Molecular biological methods in diagnosis and treatment of liver

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					Clinical Chemistry 43:8(B) 1476 –1486 (1997)

Beckman Conference

Molecular biological methods in diagnosis and treatment of liver diseases
Howard J. Worman
Molecular biology is making a tremendous impact on the diagnosis and treatment of liver diseases. Methods such as the polymerase chain reaction are changing the way physicians diagnose and monitor patients with viral hepatitis. Assays based on recombinant protein antigens allow for detection of specific autoantibodies in diseases such as primary biliary cirrhosis. The diagnosis of inherited metabolic diseases, such as hemochromatosis and Wilson disease, is being revolutionized by discovery of the defective genes involved and the development of methods to rapidly sequence DNA and identify mutations. Treatments and preventive measures are now possible with use of drugs and vaccines produced by recombinant DNA technology. Gene therapy and nucleic acid-based therapeutics are also realistic future treatment options for individuals with liver diseases. hepatitis • primary biliary cirrhosis metabolic diseases • recombinant DNA • gene therapy
INDEXING TERMS:
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and hepatitis G viruses (HCV and HGV, respectively)1 [1, 2]. PCR is used to diagnose and monitor patients with chronic viral hepatitis. Autoantibodies found in certain liver diseases can be detected with assays that use highly specific recombinant protein antigens [3]. The genes responsible for inherited disorders of metabolism that affect the liver, e.g., 1-antitrypsin deficiency [4], Wilson disease [5, 6], and hereditary hemochromatosis [7], have been identified, opening the way for molecular diagnosis. Liver-specific gene therapy has already been performed [8]. This review will summarize some of the current and potential molecular biology-based diagnostic methods and treatments for liver diseases.

Diagnosis viral hepatitis
Hepatitis C. In recent years, molecular biology has probably had its greatest clinical impact with regard to liver diseases on the diagnosis of viral hepatitis C. Before 1989, cases of chronic hepatitis presumed to be caused by a virus transmitted by blood and blood products were referred to as non-A, non-B hepatitis. This changed in 1989 when investigators at Chiron Corp. identified a positive-stranded RNA virus that was responsible for a most cases of non-A, non-B hepatitis [1]: HCV. HCV was identified by standard molecular biological techniques; the strategy is outlined in Fig. 1. DNA and RNA were extracted from the plasma of chimpanzees infected with serum from humans with non-A, non-B hepatitis. These nucleic acids were used to construct a random-primed, complementary DNA (cDNA) library in bacteriophage lambda that expressed fusion polypeptides when infected in Escherichia coli. The expression library was screened with diluted serum from patients with non-A, non-B hepatitis, and cDNA clones were isolated that encoded proteins that were recognized by serum

Molecular biology is revolutionizing clinical chemistry. The standardization and automation of molecular biological methods are making complex diagnostic procedures routine in the clinical laboratory. Extensive progress in gene discovery and DNA sequencing has already made possible the diagnosis of various inherited diseases, and new disease genes are being discovered routinely. Advances in methods for gene cloning and expression have led to production of drugs and vaccines by recombinant DNA technology. The use of genes, oligonucleotides, and ribozymes as therapeutic agents is no longer a fantasy. The liver is affected by viral, autoimmune, and metabolic disorders that can be diagnosed and treated with molecular biological methods. Standard molecular cloning methods have been used to identify the hepatitis C

Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, 630 W. 168th St., 10th Flr., Rm. 508, New York, NY 10032. Fax 212-305-6443; e-mail hjw14@columbia.edu. Received February 24, 1997; revised April 1, 1997; accepted April 7, 1997.

1 Nonstandard abbreviations: HCV, HBV, HGV, hepatitis C, B, and G viruses, respectively; RIBA, recombinant immunoblot assay; bDNA, branched DNA; RT-PCR, reverse-transcription polymerase chain reaction; RFLP, restriction fragment length polymorphism; and PBC, primary biliary cirrhosis.

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Fig. 1. Scheme for identification of HCV by molecular biological methods.
From DNA and RNA extracted from plasma of chimpanzees infected with serum from humans with non-A, non-B hepatitis, an expression library of plasma DNA and cDNA reverse-transcribed from RNA was constructed in bacteriophage gt11. The expression library was screened with diluted serum from patients with non-A, non-B hepatitis, and clones producing fusion proteins that reacted with serum antibodies were purified. DNA was sequenced that corresponded to a portion of the HCV RNA genome.

antibodies. These investigators went on to demonstrate that the cDNA clones they isolated were derived from a positive-stranded RNA virus with a genome of 10 000 nucleotides. Using a fusion protein expressed from an isolated cDNA clone, the scientists at Chiron and their colleagues [9] showed that the large majority of individuals with non-A, non-B chronic hepatitis had serum antibodies against this newly identified virus. After this pioneering work on identification of the virus, the entire HCV genome was cloned and sequenced in several laboratories [10 –13]. Sequence comparisons showed that HCV was a member of the Flaviviridae family. The genome is a positive-stranded RNA of 9500 nucleotides with a highly conserved 5 untranslated region followed by a single open-reading frame that encodes a polyprotein of 3010 to 3033 amino acids, depending on the isolate (Fig. 2). The polyprotein is processed by the host cell and virally encoded proteases into several structural and nonstructural polypeptides (Fig. 2). The major structural proteins are the core and envelope polypeptides. The

nonstructural proteins include, among others, proteases involved in the processing of the polyprotein and an RNA-directed RNA polymerase. The identification, cloning, and sequencing of HCV has led to a new era in the diagnosis of chronic viral hepatitis. The diagnostic assays that have resulted from this work can be broadly classified into those that detect antibodies against HCV polypeptides and those that detect viral nucleic acids (Table 1). Each of these assays has a role in the laboratory diagnosis of HCV infection. Because virtually all individuals infected with HCV develop antibodies against viral polypeptides, antibody detection assays provide the cornerstone of screening and initial diagnosis. The first ELISA to detect anti-HCV antibodies utilized one recombinant antigen and correctly detected 80% of individuals infected with HCV [9]. Newer ELISAs utilize several recombinant viral antigens expressed from cDNA clones, and a positive test result in an individual with risk factors for HCV infection is 99% sensitive. Because of their simplicity and high sensitivity, ELISAs can be used to screen the blood supply for HCV. One limitation of ELISAs in diagnosing patients is that test results may remain positive in individuals who have cleared HCV infection, either spontaneously or after treatment with interferon- . Antibody detection assays are also not useful in cases of acute HCV infection because of the several weeks it takes for antibodies to develop [14]. ELISAs may also be falsely positive in individuals with hypergammaglobulinemia—which is often present in patients with autoimmune hepatitis [15]. In cases where serum HCV antibody detection may be falsely positive by ELISA, a recombinant immunoblot assay (RIBA) can be helpful. In this assay, several recombinant HCV polypeptides, along with a control protein, are immobilized on a membrane, which is processed like a routine immunoblot [16]. The advantage of the RIBA is that reactivity with one or more distinct recombinant HCV polypeptides and lack of reactivity with an unrelated control can be ascertained. Nonspecific reactivity with plastic microtiter wells, e.g., those used for ELISAs, is also eliminated. Two general methods are currently available to detect HCV nucleic acids in serum. One, the branched DNA (bDNA) assay, utilizes capture of viral RNA by virusspecific nucleotide probes, followed by hybridization to bDNA molecules, which are detected by a chemiluminescent substrate system [17, 18]. The bDNA assay can be reliably quantified and is relatively simple to perform in the clinical laboratory. The analytical sensitivity of the

Fig. 2. Polyprotein and major proteolytically derived polypeptides encoded by the HCV genome.
The HCV positive-stranded RNA genome encodes a polyprotein of 3033 amino acids. The polyprotein is processed by host-cell and viral-encoded proteases into several structural (core, E1, and E2) and nonstructural (NS2, NS3, NS4, and NS5) polypeptides. Smaller fragments, such as NS4A, NS4B, NS5A, and NS5B, are obtained by further proteolytic processing by a viral protease.

Table 1. Diagnostic assays for detection of HCV infection.
Antibody tests Nucleic acids tests

Enzyme-linked immunosorbent assay (ELISA) Recombinant immunoblot (RIBA)

Branched DNA (bDNA) Reverse transcription polymerase chain reaction (RT-PCR)

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bDNA assay for detection of serum HCV RNA, however, is only 10 4 that of assays based on PCR [19]. Assays with the most analytical sensitivity currently routinely available for the detection of HCV are based on reverse transcription followed by PCR (RT-PCR). The discovery by Baltimore [20] and Temin and Mitzutani [21] of retroviral reverse transcriptase, which directs the synthesis of cDNA from a RNA template, was of fundamental significance in modern molecular biology. PCR, conceived of by Mullis [22, 23], has also revolutionized modern molecular biology by allowing the in vitro enzymatic amplification of large amounts of DNA from a small number of molecules. The use of thermostable DNA polymerases, such as Taq from Thermus aquaticus, has made PCR a rapid, reproducible, and semiautomated procedure [24]. The area of viral hepatitis diagnosis has not escaped the impact of the discoveries of reverse transcriptase and PCR. These procedures can be combined to detect very low quantities of HCV RNA in serum or tissue. RNA extracted from serum is used as the template for reverse transcription of cDNA, which is then amplified by PCR. The amplified cDNA products are separated by agarose gel electrophoresis and detected by various methods, e.g., ethidium bromide staining or Southern hybridization. The major potential drawback of RT-PCR assays is their extreme sensitivity, which makes contamination a concern. However, standardization of this method in excellent clinical laboratories has made it the gold standard for detecting HCV RNA. Several techniques, such as competitive inhibition or use of various reaction lengths and cycle numbers with known amounts of template, can make RT-PCR semiquantitative [19, 25–27]. Values for viral load can be used to assess a patient’s response to treatment. RT-PCR can also be used to determine the HCV genotype. At least 6 major genotypes and 11 subtypes of HCV have been identified on the basis of genomic sequence differences [28]. Different genotypes may differ in the severity of disease they cause and in their response to treatment [29]. Several methods exist for determining HCV genotype, including RT-PCR followed by direct sequencing, RTPCR with genotype-specific primers, restriction fragment length polymorphism (RFLP) analysis of PCR-amplified DNA sequences, and hybridization of the amplified products to oligonucleotide probes immobilized on a solidphase support [30, 31]. Some of these genotyping assays are now performed in clinical laboratories; combined with estimates of serum viral load, knowledge of the infecting genotype may be useful in predicting prognosis and response to therapy [29]. Sequence variations more subtle than those between different genotypes, e.g., point mutations that cause amino acid substitution in the NS5A gene product, may also correlate with response to interferon therapy [32]. With regards to chronic hepatitis C, molecular biology has created a new discipline for the practicing hepatologist. Before 1990, the virus that causes this disease was not

known. Now, infected individuals can be identified in the clinical laboratory with assays based on molecular biology and consequently be considered for treatment with interferon- (produced by recombinant DNA technology) and other potentially promising agents. Hepatitis B. Regarding serological diagnosis, hepatitis B differs from hepatitis C in that viral protein antigens of HBV can be readily detected in serum. Hepatitis B surface antigen is detectable in virtually all infected individuals, and the hepatitis B e antigen is detected in most patients who have high amounts of viral replication. Accordingly, nucleic acid tests are not necessary to diagnose viral infection with HBV in the clinical laboratory. In some instances, however, amplification of viral nucleic acid sequences from serum may be helpful in assessing HBVinfected individuals. HBV is a partially double-stranded DNA virus. Therefore, viral genomic sequences can be amplified directly by PCR without reverse transcription. Because viral load may be predictive of response to treatment and prognosis, it may sometimes be useful to use semiquantitative PCR to estimate serum viral concentrations. Assays utilizing bDNA can also be used to quantify HBV DNA in serum [17]. Sequencing the HBV DNA amplified by PCR can be useful for identifying mutant viruses of clinical significance. For example, mutations in the HBV precore region of the genome have been identified that prevent transcription of the e antigen but allow the continued assembly of infectious virus [33, 34]. Early reports associated these precore mutants with severe liver disease in the absence of hepatitis B e antigen [33]. A recent report has also shown that surgeons infected with precore mutant strains, without detectable serum e antigen, can transmit HBV to patients [35]. Hepatitis G. In 1995 and 1996, a new human hepatotropic virus was identified [2, 36]. In initial studies by investigators at Abbott Labs., representational difference analysis was used to identify nucleic acid sequences of two related viruses, GB-A and GB-B, in tamarin marmosets that had been infected with serum from a surgeon, “GB,” who had contracted acute hepatitis [37]. Representational difference analysis is a powerful method that had previously been used in pioneering work by investigators at Columbia University to identify a herpesvirus responsible for Kaposi sarcoma [38]. In the present instance, however, the two putative hepatitis viruses identified by this method were probably tamarin viruses and not human pathogens [39]. Nonetheless, when the Abbott investigators took RNA from human serum samples that contained antibodies against GB-A and GB-B and subjected them to RT-PCR with degenerate oligonucleotide primers, DNA sequences were amplified that were related to but distinct from GB-A, GB-B, and HCV; these sequences were determined to belong to a novel flavivirus the investigators termed GB-C [36].

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In independent work, investigators at Genelabs Technologies [2] determined the complete genomic sequences of two isolates of a flavivirus that they called HGV. HGV is essentially identical to the GB-C virus and is related to HCV, GB-B, and GB-A. To isolate HGV, the investigators at Genelabs screened a cDNA expression library constructed from the plasma of a patient with chronic hepatitis C. Immunoscreening of the expression library with the patient’s serum identified several cDNA clones encoding HCV polypeptides as well as clones encoding other related but unique polypeptides. From the unique cDNA clones, an anchored PCR method was used to amplify overlapping clones for the entire viral genome. Using RT-PCR, these workers identified HGV sequences in 13% of 38 US patients with non-A, non-B, non-C, non-D, non-E hepatitis and in 18% of patients with HCV infection. At present, the significance of HGV/GB-C virus as a cause of acute and chronic liver disease remains controversial. Although it has been associated with acute and chronic hepatitis [2], some investigators argue that this virus may not be a major cause of hepatitis in humans [39, 40]. Resolution of this controversy is important, given the presence of HGV/GB-C in the blood supply [2, 39, 40]. ELISAs based on HGV/GB-C polypeptides are able to detect antibodies against this virus in the blood supply and in infected individuals.

Table 2. Recombinant protein antigens used to detect specific autoantibodies in individuals with PBC.
Type of autoantibody Anti-mitochondrial Anti-nuclear

E2 subunit of pyruvate dehydrogenase complex E2 subunits of other oxoacid dehydrogenases “Designer” recombinant molecules constructed with predominant autoepitopes of the above

Nuclear pore membrane protein gp210 Intranuclear protein Sp100 Inner nuclear membrane protein LBR

autoimmune liver diseases
Primary biliary cirrhosis (PBC), autoimmune hepatitis, and some cases of primary sclerosing cholangitis are associated with the presence of autoantibodies against intracellular proteins. Testing for autoantibodies provides a cornerstone of diagnosis of these diseases, especially PBC. Molecular biological methods have made possible the identification of some of the intracellular protein antigens and the predominant epitopes recognized by some of the disease-specific autoantibodies. This work has led to development of assays that can be used in the clinical laboratory. Assays utilizing recombinant proteins should make tissue-based immunofluorescence assays obsolete in cases where the autoantigen is known. In PBC, cDNAs for several of the major mitochondrial and nuclear autoantigens have been cloned and sequenced, and recombinant proteins have been used to detect autoantibodies (Table 2). In almost all cases of PBC, specific autoantibodies are found that are almost never present in individuals with other diseases. For example, 90% of individuals with PBC have autoantibodies directed against the E2 subunits of mitochondrial oxoacid dehydrogenases [3, 41, 42]. In 25% of PBC cases, autoantibodies against gp210, an integral membrane protein of the nuclear pore complex, are detectable [43, 44]. Autoantibodies against the mitochondrial oxoacid dehydrogenase E2 subunits are virtually 100% specific for PBC [41, 42]. Their detection is of central importance in the diagnosis of PBC, which depends on a combination of

clinical, biochemical, immunological, and histological findings [45]. Autoantibodies that recognize these proteins have been traditionally been called anti-mitochondrial antibodies and are detected in most clinical laboratories by indirect immunofluorescence microscopy. Indirect immunofluorescence microscopy is fairly simple to perform in the clinical laboratory but has several limitations. First, the precise nature of the autoantigen cannot be established, and antibodies that label other mitochondrial proteins, not necessarily associated with PBC, will be detected. Second, antibodies against nonmitochondrial cytoplasmic antigens, including those against the cytochrome P450 isoforms found in patients with type II autoimmune hepatitis, produce labeling patterns similar to anti-mitochondrial antibodies and are often misinterpreted as such. Third, ELISAs utilizing recombinant proteins or synthetic polypeptides are easier to automate than are tissue-based immunofluorescence assays, which usually require subjective human interpretation. Screening of bacteriophage cDNA expression libraries with autoantibodies from affected patients identified the E2 subunits of the pyruvate dehydrogenase complex, the branched-chain 2-oxoacid dehydrogenase complex, and the 2-oxoglutarate dehydrogenase complex as the major mitochondrial autoantigens in PBC [41, 42, 46 – 48]. ELISAs utilizing expressed recombinant proteins or designer polypeptides have been devised that are sensitive and specific for detection of antibodies against these proteins [3, 49, 50]. Determination of the immunodominant epitopes on these three proteins has allowed construction of a “designer hybrid” clone that contains the major epitope of each [50]. An immunoassay utilizing a designer polypeptide expressed from this clone is highly sensitive and specific for the diagnosis of PBC [50]. In addition to antibodies against mitochondrial oxoacid dehydrogenase E2 subunits, some individuals with PBC also have other specific autoantibodies, e.g., as mentioned above, against gp210. Gp210 was first identified as a common autoantigen in PBC by immunoblotting assays utilizing the protein purified from cells [51]. The cDNA cloning of gp210 made possible the identification of its immunodominant epitope, which was mapped to a stretch of 15 amino acids [52]. Two ELISAs have been developed for detection of autoantibodies against the

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predominant autoepitope of gp210, one utilizing a recombinant fusion protein expressed in E. coli [53], the other a synthetic polypeptide [54]. In addition to oxoacid dehydrogenases and gp210 autoantigens in PBC, several other proteins have been identified as autoantigens in liver diseases, and for some the immunodominant epitopes have also been determined. Examples include Sp100 [55] and LBR [56, 57] in PBC and nuclear lamins [58] and cytochrome P450 isoforms in type II autoimmune hepatitis [59, 60]. Future work with expression cloning may also identify other protein autoantigens in liver diseases, such as the “atypical-ANCA” or “x-ANCA” autoantigen in primary sclerosing cholangitis. ELISAs that utilize recombinant proteins or synthetic polypeptides to detect autoantibodies in individuals with liver diseases should be useful in the clinical laboratory.

demonstrated that human 1-antitrypsin is a protein of 394 amino acids [63, 64]. The ZZ phenotype, the one most commonly associated with the development of liver disease, has a lysine substituted for a glutamic acid residue at position 342 [65, 66]. In ZZ homozygotes, the protein is not properly processed in the secretory pathway, and 85% of it accumulates in the endoplasmic reticulum. The Z mutation causes an interaction between the reactive center loop of one molecule and a portion of another [67]. The resulting protein aggregates are not secreted and their accumulation can lead to liver disease. The Z allele results from a single amino acid substitution created by a G to A transition in the gene for 1-antitrypsin. Synthetic oligonucleotide probes have been used to develop a sensitive and direct assay for the presence or absence of this mutation [68]. This assay has even been used to diagnose the mutation prenatally [69]. Wilson disease. In 1993, the gene for Wilson disease on chromosome 13 was cloned [5, 6]. The gene encodes a copper-transporting ATPase homologous to the protein that is mutated in Menkes disease, another inherited disorder of copper metabolism [5, 6]. At least 25 different mutations in the Wilson disease gene have been described, including small insertions, deletions, missense, nonsense, and splice-site mutations [70]. These different mutations may explain in part the wide phenotypic variation in individuals with Wilson disease: e.g., age of onset, neurological vs liver disease, and severity [70]. Wilson disease is at present diagnosed by measurements of 24-h urine copper and serum ceruloplasmin concentrations and by the presence of excessive copper in liver tissue obtained at biopsy. Given the wide variety of mutations that can cause this disease, molecular biological diagnosis in a random individual could require sequencing the entire gene. If a particular mutation has been characterized in one subject, however, it can be readily detected in a family member. As methods for rapid sequencing and the simultaneous detection of multiple mutations are developed, genetic tests for the laboratory diagnosis of Wilson disease should be produced. Hereditary hemochromatosis. Hereditary hemochromatosis is the most common inherited disease in persons of European descent, the prevalence of homozygosity being 3–5 per 1000 and a carrier frequency of 1 in 10 [71, 72]. Hemochromatosis is inherited as an autosomal recessive trait, and homozygous individuals have increased absorption of dietary iron resulting in excess deposition in the liver, heart, joints, and some endocrine organs. Phlebotomy is an effective treatment; however, the disease often goes undiagnosed. Increases in serum ferritin and transferrin saturation are suggestive of hemochromatosis, and diagnosis is made by measuring hepatic iron contact in liver tissue obtained at biopsy. In the 1970s, a linkage of hereditary hemochromatosis to the HLA-A locus on chromosome 6 was established

inherited metabolic diseases
The Human Genome Project and the emerging discipline of genomics is changing the way in which much of clinical medicine is practiced. The identification of disease genes by positional cloning, in which the inheritance of linked genetic markers at known chromosomal locations is examined, and other methods has already made a tremendous impact. The genes responsible for many of the major inherited metabolic diseases that affect the liver have now been identified (Table 3). These discoveries have paved the way for molecular diagnostic methods for these disorders. Molecular genetics assays will replace traditional chemical measurements and invasive procedures such as biopsy to diagnose inherited liver diseases. The molecular biology of some of the major inherited liver disorders is briefly reviewed here, followed by a discussion of some feasible molecular diagnostic methods.
1-Antitrypsin deficiency. The human gene encoding 1antitrypsin is localized to chromosome 14q31–32 [61]. Mutations in the 1-antitrypsin gene that cause the disease are of several types and include those that produce a deficiency of enzyme action, null mutations, and altered function of the gene product. Mutations producing deficiency or absence of enzyme action lead to an increased risk of developing emphysema [62]. Mutations that result in defective secretion of the protein from hepatocytes cause liver disease. Cloning and sequencing of cDNAs has

Table 3. Some inherited liver-affecting diseases and the responsible genes.
Disease
1-Antitrypsin deficiency Wilson disease Hereditary hemochromatosis Crigler–Najjar syndrome Gilbert syndrome Dubin–Johnson syndrome

Gene
1-Antitrypsin Copper-transporting ATPase HLA-H UDP-glucuronosyltransferase UDP-glucuronosyltransferase(?) cMOAT(?)

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[73]. This linkage has been useful clinically in the analysis of risk in the relative of a patient. In 1996, investigators at Mercator Genetics [7] identified a candidate gene for hemochromatosis on chromosome 6. They termed this gene HLA-H because of its homology to other MHC class I family members (recently, the name HFE has been recommended for this gene). These investigators found a G to A transition at nucleotide 845 of this gene that resulted in a cysteine to tyrosine substitution at amino acid residue 282 in the protein. Of 178 patients examined, 148 were homozygous for this mutation, 9 were heterozygous, and 21 carried only the normal allele. Several other groups have confirmed the frequency of this mutation in individuals with hereditary hemochromatosis [74 –76]. A C to G transversion that results in a histidine to aspartic acid substitution at amino acid residue 63 has also been identified in fewer individuals with hereditary hemochromatosis [7]; however, some investigators have not been able to establish a relationship between this mutation and the disease [76]. The finding that only 85% of patients carry the cysteine to tyrosine mutation suggests that other mutations in the HFE gene or in different genes may also cause hemochromatosis. Hereditary hemochromatosis is a common disorder, for which effective treatment is available. The fact that 85% of affected individuals appear to have a single mutation should make genetic testing for the majority of cases relatively simple. For these reasons, hereditary hemochromatosis may be an ideal disorder for genetic screening of the entire population. Hereditary hyperbilirubinemias. Hereditary hyperbilirubinemias are generally divided into those that cause increases in unconjugated serum bilirubin concentrations and those that cause increases in conjugated serum bilirubin. Two disorders of the former type are Crigler–Najjar syndrome and Gilbert syndrome. Patients with type I Crigler–Najjar have a complete absence of activity of the isoform of UDP-glucuronosyltransferase that conjugates bilirubin to mono- and diglucuronides in hepatocytes. Individuals with type I Crigler–Najjar syndrome have severe childhood disease. Patients with type II disease have partial deficiency of this enzyme and generally survive to adulthood without significant problems. Several different mutations in the gene that encodes the bilirubin-conjugating isoform of UDP-glucuronosyltransferase, UGT1 on chromosome 2, have been shown to cause type I and type II Crigler–Najjar [77– 82]. Gilbert syndrome is a common cause of hereditary hyperbilirubinemia. The condition is benign and of little clinical significance except that its presence can cause physicians to search for other liver diseases in patients. Mutations in the promoter region of UGT1 have been described in individuals with Gilbert syndrome [83]. These and other mutations in UGT1 and possibly other genes may be responsible for the condition.

Dubin–Johnson syndrome is characterized by conjugated hyperbilirubinemia and deposition of a dark pigment in the liver. Although rare in most of the world, Dubin–Johnson syndrome occurs with a frequency of 1 in 1300 among Iranian Jews [84]. The disorder results from the inability of conjugated bilirubin to be secreted from hepatocytes. A cDNA for rat cMOAT, a protein homologous to the multidrug resistance proteins located in the canalicular membrane of hepatocytes, has recently been characterized [85]. A single basepair deletion in the cMOAT gene, which results in a truncated protein and is associated with the impaired secretion of organic acids from hepatocytes, has been described in the TR rat, an animal model of Dubin–Johnson syndrome [85]. Cloning and sequencing the human homolog of rat cMOAT may establish the genetic defect in Dubin–Johnson syndrome. Diagnostic methods. Knowledge of the genes and mutations that cause inherited metabolic liver diseases will revolutionize the manner in which these conditions are diagnosed. Screening tests can be developed for common diseases such as hemochromatosis. Genetic tests can replace invasive procedures, such as liver biopsy, to rule out rarer conditions. Some molecular diagnostic methods that can be used, depending on the nature of the mutation, are given in Table 4. It is relatively easy to detect a specific mutation that causes a disease. Examples are the G to A transition that most commonly causes the Z-variant of 1-antitrypsin deficiency or the nucleotide change in HLA-H associated with 85% of cases of hereditary hemochromatosis. Detection of a mutation in a family member when the mutation is already known in a relative is also fairly simple. In these instances, RFLP analysis or specific oligonucleotide probes can be used to detect the mutation in total genomic DNA or DNAs amplified by PCR. It is more difficult to diagnose a genetic disorder if multiple mutations can be responsible, as in Wilson disease, and if a mutation has not been characterized in a family member. If a truncated protein is produced by most mutations, in vitro translation and examination of the product can detect an abnormal protein. An assay based on in vitro translation has been developed to detect mutations in the APC gene in individuals with familial adenomatous polyposis [86]. Amplification of cDNA or genomic regions by PCR followed by standard DNA sequencing methods can also be used to analyze genes in which many different mutations can cause disease; however, this is very time consuming.
Table 4. Some methods to detect genetic mutations.
Restriction fragment length polymorphisms Hybridization to oligonucleotide probes PCR followed by DNA sequencing In vitro translation Oligonucleotide arrays on chips

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High-density oligonucleotide arrays, such as those produced by light-directed, spatially addressable parallel chemical synthesis, can be used for rapid DNA sequence determinations [87–90]. Oligonucleotide probes synthesized and bound in ordered arrays to glass or nylon chips are used to hybridize to fluorescently labeled target DNAs. The extent and pattern of fluorescence on the chips after washing can be used to determine DNA sequences. Oligonucleotide arrays on chips have been used to simultaneously monitor the expression of multiple genes in a cell type [91], analyze the entire human mitochondrial genome [92], and simultaneously screen for 24 different heterozygous mutations or polymorphisms in a portion of the human BRCA1 gene [93]. With regard to inherited liver diseases, oligonucleotide arrays can be used to simultaneously detect the various possible mutations in the genes for disorders such as Wilson disease.

widely used [99]. Other forms of recombinant interferons are also now available. Human HBV vaccines have been produced by recombinant DNA technology [100]. Vaccines composed of hepatitis B surface antigen particles expressed from recombinant DNA in budding yeast have repeatedly been demonstrated to be effective [101]. Almost all healthy individuals younger than age 40 develop protective serum titers of anti-hepatitis B surface antigen antibodies after a series of three injections of commercially available preparations. Response rates are lower in immunocompromised and older individuals. Recombinant vaccines that protect against other hepatitis viruses may soon be developed. Preliminary results in cynomolgus monkeys suggest that vaccination with a recombinant protein representing part of the viral capsid antigen may be protective against hepatitis E [102].

Treatment
Advances in molecular biology have led to new treatments for several liver diseases (Table 5). The use of recombinant interferons to treat viral hepatitis and the use of recombinant vaccines to protect against HBV infection are currently used widely by medical practitioners. Liverdirected gene therapy is not yet routine; however, pioneering human trials have already been performed [8]. The use of recombinant polypeptides to obtain critical protein structural information for the rational design of drugs has also been applied to liver diseases [94, 95]. DNA vaccines [96], antisense oligonucleotides [97], and ribozymes [98] are being developed in many laboratories and may hold promise as agents for the treatment and prevention of liver diseases.

rational drug design
The ability to express portions of proteins from recombinant cDNA clones provides the opportunity to determine their structures. Structure determination can lead to the rational development of drugs that can, for example, inhibit an enzyme or bind to a receptor. The first steps towards rational drug design have recently been reported for the NS3 protease of HCV. This protease cleaves nonstructural polypeptides from the HCV polyprotein and is essential for viral replication. Workers at Vertex Pharmaceuticals [94] and Agouron Pharmaceuticals [95] have used x-ray crystallography to determine the structure of the NS3 protease. Standard molecular biological methods were used to obtain sufficient quantities of protein for crystallization. Based on the three-dimensional structures determined in these studies, drugs can be rationally designed to inhibit this enzyme, which is essential for viral replication. Similar methods should be useful in developing other antiviral drugs and possibly even drugs that can stimulate defective enzymes in inherited diseases.

recombinant drugs and vaccines
Recombinant DNA technology has led to the production of drugs and products for the treatment and prevention of liver diseases. Notable among these products are interferons for the treatment of viral hepatitis and vaccines to prevent hepatitis B. Recombinant interferon -2b is effective in treating chronic hepatitis B, C, and D [99]. Interferon -2b was the first recombinant drug approved by the US FDA for treatment of hepatitis and has been

gene therapy
Gene therapy holds promise as future treatment for liver diseases, including viral hepatitis, inherited metabolic diseases, and cancer. A detailed discussion of hepatic gene therapy is beyond the scope of this paper; however, this topic has recently been elegantly reviewed by Wilson and Askari [103]. In brief, two types of gene therapy strategies can be used to target the liver: ex vivo and in vivo. In ex vivo gene therapy (Fig. 3), hepatocytes are removed from the patient and cultured in vitro. The desired expression vector is introduced into the cultured hepatocytes. Several methods can be used to get the gene into the cultured cells, including transduction with a recombinant retrovirus. The transduced hepatocytes are then introduced into the portal vein and lodge in the patient’s liver. Hepatocyte-directed ex vivo gene therapy

Table 5. Available and future treatments for liver diseases, based on molecular biological methods.
Recombinant products Interferons Vaccines Rational drug design Compounds based on structure of recombinant proteins Gene therapy Ex vivo gene therapy In vivo gene therapy Antisense oligonucleotides/ribozymes DNA vaccines

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Fig. 3. General strategies for hepatic ex vivo gene therapy.
Hepatocytes are removed from the patient and cultured in vitro. An expression vector, which may be a virus, plasmid, or other construct, is introduced into the cultured hepatocytes. The recombinant hepatocytes are then introduced into the portal vein and lodge in the patient’s liver.

has already been performed in human subjects with familial hypercholesterolemia [8], a disease that results from a defect in the LDL receptor gene. In these human studies, hepatocyte cultures established from patients’ cells were transduced with recombinant retrovirus vectors that contained a functional LDL receptor gene. Once high-efficiency gene transfer was evident, the cells were introduced into the portal veins of the patients. Three of five patients treated in this fashion had persistent notable reductions in serum concentrations of cholesterol and LDL. In liver-directed in vivo gene therapy, genetic material is introduced into hepatocytes by gene transfer vectors that function after being introduced directly into the patient. Vectors for liver-directed in vivo gene therapy can be recombinant viruses taken up by hepatocytes, DNA complexed with proteins taken up by hepatocytes, or naked DNA. To develop effective in vivo gene therapies for liver diseases, investigators must devise vectors that are specific for hepatocytes and must induce hepatocytes to readily take up these vectors. Besides the transfer of human genes, other nucleic acid-based therapies currently under experimental development may be useful in the treatment of liver diseases. Antisense oligonucleotides [97] can be used to inhibit the expression of (e.g.) genes essential for the replication of hepatitis viruses. Ribozymes are catalytic RNA molecules that can be used for similar purposes as antisense oligonucleotides [98]. DNA transfer technology can also be used for vaccines [96]. In conclusion, molecular biology has already made a large impact on the diagnosis and treatment of liver diseases. In the next millennium, molecular biological methods will become increasingly important in clinical and laboratory medicine. Creative research and hard work should lead to inexpensive diagnostic tests and effective treatments for many of the liver diseases that affect people throughout the world.

References
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