Ali, Robin R.; (1993) The generation and analysis of mice bearing a human alpha-1-antitrypsin gene construct containing the Z mutation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

alpha-1-antitrypsin (AAT) is a serum protease inhibitor whose major physiological target is neutrophil elastase in the lung. The AAT Z variant is not properly secreted into the circulation, but accumulates in the endoplasmic reticulum of the hepatocytes that synthesise it. Individuals that are homozygous for the Z allele usually develop chronic emphysema and/or liver disease as adults, but a proportion (10%) develop neonatal liver disease. There is no heterogeneity in the Z allele. However, it is known that the risk of neonatal liver disease to a homozygous ZZ infant varies between families indicating that additional environmental or genetic factors may play a role in its etiology. An attempt was made to reproduce aspects of the AAT related liver disease in mice in order to investigate which additional factors may contribute to the development of the childhood disease. An AAT Z construct was introduced into mice in order to test whether this would result in liver disease despite the presence of normal endogenous mouse AAT. First the AAT Z gene construct was produced by a novel technique involving homologous recombination in E. Coli which was between a cosmid containing a normal human AAT gene and a plasmid containing an exon of the gene with the Z mutation. Secondly a facility for generating transgenic mice was established and two lines of mice carrying the AAT Z construct were produced. One of these lines expressed the transgene. The expressing Z line was compared to other lines bearing the same construct and to lines bearing the normal human AAT gene. The circulating human AAT protein in these mice was analysed by isoelectric focusing. The normal and abnormal variants were shown to be processed similarly to their human counterparts. The level of human AAT protein in the plasma was quantitated. The expressing Z line was shown to have similar levels of circulating AAT Z protein as a human homozygous ZZ individual. The AAT Z protein was shown to accumulate in the hepatocytes by immunohistochemistry. By comparing the level of circulating human AAT with the level of human AAT mRNA in transgenic M and Z mice, it was inferred that approximately 20% of the Z protein was secreted from the liver (compared with 10-15% in humans). For these reasons the expressing Z line was considered to be an appropriate line to assess whether the presence of PiZ protein was sufficient to cause liver disease. Livers taken from transgenic and nontransgenic mice were analysed for histopathology. Preliminary results suggest that moderate liver damage occurs in AAT Z mice and that this was proportional to the age of the mouse and the level of protein being produced. However, the damage seen was mild compared to that seen in either human neonates or adults. Furthermore, liver damage was not seen in juvenile mice, and equivalent damage was also was seen in mice producing such high levels of the normal AAT protein, that it was also seen to accumulate within hepatocytes. Overall, the results suggest that the liver disease that was seen in adult mice resulted from damage due to long term accumulation of the AAT protein. Although the AAT Z mice may reproduce aspects of the adult liver disease associated with the AAT Z variant, these mice do not represent a model for the neonatal liver disease.

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