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Abstract

Epidemiological studies have indicated that decreased levels of plasma HDL are associated with an increased risk of coronary artery disease. However, there are several rare familial disorders of HDL metabolism in which, despite very low levels of HDL cholesterol, affected individuals do not appear to be at an increased risk for premature atherosclerosis. Examples of such disorders include familial LCAT deficiency and fish eye disease which are rare autosomal recessive diseases associated with inherited defects within the gene coding for lecithin:cholesterol acyltransferase (LCAT). In both cases, corneal opacities and a severe HDL deficiency are characteristic features of the disease. However, unlike fish eye disease, familial LCAT deficiency is associated with severe lipoprotein abnormalities and additional clinical symptoms including hemolytic anemia, proteinuria and a progressive renal insufficiency. The basis for this segregation is believed to be a result of functional differences associated with the LCAT enzyme. The main purpose of this thesis was to define the molecular basis for the clinical and biochemical heterogeneity observed for different genetic defects of LCAT. To achieve this, the expression of recombinant LCAT (rLCAT) in mammalian cell culture was established as a model to analyse the properties of human plasma LCAT. Subsequently, a series of natural mutations associated with familial LCAT deficiency and fish eye disease were re-created and expressed in both monkey kidney COS-1 and baby hamster kidney cell lines. To determine the functional significance of each mutation, the specific activity of different mutant enzymes was analysed using both synthetic and natural substrates. A wide range of functional abnormalities were identified: (i) defects of secretion, (ii) loss of activity against HDL, (iii) loss of activity against all lipoproteins and (iv) variable reactivities for all lipoproteins. For defects which occur in the homozygous form, the properties of most mutant rLCATs were consistent with the biochemical phenotype observed in the plasma of affected probands. The recreation and analysis of single mutations associated with compound heterozygous genotypes revealed that the products of each allele pair had different characteristics. In addition, the results suggested that the presence of only a single allele coding for a partially active LCAT is sufficient to maintain normal rates of cholesterol esterification. From these biochemical analyses, it appears that LCAT has at least two functionally important domains, the catalytic center and a recognition site for HDL substrates. In conclusion, it seems that the original families identified with familial LCAT deficiency and fish eye disease represent the clinical and biochemical extremes of a wide range of defects of LCAT function. Consequently, these disorders should not be classified as separate diseases but thought of as part of a larger group of LCAT deficiency syndromes.