Recurrent intrahepatic cholestasis of pregnancy and chain-like choledocholithiasis in a female patient with stop codon in the ABDC4-gene of the hepatobiliary phospholipid transporter

We report the case of a 40-years-old female patient with recurrent cholestatic liver disease who presented twice with severe intrahepatic cholestasis of pregnancy and pronounced choledocholithiasis between pregnancies. Bile duct stones were removed endoscopically and a laparoscopic cholecystectomy was performed after the second pregnancy. Liver histology revealed intrahepatic cholestasis with portal inflammation and fibrosis, resembling progressive familial intrahepatic cholestasis (PFIC). Molecular genetic studies identified the heterozygous mutation c.957C > T in the ABCB4 gene encoding the hepatobiliary phospholipid transporter. This is the first report of this mutation that introduces a stop codon in an index patient with intrahepatic cholestasis of pregnancy and multiple bile duct stones. In addition, we detected the ABCB11 polymorphism V 444A, which is associated with a decreased expression of the bile salt export pump. Whereas homozygous carriers of the ABCB4 mutation develop PFIC type 3, the heterozygous ABC transporter mutations represent genetic risk factors for cholelithiasis and recurrent cholestatic hepatitis upon challenge with oral contraceptives or during pregnancy. Of note, the patient presented with normal serum gamma-glutamyltranspeptidase activities during pregnancy-associated cholestatic episodes but normal liver enzymes after delivery, whereas choledocholithiasis was associated with high gamma-glutamyl transpeptidase levels. It is unknown whether ursodeoxycholic acid prevents cholestasis or gallstones in patients with ABCB4 deficiency.     

Hereditäre Defekte hepatobiliärer Transportproteine

Defects in transport proteins that are expressed at the hepatocyte canalicular membrane can cause severe impairment of hepatobiliary transport processes. Progressive familial intrahepatic cholestasis (PFIC) typically manifests in early childhood. Genetic variants in the aminophospholipid transporter FIC1 (ATP8B1 gene) cause PFIC1, characterized by elevated serum bile acids but normal or only mildly elevated gamma-GT levels. Benign recurrent intrahepatic cholestasis type 1 (BRIC1) is also caused by ATP8B1 mutations. Defects in the function of the bile salt efflux pump (BSEP; ABCB11) cause PFIC2 or BRIC2, depending on the degree of BSEP impairment. A common BSEP variant, the V444A polymorphism, is commonly found in various types of cholestatic liver injury, including drug-induced liver injury. Finally, dysfunction of the multidrug resistance gene product MDR3 (ABCB4) leads to PFIC3, characterized by low biliary phospholipids and high gamma-GT levels in serum due to bile duct injury. All three transporter genes are also associated with intrahepatic cholestasis of pregnancy. Treatment options include ursodeoxycholic acid for milder forms and liver transplantation for severe pediatric cases.     

ABCB4 disease: Many faces of one gene deficiency

ATP-binding cassette (ABC) subfamily B member 4 (ABCB4), also known as multidrug resistance protein 3 (MDR3), encoded by ABCB4, is involved in biliary phospholipid secretion, protecting hepatobiliary system from deleterious detergent and lithogenic properties of the bile. ABCB4 mutations altering canalicular ABCB4 protein function and expression may have variable clinical presentation and predispose to several human liver diseases. Well-established phenotypes of ABCB4 deficit are: progressive familial intrahepatic cholestasis type 3, gallbladder disease 1 (syn. low phospholipid associated cholelithiasis syndrome), high ɣ-glutamyl transferase intrahepatic cholestasis of pregnancy, chronic cholangiopathy, and adult biliary fibrosis/cirrhosis. Moreover, ABCB4 aberrations may be involved in some cases of drug induced cholestasis, transient neonatal cholestasis, and parenteral nutrition-associated liver disease. Recently, genome-wide association studies have documented occurrence of malignant tumours, predominantly hepatobiliary malignancies, in patients with ABCB4/MDR3 deficit.The patient's age at the time of the first presentation of cholestatic disease, as well as the severity of liver disorder and response to treatment are related to the ABCB4 allelic status. Mutational analysis of ABCB4 in patients and their families should be considered in all individuals with cholestasis of unknown aetiology, regardless of age and/or time of onset of the first symptoms.     

Bile salt export pump deficiency: A de novo mutation in a child compound heterozygous for ABCB11. Laboratory investigation to study pathogenic role and transmission of two novel ABCB11 mutations

Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomal disorders. PFIC type 2 is due to mutation in ABCB11, the gene encoding the bile salt export pump (BSEP) protein. The aim of the study was to describe a child with a de novo mutation in a compound heterozygous for ABCB11 gene. We report a 1.7‐year‐old girl who presented with pruritus, jaundice and liver dysfunction of PFIC type 2. Immunohistochemistry and molecular analysis are described. Liver biopsy showed micronodular cirrhosis and immunohistochemical staining for BSEP, the protein encoded by ABCB11, displayed a patchy and faint reactivity. Molecular analysis revealed two novel mutations of ABCB11. We give details that one mutation is transmitted by the mother while the second one appears a de novo mutation as mutations or a potential mosaicism were ruled out in the natural father. We further speculate that the ABCB11 mutations do not prevent BSEP glycoprotein to be expressed at the canalicular pole of hepatocytes, but interfere with its ability to export bile salts. As in most instances, mutational analysis is performed following the histochemical demonstration of an undetectable BSEP on liver biopsy specimen. This case stresses that clinical PFIC with an attenuated rather than absent BSEP immunostaining can still be due to ABCB11 mutations presumably encoding a functionally deficient protein.     

Prenatal diagnosis of progressive familial intrahepatic cholestasis type 2

Background and Aim:  Progressive familial intrahepatic cholestasis type 2 (PFIC2) results from genetic defects of the hepatobiliary bile salt export pump (BSEP, ABCB11 ) at chromosome 2q24. Patients with progressive cholestasis and liver cirrhosis usually need liver transplantation in the first decade. Mutations in ABCB11 are also associated with benign recurrent intrahepatic cholestasis type 2 and intrahepatic cholestasis of pregnancy in adult patients. We aimed to make the prenatal diagnosis of PFIC2.
Methods:  Genetic diagnosis was performed by genomic DNA analysis. Prenatal genetic diagnosis was made by fetal amniotic DNA and chorionic DNA analysis.
Results:  We report on two families of PFIC2 with inherited compound heterozygous mutations of ABCB11 (M183V and R303K in Family 1, V284L and 1145delC in Family 2) from the parents. An infant with heterozygous M183V mutation was later born healthy in Family 1. A fetus with compound heterozygous missense mutation V284L and 1145delC was terminated in Family 2.
Conclusion:  Prenatal diagnosis of PFIC2 was helpful to prevent further affected children in families with this fatal disease.     

Familial cholestasis: progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy

Progressive familial intrahepatic cholestasis (PFIC) type 1, 2 and 3 are due to mutations in ATP8B1, ABCB11 and ABCB4, respectively. Each of these genes encodes a hepatocanalicular transporter, which is essential for the proper formation of bile. Mutations in ABCB4 can result in progressive cholestatic disease, while mutations in ATP8B1 and ABCB11 can result both in episodic cholestasis, referred to as benign recurrent intrahepatic cholestasis (BRIC) type 1 and 2, as well as in progressive cholestatic disease. This suggests a clinical continuum and these diseases are therefore preferably referred to as ATP8B1 deficiency and ABCB11 deficiency. Similarly PFIC type 3 is designated as ABCB4 deficiency. Heterozygous mutations in each of these transporters can also be associated with intrahepatic cholestasis of pregnancy. This review summarizes the pathophysiology, clinical features and current as well as future therapeutic options for progressive familial- and benign recurrent intrahepatic cholestasis as well as intrahepatic cholestasis of pregnancy.     

ABCB11 gene mutations in Chinese children with progressive intrahepatic cholestasis and low γ glutamyltransferase

Background: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe autosomal recessive liver disorder of childhood that can cause cholestasis and progress to end‐stage liver disease. ABCB11 gene mutations causing PFIC2 have been reported in some population groups, but not in mainland Chinese. Aims: To elucidate the existence of and characterize ABCB11 gene mutations in mainland Chinese with progressive intrahepatic cholestasis and low γ glutamyltransferase (GGT). Methods: Twenty‐four children presenting with progressive intrahepatic cholestasis and low GGT were admitted to a tertiary paediatric hospital in eastern China from January 2004 to July 2007. All encoding exons and flanking areas of the ABCB11 gene were sequenced. Hepatic histopathology results were obtained by review of the medical record. Results: Twelve novel mutations of ABCB11 gene were found in seven patients: three nonsense mutations, six missense mutations, two splicing mutations and one intronic mutation. Giant cell transformation of hepatocytes was demonstrated in all the four patients with ABCB11 mutations and four of 12 patients without mutations in coding sequences of ABCB11 gene who received liver needle biopsy. Conclusions: ABCB11 gene mutations play an important role in Chinese patients with progressive intrahepatic cholestasis and low GGT. The characteristics of ABCB11 gene mutations in Chinese are different from other population groups. Histological examination may be helpful in diagnosis of PFIC2.     

Reversal of advanced fibrosis after long-term ursodeoxycholic acid therapy in a patient with residual expression of MdR3

Introduction. Progressive familial intrahepatic cholestasis type 3 (PFIC-3) is a severe liver disorder associated with inherited dysfunction of multidrug resistance protein 3 (MDR3/ABCB4), which functions as a phospholipid floppase, translocating phosphatidylcholine from the inner to the outer hemileaflet of the canalicular membrane of hepatocytes. MDR3 deficiency results in a disbalanced bile which may damage the luminal membrane of cells of the hepatobiliary system. We evaluated clinical, biochemical and histological improvement in a genetically proven PFIC-3 patient after long-term ursodeoxycholic acid (UDCA) administration.Material and methods. A PFIC-3 patient and a relative with cholestatic liver disease were studied. Hepatic MDR3 expression was analyzed by immunohistochemistry and ABCB4 mutations were identified. The effect of the mutations on MDR3 expression and subcellular localization was studied in vitro.Results. A 23-year-old man presented cholestasis with severe fibrosis and incomplete cirrhosis. Canalicular staining for MDR3 was faint. Sequence analysis of ABCB4 revealed two missense mutations that reduce drastically protein expression levels. After 9 years of treatment with UDCA disappearance of fibrosis and cirrhosis was achieved.Conclusion. These data indicate that fibrosis associated with MDR3 deficiency can be reversed by long-term treatment with UDCA, at least when there is residual expression of the protein.     

Familial intrahepatic cholestasis: New and wide perspectives

BackgroundProgressive familial intrahepatic cholestasis (PFIC) includes autosomal recessive cholestatic rare diseases of childhood.AimsTo update the panel of single genes mutations involved in familial cholestasis.MethodsPubMed search for “familial intrahepatic cholestasis” alone as well as in combination with other key words was performed considering primarily original studies and meta-analyses.ResultsPFIC1 involves ATP8B1 gene encoding for aminophospholipid flippase FIC1. PFIC2 includes ABCB11 gene, encoding for protein functioning as bile salt export pump. PFIC3 is due to mutations of ABCB4 gene responsible for the synthesis of class III multidrug resistance P-glycoprotein flippase. PFIC4 and PFIC5 involve tight junction protein-2 gene and NR1H4 gene encoding for farnesoid X receptor. Benign Intrahepatic Cholestasis, Intrahepatic Cholestasis of Pregnancy and Low-phospholipid-associated cholelithiasis involve the same genes and are characterized by intermittent attacks of cholestasis, no progression to cirrhosis, reversible pregnancy-specific cholestasis and cholelithiasis in young people. Blood and liver tissue levels of bile-excreted drugs can be influenced by the presence of mutations in PFIC genes, causing drug-induced cholestasis. Mutations in PFIC genes might increase the risk of liver cancer.ConclusionThere is a high proportion of unexplained cholestasis potentially caused by specific genetic pathophysiologic pathways. The use of next generation sequencing and whole-exome sequencing could improve the diagnostic process in this setting.     

Combined features of low phospholipid‐associated cholelithiasis and progressive familial intrahepatic cholestasis 3

Adenosine triphosphate‐binding cassette, subfamily B, member 4 (ABCB4) gene alterations can cause two distinct clinical entities: progressive familial intrahepatic cholestasis type 3 (PFIC3) and low phospholipid‐associated cholelithiasis (LPAC). Based on the findings in two siblings and a review of the literature, we aimed to identify determinants of disease phenotypic traits associated with ABCB4 gene alterations. Two siblings presented, before the age of 30 years, recurrent symptomatic cholelithiasis and extensive biliary fibrosis that progressed towards portal hypertension and liver failure necessitating liver transplantation. We analysed the sequence of the ABCB4 gene and immunolocalization of the protein in the liver. Sequence analysis of ABCB11, potentially involved in similar symptoms, was also performed. Two heterozygous non‐synonymous variants of ABCB4 were found in both siblings. One of them (c.959C>T; p.Ser320Phe) was previously implicated in LPAC and the second one (c.2858C>A; p.Ala953Asp) in PFIC3. Both patients were also heterozygous for the ABCB11 variant Val444Ala, which predisposes to cholestatic disorders. ABCB4 was normally detected at the canalicular membrane of hepatocytes. The review of ABCB4 gene variants reported so far shows that the vast majority of variants causing PFIC3 and LPAC are distinct. Also as a general rule, homozygous variants cause PFIC3 while heterozygous variants lead to LPAC. Combined PFIC3 and LPAC phenotype is a rare clinical event, which may be determined by the coexistence of ABCB4 variants related to both phenotypes and also potentially to the ABCB11 variant. Thus, most of the patients presenting with LPAC are not at a particular risk of developing PFIC3 features in adulthood.     

An insertion mutation in ABCB4 is associated with gallbladder mucocele formation in dogs

Background  

ABCB4 functions as a phosphatidylcholine translocater, flipping phosphatidylcholine across hepatocyte canalicular membranes into biliary canaliculi. In people, ABCB4 gene mutations are associated with several disease syndromes including intrahepatic cholestasis of pregnancy, progressive familial intrahepatic cholestasis (type 3), primary biliary cirrhosis, and cholelithiasis. Hepatobiliary disease, specifically gallbladder mucocele formation, has been recognized with increased frequency in dogs during the past decade. Because Shetland Sheepdogs are considered to be predisposed to gallbladder mucoceles, we initially investigated ABCB4 as a candidate gene for gallbladder mucocele formation in that breed, but included affected dogs of other breeds as well.     

Benign recurrent intrahepatic cholestasis type 2 is caused by mutations in ABCB11

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are hereditary liver disorders; PFIC is characterized by severe progressive liver disease whereas BRIC patients have intermittent attacks of cholestasis without permanent liver damage. Mutations in ATP8B1 are present in PFIC type 1 and in a subset of BRIC patients. We hypothesized that a genetically distinct form of BRIC is associated with mutations in ABCB11. This gene encodes the bile salt export pump (BSEP) and is mutated in PFIC type 2. METHODS: Patients from 20 families were included; all had a normal ATP8B1 sequence. Sequencing of all 27 coding exons including the splice junctions of ABCB11 revealed 8 distinct mutations in 11 patients from 8 different families: one homozygous missense mutation (E297G) previously described in PFIC2 patients, 6 novel missense mutations, and one putative splice site mutation. RESULTS: In 12 families, no mutations in ATB8B1 or ABCB11 were detected. Pancreatitis is a known extrahepatic symptom in BRIC caused by ATP8B1 mutations, but was not present in BRIC patients with mutations in ABCB11. In contrast, cholelithiasis was observed in 7 of 11 BRIC patients with mutations in ABCB11, but has not been described in ATP8B1-affected BRIC patients. CONCLUSIONS: Mutations in ABCB11 are associated with BRIC, and consistent with the genetic classification of PFIC into 2 subtypes, we propose that this disorder be named BRIC type 2.     

Genetic cholestasis: lessons from the molecular physiology of bile formation.

Progressive familial intrahepatic cholestasis (PFIC) is a group of severe genetic cholestatic liver diseases of early life. PFIC types 1 and 2 are characterized by cholestasis and a low to normal serum gamma-glutamyltransferase (GGT) activity, whereas in PFIC type 3, the serum GGT activity is elevated. PFIC types 1 and 2 occur due to mutations in loci at chromosome 18 and chromosome 2, respectively. The pathophysiology of PFIC type 1 is not well understood. PFIC types 2 and 3 are caused by transport defects in the liver affecting the hepatobiliary secretion of bile acids and phospholipids, respectively. Benign recurrent intrahepatic cholestasis (BRIC) is linked to a mutation in the same familial intrahepatic cholestasis 1 locus at chromosome 18. Defects of bile acid synthesis may be difficult to differentiate from these transport defects. Intrahepatic cholestasis of pregnancy (ICP) appears to be related to these cholestatic diseases. For example, heterozygosity in families with PFIC type 3 is associated with ICP, but ICP has also been reported in families with BRIC. In Dubin-Johnson syndrome there is no cholestasis; only the hepatobiliary transport of conjugated bilirubin is affected. This, therefore, is a mild disease, and patients have a normal lifespan.     

A new ABCB11 mutation in two Italian children with familial intrahepatic cholestasis

Progressive familial intrahepatic cholestasis (PFIC) syndromes are characterized by defects in transporters of conjugated bile acids into the bile canaliculus. Three genes (ATP8B1, ABCB11, ABCB4) are associated with the different forms, but no easy genotype–phenotype correlations help in the prioritization for gene testing. We developed a denaturing high-performance liquid chromatography (DHPLC) method to screen patients with PFIC for mutations in ATP8B1 and ABCB11, and combined genetic analyses with immunolabeling in liver for the ABCB11 and ABCB4 gene products. Used in combination with commercially available antibodies on liver specimens, the DHPLC approach allowed us to confirm the clinical diagnosis in two Italian sisters and to identify a novel missesnse mutation in ABCB11. Our findings are expected to facilitate detection of the molecular cause of PFIC in affected families.     

A Rare BSEP Mutation Associated with a Mild Form of Progressive Familial Intrahepatic Cholestasis Type 2

Progressive Familial Intrahepatic Cholestasis type 2 (PFIC2) is a rare cholestatic disorder diagnosed in infancy or childhood that can lead to severe hepatic fibrosis and liver failure. Mutations in the ABCB11 gene result in a deficiency of the bile salt export protein (BSEP) and accumulation of bile inside the hepatocytes. Hepatocellular carcinoma is another condition associated with severe forms of deletion mutations in the ABCB11 gene. Treatment options including ursodeoxycholic acid biliary diversion have mixed outcomes and some patients require liver transplantation. Here, we describe two siblings with an extremely mild form of PFIC2 inherited from heterozygous parents. The elder sibling had acute liver failure at the age of six months and both siblings had pruritus, cholestasis, coagulopathy and fat-soluble-vitamin deficiencies in infancy but have been asymptomatic past infancy. Genetic testing of the siblings revealed that each were compound heterozygotes for two missense mutations of the ABCB11 gene: p.C68Y and p.R832H. Medical treatment typical for PFIC2 has not been necessary for either patient. This is the first report of these variants following a mild course in two affected patients.     

Acute recurrent biliary pancreatitis associated with the ABCB4 gene mutation

The ABCB4 gene codes for a protein involved in the transport of phosphatidylcholine across the canalicular membrane of the hepatocyte. ABCB4 gene defects have been associated with progressive familial intrahepatic cholestasis type 3, intrahepatic cholestasis of pregnancy, adult biliary cirrhosis and the more recently described low phospholipid associated cholelithiasis syndrome. The present paper describes 2 probands with a long history of recurrent pancreatitis and cholelithiasis and the same heterozygous, as yet undescribed del 3683>3688 within exon 28 of the ABCB4 gene resulting in a loss of function. This report shows that ABCB4 mutations may cause acute recurrent biliary pancreatitis.     

Impaired expression and function of the bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis

BACKGROUND/AIMS: Inherited dysfunction of the bile salt export pump BSEP (ABCB11) causes a progressive and a benign form of familial intrahepatic cholestasis, denominated as PFIC2 and BRIC2, respectively. We functionally characterized novel ABCB11 mutations encountered in two patients with a PFIC2 and a BRIC2 phenotype, respectively. METHODS: BSEP expression was determined in liver biopsies by immunohistochemistry. ABCB11 mutations were functionally characterized by taurocholate transport in SF9 cells transfected with human ABCB11. RESULTS: The PFIC2 patient was compound heterozygous for a splicing mutation in intron 4 ((+3)A > C) combined with an early stop codon at position 930 (R930X), while the BRIC2 patient was compound heterozygous for two nonsynonymous mutations in exon 9 (E297G) and exon 12 (R432T), respectively. Hepatic BSEP expression was absent in PFIC2 and preserved in BRIC2. In BRIC2, taurocholate transport was decreased to 13% and 20% of reference levels for R432T and E297G, respectively. CONCLUSIONS: The intron 4 (+3)A > C, R930X and R432T represent previously undescribed mutations of the ABCB11 gene that confer a PFIC2 and a BRIC2 phenotype, respectively. By combining functional in-vitro characterization with immunohistochemical detection of variant BSEP we provide direct evidence for the role of ABCB11 mutations in the pathogenesis of different forms of intrahepatic cholestasis.     

Partial Internal Biliary Diversion: A Solution for Intractable Pruritus in Progressive Familial Intrahepatic Cholestasis Type 1

Biliary diversion offers a potential option for intractable pruritus in children with chronic cholestatic disorders. Progressive familial intrahepatic cholestasis (PFIC) is an inherited disorder of impaired bile acid transport and excretion, which presents with jaundice and pruritus in the first few months of life and progresses to cirrhosis by infancy or adolescence. We report a child with PFIC type 1 who underwent internal biliary diversion for intractable pruritus and was relieved of his symptoms.