Two DNA restriction fragment length polymorphisms associated with Ag(t/z) and Ag(g/c) antigenic sites of human apolipoprotein B.

Abstract

Several high frequency restriction fragment length polymorphisms (RFLPs) associated with the human gene for apolipoprotein B have been previously reported by Priestly et al. 1 The EcoRI RFLP here was shown to be very strongly associated with the Ag(t/z) Immunochemlcal polymorphism of human low density llpoprotelns, allowing correct Ag(t/z) phenotyplng of 17 (out of 17 tested) unrelated individuals. The Xbal RFLP was associated with the Ag(g/c) Immunochemlcal polymorphism, permitting correct phenotyplng of 14 (out of 17 tested) unrelated Individuals. Its close association with an RFLP permitted localization of the Ag(t/z) polymorphism to the C-termlnal end of the apolipoprotein B peptlde, and allowed detailed discussion of Its probable molecular basis. (Arteriosclerosis 7:301-305, May/June 1987) B ecause of the central role played by apolipoprotein B (apo B) in the transport of lipids, and especially of cholesterol in low density lipoprotein (LDL), it is possible that a genetic variation in apo B could help explain why some individuals are susceptible to atherosclerosis. Apo B has been cloned, and its entire amino acid sequence has been determined.2 ' 3 This achievement permits the reexamination of previously identified genetic variations at the level of nucleotide sequence.Phenotypic expression of genetic variation in LDL has been studied extensively using antisera against allelic variants of the donors' LDL developed in the sera of multiply transfused patients. Population and family studies have confirmed that these immunochemical polymorphisms are inherited in an autosomal codominant mode, implicating the amino acid sequence variation in a protein component of LDL. The data were analyzed and summarized 4 to indicate the existence of five distinct sites of immunochemical polymorphism: Ag(a1/d), Ag(x/y), Ag(t/z), Ag(g/c), and Ag(h/i). The order of the corresponding five loci along the chromosome remains unknown. (In the terminology employed, 'Ag' means 'antigen group', and 'a1/d' are the two alternate forms of an epitope present at the first-mentioned site; 'x/y' are the two alternate epitopes at the second mentioned site, etc.) Phenotypically, individuals may be Received October 13, 1986; revision accepted January 28, 1987. homozygous or heterozygous at each of these five sites; thus, at the first site, an individual might be a1 ,a1 or d,d or a1 ,d. Therefore, 3 5 = 243 possible human Ag phenotypes exist, of which 44 have been identified. 4A monoclonal antibody against apo B which also recognizes the Ag(g/c) epitope and binds 40 times more tightly to LDL from Ag(c,c) individuals than to LDL from Ag(g,g) individuals has been developed. The existence of this antibody identified the gene for apo B as the general locus for the Ag system; possibly all of the five Ag epitopic pairs represent one or the other of two alternative amino acids at five distinct locations on the polypeptide for this apolipoprotein. 5In this communication, we demonstrate that two previously identified restriction fragment length polymorp...

Bsp 12861 restriction fragment length polymorphism detects Ag(c/g) locus of human apolipoprotein B in all 17 persons studied.

“…16 We have been attempting to map the five Ag loci onto the apo B gene using RFLP analysis as well as molecular cloning techniques. We reported a perfect correlation for 17 individuals between the Ag(t/z) locus and an EcoR I RFLP near the 3' end of apo B gene, 17 and this has been extended by 24 additional individuals. 15 Recently, we identified a T-to-C substitution at nucleorjde 1981 of apo B cDNA as being the polymorphic locus for Ag (a1/d).…”

“…An ethnically diverse subpopulatjon of 17 unrelated normal individuals previously described 17 was included in this study. DNA preparation, Southern analysis, and Ag phenotyping were also previously described.…”

“…DNA preparation, Southern analysis, and Ag phenotyping were also previously described. 17 Briefly, genomic DNA from these 17 individuals was prepared from 30 ml of peripheral blood and was digested with various restriction enzymes under the conditions suggested by the manufacturers (New England Biolabs, Beverly, MA and Bethesda Research Laboratories, Gaithersburg, MD) but with at least a threefold excess of enzyme. After digestion, 10 yjQ of DNA from each person was separated through gel electrophoresis by using 1% to 2% agarose depending on the size of the restriction fragments.…”

“…Hybridization and autoradiography were performed as described. 17 The experimental protocol was approved by the Human Subjects Protection Committee of the University of California, Los Angeles.…”

Association of DNA polymorphism at the apolipoprotein B gene locus with coronary heart disease and serum very low density lipoprotein levels.

“…This polymorphism has been associated with the Ag(x/y) and the Ag(c/g) immunochemicaJ polymorphism 10 ' 11 and with elevated serum levels of apo B, 12 cholesterol, and .triglycerides. 12 ' 1314 In patients with type III hyperlipidemia, the allele frequency for the Xba IRFLP differed significantly from the frequency in normolipidemic individuals and in those with other types of hyperiipidemias.…”