It has been reported that several DNA mutations in the receptor binding domain of apolipoprotein E (apo E) are usually involved in type III hyperlipoproteinemia. GC-clamp denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP) were used to detect DNA sequence changes. We evaluated these methods ability to determine apo E isoforms or detection of new apo E mutation. Genomic DNA for polymerase chain reaction (PCR) was extracted from whole blood of 200μl. A 244-bp fragment containing amino acid residues from 91 to 165 was amplified by first PCR. This DNA fragment included the receptor binding domain. It also included 112 and 158 amino acid residues whose mutations characterize the two common variants, apo E2 and apo E4, respectively. The second PCR product was amplified with 5'-primer or 3'-primer, which had been attached with a 40-bp G+C-rich sequence (GC-clamp), and the product was subjected to electrophoresis using a denaturing gradient gel in a bath at 60°C. The gel consisted of 7% polycrylamide with a linearly increasing gradient from 60% to 80% denaturant (100% denaturant: 7M urea/40% formamide) for 5'-GC-clamp or from 50% to 70% denaturant for 3'-GC-clamp. After electrophoresis, the gel was stained with ethidium bromide. The 5'-GC-clamp-DGGE made it possible to distinguish E4 and E3 from E2. The 3'-GC-clamp-DGGE distinguished E3 from E4. All six phenotypes of apo E could be determined by combining the 5'-and 3'-GC-clamp-DGGEs. Additionally, apo E-Kochi (145Arg->His), a rare mutation of apo E, could be detected using these methods.
SSCP was performed nonradioactively. The first PCR product was denatured to single strands by 0.5 M NaOH and heating at 42°C, and was resolved with 100% formamide. The sample was subjected to electrophoresis using 6% polyacrylamide gel at 4°C for 80 minutes. The gel was then stained using ethidium bromide. The SSCP of apo E2 homozygote showed one band, those of E3 and E4 homozygote showed two bands. The SSCP of heterozygotes showed three to four bands, whose patterns all differed. Therefore, the six phenotypes of apo E were clearly distinguished by this method. In the SSCP method, the DNA of apo E-Kochi showed yet another pattern. The SSCP method for apo E analysis was simpler and more sensitive than DGGE. PCR-SSCP took only 6 hours to analyze. Fifty patients with hyperlipoproteinemia were studied using these two methods, and another family of apo E-Kochi was found. It shoud be possible to find other mutations of apo E using these methods.
