Novel variants in human and monkey CETP

Biochimica et Biophysica Acta 1737 (2005) 69 – 75 http://www.elsevier.com/locate/bba

Novel variants in human and monkey CETP David B. Lloyd a, Jennifer M. Reynolds b, Melissa T. Cronan b, Suzanne P. Williams b, Maruja E. Lira a, Linda S. Wood a, Delvin R. Knight c, John F. Thompson a,* a

Department of Pharmacogenomics, Pfizer Global Research and Development, Eastern Point Road, MS8118D-3069, Groton, CT 06340, USA b Department of DNA Sequencing, Pfizer Global Research and Development, Groton, CT 06340, USA c Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT 06340, USA Received 11 July 2005; received in revised form 2 September 2005; accepted 4 September 2005 Available online 3 October 2005

Abstract Variation in CETP has been shown to play an important role in HDL-C levels and cardiovascular disease. To better characterize this variation, the promoter and exonic DNA for CETP was resequenced in 189 individuals with extreme HDL-C or age. Two novel amino acid variants were found in humans (V-12D and Y361C) and an additional variant (R137W) not previously studied in vitro were expressed. D-12 was not secreted and had no detectable activity in cells. C361 and W137 retained near normal amounts of cholesteryl ester transfer activity when purified but were less well secreted than wild type. Torcetrapib, a CETP inhibitor in clinical development with atorvastatin, was found to have a uniform effect on inhibition of wild type CETP versus W137 or C361. In addition, the level of variation in other species was assessed by resequencing DNA from nine cynomolgus monkeys. Numerous intronic and silent SNPs were found as well as two variable amino acids. The amino acid altering SNPs were genotyped in 29 monkeys and not found to be significantly associated with HDL-C levels. Three SNPs found in monkeys were identical to three found in humans with these SNPs all occurring at CpG sites. D 2005 Elsevier B.V. All rights reserved. Keywords: HDL-C; Pharmacogenomics; SNP; CpG

1. Introduction The cholesteryl ester transfer protein (CETP) gene was originally identified as playing a major role in modulating high-density lipoprotein cholesterol (HDL-C) in humans when individuals with a null phenotype were found to have high HDL-C levels [1]. Its role in lipid transport led to studies of its impact on cardiovascular disease. While patients with CETP deficiency were initially thought to display resistance to cardiovascular disease [2], this conclusion was later questioned among studies of those with partial [3] or complete [4] loss of CETP, setting off a debate about whether lowering CETP activity would be beneficial to health or not. However, more recent work in some of the same Japanese populations did not find a negative effect of CETP deficiency [5,6]. Furthermore, a study of Ashkenazi Jewish centenarians found a protective effect associated with a variant with lower CETP activity [7]

* Corresponding author. Tel.: +1 860 441 5139; fax: +1 860 441 0436. E-mail address: [email protected] (J.F. Thompson). 1388-1981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2005.09.007

and prospective studies generally have found variants associated with lower CETP and higher HDL-C also are associated with a reduced risk of cardiovascular disease [8]. Recent reviews of these issues are numerous [9– 11]. CETP polymorphisms have been identified and characterized in various populations. These include dozens of single nucleotide polymorphisms (SNPs) as well as other polymorphisms like variable length repeats and insertion/deletions, many of which can alter amino acid sequence, splicing, or transcription [12]. The most frequently studied SNP, TaqIB (rs708272), has been regularly found to be associated with CETP and HDL-C levels. In prospective studies, the B2 allele that is associated with lower CETP and higher HDL-C is also associated with protection from cardiovascular disease [8]. This SNP is in high linkage disequilibrium with many other SNPs in the 5V half of the gene and promoter. In addition, several amino acid variants have also been identified that have, to varying extents, been found to be associated with CETP and HDL-C levels [13]. Sequencing of the CETP gene has been carried out on a variety of individuals but extensive sequencing of more than a

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D.B. Lloyd et al. / Biochimica et Biophysica Acta 1737 (2005) 69 – 75

Table 1 SNPs identified by sequencing dbSNP

May 2004 assembly

Change

rs4783961

55552395 55552491 55552539 55552623 55552734 55552735 55552737 55552763 55552961 55553034 55553109 55553238 55553315 55553328 55553409 55553436 55553458 55553560 55553568 55553605 55553659 55554586 55560962 55561162 55561224 55561229 55561347 55561478 55561481 55562390 55562393 55562664 55562772 55562802 55562864 55562980 55563051 55563383 55563573 55564693 55564588 55564854 55564947 55564952 55566484 55569385 55569437 55569700 55572577 55572585 55572592 55572964 55573045 55573046 55573508 55573593 55573628 55574667 55574820 55574975 55575053 55575163

G 971A/Prom C 875T/Prom C 827T/Prom G 743A/Prom C 632A/Prom C 631A/Prom C 629A/Prom A 603T/Prom A 405G/Prom C 332T/Prom C 257T/Prom C 128T/Prom G 51A/Prom G 38A/Prom T + 44/Ex1 C + 71/Ex1 C + 93A/Ex1 C + 50A/In1 G + 58A/In1 C + 95A/In1 T + 149C/In1 A + 36G/In2 T + 29C/In3 G + 68A/In4 G + 130T/In4 G + 135C/In4 C + 21T/Ex5 T + 64C/In5 T + 67C/In5 G + 976A/In5 C + 979T/In5 C + 149T/In6 C + 39T/Ex7 C + 8T/In 7 G + 70A/In7 C + 186A/In7 C + 257T/In7 C + 589T/In7 G + 77A/In8 T + 1197C/In8 T + 6C/Ex9 C + 111T/Ex9 T + 24G/In9 G + 29A/In9 A + 1561G/In9 C + 2820T/In10 C + 2872T/In10 A + 197G/Ex11 G + 7A/Ex12 C + 15G/Ex12 G + 22C/Ex12 A + 326G/In12 G + 407T/In12 C + 408T/In12 C + 415T/In13 A + 16G/Ex14 G + 51A/Ex14 C + 1017T/In14 G + 82AEx15 A + 151G/In15 G + 49A/Ex16 G + 159A/Ex16

rs4783962

rs1800776 rs1800775

rs5884 rs2304475 rs3816117

rs891141

rs891142 rs891143 rs7205804 rs12708972 rs5885 rs1532625 rs1532624 rs12708974 rs12720872 rs12720873 rs9930761 rs5883 rs11076176 rs289714

rs891144

rs7192120 rs5880 rs7195984 rs7196174 rs1800774 rs5882 rs5886 rs1800777 rs289741 rs5887 rs1801706

Amino acid

Val-12Asp Ala-3Gly Thr5Thr

Arg137Trp

Ala195Ala

His235His Phe270Phe

Tyr361Cys Val368Met Thr370Thr Ala373Pro

Ile405Val Val416Val Arg451Gln Val469Met N=

Elderly Cauc

Low HDL Cauc

41 6 20 0 0 12 49 0 0 0 0 0 0 0 0

48 4 27 0 1 4 37 0 1 0 0 0 0 0 0

0

0

0 10 49

1 9 37

1 0

0 0

38 1 2 0 38 0 38 13

28 1 0 0 28 1 28 13

0 10 0 11 14 17

1 5 0 3 29 29

2 2 0 1 0 2 0

0 0 0 1 0 8 1

30 2 35 0 1 2 33 0 22 44

41 2 20 0 0 6 19 0 15 52

Low HDL Af Am

High HDL Af AM

9 5 0 2 59 1 0 0 1 0 0 7 0 7 9 3

8 6 0 0 74 0 0 1 0 1 1 14 1 14 4 0

10 1 0 12

9 2 2 10

0 9 9

4 6 6

15

10

2 17

1 16

15

10

4 0 5 26 44 0

16 2 20 22 59 1

10 1 0 6 3

8 0 1 5 0

4 31

3 15

46 4 2 2 48 0 8 43

33 4 2 0 33 1 25 50

D.B. Lloyd et al. / Biochimica et Biophysica Acta 1737 (2005) 69 – 75

few individuals has been done only in a small number of studies. Some studies have focused on the coding sequence while others have examined other parts of the gene. Those looking at the cDNA include Cargill et al. [14] who included CETP among the 106 genes they assayed in 57 ethnically diverse individuals; Thompson et al. [15] who sequenced the exons and promoter from 90 individuals from multiple ethnic groups; Morabia et al. [16] who resequenced 95 Caucasian individuals for the exons and flanking introns in 11 genes, including CETP; and Chasman et al. [17] who resequenced several genes, including CETP, in 32 to 96 individuals from multiple ethnic groups. Those examining other parts of the gene include Klerkx et al. [18] who resequenced 3000 bp in the promoter and 1000 bp of the gene in ten Europeans, Lu et al. [19] who examined 3300 bp of the promoter in 60 Japanese, and Stanssons et al. [20] who examined 96 Caucasians for about 5000 bases spanning exons 9 to 11. All of these studies focused on healthy individuals who would be expected to have a normal degree of genetic variation. To identify polymorphisms that might be more likely to have causal effects, promoter and exonic DNA from 189 individuals with extreme HDL-C or age was resequenced. In addition, the degree of CETP variation in other species was assessed by resequencing the CETP exons of nine cynomolgus monkeys. 2. Materials and methods 2.1. DNA samples The Caucasian individuals with low HDL-C whose DNA was resequenced were selected from the Atorvastatin Comparative Cholesterol Efficacy and Safety Study (ACCESS) clinical trial [21]. All of the other human DNA samples that were sequenced were obtained from commercial sources with most coming from Genomics Collaborative Inc. Among those individuals selected for resequencing, the cutoff was 37 mg/dl for African Americans with low HDL-C and below and 74 mg/dl and above for African Americans with high HDL-C. After resequencing, the remainder of the population was also genotyped and the cutoffs for the larger population were 45 mg/ml and 60 mg/ml HDL-C, respectively. In all trials, whole blood from subjects participating was obtained with appropriate institutional review and appropriate informed consent documentation that defined the study design and provided an assessment of the risks and benefits associated with study participation. Whole blood from cynomolgus monkeys (Macaca fascicularis) was obtained as aliquots from samples being taken for other studies. DNA preparation was as described previously [15]. Sequencing of the DNA from Caucasian samples was carried out at Genaissance and sequencing of African American and cynomolgus samples was carried out at Pfizer using an ABI 3730xl DNA Analyzer. TaqMan assays obtained from ABI were used for all human genotyping as described previously [8]. Genotypes for monkeys were obtained by direct sequencing. Experimental procedures in this study complied with the guidelines of the Animal Care and Use Committee of Pfizer Central Research and with the Guide for the Care and Use of Laboratory Animals (DHHS Publication No. (NIH) 86-23, revised 1985, Animal Resources Program, DRR/NIH, Bethesda, MD 20205).

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2.2. Expression constructs CETP W137 and C361 missense variants were generated by overlapping PCR and expressed in pSecTag2 from Invitrogen as described previously [13]. C-terminal tags were eliminated by double stop codons at the C terminus of CETP. Internal variants had the vector Igk signal peptide and a His tag that allowed purification. Signal peptide proteins V-12 and D-12 were created by replacing the Igk leader and tag sequences in the expression vector with synthetic linkers matching the leader sequence at the amino terminus of wildtype CETP.

2.3. Cell culture/Western blotting HEK293S cells were cultured and transfected as described [13]. Transfected cells were harvested in physiologically buffered saline (PBS) and whole-cell lysates prepared in lysis buffer (50 mM Tris HCl (pH 8.0), 150 mM NaCl, 24 mM MgCl2, 0.5 mM Na-o-vanadate, 10% glycerol, 0.1% NP-40). Protein concentrations were estimated using a Bradford assay. Ten micrograms of whole cell lysate and 10 Al of concentrated serum-free media were electrophoresed on a 4 – 12% bis-acrylamide NuPage gel (Invitrogen) in 1 MES buffer, and transferred to nitrocellulose using a semi-dry transfer cell (BioRad). Membranes were probed with a mouse anti-V5 primary antibody (Invitrogen) followed by a goat anti-mouse peroxidase conjugated secondary antibody, and developed using Super Signal West Pico chemiluminescent substrate (Pierce). Blots were analyzed and quantitated on a GeneGnome chemiluminescence detection system (Syngene).

2.4. CETP assays Cholesteryl ester transfer activity was measured in concentrated media from transfected HEK293S cells using a fluorescence-based assay as described [13]. Reactions were read (excitation 485 nm, emission 520 nm) at To and again after a 1 h incubation at 37 -C in a Victor 2 plate reader (Perkin-Elmer). Data were plotted as percent DMSO control and IC50 values were estimated by linear regression using LabStats.

3. Results Because CETP variation has already been associated with phenotypic consequences, additional variation might be present in extreme populations so all exons and some of the adjacent promoter, 3VUTR, and varying amounts of adjacent intronic sequence were resequenced in 145 individuals with extreme HDL-C levels. Additionally, because one CETP SNP was previously identified as playing a role in longevity [7], resequencing of DNA from 44 elderly Caucasians (>90 years old) was also carried out. A listing of all SNPs identified is shown in Table 1. Each of the subsets of individuals, 44 elderly Caucasians, 52 Caucasians with HDL-C30 mg/dl, 43 African Americans with HDL-C37 mg/dl, and 50 African Americans with HDL-C74 mg/dl, is listed in its own column. When available, dbSNP identifiers are provided. Given the depth of sequencing already carried out with the CETP gene, it is not surprising that all of the most common SNPs that we identified had been previously found

Notes to Table 1: All SNPs identified by sequencing are listed. If available, dbSNP reference numbers are provided in the first column. The location of the SNP on chromosome 16 in the May 2004 genome assembly is in the second column and the location using gene-specific nomenclature [15] is provided in the third column. If the SNP was exonic, its impact on amino acid sequence is listed in column 4 using the mature peptide numbering. The percent allele frequency for each population (elderly Caucasians, low HDL-C Caucasians, low HDL-C African Americans, and high HDL-C African Americans) is listed in the final four columns. If an entry is blank, the sequence was not examined in enough individuals to determine frequency.

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Fig. 1. Alignment of CETP sequences for humans and monkeys. Protein sequences for humans, chimpanzees, and cynomolgus monkeys is shown and aligned. The chimpanzee sequence is used as a reference for the other two with amino acids numbered using the mature protein sequence. Shaded amino acids vary between the species and amino acids in reverse print are variable within a species with the variant not matching the chimpanzee sequence shown. At position 438, neither human amino acid allele matches chimpanzee so both are shown.

in other populations. Indeed, 30 of the 46 SNPs we found in multiple individuals were already deposited in dbSNP. Only 4 of the 16 SNPs we found in just a single individual were already in dbSNP. The two novel amino acid variants, V12D and Y361C (numbering based on mature peptide), were initially identified among African Americans. All other variants had been noted in multiple previous studies except for R137W, which had been identified by one other group [17] at low frequency (0.2%). The common allele for each of these protein variants was identical to the amino acid found in cynomolgus monkeys (Fig. 1). Because these amino acid variants had not been studied previously, they were genotyped in a large, multi-ethnic population in which HDL-C levels were available [21] as well as a smaller, African American population chosen based on extreme HDL-C levels (45 mg/dl or 60 mg/dl). As shown in Table 2, C361 is the rarest of these variants, being found only once in 2994 individuals. D-12 is also quite rare, found once among 2463 Caucasians and three times among the high HDL-C African Americans. The variants at positions 12 and 361 are too rare to draw conclusions about any association with HDL-C although D-12Vs higher prevalence in the high HDL-C group is certainly suggestive. R137W is the most common variant characterized here with an allele frequency of about 3% among African Americans but

found in only 2 individuals among 2463 Caucasians. African American individuals heterozygous at position 137 trend toward higher HDL-C. When the African American population chosen based on high HDL-C is examined, there is a significant difference in HDL-C between individuals homozygous for the common allele versus heterozygotes (Table 3, P < 0.001). There are fewer heterozygotes in the low HDL-C population and no Table 2 Frequency of SNPs across ethnic groups Position

Af. Am.

Asian

Cauc.

Af. Am. High HDL

Af. Am. Low HDL

N= V-12D R137W Y361C Age % Male HDL-C % No alcohol % 1 – 10 Drink/week % >10 drinks/week

166 0/0 8/1 0/0 58.7 53.0 50.5 75.9 21.7 2.4

36 0/0 0/0 0/0 60.4 55.6 52.9 80.6 11.1 8.3

2413 1/0 2/0 0/0 61.6 60.8 47.3 60.9 32.5 6.6

123 3/0 7/0 0/0 64.4 30.1 75.5 76.4 21.1 2.4

119 0/0 2/0 1/0 57.7 64.7 37.5 76.5 21.8 1.7

For African Americans (Af. Am.), Asians, and Caucasians (Cauc.), the number of heterozygotes/homozygotes for each of the three amino acid variants was determined using TaqMan in the ACCESS population [21] and the high and low HDL-C African American populations. The number of individuals genotyped is listed in the second row.

D.B. Lloyd et al. / Biochimica et Biophysica Acta 1737 (2005) 69 – 75 Table 3 W137 Association with HDL-C

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Table 4 Exonic SNPs in Monkey CETP

Population

Rare alleles

N

HDL-C

SD

P

Exonic SNPs

Frequency

Amino acid

High HDL

0 1 0 1 0 1/2

116 7 117 2 138 9

73.8 102.9 37.4 43.5 50.7 58.7

15.7 63.8 7.6 0.7 13.3 23.6

<0.001

A + 39G/Ex9 C + 57T/Ex9 T + 152C/Ex9 G + 59A/Ex11 G + 125A/Ex11

0.22 0.22 0 0.31 0.03

P246P P252P M284T R330K R352H

Low HDL ACCESS

ns ns

For each of the three African American populations, the mean HDL-C and standard deviation for it are listed. The P value for the difference between individuals homozygous for the common allele and those heterozygous is listed in the final column.

Variations found in the exonic regions of cynomolgus monkeys are listed (n = 29 for exon 11, n = 9 for exon 9). The frequency of the SNPs is shown. One SNP was not variable within the animals we sequenced but differed from the reference sequence [27].

conclusions can be drawn. Similarly, the difference in HDL-C among African American individuals is not significant in the ACCESS population but trends in the same direction as in the high HDL-C population. The single individual (male) homozygous for the variant has higher than average HDL-C (61 mg/ dl) but not extraordinarily so. To better understand these in vivo results, further studies were carried out in vitro. Each of the three variants was cloned and then expressed in HEK293 cells. Media from transfected cells was collected and concentrated. Whole cells were also collected for generation of lysates for examination of intracellular protein. Media and whole cell lysates were examined via Western blotting from duplicate transient transfections. As shown in Fig. 2, wild-type, W137, and C361 were all found at similar levels intracellularly, but D-12 was barely detectable and a smaller size than the other isoforms. Furthermore, none of the D-12 protein could be detected in the media, suggesting that its processing was prevented by the charged residue in its signal sequence. W137 and C361 could both be detected in the media, although at lower levels than wild type. Protein secreted into the media and remaining in cell lysates was tested for cholesteryl ester transfer activity using artificial substrates [13]. With D-12, no activity could be detected either in the media or from cell lysates. In contrast, both W137 and C361 activity could be detected. Based on Western blotting intensity

and CETP activity at 1 h, both C361 and W137 were estimated to have approximately the same or possibly slightly higher specific activity than wild-type protein (data not shown). W137 is sufficiently common that it would be seen among typical clinical populations and the variability in HDL-C among individuals bearing it means that some of these individuals would have low HDL-C. Thus, it is of interest to see if torcetrapib, the CETP inhibitor currently in clinical trials with atorvastatin [22], would inhibit W137 to the same extent as R137. Even though C361 is extremely rare, it was tested also. As shown in Fig. 3, torcetrapib inhibits all isoforms to the same extent, indicating that these target polymorphisms should not affect response to the drug among patients taking it. To determine the variability of the CETP protein in other species, DNA from nine cynomolgus monkeys was resequenced across all exons. Within exons, four sites were found to be polymorphic with two SNPs being silent and two causing amino acid changes. A fifth site was uniform in the animals that we sequenced but differed from the reference sequence, M86343 (Table 4). Because the two amino acid variants detected in cynomolgus monkeys were in exon 11, both could be analyzed by resequencing a single PCR product. DNA from 29 monkeys was sequenced with 2 found to be heterozygous R352H. Twelve monkeys were heterozygous R330K while 14 were homozygous R330 and 3 were homozygous K330. HDL-C levels among the monkeys were similar (45.0 mg/dl T 15.4) to humans. Neither

Fig. 2. Expression of variants in media and whole cell lysates. CETP was expressed as described. Media was concentrated and cells were lysed and the resultant proteins run on gels for Western blotting. All CETP variants included a HisV5 tag at the amino terminus except D-12 and V-12 that contained the natural CETP leader sequence. Duplicate transfections are included for W137 and C361. The two panels are identical except for the exposure time that was extended in the lower panel in order to detect any faint bands for D-12.

Fig. 3. Inhibition of CETP by torcetrapib. Wild type CETP, W137, and C361 were purified from media after transient transfection of HEK293S cells. An in vitro fluorescence-quenching assay was run for each variant for 1 h at 37- and relative activity plotted as a function of torcetrapib concentration.

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cSNP was found to be significantly associated with HDL-C levels but this may be due to the small number of individuals tested. In addition to these cSNPs, 38 intronic SNPs, 3 silent cSNPs, and 2 deletions were also found. Each of these was unique to cynomolgus monkeys except for the equivalents to the human SNPs rs12720872, rs12691052, and rs289741. 4. Discussion CETP has been the subject of much study with respect to both genetic variation and disease. The divergent conclusions on its role highlight the difficulties in trying to understand a complex, high mortality condition like cardiovascular disease. The environmental variation superimposed on the genetic combinations found among different ethnic groups makes comparisons across studies challenging. As a more detailed understanding of CETP’s genetic variation and the role of different polymorphisms in affecting activity and expression has emerged, some questions have been resolved but the apparent disconnect between HDL-C levels and cardiovascular disease has been, at times, puzzling. Part of the difficulty clearly arises from clinical studies that were either too small or not run prospectively. When large, prospective studies are analyzed [12,23], the association of lower CETP activity with reduced cardiovascular risk is apparent. This effect is generally assumed to be caused by the beneficial effects on HDL-C but other factors could also contribute. The I405V CETP amino acid variant that is associated with longevity among Ashkenazi Jewish centenarians is not one that is noted for an extreme phenotype relative to other variations. Other variants with no activity or altered expression have been found with much less pronounced effects on longevity, suggesting that the actual causal variation might be in linkage disequilibrium with I405V. Furthermore, efforts to replicate this finding in a population of Italian centenarians were unsuccessful [24]. For these reasons, the CETP gene from elderly individuals was resequenced, including at least one Ashkenazi Jewish centenarian. There were no novel SNPs found in that individual and the elderly group as a whole had only a handful of rare SNPs that were unique to that group, a number not significantly different than the other groups sequenced. Thus, coding variation in CETP does not appear to play a marked role in longevity for most individuals but variation that could change splicing or expression patterns, possibly in a tissuespecific manner, could still be responsible. With increasing interest in CETP as a therapeutic target for raising HDL-C, it is important to understand its natural variation and whether that variation could cause issues when individuals receive a drug inhibiting it. Other common variants have already been examined for whether they might respond differentially to an inhibitor but the variants described here had not. Similar to previous work [13], the variants we examined in CETP do not affect its ability to be inhibited by torcetrapib, a finding consistent with the relatively uniform clinical response thus far observed. The amino acid variants found in cynomolgus monkeys are unique to that species and, like most variants found in human

CETP, do not appear to have a large impact on function. The small number of monkeys available relative to the size of human populations studied makes any conclusions on the functional impact of the SNPs preliminary. With sequence available from only nine monkeys, it is not possible to do a thorough comparison of amino-acid changing SNPs but the frequency we found with even this small sampling is similar to what has been observed in humans. In both species, CETP has one very common SNP (I405V in humans, >30% frequency; R330K in monkey, 31% frequency) and some uncommon SNPs (A373P, R451Q in humans; 1 –5%, R352H in monkeys, 3%). Previous work [25] has shown that most SNPs are not shared among species with the prime exceptions among primates being those that occur at CpG sites. Since these sites are frequently methylated, they are much more prone to de novo mutation and hence independent occurrence. Indeed, the three SNPs that are shared between humans and cynomolgus monkeys all occur at CpG sites, further supporting earlier conclusions about SNPs that are present in multiple species. Furthermore, each of the alleles at the human I405V variant matches a different monkey species but this variation also occurs at a CpG site. These additional sequences bring the total number of single amino acid variants reported in the literature for CETP to fifteen, V-12D, A-3G, R137W, L151P, R282C, L296Q, G314S, Y361C, V368M, A373P, I405V, V438M, D442G, R451Q, V469M [11,17,26]. Only two of these, V-12D and L151P, appear to completely knock out activity. More drastic changes in sequence induced by splicing and frameshifts bring about a complete loss of activity, but CETP seems fairly resilient with respect to small changes in sequence. The fact that these variants are also similar with respect to inhibition by torcetrapib suggests that inhibiting CETP will be a predictable target for generating increases in HDL-C. While the number of individuals examined here is too small to draw conclusions about either longevity or cardiovascular disease, these data further support earlier conclusions about the important role that CETP plays in affecting human HDL-C levels and provide additional SNPs that can be examined across different populations. With this depth of sequencing, no additional common coding or proximal promoter SNPs will be found that are shared across most populations. Acknowledgement We wish to acknowledge the technical assistance of David M. Flynn in Cardiovascular and Metabolic Diseases, Pfizer, Groton, for supplying the blood samples and HDL-C levels from the cynomolgus monkeys used in this study and the assistance of John Hearst and Sohel Talib in DNA preparation. References [1] M.L. Brown, A. Inazu, C.B. Hesler, L.B. Agellon, C. Mann, M.E. Whitlock, Y.L. Marcel, R.W. Milne, J. Koizumi, H. Mabuchi, R. Takeda, A.R. Tall, Molecular basis of lipid transfer deficiency in a family with increased high-density lipoproteins, Nature 342 (1989) 448 – 451.

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