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Apolipoprotein A4 Gene (APOA4) (Chromosome 11/Haplotypes/Intron Loss/Coronary Artery Disease/Apoal-APOC3 Deficiency) Sotirios K

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Apolipoprotein A4 Gene (APOA4) (Chromosome 11/Haplotypes/Intron Loss/Coronary Artery Disease/Apoal-APOC3 Deficiency) Sotirios K Proc. Natl. Acad. Sci. USA Vol. 83, pp. 8457-8461, November 1986 Biochemistry Structure, evolution, and polymorphisms of the human apolipoprotein A4 gene (APOA4) (chromosome 11/haplotypes/intron loss/coronary artery disease/APOAl-APOC3 deficiency) SOTIRios K. KARATHANASIS*t, PETER OETTGEN*t, ISSAM A. HADDAD*t, AND STYLIANOS E. ANTONARAKISt *Laboratory of Molecular and Cellular Cardiology, Department of Cardiology, Children's Hospital and tDepartment of Pediatrics, Harvard Medical School, Boston, MA 02115; and tDepartment of Pediatrics, Genetics Unit, The Johns Hopkins University, School of Medicine, Baltimore, MD 21205 Communicated by Donald S. Fredrickson, July 11, 1986 ABSTRACT The genes coding for three proteins of the APOC3 deficiency and premature coronary artery disease plasma lipid transport system-apolipoproteins Al (APOAI), (13-15), hypertriglyceridemia (16), and hypoalphalipopro- C3 (APOC3), and A4 (APOA4)-are closely linked and teinemia (17). tandemly organized on the long arm ofhuman chromosome 11. In this report the nucleotide sequence of the human In this study the human APOA4 gene has been isolated and APOA4 gene has been determined. The results suggest that characterized. In contrast to APOAl and APOC3 genes, which the APOAI, APOC3, and APOA4 genes were derived from a contain three introns, the APOA4 gene contains only two. An common evolutionary ancestor and indicate that during intron interrupting the 5' noncoding region of the APOA1 and evolution the APOA4 gene lost one of its ancestral introns. APOC3 mRNAs is absent from the corresponding position of Screening of the APOA4 gene region for polymorphisms the APOA4 mRNA. However, similar to APOAI and APOC3 showed that two different Xba I restriction endonuclease genes, the introns of the APOA4 gene separate nucleotide sites are polymorphic in Mediterranean and Northern Euro- sequences coding for the signal peptide and the amphipathic pean populations. Haplotype analysis indicated that these domains in APOA4. These results suggest that the APOAI, polymorphisms do not display significant nonrandom asso- APOC3, and APOA4 genes were derived from a common ciation. Finally, restriction mapping of the APOA4 gene evolutionary ancestor and indicate that during evolution the region in a patient with combined APOA1-APOC3 deficiency APOA4 gene lost one of its ancestral introns. Two restriction and premature coronary artery disease indicated that this endonuclease sites, an Xba I located in the second intron of the patient has a structurally normal APOA4 gene. APOA4 gene and a different Xba I located 9 kilobases 3' to the APOA4 gene, are polymorphic in Mediterranean and Northern MATERIALS AND METHODS European populations. Haplotype analysis indicated that even Nucleotide Sequencing Determinations. DNA sequencing though these polymorphic sites are located within 9 kilobases was carried out either by the base-specific chemical cleavage they do not display significant nonrandom association. Finally, method (18) or by subcloning various DNA fragments in M13 restriction mapping analysis of DNA from a patient with vectors (19) and using the resulting single-stranded DNA combined APOAI-APOC3 deficiency and premature coronary templates for nucleotide sequence determination by the artery disease indicated that this patient has a structurally dideoxy chain-termination method (20). normal APOA4 gene. Isolation of DNA Fragments and Radioactive Labeling of Hybridization Probes. Cloned DNA fragments were isolated Apolipoproteins are lipid-binding proteins involved in the by electrophoresis in low-melting (Bio-Rad) agarose gels and transport of triglycerides, phospholipids, and cholesterol in purified by phenol extraction and ethanol precipitation. the plasma. Association ofapolipoproteins with lipids results These fragments were labeled using [a-32P]dCTP (Amer- in formation of lipid-protein particles (lipoproteins), the sham, 3000 Ci/mmol; 1 Ci = 37 GBq) as detailed (21). major carriers of lipids in the intra- and extravascular space. S1 Nuclease Mapping and Primer-Extension. Purified re- Apolipoprotein A4 (APOA4) is synthesized primarily in the striction DNA fragments were treated with bacterial alkaline intestine (1, 2) and represents a major protein constituent of phosphatase according to the specifications of the vendor newly secreted intestinal triglyceride-rich lipoproteins (IBI), extracted with phenol, precipitated with ethanol, and (chylomicrons) (3). The significant increase in APOA4 syn- 5' end labeled with [y-32P]ATP (Amersham, 3000 Ci/mmol) thesis and secretion by the intestine during fat absorption (4) using polynucleotide kinase (New England Biolabs). These 5' and the rapid dissociation of this protein from the surface of end-labeled fragments were used for S1 nuclease mapping lymph chylomicrons during catabolism of these particles (5, (22) and primer-extension analysis (23). 6) suggest that APOA4 plays a significant role in triglyceride Genomic Restriction Mapping Analysis and Statistical Meth- metabolism. Similarly, the possible involvement of APOA4 ods. Chromosomal DNA was prepared from peripheral blood in activation of lecithin-cholesterol acyltransferase [(LCAT) of normal [with regard to coronary artery disease (17)] phosphatidylcholine-sterol acyltransferase; EC 2.3.1.43] (7) individuals digested with restriction enzymes (New England and the influence of the LCAT reaction on the distribution of Biolabs), electrophoresed in 1% agarose gels, transferred onto APOA4 among different lipoproteins (8) may indicate that nitrocellulose filters (24), and hybridized with 32P-labeled DNA APOA4 is also involved in cholesterol metabolism. probes. These filters were washed and used for autoradiog- The genes coding for apolipoproteins Al (APOAJ), C3 raphy. Estimation of the nucleotide diversity (ir), heterozy- (APOC3), and A4 (APOA4) are closely linked and tandemly gosity (h), standardized nonrandom association (A), recombi- organized in the long arm of the human chromosome 11 nation frequency per kilobase (kb) ((D), and polymorphic infor- (9-12). Several different DNA polymorphisms in this region mation content (PIC value) was as described (25-28). of the human genome are associated with combined APOA1- Abbreviations: PIC, polymorphic information content; APO-, apo- The publication costs of this article were defrayed in part by page charge lipoprotein; kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); payment. This article must therefore be hereby marked "advertisement" LCAT, lecithin-cholesterol acyltransferase; IVS, intervening se- in accordance with 18 U.S.C. §1734 solely to indicate this fact. quence; df, degree(s) of freedom. 8457 Downloaded by guest on September 25, 2021 8458 Biochemistry: Karathanasis et al. Proc. Natl. Acad. Sci. USA 83 (1986) APOA 1 APOC3 APOA4 3' 2). The start site (cap site) of this gene was determined by S1 513'iS'5pJ3' nuclease mapping analysis of K KH3 SR B R K RSKR H3 H3 8 adult human intestine total | 1Kbp e_ RNA using as probe a 468-bp Msp I DNA fragment extending between nucleotides (nt) -334 and 134 (Fig. 2). In addition, - AATAAA primer-extension analysis of this RNA was carried out by X P R P KAP - P P BPASmSe BX PSTSRSP - - using as primer a 91-bp I II 100bp ]- IY .. HIS_ BamHI-Msp DNA fragment I I 1 /-77'I f _-- I extending between nt 45 and 134 (Fig. 2). The resulting liV1/ IVS-2 k I// -- autoradiogram (Fig. 3) shows that S1 nuclease mapping (lane III I10b APO -5CAP -/ Poy-A 3' MRNA I' S) and primer-extension analysis (lane P) result in a 134-nt AUG UGA product. These data indicate that the start site of the human X P PHTSmH P88H P RHBHX P KP H PSTSRSPH HP || A APOA4 gene is located 54 residues 3' to the last nucleotide of IV lOObP 'i Y'(( r the TATA box (TTTAAAT) in this gene (Fig. 2). DNA Polymorphisms and Nucleotide Diversity of theAPOA4 Gene Region. Two DNA fragments containing APOA4 gene sequences, a 724-bp BamHI fragment extending between nt FIG. 1. (1) Restriction map of the human APOAJ, APOC3, and 39 and 763 (-probe I) and 1812-bp Sac I fragment extending APOA4 genes (open boxes). (H) Restriction map of the human between nt -3 and 1809 (probe II), were used as probes for APOA4 gene. "TATA box" and poly(A) signal are boxed. Introns restriction site polymorphism screening of Mediterranean [intervening sequence (IVS)] and exons (filled boxes) are indicated. and Northern European populations (Fig. 4). Of the total 27 (III) Structure of the APOA4 mRNA. The 5' (5' CAP) and 3' (Poly-A sites screened, only 2, an Xba I (XbaIA) 3') ends and initiation (AUG) and termination (UGA) codons are located in the IVS2 indicated. (IV) Direction and extent of nucleotide sequencing deter- of the APOA4 gene and a different Xba I (XbaIB) located 9 minations are shown by arrows below a map of the human APOA4 kb 3' to XbaIA, are polymorphic in both of these populations gene; bars on the left side of the maps indicate the nucleotide scale. (Table 1). The sizes of hybridization fragments obtained with Restriction enzymes are shown as B, BamHI; S, Sac I; R, EcoRI; each of the nonpolymorphic sites (Table 1) are identical for H3, HindIII; K, Kpn I; P, Pst; X, Xba I; Sm, Sma; T, Taq I; A, Acc every DNA sample studied (data not shown). Thus, the I; and H, Hpa II. Kbp, kilobase pairs; bp, base pairs. observed polymorphisms are most likely due to base substi- tutions rather than large deletions or insertions. Assuming RESULTS that these base substitutions are selectively neutral it is possible to estimate the nucleotide diversity. (ir; refs. 25-27) Isolation and Characterization of the Human APOA4 Gene. over the Genomic clones with overlapping DNA inserts spanning the 38-kb DNA segment containing the human APOA4 human APOAI, APOC3, and APOA4 genes have been gene. The or values for the Mediterranean and Northern isolated and characterized (12). These clones were used to European populations are 0.0022 and 0.0020, respectively. construct a restriction endonuclease map of the human The combined data from both populations indicate a ir value APOA4 gene (Fig. 1). The nucleotide sequence of this gene of 0.0021.
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