Locus heterogeneity in progressive familial intrahepatic cholestasis.

Progressive familial intrahepatic cholestasis (PFIC or Byler disease) is a rare autosomal recessive form of severe and fatal cholestatic liver disease. A locus for PFIC has recently been mapped to chromosome 18q21-q22 in the original Byler pedigree. This region harbours the locus for a related phenotype, benign recurrent intrahepatic cholestasis (BRIC), suggesting that these traits are allelic. Linkage analysis was undertaken in five consanguineous PFIC pedigrees from Saudi Arabia using marker loci (D18S69, D18S41, D18S64, D18S38, D18S42, D18S55, D18S68, and D18S61) which span the Byler disease/BRIC region on 18q21-q22. In this family set the disease locus was excluded from this region, showing that locus heterogeneity exists for the PFIC phenotype.     

Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking

Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy with serious consequences for the mother and fetus. Two pedigrees have been reported with ICP in the mothers of children with a subtype of autosomal recessive progressive familial intrahepatic cholestasis (PFIC) with raised serum gamma-glutamyl transpeptidase (gamma-GT). Affected children have homozygous mutations in the MDR3 gene (also called ABCB4 ), and heterozygous mothers have ICP. More frequently, however, ICP occurs in women with no known family history of PFIC and the genetic basis of this disorder is unknown. We investigated eight women with ICP and raised serum gamma-GT, but with no known family history of PFIC. DNA sequence analysis revealed a C to A transversion in codon 546 in exon 14 of MDR3 in one patient, which results in the missense substitution of the wild-type alanine with an aspartic acid. We performed functional studies of this mutation introduced into MDR1, a closely related homologue of MDR3. Fluorescence activated cell sorting (FACS) and western analysis indicated that this missense mutation causes disruption of protein trafficking with a subsequent lack of functional protein at the cell surface. The demonstration of a heterozygous missense mutation in the MDR3 gene in a patient with ICP with no known family history of PFIC, analysed by functional studies, is a novel finding. This shows that MDR3 mutations are responsible for the additional phenotype of ICP in a subgroup of women with raised gamma-GT.     

Autosomal dominant benign recurrent intrahepatic cholestasis (BRIC) unlinked to 18q21 and 2q24

Benign recurrent intrahepatic cholestasis (BRIC) is an autosomal recessive liver disease characterized by multiple episodes of cholestasis without progression to chronic liver disease. On the basis of recent evidence of locus heterogeneity, we studied 19 subjects (7 affected members) of a BRIC family. Male-to-male transmission and the presence of affected females suggested autosomal dominant inheritance. Blood samples were collected after informed consent. Subjects were genotyped by using markers mapping to 18q and 2q24 region, respectively, where the genes FIC1 and FIC2 have been mapped. Segregation of haplotypes excluded the two regions in our family. These findings suggest further genetic heterogeneity of the origin of BRIC.     

Molecular characterization and structural implications of 25 new ABCB4 mutations in progressive familial intrahepatic cholestasis type 3 (PFIC3)

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is an autosomal-recessive disorder due to mutations in the ATP-binding cassette, subfamily B, member 4 gene (ABCB4). ABCB4 is the liver-specific membrane transporter of phosphatidylcholine, a major and exclusive component of mammalian bile. The disease is characterized by early onset of cholestasis with high serum gamma-glutamyltranspeptidase activity, which progresses into cirrhosis and liver failure before adulthood. Presently, about 20 distinct ABCB4 mutations associated to PFIC3 have been described. We report the molecular characterization of 68 PFIC3 index cases enrolled in a multicenter study, which represents the largest cohort of PFIC3 patients screened for ABCB4 mutations to date. We observed 31 mutated ABCB4 alleles in 18 index cases with 29 distinct mutations, 25 of which are novel. Despite the lack of structural information on the ABCB4 protein, the elucidation of the three-dimensional structure of bacterial homolog allows the three-dimensional model of ABCB4 to be built by homology modeling and the position of the mutated amino-acids in the protein tertiary structure to be located. In a significant fraction of the cases reported in this study, the mutation should result in substantial impairment of ABCB4 floppase activity. The results of this study provide evidence of the broad allelic heterogeneity of the disease, with causative mutations spread along 14 of the 27 coding exons, but with higher prevalence on exon 17 that, as recently shown for the closely related paralogous ABCB1 gene, could contain an evolutionary marker for mammalian ABCB4 genes in the seventh transmembrane segment.     

Canalicular ABC transporters and liver disease

Bile is a complex mixture that includes bile salts, the membrane phospholipid phosphatidylcholine (PC), cholesterol and various endobiotic and xenobiotic toxins, each of which is secreted across the canalicular membrane of the hepatocyte by different ATP-binding cassette (ABC) transporters. The bile salts are essential for the emulsification of dietary fat and lipophilic vitamins. They are synthesized from cholesterol in the hepatocyte and their secretion by the bile salt export pump (BSEP or ABCB11) drives bile flow and is the starting point for the enterohepatic cycle. The detergent nature of bile salts that is key to their physiological role also means that they are inherently cytotoxic, and failure to secrete bile (intraheptic cholestasis) can precipitate severe liver disease and mortality. Such progressive familial intrahepatic cholestasis (PFIC) comes in three types of autosomal recessive disease. PFIC2 is caused by mutation to ABCB11. PFIC3 is caused by mutation of a closely related ABC transporter, ABCB4, which flops PC into the outerleaflet of the canalicular membrane. The flopped PC is extracted by the bile salts in the canaliculus to form a mixed micelle that reduces bile salt detergent activity. The third protein that is essential for bile flow from the hepatocyte is a member of a different class of transporter protein, a P-type ATPase, ATP8B1. Mutation of ATP8B1 causes PFIC1, but ATP8B1 does not transport a component of bile into the canaliculus. Data from different laboratories, published this year, suggests two different roles for ATP8B1 in the hepatocyte: a lipid flippase, that counterbalances the deleterious effects of ABCB4 on barrier function of the canalicular membrane; and an anchor of the actin cytoskeleton necessary to form the microvilli of the brush border. These latest discoveries are described, along with a spectrum of cholestatic disorders whose aetiologies lie in these and other transporters of the canalicular membrane.     

Sequence variation in the ATP8B1 gene and intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic condition that may affect women during the third trimester of pregnancy. Symptoms experienced by these women generally resolve spontaneously following delivery, but prior to delivery the fetus is at increased risk of intrauterine distress and sudden intrauterine death. The genetic etiology of most cases of ICP is unknown, although heterozygous carriers of mutations causing progressive familial intrahepatic cholestasis (PFIC) diseases may experience ICP. When examining linkage to known cholestasis genes, affected members of four Finnish ICP families shared haplotypes around ATP8B1, the gene responsible for PFIC1. This gene was subsequently screened in 176 familial and sporadic ICP patients. A total of 17 sequence changes were detected, five exonic and 12 intronic. No intronic change was associated with ICP in sporadic cases. Four intronic changes segregated with ICP in three families, a different change in each of two families and three changes in another family, although the significance of this is currently unknown. Three exonic changes were nonsynonymous, one (in exon 23) is probably a polymorphism while two predict novel amino-acid replacements (N45T and K203R). These changes, in exons 2 and 7, were detected in one individual each, and may have predisposed these individuals to ICP. In conclusion, although the exon 2 and 7 changes may have functioned as risk alleles, ATP8B1 is probably not a major gene contributing to the occurrence of ICP.     

The spectrum of Progressive Familial Intrahepatic Cholestasis diseases: Update on pathophysiology and emerging treatments

The Progressive Familial Intrahepatic Cholestasis (PFIC) disease spectrum encompasses a variety of genetic diseases that affect the bile production and the secretion of bile acids. Typically, the first presentation of these diseases is in early childhood, frequently followed by a severe course necessitating liver transplantation before adulthood. Except for transplantation, treatment modalities have been rather limited and frequently only aim at the symptoms of cholestasis, such as cholestatic pruritus. In recent years, progress has been made in understanding the pathophysiology of these diseases and new treatment modalities have been emerging. Herewith we summarize the latest developments in the field and formulate the current key questions and opportunities for further progress.     

First description of ABCB4 gene deletions in familial low phospholipid-associated cholelithiasis and oral contraceptives-induced cholestasis

The wide clinical spectrum of the ABCB4 gene (ATP-binding cassette subfamily B member 4) deficiency syndromes in humans includes low phospholipid-associated cholelithiasis (LPAC), intrahepatic cholestasis of pregnancy (ICP), oral contraceptives-induced cholestasis (CIC), and progressive familial intrahepatic cholestasis type 3 (PFIC3). No ABCB4 mutations are found in a significant proportion of patients with these syndromes. In the present study, 102 unrelated adult patients with LPAC (43 patients) or CIC/ICP (59 patients) were screened for ABCB4 mutations using DNA sequencing. Heterozygous ABCB4 point or short insertion/deletion mutations were found in 37% (16/43) of the LPAC patients and in 27% (16/59) of the ICP/CIC patients. High-resolution gene dosage methodologies were used in the 70 negative patients. Here, we describe for the first time ABCB4 partial or complete heterozygous deletions in 7% (3/43) of the LPAC patients, and in 2% (1/59) of the ICP/CIC patients. Our observations urge to systematically test patients with LPAC, ICP/CIC, and also children with PFIC3 for the presence of ABCB4 deletions using molecular tools allowing detection of gross rearrangements. In clinical practice, a comprehensive ABCB4 alteration-screening algorithm will permit the use of ABCB4 genotyping to confirm the diagnosis of LPAC or ICP/CIC, and allow familial testing. An early diagnosis of these biliary diseases may be beneficial because of the preventive effect of ursodeoxycholic acid on biliary complications. Further comparative studies of patients with well-characterized genotypes (including deletions) and phenotypes will help determine whether ABCB4 mutation types influence clinical outcomes.     

Acute intrahepatic cholestasis accompanied with Chlamydophila pneumoniae infection

We report a case of Chlamydophila (C.) pneumoniae infection presenting with fever and rapid intrahepatic cholestasis. A 63-year-old man had a week-long history of intermittent high fever and rapidly progressive jaundice with atypical erythema. The results of liver function tests were recorded. The results of all serological tests were negative; the IgM, IgG, and IgA titers for C. pneumoniae had increased, which indicates a C. pneumoniae infection. The patient's fever and liver dysfunction improved upon administration of minocycline. Light microscopic findings showed the presence of enlarged liver cells with clear cytoplasm, a few mitotic figures, multinucleated cells, and bile cholestasis. The electron microscopic appearance of liver biopsy showed that bile canaliculi exhibited intrahepatic forms of cholestasis. From the results of light and electron microscopy, we inferred atypical intrahepatic cholestasis, probably resulting from the C. pneumoniae infection.     

Phenobarbital-induced alterations of bile acid metabolism in cases of intrahepatic cholestasis

Summary Bile acid profiles from plasma and urine of six patients suffering from intrahepatic cholestasis were studied before and during treatment with phenobarbital. All patients responded to this treatment by decreasing their plasma bile acid levels. Using gas chromatographic and mass spectrometric methods for separation and identification of the bile acids, especially the occurrence of major atypical bile acids was quantitatively evaluated.The plasma bile acid lowering effect of phenobarbital in intrahepatic cholestasis can be partly explained by an increased formation of tetrahydroxylated bile acids, which are rapidly excreted by renal pathways. These tetrahydroxylated bile acids, present as nonsulfated compounds, have high renal excretory flow rates exceeding those of bile acid sulfates. Their enterohepatic circulation, however, seems to be low, since only small amounts of tetrahydroxylated bile acids can be found in bile. It is mainly the 1- and 6-hydroxylation that is stimulated by phenobarbital. The exact site of formation of tetrahydroxylated bile acids, however, is still unknown. These findings may provide a rationale for the institution of a phenobarbital treatment in cases of intrahepatic cholestasis.Dedicated to Prof. Dr. G. W. Löhr on his 60th birthday     

Clinical heterogeneity of neonatal intrahepatic cholestasis caused by citrin deficiency: case reports from 16 patients

A deficiency of citrin, which is encoded by the SLC25A13 gene, causes both adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). We analyzed 16 patients with NICCD to clarify the clinical features of the disease. Severe intrahepatic cholestasis with fatty liver was the most common symptom, but the accompanying clinical features were variable, namely; suspected cases of neonatal hepatitis or biliary atresia, positive results from newborn screening, tyrosinemia, failure to thrive, hemolytic anemia, bleeding tendencies and ketotic hypoglycemia. Laboratory data showed elevated serum bile acid levels, hypoproteinemia, low levels of vitamin K-dependent coagulation factors, and hypergalactosemia. Hypercitrullinemia was detected in 11 out of 15 patients examined. Most of the patients were given a lactose-free and/or medium chain triglycerides-enriched formula and lipid-soluble vitamins. The prognosis of the 16 patients is going fairy well at present, but we should observe these patients carefully to see if they manifest any symptom of CTLN2 in the future.     

105例妊娠期肝内胆汁淤积症临床分析

目的探讨妊娠期肝内胆汁淤积症(ICP)对母儿的不良影响.方法对105例ICP病人进行回顾性分析.结果与孕28w以后出现搔瘁的患者比较,孕28w以前出现搔瘁的患者的胆汁酸、转氨酶及胆红素值显著性升高(P＜0.05),孕妇各种妊娠结局、围生儿结局的发生率及羊水粪染率显著性升高(P＜0.05).结论ICP易引起早产、胎儿宫内窘迫、胎儿突死官内及母亲产后出血,病人发病时间越早,围产儿预后越差,母亲产后出血发生率及羊水粪染率越高.     

Biochemical features of intrahepatic cholestasis.

A pattern of results is reported which was found to be common among patients who had intrahepatic cholestasis (IHC) which was rarely found in patients with other hepatic conditions. The pattern was recognized from over 1000 cases suspected of hepatobiliary disease. 29 were diagnosed with IHC, and excluding 4, 25 revealed the following etiological pattern: chlorpromazine (12 patients); pregnancy and oral contraceptive use (8); and other (5). As opposed to patients with acute and chronic hepatic disease, IHC sufferers had relatively normal values for immunoglobulins and antibody titers. A disproportionate elevation of serum bilirubin vis-a-vis serum enzymatic activities separated potential IHC cases into intra- and extrahepatic cholestasis. The following factorial evaluations were useful in distinguishing hepatic disease states: 1) when the sum of the activities of serum alkaline phosphatase, 5'-nucleotidase, aspartate and alanine amiotransferases, and isocitrate dehydrogenase was divided by the serum bilirubin concentration, there was good resolution of the distinction between patients with IHC and those with primary biliary cirrhosis, early and late viral hepatitis, cholelithiasis, and pancreatic and bile duct cancers. 2) Resolution was also achieved when the numerator included alkaline phosphatase, 5'-nucleotidase, and aspartate aminotransferase, but not when alkaline phosphatase alone, or alkaline phosphatase combined with 5'-nucleotidase, was used. The essential lesion in IHC is an excretory defect.     

Identification of MIC 11 antigen as an epitope of the CD59 molecule.

The MIC 11 antigen is expressed on human cells and is characterized by reaction with a monoclonal antibody (mAb), 16.3A5. The gene controlling MIC 11 was recently mapped to the p13 region of chromosome 11 within 500 kb of the gene encoding CD59, a complement regulatory protein. The present report investigates the antigenic relationship between these cell-membrane determinants and sets out evidence that MIC 11 and CD59 are encoded by the same gene. Western blotting of human erythrocyte membrane proteins and purified membrane CD59 showed that 16.3A5 anti-MIC 11 antibody bound to a 19-24,000 MW band with the characteristic appearance of CD59 protein, and gave staining patterns identical to those obtained with the CD59 antibody, BRIC 229. The binding of 16.3A5 monoclonal IgG to purified urine-derived CD59 in enzyme-linked immunosorbent assay (ELISA) was inhibited by YTH 53.1 rat CD59 antibody, indicating that the MIC 11 epitope is the same as, or close to, that recognized by CD59 antibodies such as YTH 53.1, BRIC 229 and 2/24. Prior exposure of erythrocytes to 16.3A5 anti-MIC 11 also reduced the ability of the CD59 antibodies, BRIC 229 and YTH 53.1, to block the complement-inhibiting function of membrane CD59. Anti-MIC 11 antibody alone, however, had no inhibitory effect on CD59 function. This may be due to its relatively low binding affinity or to some slight difference in epitope specificity. Further studies using immunofluorescence showed that the MIC 11 epitope, like CD59, is absent from EBV-B cells lacking GPI-anchored proteins and from a B-cell line specifically deficient in CD59 protein. Overall, the results provide strong evidence that MIC 11 is a determinant on the CD59 molecule.     

Effect of maternal cholestasis on the kinetics of bile acid transport across the canalicular membrane of infant rat livers

A reversible impairment in the ability of the liver to secrete cholephilic compounds has been reported to exist in infant rats born from mothers with surgically induced complete cholestasis during the last third of the pregnancy. Canalicular plasma membranes (CPM) were purified from livers obtained from 4 and 8 week-old offspring of healthy or cholestatic rats. Using radiolabelled glycocholic acid (GC) and a rapid filtration technique, bile acid transport by CPM vesicles in the presence of 3 mM ATP plus an ATP-regenerating system was measured at varying substrate concentrations. Kinetic parameters were calculated by nonlinear regression analysis. Similar values for the apparent affinity constant (Kt) were found in all experimental groups (approximately 350 μM ). The value of the maximal velocity of the transport (V_max) was similar for CPM obtained from control animals at 4 or 8 weeks of age (approximately 1.5 nmol/20 s/mg protein). In the offspring of cholestatic mothers the V_max value was not different from that found in control animals as far as 4 week-old rats were concerned. However, V_max in the 8 week-old group from cholestatic mothers was two-fold higher than that found in the rest of the experimental groups. Thus, the efficiency of transport, defined as V_max/Kt, was very similar in all experimental groups, except in the group of 8 week-old offspring of cholestatic mothers, where this value was 60% higher. Isolated livers obtained from this group were able to secrete a tracer dose of radiolabelled GC (11.25 nmol) into bile significantly faster than isolated livers obtained from control animals of the same age (8 weeks). In sum, these results indicate that, in young infant rats (4 week-old), in which the maximal secretion rate for bile acids was reduced by maternal cholestasis during pregnancy, the kinetics of ATP-dependent bile acid transport across the canalicular membrane were not affected. By contrast, in older infant rats (8 week-old), in which the overall ability of the liver to secrete bile acids seems to be restored to normality, the efficiency of the canalicular transport system was actually enhanced. This suggests the existence of compensation at the level of the canalicular membrane transfer and thus that there is another hitherto unidentified mechanism involved in bile acid secretion.     

Activity of lysosomal enzymes in the bile and serum of mice with intrahepatic cholestasis

Lysosomal enzyme activity in the bile and blood serum was compared in mice with experimental intrahepatic cholestasis induced by α-naphthyl isothiocyanate and Triton WR 1339. Triton WR 1339 increases the synthesis of cholesterol (fatty acid precursor) in liver cells. The development of intrahepatic cholestasis was confirmed by the increase in activities of alkaline phosphatase and γ-glutamyltransferase in blood serum. Administration of Triton WR 1339 in a dose of 100 mg/100 g was followed by a 10-fold increase in β-galactosidase activity (hepatocyte lysosomal enzyme) in the bile, but not in the serum of mice. β-Galactosidase activity significantly increased in the bile, but decreased in the serum of mice after treatment with α-naphthyl isothiocyanate in a dose of 200 mg/kg. Our results indicate that intrahepatic cholestasis is manifested in increased secretion of lysosomal glycosidases into the bile. Bile components can aggravate damage to liver cells by affecting the processes of hepatocyte apoptosis and necrosis. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 496–499, May, 2008     

Concomitant congenital CMV infection and inherited liver diseases

Inherited liver diseases may present in infancy as cholestatic jaundice progressing to severe hepatic dysfunction. Congenital cytomegalovirus (cCMV) infection may initially involve the liver, yet in otherwise healthy hosts rarely leads to long-term hepatic disease. We report a series of three patients, diagnosed with hereditary liver diseases: progressive familial intrahepatic cholestasis (PFIC) type IV, alpha 1 anti-trypsin deficiency (A1ATD) and Alagille syndrome (ALGS), who were also diagnosed with cCMV infection. All patients were treated with valgancilovir for symptomatic cCMV infection (6–12 months), followed by suppressive dosing in the 2 patients with PFIC and A1ATD. Following 15–24 months of follow-up – the patients with PFIC and A1ATD developed severe liver failure, and the third had ongoing cholestatic disease with stable synthetic function. We propose a significant contribution of cCMV infection to the course of the inherited primary disease, possibly leading to further compromise of the liver. We recommend screening patients with inherited liver disease for cCMV, and considering anti-viral treatment with valganciclovir to delay hepatic disease progression.     

低血清谷氨酸转肽酶进行性家族性肝内胆汁淤积症23例临床分析

目的总结以低血清谷氨酸转肽酶（GGT）为特征的进行性家族性肝内胆汁淤积症（PFIC）各型的临床特征,提高对该病的认识。方法回顾性分析2004年1月至2007年6月在复旦大学附属儿科医院临床诊断为低血清GGT的PFIC患儿的临床症状、实验室检查、肝脏组织病理学检查和随访等资料,根据ATP881和ABCB11全部基因外显子测序结果分为PFIC-Ⅰ、PFIC—Ⅱ和未分型3组,分析3组间临床特征的差异。结果23例低血清GGT的PFIC患儿进入分析,PFIC-Ⅰ、PFIC—Ⅱ和未分型组分别为9、7和7例。23例PFIC患儿出生体重均正常,以黄疸、瘙痒和肝功能损害起病。中位发病年龄40d（0d至7个月）,中位就诊年龄6（2—36）个月。①13例（56．5％）病程中出现过白陶土样大便,有明显腹泻症状3例。6例（26．1％）有营养不良、佝偻病和生长发育落后。2例（8．7％）随访中发现胆结石。②15例肝组织病理活检示：7例（46．7％）肝细胞多核巨细胞样转化,14例（93．3％）肝细胞和毛细胆管胆汁淤积明显,未发现脂肪变性病例。③未分型组ALT水平较PFIC—Ⅰ型和PFIC—Ⅱ型组显著升高（P〈0．05）;PFIC—Ⅱ型组ALT、TBA水平显著高于PFIC—Ⅰ型组（P〈0．05）;PFIC—Ⅱ型和未分型组肝细胞多核巨细胞转化发生率显著高于PFIC—Ⅰ型组（P〈0．05）。结论低血清GGT的PFIC患儿以黄疸、瘙痒和肝功能损害为主要临床表现,PFIC—Ⅰ型肝穿刺病理活检未见肝细胞多核巨细胞转化,PFIC—Ⅱ型血清ALT、TBA水平明显高于PFIC-Ⅰ型,有助于临床分型和治疗方法的选择。