RESUMEN
We have characterized the molecular defect causing lecithin:cholesterol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in three siblings of Austrian descent. The patients presented with typical symptoms including corneal opacity, hemolytic anemia, and kidney dysfunction. LCAT activities in the plasma of these three patients were undetectable. DNA sequence analysis of polymerase chain reaction (PCR)-amplified 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 converting 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 homozygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids containing a wild-type or a mutant LCAT cDNA revealed that, in contrast to the cell medium containing wild-type enzyme, no enzyme activity was detectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT deficiency in affected individuals and supports the conclusion that Arg140 is crucial for the structure of an enzymatically active LCAT protein.
Asunto(s)
Exones , Fosfatidilcolinas/genética , Esterol O-Aciltransferasa/deficiencia , Esterol O-Aciltransferasa/genética , Adenosina , Arginina , Secuencia de Bases , Femenino , Expresión Génica/genética , Guanosina , Histidina , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Linaje , Mutación Puntual , Reacción en Cadena de la PolimerasaRESUMEN
The composition of lipoproteins in the plasma of patients with LCAT deficiency (LCAT-D) is grossly altered due to the lack of cholesteryl esters which form the core of normal lipoproteins. When plasma from LCAT-D patients and their relatives was examined we found that nine heterozygotes had plasma Lp(a) levels of 2-13 mg/dl whereas none of 11 affected homozygous individuals from different families contained detectable amounts of Lp(a) in their plasma. Therefore, the binding of apo(a) to LDL density particles was studied in vitro using LDL density fractions prepared from patients, and recombinant apo(a) [r-apo(a)], which was expressed and secreted by transfected COS-7 cells. The LDL from heterozygotes were chemically indistinguishable from normal LDL and homogeneous with regard to morphology, whereas the crude LDL floating fraction from homozygotes consisted of a heterogeneous mixture of large vesicles, and small spheres resembling normal LDL. The LDL density fraction from the LCAT-D patient lacked almost completely cholesteryl esters. Incubation of LCAT-D plasma with active LCAT caused a substantial augmentation of the original subfraction which morphologically resembled normal LDL. Using r-apo(a) and normal LDL or LDL of heterozygous individuals, apoB:r-apo(a) complexes were formed when incubated at 37 degrees C in vitro for 20 h. In contrast, the total LDL floating fraction from a homozygous LCAT-D patient failed to form apoB:r-apo(a) complexes. After treatment with active LCAT, a significant apoB:r-apo(a) association was observed with LCAT-D LDL-density particles. Our data emphasize the importance of the integrity of LDL structure and composition for the formation of Lp(a). In addition, we demonstrate that the absence of LCAT activity has a fundamental impact on the regulation of plasma Lp(a) levels.
Asunto(s)
Apolipoproteínas A/biosíntesis , Deficiencia de la Lecitina Colesterol Aciltransferasa/metabolismo , Lipoproteínas LDL/biosíntesis , Apolipoproteínas A/sangre , Apolipoproteínas A/metabolismo , Apolipoproteínas B/metabolismo , Austria/epidemiología , Femenino , Homocigoto , Humanos , Deficiencia de la Lecitina Colesterol Aciltransferasa/epidemiología , Lipoproteínas LDL/química , Lipoproteínas LDL/ultraestructura , Masculino , Linaje , Unión ProteicaAsunto(s)
Granulocitos/fisiología , Diálisis Renal , Fosfatasa Alcalina/metabolismo , Creatinina/sangre , Granulocitos/inmunología , Humanos , Recuento de Leucocitos , Neutrófilos , Nitroazul de Tetrazolio/metabolismo , Reacción del Ácido Peryódico de Schiff , Peroxidasa/metabolismo , Fagocitosis , Polisacáridos/análisisRESUMEN
9 fluid overloaded patients, 8 of them with congestive heart failure and 1 patient with acute renal failure, were treated by ultrafiltration without hemodialysis. Using Gambro-major- and Rhone-Poulenc-6-dialysers the patients were deprived of 6,907.14+/-3,586.13 ml or 652.83 ml/h of extracellular fluid, on an average. During the withdrawal of fluid the plasma volume and central venous pressure decreased, whereas the cardiac output slightly increased. After the ultrafiltration, which did not show any undesirable side effects, the patients could be held in a compensative state by conservative therapy (digitalis and diuretics).