Screening For The New Variants Of Apolipoprotein B-100 With Defective Receptor Binding In Hyperlipidemic Chinese

Objective: ApoB- 100 is the only protein that LDL has, and there is only one copy of this protein in each LDL granule. If the receptor-binding activity of ApoB- 100 was damaged by any reason, the clearance rate of LDL would decrease seriously and the plasma LDL-C level would increase, which can lead to hypercholesterolemia and premature atherosclerosis. This metabolic disorder causing by destroyed receptor-binding ability of ApoB-100 was designated as FDB. Up to now the precise region of Apo B responsible for binding to the LDL receptor remain to be determined. According to the researches other scientists have done before, we can conclude that receptor-binding areas can be orientated between the amino acid residues 2980-3780, among them there are four areas such as 2980-3084, 3359-3369, 344 1-3569 and 3663-3782 which are mostly like to be the receptor-binding areas. Our research is to detect the putative receptor-binding domain from amino acid residue 2980 to 3084, and try to find out if there is any unknown point mutation leading to Familial defective ApoB-100. From this research I try to provide people more experimental information to elucidate the exact receptor-binding area and to make clear the genetic background of Familial Defective ApoB-100 and Hypercholesterolemia. Methods: Genomic DNA was extracted from leucocyte of 341 patients with primary hypercholesterolemia. A DNA segment of 351bp including codons 2980-3084 of ApoB-l00 gene was amplified by PCR. The PCR product was confirmed by restriction enzyme digesting. All the PCR products were analyzed by single-strand conformation polymorphism (SSCP). Results: The mean level of serum total cholesterol of 341 patients was 6.75 ? 0.9lmmolIL, and that of serum triglyceride was 1.45 ?.8lmmoIIL. The target segment of these genomic DNA were amplified successfully. All the PCR product were analyzed by SSCP in optimized conditions, but no abnormal figure was found. 2 Conclusion: l)Point mutation causing hypercholesterolemia is unlildy to exist or rare in the codons 2980-3084of ApoB-l00. 2)Aniino acid residue 2980-3084 may not be involved in the receptor-binding area of ApoB-l00. 3) I have found a DNA polymerase-mediated PCR method for the generation of a positive control to optimize the analyzing conditions of SSCP.