Molecular genetics of chronic liver diseases

13 Molecular genetics of chronic liver diseases A. M. BRIND M. F. BASSENDINE Many adult chronic liver disorders are either wholly (‘single gene’) or in some part (‘polygenic’) genetically determined. The application of recombinant DNA technology has already told us a great deal about the molecular pathology of some single gene disorders affecting the liver. For example, in olr-antitrypsin deficiency we now have a good idea of the repertoire of different mutations that underlies this disease. In others, such as Wilson’s disease, we know the chromosomal location of the disease allele and polymorphic deoxyribonucleic acid (DNA) markers are available to assist in diagnosis. In many single gene disorders, such as Wilson’s disease and haemochromatosis, the chromosomal location of the disease allele has been determined but the biochemical abnormality responsible for the disease is not known. In these conditions it is likely that ‘reverse genetics’ will ultimately determine the exact cause. Thus it is possible, by various manoeuvres of recombinant DNA technology, to move gradually closer to a gene, clone it, and work back to the protein sequence. A spectacular example of this approach is provided by cystic fibrosis, where the recent cloning of the gene is already providing valuable new insights into disease pathogenesis. When it comes to the genetic analysis of chronic liver conditions that are more commonly seen in clinical practice, such as alcoholic cirrhosis or autoimmune disease, the position is much more complicated. Recent developments in molecular biology, however, enable a start to be made in dissecting the complex interactions of genotype and environment that contribute to these disorders. It may soon be possible to make useful predictions about high-risk groups of individuals for these diseases and, ultimately, the molecular approach should teach us more about their basic pathophysiology. SINGLE

GENE DISORDERS

These conditions can be traced through families and are clearly defined as following an autosomal dominant or recessive (or sex linked) pattern of inheritance. A dominant allele is one which manifests its phenotypic (recognizable) effect in heterozygotes, whereas a recessive allele only causes Baillidre’s

Clinical

Gastroenterology-

Vol. 4, No. 1, March 1990 ISBNO-7020-1504-O

233 Copyright 0 1990, by Baillit?re Tindall All rights of reproduction in any form reserved

Wilson’s

Polycystic

disease

disease

Variable: 100000

1 in 1000

1 in 50to 1 in 3000

HS in 1000

Haemochromatosis

Autosomal

Autosomal

Autosomal

Autosomal

lin2000

_

Autosomal

Inheritance

single

lin3000

Disease frequency in Caucasians

1. Common

or-Antitrypsin deficiency (Pi2 allele) Cystic fibrosis

Condition

Table

recessive

dominant

recessive

recessive

recessive

gene disorders

adult

liver location

disease.

Long arm (q31) Short arm (P21) Short arm (pl2-pter) Long arm (ql4-21) of chromosome

of chromosome

of chromosome

of chromosome

Long arm of chromosome (q31-32.3)

Chromosomal

causing

13

16

6

7

14

of gene

product acids,

antiprotease

?

?

1480 amino acids, membrane protein superfamily ?

394 amino

Gene

3

3

$ z

F ”

5

3

E

3

?

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its phenotypic effect when present in the homozygous state. The majority of adult chronic liver disease caused by single gene defects are autosomal recessive, and we will consider the impact of molecular biology on our understanding of some of these. aI-Antitrypsin ai-Antitrypsin (AAT), a 52 kDa glycoprotein, is the major serum protease inhibitor. In normal individuals, more than 2g AAT is produced daily, primarily in hepatocytes but also in mononuclear cells. Serum deficiency of AAT results from mutations in the single copy 12.2 kilobase (kb) gene located on chromosome 14q31-32.3 (Schroeder et al, 1985) (Table 1). As the gene is known, there has been considerable research into the molecular biology of the liver disease with which it is associated. The cYI-antitrypsin alleles

Extensive genetic heterogeneity is seen at the AAT locus, with approximately 75 alleles identified (Brantly et al, 1988b). Classically, the phenotype (Pi type) is determined by isoelectric focusing (IEF) of serum; over 30 phenotypes have been described but far more alleles can be differentiated by direct analysis of the gene. Each individual expresses both parentally acquired alleles; the allelic nomenclature is based on letter assignments corresponding to the position of migration of the serum protein on IEF. The commonest phenotype in the UK population is PiM (including all subtypes) with a frequency of 80.5% ; PiS has a frequency of 0.25%, PiZ of 0.03%) PiMZ of 3% and PiMS of 9% (Kalsheker, 1985). Low serum AAT levels are seen in some phenotypes; PiZ is associated with 15% and PiS with 60% normal PiM serum levels. There are rare Pi ‘null’ variants associated with no detectable serum AAT. Several of the genes encoding for these protein variants have been sequenced (Brantly et al, 1988b). The M and the Z variants differ by a single base substitution in exon V of the normal gene, causing a glutamic acid-, lysine substitution at position 342 in the molecule (Jeppsson, 1976). In addition a valine- alanine substitution at position 213 occurs but this is also seen in around 40% of subtype Ml (Nukiwa et al, 1986). Molecular biological techniques to identify the Z allele either use oligonucleotide probes specific for the codon change at 342 on digested DNA or amplified DNA (Kidd et al, 1983; Brind et al, 1990b), or analyse for the presence or absence of the restriction site for Bst EII (PiZ and 40% of PiMl) (Abbott et al, 1988). These techniques have mainly been applied in prenatal diagnosis to detect Z homozygotes but can also be used in adult liver disease patients to detect carriers of the Z allele (Brind et al, 1990b). The new technique of polymerase chain reaction (PCR) to amplify DNA means that DNA from nearly every source, including formalin-fixed liver biopsy sections, may be analysed. Liver disease associated with al-antitrypsin

Two major patterns

variants

of liver disease are described in association with

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abnormalities of AAT: neonatal cholestatic jaundice and adult chronic liver disease. Around 10% of individuals homozygous for the Z allele develop cholestatic jaundice within the first 6 weeks of life; 25% of these progress to cirrhosis but many recover completely or show minor abnormalities in liver function (Sharp, 1982). PiZ adults are at increased risk of cirrhosis and hepatocellular carcinoma, the estimated lifetime relative risk, compared with the normal population, of cirrhosis is 7.4 and of hepatocellular carcinoma 20 (interestingly the relative risks are only significant in males) (Eriksson et al, 1986). There is still some controversy as to whether individuals heterozygous for the Z allele are also at increased risk of adult chronic liver disease, as several studies have come to conflicting conclusions (Morin et al, 1975; Hodges et al, 1981; Roberts et al, 1984; Carlson and Eriksson, 1985). It seems likely, however, that Z heterozygotes are at a small increased risk of cryptogenic chronic liver disease which has not been detected in all studies due to insufficient sample numbers and technical difficulties in detecting the Z phenotype. Pathophysiology

of liver disease

The basic characteristic of AAT-related liver disease has been the finding on liver biopsy sections of periportal AAT inclusion granules by either periodic acid-Schiff diastase (PAS-d) or AAT specific immunoperoxidase staining. These granules represent AAT in the high mannose form in the rough endoplasmic reticulum (rER); this is the form appropriate to the rER but normally the AAT is transported to the Golgi for further modification to its carbohydrate side chains prior to secretion (Bathurst et al, 1984). The granules are seen mainly in association with the PiZ, PiM Malton, PiM Elemberg, PiM Duarte and have even been described in individuals who appear to be PiM (Carlson et al, 1988). The molecular pathology leading to the accumulation oi AAT inclusion granules has been extensively investigated. Figure 1 shows the processes involved in the biosynthesis of AAT. Transcription of the Z gene is normal; the relative abundance of Z AAT messenger ribonucleic acid (mRNA) is not different from M AAT mRNA in hepatocytes (Schwarzenberg et al, 1986) and in Xenopus oocyte expression systems translation is not different for M and Z genes (Verbanac and Heath, 1986). The secretion defect is not dependent on glycosylation, as agents inhibiting this process do not affect the differences in secretion observed (Foreman, 1987). The deficit in AAT secretion appears to be at the point of transport from rER to Golgi. Extrapolating from crystallographic studies it has been hypothesized that the Z specific mutation disrupts an intramolecular salt bridge and causes abnormal aggregation of AAT in the rER (Loeberman et al, 1984). One study involving site specific mutagenesis of the M and Z gene to disrupt and reform the salt bridge, and subsequent expression of the mutant genes in mammalian cells, has been compatible with this hypothesis (Brantly et al, 1988a), but two studies have shown that it is not the salt bridge alone that is responsible for the abnormal secretion but the actual amino acid at position 342 in the molecule (McCracken et al, 1989; Sifers et al, 1989). It has been

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CHRONIC LIVER DISEASES HEPATOCYTE

Transcription

Translation

Carbohydrate addition of mannase chains and protein folding

I

/\ /

Carbohydrate trimming to complex

chains

Secretion

Figure 1. Schematic representation of steps in the biosynthesis of cY1-antitrypsin (AAT). rough endoplasmic reticulum.

rER,

suggested that there may be a problem in molecular recognition of the Z variant leading to failure of transport from rER to Golgi prior to secretion (McCracken et al, 1989). Using DNA sequence data, the molecular mechanisms whereby other mutants are sequestered in hepatocytes have been proposed but the exact mechanisms remain unproven (Huber and Carrell, 1989). The relationship between intrahepatic granules and disease is uncertain. Serum deficiency is not thought to be pathogenic, as individuals with the null-null phenotype do not apparently develop liver disease. It has been hypothesized that inclusion granules are themselves pathogenic (the ‘engorgement hypothesis’) and one theory suggests that their presence leads to the induction of the heat shock proteins and liver damage (Carrell, 1986). Evidence supporting this hypothesis comes from experiments involving transgenic mice who had been transfected with either the human Z AAT gene or the M AAT gene. Animals transfected with the Z gene developed AAT granules in the rER of their hepatocytes and liver damage (Dycacio et al, 1988; Carlson et al, 1989). However, in adult clinical practice, periportal AAT granules are seen in the absence of liver disease and in liver disease of recognized alternative aetiology (Brind et al, 1990a). Factors leading to liver disease in PiZ carriers have not been identified but siblings of affected neonates have an increased risk of cholestatic jaundice and breast feeding is protective (Sharp, 1982). Children with HLA-B5 have been reported to be at increased risk of chronic liver disease (Mieli-Vergani et al, 1987). Although much is known about the molecular biology of AAT, there are still important questions to be answered in relation to its association with liver disease.

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Cystic fibrosis

Cystic fibrosis (CF) is a generalized disorder characterized by membrane dysfunction, producing bile duct hypoplasia, focal biliary fibrosis (cirrhosis), micro-gallbladder and biliary sludging (Bodian, 1953); steatosis is a common feature (Hultcranz et al, 1986). More recently, sclerosing cholangitis has been described (Strandvik et al, 1988). The frequency of CF varies considerably between populations, being common in North Europeans (1 in 2000 live births) compared to Afro-Americans or Orientals (1 in 100 000). The disease is inherited as an autosomal recessive trait and the variation in prevalence may reflect heterozygous advantage for the CF gene in a particular environment. As one of the most common lethal hereditary diseases in Caucasians (Boat et al, 1989), it has been the subject of a concerted research effort. The basic defect has been associated with decreased chloride ion conductance across the apical membrane in epithelial cells (Widdicombe et al, 1985) but the underlying biochemical defect remained unknown. In view of this the ‘reverse genetic’ approach was used in an attempt to understand the molecular defect through direct cloning of the responsible gene on the basis of its chromosomal location. Identification

of the CF gene

Extensive studies using a large number of polymorphic DNA markers in multiple kindred with the CF gene provided evidence for a single CF locus on the long arm of chromosome 7, band q31 (Spence and Tsui, 1988) (Table 1). Chromosome walking and jumping and complementary DNA (cDNA) hybridization were used to isolate DNA sequences, encompassing more than 500 000 base pairs (bp) from this region of chromosome 7. Ten genomic libraries were constructed during the course of the experiments. Several transcribed sequences and conserved segments were identified, one of which corresponded to a portion of the CF gene locus (Rommens et al, 1989), which has now been shown to be large, spanning about 250 kb and containing a minimum of 24 exons. A putative mRNA of about 6.5 kb was identified and overlapping cDNA clones isolated. Together, these clones span about 6.1 kb and contain an open reading frame capable of encoding a protein of 1480 amino acids (Riordan et al, 1989). The predicted protein structure of the normal CF gene has similar characteristics to the mammalian multi-drug resistance P-glycoprotein with membrane bound and ATP binding domains. It is probably a member of a membrane protein superfamily and has been called the CF transmembrane conductance regulator (CFTR). It remains unclear how the CFTR is involved in the regulation of ion conductance across the apical membrane of epithelial cells. It may serve as an ion channel itself or serve to regulate ion channel activities. Implications

for diagnosis and therapy

A single mutational event may account for most CF mutations in Northern European populations (Estivill et al, 1987). Another mutation appears to be prevalent in some Southern European populations (Estivill et al, 1988) and

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haplotype data indicate there could be several other mutations. Genetic analysis has shown that the most common mutation in CF individuals is a 3 bp deletion in the gene, resulting in the loss of a single phenylalanine residue (position 508) in the predicted amino acid sequence (Kerem et al, 1989). Since approximately 70% of CF individuals carry this mutation, molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis and PCR amplification may be used in diagnosis in the near future. When additional CF mutations are identified clearly, a panel of molecular diagnostic procedures will be developed. This single amino acid loss in a functional domain of the protein suggests that the CF phenotype is not likely to be due to complete loss of the gene product but rather may give rise to an altered protein with unusual behaviour. A complete molecular description of all the CFmutations should help to elucidate the pathophysiology of the disease and form the basis for the development of improved treatment. Haemochromatosis An inborn error of iron metabolism was first postulated as the cause of idiopathic haemochromatosis (IH) more than 50 years ago (Sheldon, 1935). For some years the relative contributions of genetic and environmental factors remained controversial, until Saddi and Feingold (1974) provided strong evidence in favour of recessive inheritance. The subsequent discovery of an association between haemochromatosis and certain HLA antigens (Walters et al, 197.5; Simon et al, 1976) allowed a new approach to the topic that has established the genetic nature of IH and its recessive mode of inheritance. Association

with the HLA

system

IH is closely linked to HLA phenotypes: HLA-A3 occurs in 55-100% (average prevalence 73%) of affected individuals, compared with 19-31% of controls; HLA-B7 in 28-86% (average prevalence 47%), as opposed to 9-34% in controls. HLA-B14 is also seen more frequently, 29% in IH compared to 10% in controls in one study. Certain HLA haplotypes are in positive linkage disequilibrium with the IH gene; A3-B7 (21.2 versus 6.1% in controls, P< 10-l’) and A3-B14 (13.8 versus 1.5% in controls, P< 10-l’) are the two most frequent (Simon et al, 1988). From data like this it has been shown that the disorder is due to a mutation (Simon and Brissot, 1988) or mutations (Summers et al, 1989) in a recessive gene on chromosome 6 in close proximity to the HLA locus. From the estimated rate of recombination with the HLA alleles, the putative IH gene has been mapped to lcM, probably telomeric, to the HLA-A locus (Figure 2a). The HLA linkage is considered to be purely one of genetic distance and does not implicate the HLA antigens in pathogenesis (Simon and Brissot, 1988). Disease only occurs in homozygotes for the abnormal gene, although heterozygotes show biochemical evidence of iron overload. Homozygous individuals do not show uniform disease expression; evidence to date

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favours the interpretation that this variation is due to environmental factors and not different mutant genes. Interestingly, evidence suggests that carriage of the haemochromatosis gene does not increase susceptibility to the states of iron overload associated with alcoholic liver disease or transfusion. However, it is possible that heterozygosity for the defective IH allele may be a factor in the development of porphyria cutanea tarda (Simon and Brissot, 1988) and iron overload in sideroblastic anaemia (Barron et al, 1989). Frequency and screening

Extensive familial studies in Brittany (Lalouel et al, 1985), Sweden (Olsson et al, 1983), Australia (Bassett et al, 1982a) and Utah (Dadone et al, 1982) have established the global nature of IH and yielded estimates of the frequency of the defective allele of 0.05-0.088. This corresponds to a heterozygous frequency of lO-16% and a disease frequency of 3-8 per thousand. The question of why the haemochromatosis gene is so prevalent is a matter of speculation, but possibly a scarcity of iron in the diet of ancestral European populations may have favoured IH carriers by enhancing their ability to endure pregnancies. Early diagnosis is important in the management of haemochromatosis so that prophylactic phlebotomy can commence before tissue damage occurs. All first degree, and possibly second degree relatives of an affected proband must be screened and, given the high frequency of the disorder, population screening or screening of diabetic patients has been advocated (Edwards et al, 1988; Phelps et al, 1989). Diagnosis is currently based upon the biochemical evidence of elevated iron stores: a transferrin saturation of more than 62% (reflecting excess parenchymal iron) and a raised ferritin (reflecting excess body stores). Unfortunately, these parameters are of limited sensitivity and specificity; the sensitivity in early asymptomatic disease is around 94% and the specificity 86% (Bassett et al, 1984). Genetic techniques involving HLA haplotype analysis are vital to family studies. HLA haplotype linkage can be used, because of recombination events, to follow the probable inheritance of the haemochromatosis gene. If a relative shares both HLA haplotypes there is a more than 90% probability of homozygosity, but if he or she shares neither, it excludes homozygosity for the haemochromatosis gene. Simpler molecular genetic techniques for screening must await identification of the actual gene involved. Pathophysiology

and candidate genes

The substance for which the haemochromatosis gene codes which would be responsible for the phenotypic expression of the disease is unknown. However, as haemochromatosis is a disorder of iron metabolism, potential candidate genes can be identified by studying the movement of iron, both into and out of the body and into and out of cells. Intestinal iron absorption is regulated and related to the body’s iron stores; intestinal macrophages are ’ thought to be important in this regulation. Iron is transported mainly linked

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to transferrin and taken up by cells, either by receptor mediated endocytosis of ferric-transferrin complexes or, at high serum iron levels (as seen in haemochromatosis), by non-specific absorptive or fluid phase endocytosis of transferrin bound or non-transferrin bound iron (Anderson et al, 1987). Storage in the form of ferritin and haemosiderin occurs in parenchymal cells of the liver and in reticuloendothelial cells in the bone marrow, spleen and skeletal muscle. In IH there is inappropriately high passage of iron through the intestinal mucosa and it is possible that the primary defect lies in the intestinal mucosa (Anderson et al, 1987). It is hypothesized that iron retention is abnormal in the intestinal macrophage, disturbing the regulation of iron absorption. Evidence for this hypothesis includes the observation that there is rarely iron overload in reticuloendothelial cells in haemochromatosis (Brink et al, 1976). A second possibility is that there is an abnormality of transferrin disturbing transport and uptake of iron. There is little to support this notion: no phenotypic differences in the electrophoretic pattern of transferrin and its function in iron turnover and receptor binding are observed in haemochromatosis. The gene for transferrin and its receptor are located on chromosome 3, not chromosome 6, but it is possible that regulatory factors for their expression may be at the haemochromatosis locus. However, studies of transferrin receptor expression have found it to be appropriate to the iron status of hepatocytes and monocytes in haemochromatosis (Anderson et al, 1987). A more recent, intriguing hypothesis is that ferritin handling is abnormal. Sequences encoding the H-subunit of ferritin, both genes and pseudogenes, have been found on 10 chromosomes, including chromosome 6, at 6~21.3 near the HLA locus (McGill et al, 1987). There has been an uncorroborated report of an increased frequency of a Hind III RFLP, known to be located on chromosome 6, detected with a ferritin H subunit cDNA probe in patients with haemochromatosis compared with normal controls (David et al, 1989). The site and cell in which this mutation may act is conjectural. Iron release by Kupffer cells is ill understood but it is thought to be mediated by ferritin; ferritin synthesis appears to be normal (Bassett et al, 1982b) but ferritin release by peripheral blood mononuclear cells is enhanced in IH (Flanagan et al, 1989). Hepatocytes are known to take up iron from Kupffer cells, and a hepatic ferritin receptor has been identified (Adams et al, 1988). The H subunit of ferritin may have a regulatory role in binding to this receptor and this may be important in the iron overload of haemochromatosis. If the molecular pathology of iron metabolism in haemochromatosis is uncertain, so are the specific pathophysiological mechanisms leading to hepatocyte damage in iron overload. Iron deposition is mainly hepatocellular, in the form of ferritin, usually free in the cytoplasm, and haemosiderin, often in secondary lysosomes. This is demonstrated in liver biopsy sections as diffuse cytoplasmic staining and granular deposits along the biliary canaliculi on Perls’ Prussian blue staining. A definite relationship between iron and damage exists, the threshold for fibrosis and cirrhosis is approximately 22 000 kg/g dry weight. Normally, tissue levels are less than 1800 &g, levels up to 5000 pg/g are seen in alcoholic liver disease with iron

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overload, and levels greater than 10 000 kg/g are considered specific for homozygotes for the haemochromatosis gene (Bassett et al, 1986). There is even a linear relationship between hepatic iron concentration and age which can be used to predict fibrosis and cirrhosis (Bassett et al, 1986). One theory suggests that iron overload leads to free radical formation and lipid peroxidation of the phospholipid of cellular membranes, with damage to lysosomal membranes causing release of damaging hydrolytic enzymes and damage to mitochondria and microsomes, disturbing enzyme function (Britton et al, 1987). An alternative hypothesis is that iron overload itself stimulates collagen synthesis (Weintraub et al, 1985). The pathophysiology of haemochromatosis is thus only partly understood but, even at the present level of knowledge, the molecular biology is helpful in diagnosis. It seems likely that reverse genetics, where identification of a mutation follows the identification of the genomic region where the liability to a clinical condition resides, will be the approach that will play a determinant role in clarifying the abnormality of iron metabolism in haemochromatosis. Wilson’s disease S. A. K. Wilson described the disease (hepatolenticular degeneration) that bears his name in 1912 (Wilson, 1912) and established its familial nature. It is an autosomal recessive disorder of copper metabolism, characterized by copper accumulation predominantly in the liver, leading to progressive liver damage, with central nervous system involvement resulting in extrapyramidal syndromes and psychiatric disorders. Heterozygotes do not develop disease but may exhibit biochemical abnormalities of copper metabolism, which cause diagnostic confusion. Early asymptomatic diagnosis is vital in tackling the disease as treatment with either n-penicillamine or triethylene tetramine prevents disease but only reverses some of the disease manifestations, not irreversible liver and brain damage (Marsden, 1987). Molecular tests to detect the Wilson’s disease (WND) gene would greatly improve management of this disorder. Assignment of gene to chromosome

13

The first step to localization of the WND gene came from classical family studies in Middle-Eastern kindred, in which linkage with a polymorphic red cell enzyme, esterase D (ESD), which is encoded on chromosome 13, was observed (Frydman et al, 1985; Bonne-Tamir et al, 1986). Until recently, only a few useful genetic markers from chromosome 13 were available. Partly because of the localization of hereditary retinoblastoma (RB) to chromosome 13 by linkage to ESD, several probes revealing polymorphic loci have been isolated and several groups have constructed genetic maps for the long arm of chromosome 13, with some dispute about exact ordering of loci (Leppert et al, 1986; Bowcock et al, 1988; Haines et al, 1988). Using these DNA probes, the Wilson’s disease locus has now been more accurately localized to 13q14-22 and its position relative to the polymorphic markers

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A

IH

D13510

A .-II

HLAAf

ICM

ESTERASE

P

HLA

B

+

RB

HLA C

4.4cM q

WND

HLA D D13526 6

13

2. Genetic mapping of the putative loci for (a) haemochromatosis (IH) approximately 1cM from HLA-A locus; (b) Wilson’s disease (WND) approximately 4.4cM from retinoblastoma (RB) gene, 4.0cM from DNA probe D13526 and 9.4cM from the esterase D locus. Figure

delineated, 4.4 CM distal to the RB gene and 9.4 CM distal to the ESD gene (Bowcock et al, 1988; Yuzbasiyan-Gurkan et al, 1988; Figus et al, 1989) (Figure 2b). Probes with a high degree of polymorphism with restriction enzymes have been described, which might be used as markers for the WND gene in prenatal, presymptomatic diagnosis and carrier detection (Farrer et al, 1988; Scheffer et al, 1987; Yuzbasiyan-Gurkan et al, 1988; Figus et al, 1989). Because of the variable clinical manifestations of Wilson’s disease, it has been suggested that more than one genetic locus may be involved in its pathogenesis. However, linkage to the same region of chromosome 13 is observed in all cases of Wilson’s disease, irrespective of ethnic origin and clinical manifestations (Bowcock et al, 1988; Yuzbasiyan-Gurkan et al, 1988; Figus et al, 1989). However, this does not exclude heterogeneity in the possible mutations at the Wilson’s disease locus or a cluster of genes involved in copper handling. Frequency and diagnosis

Scheinberg and Sternlieb (1984) have calculated the worldwide prevalence of Wilson’s disease to be 30 per million of the general population and the prevalence of heterozygous carriers one in a hundred. The incidence of the disease is believed to be higher in certain communities, including Arab, Druze and Oriental Jews in Israel, and in Sardinia (Giagheddu et al, 1985). Biochemical diagnosis is not straightforward. Serum caeruloplasmin is <20 mg/dl in 95% of cases, but may be normal with active liver involvement and low in heterozygotes for the Wilson’s disease gene and in disorders of low serum proteins. Urinary copper excretion is > 100 pg/day in affected individuals but may be normal in early disease and is often raised in chronic liver disease. Hepatic copper concentration is always high in untreated cases, > 250 pg/g dry weight, but elevated levels are also seen in cholestatic liver disorders. A test which may be useful to distinguish individuals who are homozygotes from heterozygotes and those with chronic liver disease and secondary abnormalities in copper metabolism involves determination of

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the incorporation of oral radioactive copper into caeruloplasmin; this is deficient in Wilson’s disease homozygotes (Sternlieb and Scheinberg, 1979). The simplest diagnostic method remains clinical examination of the eyes for Kayser-Fleischer rings, some of which may be apparent only on slit-lamp examination. It is to be expected, however, that molecular genetic techniques will identify the WND gene in the near future, opening up the possibility of simple molecular tests for the defective allele.

Pathophysiology

and candidate genes

The molecular pathology involved in the excessive accumulation of tissue copper in Wilson’s disease is poorly understood. However, the apparent reversal of abnormal copper metabolism in patients following liver transplantation strongly suggests that the primary defect is located in the liver. Caeruloplasmin metabolism is not thought to be of primary importance as serum levels do not correlate with disease activity; levels may be normal in affected homozygotes and are low in normal heterozygotes. In addition, the genetic locus of caeruloplasmin has been localized to chromosome 3 (Naylor et al, 1985). The mechanism of reduced caeruloplasmin levels seen in Wilson’s disease is due to a reduction in hepatic caeruloplasmin mRNA transcription (Czija et al, 1987). The biliary system is the major excretory pathway for copper which plays a major role in copper homeostasis. Wilson’s disease involves defective biliary secretion of copper. An attractive hypothesis is that this inability to excrete copper into bile is due to the absence of a high molecular weight copper-binding protein in the bile in affected individuals (Iyengar et al, 1988; McClain and Shedlofsky, 1988). Another intriguing theory is that the abnormality may reside in controller gene function rather than a structural gene as the pattern of liver copper metabolism seen in Wilson’s disease resembles that seen in the neonate. The hypothesis is that there is a failure to switch from fetal to adult gene expression. The mechanisms of hepatic damage secondary to the copper overload and the explanation for the variable manifestations of liver disease are poorly understood. Copper will cause lipid peroxidation of cellular membranes, and lipid peroxidation has been found in copper overload states. It is postulated that damaged cellular membranes leads to abnormal mitochondrial, lysosomal and nuclear function (Sokol et al, 1987). A reduced lysosomal pH has been demonstrated; this may be an effect of copper on membrane hydrogen pumps (Myers et al, 1987). Polymerization of some cytosolic proteins may be induced by copper, whilst copper loading leads to an inhibition of protein synthesis (Sternlieb, 1980). It remains a matter of speculation as to which, if any, of these processes may be involved in Wilson’s disease. Wilson’s disease is a serious, preventable disorder which is difficult to diagnose. The gene has nearly been identified and with its identification will come easier diagnosis and valuable insights into normal copper handling and disease pathogenesis.

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Adult polycystic liver disease Adult polycystic liver disease is associated with adult polycystic kidney disease (PKD); renal cysts are present in 50% of patients with liver cysts (Melnick, 1955) and, conversely, liver cysts have been found in approximately 50% of PKD patients at autopsy (Zeier et al, 1988). The hepatic cysts are often asymptomatic, although they frequently present as abdominal pain. The condition can produce obstructive jaundice, ascites or oesophageal varices (van Erpecum et al, 1987). Polycystic liver disease is increasingly becoming more clinically important as improvements in dialysis and transplantation prolong the survival of polycystic kidney patients. Cysts in the liver continue to grow and may appear de novo after renal transplantation for PKD. It has been found that there is a correlation between age and frequency of hepatic cysts in PKD (Thomson and Thayson, 1988). Adult polycystic kidney disease is inherited as an autosomal dominant disorder with a gene frequency of 1 in 1000 (Danovitch, 1976). The genetic locus for the disease has been assigned to the short arm of chromosome 16 by the finding of genetic linkage to the a-globin gene cluster (Reeders et al, 1985). Linkage to the phosphoglycolate phosphatase locus on chromosome 16 has also been reported (Reeders et al, 1986); this has less polymorphism than the a-globin locus, so has limited diagnostic use. In contrast, the 3’ hypervariable region polymorphism (8 kb downstream of the cw-globin cluster) is of particular value in diagnosis by linkage analysis because of the high heterozygosity of this locus. Little is known about the pathogenesis of polycystic liver and kidney disease but it is thought to involve the cell basement membrane (Wilson et al, 1986), possibly a regulatory defect of extracellular matrix production. Once the gene is identified and the mutation(s) producing the disease defined, the pathogenesis and the extent of genetic homogeneity of adult polycystic disease will become clearer. In particular, it should help to determine whether the same gene is involved in the related recessive disorders of infantile polycystic liver and kidney disease and congenital hepatic fibrosis.

POLY GENIC

DISEASE

Several common conditions which may affect the liver, such as alcoholism and autoimmune disease, tend to run in families but do not follow any clear-cut pattern of inheritance. The familial aggregation is not caused by a single gene defect but is thought to result from the cumulative interaction of a number of genes with environmental factors. Analysis of these complex polygenic diseases is not easy but ‘candidate genes’ can be identified and recombinant DNA techniques used to investigate whether any particular genotype is associated with increased risk of disease. Such studies may ultimately help to sort out the basic pathogenesis of the disease in question. As an example, the puzzle of who gets alcoholic cirrhosis will be considered.

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Alcoholic cirrhosis

A classic early study by Crothers (1909) suggested that susceptibility to alcohol abuse is, in part, genetically determined. This has been confirmed recently by studies in adopted out children of alcoholics, who are four times more likely to become alcoholic than ‘control’ adoptees, and show an earlier age of onset of alcohol related problems (Goodwin, 1987). Evidence for genetic predisposition to alcoholic abuse is provided by twin studies. In one large study of 15 924 male twin pairs, the twin concordance rates for alcoholism were 26.3% for monozygous twins and 11.9% for dizygous twins and for alcoholic cirrhosis were 14.6% for monozygous and 5.4% for dizygous twins (Hrubec and Omenn, 1981). In both cases the difference is statistically significant and is taken to indicate genetic factors are operating in the risk of alcoholism and alcoholic cirrhosis. This is corroborated by prospective studies of alcoholics which demonstrate that the rate of development of cirrhosis is independent of duration of alcohol abuse and amount of alcohol consumed (above a minimum level of 50g alcohol/day). The studies conclude that the risk of an alcoholic developing cirrhosis is about 15% (Sorensen et al, 1984). Such results have served to stimulate searches for underlying biochemical mechanisms and/or genetic differences. Pathophysiology

and candidate genes (Table 2)

Genes involved in the metabolism of alcohol need to be considered as potential candidates in studies of genetic predisposition to alcoholism and/or cirrhosis. About 90% of ethanol metabolism occurs in the liver. The main pathway is its oxidation to acetaldehyde which is catalysed by NAD+ dependent alcohol dehydrogenases. Another enzyme system that has been Table 2. Candidate genes in alcoholic cirrhosis. Gene

Chromosomal location

Alcohol dehydrogenases (ADH2 and ADH3) Mitochondrial aldehyde dehydrogenase (ALDH2) Ethanol-inducible cytochrome P450 Type 1 collagen (COLlAl and COLlA2) HLA Class I and II

4q21-25 12 10 17q21-qter and 7q22 6~21

implicated in ethanol metabolism is the cytochrome P4sadependent microsomal ethanol metabolizing system (MEOS). This form of cytochrome Pd5ais induced after chronic ethanol consumption and the contribution of MEOS to ethanol metabolism is probably substantial under these circumstances (Lieber, 1987). After a moderate dose of ethanol, the blood acetaldehyde concentration is maintained about one thousandfold lower than the blood ethanol concentration, suggesting that the alcohol dehydrogenase catalysed step is rate limiting. As many as 20 different molecular forms of alcohol dehydrogenase have been identified in human liver by gel electrophoresis or isoelectric focusing (Bosron and Li, 1986). Alcohol dehydrogenases (ADH)

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are dimeric zinc metalloenzymes with subunits of 40 000 Da ((.u, p, y, n and x), encoded at five different genes (ADHl, ADH2, ADH3, ADH4 and ADHS) (Agarwal and Goedde, 1987). Studies to date indicate that there is genetic polymorphism at two loci, ADH2 and ADH3, but no polymorphic variants have been identified at ADHl. Multiple forms of ‘TF-ADH and X-ADH have been observed but it is not clear that these differences are due to genetic polymorphism at ADH4 or ADHS (Jomvall et al, 1987). The investigation of the potential relationships between ADH polymorphism and genetic predisposition to alcoholism and/or alcoholic cirrhosis has been hampered in the past by the need to use liver biopsy tissue to determine the enzyme phenotype. Advances in molecular biology have now overcome this as direct genotyping of all three of the ADH2 and the two ADH3 alleles is possible using amplified leukocyte DNA and specific oligonucleotide probes (Xu et al, 1988). Genetic epidemiological studies are currently in progress to investigate whether any ADH genotype is associated with increased risk of alcoholic cirrhosis (T. K. Li, personal communication). Four different groups of aldehyde dehydrogenase isoenzymes have been identified by isoelectric focusing of human liver. The mitochondrial enzyme acetaldehyde dehydrogenase 2 (ALDH2) is responsible for the majority of the oxidation of acetaldehyde produced during ethanol metabolism. The gene for ALDH2 has been cloned and sequenced; it maps to chromosome 12 (Hsu et al, 1985, 1986). About half of Orientals lack the active form of ALDH2 and exhibit facial flushing, tachycardia, nausea, hypotension and dysphoria due to acetaldehyde accumulation when they drink (Harada et al, 1981). Amino acid sequencing has shown that Glu at position 487 in the normal active isoenzyme is replaced by Lys in the inactive form due to a single base pair change. This can be detected by probing leukocyte DNA or amplified DNA with a specific oligonucleotide probe (Hsu et al, 1987; Crabb et al, 1989). Interestingly, the inactive ALDH2 allele is dominant. It has been shown that only 2.3% of alcoholics and 2.8% of alcoholics with alcoholic liver disease possess this allele, as detected by phenotyping, compared with 41% of the general population in Japan (Harada et al, 1982). It seems likely the alcohol flush reaction, similar to the alcohol disulfiram reaction, discourages alcohol ingestion and thereby markedly reduces the risk of alcoholism. This is the first demonstration of a relationship between alcohol drinking behaviour and genetic variability of an enzyme involved in alcohol metabolism. To date, no linkage studies of alcoholism and/or alcoholic cirrhosis have been performed with the other ALDH loci. However, these enzymes, ALDHl, 3 and 4, have a higher Michaelis constant (K,) for acetaldehyde than ALDH2 and may not participate in acetaldehyde oxidation. The ethanol-inducible human cytochrome P45a has been cloned and sequenced (Song et al, 1986), but the role of genetic variants in predisposition to alcoholism or end-organ damage has not yet been examined. An alternative approach to studying the enzymes involved in ethanol metabolism has been to look at the mechanism(s) of end-organ damage. For example, type 1 collagen is increased in cirrhotic livers, and in the baboon model of alcohol fibrosis, ethanol consumption results in an increase in type 1

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procollagen mRNA (Zern et al, 1985). Type 1 collagen contains two identical polypeptide chains, al(l) and a slightly different chain, a2(1). The structural genes encoding these chains have been assigned to different chromosomes, the al(l) COLlAl gene is on the long arm of chromosome 17q21-qter, whereas the CY~(1) COLlA2 gene is on the long arm of chromosome 7q22. Informative RFLPs exist for both loci (Sykes et al, 1986). A preliminary small study has suggested that one haplotype at the a2( 1) locus is present in a higher percentage of alcoholics with cirrhosis than in controls (Eskreis et al, 1986), but this has yet to be confirmed. Another area that has been studied in alcoholic liver disease is immunogenetics, in particular the association of the major histocompatibility complex with disease. An early study showed that HLA-B40 was significantly increased in patients with alcoholic cirrhosis compared with controls (Bell and Nordhagen, 1980), although other studies have implicated different antigens (Devor et al, 1988). The HLA system at chromosome 6~21 has been extensively studied in other diseases, such as diabetes mellitus, at the molecular level but, given the inconsistency of the tissue type associations to alcoholic cirrhosis, it is perhaps not surprising that no studies have yet been reported using molecular techniques such as RFLP linkage. SUMMARY The molecular genetics of five common single gene and one polygenic chronic liver disease is discussed. In two of the single gene disorders, al-antitrypsin deficiency and cystic fibrosis, the gene responsible is now known and the repertoire of different mutations underlying the disease is being defined. In the other three single gene defects (haemochromatosis, polycystic liver disease and Wilson’s disease) the chromosomal location of the disease allele is known. It is anticipated that recombinant DNA techniques will enable the genes responsible for these diseases to be cloned in the near future, thus allowing the biochemical abnormalities to be defined through reverse genetics. In many chronic liver diseases the relative contribution of genetic and environmental factors remains unclear. Evidence suggests there is a definite genetic component in predisposition to alcoholic cirrhosis; the role of putative candidate genes is discussed. It is hoped that the definition of a genetic locus linked to alcoholic cirrhosis will ultimately teach us more about the basic pathogenesis of this disease.

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