A SINGLE G-NUCLEOTIDE TO A-NUCLEOTIDE TRANSITION IN EXON IV OF THE LECITHIN - CHOLESTEROL ACYLTRANSFERASE (LCAT) GENE RESULTS IN AN ARG(140) TO HIS SUBSTITUTION AND CAUSES LCAT-DEFICIENCY

We have characterized the molecular defect causing lecithin: cholester ol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in thre e siblings of Austrian descent. The patients presented with typical sy mptoms including corneal opacity, hemolytic anemia, and kidney dysfunc tion. LCAT activities in the plasma of these three patients were undet ectable. DNA sequence analysis of polymerase chain reaction (PCR)-ampl ified DNA of all six LCAT exons revealed a new point mutation in exon IV of the LCAT gene, i.e., a G to A substitution in codon 140 converti ng Arg to His. This mutation caused the loss of a cutting site for the restriction endonuclease HhaI within exon IV: Upon digestion of a 629 -bp exon IV PCR product with HhaI, the patients were found to be homoz ygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids conta ining a wildtype or a mutant LCAT cDNA revealed that, in contrast to t he cell medium containing wild-type enzyme, no enzyme activity was det ectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT de ficiency in affected individuals and supports the conclusion that Arg( 140) is crucial for the structure of an enzymatically active LCAT prot ein.