DETERMINATION OF APOLIPOPROTEIN (APO) E GENE POLYMORPHISM IN 144 CHINESE BY PCR AMPLIFICATION/HhaI RESTRICTION
MU H, LIU MZ, DENG ZH.
Laboratory Center, Tianjin First Central Hospital, Tianjin, P.R. China
The structure gene locus for human apo E is polymorphic. The common alleles encoding for the three major plasma apoE isoforms are apo E4, apo E3 and apo E2. These proteins differ by amino acid substitution at one or both of 112 and 158 positions. The most common isoform is E3 polypeptide [Cys112/Arg158], for these amino acids DNA codons are GTGC and GCGC. The E4 isoform [Arg112/Arg158] is often associated with increased concentrations of cholesterol and a higher risk of coronary artery disease. The E2 isoform is [Cys112/Cys158]. The homozygous of E2 is associated with type III hyperlipoproteinemia. We extract DNA from blood cells and amplify a specific sequence of apo E gene by PCR with primer 5'-AGAATTCGCCCCGGCCTGGTACAC-3' and 5'-TAAGATTGGCACGGTCGTCCAAGGA-3', resulting in a 244 bp fragment which encode 112 and 158 amino acids codon. The amplification product was digested with HhaI restriction endonuclease which cut site is GCG↑C. After separating by polyacrylamide gel and staining with ethidium bromide, the apo E genotypes were determined. Blood samples obtained from 144 normal Chinese subjects were genotyped. The frequency distribution of apo E geneotypes and alleles are E2/2, 0%, E3/3, 68.75%; E4/4, 2.78%; E2/3, 14.58%, E2/4, 2.7%, E3/4, 11.11%; and ε2, 8.68%, ε3, 81.6%, ε4, 9.72%, respectively. This method is easy to operate in the clinical laboratory.
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