Genetic Liver Disease
Introduction
A number of presentations held during the 54th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD) addressed important advances in genetic/metabolic liver disease. A clearer understanding of genetic liver disease will help guide new diagnostic and management strategies for treating liver diseases that affect patients of all ages. The observations presented at this year's meeting will therefore open new avenues for research into hepatobiliary physiology and pathophysiology.
This report discusses those studies that provided new insights into the pathophysiology, diagnosis, and treatment of 3 important genetic/ metabolic disorders of the liver: Wilson's disease, alpha-1-antitrypsin (alpha-1-AT) deficiency, and progressive familial intrahepatic cholestasis (PFIC), a heterozygous group of conditions.
Wilson's Disease Clinical Features
Wilson's disease is an autosomal recessive copper storage disorder caused by mutations of the ATP7B gene on chromosome 13. The disease is characterized by abnormal biliary copper excretion and hypoceruloplasminemia. The clinical presentation of Wilson's disease is highly variable, ranging from acute or chronic liver disease to a diverse pattern of neurologic disease. Previous genotype/phenotype correlations have been hampered by extreme heterogeneity in the populations studied and of ATP7B mutations. Two studies presented during these meeting proceedings revisited this issue.
Genotype-phenotype correlations. Genotype-phenotype correlations were investigated in a large number of patients with Wilson's disease by Ferenci and colleagues. Samples were obtained from 721 patients from various regions in the world (Germany, Austria, Benelux, Hungary, Turkey, Slovakia, Balkan countries, and other European and non-European areas). Overall, 620 subjects were index patients and 101 were detected during family screening. In this large study cohort, the H1069Q/H1069Q mutation was found in 28% of patients; all other mutations were rare. The only significant phenotypic-genotypic correlations were found for H1069Q homozygotes and H1069Q/3400delC, which was more frequently associated with late-onset neurologic disease. In addition, exon 8 mutations were more frequently associated with hepatic Wilson's disease. Among siblings, one third presented with the same symptoms. The remaining siblings were asymptomatic, including the monozygous twin sister of a patient with severe neurologic disease. Thus, most mutations in patients with Wilson's disease are rare, and no clear phenotype-genotype correlations were documented.
Seela and colleagues delineated the clinical and genetic characteristics of a large cohort of patients with Wilson's disease (n = 220) from a single ethnic group in Bangalore, South India. Mean age of presentation was 14 ± 8 years in men and 12 ± 8 years in women. The majority (65%) presented with neurologic symptoms; 15% had hepatic manifestations. The remainder of the patients were diagnosed through family screening. The mean serum ceruloplasmin level was 6 ± 0.91 mg/dL (normal range, 15-35 mg/dL) and the mean urinary copper excretion was 399 ± 44 micrograms[mcg]/24 hours (normal, < 70 mcg/24 hours). Kayser-Fleischer rings were present in 95% of patients, but overt signs of chronic liver disease were found in only 16%. Hepatic presentation occurred earlier, at mean age 11.8 ± 4.9 years, compared with neurologic presentation at 16 ± 9 years (P < .05). Neurologic presentation in the Bangalore cohort occurred significantly earlier than in other populations described in the literature (at 30-50 years of age). There was a diverse pattern of neurologic manifestations: 65% with parkinsonism, 23% with tremors, 20% had dysarthria, 3% had mental retardation, 2% had gait disturbances, and 2% had delayed milestones.
Apparently factors other than ATP7B mutations may modify the phenotypic presentation of Wilson's disease.
Pathophysiology
Copper transport in hepatocytes is mediated by the ATP7B copper pump, which is required for cuproenzyme synthesis and biliary copper excretion. Mutations in the ATP7B gene lead to impaired biliary excretion of copper and multiorgan copper toxicosis.
Modulation of copper transport. In yeast, copper transport processes are dependent on the activity of an intracellular chloride channel that shunts the voltage gradient generated by electrogenic copper pumping. Whether chloride channels modulate copper transport in hepatocytes and other mammalian cells is not known.
Wang and colleagues sought to determine if intracellular chloride channels can promote copper incorporation into ceruloplasmin. Copper incorporation into ceruloplasmin is chloride-dependent because chloride channels are required to shunt the potential of electrogenic copper transport. Chloride channel protein (ClC)-4 is the intracellular chloride channel most abundantly expressed in hepatocytes; its expression increases copper incorporation into ceruloplasmin, particularly when copper availability is limiting. The investigators proposed that intracellular chloride channels may function to modulate copper transport rates under conditions of abnormal hepatic copper processing, such as in Wilson's disease. This observation offers a novel therapeutic strategy -- pharmacologic modulation of chloride channels.
Molecular mechanisms. In Wilson's disease, mutations in the ATP7B gene lead to hepatic accumulation of copper. The accumulated copper becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and ultimately to hepatic injury and acute liver failure. Several proteins are presumably involved in dealing with the excess copper and the oxidative stress. Using a proteomics approach, Roelofsen and colleagues characterized copper-induced changes in protein expression in human HepG2 hepatoma cells as an in vitro model of copper overload. They demonstrated that both the intracellular protein profile as well as the excreted protein pattern is substantially altered as a reaction to a high extracellular copper concentration. In addition, 9 proteins with high-affinity copper-binding characteristics were discovered; however, their exact cellular roles are undetermined. These findings indicate that HepG2 cells provide a sensitive human in vitro model to study effects of copper overload on hepatic protein expression.
Mitochondrial injury in Wilson's disease. Diverse structural changes in liver cell mitochondria are typical of Wilson's disease, including variability in size and shape, increased density of matrix, pleomorphic inclusions, and prominent cystic dilatation of the tips of the cristae. Roberts and colleagues examined the functional basis for these structural abnormalities and assessed whether they were pathognomonic of Wilson's disease. These investigators used the tx-j mouse (G712D mutation in Atp7b gene on C3H/FeJ background), an accepted murine model for Wilson's disease, in which the hepatic histology becomes abnormal at about 5 months of life. Hepatic parenchymal copper concentration was found to be 40-fold above normal (424 mcg/g dry weight) at 1 month of age, was 789 mcg/g at 2-4 months of age, and was 538 mcg/g at 5-6 months. Electron microscopy showed typical mitochondrial abnormalities, specifically cystic dilatation of tips of cristae, at 4 and 6 months. The amount of mitochondrial DNA (mtDNA) was similar to that in control mice at 1-2 months and then progressively declined over ages 3-6 months. Thus, the study authors concluded that Wilson's disease is a mtDNA depletion disorder. Ultrastructural changes in Wilson's disease are not strictly pathognomonic for the disorder; instead, they reflect the mtDNA depletion. These investigators postulated that Wilson's disease is an example of acquired mtDNA depletion due to copper toxicity.
These data may provide new insights into the molecular mechanisms leading to hepatic dysfunction in Wilson's disease.
Diagnosis
Because Wilson's disease is extremely variable in its presenting manifestations and in age of onset, the clinical and biochemical features do not always confirm the diagnosis for this treatable disease.
Role of mutation analysis in diagnosis. Cox and colleagues examined the utility of molecular diagnosis, using mutation and marker analysis, as an aid in the diagnosis of clinically affected individuals with Wilson's disease and for asymptomatic siblings. They compared the traditional diagnostic features and mutation results in 99 definitively diagnosed patients with Wilson's disease. In 60 patients homozygous for 1 of the more than 250 described mutations, they examined the association between mutation type and clinical expression. Presymptomatic siblings were studied in 55 families in which the patient had a firm diagnosis of Wilson's disease. At least 1 mutation in the ATP7B gene was identified in 95% of patients with hepatic onset and in 86% with neurologic onset; mean age of hepatic onset was 13.4 years, and mean age of neurologic onset was 20.2 years. Kayser-Fleischer rings were reported in 89% of definitively diagnosed patients (81% in hepatic onset, 100% in neurologic onset). However, in patients whose diagnosis was initially queried, then confirmed by mutation identification, only 57% had Kayser-Fleischer rings (50% in hepatic onset, 69% in neurologic onset). Urinary copper excretion was > 1.6 micromoles/day in all "definite" patients, but was below this value in 3 of 25 "queried" patients. Serum ceruloplasmin concentration was below normal in all 28 definite neurologic cases, but was normal in 3 of 31 definite hepatic cases. In query cases, 4 of 16 patients with neurologic onset and 11 of 32 with hepatic onset had a normal serum ceruloplasmin level.
The type of mutation influences the age and type of onset of the disease. Among 60 patients homozygous for the same mutation, the only individuals for whom clinical phenotype-genotype correlation could be reliably examined, the age of onset was found to be 11.4 years for those with the most deleterious mutations (deletions, insertions, and nonsense), with 11 patients showing hepatic and 4 showing neurologic onset. Two patients with splice-site mutations had age of onset of 3 and 45 years. In 16 subjects with various missense mutations, the mean age of onset was 17.8 years. Patients with the common His1069Gln mutation had a mean age of onset of 20.3 years. In 54 families studied, 22 siblings of patients were confirmed or newly diagnosed as affected, and in many cases, the biochemical results had not been definitive.
The conclusion that one must draw from these important data is that DNA mutation analysis is an important adjunct to clinical and biochemical assessment because traditionally used features are not consistently abnormal. Mutation analysis is not trivial, as there are currently more than 250 mutations known. Because of the uncertainty of biochemical signs in asymptomatic patients, DNA analysis using markers flanking the ATP7B gene is essential for reliable diagnosis of Wilson's disease in the presymptomatic phase in siblings, and does not require knowledge of the specific mutation present.
Treatment
The management of patients with Wilson's disease follows the rules established for many metabolic liver diseases. The goal is to decrease the intake of the substrate by limiting copper in the diet, alter copper absorption via competition, shunt copper from its usual metabolic pathway by chelation, and limit the toxic effects of copper by supplying antioxidants to the depleted cells.
Chelation therapy. In the Bangalore Wilson's disease phenotype reported by Seela and colleagues, chelation therapy resulted in complete remission of symptoms in 70% of cases. Exacerbation of neurologic symptoms occurred in only 4% of patients. There was no response to therapy in 15% of individuals, but neurologic symptoms remained stable. Further delineation of this cohort may provide important insights into aberrant copper metabolism and allow more effective management.
Gene therapy. The long-term approach to the management of patients with metabolic liver disease is gene therapy. Merle and colleagues examined the feasibility of lentiviral vector-mediated gene therapy for Wilson's disease in an animal model (LEC rat). A recombinant lentiviral vector carrying a human Wilson's disease gene under control of the phosphoglycerokinase (PGK) promoter was cloned (PGK-hWD) and evaluated both in vitro and in vivo. Primary LEC rat hepatocytes transduced with PGK-hWD expressed ATP7B, demonstrating the feasibility of ATP7B transfer to primary rat hepatocytes by lentiviral in vitro transduction. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on liver-tissue of LEC rats at different time-points after systemic lentiviral PGK-hWD transduction and after transplantation of ex vivo transduced LEC-rat hepatocytes. For both tested approaches, these investigators found stable mRNA expression in the treated rats. ATP7B expression could be observed in hepatocytes of all treated rats up to 2 months. However, in hepatocyte-transplanted rats, the expression level diminished with time after transplantation. These results demonstrate the feasibility of lentiviral vector-mediated gene therapy of Wilson's disease in an animal model. Systemic vector administration leads to stable gene transfer and expression. In contrast, gene expression after transplantation of transduced hepatocytes reaches higher maximal levels, but diminishes with time.
Therefore, the key to managing patients with Wilson's disease is early diagnosis and prompt institution of chelation therapy. It is also clear that gene therapy is feasible and represents the hope for the future of treatment.
Alpha-1-AT Deficiency Clinical Features
Alpha-1-AT deficiency, one of the most common inherited metabolic disorders, has the potential to cause severe liver and lung injury. More than 70 genetic variants of alpha-1-AT have been described; these differ by point mutations comprising the so-called Pi (proteinase inhibitor) system. Pi*M is the most common allele and is associated with the normally-functioning alpha-1-AT molecule. Pi*Z is the most common deficient type, with decreased serum alpha-1-AT concentrations secondary to retention of the mutant Z protein in the endoplasmic reticulum in the hepatocyte. Homozygous (ZZ) alpha-1-AT deficiency is an important cause of liver disease in children, and can also cause chronic liver disease and hepatocellular carcinoma in adults. There is increasing evidence that carriers of a heterozygous alpha-1-AT phenotype (PiMZ or PiSZ) are also at an increased risk of developing cirrhosis and liver failure. These issues were addressed during the core proceedings of this year's AASLD meeting.
Pathogenetic role of alpha-1-AT polymorphisms. Berg and colleagues set out to elucidate the pathogenetic role of alpha-1-AT polymorphisms by investigating alpha-1-AT allele frequencies in a large group of patients (n = 1600) with compensated and decompensated liver diseases of different etiologies as well as in 185 healthy control subjects. Their study included 215 patients with alcoholic liver disease, 647 patients with hepatitis C, 199 with hepatitis B, 104 with autoimmune hepatitis, 216 patients with primary biliary cirrhosis or primary sclerosing cholangitis, 62 with cryptogenic cirrhosis, 52 with nonalcoholic liver disease, and 105 patients with various other liver diseases.
Hepatocellular carcinoma was present in 78 of the 1600 patients studied (5%). Liver transplantation was performed in 757 patients (47%) because of decompensated liver disease. Pi*M, Z and S allele distribution did not differ significantly between patients with liver diseases and healthy controls (96%, 2%, and 2% vs 95%, 2%, and 3%, respectively). There was also no significant difference in the Pi*Z allele frequency between patients with decompensated cirrhosis compared with patients with compensated liver disease (2.6% vs 1.8%; P = .22) or patients with and without hepatocellular carcinoma (2.6% vs 2.2%; P = .9). However, alpha-1-AT allele distribution differed significantly between the patients with different liver disorders (P = .02). The latter was primarily due to a significant over representation of Pi*Z allele in the group of patients with alcoholic and cryptogenic liver disease (4.9% and 5.6% Pi*Z allele, respectively). When they compared all patients with histologically proven cirrhosis (n = 687) with those classified as having no cirrhosis (n = 913), a trend for a higher Pi*Z allele frequency in the cirrhosis group was observed (2.9% vs 1.7%; P = .057). However, this association could not be confirmed when patients with alcoholic cirrhosis were excluded from the analysis.
This study confirms previous reports showing higher Pi*Z allele frequencies in patients with alcoholic and cryptogenic cirrhosis. These data indicate that individuals bearing the Pi*Z allele are more prone to develop severe alcoholic liver disease. There is, however, no general association of chronic liver disease development or severity of liver disease in carriers of a single Pi*Z allele. This study offers guidelines for counseling.
Pathophysiology
The alpha-1-AT mutant Z gene encodes a mutant protein that accumulates in the endoplasmic reticulum of hepatocytes rather than being secreted into the serum. Liver injury is presumably caused by accumulation of the alpha-1-AT mutant Z protein within hepatocytes; this accumulation triggers downstream intracellular injury pathways. However, the development of clinical liver disease among ZZ homozygotes is highly variable, suggesting that there is a significant influence of other genetic or environmental factors that contribute to liver injury.
Mechanism of liver injury. Rudnick and colleagues tested the hypothesis that nonsteroidal anti-inflammatory drugs (NSAIDs) could be cofactors in the development of liver injury in alpha-1-AT deficiency. They used the Pi*Z mouse, a model transgenic for the human alpha-1-AT mutant Z gene in which gene expression is regulated by the human alpha-1-AT promoter sequences. These investigators showed that indomethacin administered in typically nontoxic doses to Pi*Z mice was associated with increased alpha-1-AT gene transcription as determined by RT-PCR analysis of alpha-1-AT mRNA levels. Indomethacin also increased hepatic alpha-1-AT mutant Z protein content, as shown by increased globular accumulations of alpha-1-AT in histopathologic sections and by quantitative immunoblot analysis of liver lysates for human alpha-1-AT protein. Furthermore, indomethacin treatment in Pi*Z mice was associated with increased hepatic injury and increased mortality compared with that seen in vehicle-treated Pi*Z mice and indomethacin-treated wild-type mice. Evidence of hepatic injury included focal hepatocellular necrosis, apoptosis, and increased hepatocellular proliferation as a compensatory response to increased cell death.
These data suggest that environmental factors, such as exogenous medication administration, can significantly potentiate the liver injury associated with alpha-1-AT mutant Z hepatic accumulation, and that NSAIDs may be especially injurious to ZZ patients, possibly by mediating increased alpha-1-AT synthesis. This is another opportunity for prevention.
Treatment
In human alpha-1-AT deficiency, the associated lung disease is thought to reflect insufficient normal alpha-1-AT activity in the circulation, whereas the related liver disease occurs because abnormal alpha-1-AT accumulates in hepatocytes. Are there strategies that could limit the accumulation or enhance the degradation of the stored protein and thereby reduce liver injury?
Accelerated destruction. Duan and colleagues evaluated the efficacy of ribozyme-mediated destruction of targeted human Pi*Z transcripts in vivo. Quantitative RT-PCR analysis revealed that the average reduction of human Pi*Z transcripts in livers was 57 ±18% (P = .05) in mice that were killed between 6 and 16 weeks after transduction with the ribozyme construct (recombinant SV40 virus containing a ribozyme designed to target human alpha-1-AT mRNA). The administration of the ribozyme lowered serum levels of human alpha-1-AT to 42 ± 12% of pretreatment values (P < .01) 3-25 weeks post transduction, whereas serum human alpha-1-AT levels in transgenic mice not treated with the ribozyme were unchanged. Serum human alpha-1-AT level was reduced by 99% at 6 weeks, and human alpha-1-AT Pi*Z transcripts were undetectable by quantitative RT-PCR from the mouse liver. Moreover, quantitative RT-PCR showed that the levels of mouse alpha-1-AT, albumin, and beta-actin mRNA remained the same as in control mice despite the complete loss of the human alpha-1-AT transcripts.
These findings demonstrate that an SV40-derived construct containing a ribozyme is highly effective in lowering human alpha-1-AT mRNA and protein levels in vivo. This represents the first step in the development of a clinically valuable gene therapy approach for alpha-1-AT deficiency.
Progressive Familial Intrahepatic Cholestasis (PFIC)
There is a wide spectrum of inherited intrahepatic cholestatic disease, ranging from neonatal cholestasis to cirrhosis in adults. These syndromes are of great theoretic interest; detailed study of these "experiments of nature'' has enhanced our understanding of hepatic excretory function and bile acid metabolism.
Clinical Features
The PFIC syndromes are a heterozygous group of conditions that typically lead to end-stage liver disease by the second decade of life. These syndromes are caused by dysfunction of specific hepatocanalicular transport systems:
Although traditionally thought of as "pediatric liver diseases," various forms of PFIC can appear at all ages and may underlie/predispose to adult-onset disease, such as:
Epidemiology of PFIC types 1 and 2. FIC1 deficiency (also known as PFIC type 1) and BSEP deficiency (also known as PFIC type 2) comprise the 2 main forms of PFIC. Both of these forms of PFIC are endemic in certain regions of Israel and Saudi Arabia, but to date, few mutations have been described in these populations.
In a collaborative study, Antoniou and colleagues defined the genetic basis of PFIC. Linkage analysis was carried out on a group of 42 consanguineous families, 33 Saudi Arabian and 9 Israeli. Closely linked and intragenic microsatellite markers were used to generate haplotypes across the ATP8B1 (FIC1) and ABCB11 (BSEP) chromosomal regions. Segregation analysis was consistent with 23 families being linked to ABCB11 and 5 families to ATP8B1; 4 were uninformative and 10 were linked to neither locus. Three families were found to have the only recurring ABCB11 mutation identified in this population, G982R. Mutation screening of linked families by single-strand conformation polymorphism and direct sequencing suggested defects in 13 families. These included 2 novel missense changes in exons 13 and 25 of ABCB11.
These observations are important because the response to both medical and surgical intervention has been shown to be dependent not only on the type of PFIC present, but also on the specific mutation.
Role of mutations in specific hepatocanalicular transporters in ICP. ICP is a liver disorder associated with increased risk of intrauterine fetal death and prematurity. ICP usually occurs in late pregnancy and resolves after delivery. The main biochemical finding is an increase in total serum bile acid concentrations; other laboratory findings reflecting cholestasis are an elevation in serum alkaline phosphatase and total bilirubin, while gamma-glutamyltransferase (gamma-GT) levels are normal.
The cause of ICP is unknown, but there is increasing evidence that genetically determined dysfunction in the canalicular ATP-binding cassette (ABC)-transporters bile salt export pump (BSEP, ABC11) and/or MDR3 (ABCB4) might be risk factors for the development of ICP. Impaired expression and function of these transporters, which are the main factors in hepatocellular bile formation, have been shown to be the cause of different hereditary cholestatic syndromes, such as PFIC1, 2, or 3.
Pauli-Magnus and colleagues described the extent of genetic variability in BSEP and MDR3 in 42 women with ICP and 80 healthy pregnant controls, and searched for disease-causing mutations. Criteria for diagnosis of ICP included pruritus, increased bile acid concentrations in serum, and spontaneous resolution of symptoms after delivery. BSEP and MDR3 sequencing revealed several new variant sites in both genes. For BSEP, 23 of 26 (88%) variant sites detected in ICP patients were also present in the control group. However, in the case of MDR3, only 30 of 46 (65%) variant sites were present in the control group. Variant sites included 6 nonsynonymous variants in BSEP and 8 in MDR3, 3 of which were specific for the ICP group (in BSEP: exon 18, R698H; in MDR3: exon 9, S320F; and in exon 18, G762E). Furthermore, in MDR3, 5 new heterozygous splicing-consensus mutations were detected, all of which were unique to the ICP subgroup. One patient with ICP and a new MDR3 splicing mutation had a familial cholestatic syndrome associated with this mutation.
This is the first study to determine the extent of genetic variation in BSEP and MDR3 in a large collective of women with ICP and healthy control women. Although most of the mutations detected in BSEP were found in both populations, a greater number of ICP-specific variants were detected in MDR3, including 2 highly conserved nonsynonymous and 5 splicing-consensus sites.
These findings further support a pathogenic role for MDR3 genetic variability in ICP. Analysis of population-specific variant frequencies as well as the BSEP and MDR3 haplotype structure in these groups will allow a more detailed analysis of mutations that might represent a susceptibility factor for the development of ICP.
Role of mutations in specific hepatocanalicular transporters in primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). There is ongoing discussion regarding whether genetically determined dysfunction may also play a pathogenetic role in PBC and PSC. PBC and PSC are characterized by a cholestatic pattern of liver damage with elevated serum bile acids. Such a pattern is similar to that observed in hereditary or acquired dysfunction of the hepatic canalicular membrane transporters BSEP and MDR3, which secrete cholephilic compounds into the bile canaliculus.
Pauli-Magnus and colleagues tested whether sequence diversity and haplotype structure of BSEP and MDR3 in patients with PBC and PSC were different from that observed in healthy volunteers with the same ethnic background. DNA samples from 93 healthy white individuals, 70 patients with PBC, and 46 patients with PSC were screened for genetic variations in BSEP and MDR3. The total number of different haplotypes as well as the relative fraction of the most common haplotypes were similar in all 3 groups for BSEP and MDR3. Also, the number of population-specific haplotypes accounted for a minority of alleles and did not differ between the 3 groups. Furthermore, similar patterns of linkage disequilibrium were found in all 3 patient populations. These investigators concluded that patients with PBC and patients with PSC did not show a significant deviation in haplotype structure in BSEP and MDR3 from that observed in healthy white controls.
These data do not support a role for BSEP and MDR3 genetic variation in the pathogenesis of PBC and PSC, because variant segregation and haplotype structure of both genes did not differ from the pattern observed in healthy controls. Although a functional impact of some of these rare variants or haplotypes cannot be ruled out, they cannot serve as a common denominator of disease pathogenesis in PBC and PSC. Alternatively, the cumulative data provide a basis for the study of phenotypic differences in BSEP and MDR3 genetic variation in the normal population, which may help to define genotypes associated with an increased risk of developing cholestasis.
Pathophysiology
PFIC-1 and PFIC-2. The genetic defect in PFIC-1 has been mapped to a locus on chromosome 18q21-q22, a region encoding FIC1 (ATP8B1), a P-type ATPase. Thus ATP8B1 is mutated in a form of low gamma-GT intrahepatic cholestasis formerly called "Byler's syndrome."
PFIC-2, another form of low gamma-GT intrahepatic cholestasis, is caused by deficiency in BSEP, the major bile salt transporter in human liver, due to mutations in the ABCB11 (BSEP) gene. The chromosomal locus is 2q24
Molecular mechanism of PFIC-1 and PFIC-2. Bile sampled from gallbladders of individuals with intrahepatic cholestasis due to mutation in ABCB11 (PFIC-2) is deficient in primary bile acids. This can be ascribed to the fact that the ABCB11 gene encodes the bile salt export protein, BSEP. Similar deficiency of biliary bile acids in mature bile is found in intrahepatic cholestasis owing to mutation in the ATP8B1 gene (PFIC-1). The ATP8B1 gene encodes a putative aminophospholipid "flippase," FIC1, hypothesized to maintain compositional asymmetry between leaflets of cell membranes. FIC1 may be necessary for optimal function or trafficking of membrane-inserted proteins. Some canalicular proteins appear deficiently expressed in persons with ATP8B1 disease. The question, therefore, is whether deficiency in BSEP expression at the canaliculus underlies the lack of primary bile acids in bile.
Knisely and colleagues evaluated expression at the canaliculus of BSEP in formalin-fixed, paraffin-embedded archival liver-biopsy or hepatectomy materials from 20 patients of varying ages with intrahepatic cholestasis. Ten had documented ATP8B1 (PFIC-1) gene mutations and 10 had documented ABCB11 (PFIC-2) gene mutations. In the 10 patients with ATP8B1 gene mutations, expression of BSEP was indistinguishable from that in controls; in the 9 patients with ABCB11 gene mutations, expression of BSEP was absent. Function of BSEP was intact in persons with ATP8B1 gene disease, as judged by findings at liver biopsy on presentation in infancy. While individuals with ABCB11 gene mutation have giant-cell hepatitis with hepatocellular cholestasis and necrosis, those with ATP8B1 mutation have bland canalicular cholestasis and remarkably little hepatocellular disarray. This suggests that in ATP8B1 gene disease, toxic bile acids do not accumulate within hepatocytes to the extent that it occurs in ABCB11 gene disease. These findings indicate that not all canalicular proteins are abnormally expressed in ATP8B1 gene disease, and that cholestasis in ATP8B1 gene disease is not due to disruption of canalicular transporter-protein expression. The study authors speculated that individuals with ATP8B1 gene disease do not downregulate the sodium-dependent apical bile acid transporter (ASBT) in the terminal ileum. If in ATP8B1 gene disease bile acids are normally exported into the canaliculus, yet are deficient in mature bile, it may be due in part to their absorption in excess during transit past cholangiocytes in which ASBT is abnormally regulated. The clinical implication of these findings is unclear at present.
PFIC-3. PFIC-3 is associated with mutations in the MDR3 gene, also designated ABCB4, localized on chromosome 7q21, and is a phospholipid (PL) transporter. The clinical manifestations of MDR3 mutations are highly variable: heterozygous mutations are associated with ICP, drug-induced cholestasis, and cholesterol lithiasis (caused by the high lithogenicity of bile due to the low PL content). Rosmorduc and colleagues described an MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Thus, MDR3 gene mutations represent a genetic factor involved in a form of symptomatic cholesterol cholelithiasis in adult patients (low phospholipid-associated cholelithiasis [LPAC]).
Lucena and colleagues reported a woman who presented as an adolescent with cholelithiasis. She later developed recurrent ICP and finally biliary cirrhosis at age 47. She and her daughter (who also had ICP) were heterozygous for an MDR3 mutation. Thus, 3 consecutive diseases were associated with an MDR3 mutation; this led to end-stage cirrhosis after the fourth decade of life.
Treatment
Because the efficacy of medical therapy is poor, a number of surgical procedures have been successfully used in the management of patients with PFIC-1 and PFIC-2. Biliary diversion and ileal exclusion are increasingly being used as pretransplant surgical interventions. It is proposed that these surgical procedures may significantly delay, or even obviate, the necessity of liver transplantation. However, results are contradictory and center-specific, and a sufficient attempt has not been made to correlate outcome with the type of PFIC present.
Pawlikowska and colleagues presented data emerging from a multicenter study of over 100 patients to correlate clinical and biochemical data (including information on surgical treatment outcomes) with genotype in these disorders. Biliary diversion has been performed in 18 patients with BSEP (mean age at surgery, 5.5 years; none had progressed to cirrhosis at time of surgery). Fifteen patients had a positive outcome with biliary diversion, including significant and sustained improvement in pruritus (n = 13), reduction in hyperbilirubinemia (n = 10), and improved growth (n = 9). Three patients are on ursodeoxycholic acid and/or rifampicin following biliary diversion for control of pruritus; 11 have required no medications. Eleven of the 15 patients with a good outcome carry at least 1 of the common European mutations, D482G and E297G. Sixteen patients with BSEP (including 2 with failed biliary diversion) have undergone liver transplantation (mean age at liver transplantation was 5.5 years). Fifteen patients had excellent outcomes, including complete relief of jaundice and pruritus. Biliary diversion has been performed in 16 patients with FIC1 (mean age at surgery, 3.1 years); 3 subsequently underwent liver transplantation. In the remainder of patients, outcomes have been variable. Four are doing well, with minimal pruritus, improved growth, and reduced hyperbilirubinemia; 3 have minimal benefit; and in another 3 patients, initial improvement was followed by symptom recurrence. Twenty-two patients with FIC1 have had liver transplantation (at a mean age of 6.9 years); 10 showed signs of cirrhosis at liver transplantation, including 3 patients who previously had biliary diversion.
Biliary diversion can be a useful treatment in some cases of BSEP and FIC1 disease. These data indicate that carrying at least 1 copy of the common D482G BSEP mutation is predictive of a positive outcome with biliary diversion. Studies to determine whether biliary diversion may be more reliably successful in patients with BSEP than in those with FIC1 are under way.
Concluding Remarks
Studies presented during this year's AASLD meeting provide a great stimulus to the further study of genetic liver disease. These studies offer new diagnostic and management strategies for liver diseases that affect diverse patient populations. The era of definitive therapy for these disorders is near.
References
A number of presentations held during the 54th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD) addressed important advances in genetic/metabolic liver disease. A clearer understanding of genetic liver disease will help guide new diagnostic and management strategies for treating liver diseases that affect patients of all ages. The observations presented at this year's meeting will therefore open new avenues for research into hepatobiliary physiology and pathophysiology.
This report discusses those studies that provided new insights into the pathophysiology, diagnosis, and treatment of 3 important genetic/ metabolic disorders of the liver: Wilson's disease, alpha-1-antitrypsin (alpha-1-AT) deficiency, and progressive familial intrahepatic cholestasis (PFIC), a heterozygous group of conditions.
Wilson's Disease Clinical Features
Wilson's disease is an autosomal recessive copper storage disorder caused by mutations of the ATP7B gene on chromosome 13. The disease is characterized by abnormal biliary copper excretion and hypoceruloplasminemia. The clinical presentation of Wilson's disease is highly variable, ranging from acute or chronic liver disease to a diverse pattern of neurologic disease. Previous genotype/phenotype correlations have been hampered by extreme heterogeneity in the populations studied and of ATP7B mutations. Two studies presented during these meeting proceedings revisited this issue.
Genotype-phenotype correlations. Genotype-phenotype correlations were investigated in a large number of patients with Wilson's disease by Ferenci and colleagues. Samples were obtained from 721 patients from various regions in the world (Germany, Austria, Benelux, Hungary, Turkey, Slovakia, Balkan countries, and other European and non-European areas). Overall, 620 subjects were index patients and 101 were detected during family screening. In this large study cohort, the H1069Q/H1069Q mutation was found in 28% of patients; all other mutations were rare. The only significant phenotypic-genotypic correlations were found for H1069Q homozygotes and H1069Q/3400delC, which was more frequently associated with late-onset neurologic disease. In addition, exon 8 mutations were more frequently associated with hepatic Wilson's disease. Among siblings, one third presented with the same symptoms. The remaining siblings were asymptomatic, including the monozygous twin sister of a patient with severe neurologic disease. Thus, most mutations in patients with Wilson's disease are rare, and no clear phenotype-genotype correlations were documented.
Seela and colleagues delineated the clinical and genetic characteristics of a large cohort of patients with Wilson's disease (n = 220) from a single ethnic group in Bangalore, South India. Mean age of presentation was 14 ± 8 years in men and 12 ± 8 years in women. The majority (65%) presented with neurologic symptoms; 15% had hepatic manifestations. The remainder of the patients were diagnosed through family screening. The mean serum ceruloplasmin level was 6 ± 0.91 mg/dL (normal range, 15-35 mg/dL) and the mean urinary copper excretion was 399 ± 44 micrograms[mcg]/24 hours (normal, < 70 mcg/24 hours). Kayser-Fleischer rings were present in 95% of patients, but overt signs of chronic liver disease were found in only 16%. Hepatic presentation occurred earlier, at mean age 11.8 ± 4.9 years, compared with neurologic presentation at 16 ± 9 years (P < .05). Neurologic presentation in the Bangalore cohort occurred significantly earlier than in other populations described in the literature (at 30-50 years of age). There was a diverse pattern of neurologic manifestations: 65% with parkinsonism, 23% with tremors, 20% had dysarthria, 3% had mental retardation, 2% had gait disturbances, and 2% had delayed milestones.
Apparently factors other than ATP7B mutations may modify the phenotypic presentation of Wilson's disease.
Pathophysiology
Copper transport in hepatocytes is mediated by the ATP7B copper pump, which is required for cuproenzyme synthesis and biliary copper excretion. Mutations in the ATP7B gene lead to impaired biliary excretion of copper and multiorgan copper toxicosis.
Modulation of copper transport. In yeast, copper transport processes are dependent on the activity of an intracellular chloride channel that shunts the voltage gradient generated by electrogenic copper pumping. Whether chloride channels modulate copper transport in hepatocytes and other mammalian cells is not known.
Wang and colleagues sought to determine if intracellular chloride channels can promote copper incorporation into ceruloplasmin. Copper incorporation into ceruloplasmin is chloride-dependent because chloride channels are required to shunt the potential of electrogenic copper transport. Chloride channel protein (ClC)-4 is the intracellular chloride channel most abundantly expressed in hepatocytes; its expression increases copper incorporation into ceruloplasmin, particularly when copper availability is limiting. The investigators proposed that intracellular chloride channels may function to modulate copper transport rates under conditions of abnormal hepatic copper processing, such as in Wilson's disease. This observation offers a novel therapeutic strategy -- pharmacologic modulation of chloride channels.
Molecular mechanisms. In Wilson's disease, mutations in the ATP7B gene lead to hepatic accumulation of copper. The accumulated copper becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and ultimately to hepatic injury and acute liver failure. Several proteins are presumably involved in dealing with the excess copper and the oxidative stress. Using a proteomics approach, Roelofsen and colleagues characterized copper-induced changes in protein expression in human HepG2 hepatoma cells as an in vitro model of copper overload. They demonstrated that both the intracellular protein profile as well as the excreted protein pattern is substantially altered as a reaction to a high extracellular copper concentration. In addition, 9 proteins with high-affinity copper-binding characteristics were discovered; however, their exact cellular roles are undetermined. These findings indicate that HepG2 cells provide a sensitive human in vitro model to study effects of copper overload on hepatic protein expression.
Mitochondrial injury in Wilson's disease. Diverse structural changes in liver cell mitochondria are typical of Wilson's disease, including variability in size and shape, increased density of matrix, pleomorphic inclusions, and prominent cystic dilatation of the tips of the cristae. Roberts and colleagues examined the functional basis for these structural abnormalities and assessed whether they were pathognomonic of Wilson's disease. These investigators used the tx-j mouse (G712D mutation in Atp7b gene on C3H/FeJ background), an accepted murine model for Wilson's disease, in which the hepatic histology becomes abnormal at about 5 months of life. Hepatic parenchymal copper concentration was found to be 40-fold above normal (424 mcg/g dry weight) at 1 month of age, was 789 mcg/g at 2-4 months of age, and was 538 mcg/g at 5-6 months. Electron microscopy showed typical mitochondrial abnormalities, specifically cystic dilatation of tips of cristae, at 4 and 6 months. The amount of mitochondrial DNA (mtDNA) was similar to that in control mice at 1-2 months and then progressively declined over ages 3-6 months. Thus, the study authors concluded that Wilson's disease is a mtDNA depletion disorder. Ultrastructural changes in Wilson's disease are not strictly pathognomonic for the disorder; instead, they reflect the mtDNA depletion. These investigators postulated that Wilson's disease is an example of acquired mtDNA depletion due to copper toxicity.
These data may provide new insights into the molecular mechanisms leading to hepatic dysfunction in Wilson's disease.
Diagnosis
Because Wilson's disease is extremely variable in its presenting manifestations and in age of onset, the clinical and biochemical features do not always confirm the diagnosis for this treatable disease.
Role of mutation analysis in diagnosis. Cox and colleagues examined the utility of molecular diagnosis, using mutation and marker analysis, as an aid in the diagnosis of clinically affected individuals with Wilson's disease and for asymptomatic siblings. They compared the traditional diagnostic features and mutation results in 99 definitively diagnosed patients with Wilson's disease. In 60 patients homozygous for 1 of the more than 250 described mutations, they examined the association between mutation type and clinical expression. Presymptomatic siblings were studied in 55 families in which the patient had a firm diagnosis of Wilson's disease. At least 1 mutation in the ATP7B gene was identified in 95% of patients with hepatic onset and in 86% with neurologic onset; mean age of hepatic onset was 13.4 years, and mean age of neurologic onset was 20.2 years. Kayser-Fleischer rings were reported in 89% of definitively diagnosed patients (81% in hepatic onset, 100% in neurologic onset). However, in patients whose diagnosis was initially queried, then confirmed by mutation identification, only 57% had Kayser-Fleischer rings (50% in hepatic onset, 69% in neurologic onset). Urinary copper excretion was > 1.6 micromoles/day in all "definite" patients, but was below this value in 3 of 25 "queried" patients. Serum ceruloplasmin concentration was below normal in all 28 definite neurologic cases, but was normal in 3 of 31 definite hepatic cases. In query cases, 4 of 16 patients with neurologic onset and 11 of 32 with hepatic onset had a normal serum ceruloplasmin level.
The type of mutation influences the age and type of onset of the disease. Among 60 patients homozygous for the same mutation, the only individuals for whom clinical phenotype-genotype correlation could be reliably examined, the age of onset was found to be 11.4 years for those with the most deleterious mutations (deletions, insertions, and nonsense), with 11 patients showing hepatic and 4 showing neurologic onset. Two patients with splice-site mutations had age of onset of 3 and 45 years. In 16 subjects with various missense mutations, the mean age of onset was 17.8 years. Patients with the common His1069Gln mutation had a mean age of onset of 20.3 years. In 54 families studied, 22 siblings of patients were confirmed or newly diagnosed as affected, and in many cases, the biochemical results had not been definitive.
The conclusion that one must draw from these important data is that DNA mutation analysis is an important adjunct to clinical and biochemical assessment because traditionally used features are not consistently abnormal. Mutation analysis is not trivial, as there are currently more than 250 mutations known. Because of the uncertainty of biochemical signs in asymptomatic patients, DNA analysis using markers flanking the ATP7B gene is essential for reliable diagnosis of Wilson's disease in the presymptomatic phase in siblings, and does not require knowledge of the specific mutation present.
Treatment
The management of patients with Wilson's disease follows the rules established for many metabolic liver diseases. The goal is to decrease the intake of the substrate by limiting copper in the diet, alter copper absorption via competition, shunt copper from its usual metabolic pathway by chelation, and limit the toxic effects of copper by supplying antioxidants to the depleted cells.
Chelation therapy. In the Bangalore Wilson's disease phenotype reported by Seela and colleagues, chelation therapy resulted in complete remission of symptoms in 70% of cases. Exacerbation of neurologic symptoms occurred in only 4% of patients. There was no response to therapy in 15% of individuals, but neurologic symptoms remained stable. Further delineation of this cohort may provide important insights into aberrant copper metabolism and allow more effective management.
Gene therapy. The long-term approach to the management of patients with metabolic liver disease is gene therapy. Merle and colleagues examined the feasibility of lentiviral vector-mediated gene therapy for Wilson's disease in an animal model (LEC rat). A recombinant lentiviral vector carrying a human Wilson's disease gene under control of the phosphoglycerokinase (PGK) promoter was cloned (PGK-hWD) and evaluated both in vitro and in vivo. Primary LEC rat hepatocytes transduced with PGK-hWD expressed ATP7B, demonstrating the feasibility of ATP7B transfer to primary rat hepatocytes by lentiviral in vitro transduction. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on liver-tissue of LEC rats at different time-points after systemic lentiviral PGK-hWD transduction and after transplantation of ex vivo transduced LEC-rat hepatocytes. For both tested approaches, these investigators found stable mRNA expression in the treated rats. ATP7B expression could be observed in hepatocytes of all treated rats up to 2 months. However, in hepatocyte-transplanted rats, the expression level diminished with time after transplantation. These results demonstrate the feasibility of lentiviral vector-mediated gene therapy of Wilson's disease in an animal model. Systemic vector administration leads to stable gene transfer and expression. In contrast, gene expression after transplantation of transduced hepatocytes reaches higher maximal levels, but diminishes with time.
Therefore, the key to managing patients with Wilson's disease is early diagnosis and prompt institution of chelation therapy. It is also clear that gene therapy is feasible and represents the hope for the future of treatment.
Alpha-1-AT Deficiency Clinical Features
Alpha-1-AT deficiency, one of the most common inherited metabolic disorders, has the potential to cause severe liver and lung injury. More than 70 genetic variants of alpha-1-AT have been described; these differ by point mutations comprising the so-called Pi (proteinase inhibitor) system. Pi*M is the most common allele and is associated with the normally-functioning alpha-1-AT molecule. Pi*Z is the most common deficient type, with decreased serum alpha-1-AT concentrations secondary to retention of the mutant Z protein in the endoplasmic reticulum in the hepatocyte. Homozygous (ZZ) alpha-1-AT deficiency is an important cause of liver disease in children, and can also cause chronic liver disease and hepatocellular carcinoma in adults. There is increasing evidence that carriers of a heterozygous alpha-1-AT phenotype (PiMZ or PiSZ) are also at an increased risk of developing cirrhosis and liver failure. These issues were addressed during the core proceedings of this year's AASLD meeting.
Pathogenetic role of alpha-1-AT polymorphisms. Berg and colleagues set out to elucidate the pathogenetic role of alpha-1-AT polymorphisms by investigating alpha-1-AT allele frequencies in a large group of patients (n = 1600) with compensated and decompensated liver diseases of different etiologies as well as in 185 healthy control subjects. Their study included 215 patients with alcoholic liver disease, 647 patients with hepatitis C, 199 with hepatitis B, 104 with autoimmune hepatitis, 216 patients with primary biliary cirrhosis or primary sclerosing cholangitis, 62 with cryptogenic cirrhosis, 52 with nonalcoholic liver disease, and 105 patients with various other liver diseases.
Hepatocellular carcinoma was present in 78 of the 1600 patients studied (5%). Liver transplantation was performed in 757 patients (47%) because of decompensated liver disease. Pi*M, Z and S allele distribution did not differ significantly between patients with liver diseases and healthy controls (96%, 2%, and 2% vs 95%, 2%, and 3%, respectively). There was also no significant difference in the Pi*Z allele frequency between patients with decompensated cirrhosis compared with patients with compensated liver disease (2.6% vs 1.8%; P = .22) or patients with and without hepatocellular carcinoma (2.6% vs 2.2%; P = .9). However, alpha-1-AT allele distribution differed significantly between the patients with different liver disorders (P = .02). The latter was primarily due to a significant over representation of Pi*Z allele in the group of patients with alcoholic and cryptogenic liver disease (4.9% and 5.6% Pi*Z allele, respectively). When they compared all patients with histologically proven cirrhosis (n = 687) with those classified as having no cirrhosis (n = 913), a trend for a higher Pi*Z allele frequency in the cirrhosis group was observed (2.9% vs 1.7%; P = .057). However, this association could not be confirmed when patients with alcoholic cirrhosis were excluded from the analysis.
This study confirms previous reports showing higher Pi*Z allele frequencies in patients with alcoholic and cryptogenic cirrhosis. These data indicate that individuals bearing the Pi*Z allele are more prone to develop severe alcoholic liver disease. There is, however, no general association of chronic liver disease development or severity of liver disease in carriers of a single Pi*Z allele. This study offers guidelines for counseling.
Pathophysiology
The alpha-1-AT mutant Z gene encodes a mutant protein that accumulates in the endoplasmic reticulum of hepatocytes rather than being secreted into the serum. Liver injury is presumably caused by accumulation of the alpha-1-AT mutant Z protein within hepatocytes; this accumulation triggers downstream intracellular injury pathways. However, the development of clinical liver disease among ZZ homozygotes is highly variable, suggesting that there is a significant influence of other genetic or environmental factors that contribute to liver injury.
Mechanism of liver injury. Rudnick and colleagues tested the hypothesis that nonsteroidal anti-inflammatory drugs (NSAIDs) could be cofactors in the development of liver injury in alpha-1-AT deficiency. They used the Pi*Z mouse, a model transgenic for the human alpha-1-AT mutant Z gene in which gene expression is regulated by the human alpha-1-AT promoter sequences. These investigators showed that indomethacin administered in typically nontoxic doses to Pi*Z mice was associated with increased alpha-1-AT gene transcription as determined by RT-PCR analysis of alpha-1-AT mRNA levels. Indomethacin also increased hepatic alpha-1-AT mutant Z protein content, as shown by increased globular accumulations of alpha-1-AT in histopathologic sections and by quantitative immunoblot analysis of liver lysates for human alpha-1-AT protein. Furthermore, indomethacin treatment in Pi*Z mice was associated with increased hepatic injury and increased mortality compared with that seen in vehicle-treated Pi*Z mice and indomethacin-treated wild-type mice. Evidence of hepatic injury included focal hepatocellular necrosis, apoptosis, and increased hepatocellular proliferation as a compensatory response to increased cell death.
These data suggest that environmental factors, such as exogenous medication administration, can significantly potentiate the liver injury associated with alpha-1-AT mutant Z hepatic accumulation, and that NSAIDs may be especially injurious to ZZ patients, possibly by mediating increased alpha-1-AT synthesis. This is another opportunity for prevention.
Treatment
In human alpha-1-AT deficiency, the associated lung disease is thought to reflect insufficient normal alpha-1-AT activity in the circulation, whereas the related liver disease occurs because abnormal alpha-1-AT accumulates in hepatocytes. Are there strategies that could limit the accumulation or enhance the degradation of the stored protein and thereby reduce liver injury?
Accelerated destruction. Duan and colleagues evaluated the efficacy of ribozyme-mediated destruction of targeted human Pi*Z transcripts in vivo. Quantitative RT-PCR analysis revealed that the average reduction of human Pi*Z transcripts in livers was 57 ±18% (P = .05) in mice that were killed between 6 and 16 weeks after transduction with the ribozyme construct (recombinant SV40 virus containing a ribozyme designed to target human alpha-1-AT mRNA). The administration of the ribozyme lowered serum levels of human alpha-1-AT to 42 ± 12% of pretreatment values (P < .01) 3-25 weeks post transduction, whereas serum human alpha-1-AT levels in transgenic mice not treated with the ribozyme were unchanged. Serum human alpha-1-AT level was reduced by 99% at 6 weeks, and human alpha-1-AT Pi*Z transcripts were undetectable by quantitative RT-PCR from the mouse liver. Moreover, quantitative RT-PCR showed that the levels of mouse alpha-1-AT, albumin, and beta-actin mRNA remained the same as in control mice despite the complete loss of the human alpha-1-AT transcripts.
These findings demonstrate that an SV40-derived construct containing a ribozyme is highly effective in lowering human alpha-1-AT mRNA and protein levels in vivo. This represents the first step in the development of a clinically valuable gene therapy approach for alpha-1-AT deficiency.
Progressive Familial Intrahepatic Cholestasis (PFIC)
There is a wide spectrum of inherited intrahepatic cholestatic disease, ranging from neonatal cholestasis to cirrhosis in adults. These syndromes are of great theoretic interest; detailed study of these "experiments of nature'' has enhanced our understanding of hepatic excretory function and bile acid metabolism.
Clinical Features
The PFIC syndromes are a heterozygous group of conditions that typically lead to end-stage liver disease by the second decade of life. These syndromes are caused by dysfunction of specific hepatocanalicular transport systems:
Familial intrahepatic cholestasis gene 1 (FIC1; also known as ATP8B1);
Bile salt export pump (BSEP); and
Multidrug resistance 3 glycoprotein (MDR3; also known as ABCB4).
Although traditionally thought of as "pediatric liver diseases," various forms of PFIC can appear at all ages and may underlie/predispose to adult-onset disease, such as:
Progressive familial intrahepatic cholestasis (PFIC);
Benign recurrent intrahepatic cholestasis(BRIC);
Intrahepatic cholestasis of pregnancy (ICP); and
Cholesterol cholelithiasis.
Epidemiology of PFIC types 1 and 2. FIC1 deficiency (also known as PFIC type 1) and BSEP deficiency (also known as PFIC type 2) comprise the 2 main forms of PFIC. Both of these forms of PFIC are endemic in certain regions of Israel and Saudi Arabia, but to date, few mutations have been described in these populations.
In a collaborative study, Antoniou and colleagues defined the genetic basis of PFIC. Linkage analysis was carried out on a group of 42 consanguineous families, 33 Saudi Arabian and 9 Israeli. Closely linked and intragenic microsatellite markers were used to generate haplotypes across the ATP8B1 (FIC1) and ABCB11 (BSEP) chromosomal regions. Segregation analysis was consistent with 23 families being linked to ABCB11 and 5 families to ATP8B1; 4 were uninformative and 10 were linked to neither locus. Three families were found to have the only recurring ABCB11 mutation identified in this population, G982R. Mutation screening of linked families by single-strand conformation polymorphism and direct sequencing suggested defects in 13 families. These included 2 novel missense changes in exons 13 and 25 of ABCB11.
These observations are important because the response to both medical and surgical intervention has been shown to be dependent not only on the type of PFIC present, but also on the specific mutation.
Role of mutations in specific hepatocanalicular transporters in ICP. ICP is a liver disorder associated with increased risk of intrauterine fetal death and prematurity. ICP usually occurs in late pregnancy and resolves after delivery. The main biochemical finding is an increase in total serum bile acid concentrations; other laboratory findings reflecting cholestasis are an elevation in serum alkaline phosphatase and total bilirubin, while gamma-glutamyltransferase (gamma-GT) levels are normal.
The cause of ICP is unknown, but there is increasing evidence that genetically determined dysfunction in the canalicular ATP-binding cassette (ABC)-transporters bile salt export pump (BSEP, ABC11) and/or MDR3 (ABCB4) might be risk factors for the development of ICP. Impaired expression and function of these transporters, which are the main factors in hepatocellular bile formation, have been shown to be the cause of different hereditary cholestatic syndromes, such as PFIC1, 2, or 3.
Pauli-Magnus and colleagues described the extent of genetic variability in BSEP and MDR3 in 42 women with ICP and 80 healthy pregnant controls, and searched for disease-causing mutations. Criteria for diagnosis of ICP included pruritus, increased bile acid concentrations in serum, and spontaneous resolution of symptoms after delivery. BSEP and MDR3 sequencing revealed several new variant sites in both genes. For BSEP, 23 of 26 (88%) variant sites detected in ICP patients were also present in the control group. However, in the case of MDR3, only 30 of 46 (65%) variant sites were present in the control group. Variant sites included 6 nonsynonymous variants in BSEP and 8 in MDR3, 3 of which were specific for the ICP group (in BSEP: exon 18, R698H; in MDR3: exon 9, S320F; and in exon 18, G762E). Furthermore, in MDR3, 5 new heterozygous splicing-consensus mutations were detected, all of which were unique to the ICP subgroup. One patient with ICP and a new MDR3 splicing mutation had a familial cholestatic syndrome associated with this mutation.
This is the first study to determine the extent of genetic variation in BSEP and MDR3 in a large collective of women with ICP and healthy control women. Although most of the mutations detected in BSEP were found in both populations, a greater number of ICP-specific variants were detected in MDR3, including 2 highly conserved nonsynonymous and 5 splicing-consensus sites.
These findings further support a pathogenic role for MDR3 genetic variability in ICP. Analysis of population-specific variant frequencies as well as the BSEP and MDR3 haplotype structure in these groups will allow a more detailed analysis of mutations that might represent a susceptibility factor for the development of ICP.
Role of mutations in specific hepatocanalicular transporters in primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). There is ongoing discussion regarding whether genetically determined dysfunction may also play a pathogenetic role in PBC and PSC. PBC and PSC are characterized by a cholestatic pattern of liver damage with elevated serum bile acids. Such a pattern is similar to that observed in hereditary or acquired dysfunction of the hepatic canalicular membrane transporters BSEP and MDR3, which secrete cholephilic compounds into the bile canaliculus.
Pauli-Magnus and colleagues tested whether sequence diversity and haplotype structure of BSEP and MDR3 in patients with PBC and PSC were different from that observed in healthy volunteers with the same ethnic background. DNA samples from 93 healthy white individuals, 70 patients with PBC, and 46 patients with PSC were screened for genetic variations in BSEP and MDR3. The total number of different haplotypes as well as the relative fraction of the most common haplotypes were similar in all 3 groups for BSEP and MDR3. Also, the number of population-specific haplotypes accounted for a minority of alleles and did not differ between the 3 groups. Furthermore, similar patterns of linkage disequilibrium were found in all 3 patient populations. These investigators concluded that patients with PBC and patients with PSC did not show a significant deviation in haplotype structure in BSEP and MDR3 from that observed in healthy white controls.
These data do not support a role for BSEP and MDR3 genetic variation in the pathogenesis of PBC and PSC, because variant segregation and haplotype structure of both genes did not differ from the pattern observed in healthy controls. Although a functional impact of some of these rare variants or haplotypes cannot be ruled out, they cannot serve as a common denominator of disease pathogenesis in PBC and PSC. Alternatively, the cumulative data provide a basis for the study of phenotypic differences in BSEP and MDR3 genetic variation in the normal population, which may help to define genotypes associated with an increased risk of developing cholestasis.
Pathophysiology
PFIC-1 and PFIC-2. The genetic defect in PFIC-1 has been mapped to a locus on chromosome 18q21-q22, a region encoding FIC1 (ATP8B1), a P-type ATPase. Thus ATP8B1 is mutated in a form of low gamma-GT intrahepatic cholestasis formerly called "Byler's syndrome."
PFIC-2, another form of low gamma-GT intrahepatic cholestasis, is caused by deficiency in BSEP, the major bile salt transporter in human liver, due to mutations in the ABCB11 (BSEP) gene. The chromosomal locus is 2q24
Molecular mechanism of PFIC-1 and PFIC-2. Bile sampled from gallbladders of individuals with intrahepatic cholestasis due to mutation in ABCB11 (PFIC-2) is deficient in primary bile acids. This can be ascribed to the fact that the ABCB11 gene encodes the bile salt export protein, BSEP. Similar deficiency of biliary bile acids in mature bile is found in intrahepatic cholestasis owing to mutation in the ATP8B1 gene (PFIC-1). The ATP8B1 gene encodes a putative aminophospholipid "flippase," FIC1, hypothesized to maintain compositional asymmetry between leaflets of cell membranes. FIC1 may be necessary for optimal function or trafficking of membrane-inserted proteins. Some canalicular proteins appear deficiently expressed in persons with ATP8B1 disease. The question, therefore, is whether deficiency in BSEP expression at the canaliculus underlies the lack of primary bile acids in bile.
Knisely and colleagues evaluated expression at the canaliculus of BSEP in formalin-fixed, paraffin-embedded archival liver-biopsy or hepatectomy materials from 20 patients of varying ages with intrahepatic cholestasis. Ten had documented ATP8B1 (PFIC-1) gene mutations and 10 had documented ABCB11 (PFIC-2) gene mutations. In the 10 patients with ATP8B1 gene mutations, expression of BSEP was indistinguishable from that in controls; in the 9 patients with ABCB11 gene mutations, expression of BSEP was absent. Function of BSEP was intact in persons with ATP8B1 gene disease, as judged by findings at liver biopsy on presentation in infancy. While individuals with ABCB11 gene mutation have giant-cell hepatitis with hepatocellular cholestasis and necrosis, those with ATP8B1 mutation have bland canalicular cholestasis and remarkably little hepatocellular disarray. This suggests that in ATP8B1 gene disease, toxic bile acids do not accumulate within hepatocytes to the extent that it occurs in ABCB11 gene disease. These findings indicate that not all canalicular proteins are abnormally expressed in ATP8B1 gene disease, and that cholestasis in ATP8B1 gene disease is not due to disruption of canalicular transporter-protein expression. The study authors speculated that individuals with ATP8B1 gene disease do not downregulate the sodium-dependent apical bile acid transporter (ASBT) in the terminal ileum. If in ATP8B1 gene disease bile acids are normally exported into the canaliculus, yet are deficient in mature bile, it may be due in part to their absorption in excess during transit past cholangiocytes in which ASBT is abnormally regulated. The clinical implication of these findings is unclear at present.
PFIC-3. PFIC-3 is associated with mutations in the MDR3 gene, also designated ABCB4, localized on chromosome 7q21, and is a phospholipid (PL) transporter. The clinical manifestations of MDR3 mutations are highly variable: heterozygous mutations are associated with ICP, drug-induced cholestasis, and cholesterol lithiasis (caused by the high lithogenicity of bile due to the low PL content). Rosmorduc and colleagues described an MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Thus, MDR3 gene mutations represent a genetic factor involved in a form of symptomatic cholesterol cholelithiasis in adult patients (low phospholipid-associated cholelithiasis [LPAC]).
Lucena and colleagues reported a woman who presented as an adolescent with cholelithiasis. She later developed recurrent ICP and finally biliary cirrhosis at age 47. She and her daughter (who also had ICP) were heterozygous for an MDR3 mutation. Thus, 3 consecutive diseases were associated with an MDR3 mutation; this led to end-stage cirrhosis after the fourth decade of life.
Treatment
Because the efficacy of medical therapy is poor, a number of surgical procedures have been successfully used in the management of patients with PFIC-1 and PFIC-2. Biliary diversion and ileal exclusion are increasingly being used as pretransplant surgical interventions. It is proposed that these surgical procedures may significantly delay, or even obviate, the necessity of liver transplantation. However, results are contradictory and center-specific, and a sufficient attempt has not been made to correlate outcome with the type of PFIC present.
Pawlikowska and colleagues presented data emerging from a multicenter study of over 100 patients to correlate clinical and biochemical data (including information on surgical treatment outcomes) with genotype in these disorders. Biliary diversion has been performed in 18 patients with BSEP (mean age at surgery, 5.5 years; none had progressed to cirrhosis at time of surgery). Fifteen patients had a positive outcome with biliary diversion, including significant and sustained improvement in pruritus (n = 13), reduction in hyperbilirubinemia (n = 10), and improved growth (n = 9). Three patients are on ursodeoxycholic acid and/or rifampicin following biliary diversion for control of pruritus; 11 have required no medications. Eleven of the 15 patients with a good outcome carry at least 1 of the common European mutations, D482G and E297G. Sixteen patients with BSEP (including 2 with failed biliary diversion) have undergone liver transplantation (mean age at liver transplantation was 5.5 years). Fifteen patients had excellent outcomes, including complete relief of jaundice and pruritus. Biliary diversion has been performed in 16 patients with FIC1 (mean age at surgery, 3.1 years); 3 subsequently underwent liver transplantation. In the remainder of patients, outcomes have been variable. Four are doing well, with minimal pruritus, improved growth, and reduced hyperbilirubinemia; 3 have minimal benefit; and in another 3 patients, initial improvement was followed by symptom recurrence. Twenty-two patients with FIC1 have had liver transplantation (at a mean age of 6.9 years); 10 showed signs of cirrhosis at liver transplantation, including 3 patients who previously had biliary diversion.
Biliary diversion can be a useful treatment in some cases of BSEP and FIC1 disease. These data indicate that carrying at least 1 copy of the common D482G BSEP mutation is predictive of a positive outcome with biliary diversion. Studies to determine whether biliary diversion may be more reliably successful in patients with BSEP than in those with FIC1 are under way.
Concluding Remarks
Studies presented during this year's AASLD meeting provide a great stimulus to the further study of genetic liver disease. These studies offer new diagnostic and management strategies for liver diseases that affect diverse patient populations. The era of definitive therapy for these disorders is near.
References
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