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Detection of a mutation in a human LCAT gene Hornby, Ann Elizabeth


LCAT deficiency is a rare autosomal recessive disease characterized by low levels of plasma HDL and an inability of the enzyme lecithin:cholesterol acyltransferase (LCAT) to esterify cholesterol. An understanding of the structure and function of the LCAT protein will add significantly to the understanding of reverse cholesterol transport. This understanding can be gained, in part, by studying different mutations within the LCAT gene and their resultant phenotypes. Recombinant DNA technology has been used to determine the nature of a mutation in an LCAT gene of a previously described homozygote with this disorder. Southern blot analysis determined there were no major rearrangements in the genomic DNA at the LCAT locus. An attempt was made to follow segregation of the mutant alleles in three generations of a large pedigree by linkage analysis. There are known polymorphisms at the haptoglobin (Hp) locus, which is linked to LCAT on the long arm of chromosome 16, and in the adenosine phosphoribotransferase (APRT) and choesterol ester transfer protein (CETP) loci which are also on the long arm of chromosome 16, but have not been shown linked to LCAT. The information gained was uninformative in this pedigree. An extensive restriction fragment length polymorphism (RFLP) search in the immediate vicinity of the LCAT gene did not reveal any polymorphic sites. 2.4 kb of the ⋋ phage clone SF1020, obtained from one of the homozygotes, containing exons 1-5 plus 0.5 kb of DNA 5¹ to the LCAT gene, but not exon 6, was subcloned into M13 and sequenced. A cytosine to thymidine (C->T) transition was discovered in exon 4. This would result in a substitution of tryptophan for arginine at amino acid 135. The amino acid arginine is positively charged and resides in one of the most highly charged segments along the amino acid chain of the LCAT protein indicating that this region is likely involved in protein folding. Tryptophan, on the other hand is the most hydrophobic of the amino acids and would, therefore, severely disrupt the interaction of charged amino acids in that region, preventing normal folding of the LCAT protein.

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