassette (ABC) transporter family members, ABCB11, ABCB4 and ABCG5/ABCG8, mediate the biliary secretion of bile salts, phospholipids and cholesterol, respectively. Mutations in ABCB4 result in severe cholestatic diseases, and the biliary phospholipids are necessary for the attenuation of bile salt cytotoxicity. On the other hand, cholesterol reverses the cytoprotective effects of phospholipids against bile salts. In addition, phosphatidylethanolamine N-methyltransferase increases the cell resistance to bile salts by changing the phospholipid composition and structures of the apical membranes. In this review, we focus on the molecular mechanisms for the protection of hepatocytes against bile salt cytotoxicity. Further understanding of these mechanisms will help to develop new therapeutic strategies for cholestatic liver diseases.

Severe and protracted cholestasis in 44 young men taking bodybuilding supplements: assessment of genetic, clinical and chemical risk factors.

Bodybuilding supplements can cause a profound cholestatic syndrome. To describe the drug-Induced liver injury network's experience with liver injury due to bodybuilding supplements. Liver injury pattern, severity and outcomes, potential genetic associations, and exposure to anabolic steroids by product analysis were analysed in prospectively enrolled subjects with bodybuilding supplement-induced liver injury with causality scores of probable or higher. Forty-four males (mean age 33years) developed liver injury with a median latency of 73 days. Forty-one per cent presented with hepatocellular pattern of liver injury as defined by the R>5 ([Fold elevation of ALT]÷[Fold elevation of Alk Phos] (mean, range=6.4, 0.5-31.4, n=42) despite all presenting with clinical features of cholestatic liver injury (100% with jaundice and 84% with pruritus). Liver biopsy (59% of subjects) demonstrated a mild hepatitis and profound cholestasis in most without bile duct injury, loss or fibrosis. Seventy-one per cent were hospitalised, and none died or required liver transplantation. In some, chemical analysis revealed anabolic steroid controlled substances not listed on the label. No enrichment of genetic variants associated with cholestatic syndromes was found, although mutations in ABCB11 (present in up to 20%) were significantly different than in ethnically matched controls. Patients with bodybuilding supplements liver injury uniformly presented with cholestatic injury, which slowly resolved. The ingested products often contained anabolic steroids not identified on the label, and no enrichment in genetic variants was found, indicating a need for additional studies.

ABCB4/MDR3 in health and disease - at the crossroads of biochemistry and medicine.

Several ABC transporters of the human liver are responsible for the secretion of bile salts, lipids and cholesterol. Their interplay protects the biliary tree from the harsh detergent activity of bile salts. Among these transporters, ABCB4 is essential for the translocation of phosphatidylcholine (PC) lipids from the inner to the outer leaflet of the canalicular membrane of hepatocytes. ABCB4 deficiency can result in altered PC to bile salt ratios, which led to intrahepatic cholestasis of pregnancy, low phospholipid associated cholelithiasis, drug induced liver injury or even progressive familial intrahepatic cholestasis type 3. Although PC lipids only account for 30-40% of the lipids in the canalicular membrane, 95% of all phospholipids in bile are PC lipids. We discuss this discrepancy in the light of PC synthesis and bile salts favoring certain lipids. Nevertheless, the in vivo extraction of PC lipids from the outer leaflet of the canalicular membrane by bile salts should be considered as a separate step in bile formation. Therefore, methods to characterize disease causing ABCB4 mutations should be considered carefully, but such an analysis represents a crucial point in understanding the currently unknown transport mechanism of this ABC transporter.

The Nowhere Stone: Unusually Located Intrahepatic Calculi in a Patient with Low Phospholipid-associated Cholelithiasis Syndrome

ABSTRACTIntrahepatic calculi or hepatolithiasis is an uncommon phenomenon which is found in relatively higher rates in eastern Asian countries. Patients usually present with features of cholangitis or intrahepatic abscess and are at risk for developing cholangiocarcinoma. Correct diagnosis and management often pose challenges. A 42-year-old female presented with a history of episodic upper abdominal pain since 8 years. Initial imaging studies showed evidence of biliary calculi, but were confounding in their localization. Magnetic resonance cholangiopancreatography showed two large calculi in the right biliary system proximal to the confluence, along with choledocholithiasis. Endoscopic retrograde cholangiopancreatography and common bile duct clearance were done; however, the patient underwent hepatic sectionectomy as a curative procedure. ABCB4 mutation was detected on DNA analysis and hence aided in making the diagnosis of low phospholipid-associated cholelithiasis. Additional imaging studies are warranted in doubtful cases. Difficult accessibility of the calculi may often limit endoscopic therapeutic options and pave the way for surgical interventions. As there is an increased risk of cholangiocarcinoma in these patients, regular follow-up is essential.

Comprehensive bile acid profiling in hereditary intrahepatic cholestasis: Genetic and clinical correlations.

Genetic defects causing dysfunction in bile salt export pump (BSEP/ABCB11) lead to liver diseases. ABCB11 mutations alter the bile acid metabolome. We asked whether profiling plasma bile acids could reveal compensatory mechanisms and track genetic and clinical status. We compared plasma bile acids in 17 ABCB11-mutated patients, 35 healthy controls and 12 genetically undiagnosed cholestasis patients by ultra-high-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS). We developed an index to rank bile acid hydrophobicity, and thus toxicity, based on LC retention times. We recruited 42 genetically diagnosed hereditary cholestasis patients, of whom 12 were presumed to have impaired BSEP function but carried mutations in genes other than ABCB11, and 8 healthy controls, for further verification. The overall hydrophobicity indices of total bile acids in both the ABCB11-mutated group (11.89±1.07min) and the undiagnosed cholestasis group (11.46±1.07min) were lower than those of healthy controls (13.69±0.77min) (both p<0.005). This was owing to increased bile acid modifications. Secondary bile acids were detected in patients without BSEP expression, suggesting biliary bile acid secretion through alternative routes. A diagnostic panel comprising lithocholic acid (LCA), tauro-LCA, glyco-LCA and hyocholic acid was identified that could differentiate the ABCB11-mutated cohort from healthy controls and undiagnosed cholestasis patients (AUC=0.946, p<0.0001) and, in non-ABCB11-mutated cholestasis patients, could distinguish BSEP dysfunction from normal BSEP function (9/12 vs 0/38, p<0.0000001). Profiling of plasma bile acids has provided insights into cholestasis alleviation and may be useful for the clinical management of cholestatic diseases.

Zebrafish abcb11b mutant reveals strategies to restore bile excretion impaired by bile salt export pump deficiency.

Collectively, these data suggest a model that rapamycin rescues BSEP-deficient phenotypes by prompting alternative transporters to excrete bile salts; multidrug resistance protein 1 is a candidate for such an alternative transporter. (Hepatology 2018;67:1531-1545).

Can genetic testing guide the therapy of cholestatic pruritus? A case of benign recurrent intrahepatic cholestasis type 2 with severe nasobiliary drainage-refractory itch.

Benign recurrent intrahepatic cholestasis (BRIC) is a peculiar familial disease caused by mutations of the genes encoding hepatocanalicular flippase for phosphatidylserine (ATP8B1; BRIC type 1) or the bile salt export pump (ABCB11; BRIC type 2). Here, we report on a patient with nasobiliary drainage‐refractory BRIC type 2 who improved under plasma separation and anion absorption therapy. We also suggest that nasobiliary drainage might be an ineffective approach in carriers of severe loss‐of‐function mutations of the bile salt export pump ABCB11. (Hepatology Communications 2018;2:152–154)

Cryptogenic cholestasis in young and adults: ATP8B1, ABCB11, ABCB4, and TJP2 gene variants analysis by high-throughput sequencing.

Mutations in ATP-transporters ATPB81, ABCB11, and ABCB4 are responsible for progressive familial intrahepatic cholestasis (PFIC) 1, 2 and 3, and recently the gene for tight junction protein-2 (TJP2) has been linked to PFIC4. As these four genes have been poorly studied in young people and adults, we investigated them in this context here. In patients with cryptogenic cholestasis, we analyzed the presence of mutations by high-throughput sequencing. Bioinformatics analyses were performed for mechanistic and functional predictions of their consequences on biomolecular interaction interfaces. Of 108 patients, 48 whose cause of cholestasis was not established were submitted to molecular analysis. Pathogenic/likely pathogenic mutations were found in ten (21%) probands for 13 mutations: two in ATP8B 1, six in ABCB11, two in ABCB4, three in TJP2. We also identified seven variants of uncertain significance: two in ATP8B1, one in ABCB11, two in ABCB4 and two in TJP2. Finally, we identified 11 benign/likely benign variants. Patients with pathogenic/likely pathogenic mutations had higher levels of liver stiffness (measured by FibroScan®) and bile acids, as well as higher rates of cholestatic histological features, compared to the patients without at least likely pathogenic mutations. The multivariate analysis showed that itching was the only independent factor associated with disease-causing mutations (OR 5.801, 95% CI 1.244-27.060, p=0.025). Mutations in the genes responsible for PFIC may be involved in both young and adults with cryptogenic cholestasis in a considerable number of cases, including in heterozygous status. Diagnosis should always be suspected, particularly in the presence of itching.

The Identification of Two New ABCB11 Gene Mutations and the Treatment Outcome in a Young Adult with Benign Recurrent Intrahepatic Cholestasis: A Case Report

Introduction: Benign recurrent intrahepatic cholestasis (BRIC) is a rare autosomal recessive disease characterized by recurrent episodes of severe pruritus and jaundice. Although the disease symptom will relieve spontaneously without leaving any hepatic injury, the ceaseless attacks would reduce the life quality of patients. However, there is not a validated treatment for BRIC yet. Considering the limited cases and the unpredictability of this disease, the publications of well-described case reports are necessary for the investigation of disease development and treatment efficacy. Case Presentation: A 26-year-old Chinese male, with clinical approved and genetic diagnosis of BRIC, experienced 3 attacks of recurrent intrahepatic cholestasis from 2010 to 2016. During hospitalizations, he received symptomatic treatments and plasmapheresis therapies. Both plasma exchange (PE) and plasma bilirubin adsorption (PBA) were conducted for him. The whole-exome sequencing revealed several single nucleotide polymorphisms (SNPs) as well as 2 novel mutations in ABCB11 (c.70A > T, p.Lys24*, exon2 and c.1417G > A, p.Asp473Asn, exon13). These SNPs and mutations might be associated with the BRIC development. Conclusions: Both medications and plasmapheresis interventions could relieve the patient’s symptoms, however, neither could shorten the natural process of the disease. The 2 mutations (c.70A > T and c.1417G > A) in ABCB11 were first reported in a BRIC patient.

An expanded role for heterozygous mutations of ABCB4, ABCB11, ATP8B1, ABCC2 and TJP2 in intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) affects 1/140 UK pregnancies; with pruritus, hepatic impairment and elevated serum bile acids. Severe disease is complicated by spontaneous preterm delivery and stillbirth. Previous studies have reported mutations in hepatocellular transporters (ABCB4, ABCB11). High throughput sequencing in 147 patients was performed in the transporters ABCB4, ABCB11, ATP8B1, ABCC2 and tight junction protein 2 (TJP2). Twenty-six potentially damaging variants were identified with the following predicted protein changes: Twelve ABCB4 mutations - Arg47Gln, Met113Val, Glu161Gly, Thr175Ala, Glu528Glyfs*6, Arg590Gln, Ala601Ser, Glu884Ter, Gly722Ala, Tyr775Met (x2), Trp854Ter. Four potential ABCB11 mutations - Glu297Gly (x3) and a donor splice site mutation (intron 19). Five potential ATP8B1 mutations - Asn45Thr (x3), and two others, Glu114Gln and Lys203Glu. Two ABCC2 mutations - Glu1352Ala and a duplication (exons 24 and 25). Three potential mutations were identified in TJP2; Thr62Met (x2) and Thr626Ser. No patient harboured more than one mutation. All were heterozygous. An additional 545 cases were screened for the potential recurrent mutations of ATP8B1 (Asn45Thr) and TJP2 (Thr62Met) identifying three further occurrences of Asn45Thr. This study has expanded known mutations in ABCB4 and ABCB11 and identified roles in ICP for mutations in ATP8B1 and ABCC2. Possible novel mutations in TJP2 were also discovered.

Hepatic MDR3 expression impacts lipid homeostasis and susceptibility to inflammatory bile duct obstruction in neonates.

BackgroundHeterozygous mutations in the gene ABCB4, encoding the phospholipid floppase MDR3 (Mdr2 in mice), are associated with various chronic liver diseases. Here, we hypothesize that reduced ABCB4 expression predisposes to extrahepatic biliary atresia (EHBA).MethodsLivers from neonatal wildtype (wt) and heterozygous Mdr2-deficient mice were subjected to mass-spectrometry based lipidomics and RNAseq studies. Following postnatal infection with rhesus rotavirus (RRV), liver immune responses and EHBA phenotype were assessed. Hepatic microarray data from 40 infants with EHBA were mined for expression levels of ABCB4.ResultsPhosphatidylcholine (PC) and phosphatidylethanolamine (PE) were increased while the PC/PE ratio was decreased in neonatal Mdr2+/− compared with wt mice. Following RRV challenge, hepatic expression of IFNγ and infiltration with CD8+ and NK+ lymphocytes were increased in Mdr2+/− mice. Plasma total bilirubin levels and prevalence of complete ductal obstruction were higher in these mice. In infants with EHBA, hepatic gene expression of ABCB4 was downregulated in those with an inflammatory compared with a fibrosing molecular phenotype.ConclusionsDecreased expression of ABCB4 causes dysregulation in (phospho)lipid homeostasis, and predisposes to aberrant pro-inflammatory lymphocyte responses and an aggravated phenotype of EHBA in neonatal mice. Downregulated ABCB4 is associated with an inflammatory transcriptome signature in infants with EHBA.

Chronic elevated liver enzymes with ABCB11 mutation in healthy adult twin males: A case report

Chronic elevated liver enzymes with ABCB11 mutation in healthy adult twin males: A case report

Early Diagnosis of ABCB11 Spectrum Liver Disorders by Next Generation Sequencing.

PurposeThe goal of this study was the early diagnosis of ABCB11 spectrum liver disorders, especially those focused on benign recurrent intrahepatic cholestasis and progressive familial intrahepatic cholestasis.MethodsFifty patients presenting neonatal cholestasis were evaluated to identify underlying etiologies. Genetic analysis was performed on patients suspected to have syndromic diseases or ABCB11 spectrum liver disorders. Two families with proven ABCB11 spectrum liver disorders were subjected to genetic analyses to confirm the diagnosis and were provided genetic counseling. Whole exome sequencing and Sanger sequencing were performed on the patients and the family members.ResultsIdiopathic or viral hepatitis was diagnosed in 34%, metabolic disease in 20%, total parenteral nutrition induced cholestasis in 16%, extrahepatic biliary atresia in 14%, genetic disease in 10%, neonatal lupus in 2%, congenital syphilis in 2%, and choledochal cyst in 2% of the patients. The patient with progressive familial intrahepatic cholestasis had novel heterozygous mutations of ABCB11 c.11C>G (p.Ser4*) and c.1543A>G (p.Asn515Asp). The patient with benign recurrent intrahepatic cholestasis had homozygous mutations of ABCB11 c.1331T>C (p.Val444Ala) and heterozygous, c.3084A>G (p.Ala1028Ala). Genetic confirmation of ABCB11 spectrum liver disorder led to early liver transplantation in the progressive familial intrahepatic cholestasis patient. In addition, the atypically severe benign recurrent intrahepatic cholestasis patient was able to avoid unnecessary liver transplantation after genetic analysis.ConclusionABCB11 spectrum liver disorders can be clinically indistinguishable as they share similar characteristics related to acute episodes. A comprehensive genetic analysis will facilitate optimal diagnosis and treatment.

Current and future therapies for inherited cholestatic liver diseases.

Familial intrahepatic cholestasis (FIC) comprises a group of rare cholestatic liver diseases associated with canalicular transport defects resulting predominantly from mutations in ATP8B1, ABCB11 and ABCB4. Phenotypes range from benign recurrent intrahepatic cholestasis (BRIC), associated with recurrent cholestatic attacks, to progressive FIC (PFIC). Patients often suffer from severe pruritus and eventually progressive cholestasis results in liver failure. Currently, first-line treatment includes ursodeoxycholic acid in patients with ABCB4 deficiency (PFIC3) and partial biliary diversion in patients with ATP8B1 or ABCB11 deficiency (PFIC1 and PFIC2). When treatment fails, liver transplantation is needed which is associated with complications like rejection, post-transplant hepatic steatosis and recurrence of disease. Therefore, the need for more and better therapies for this group of chronic diseases remains. Here, we discuss new symptomatic treatment options like total biliary diversion, pharmacological diversion of bile acids and hepatocyte transplantation. Furthermore, we focus on emerging mutation-targeted therapeutic strategies, providing an outlook for future personalized treatment for inherited cholestatic liver diseases.

Functional characterization of ABCB4 mutations found in progressive familial intrahepatic cholestasis type 3.

Multidrug resistance 3 (MDR3), encoded by the ATP-binding cassette, subfamily B, member 4 gene (ABCB4), localizes to the canalicular membrane of hepatocytes and translocates phosphatidylcholine from the inner leaflet to the outer leaflet of the canalicular membrane. Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare hepatic disease caused by genetic mutations of ABCB4. In this study, we characterized 8 ABCB4 mutations found in PFIC3 patients, using in vitro molecular assays. First, we examined the transport activity of each mutant by measuring its ATPase activity using paclitaxel or phosphatidylcholine. Then, the pathogenic mechanisms by which these mutations affect MDR3 were examined through immunoblotting, cell surface biotinylation, and immunofluorescence. As a result, three ABCB4 mutants showed significantly reduced transport activity. Among these mutants, one mutation A364V, located in intracellular domains, markedly decreased MDR3 expression on the plasma membrane, while the others did not affect the expression. The expression of MDR3 on the plasma membrane and transport activity of A364V was rescued by a pharmacological chaperone, cyclosporin A. Our study provides the molecular mechanisms of ABCB4 mutations and may contribute to the understanding of PFIC3 pathogenesis and the development of a mutation-specific targeted treatment for PFIC3.

Clinical and ABCB11 profiles in Korean infants with progressive familial intrahepatic cholestasis.

To investigate clinical profiles and mutations of ABCB11 in Koreans with progressive familial intrahepatic cholestasis 2 and review the differences between Koreans and others. Of 47 patients with neonatal cholestasis, five infants had chronic intrahepatic cholestasis with normal γ-glutamyl transpeptidase. Direct sequencing analyses of ABCB11, including exons and introns, were performed from peripheral blood. Living donor-liver transplantation was performed in four patients because of rapidly progressive hepatic failure and hepatocellular carcinoma. Three missense mutations were found in two patients: compound heterozygous 677C>T (S226L)/3007G>A (G1003R) and heterozygous 2296G>A (G766R). The mutations were located near and in the transmembranous space. Alterations in the transmembrane of the bile salt export pump in the Korean infants were different from those previously reported in Chinese, Japanease, Taiwanese, and European patients.