Comprehensive analysis of genomic variation in the LPA...

Manuscript #: CIRCULATIONAHA/2009/894626

Comprehensive analysis of genomic variation in the LPA locus and its relationship to plasma Lipoprotein(a) in South Asians, Chinese

and European Caucasians

Lanktree et al. Multiethnic Genomic Study of LPA

Matthew B. Lanktree, BSc; Sonia S. Anand, MD, PhD, FRCPC; Salim Yusuf, DPhil, FRCPC; Robert A. Hegele, MD, FRCPC, FACP; on behalf of the SHARE Investigators

From the Departments of Medicine and Biochemistry (M.B.L., R.A.H), Robarts Research Institute and Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada; Population Health Research Institute, Hamilton Health Sciences (S.S.A., S.Y.) and Departments of Medicine and Clinical Epidemiology (S.S.A., S.Y.), McMaster University, Hamilton, Ontario, Canada.

Correspondence to: Robert A. Hegele, MD, FRCPC, FACP Blackburn Cardiovascular Genetics Laboratory Robarts Research Institute University of Western Ontario London, Ontario, Canada, N6A 5K8 Tel: 519-931-5271 Fax: 519-931-5218; Email: [email protected] Journal Subject Codes: [8] Epidemiology; [109] Clinical genetics; [135] Risk Factors; [89] Genetics of cardiovascular disease; [146] Genomics; [90] Lipid and lipoprotein metabolism; [134] Pathophysiology.

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Abstract

Background - Functional copy number variation in the apolipoprotein(a) gene (LPA) underlies a

variable number of protein “kringle” domains repeated in tandem in the lipoprotein(a) [Lp(a)]

particle. Genomic analysis of LPA, including both single nucleotide polymorphisms (SNPs) and

kringle IV type 2 (KIV-2) copy number, has yet to be performed.

Methods and Results - First, we genotyped 49 SNPs within 100 kb of LPA in a multiethnic

sample comprised of South Asians (n=330), Chinese (n=304), and European Caucasians

(n=272). Second, using quantitative PCR, we estimated the KIV-2 copy number in each sample.

European Caucasians had the lowest KIV-2 copy number, but displayed the strongest correlation

between KIV-2 copy number and plasma Lp(a) concentration (rs =-0.31, P=4.2 x 10-7). SNP

rs10455872, only prevalent in European Caucasians, was strongly associated with both plasma

Lp(a) concentration (P=4.2 x 10-29) and KIV-2 (P=7.2 x 10-5). LPA SNP rs6415084, within the

same haplotype block as the KIV-2 variation, was significantly associated with both Lp(a)

concentration and KIV-2 copy number, in the same direction in all three ethnicities (Lp(a): P=5.3

x 10-7; KIV-2: P=2.6 x 10-4). SNPs and KIV-2 copy number together explain a larger proportion

of variation in plasma Lp(a) concentrations in European Caucasians (36%) than in Chinese

(27%) or South Asians (21%).

Conclusions – LPA SNPs are in linkage disequilibrium with KIV-2 copy number, but KIV-2

copy number explains an increment in plasma Lp(a) variation over SNPs alone. Thus, both

SNPs and KIV-2 should be included in future genetic epidemiology studies of Lp(a).

Keywords: genetics, apolipoproteins, atherosclerosis, risk factors, lipoproteins

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An intricate system of lipoproteins is required for the transport of lipids through the

aqueous plasma milieu to ensure lipid delivery for a wide variety of metabolic functions.1

Contrary to most lipoproteins, the physiological function of lipoprotein(a) [Lp(a)], and the

consequence of an extreme concentration of Lp(a), remains incompletely understood. Increased

cardiovascular disease (CVD) risk has been associated with elevated plasma Lp(a)

concentration,2-4 though somewhat inconsistently between populations.5, 6 Although the precise

pathogenic mechanisms underlying the association remain unknown, both atherogenic and

prothrombotic pathways have been suggested, possibly involving inflammation,7 endothelial

dysfunction,8 interaction with platelet function9 or plasminogen activation.10

Lp(a) is composed of a single large apolipoprotein(a) [apo(a)] connected via a disulfide

bond to the apolipoprotein (apo) B moiety of a cholesterol-rich low-density lipoprotein (LDL)

cholesterol particle.11 Apo(a) is extremely heterogeneous in size, which is the result of a

genetically-determined functional copy number variation (CNV) within the LPA gene (OMIM:

15220) on chromosome 6q27.11 Each additional genomic copy of the tandem repeat region

contains two exons which code for a protein domain called kringle IV type 2 (KIV-2).11 The

mature apo(a) particle has a KIV-2 number range of 5 to >50 domains across human populations

with larger protein isoforms being compromised with respect to protein folding, transport, and

secretion.11 Association between plasma Lp(a) concentration and KIV-2 repeat number has been

identified.12 Null alleles, in which the LPA gene contains either a truncation mutation or an

extremely large number of KIV-2 repeats - and thus does not produce a secreted protein - have

been identified, but are uncommon.13 Beyond its effect on the concentration of plasma Lp(a),

other functions of the KIV-2 repeat are unknown, but associations between low KIV-2 copy

number and CVD have been consistently reported.14-16 Recently, a large Mendelian

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randomization study reported association between low KIV-2 copy number and myocardial

infarction in a sample of European Caucasians, but did not include single nucleotide

polymorphism (SNP) genotypes.17

To date, KIV-2 copy number has generally been identified either at the biochemical level

by examining the protein size using electrophoresis and immunoblotting18, 19 or at the genomic

level using pulse-field gel electrophoresis with Southern-blotting of unamplified genomic

DNA.19 Due to the requirement of either large amounts of fresh plasma or genomic DNA,

respectively for these methods, candidate-gene and genome-wide association studies of Lp(a)

concentration have not included the apo(a) KIV-2 copy number as a covariate or independent

variable, nor has there been any opportunity to study association or linkage disequilibrium

between LPA SNPs and KIV-2 copy number. To address these issues, we genotyped a

multiethnic sample for both SNPs, using a targeted CVD SNP microarray, and for KIV-2 copy

number, using a quantitative PCR method.20

Methods

Subjects

The Study of Health Assessment and Risk in Ethnic Groups (SHARE) cohort was

collected for a prospective population-based assessment of traditional and novel CVD risk

factors in Canada.21 As previously described, all participants resided in Hamilton, Toronto or

Edmonton for at least five years and were classified as South Asian (n = 330) if their ancestors

originated from India, Pakistan, Sri Lanka, or Bangledesh; Chinese (n = 304) if their ancestors

originated from China, Taiwan, or Hong Kong; and European (n = 272) if their ancestors

originated from Europe.21, 22 Description of participant ascertainment, selection, and exclusion,

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as well as biochemical and anthropometric measurement techniques, have been previously

described.21 For Lp(a) specifically, quantitative measurements were made with automated

immunoprecipitation and turbidimetric detection using the Incstar Lp(a) test kit (Stillwater, MN,

USA) on the Ciba-Corning 550 EXPRESS (Oberlin, OH, USA).21 Baseline anthropometric

measures are presented in Table 1. The study was approved by the ethics review boards of both

McMaster University and the University of Western Ontario. All participants provided informed

consent for genetic analysis. The authors had full access to and take full responsibility for the

integrity of the data. All authors have read and agree to the manuscript as written.

LPA SNP Genotyping

Genomic DNA was extracted from leukocytes as previously described.23 SNP genotypes

were produced using the Illumina Human CVD beadchip,24 scanned on the Illumina BeadStation

500G (San Diego, CA, USA; www.illumina.com), at the Centre for Applied Genomics (Hospital

for Sick Children, Toronto, Ontario, Canada; www.tcag.ca). The Illumina CVD beadchip

contains ~50000 SNPs, densely mapping ~2000 genes with potential roles in cardiovascular,

metabolic and inflammatory phenotypes.24 Genotyping and quality control were performed in the

Illumina BeadStudio Genotyping Module v3.2. Eight replicate chip assays were performed, with

an average of 5.7 non-conforming calls per replicate, giving an overall accuracy of >99.99%.

Sixteen individuals (1.8%) were excluded due to SNP call rates <95% and 1151 (2.3%) SNPs

were excluded due to genotype call rates <95%. SNPs that were not in Hardy-Weinberg

equilibrium (P < 0.0001) or with a minor allele frequency (MAF) < 0.01 were excluded, leaving

35303, 31751, and 35018 SNPs genotyped in South Asian, Chinese and European Caucasian

samples respectively.

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LPA KIV-2 Copy Number Genotyping

The number of KIV-2 repeats was determined using a previously-described quantitative

PCR-based approach.20 Briefly, a multiplexed quantitative PCR was performed containing

alternatively-labeled fluorescent probes for an exon in the KIV-2 domain and in a control region

of the genome. The relative difference in fluorescence between probes, as measured by the

difference in PCR cycle threshold ( CT), can be used to estimate the kringle-repeat number.

Taqman RNase P (RNAP) control reagent was used as the single copy reference gene (Part

Number 4316844) and the quantitative PCR was performed in the Applied Biosystems 7900HT

Fast Real-Time PCR system (Foster City, CA, USA; www.appliedbiosystems.com). To ensure

accuracy of results, all reactions were performed in triplicate; primers and fluorescent-labeled

probes were designed for exonic sequence containing no reported SNPs, and the experiment was

performed for probes targeted to exons 4 and 5 of LPA, each of which is found once within every

KIV-2 repeat at the genomic level.20 Small modifications to the original protocol20 included a

reduction of total reaction volume to 10 uL, a switch to Applied Biosystems Taqman Genotyping

Master Mix (Part Number 4371357), and the use of a fixed cycle threshold of 0.2. As expected, a

correlation was observed between the cycle threshold for the probe for exon 4 ( CT4) and the

cycle threshold for the probe for exon 5 ( CT5) (South Asian: rs = 0.69, P < 1 x 10-31; Chinese: rs

= 0.79, P < 1 x 10-31 ; European Caucasian: rs = 0.69, P < 1 x 10-31). A discrepancy between

CT4 and CT5, defined as a difference between CT4 and CT5 greater than 2 standard

deviations from the mean difference, was identified in 64 individuals (7%), all of which were

resolved when retested. A collection of calibration samples could theoretically be used to

approximate the number of KIV-2 protein domains, however no distinct clusters of CT values

were identified and we considered that rounding to estimated KIV-2 protein domain number

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might result in loss of signal. Thus the KIV-2 copy number determination cited henceforth refers

to the mean of CT4 and CT5, as previously described.20

Statistical Analysis

To obtain a normal distribution of plasma Lp(a) concentration, the square root of the

plasma Lp(a) concentration was calculated.17 KIV-2 copy number and square root transformed

Lp(a) concentration were compared between ethnicities using the Student’s t test. Correlation

between CT4 and CT5, and between KIV-2 copy number and Lp(a) concentration was

performed using the non-parametric Spearman’s rank correlation coefficient as implemented in

SAS v9.1 (SAS Institute, Cary, NC, USA). Linear regression was used to test the association

between the number of minor alleles at a SNP and both plasma Lp(a) concentration and KIV-2

copy number, as implemented in PLINK, in each ethnicity independently.25 Age and sex were

included as covariates for all tests of association with plasma Lp(a) concentration. Population

structure in the SHARE cohort was assessed using principal components in the program

EIGENSTRAT,26 and pairwise identity-by-state followed by multi-dimensional scaling as

implemented in PLINK.25 Three distinct homogeneous clusters were observed using either

analytical technique. All of the study participants clustered with their self-reported ethnicity with

the exception of two (0.2%) individuals who were excluded from further analysis.22 Within each

of the ethnic subgroups of the SHARE cohort, further correction for population stratification was

not required as the genomic control inflation factor was 1.00 when using all SNPs found on the

CVD beadchip and plasma Lp(a) concentration as the trait, including age and sex as covariates.

Meta-analysis of the effect-size in the independent ethnicities was performed using the METAL

software package (www.sph.umich.edu/csg/abecasis/metal/). Analysis was performed using all

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SNPs that passed quality control, however as virtually all previous genetic associations with

Lp(a) have been reported to the LPA locus, the focus of this analysis included 49 SNPs within

100 kb up- or down-stream of LPA, including 28 SNPs within the LPA locus, which passed

quality control on the CVD beadchip in at least one of the ethnicities. Thus, for de novo

associations a Bonferroni corrected significance threshold would be P = 1.4 x 10-6 (0.05 / 35303),

and for the 49 SNPs surrounding LPA a Bonferroni corrected significance would be P = 0.001

(0.05 / 49). Multivariate multiple regression with a forward stepwise modeling approach was

performed in SAS v9.1 (SAS Institute, Cary, NC, USA) to determine the degree of variation

explained by LPA SNP genotype, KIV-2 copy number, sex, LDL cholesterol, and apo B (see

supplementary online content).

Results

Association between kringle-repeat number and Lp(a)

Differences of both KIV-2 copy number distribution and plasma Lp(a) concentration

were observed between the three ethnicities (Table 2, Figure 1). In the SHARE sample, similar

median Lp(a) concentrations were observed in Chinese (Lp(a) = 159 mg/dL) and European

Caucasians (Lp(a) = 156 mg/dL, P = 0.53), while South Asians had a significantly higher

median Lp(a) concentration (Lp(a) = 203, versus Chinese P = 9.2 x 10-5, versus European

Caucasian P = 0.015). Paradoxically, European Caucasians have the lowest average KIV-2 copy

number (mean KIV-2 copy number = 6.19), followed by the Chinese (mean KIV-2 copy number

= 6.47, versus European Caucasians P = 3.9 x 10-5), then the South Asians with the highest

average KIV-2 copy number (mean KIV-2 copy number = 6.69, versus Chinese P = 7.4 x 10-4,

versus European Caucasians P = 3.0 x 10-14) (Table 2, Figure 1). Significant correlation was

observed between KIV-2 copy number and plasma Lp(a) concentration in all three ethnicities,

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and was marginally stronger in European Caucasians (South Asian: rs = -0.25, P = 4.7 x 10-6;

Chinese: rs = -0.25, P = 1.5 x 10-5; European Caucasian: rs = -0.31, P = 4.2 x 10-7; Figure 2).

Genetic structure of LPA

In total, 49 SNPs were within 100 kb of either side of LPA. A consistent haplotype block

structure across the three ethnicities was observed (Figure 3). One large haplotype block contains

the 5’ end of the LPA locus, including the hypervariable KIV-2 copy number variation, and the

first 35 kb immediately upstream of LPA. A second haplotype block contains the 3’ end of LPA,

including the peptidase domain and the kringle type V domain. The plasminogen gene (PLG),

and potentially additional cis-acting elements >35 kb upstream of LPA, lie within a separate

upstream haplotype block.

To estimate linkage disequilibrium between SNPs in LPA and KIV-2 copy number,

association testing was performed with KIV-2 copy number as a continuous trait. SNP

rs10455872, which was present only in European Caucasians, was significantly associated with

KIV-2 copy number (P = 7.2 x 10-5; Table 3). SNP rs6415084 was prevalent in all three

ethnicities and was associated with KIV-2 copy number in the same direction (P = 2.6 x 10-4;

Table 3). Two additional SNPs in the 5’ haplotype block of LPA were marginally associated with

KIV-2 copy number, with the direction of the association being the same in all three ethnicities

(0.0018 < P < 0.0076; Table 3).

Association between SNPs and Lp(a)

Of the 14 SNPs within the 5’ LPA haplotype block that were prevalent in all three

ethnicities, 10 SNPs were associated with plasma Lp(a) concentration in the same direction in all

three ethnicities (0.001 < P < 2.5 x 10-9; Table 3). The minor alleles of particular SNPs within the

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5’ block were associated with both increased and decreased plasma Lp(a) concentration. One

SNP, rs10455872, only present in European Caucasians, was very strongly associated with

plasma Lp(a) concentration (P = 4.2 x 10-23). Two SNPs within the 3’ LPA haplotype block,

rs11751605, found only in European Caucasians, and rs6919346, found in European Caucasians

and South Asians, were observed to be associated with plasma Lp(a) concentration (P = 6.0 x 10-

6 and P = 7.6 x 10-6 respectively). Excluding the 100 kb region surrounding LPA, no SNPs found

within the 20 Mb (150-170 Mb) region on 6q previously reported to be associated with Lp(a)

concentration27 were associated with Lp(a) concentration (P > 1 x 10-3). As well, none of the

other tested SNPs on the CVD beadchip were associated with plasma Lp(a) concentration (P > 1

x 10-5).

To assess how KIV-2 copy number might modulate the association between SNPs and

plasma Lp(a) concentration, regression analysis was performed with KIV-2 included as a

covariate (Table 3, right column). For all SNPs that were significantly associated with plasma

Lp(a) concentration, the significance was reduced with KIV-2 copy number included as a

covariate, but all remained significant, even after multiple-testing correction (1.8 x 10-5 < P <

2.8x 10-19).

Genomic contribution to plasma Lp(a) variation

In order to measure the total genetic contribution of the genotyped variants in the LPA

locus to plasma Lp(a) concentration variation, we performed a multivariate multiple regression

with a forward stepwise modeling approach. Including sex, LDL cholesterol, and apo B, a

substantially larger proportion of plasma Lp(a) concentration variation could be explained in

European Caucasians (r2 = 0.36; F4 = 34.96) compared to Chinese (r2 = 0.27; F7 = 15.44) or

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South Asians (r2 = 0.21; F5 = 16.35) (Figure 4; online supplementary table 1). Likely

contributing to this discrepancy was the strong association observed between rs10455872 and

plasma Lp(a) concentration prevalent only in European Caucasians. For each group, the KIV-2

copy number explained an additional proportion of the variation in plasma Lp(a) than the SNP

genotype alone.

Discussion

In a multiethnic sample of ~900 individuals, we examined the genomic structure of the

LPA locus and quantified its contribution to Lp(a) concentrations. The haplotype block structure

was similar across the three studied ethnicities, and we identified SNPs within a large block over

the 5’ end of LPA that were significantly associated with both Lp(a) concentration and number of

KIV-2 repeats. One SNP, rs10455872, only observed in European Caucasians, was very strongly

associated with both KIV-2 copy number and plasma Lp(a) concentration. Two SNPs within the

haplotype block covering the 3’ end of LPA were also associated with plasma Lp(a)

concentration.

Two previous studies have shown no correlation, or linkage disequilibrium, between LPA

SNPs and KIV-2 copy number.27, 28 One study used Southern blotting,28 while the other used

immunoblotting,27 and thus the sample sizes were very small (n=47 and n=63 respectively) and

were subject to the technical limitations of the methods discussed above. Without measurement

of KIV-2 copy number, a recent study identified linkage disequilibrium between the (TTTTA)n

promoter polymorphism and sequence variation 3’ to the KIV-2 repeat in kringle IV types 8 and

10, all of which lie within the 5’ LPA haplotype block.29 Using a quantitative PCR KIV-2

genotyping technique, we estimated KIV-2 copy number across our complete sample, and

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identified strong association between KIV-2 copy number and rs10455872 in European

Caucasians. An additional SNP, rs6415084, is prevalent in all three ethnicities, located in the 5’

haplotype block, and associated with both KIV-2 copy number and Lp(a) concentration. Two

additional SNPs (rs13202636 and rs3798221) were associated with Lp(a) concentration and were

marginally associated with KIV-2 copy number in all three ethnicities in the same direction.

Following expectations given the known biology of large KIV-2 copy number and Lp(a)

secretion, in all four cases, the particular SNPs were either associated with an increase in KIV-2

copy number together with a decrease in plasma Lp(a) concentration, or with a decrease in KIV-

2 copy number and an increase in plasma Lp(a) concentration. Further development of a method

for quantification of KIV-2 copy number in an allele-specific manner would allow for

determination of phase between KIV-2 copy number and surrounding alleles, allowing

traditional linkage disequilibrium calculations (including the calculation of r2 values).

Eight SNPs that were not associated with KIV-2 copy number, were associated with

Lp(a) concentration, suggesting that the SNPs are associated with an alternative mechanism for

modifying Lp(a) concentration, such as efficiency of transcription or expression. Previous work

has identified promoter polymorphisms that would be in some degree of linkage disequilibrium

with SNPs in the 5’ block.29, 30 As well, rs6919346 in the 3’ haplotype block is associated with

Lp(a) concentrations in the same direction with similar effect size as seen in a previous report.27

No SNPs outside of LPA were associated with Lp(a), suggesting that previous associations

between more distant regions of 6q and Lp(a) might have been related to longer stretches of

linkage disequilibrium in Hutterite chromosomes.27

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It has long been recognized that elevated plasma Lp(a) concentration is associated with

increased CVD risk,2-4 and that Lp(a) concentration is largely determined by genetic variation

within the LPA locus, including both SNPs and CNVs.11, 13 In studies largely in Caucasians,

SNPs within LPA have been associated with CVD endpoints. 31, 32 Furthermore, KIV-2 copy

number has been associated with CVD endpoints in case-control studies,14-16 and recently in a

prospective Mendelian randomization study.17 However, no multiethnic studies to date have

generated both SNP genotypes and the KIV-2 copy number genotype. Including KIV-2 copy

number as a covariate in regression analysis reduced the significance of association between LPA

SNP genotype and plasma Lp(a) concentration, but did not altogether ablate the signal. LPA

SNP genotypes, and rs10455872 specifically, explained a much larger extent of Lp(a) variation

in European Caucasians than that explained by genetic factors in South Asians and Chinese. The

addition of an assay to query KIV-2 copy number produces a meaningful increment over simple

SNP genotypes in the explanation of plasma Lp(a) concentration variation, and should be

included in future studies of Lp(a), especially in epidemiologic samples for which DNA but not

plasma Lp(a) concentration is available.

The Mendelian randomization approach, in which the association between a disease

endpoint and a risk factor is assessed in the presence or absence of a genetic variant associated

with the risk factor, has advantages over even the best designed prospective observational

study.33 As further Mendelian randomization studies of Lp(a) and atherosclerosis susceptibility

are performed, including more SNPs and ethnicities, the selection of the genetic markers, and the

degree of trait variation they explain, will greatly affect results. Multiple explanations are

possible for the large difference in explained Lp(a) concentration variation observed between

ethnicities. Perhaps plasma Lp(a) concentration is not as strongly determined by genetic factors

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14

in South Asians and Chinese compared to European Caucasians. Possibly SNPs represented on

the CVD beadchip are present at higher allele frequencies (thus affording greater statistical

power) due to a bias in the initial beadchip design towards European Caucasian samples. The

potentially most likely scenario is that additional genetic variants, either not queried on the CVD

beadchip, or outside the LPA locus, or too rare or with effects too small to be detected in the

SHARE sample, play a greater role in South Asian and Chinese ethnicities than in European

Caucasians. Indeed, in a preliminary post-hoc imputation-based analysis using Hapmap 3 release

2 data, it appears that an untyped LPA SNP, rs7770628, is strongly associated with both KIV-2

copy number and Lp(a) concentrations in Chinese SHARE participants (data not shown). Further

identification and categorization of rare and common variants is required in South Asians and

Chinese, as well as validation of imputation-based methods in de novo investigations of non-

European Caucasian populations.

The inclusion of CVD endpoints would have provided an additional dimension to this

work, allowing for assessment of association between LPA genomic SNPs and CNVs and plasma

Lp(a) concentration and CVD events. However, SHARE was a cross-sectional study of risk

factor prevalence in healthy Canadians, with very few end points recorded. Many significant

associations, both genetic and biochemical, have been observed in the SHARE sample,21, 22, 34, 35

but many more participants would have been required to collect enough end points to provide

sufficient power to detect genetic associations of LPA SNPs or KIV-2 copy number with

cardiovascular events. As DNA sequencing was not performed and we restricted analysis to

genetic variants with a MAF > 1%, no null alleles were determined.

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In conclusion, we have quantified the proportion of plasma Lp(a) variation that can be

explained using sex, LDL cholesterol, apo B and both LPA SNPs and KIV-2 copy number,

provided evidence of linkage disequilibrium between certain LPA SNPs and genomic KIV-2

copy number, and identified similar LPA haplotype block structure in South Asians, Chinese and

European Caucasians. Future work will be required to uncover genetic factors explaining

additional plasma Lp(a) concentration variation, especially in South Asians and Chinese

ethnicities, and to further validate Lp(a) as a CVD risk factor through Mendelian randomization

studies including multiple ethnicities. Both SNPs and KIV-2 copy number are thus genomic

determinants of plasma Lp(a) concentration, but relationships differ across ethnic groups, and

this will need to be considered in future epidemiological studies that incorporate LPA genetic

variation.

Acknowledgements: The authors would like to thank Christopher Johansen and Piya Lahiry for their helpful discussions. Finally, this research would not have been possible without the SHARE study participants.

Funding Sources: Mr. Lanktree is supported by the Canadian Institutes of Health Research (CIHR) MD/PhD Studentship Award, the University of Western Ontario MD/PhD Program, and is a CIHR Fellow in Vascular Research. Dr. Anand holds the Michael G. DeGroote/Heart and Stroke Foundation of Ontario endowed Chair in Population Health Research and the Eli Lilly –May Cohen Chair in Women’s Health Research. Dr. Yusuf holds an endowed chair in Cardiovascular Research from the Heart and Stroke Foundation of Ontario. Dr. Hegele is a Career Investigator of the Heart and Stroke Foundation of Ontario, holds the Edith Schulich Vinet Canada Research Chair (Tier I) in Human Genetics, the Martha G. Blackburn Chair in Cardiovascular Research, and the Jacob J. Wolfe Distinguished Medical Research Chair at the University of Western Ontario. This work was supported by CIHR, the Heart and Stroke Foundation of Ontario, Genome Canada through Ontario Genomics Institute, and the Pfizer Jean Davignon Distinguished Cardiovascular and Metabolic Research Award.

Conflict of Interest Disclosures: None.

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a aaaaaaaaaaaaaaa SMSMSMSMSMSMSMSMSMSMSMSMSMSMSMSMSMMMSMSMSMMMMM, ,,,,,,,,,,,,, DuDuDuDuDuuDuDuDuDuDDuDuDuDuDDuDuDuDuDDuDDuuDubebebebebebebebebebbebebebebebebebebbbbeeb GGGGGGGGGGGGGGGGGGG, AAAAAAAAAAAAAAAAAAAAAronarrrrrrrrrrrrrrrry y y yy y y y y yy y y yy y y y y y yyy ataataataaaaaataatatatatatatathehehehehehehehehehehehehehehehehehheh rorororoororrororororororoooroooorromammmmmmmmmmmmmmmmm

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plasma lipoprotein(a) levels identifies multiple genes on chromosome 6q. J Lipid Res. 2009; 50:798-806.

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29. Hirschfeldova K, Lipovska D, Skrha P, Ceska R. The apo(a) gene (TTTTA)n promoter polymorphism and its association with variability in exons of the kringle IV types 8 to 10. Clin Chim Acta. 2009; 405:39-42.

30. Zidkova K, Zlatohlavek L, Ceska R. Variability in apo(a) gene regulatory sequences, compound genotypes, and association with Lp(a) plasma levels. Clin Biochem. 2007; 40:802-805.

31. Shiffman D, Kane JP, Louie JZ, Arellano AR, Ross DA, Catanese JJ, Malloy MJ, Ellis SG, Devlin JJ. Analysis of 17,576 potentially functional SNPs in three case-control studies of myocardial infarction. PLoS One. 2008; 3:e2895.

32. Shiffman D, O'Meara ES, Bare LA, Rowland CM, Louie JZ, Arellano AR, Lumley T, Rice K, Iakoubova O, Luke MM, Young BA, Malloy MJ, Kane JP, Ellis SG, Tracy RP, Devlin JJ, Psaty BM. Association of gene variants with incident myocardial infarction in the Cardiovascular Health Study. Arterioscler Thromb Vasc Biol. 2008; 28:173-179.

33. Thanassoulis G, O'Donnell CJ. Mendelian randomization: nature's randomized trial in the post-genome era. JAMA. 2009; 301:2386-2388.

34. Burman D, Mente A, Hegele RA, Islam S, Yusuf S, Anand SS. Relationship of the ApoE polymorphism to plasma lipid traits among South Asians, Chinese, and Europeans living in Canada. Atherosclerosis. 2009; 203:192-200.

35. Kelemen LE, Anand SS, Hegele RA, Stampfer MJ, Rosner B, Willett WC, Montague PA, Lonn E, Vuksan V, Teo KK, Devanesen S, Yusuf S. Associations of plasma homocysteine and the methylenetetrahydrofolate reductase C677T polymorphism with carotid intima media thickness among South Asian, Chinese and European Canadians. Atherosclerosis. 2004; 176:361-370.

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Table 1. Baseline characteristics SHARE cohort participants

Measure South Asian Chinese European Caucasian Total

N 330 304 272 906 Male (%) 180 (54%) 155 (51%) 130 (48%) 465 (51%) Age, years 49.6 (9.3) 47.7 (8.9) 51.2 (11.0) 49.4 (9.8) BMI, kg/m2 26.3 (4.2) 24.0 (3.6) 27.5 (4.6) 25.9 (4.4) Current smoker (%) 33 (10%) 17 (6%) 43 (16%) 93 (10%) Dyslipidemia on treatment (%) 30 (9%) 17 (6%) 16 (6%) 64 (7%) LDL cholesterol, mmol/L 3.30 (0.82) 3.13 (0.79) 3.18 (0.81) 3.21 (0.81) Apo B, g/L 1.08 (0.26) 0.99 (0.26) 1.00 (0.25) 1.02 (0.26) HDL cholesterol, mmol/L 1.03 (0.30) 1.18 (0.36) 1.20 (0.38) 1.12 (0.35) Triglycerides, mmol/L 1.99 (1.29) 1.78 (1.41) 1.61 (0.96) 1.81 (1.26) Mean with standard deviation is given in brackets, unless median or percentage is indicated


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Table 2. Lp(a) and KIV-2 in SHARE participants

Between ethnicity comparisons

SA CH EC SA vs. CH* SA vs. EC* CH vs. EC*

Lp(a), mg/dL median (IQR) range†

203

(101-425) 15-2338

156

(69-308) 15-1557

159

(61-376) 15-1246

0.00092 0.015 0.53

KIV-2, CT mean (SD) range

6.69 (0.78)

4.68-9.48

6.47 (0.84)

2.65-8.69

6.19 (0.77)

4.37-8.51

7.4 x 10-4 3.0 x 10-14 3.9 x 10-5

Abbreviations: SA, South Asians; CH, Chinese; EC, European Caucasian; KIV-2, kringle IV type 2 repeat number; IQR, interquartile range; SD, standard deviation; CT, change in cycle threshold. *significance of Student’s t test between groups. †15 mg/dL was the lowest-limit of detection of the Lp(a) assay.


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Table 3. SNPs associated with Lp(a)

SNP

Physical Position

Location

Allele

MAF SA CH EC

Lp(a)* P

KIV-2† P

Lp(a) w/ KIV-2‡ P

rs10455872 160930108 5’ block G <0.01 <0.01 0.07 4.2 x 10-23 10.74 7.2 x 10-5 -0.50 2.8 x 10-19 9.78 rs10945682 160989931 5’ block A 0.41 0.40 0.35 2.5 x 10-9 -2.13 NS 5.3 x 10-9 -2.02 rs6923877 160916787 5’ block G 0.40 0.37 0.33 6.2 x 10-8 -2.00 NS 3.2 x 10-7 -1.83 rs7765781 160927486 5’ block C 0.40 0.37 0.33 8.1 x 10-8 -1.98 NS 4.0 x 10-7 -1.81 rs7765803 160927528 5’ block G 0.39 0.37 0.33 8.5 x 10-8 -1.98 NS 4.9 x 10-7 -1.80 rs1950562 161043175 5’ block G 0.41 0.15 0.57 4.8 x 10-7 2.02 NS 1.2 x 10-6 1.88 rs6415084 160900320 5’ block A 0.31 0.10 0.47 5.3 x 10-7 2.11 0.00026 -0.16 3.9 x 10-5 1.69

rs13202636 160949718 5’ block G 0.30 0.40 0.23 1.3 x 10-6 -1.88 0.0076 0.11 2.6 x 10-5 -1.59 rs3798221 160918138 5’ block A 0.29 0.37 0.20 2.5 x 10-6 -1.87 0.0018 0.13 9.4 x 10-5 -1.51 rs9365171 160901726 5’ block A 0.41 0.32 0.34 2.9 x 10-6 -1.74 NS 1.5 x 10-5 -1.56 rs6919346 160930108 3’ block A 0.05 <0.01 0.19 6.0 x 10-6 -3.21 NS 1.3 x 10-5 -2.98

rs11751605 160883220 3’ block G <0.01 <0.01 0.17 7.6 x 10-6 3.58 NS 1.8 x 10-5 3.28 *meta-analysis of regression analysis of Lp(a) performed in each ethnicity including age and sex as covariates †meta-analysis of regression analysis using the KIV-2 as the trait performed in each ethnicity ‡meta-analysis of regression analysis of Lp(a) performed in each ethnicity including age, sex, and KIV-2 as covariates. Abbreviations: SNP, single nucleotide polymorphism; MAF, minor allele frequency; SA, South Asian; CH, Chinese; EC, European Caucasian; Lp(a), lipoprotein(a), P, regression significance; , percent of a single standard deviation change with each additional risk allele; NS, not significant; w/, with.

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Figure Legends

Figure 1. Distribution of plasma lipoprotein(a) concentration (A), and KIV-2 copy number as

measured by CT (B).

Figure 2. The inverse correlation between KIV-2, as measured by CT, and plasma

lipoprotein(a) concentration in South Asians, Chinese, and European Caucasians.

Figure 3. Pairwise linkage disequilibrium map, depicting the three large haplotype blocks

surrounding LPA in the South Asian, Chinese, and European Caucasian SHARE participants.

The intensity of each box is proportional to the degree of linkage disequilibrium between the pair

of polymorphisms. Blue boxes were uninformative. The physical position of each polymorphism

is shown to scale on the white line above each map. A map depicting the physical position of

LPA exons and the kringle-repeats is shown at the top of the figure.

Figure 4. Percentage of plasma lipoprotein(a) concentration explained by LPA SNP variation

shown in Table 3, KIV-2 copy number, and the cumulative effect of sex, low-density lipoprotein

(LDL) cholesterol, and apolipoprotein B (apoB), as determined by multivariate multiple

regression with a forward stepwise modeling approach. See supplementary online content for

detailed methods and results.

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Matthew B. Lanktree, Sonia S. Anand, Salim Yusuf and Robert A. HegelePlasma Lipoprotein(a) in South Asians, Chinese and European Caucasians

Locus And Its Relationship toLPAComprehensive Analysis of Genomic Variation in the

Print ISSN: 1942-325X. Online ISSN: 1942-3268 Copyright © 2009 American Heart Association, Inc. All rights reserved.

TX 75231is published by the American Heart Association, 7272 Greenville Avenue, Dallas,Circulation: Cardiovascular Genetics

published online December 30, 2009;Circ Cardiovasc Genet.

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SUPPLEMENTAL MATERIAL Online supplemental materials for “Comprehensive analysis of genomic variation in the LPA locus and its relationship to plasma Lipoprotein(a) in South Asians, Chinese and European Caucasians.” Lanktree et al. CIRCULATIONAHA/2009/907642

Supplemental Methods:

Multivariate multiple regression with a forward modeling approach was performed in SAS v9.1 (SAS Institute, Cary, NC, USA) to determine the degree of variation explained by LPA SNP genotypes, KIV-2 copy number, and covariates for elevated Lp(a) including sex, LDL cholesterol, and apo B. In the unbiased, iterative forward selection approach, linear regression is performed with all available independent variables individually. The variable explaining the greatest extent of variation in the dependent variable (measured by the largest r2) is entered into the model. Regression is then performed again, including the selected variable from the first round, testing all other independent variables individually, and the new largest predictor is selected for inclusion in the model. The procedure is repeated until no remaining variables significantly contribute to the model (P < 0.05).

Clinical characteristics available for inclusion in the model included sex, LDL cholesterol and apoB. Available genetic variables included the KIV-2 copy number and all SNPs shown in Table 2. No two-way or higher interactions were included as possible terms in the multivariate multiple regression. The results are visually depicted in Figure 4 and the selected model is described in Supplementary Table 1.

Supplemental Table:

Supplemental Table 1 - Forward stepwise multivariate multiple regression results

Supplemental Table 1: Forward stepwise multivariate multiple regression results

Ethnicity Variable Partial r2 Cumulative

Model r2 F P > F* South Asian KIV-2 0.067 0.067 22.68 2.9 x 10-6

LDL 0.058 0.125 20.61 8.0 x 10-6 rs10945682 0.045 0.170 16.71 5.5 x 10-5 rs6919346 0.023 0.193 8.99 2.9 x 10-3 rs6415084 0.016 0.209 6.22 0.013 rs1950562 NS rs13202636 NS Sex NS ApoB NS rs3798221 NS rs9365171 NS rs6923877 NS rs7765781 NS rs7765803 NS

Chinese rs6415084 0.082 0.082 26.41 5.0 x 10-7 LDL 0.067 0.149 23.17 2.4 x 10-6 rs3798221 0.049 0.198 17.93 3.1 x 10-5 KIV-2 0.030 0.228 11.18 9.3 x 10-4 Sex 0.017 0.245 6.87 0.0092 rs1950562 0.014 0.259 5.38 0.021 apoB 0.013 0.272 5.09 0.024 rs10945682 NS rs9365171 NS rs6923877 NS rs7765781 NS rs7765803 NS rs13202636 NS

European Caucasian rs10455872 0.279 0.279 98.12 9.0 x 10-20 KIV-2 0.042 0.320 15.55 1.0 x 10-4 rs6919346 0.022 0.343 8.50 0.0039 Sex 0.015 0.358 5.94 0.016 apoB NS rs11751605 NS LDL NS rs10945682 NS rs7765781 NS rs13202636 NS rs3798221 NS rs1950562 NS rs9365171 NS rs6415084 NS rs6923877 NS rs7765803 NS

*- statistical significance of model improvement with addition of variable. NS, not significant.

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