2 NOVEL POINT MUTATIONS IN THE LECITHIN-CHOLESTEROL ACYLTRANSFERASE (LCAT) GENE RESULTING IN LCAT DEFICIENCY - LCAT (G(873) DELETION) AND LCAT (GLY(344)-]SER)

We investigated the genetic defects in two patients with familial leci thin:cholesterol acyltransferase (LCAT) deficiency. Their clinical man ifestations including corneal opacities, anemia, proteinuria, and hypo alphalipoproteinemia were identical for familial LCAT deficiency. Thei r LCAT activities and the cholesterol esterification rate (CER) were n early zero, and their LCAT masses were below 10% of normal control val ues. Sequence analysis of the amplified DNA of case 1 revealed one bas e deletion of G at base 873 (first position of Val(264)) in exon 6, le ading to a premature termination by frameshift. Sequence analysis of a mplified DNA of case 2 revealed a single G to A converting Gly (GGT) t o Ser (AGT) substitution at residue 344. When COS-1 cells were transfe cted with these mutants, LCAT activity in the medium was nearly zero, and the LCAT mass was undetectable (<0.01 mu g/ml). In contrast, LCAT activity in the medium of COS-I cells, transfected with wild-type LCAT , was 1.7 nmol/h per mi and the LCAT mass was 0.09 mu g/ml. The LCAT m ass in the cell lysates of the mutants was less than 12% of control fo r case 1 and 18% of control for case 2. Northern blot analysis of the mRNA of COS-1 cells transfected with the mutants showed the same amoun ts of LCAT mRNA as compared with wild-type LCAT. Biosynthesis of mutan t LCATs was analyzed by pulse-chase and immunocytochemistry in transfe cted baby hamster kidney cells. SDS-PAGE/fluorography demonstrated tha t wild-type LCAT was synthesized as a high-mannose type of 56 kDa, whi ch was very slowly converted to a mature form of 67 kDa and was se cre ted into the media. In contrast to the wild-type LCAT, the mutant prec ursors were not processed into the mature form but slowly degraded alo ng with chase times. On steady and continuous labeling in the case of wild-type LCAT, the mature 67 kDa form was observed in both the cell l ysate and media, whereas no mature form was detected in the cell lysat es and media which were transfected mutant LCATs. These data suggest t hat the mutant LCATs are actually synthesized in an amount comparable to that of wild-type, but they are slowly degraded without being proce ssed into the mature form. The immunocytochemistry revealed that mutan t LCATs were mainly retained in the endoplasmic reticulum. These data suggest that these two mutations may disrupt the mutant LCATs' transpo rt from the endoplasmic reticulum into Golgi apparatus, resulting in L CAT deficiency.