s
The role of abca1 in atherosclerosis: lessons from in vitro and in vivo models
Singaraja, R.R.
Publication date
2003
Link to publication
Citation for published version (APA):
Singaraja, R. R. (2003). The role of abca1 in atherosclerosis: lessons from in vitro and in vivo
models.
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Chapter r
Effluxx and Atherosclerosis: The clinical and
biochemicall impact of variations in the ABCA1 gene
Roshnii R. Singaraja
1*, Liam R. Brunham
1*, Henk Visscher
1, John J.P.
Kastelein
22and Michael R. Hayden
1"Thee Centre for Molecular Medicine and Therapeutics, University of British Columbia and Children'ss and Women's Hospital, Vancouver, B.C., V5Z4H4, Canada
:
Thee Department of Vascular Medicine, Academic Medical Centre, Amsterdam,, the Netherlands 'Thesee authors contributed equally to this work.
Abstract t
Approximatelyy 50 mutations and many SNPs have been described in the ABCA1 gene, with mutationss leading to Tangier Disease and Familial Hypoalphalipoprotememia- Homozygotes andd heterozygotes for mutations in ABCA1 display a wide range of phenotypes. Identification off ABCA1 as the molecular defect in these diseases has allowed for ascertainment based on geneticc status, determination of genotype-phenotype correlations, and has permitted us to identifyy mutations conferring a range of severity of cellular, biochemical and clinical phenotypes Heree we review how genetic variation at the ABCA1 locus affects its role in the maintenance off lipid homeostasis and the natural progression of atherosclerosis.
Abbreviations: :
(HDL)) high density lipoprotein, (SNP) single nucleotide polymorphism, (CAD) coronary artery disease,, (TD) tangier disease, (FHA) familial hypoalphalipoproteinemia, (NBF) nucleotide binding fold d
Partt I: The ABCA1 gene and its biological role
Atheroscleroticc coronary artery disease (CAD) constitutes the major public health burden in developedd countries, and by 2020 is predicted to be the single greatest cause of death worldwide ff I 2). Dc-ctc-aseJ HDLC is the must common iipiü abnormality in patients with premature CAD (4,5).. ABCA1 encodes the key protein regulating the efflux of lipids from peripheral cells to HDL,, following which these lipids are transported back to the liver and excreted as bile in a processs termed reverse cholesterol transport (6). At least 50 mutations have been identified in thee ABCA1 gene, leading t o the allelic disorders Tangier Disease (TD) and Familial Hypoalphalipoprotememiaa (FHA), which are associated with a wide range of phenotypic consequencess and putative biochemical defects. Here we review how genetic alterations of thee ABCA1 gene highlight its role in lipid homeostasis and atherosclerosis. Although TD is exceedinglyy rare, and ABCA1 mutations appear to be an infrequent cause of FHA, the study of m u t a t i o n ss in this gene has shed new light on a key pathway in the pathogenesis of atherosclerosis,, and opened up new approaches for its prevention and treatment.
1.11 Super-family: The ATP-binding cassette (ABC) transporters
Thee transport of specific molecules across membranes is critical for survival, and the ABC proteinss transport a wide variety of substances including lipids and sterols, metabolic products andd drugs across both intra and extracellular membranes (7). The first ABC transporter was clonedd in 1982 (8). ABC transporters are the largest membrane transporter family, consisting off 48 members in humans (9), 52 members in the mouse (10), 56 in Drosophila (9), 58 in C
eleganselegans (10), 31 in yeast (11) and 129 in Arabidopsis (10). The genome of E. coli contains 80
ABCC transporters, corresponding to 2 % of its genome (12). Despite their large numbers and substratee diversity, all ABC proteins bind and hydroiyze ATP and use the derived energy for transportt of the various molecules (13-15).
1.22 Classification and topology of ABC proteins and ABCA1
Knowledgee of the normal topology and organization of ABC proteins and ABCA1 is important ass it provides insight into potential functional domains. ABC transporters are defined based on thee presence of ATP binding domains, also known as nucleotide binding folds (NBFs) which conlamm three characteristic conserved regions, the Walker A and B domains, which are separated byy approximately 90-110 amino acids, and a signature (C) motif, located just upstream of the Walkerr B site (16,17) (Figure 1). Using flag tags and antibodies, and also by deciphering the glycosylationn status of ABCA1, the most current topological analysis reveals that ABCA1 consists off t w o large extracellular loops, one between the first and second transmembrane domains, andd the other following the intracellular NBF1 domain (18,19) (Figure 1),
Figuree 1. Schematic diagram of ABCA1 with each octagon representing 2 amino acids. The location of the
transmembranee domains (blue), extracellular loops (purple), Walker A motifs (green), Walker B motifs (royal) and Walkerr C signatures (red) and their residues are indicated.
domainss (MSD), each usually comprised of six membrane spanning a-helices which provide substratee specificity (20)
Thee mammalian ABC genes are divided into seven subfamilies, ABCA to ABCG, based on similarityy in gene structure, order of the domains, and sequence homology in the NBF and TM domains.. Figure 2 shows the phylogenetic relationship of selected ABC family members, and thee percent identity and similarity of these proteins.
1.33 Mutations in ABC genes cause many human genetic diseases:
Geneticc variation in ABC genes have been shown to be the cause of at least 12 genetic diseasess (Table 1). The ABCA1 gene is highly conserved between species (Figure 3). Human ABCA11 is 95.2% identical to mouse, 85.3% to chicken, 25.5% to drosophila, 21.6% to C.
eleganselegans and 10 2 % identical to fugu ABCA1. We have performed identity and similarity
searches,, and generated a phylogenetic tree (Figure 3). Although the C elegans gene CED7 wass proposed as the orthologue of ABCA1 based on similarities of function (36), the C.
eleganselegans t r a n s p o r t e r CE2 ( Q 9 T X V 8 ) is t h e closest m e m b e r t o A B C A 1 , as s u p p o r t e d by similar
analysiss by Peelman a n d colleagues (37). Thus A B C A 1 a n d CED7 are likely t o be paralogs a n d n o tt o r t h o l o g s .
similarity y
ABCA11 ABCA4 ABCA7 ABCA2 A B C B 1 ABCC1 ABCC7 ABCG5
A B C A 11 ABCA1 A B C A 4 4 6 0 0 0 A B C A 2 2 ABCA7 7 ABCG5 5 ABCA4 4 ABCA2 2 ABCC11 ABCB1 ABCC77 . „ „ „ . . ABCC1 1 ABCC7 7 4 8 2 2 544 5 4 5 9 177 3 1788 15 1 1 7 22 141 1800 15.1 9.4 4 244 7 23 0 134 300 9 10 6 identity y
Figuree 2. (A) The phylogenetic relationship of selected ABC Family Proteins. Sequences from ABCA1 (Genbank
accessionn number NM_00502), ABCA2 (NM_001606), ABCA4 (NM_000350): ABCA7 (AF_328787), ABCB1
(NM_000927),, ABCC1 (NM_004996), ABCC7 (NM_000492) and ABCG5 (NM_022436) were aligned using Clustall X. 1.8. A Neighbourhood-Joining tree was generated in ClustalX and viewed using phylodendron webservice att http://iubio.bio.indiana.edu/treeapp/treeprint-form,html. (B) Identity/similarity table of selected ABC family memberss generated using Clustal X 1.8. The same Genbank sequences as above were used.
Tablee 1 . Mutations in ABC genes are associated with several genetic diseases.
Genee Disease ABCA1 1 ABCA4 4 ABCB4 4 ABCB7 7 ABCB11 1 ABCC6 6 ABCC8 8 ABCD1 1 ABCC2 2 ABCC7 7 ABCG5andABCG8 8
Tangierr disease, familial Hypoalphalipoproteinemia (21)
Stargardtt disease, Retinitis pigmentosum 19, cone-rod dystrophy, age-related macularr degeneration (22, 23)
Progressivee familial intrahepatic cholestasis type 3 (PFIC) (24) X-linkedd Sideroblastic anemia and cerebellar ataxia (25) Progressivee familial intrahepatic cholestasis type 2 (26, 27, 28) Pseudoxanthomaa elasticum (29)
Persistentt hypermsulinemic hypoglycemia of Infancy (PHHI) (30) X-lmkedd adrenoleukodystrophy (31)
Dubin-Johnsonn syndrome (32) Cysticc fibrosis (33, 34) Sitosterolemiaa (35;
AA B s i m i l a r i t y y ii D. M e l a n o g a s t e r H.. M. G. D. C F. S a p i e n ss M u s c u l u s G a l l u s M e l a n o g a s t e r E l e g a n s R u b r i p e s r-- H. S a p i e n s H. S a p i e n s — 96 6 89 9 36.9 28 0 19 8 II | i-j M. M u s c u l u s 95 2 — 89.5 37.0 33 9 19 6 —— M. M u s c u l u s :: a G a l l u s 85 3 85 1 — 37 4 33 7 19 7
i i
G.. G a l l u s D. M e l a n o g a s t e r 25 5 25 6 25 8 — 4u 7 1 5 ' C.. E l e g a n s 2 1 6 jj C. E l e g a n s F.. R u b r i p e s 10 2 11 F R u b r i p e s i d e n t i t y yFiguree 3. (A) A phylogenetictreeof ABCA1 orthologuesfromH. sapiens (NM_005502), M. musculus (NMJ313454),
GG gallus (AF 362377), C. elegans (AF_ 101313), D melanogaster (NM_1 34601) and F lubripes (Hayden laboratory, unpublishedd data) were aligned using Clustal X 1.8, Neighbourhood-Joining tree was generated in ClustalX and viewedd using phylodendron webservice at httpy/iubio. bio.indiana.edu/treeapp/treeprint-form html as above. (B) Ann identity/similarity matrix consisting of the orthologues of ABCA1 was generated as described above.
Partt II: Variation in ABCA1:
Insightss into protein function and its contribution to atherosclerosis
2.11 Mutations in ABCA1
Att least 50 mutations in the ABCA1 gene have been identified (21,38-54,Hayden group unpublishedd data). These include 23 missense, 6 nonsense and 21 insertions or deletions. Forty-ninee of the reported mutations occur in exons. One mutation in intron 2 leads to abnormally splicedd transcripts lacking exon 2 or exon 4, or both (48). All mutations by definition result in decreasedd lipid efflux. The extremely high correlation between phospholipid and cholesterol effluxx (r = 0.86, p <0 0001 ) in more than 15 mutations tested (Singaraja and Hayden, unpublishedd data) indicates that ABCA1 influences efflux of both lipid types
2.22 Non-random distribution of mutations in t h e ABCA1 gene
Whilee mutations do occur throughout the gene, mutations in ABCA1 are not in random distribution.. Four mutations cluster between amino acids 230 and 282, 6 between residues 5877 and 635, 8 occur between amino acids 909 and 1099, 5 between residues 1145 and 12899 and 5 mutations occur between 2144 and 2215 (Figure 4). Conversely, with the exception off a large deletion, only one mutation occurs in the transmembrane regions between residues 6366 and 908, and no mutations occur in the second set of transmembrane domains. Many of thee residues harboring mutations are highly conserved with C elegans, a nematode that is
21.77 2 1 6 28 0 — 16 6
IVS22 +5 G>C —-R282X X
Figuree 4. Location of mutations in the ABCA1 protein. At least 50 mutations have been reported thus far in the
ABCA11 gene, many of which show a non-random distribution and cluster in specific regions of the protein (yelloww boxes).
estimatedd to have diverged from other metazoans 600-1,200 million years ago (55,56), thus indicatingg their functional importance (Table 2).
2.33 Functional effects of mutations in the extracellular loops of ABCA1
Approximatelyy half of the missense mutations in the ABCA1 gene associated with TD and FHA falll within the t w o extracellular loops. Mutations in the first and second extracellular loops mightt be expected to result in a lack of lipid efflux caused by dysfunctional interaction of ABCA11 with ApoA-l, since lipid poor pre-B HDL particles would require either direct interaction orr close proximity to ABCA1 in order for the lipid transfer to occur. Thus the extracellular loops off ABCA1 might provide a potential binding target for ApoA-l. Impaired transport of ABCA1 to thee plasma membrane could prevent interaction with ApoA-l. It Is also possible that mutations inn the extracellular domains will result in a disruption of the tertiary structure of ABCA1, therebyy preventing its function at the plasma membrane where it is normally localized. This conceptt is further supported by studies of ABCR, which shows very similar topology to ABCA1 (57).. The t w o halves of ABCR each contain a large extracellular loop, a transmembrane domain regionn and a nucleotide binding domain that interact together through one or more disulfide bonds,, putatively involving cysteines located in the extracellular loops in ABCR (57). Interestingly, mostt of the cysteines are conserved in these regions between ABCA1 and ABCR (57) suggesting
Tablee 2. Conservation of amino acid residues mutated in the human ABCA1 gene. MUTATION N P8BL L R230C C A255T T R587W W W590S S Q597R R \ L 6 9 3 3 T929I I N935S/H H A937V V A1046D D M1091T T D1099Y Y D1289L/N N C1477R R S1506L L N161ID D R1680W W N1800H H F20095 5 R2081W W P2150L L AE1893 3 ADD 1894 H,Sapiens s P P R R A A R R W W Q Q L L T T N N A A A A M M D D D D C C S S N N R R N N F F R R P P E E D D M.Musculus s P P R R A A R R W W Q Q L L T T N N A A A A M M D D D D C C
s s
N N R R N N F F R R P P E E D D G.Gallus s P P R R S S R R W W 0 0 L L T T N N A A A A M M D D D D C Cs s
N N R R N N F F R R P P E E D D D.Meianogaster r P P R R Q Q L L T T N N A A A A M M D D D D N N R R A A 1 1 R R R R D D D D C.EIeg g P P G G 0 0 Q Q L L T T N N A A A A M M D D D D S S R R W W M M R R N N S S D D23/244 (95.83%) mutations are conserved down to the chicken
thatt these residues are essential for folding and interactions between the different domains. Of note,, ABCA1 contains a cysteine at position 1477 that is mutated to an argmme residue (21) andd could thereby disrupt proper three dimensional folding of the ABCA1 protein necessary forr its ability to efflux lipids.
Furtherr insights into how the mutations R587W, W590S and Q597R that occur in the extracellularr loops affect ABCA1 function have recently been described (58-60). Two studies havee reported that ABCA1 containing the point mutation Q597R, which occurs in the first extracellularr loop, does not localize to the plasma membrane (59,60). However, other studies havee reported that this mutant is expressed at the plasma membrane, but at reduced levels relativee to wildtype ABCA1 (58,44). R587W, another missense mutation in the first extracellular loop,, also prevents the trafficking of ABCA1 to the plasma membrane, although results with thiss mutant have been variable (58-60). Both the R587W and Q597R mutants are resistant to PNGasee digestion indicating that they are not glycosylated, suggesting that ABCA1 harboring thesee mutations does not traverse the medial and trans Golgi network. However, not all mutations inn the extracellular loops prevent the export of ABCA1 to the plasma membrane. ABCA1
harboringg the W590S mutation does reach the cell surface and cross-linking studies reported normall interaction of the W590S mutant with ApoA-l despite defective efflux, suggesting that interactionn with ApoA-l may not be sufficient for lipid efflux (58),
ABCA11 that dues nol leach the plasma membrane cannot induce the binding of ApoA-l. This iss indeed the case with mutant Q597R (58,60) which shows no ApoA-l binding. However, failuree of binding may also occur due to disruption of residues crucial for this function. Indeed, thee variants C1477R and S1506L which are both localized in the second large extracellular loopp are normally translocated to the plasma membrane but show no ApoA-l binding, indicating thatt specific amino acids in the large extracellular loops are also necessary for ApoA-l binding. A l t h o u g hh thus far only a small subset of the naturally occurring mutations have been biochemicallyy characterized, these in vitro studies of ABCA1 mutations have begun to provide valuablee information on structure-function relationships of the protein.
2.44 Mutations in t h e transmembrane domain and impact on ABCA1 function
Onlyy one mutation has been described in the transmembrane domain of ABCA1. five mutations havee been described in the transmembrane domains of the ABCR gene. Small deletions and mutationss that introduce charged amino acids into transmembrane regions of ABCR result in greatlyy reduced amounts of ABCR protein (22,61). Mutations in the transmembrane domain regionn of ABCA1 might also disrupt ABCAI's integration into membranes and therefore prevent itt from exiting the endoplasmic reticulum and the golgi, or prevent its integration into the plasmaa membrane. This could result in the rapid turnover of the mutant ABCA1 protein. The onlyy described mutation in ABCA1 that occurs in the transmembrane domain, AL693, results inn the deletion of one amino acid. ABCA1 with this mutation does not exit from the endoplasmic reticulumm and therefore it also shows no ApoA-i binding (60).
2.55 The integrity of the nucleotide binding folds is essential for ABCA1 function
Severall mutations have been described in the NBF region of the ABCR gene, and these mutants aree defective in ATP binding (22,61). Thus ABCA1 harboring mutations in the NBFs may not generatee the ATP necessary for active transport of substrates. Although ABCA1 harboring thesee mutations would be expected to show defects in lipid efflux which may be energy requiring,, no detect in localization to the plasma membrane is expected in these mutants. A totall of 6 missense mutations have been described in the NBF region between the Walker A andd B motifs. None of these mutations have yet been characterized biochemically for their abilityy to bind ATP, their localization, their ability to bind ApoA-l, or their ability to induce lipid efflux.. Interestingly, of the six mutations in the NBFs, five occur in the first NBF but only one occurss in the second NBF. The t w o NBFs in ABCR show significantly different ATP binding and hydrolysiss properties, with the NBF1 being active as an ATPase and binding ATP, CTP, GTP and UTPP (62). The NBD1 of the CFTR protein also shows greater affinity for ATP than does NBD2
(63,64).. Studies of the MRP1 molecule have shown that ATP binding at NBD1 induces conformationall changes in the protein and enhances ADP trapping at NBD2 (65). These data suggestt that the two NBD domains in ABCA1 have differential function, with NBD1 being rate-limitingg for proper function.
2.66 Critical role of the C-terminus of ABCA1
Thee CFTR protein, another homolog of ABCA1, is usually targeted to the apical surface of cells Whenn the C-terminal portions of CFTR are disrupted, there is a redistribution of the protein to bothh the apical and basolateral surfaces of cells, as well as a reduction in its half-life (66). In additionn the C-terminus of the CFTR protein contains a PDZ binding domain that when mutated causess redistribution of the protein (67).
ABCA11 also contains a PDZ binding domain in its C-terminus (43,68) which when mutated couldd lead to protein mislocalization. The functional significance of the PDZ domain remains to bee determined, though binding of PDZ proteins has been shown to occur (68). Mutations in thee C-terminus of ABCA1 might similarly impact the normal targeting of ABCA1 to the basolateral surfacee in polarized cells and influence its stability. The five naturally occurring mutations in thee C-terminal region of ABCA1 have yet to be functionally characterized.
2.77 The nature of mutations in ABCA1 contributes t o t h e biochemical, cellular and clinicall phenotype
Itt is likely that the phenotypic heterogeneity in TD patients or in those heterozygous for ABCA11 deficiency might at least in part be accounted for by the nature of the mutation and its effectt on the protein. Until the genetic basis for TD and FHA were discovered, these patients weree diagnosed based on their phenotype. Since the genetic defect underlying both diseases hass been discovered, the assignment of disease has been mainly based on genotype with heterozygotess for ABCA1 mutations being classified as having FHA, and those homozygous forr ABCA1 mutations being categorized as having TD. Thus, in the past there was a potential forr underascertainment, with TD patients carrying mutations conferring milder phenotypes beingg classified as having FHA, and those heterozygous for very mild mutations not being recognizedd at all. The converse is also true, with those carrying severe heterozygous mutations beingg designated as having TD. Indeed, phenotypic variability of TD is now readily apparent withh some TD patients having very low HDL levels (<1%), and others having >10% compared too age and sex matched controls.
Basedd on the knowledge of the mutations in affected individuals, it is now possible to ascertain functionall deficits for a spectrum of phenotypes associated w i t h either heterozygosity or homozygosityy for mutations in ABCA1 (Figure 5). For TD, individuals with severe clinical phenotypes mayy show no ABCA1 protein at the plasma membrane or have ABCA1 at the plasma membrane thatt is completely lacking in function. This could result from two null alleles for ABCA1 preventing
normal l C A DD Incidence A B C A 11 Activity A B C A 11 Allele 1 A B C A 11 Allele 2 normal l normal l FHA A normal l >50% % HDL-C C normal l -50% % HDL-C C normal l <50% % HDL-C C TD D 10-15% % HDL-C C 10-15% % HDL-C C 1-4% % HDL-C C 1-4% % HDL-C C A255T T W590S S T929I I A937V V R1680W W P85L L R587W W \L693 3 R909X X N935S S A1046D D D1099Y Y C1477R R \E,DD 1893/94 R2081W W 2145X X 2203X X M1091T T A255T T Q597R R R1680W W 635X X N935S S N1800H H 1851X X 2203X X C-termm deletion
Figuree 5. A model showing genotype/phenotype correlations for heterozygous and homozygous mutations in
ABCA1.. HDL-C levels reported in the literature for patients harboring specific mutations in ABCA1 were compared too age and sex matched control HDL-C levels from the LRC database. Increasing CAD incidence and decreasing HDL-CC levels correlating to decreasing ABCA1 activity is modeled.
exportt of the protein to the plasma membrane, or from ABCA1 at the plasma membrane harboring mutationss in residues crucial for its function. Indeed patients harboring the mutations 635X, N935S,, N1800H, 1851X, 2203X and the large C-terminal deletion all have below 1 % of HDL-C levelss of age and sex matched controis from the LRC population.
Patientss homozygous for the mutations A255T and R1680W show HDL-C levels that are greater thann 10% of age and sex matched population controls. These patients with a less severe clinicall phenotype imply that the ABCA1 protein shows some residual activity.
Inn general, of those patients heterozygous for ABCA1 mutations, those with a mutation resulting inn completely dysfunctional protein w o u l d be expected to show HDL-C levels that are approximatelyy 50% of those for age and sex matched controls. The protein generated from thiss allele either would not be expected to reach the plasma membrane or would affect a residuee essential for its function. Most patients harboring heterozygous mutations do show HDL-CC levels that are close to 50% of those of normal age and sex matched controls. Somee patients heterozygous for ABCA1 mutations show >50% HDL-C levels compared to controlss when environmental effects such as age are controlled for. These patients would be hypothesizedd to synthesize a mutant ABCA1 protein that shows some residual activity. In this case,, mutant ABCA1 would be localized at the plasma membrane, and show a mild defect.
Thiss is indeed the case in heterozygous patients harboring mutations A255T, W590S, T929I, R1680WW and A937V who all show HDL-C levels >75% of normal age and sex matched controls. Anotherr possibility is that heterozygotes for mutations in ABCA1 show <50% of normal levels off HDL-C. This could be caused by dominant negative effects of ABCA1, as previously shown forr truncation mutations (69). Patients harboring the mutation M1091T show HDL-C levels thatt are 30% of age and sex matched controls (70). This finding suggests that ABCA1 acts as aa dimer or as part of a complex in the exertion of its function.
2.88 ABCA1 heterozygotes have increased atherosclerosis
Priorr to the cloning of the ABCA1 gene, studies of obligate heterozygotes had reported conflicting findingss on whether individuals heterozygous for mutations in ABCA1 are at an increased risk off developing CAD (71,72). This is not surprising considering that patients harboring mutations inn ABCA1 show a wide range of phenotypes, and thus misclassification of patients was likely. Cloningg of the gene and descriptions of the mutations allowed for the direct assessment of atherosclerosiss in heterozygotes. In one large study of 13 different mutations in 11 families, bothh w i t h TD and FHA, phenotypic analysis in a cohort of heterozygous individuals was undertakenn (70). The control cohort consisted of unaffected family members. A greater than three-foldd increase in CAD in adult heterozygotes compared to controls, with earlier age of onsett (by 10 years) was evident. Intriguingly, the relative cholesterol efflux levels were directly relatedd to CAD, with families with the clearest evidence for premature CAD having individuals withh the lowest cholesterol efflux.
However,, several caveats were evident in this study. Firstly, the collection of the kindreds may havee been biased by clinical sampling, since only families with the most severe phenotypes mayy have presented at clinics. Secondly, a very low number of events were seen. Thirdly, using ann endpoint of CAD as an outcome measure might have underestimated the effects. CAD is an insensitivee marker for atherosclerosis and does not address the effects of mutations on the naturall history of presymptomatic atherogenesis.
Too address these issues, a second study elucidating the association between mutations in ABCA11 and surrogate markers, namely, increased arterial wall thickness and ABCA1 mediated cholesteroll efflux was performed (73). The study group consisted of 30 individuals heterozygous forr four different missense mutations in the ABCA1 gene, C1477R, M1091T, P2150L and T929I.. Importantly, the mean intima-media thickness in carriers was higher than in controls andd carriers for mutations in ABCA1 also showed increased progression of arterial thickening thatt reached the upper limit of normal (0.8mm) much earlier (55 years) when compared to controlss (80 years, p<0.0001). Similar to the previous study, regression analysis of the data fromm this study indicated that a 50% increase in ABCA1 mediated cholesterol efflux would resultt in a 3 0 % increase in HDL-C concentrations, and that this could translate into a 35-50% reductionn in the risk of CAD.
Interestingly,, mutations in ABCA1 do affect susceptibility to atherosclerosis not only by influencingg lipids but by direct effects on the vessel wall (74). Nitric oxide is derived from the vascularr endothelium, and is a crucial antiatherogenic agent that maintains vascular homeostasis. Diminishedd NO availability represents an e-aily step in atheiuscleiusis. ABCA1 hetetozygotes showw significantly impaired endothelial function with impaired basal and stimulated nitric oxidee activity when compared to controls, indicating that ABCA1 affects vessel wall function.
3.11 Single Nucleotide Polymorphisms (SNPs) in ABCA1
Tenn coding single nucleotide polymorphisms (cSNPs) along w i t h hundreds of non-coding SNPss have been described in the ABCA1 gene (see Table 3) (41,75-80). Among the non-coding SNPss are 9 promoter and 5' UTR variants that have been analyzed for functional significance (76,78,79,81).. Most cSNPS are found distal to known functional domains (Figure 6). In addition, thee amino acid residues affected by cSNPs are less conserved compared to those affected by mutationss (50% compared to >95%, Table 4).
Tablee 3. Regulatory and coding single nucleotide polymorphisms described thus far in the ABCA1 gene, and
theirr relative frequencies in the general population. Nucleotide e -1095A/G G -477C/T T -419A/C C -320G/C C -191G/C C C69T T C117G G lnsG319 9 G378C C G1051A A T1591C C G2706A A A2715C C G2723C C G2826A A A3044G G G3911C C G5255A A C5587G G ndd = not determined Aminoo Acid promoter r promoter r promoter r promoter r promoter r 5'UTR R 5'UTR R 5'UTR R 5'UTR R R219K K V399A A V771M M T774P P K776N N V825! ! I883M M E1172D D R1587K K S1731C C Exon n
— —
--1 --1 1 1 2 2 2 2 7 7 11 1 16 6 16 6 16 6 17 7 18 8 24 4 35 5 38 8 Frequency y nd d 0.42 2 0.01 1 0.42 2 0.42 2 0.138 8 0.065 5 0.085 5 nd d 0.254 4 0.008 8 0.029 9 0 0 0 3 3 0 0 0 3 3 0.0811 - 0.541 0.0799 - 0 737 0.026 6 0.259 9 nd d
3.22 Association of ABCA1 cSNPs and regulatory SNPs w i t h HDL and atherosclerosis
Soonn after mutations in ABCA1 were found to be causative of TD and FHA, our laboratory and otherss investigated whether common variation in ABCA1 could contribute to variation in HDL-CC levels and atherosclerosis in the general population (41,75,77).
I883M M
E1172D D
Figuree 6. Distribution of cSNPs in the ABCA1 gene. Interestingly, no cSNPs have been found thus far in the
c-terminuss of the ABCA1 gene.
Tablee 4. Conservation of amino acid residues polymorphic in humans
cSNP P R219K K V399A A V771M M T774P P K776N N V825I I I883M M E1172D D R1587K K S1731C C apiens s R R V V V V T T
< <
V V I I E E R R 5 5 M.Musculus s R R V V V V S S K K V V V V E E K K S S G.Gallus s K K V V V Vs s
K K A A P P E E K K S S D.M M elanogaster r A A L L 5 5 K K M M R R E E T T C.EIegans s L L Y Y G G R R_ _
A A--V --V H H
5/100 (50%) of cSNPs are conserved to the chicken
Remarkably,, of the 10 cSNPs described, 6 are associated with potential functional effects, includingg alterations of lipid levels or measures of atherosclerosis (see Table 5). However, it is importantt to note that few of these findings have been replicated, and some results are inconsistent.. Still, it is remarkable that so many cSNPs have been associated with functional effects,, suggesting that ABCA1 may be a major atherosclerosis susceptibility iocus in the generall population. The R219K, V771M and I883M variants have been recognized as putative
Tablee 5. Clinical and biochemical consequences of ABCA1 cSNPs
Nucleotidee Ammo Acid/position Lipids s CAD/Atherosclerosis s Anti-atherogpnirr SNPs C-17G G IN5G319 9 G1051A A A3044G G promoterr no change j 5'UTRR no change J, R219KK JTG, THDL, fApoAl, TApoB, | LDL j I883MM JTG, f HDL f Pro-atherogenicc SNPs -191C/-320 0 G-19K K A-1095G G C117G G G2706A A G2868A A G3911C C G5155A A C/-477TT haplotype promoter r promoter r promoter r 5'UTR R V771M M V825I I E1172D D R1587K K noo change noo change noo change | T G G noo change noo change JJ HDL, JApoB JJ HDL
t t
t t
I I
noo changet t
t t
T T
noo changeAssociationss with lipid levels and coronary artery disease (CAD) are shown by arrows to represent the direction of association. .
anti-atherogenicc polymorphisms, associated with increased HDL-C and decreased TG levels (K2199 and M883), and increased HDL-C and ApoA-l (M771) (41,75,82). The E1172D, R1587K cSNPss have been reported to be associated with decreased HDL-C (75).
Fivee promoter SNPs are associated with increased severity of atherosclerosis, including the -191C/-320C/-477TT haplotype (76,78) as well as the G-191C and A-1096G SNPs. In contrast thee C-17G variant was associated with less atherosclerosis (78). Interestingly, these SNPs are nott associated with changes in lipid levels, suggesting that changes in ABCA1 activity can occurr without changes in steady state plasma lipid levels. The V825I, I883M and E1172D SNPs havee also been associated with increased clinical events and severity of atherosclerosis (75,77).
3.33 The R219K Variant
Thee R219K SNP has been most studied, and highlights many of the difficulties associated with thee study of SNPs in general. At least 8 studies have examined the role of this SNP in lipid homeostasiss or atherogenesis (75,77,79,81-84) (Table 6). Of these, 5 have reported positive associationn with either increased HDL-C or reduced severity of atherosclerosis. This number of positivee replications in independent studies consistently in the same direction indicates that thiss is truly an important variant with a significant athero-protective effect.
Inn 3 studies, the athero-protective effect of the K219 allele was observed only in certain circumstances,, for example in w o m e n (83), in individuals with elevated Lp(a) or with the ApoE3/E33 genotype (82), or in smokers (84). These findings indicate the functional effect of K2199 may be particularly significant in certain genetic and environmental backgrounds.
Tablee 6. Functional associations reported for the R219K variant. HDL-C C Atherosclerosis s 3rousseauu et al '.,85; Cleeett al. (83) Takagii et al t.87) Lutucutaa et al. (89) Yamakawa-Kobayashii eta (personall communication] Evanss et al. (90) nc c T T Kakkoetal.(91)) t Cenarroo et al (92) nc
ncc - no change - - not assesed
Tablee 7. Ethnic variation of SNP frequency in the ABCA1 gene.
TG G
nc c nc c
comments s
Differencess in changes of HDL-C withh age observed
Onlyy in ApoE3/E3(TG)and elevatedd Lpiaj (atherosclerosis)
ndividuals s Onlyy in women
FHH patients, K allele more protectivee in smokers R219K K V825I I I883M M E1172D D R1587K K Dutch h 0.254 4 0.081 1 0.136 6 0.026 6 0.259 9 Japanese e 11 51 0.54 4 1.60 0 nd d 0.71 1 German n 0.26 6 nd d nd d nd d nd d Finn n 0.22 2 nd d 0.112 2 0.026 6 nd d Inuit t nd d 0.200 0 0.294 4 nd d 0.561 1 Oji-Cree e nd d 0.250 0 0.690 0 nd d 0.378 8 South-Asian n nd d 0.053 3 0.145 5 nd d 0.303 3 ndd - not determined
Thee 3 reports that found no association with HDL-C or severity of atherosclerosis may have beenn confounded by population admixture, as t w o of these studies examined ethnically mixed urbann U.S. populations (77,81). The frequencies of ABCA1 cSNPs are highly divergent across populationss (Table 7), and interestingly, the 'minor' K219 allele is actually the wildtype allele in aa cohort of 327 Japanese school-aged children (Yamakawa-Koboyashi, personal communication). Linkagee disequilibrium among ABCA1 cSNPs is a further confounding variable, which to date hass not been adequately addressed. Clee et al. reported significant LD between the R219K and thee V771M, K776N, I883M, and R1587K cSNPs, but found that after carriers of these variants weree excluded, R219K remained significantly associated with degree of atherosclerosis and TG levelss (75). Further study of population-specific patterns of LD among these SNPs and haplotype analysiss should clarify these results. In addition, biochemical and functional assessment of thesee SNPs is needed for definitive clarification of their effects.
3.44 ABCA1 SNPs may be associated w i t h changes in atherosclerosis independent of changess in HDL-C levels
Off the 12 cSNPs and regulatory SNPs associated with alterations in plasma lipid levels or atherosclerosis,, 7 display altered severity of atherosclerosis without detectable changes in lipid levels.. This suggests that while ABCA1 may be an important atherosclerosis susceptibility locus,, the mechanisms by which it exerts this effect is not necessarily by altering steady state HDL-CC levels. The non-coding SNPs G-191C, C-69T, C-17G and lnsG319 and the cSNPs R219K, V 7 7 1 M ,, and V825I have all been found to be associated w i t h differences in severity of atherosclerosiss but not with changes in HDL-C levels in at least one study. The implication is that HDLL quality and composition, as determined by ABCA1-mediated efflux, may be a determinant of thee efficiency of reverse cholesterol transport, without actually affecting the levels of circulating HDL-C.. These results are consistent with efflux influencing atherogenesis without necessarily changingg lipid levels. Several studies have recently provided evidence into how this may occur. Bonee marrow transplant experiments between ABCA1 null and wildtype mice have demonstrated thatt deficiency of macrophage ABCA1 is associated with small changes in lipid levels, but significant increasess in atherosclerosis (85,86). This concept has been recapitulated by the study of ABCA1 BACC transgenic mice, in which a significant protection f r o m atherosclerosis is evident with minimall changes in HDL-C levels (87). Taken together, these studies indicate that ABCA1 can influencee atherogenesis independent of steady state HDL-C levels.
Conclusion n
Thee study of TD, a rare disorder of lipoprotein metabolism with less than 60 reported cases wondwiue,, ied to the identhication Oi the lunctionai impact oi the ABCA1 gene and protein. Variationn of this protein has now been shown to confer a risk for atherosclerosis in the general population,, and has provided an answer to a question asked for many years, namely, how lipidss are effluxed from cells in the first step of reverse cholesterol transport. The study of this raree disorder in a few has led to the identification of a validated drug target that offers hope forr raising HDL and prevention of atherosclerosis in many.
Acknowledgements s
Thiss work was supported by grants from the Canadian Institutes of Health Research and the Heartt and Stroke Foundation of British Columbia and Yukon. M.R.H. is an established investigator off the University of British Columbia and the Children and Women's Hospital and is a holder off a Canada Research Chair in human genetics. Prof. John J. Kastelein is an established investigatorr of the Netherlands Heart Foundation (2000D039)
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