Apolipoprotein E polymorphism: automated determination of apolipoprotein E2, E3, and E4 isoforms.

Apolipoprotein E (apo E) plays an essential role in lipoprotein metabolism, where it is involved in the clearance of chylomicrons and very low density lipoproteins. Apart from some rare variants, apo E exists in three common isoforms (E2, E3, and E4). The different isoforms have not only been associated with different plasma lipid levels but have also been correlated with certain pathological conditions, such as lipid disorders (dysbetalipoproteinemia, hypercholesterolemia), cardiovascular diseases, and Alzheimer's disease. Here we describe a rapid, automated test for the determination of the most frequent polymorphisms (E2, E3, and E4). This polymerase chain reaction-based test allows the reliable discrimination of all six genotypes. The assay has been developed especially for the nonspecialized routine clinical laboratory by employing an analyzer and chemistry often present in this type of laboratory. Because of its low costs and easy handling, the assay can be performed on a daily basis.

Activated protein C (APC)-resistance: automated detection of the point mutation at position 1691 in the factor V gene.

Proteolytic cleavage of factor Va, caused by activated protein C, is an important mechanism in limiting clot formation in normal haemostasis. A single point mutation in the factor V gene has been demonstrated to cause resistance of factor Va to proteolytic cleavage by activated protein C. With an 8-fold increased risk of thrombosis and a 2 to 13% prevalence in the Caucasian population for the heterozygous state of this mutation, knowledge of the patient's genetic disposition is of great importance in conditions such as pregnancy, surgery, use of oral contraceptives and immobilization. Therefore we have developed an automated test for the detection of the factor V mutation. This PCR based test makes use of the disappearance of an Mnl 1 restriction site if the mutation is present. The assay has been developed for the widely used ES-systems of Boehringer Mannheim. The test discriminates between the heterozygous and the homozygous state. Because of its low costs and easy handling the assay can be used as a screening test and can be performed in routine clinical laboratories.

Screening for mutations in the LDL receptor gene and apolipoprotein B-100 gene in 218 patients with severe hypercholesterolemia.

A group of 218 patients with severe hypercholesterolemia (LDL cholesterol > 260 mg/dl) living in the Cologne area were screened for mutations in the LDL receptor gene and apolipoprotein B-100 gene. In the LDL receptor gene Southern blotting was used for detection of major DNA rearrangements and the single-strand conformation polymorphism (SSCP) method was used to screen for micro-deletions and insertions and single base alterations. The Arg3500-->Glu mutation, which is the only relevant mutation in the apolipoprotein B-100 gene causing hypercholesterolemia, was detected by a modified PCR and restriction enzyme digestion. Three different major rearrangements, all of which were deletion, were found in the LDL receptor gene. The SSCP screening was started with exon 4. In 20 cases an abnormal fragment pattern was observed. The apolipoprotein B-100 mutation was detected in 15 patients. In summary, by the combined analysis of major rearrangements, micro-deletions, insertions and single base alterations in the LDL receptor gene and the Arg3500-->Glu mutation in the apolipoprotein B-100 gene, mutations causing or probably causing hypercholesterolemia could be detected in 38 of the 218 studied patients. The expansion of SSCP screening to other exons of the LDL receptor gene will greatly increase the identification of mutations causing hypercholesterolemia.

Screening for mutations in exon 4 of the LDL receptor gene in a German population with severe hypercholesterolemia.

A group of 218 patients with severe hypercholesterolemia (LDL cholesterol > 260 mg/dl) living in the Cologne area were screened for mutations in the 3 half of exon 4 of the low density lipoprotein (LDL) receptor gene by the single-strand conformation polymorphism (SSCP) method. The analysed fragment was 242 bp in length and comprised approximately 6% of the coding region. In 11 patients an abnormal SSCP pattern was observed. Two of the abnormal fragment patterns were identical. The results of the SSCP screening could be confirmed by direct DNA sequencing. Three of the ten different mutations were previously described (3 bp deletion: codon 197; Asp200-->Gly; Glu207-->stop). Of the newly identified mutations there were two deletions, two insertions, one combined insertion and deletion mutation and two single base pair substitutions [1 bp deletion: G in codon 197; 37 bp deletion: T in codon 196-208 or AT in 196-207 and GA in codon 208; 18 bp insertion: codon 201-206; 8 bp insertion: codon 155-156 and GA in codon 157; 6 bp insertion (codon 196-197) and 5 bp deletion (codon 199, C in codon 198 and G in codon 198 or 200); Asp200-->Tyr; Asp203-->Val]. The 8-bp insertion was detected in a second unrelated individual. The analysis of the functional consequences of the mutations indicates that all mutations were causative of the LDL cholesterol elevation.