Three possible sources of genetic heterogeneity that may result in variation in severity of liver disease have been investigated. One source is heterogeneity in the PiZ allele itslf. Another is heterogeneity in genes linked to Pi, and the third source is heterogeneity in unlinked genes.
1.2.9.1 Heterogeneity of the PiZ allele.
Although all PiZ alleles have the same point mutation in exon V there was a formal possiblity that some alleles could contain additional mutations that affect the production or nature of the PiZ protein and hence influence disease. Since the overwhelming majority of Z mutations are found on the same haplotype (see section 1.1.6) it is thought unlikely that heterogeneity of PiZ alleles is responsible for variation in liver pathology (Povey, 1990).
1.2.9.2 Genes linked to Pi.
Pi, which is located on 14q31-32.3, is linked to PiL, AACT, and the Gm locus (a cluster of genes encoding IgG heavy chains which specify Gm markers). PiL and AACT are members of the serpin gene family and share sequence homology with AAT. AACT and Pi are found within 250 kb of each other (Sefton et al., 1989), whilst PiL
is located approximately 10 kb downstream of Pi (Kelsey et al.,
1988a). The Gm locus is distal to Pi, PiL and AACT and is located on 14q32.33-qter (Cox e ta !., 1984).
PiL has a common polymorphism which consists of a 2 kb deletion that encompasses the whole of exon IV and part of exon V. This deleted allele (D) is present in the Dutch population at a frequency of 0.3 (Hofker at a/., 1988). Sequencing of the undeleted PiL allele (U) has shown extensive homology in the introns (65%) as well as the exons (70%) (Bao at a/., 1988). An accumulation of mutations in the promoter, start codon and in an intron/exon junction would suggest that the D allele is non-functional (Hofker at a/., 1988). It has been suggested that the structure of the U allele is not that of a pseudogene because it has conserved RNA splice sites and there are no internal termination codons (Bao at a/., 1988). If expressed, the PiL protein is predicted to have a different active site to that of human AAT (Tryp-Ser instead of Met-Ser), but the same as that of murine AAT (Bao at a/., 1988; Hill at a/., 1984). However, Kelsey a t
a/., (1988a) were unable to demonstrate expression of this gene in a range of tissues using a sensitive RNase protection assay. Almost all the PiZ alleles, that have been examined, have been shown to be associated with the U allele (Povey, 1990; Meisen at a/., 1988; Cox and Mansfield, 1987). The two genes are very close and recombination between them is unlikely. Therefore variation in the U allele is not thought to contribute to variation in the severity of liver disease in families (Povey, 1990).
The AACT gene has about 40% sequence homology with Pi (Chandra
al., 1984) as well as in the liver. Its product is a serum protease inhibitor, whose target substrates include mast cell chymase and neutrophil cathepsin G (Travis et a!., 1978). The physiological function of the antiprotease has not been clearly defined. However, cathepsin G enhances the rate of elastin digestion by elastase (Travis, 1978), and AACT is found at elevated levels in the bronchial lumen of patients with chronic bronchitis (Ryley and Brogan, 1973). The antiprotease has also been shown to markedly reduce the natural cytotoxic activity of T-cell killer lymphocytes in vitro (Gravagna, at a/., 1982), and to enhance the antibody response in vivo (Matsumoto at a/., 1982). Thus, AACT could be involved in the overall protease/antiprotease balance in the lung and may modify the immune response. It has been suggested that a genetic AACT partial deficiency (for which the molecular basis has not been established) is associated with liver and lung disease (Eriksson a t
a /., 1986). Although the AACT gene is fairly close to Pi, in one family recombination between the two genes has been observed. However, from limited data it appears that there is no linkage disequilibrium between a common Taq I polymorphism in the AACT gene and PiZ related liver disease (Kelsey at a/., 1988b).
Linkage between Pi and Gm was first reported by Gedde-Dahl at a/. (1972) in a study of 68 families where the recombination fraction was estim ated to be 0.25. They suggested, however, that recombination was decreased in carriers of the PiZ allele (PiMZ individuals) compared with non-carriers. This has been confirmed in a number of subsequent reports (Weitkamp at a/., 1978; Gedde- Dahl at a/., 1975, 1981; Babron at a/., 1990). Although it has been suggested that the reduction in recombination rate for PiZ alleles is
confined to males (Gedde-Dahl et al., 1975, 1981), this has not been confirmed in the largest study to date involving 843 families (Babron at a!., 1990). In addition, this study did not confirm an earlier finding of heterogeneity in the recombination rate of PiS versus non-PiS alleles (Chepuis-Cellier at a!., 1981).
Possible explanations of the reduced recombination rate between the PiZ allele and Gm include a linkage disequilibrium between the PiZ allele and a large chromosomal inversion or deletion between the two loci. It is not clear whether this phenomenon is true for PiZ alleles in all populations. The possibility that heterogeneity in the Gm locus is responsible for variation in severity of PiZ related liver disease exits, and is being explored (Povey, 1990).
1.2.9.3 Genes not linked to PI.
The only other source of genetic heterogeneity that has been investigated is the HLA complex. This cluster of genes has been associated with susceptibility to a number of human diseases, especially ones considered to have an autoimmune basis (see Kostyu (1991) for review). Doherty at at. (1990) assessed 140 PiZZ subjects for HLA type. Of these individuals, 92 had juvenile liver disease. They found an increase in HLA type DR4 in patients without liver disease (60% of individuals without liver disease, compared to 39% with). In addition, DR3 was significantly more frequent in PiZZ individuals with liver disease (46% ), compared to those without (17% ) and to a random control population (23% ). Since, however, there was a lack of concordance with HLA and disease status in
siblings, they concluded that other genetic or environmental factors must also be involved in the pathogenesis of neonatal liver disease.
Variation in other genes may also play a role in the heterogeneity of disease in PiZZ inividuals. For example, variation in the gene encoding elastase (the physiological target of AAT) may result in differences in antiprotease/protease physiology and hence lead to differences in pathology. Initial results, however, from lEF of the protein from serum suggests it is not very polymorphic (Dr. D. W hitehouse, personal communication). Conceivably variation in genes encoding proteins that regulate the expression of the AAT gene might also affect severity of liver disease. Thus if the severity is dependent upon the level of AAT accumulating in the liver, this might be revealed by polymorphisms in transcription factors or IL6 which upregulate the level of expression of the AAT gene. Since hsps have been shown to be raised in individuals with liver disease, it may be interesting to determine whether this is linked directly to variation in the hsps themselves.
PART THREE: A MOUSE MODEL OF PiZ RELATED LIVER DISEASE?