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Genetic contribution of SCARB1 variants to lipid traits in African Blacks: a candidate gene association study

  • Vipavee Niemsiri1,
  • Xingbin Wang1,
  • Dilek Pirim1,
  • Zaheda H. Radwan1,
  • Clareann H. Bunker2,
  • M. Michael Barmada1,
  • M. Ilyas Kamboh1Email author and
  • F. Yesim Demirci1Email author
Contributed equally
BMC Medical Genetics201516:106

https://doi.org/10.1186/s12881-015-0250-6

Received: 9 April 2015

Accepted: 30 October 2015

Published: 12 November 2015

Abstract

Background

High-density lipoprotein cholesterol (HDL-C) exerts many anti-atherogenic properties including its role in reverse cholesterol transport (RCT). Scavenger receptor class B member 1 (SCARB1) plays a key role in RCT by selective uptake of HDL cholesteryl esters. We aimed to explore the genetic contribution of SCARB1 to affecting lipid levels in African Blacks from Nigeria.

Methods

We resequenced 13 exons and exon-intron boundaries of SCARB1 in 95 individuals with extreme HDL-C levels using Sanger method. Then, we genotyped 147 selected variants (78 sequence variants, 69 HapMap tagSNPs, and 2 previously reported relevant variants) in the entire sample of 788 African Blacks using either the iPLEX Gold or TaqMan methods. A total of 137 successfully genotyped variants were further evaluated for association with major lipid traits.

Results

The initial gene-based analysis demonstrated evidence of association with HDL-C and apolipoprotein A-I (ApoA-I). The follow-up single-site analysis revealed nominal evidence of novel associations of nine common variants with HDL-C and/or ApoA-I (P < 0.05). The strongest association was between rs11057851 and HDL-C (P = 0.0043), which remained significant after controlling for multiple testing using false discovery rate. Rare variant association testing revealed a group of 23 rare variants (frequencies ≤1 %) associated with HDL-C (P = 0.0478). Haplotype analysis identified four SCARB1 regions associated with HDL-C (global P < 0.05).

Conclusions

To our knowledge, this is the first report of a comprehensive association study of SCARB1 variations with lipid traits in an African Black population. Our results showed the consistent association of SCARB1 variants with HDL-C across various association analyses, supporting the role of SCARB1 in lipoprotein-lipid regulatory mechanism.

Keywords

African continental ancestry group Candidate gene association study Genetic variation Haplotypes Lipids SCARB1 protein, human Sequence analysis, DNA

Background

Abnormal lipid and lipoprotein levels are a major risk factor for coronary heart disease (CHD) [1], the leading cause of death worldwide [2]. Elevated low-density lipoprotein cholesterol (LDL-C) levels and decreased high-density lipoprotein cholesterol (HDL-C) levels are correlated with the development of CHD. There is a strong genetic basis for lipoprotein-lipid levels with heritability estimates of 40–80 % [3]. A large number of genes and genetic variants associated with lipid traits have been discovered in genome-wide association studies (GWAS) [46]. Most of the common variants (minor allele frequency [MAF] ≥5 %) identified by GWAS have modest effects on lipid levels, and have overall a small contribution to total genetic variance of lipid traits (~25–30 % of the heritability) [48]. A portion of the missing heritability of lipid traits could be explained by low frequency (LoF)/rare variants (MAF <5 %) as suggested by recent studies [911].

HDL, the smallest and densest (d = 1.063–1.21 g/mL) class of lipoprotein particles, has a variety of antiatherogenic properties [12]. One of the HDL properties to protect against CHD is mediated by reverse cholesterol transport (RCT) from peripheral tissues back to the liver [13]. Scavenger receptor class B member 1 (SCARB1, protein; SCARB1, gene) serves as a HDL-C receptor in RCT that mediates selective uptake of HDL-C cholesteryl esters (CE) by the liver and free cholesterol efflux from cells to HDL-C [14]. SCARB1 is also implicated in the metabolism of apolipoprotein B (ApoB)-containing particles [1521].

The SCARB1 gene (Entrez Gene ID: 949) is located on human chromosome 12, and is abundantly expressed in liver and steroidogenic tissues [22, 23]. The role of SCARB1 in HDL-C and ApoB-containing lipoproteins metabolism has been established in animal studies. The disruption of SCARB1 is associated with increased HDL-C levels and decreased CE uptake [2426]. Whereas the overexpression of SCARB1 reduces levels of HDL-C, ApoA-I, very low-density lipoprotein cholesterol (VLDL-C), LDL-C, and ApoB [1517, 19] and promotes the hepatic uptake of CE as well as the biliary secretion of HDL-C [15, 27]. The SCARB1 expression is also significantly associated with hepatic VLDL-triglycerides (TG) and VLDL-ApoB production. Hepatic VLDL cholesterol production together with VLDL clearance is enhanced in response to SCARB1 overexpression [21]. In contrast, reduced hepatic VLDL-TG and VLDL-ApoB production is associated with SCARB1 knockout status [18, 20, 21].

In humans, three SCARB1 mutations (rs397514572 [p.Ser112Phe], rs187831231 [p.Thr175Ala], and rs387906791 [p.Pro297Ser]; MIM: 601040) have been reported to be associated with significantly increased HDL-C levels [28, 29]. Moreover, several genetic studies have demonstrated the association of common SCARB1 variation with lipoprotein-lipid levels [5, 2839] and subclinical atherosclerosis [40].

To our knowledge, no genetic study has exclusively investigated the association between SCARB1 and lipid traits in native African populations to date. The objective of this study was to resequence all 13 exons and exon-intron boundaries of SCARB1 in 95 African Blacks from Nigeria with extreme HDL-C levels for variant discovery and then to genotype selected variants in the entire sample of 788 African Blacks, followed by genotype-phenotype association analyses with five major lipid and apolipoprotein (Apo) traits (HDL-C, LDL-C, TG, ApoA-I and ApoB). Because our initial gene-based analysis demonstrated evidence of association with HDL-C and ApoA-I, our subsequent analyses focused on these two traits.

Methods

Study population

The present study was carried out on 788 African Black subjects from Benin City, Nigeria, who were recruited as part of a population-based epidemiological study on CHD risk factors. Detailed information on the study design and population description is provided elsewhere [41]. In brief, 788 recruited subjects were healthy civil servants (37.18 % females) from three government ministries of the Edo state in Benin City, Nigeria, aged between 19 and 70 years, including 464 junior staff (non-professional staff with salary grades 1–6), and 324 senior staff (professional and administrative staff with salary grades 7–16). The summary features, including biometric and quantitative data of the entire sample of 788 subjects are given in Table 1 and Additional file 1: Table S1.
Table 1

Characteristics and lipid profile of 95 individuals with extremea HDL-C levels and of the entire sample of 788 African Blacks

 

95 Individuals with Extremea HDL-C Levels

The Entire Sampleb

Variables

High HDL-C Group

Low HDL-C Group

P d

 

(HDL-C rangec: 68.30–99.00 mg/dL)

(HDL-C rangec: 10.30–35.00 mg/dL)

N (Females, n)

48 (24)

47 (24)

1.00

788 (293)

Age, years

41.29 ± 8.72

40.87 ± 7.12

0.80

40.95 ± 8.39

BMI, kg/m2

22.06 ± 4.70

23.91 ± 5.51

0.08

22.87 ± 4.04

Total Cholesterol, mg/dL

201.00 ± 39.68

141.68 ± 31.03

2.40E-12

172.01 ± 38.47

LDL-Cholesterol, mg/dL

112.55 ± 39.75

95.04 ± 28.28

0.02

109.25 ± 34.40

HDL-Cholesterol, mg/dL

76.05 ± 7.53

25.51 ± 5.66

2.20E-16

47.88 ± 12.87

Triglycerides, mg/dL

61.98 ± 19.85

95.79 ± 73.21

0.004

72.96 ± 39.32

Apolipoprotein A-I, mg/dL

166.04 ± 28.19

103.84 ± 27.23

2.20E-16

137.03 ± 28.46

Apolipoprotein B, mg/dL

66.00 ± 20.22

69.64 ± 21.46

0.40

66.98 ± 22.19

BMI body mass index, HDL-C/HDL-Cholesterol high-density lipoprotein cholesterol, LDL-Cholesterol low-density lipoprotein cholesterol

Values are presented as unadjusted means ± standard deviation (SD), unless otherwise mentioned

aDistribution of HDL-C was adjusted for sex and age: HDL-C levels ≥90th % tile defined as the “High HDL-C group”, and HDL-C levels ≤10th % tile defined as the “Low HDL-C group”

bAll data were unadjusted and included individuals with missing values or outliers (values beyond mean ± 3.5 SD)

cUnadjusted range values

dUnadjusted P-values were calculated with t-test or χ2 test depending on types of variables

For resequencing, 95 individuals with extreme HDL-C levels (within the upper and lower 10th percentiles of HDL-C distribution) were chosen from the entire sample of 788 African Blacks. Resequencing sample comprised of 48 individuals with high HDL-C levels (≥90th percentile, range 68.30–99.00 mg/dL; Table 1) and 47 individuals with low HDL-C levels (≤10th percentile, range 10.30–35.00 mg/dL; Table 1). The University of Pittsburgh Institutional Review Board approved the study protocol. All participants gave their informed consent.

Lipid and apolipoprotein measurements

At least 8-hour fasting blood samples were collected from all participants. Serum specimens were separated by centrifugation of blood samples and then stored at −70 °C for 6–12 months until ready for testing. Lipid and apolipoprotein measurements included total cholesterol, HDL-C, TG, ApoA-I, and ApoB and were done with standard assays at the Heinz Nutrition Laboratory, University of Pittsburgh under the Centers for Disease Control Lipid Standardization Program [41]. LDL-C was calculated with the Friedewald equation [42] when TG levels were less than 400 mg/dL.

PCR and sequencing

Genomic DNA was isolated from clotted blood using the standard DNA extraction procedure. All 13 SCARB1 exons (isoform 1, NM_005505), exon-intron boundaries, and 1 kb of each of 5′ and 3′ flanking regions on chromosome 12 (hg19, chr12: 125,262,175-125,348,519) were polymerase chain reaction (PCR) amplified and sequenced. Specific primers were designed using the Primer3 software (Whitehead Institute for Biomedical Research, http://bioinfo.ut.ee/primer3-0.4.0/) to cover 13 target regions, resulting in 14 amplicons, including two overlapping amplicons for the largest last exon 13. PCR reaction and cycling conditions are available upon request. The primer sequences and amplicon sizes are given in Additional file 2: Table S2.

Automated DNA sequencing of PCR products was performed in a commercial lab (Beckman Coulter Genomics, Danvers, MA, USA) using Sanger method and ABI 3730XL DNA Analyzers (Applied Biosystems, Waltham, MA, USA). Variant analysis was performed using Variant Reporter (version 1.0, Applied Biosystems, Waltham, MA, USA) and Sequencher (version 4.8, Gene Codes Corporation, Ann Arbor, MI, USA) software in our laboratory.

Variant selection for genotyping

Of 83 variants identified in the discovery step (see Additional file 3: Table S3, Additional file 4: Table S4, Additional file 5: Figure S1, and Additional file 6: Figure S2), 78 (28 with MAF ≥5 % and 50 with MAF <5 %) were selected based on the pairwise linkage disequilibrium (LD) and Tagger analysis using an r 2 threshold of 0.90 (5 were excluded due to high LD) in Haploview (Broad Institute of MIT and Harvard, https://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview) [43] for follow-up genotyping in the entire sample (n = 788). Since our sequencing was focused primarily on coding regions, in addition we selected 69 HapMap tag single nucleotide polymorphisms [SNPs] (out of total 108 HapMap tagSNPs; see Additional file 7: Table S5 and Additional file 8: Figure S3) based on Tagger analysis (MAF ≥5 % and r 2  ≥ 0.80) of HapMap data (Release #27) from the Yoruba people of Ibadan, Nigeria (YRI), in order to cover the entire gene for common genetic variation information. Moreover, we selected two SCARB1 variants previously reported to be significantly associated with lipid traits in the literature (Additional file 9: Table S6). Conclusively, a total of 149 variants, comprising of 78 sequence variants, 69 common HapMap-YRI tagSNPs, and two relevant associated variants, were selected for follow-up genotyping.

Genotyping

Genotyping of selected variants in the total sample of 788 individuals was performed by using either iPLEX Gold (Sequenom, Inc., San Diego, CA, USA) or TaqMan (Applied Biosystems, Waltham, MA, USA) methods and following the manufacturers’ protocols.

Out of 149 selected variants, two failed assay designs and nine failed genotyping runs (see details in Additional file 3: Table S3, Additional file 7: Table S5, and Additional file 9: Table S6). Quality control (QC) measures for successfully genotyped variants were as follow: a genotype call rate of ≥90 %, a discrepancy rate of <1 in 10 % replicates, and no deviation from Hardy-Weinberg equilibrium [HWE] (P >3.62 × 10−4 after Bonferroni correction). Ultimately, a total of 137 QC-passed genotyped variants were included in genetic association analyses (see Additional file 9: Table S6, Additional file 10: Table S7, Additional file 11: Figure S4, and Additional file 12: Figure S5).

Statistical analysis

We used the Haploview program to determine allele frequencies, to test HWE for genotype distribution, and to evaluate the LD and pairwise correlations (r2) between variants [43].

The values of each lipid phenotype outside the mean ± 3.5 standard deviation (SD) were excluded from downstream gene-based, single-site, and haplotype analyses. However, the extreme phenotypic values associated with rare variants (MAF ≤1 %) were maintained during rare variant analysis, as was the case for the p70201/chr12:125279319 variant (see study workflow in Fig. 1). Values of the five lipid and apolipoprotein traits—HDL-C, LDL-C, TG, ApoA-I, and ApoB—were transformed using the Box-Cox transformation. For each trait, we used stepwise regression method to select the most parsimonious set of covariates from the following list: sex, age, body mass index, waist, current smoking (yes/no), minutes of walking or biking to work each day (jobmin), and occupational status (staff: junior [non-professional staff]/senior [professional and administrative staff]). Genetic association analyses, including gene-based, single-site, LoF/rare variant, and haplotype association tests, were performed using linear regression models that included significant covariates for each variable (Additional file 13: Table S8).
Fig. 1

Summary of the study design and flow. Chart presents an overview of the study design and flow, including sequencing and genotyping stages and analysis approaches. ApoA-I, apolipoprotein A-I; ApoB, apolipoprotein B; HDL-C, high-density lipoprotein cholesterol; LD, linkage disequilibrium; LDL-C, low-density lipoprotein cholesterol; LoF, low-frequency; MAF, minor allele frequency; SD, standard deviation; SKAT-O, an optimal sequence kernel association test; SNP, single nucleotide polymorphism; TG, triglycerides; YRI, Yoruba people of Ibadan from Nigeria

The gene-based association analysis was conducted under linear additive model for the combined evaluation of common and LoF/rare variants (n = 136, excluding p70201/chr12:125279319; see details above in paragraph two of this section) for five major lipid traits using the versatile gene-based association study [VEGAS] (http://gump.qimr.edu.au/VEGAS/) software [44]. The significance threshold for the gene-based test was set at P-value of 0.05.

Following gene-based analysis, which primarily implicated SCARB1 in regulation of HDL-C and ApoA-I levels, we further elucidated the association of SCARB1 variants with these two traits using additional tests. In single-site association analysis, P-values for each trait were adjusted for multiple testing using Benjamini-Hochberg procedure [45] to determine the false discovery rate [FDR] (q-value). For common variants (MAF ≥5 %), a nominal P-value of <0.05 was considered to be suggestive evidence of association, and an FDR cut-off of 0.20 was used to define statistical significance. For LoF/rare variants (MAF <5 %), the single-site association results were interpreted separately because of inadequate power of our study to detect individual statistical significance for these variants.

We conducted an optimal sequence kernel association test (SKAT-O) [46] to evaluate the association between a total of 43 LoF/rare variants (MAF <5 %) and the two lipid traits (HDL-C and ApoA-I) by using three different MAF thresholds: <5 % (n = 43), ≤2 % (n = 26), and ≤1 % (n = 23). A significant SKAT-O test was set at a P-value of <0.05.

Haplotype association analysis was performed using the generalized linear model. We applied a fixed sliding window approach that included four variants per window and sliding for one variant at a time. For each window, a global P-value was used to assess the association between the haplotypes with frequency >1 % and a given trait. A global P-value threshold of 0.05 was used to define significant haplotype association.

All analyses, except for VEGAS, were performed using the R statistical software (http://www.r-project.org/) and relevant R packages (i.e., Haplo.Stats for haplotype analysis and SKAT for SKAT-O analysis).

Results

Identification and distribution of SCARB1 sequence variants in 95 individuals with extreme HDL-C levels

Resequencing of SCARB1 exons and exon-intron boundaries plus flanking regions in 95 African Blacks with extreme HDL-C levels identified 83 variants, of which 51 had MAF <5 % (Additional file 3: Table S3 and Additional file 5: Figure S1). The majority of 83 variants (n = 73) were previously identified (dbSNP build 139: GRCh37.p10). Most variants (n = 80) were singlenucleotide variations [SNVs] (67 transitions and 13 transversions); the rest (n = 3) were short insertion and deletion variations (indels).

Tagger analysis using an r 2 cutoff of 0.9 identified 28 bins for 32 common variants (MAF ≥5 %), of which three included more than one variant (r2 ranging from 0.95 to 1.0) (Additional file 6: Figure S2). One of these three bins contained two variants (rs204901986 and rs34339961) in complete LD (r2 = 1.0). Of 51 LoF/rare variants (MAF between 1 and 5 %, n = 31; MAF ≤1 %, n = 20), 17 were present only in the high HDL-C group (MAF ranging between 0.010 and 0.042) and eight were observed only in the low HDL-C group (MAF ranging between 0.011 and 0.033). In the high HDL-C group, 29 of 48 (~60 %) individuals cumulatively carried at least one LoF/rare variant, ranging from 1 to 7 variants. Similarly, in the low HDL-C group, 27 of 47 (~57 %) individuals carried at least one LoF/rare variant, ranging from 1 to 9 variants.

Most variants (n = 60) from our sequencing were located in intronic regions, of which two (rs113910315, MAF = 0.005 and rs10396210, MAF = 0.138) were within splice sites (defined as ± 20 bp from the start or end of an exon). The former splice site variant was observed only in the low HDL-C group.

Of the total eight coding variants observed, four were common variants (rs2070242 [p.Ser4Ser], rs10396208 [p.Cys21Cys], and rs5888 [p.Ala350Ala], and rs701103 [p.Gly499Arg]—3′ untranslated region [UTR] in isoform 1 and exon 13 in isoform 2), and the remaining four were LoF/rare variants (rs4238001 [p.Gly2Ser], rs5891 [p.Val135Ile], rs5892 [p.Phe301Phe], and rs141545424 [p.Gly501Gly]). Of note, two LoF/rare coding variants, (rs5891 [p.Val135Ile] and rs141545424 [p.Gly501Gly]), were found only in the high HDL-C group.

Fifteen variants were located in either UTRs (n = 5) or flanking regions (n = 10). One 3′ UTR variant (rs150512235, MAF = 0.006) was very close to a predicted microRNA-145 (miR-145) target site (TargetScanHuman version 6.2, http://www.targetscan.org/). One 5′ flanking variant (rs181338950, MAF = 0.048) was located in the putative promoter region [47].

All 10 novel variants (9 SNVs and 1 insertion) identified in this study have been submitted to dbSNP database ([batch ID: SCARB1_AB]:

http://www.ncbi.nlm.nih.gov/SNP/snp_viewTable.cgi?handle=KAMBOH) and were non-coding with MAF <5 % (ranging between 0.005 and 0.011; Additional file 4: Table S4). Of these novel variants, six and four were present only in the high and low HDL-C groups, respectively.

Genotyping of SCARB1 variants in the entire sample of 788 individuals

Since our sequencing was focused primarily on coding regions, we selected additional HapMap tagSNPs from the HapMap-YRI data in order to cover the entire SCARB1 gene for common genetic variation in SCARB1. Altogether we selected 149 variants for genotyping in our entire African Black sample as follows: 78 variants (28 common variants and 50 LoF/rare variants) discovered in the sequencing step (Additional file 3: Table S3, Additional file 5: Figure S1, and Additional file 6: Figure S2), 69 common HapMap-YRI tagSNPs (Additional file 7: Table S5), and two additional variants with reported association in the literature (Additional file 9: Table S6).

Of these 149 variants, 11 (10 from sequencing, including one promoter [rs181338950], one coding (rs4238001 [p.Gly2Ser]), and one novel [p87459/chr12:125262061], and 1 from HapMap tagSNPs [rs4765180]) failed genotyping, and one (rs866793 from HapMap tagSNPs) failed QC measures. Thus, a total of 137 variants (Additional file 9: Table S6 and Additional file 11: Figure S4) that passed QC were advanced into association analyses with five lipoprotein-lipid traits.

The majority of 137 genotyped variants (n = 120) were located in introns, 11 were in exons, and six were in 3′ flanking region (Table 2 and Additional file 12: Figure S5). Ninety-four of 137 variants had MAF ≥5 %, including four coding variants, one UTR variant, two deletions, and one splice site variant. The remaining 43 variants had MAF <5 % (MAF between 1 and 5 %, n = 20; MAF ≤1 %, n = 23), including three coding variants, three UTR variants, one insertion, and one splice variant.
Table 2

Distribution of 137 SCARB1 genotyped variants

 

Total

MAF ≥5 %

MAF between 1-5 %

MAF ≤1 %

N (%)

n (%)

n (%)

n (%)

Total variants

137 (100.00)

94 (68.61)

20 (14.60)

23 (16.79)

By known/novela

    

 Known

128 (93.43)

94 (68.61)

20 (14.60)

14 (10.22)

  Single-nucleotide variation

126

92

20

14

  Short indels

2

2

  

 Novel

9 (6.57)

  

9 (6.57)

  Single-nucleotide variation

8

  

8

  Short indels

1

  

1

By location

    

 Exons-codingc

7

4c

1

2

 Exons-UTRs

4

1

1

2

 Introns

118

85

16

17

 Introns-splice sitesb

2

1

 

1

 3′ flanking

6

3

2

1

By amino acid change

    

 Non-synonymousc

2

1c

 

1

 Synonymous

5

3

1

1

Indels insertion and deletion variations, MAF minor allele frequency, UTR untranslated region

The list of 137 genotyped variants is shown in Additional file 9: Table S6

The list of 10 novel variants is shown in Additional file 4: Table S4

adbSNP build 139: GRCh37.p10. All 10 novel variants identified in this study have been submitted to dbSNP (batch ID: SCARB1_AB): http://www.ncbi.nlm.nih.gov/SNP/snp_viewTable.cgi?handle=KAMBOH

bSplice site, defined as ± 20 bp from the start or end of an exon

cIncluding rs701103 (p.Gly499Arg; MAF = 0.2451) that is located in exon 13-3′ UTR and translated only in isoform 2

Of the 10 novel variants discovered in the sequencing step, nine (8 SNVs and 1 insertion) with MAF <1 % were successfully genotyped (Additional file 4: Table S4). There was one individual with plasma HDL-C levels above the mean + 3.5 SD carrying one novel variant—p70201/chr12:125279319 (MAF = 0.0010). Although this extreme HDL-C value was excluded as outlier from the gene-based, single-site, and haplotype analyses, it was included in the SKAT-O rare variant analysis considering a possible large effect size of this variant (Fig. 1).

Gene-based association analyses

Gene-based tests revealed a nominally significant association (P = 0.0421; Table 3) of SCARB1 variants with HDL-C levels (best SNP: rs141545424 [p.Gly501Gly], exon 12, MAF = 0.0007, P = 0.0016). Additionally, a trend for association (P = 0.1016) was also observed for ApoA-I levels (best SNP: rs7134858, intron 6, MAF = 0.1560, P = 0.0052).
Table 3

Gene-based association analysis results

Trait

Variants

Test Statistics

P

Best SNP

(n)

SNP Namea-SNP IDb

MAF

P

HDL-C

136

207.5483

0.0421

p82264-rs141545424

0.0007

0.0016

LDL-C

136

134.1860

0.4640

p32777-rs11057841

0.2805

0.0047

TG

136

118.1598

0.6700

p86316-rs701104

0.0487

0.0357

ApoA-I

136

183.5565

0.1016

p55963-rs7134858

0.1560

0.0052

ApoB

136

143.7284

0.3760

p22116-rs12370382

0.0645

0.0153

ApoA-I apolipoprotein A-I, ApoB apolipoprotein B, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, MAF minor allele frequency, SNP single nucleotide polymorphism, TG triglycerides

All results were adjusted for covariates: sex, age, body mass index, waist, current smoking (yes/no), minutes of walking or biking to work each day (jobmin), and occupational status [staff: junior (non-professional staff)/senior (professional and administrative staff)]

Nominally significant gene-based P-values (P < 0.05) are shown in bold

aRefSeq of SCARB1: hg19, NM_005505 (CHIP Bioinformatics)

bdbSNP build 139: GRCh37.p10

Since the gene-based tests showed evidence of associations with HDL-C and ApoA-I, we primarily focused on these two traits to further examine the SCARB1 variants in the entire sample of 788 African Blacks.

Single-site association analyses of common SCARB1 variants

Of 94 common SCARB1 variants with MAF ≥5 %, 10 showed nominal associations (P < 0.05) with HDL-C and/or ApoA-I (Table 4; see results for each trait in Additional file 14: Table S9 and Additional file 15: Table S10), of which three (rs11057851, rs4765615, and rs838895) exhibited associations with both HDL-C and ApoA-I.
Table 4

Nominally significant single-site associations (P < 0.05) of common SCARB1 variants

SNP Namea

SNP IDb

Chr12 Positionc

Location

Amino Acid Change

RegDB Scored

Major/Minor Alleles

MAF

β

SE

R2 (%)

P

FDR

Secondary Trait (Effect)

Top 3 Variants

HDL-C

              

 p20207

rs11057853

125329313

Intron 1

 

5

G/A

0.4484

0.4082

0.1925

1.0650

0.0343

0.4235

  

 p20741

rs11057851

125328779

Intron 1

 

5

C/T

0.3237

−0.5924

0.2067

1.3010

0.0043

0.1465

ApoA-I (↓)

Top 1

 p45516

rs1902569

125304004

Intron 1

 

5

G/A

0.1544

0.5447

0.2629

0.6390

0.0386

0.4375

  

 p49690

rs4765615

125299830

Intron 2

 

5

G/A

0.4426

−0.4646

0.1866

0.9330

0.0130

0.2526

ApoA-I (↓)

 

 p79828

rs838895

125269692

Intron 11

 

5

C/G

0.3171

0.4961

0.2059

0.8220

0.0162

0.2756

ApoA-I (↑)

 

ApoA-I

              

 p20741

rs11057851

125328779

Intron 1

 

5

C/T

0.3237

−1.2331

0.5117

0.8600

0.0162

0.3186

HDL-C (↓)

 

 p49690

rs4765615

125299830

Intron 2

 

5

G/A

0.4426

−0.9139

0.4614

0.6770

0.0480

0.5022

HDL-C (↓)

 

 p55963

rs7134858

125293557

Intron 6

 

6

C/T

0.1560

1.7537

0.6260

1.0710

0.0052

0.2918

 

Top 2

 p63483

rs838912

125286037

Intron 7

 

7

G/A

0.0867

1.8700

0.8230

0.6880

0.0234

0.3972

  

 p64772

rs5888

125284748

Exon 8

Ala350Ala

3a

C/T

0.0961

2.0962

0.7888

0.9460

0.0080

0.2918

 

Top 3

 p79721

rs838896

125269799

Intron 11

 

5

G/C

0.3104

1.1147

0.5056

0.7270

0.0278

0.4197

  

 p79828

rs838895

125269692

Intron 11

 

5

C/G

0.3171

1.2206

0.5074

0.7800

0.0164

0.3186

HDL-C (↑)

 

 p83884

rs701106

125265636

Intron 12

 

5

C/T

0.2597

1.2967

0.5352

0.7770

0.0156

0.3186

  

ApoA-I apolipoprotein A-I, FDR false discovery rate, HDL-C high-density lipoprotein cholesterol, MAF minor allele frequency, RegDB RegulomeDB, SE standard error, SNP single nucleotide polymorphism, UTR untranslated region, R 2 , the proportion of the phenotypic variance explained by the variant; ↓, decreased; ↑, increased

Alleles on reverse strand. HDL-C and ApoA-I variables were in mg/dL and Box-Cox transformed

Results were adjusted for covariates: sex, age, waist, current smoking (yes/no), and minutes of walking or biking to work each day (jobmin) for HDL-C; sex and age for ApoA-I

The most significant P-value for each trait is shown in bold, see the single-site association (−log10 P) plot and pairwise correlations (r 2 ) in Fig. 2

FDR that reached a threshold of <0.20 is shown in bold

a, cRefSeq of SCARB1: hg19, NM_005505 (CHIP Bioinformatics)

bdbSNP build 139: GRCh37.p10

dDetailed RegulomeDB (version 1.0) scoring scheme is described in Additional file 17: Table S12 or at http://regulome.stanford.edu/help, see functional assignments in Additional file 18: Table S13

The most significant association was found between rs11057851 and HDL-C (β = −0.5924, P = 0.0043, FDR = 0.1465). The second best association was between rs7134858 and ApoA-I (β = 1.7537, P = 0.0052, FDR = 0.2918), followed by the association of rs5888 (p.Ala350Ala) with ApoA-I (β = 2.0962, P = 0.0080, FDR = 0.2918).

Of 10 variants that showed nominal associations, high LD (r2 > 0.80) was observed for two pairs of variants (Fig. 2), between rs8388912 and rs5888 (p.Ala350Ala; r2 = 0.86), and between rs838896 and rs838895 (r2 = 0.84).
Fig. 2

Single-site P-values of 94 SCARB1 common variants for HDL-C and ApoA-I. Top: The -log10 P-values are presented in the Y-axis. A total of 94 genotyped variants with MAF ≥5 % are shown on SCARB1 gene (5′ → 3′; RefSeq: hg19, NM_005505) in the X-axis. The dash line indicates the nominal significance threshold (P = 0.05). Middle: Gene structure of SCARB1. Bottom: Linkage disequilibrium (LD) plot of 10 SCARB1 variants with P-values <0.05. Shades and values (r 2  × 100) in each square of LD plot indicate pairwise correlations: black indicating r2 = 1, white indicating r2 = 0, and shade intensity indicating r2 between 0 and 1. Marker names are shown as “SNP name-SNP ID”. SNP ID is based on dbSNP build 139. ApoA-I, apolipoprotein A-I; FDR, false discovery rate; HDL-C, high-density lipoprotein cholesterol; LD, linkage disequilibrium; MAF, minor allele frequency; SNP, single nucleotide polymorphism; UTR, untranslated region

Association analyses of low-frequency/rare SCARB1 variants

The LoF/rare variants (n = 43) were categorized into three groups based on their frequencies for association analysis with HDL-C and ApoA-I using SKAT-O: MAF <5 % (n = 43), MAF ≤2 % (n = 26), and MAF ≤1 % (n = 23). Although no association between LoF/rare variants and ApoA-I was detected, the group of 23 variants with MAF ≤1 % yielded nominal association with HDL-C levels (P = 0.0478; Table 5).
Table 5

Association results for low-frequency and rare SCARB1 variants (MAF <5 %)

MAF

No of Variants

No of Samples with/without Variants

HDL-C

ApoA-I

Stat

P

Stat

P

≤0.01

23a

93/694

126653.8207

0.0478

60151.0985

0.3707

≤0.02

26

134/653

123009.0805

0.1324

48439.6697

0.5166

<0.05

43

442/346

135697.1974

0.0737

298813.0544

0.1517

ApoA-I apolipoprotein A-I, HDL-C high-density lipoprotein cholesterol, MAF minor allele frequency, SD standard deviation, SNP single nucleotide polymorphism

Results were adjusted for covariates: sex, age, waist, current smoking (yes/no), and minutes of walking or biking to work each day (jobmin) for HDL-C; sex and age for ApoA-I

Nominally significant P-values (P < 0.05) are shown in bold

aIncluding p70201/chr12:125279319 that was observed in one individual with an outlier value (above the mean + 3.5 SD). See details in Result Section 3.5

We then individually examined the association of 23 variants with MAF ≤1 % with HDL-C and ApoA-I. Six of these rare variants showed association with either HDL-C levels or both HDL-C and ApoA-I levels (Table 6). While three of them are known variants (rs115604379, rs377124254, and rs141545424 [p.Gly501Gly]), the other three are novel (p52919/chr12:125296601, p54611/chr12:125294909, and p54856/chr12:125294664). Moreover, four of these six rare variants (rs377124254, rs141545424 [p.Gly501Gly], p54611/chr12:125294909, and p54856/chr12:125294664) were present in individuals with extreme phenotypic values (above or below the 3rd percentile). Two of these variants (rs377124254: β = 11.5518, P = 0.0016; rs141545424 [p.Gly501Gly]: β = 11.585, P = 0.0016) were found in a single subject who had very high HDL-C level. Whereas the other two were observed in one individual each, who had extremely low HDL-C levels (p54611/chr12:125294909: β = −9.5243, P = 0.0097; p54856/chr12:125294664: β = −8.4305, P = 0.0215) and ApoA-I levels (p54611/chr12:125294909: β = −19.3821, P = 0.0344; p54856/chr12:125294664: β = −24.0757, P = 0.0082). This rare variant group also included a novel variant (p70201/chr12:125279319) that was observed in one individual with an unusually high plasma HDL-C level (above the mean + 3.5 SD).
Table 6

Characteristics and effects of 6 SCARB1 rare variants of interest

SNP Namea

SNP IDb

Chr12 Positionc

Location

Amino Acid Change

RegDB Scored

Major/Minor Alleles

MAF

GT

GT Count (Carrier Freq)

Adjusted Mean ± SD (mg/dL)

β

SE

R2 (%)

P

FDR

Second Assoc Trait (Effect)

HDL-C

                

 p52919

 

125296601

Intron 4

 

5

G/T

0.0013

GG

734

47.87 ± 12.71

−7.4063

2.5863

1.1050

0.0043

0.1465

ApoA-I (↓)

        

GT

2 (0.27)

24.67 ± 9.26

      

 p53372

rs115604379

125296148

Intron 5

 

5

C/T

0.0066

CC

729

47.68 ± 12.64

3.0372

1.1642

0.9140

0.0093

0.2190

 
        

CT

10 (1.35)

58.2 ± 13.03

      

 p54611

 

125294909

Intron 5

 

4

T/C

0.0007

TT

742

47.86 ± 12.68

−9.5243

3.6710

0.8920

0.0097

0.2190

ApoA-I (↓)

        

TC

1 (0.13)

19.59 ± NA

      

 p54856

 

125294664

Intron 6

 

4

C/T

0.0007

CC

742

47.85 ± 12.70

−8.4305

3.6579

0.7130

0.0215

0.3243

ApoA-I (↓)

        

CT

1 (0.13)

21.48 ± NA

      

 p77620

rs377124254

125271900

Intron 10

5

G/A

0.0007

GG

735

47.77 ± 12.67

11.5518

3.6514

1.3500

0.0016

0.1104

 
        

GA

1 (0.14)

90.2 ± NA

      

 p82264

rs141545424

125267256

Exon 12

Gly501Gly

5

C/A

0.0007

CC

739

47.77 ± 12.66

11.5850

3.6469

1.3530

0.0016

0.1104

 
        

CA

1 (0.14)

90.31 ± NA

      

ApoA-I

                

 p52919

 

125296601

Intron 4

 

5

G/T

0.0013

GG

741

136.81 ± 27.74

−13.4137

6.4689

0.5750

0.0385

0.4359

HDL-C (↓)

        

GT

2 (0.27)

97.42 ± 18.38

      

 p54611

 

125294909

Intron 5

 

4

T/C

0.0007

TT

748

136.83 ± 27.66

−19.2831

9.0970

0.5980

0.0344

0.4359

HDL-C (↓)

        

TC

1 (0.13)

80.62 ± NA

      

 p54856

 

125294664

Intron 6

 

4

C/T

0.0007

CC

748

136.87 ± 27.61

−24.0757

9.0781

0.9330

0.0082

0.2918

HDL-C (↓)

        

CT

1 (0.13)

67.98 ± NA

      

ApoA-I apolipoprotein A-I, FDR false discovery rate, GT genotype, HDL-C high-density lipoprotein cholesterol, MAF minor allele frequency, RegDB RegulomeDB, SD standard deviation, SE standard error, SNP single nucleotide polymorphism; R 2 , the proportion of the phenotypic variance explained by the variant; ↓, decreased

All alleles were on reverse stand. HDL-C and ApoA-I variables were in mg/dL and Box-Cox transformed

Results were adjusted for covariates: sex, age, waist, current smoking (yes/no), and minutes of walking or biking to work each day (jobmin) for HDL-C; sex and age for ApoA-I.

Detailed single-site association results are shown in Additional file 14: Table S9 and Additional file 15: Table S10.

a, cRefSeq of SCARB1: hg19, NM_005505 (CHIP Bioinformatics)

bdbSNP build 139: GRCh37.p10. All 10 novel variants identified in this study have been submitted to dbSNP (batch ID: SCARB1_AB): http://www.ncbi.nlm.nih.gov/SNP/snp_viewTable.cgi?handle=KAMBOH

dThe RegulomeDB (version 1.0) scoring scheme and functional assignments are described in Additional file 17: Table S12 and Additional file 18: Table S13, respectively

Haplotype association analyses

The 4-SNP sliding window haplotype analyses revealed associations of 32 haplotype windows with HDL-C and/or ApoA-I (global P < 0.05; Table 7; see results for each trait in Additional file 16: Table S11), of which five (windows #47, #72, #111, #112, and #123) were associated with both.
Table 7

Significant haplotype association (global P < 0.05) of 136 SCARB1 genotyped variants with HDL-C and ApoA-I

Wind #

SNP 1 - SNP 4

Chr12 Positionc

Location

Amino Acid Change

Major/ Minor Alleles

MAF

β

Single-site P

Haplotype #

Hap Seq

Hap Freq

Coef

SE

t.stat

Hap P

Global P

(SNP Namea-SNP IDb/Chr12 Posc)

HDL-C

               

39

p41632-rs6488943

125307888

Intron 1

 

A/C

0.2954

−0.2195

0.3244

h39.1

CCGG

0.0315

0.4305

0.6471

0.6654

0.5060

0.0207

39

p42467-rs11057830

125307053

Intron 1

 

C/T

0.1523

−0.2810

0.3015

h39.2

CCGA

0.2508

−0.5918

0.2725

−2.1713

0.0302

 

39

p45516-rs1902569

125304004

Intron 1

 

G/A

0.1544

0.5447

0.0386

h39.3

ATGA

0.1414

−0.6841

0.3192

−2.1433

0.0324

 

39

p45627-rs12297372

125303893

Intron 1

 

A/G

0.0487

−0.0483

0.9156

h39.4

ACAA

0.1514

0.1991

0.2963

0.6720

0.5018

 
         

h39.5

ACGG

0.0155

−1.7144

0.9080

−1.8880

0.0594

 
         

h39.6 (rare)

****

0.0148

2.5239

1.0902

2.3151

0.0209

 
         

hap.base39

ACGA

0.3946

NA

NA

NA

NA

 

44

p48969-rs2343394

125300551

Intron 2

 

C/T

0.1898

0.3165

0.1788

h44.1

TCWG

0.1855

0.5292

0.2523

2.0977

0.0363

0.0271

44

p49537-rs7305310

125299983

Intron 2

 

C/T

0.1007

−0.3396

0.2566

h44.2

CCDG

0.2244

0.4676

0.2429

1.9249

0.0546

 

44

p49570delC-rs145376237

125299950

Intron 2

 

W/D

0.2276

0.3121

0.1773

h44.3

CCWG

0.0446

1.0491

0.4882

2.1489

0.0320

 

44

p49690-rs4765615

125299830

Intron 2

 

G/A

0.4426

−0.4646

0.0130

h44.4

CTWG

0.1018

−0.1197

0.3121

−0.3835

0.7015

 
         

h44.5 (rare)

****

0.0089

−0.9887

1.0998

−0.8990

0.3689

 
         

hap.base44

CCWA

0.4348

NA

NA

NA

NA

 

45

p49537-rs7305310

125299983

Intron 2

 

C/T

0.1007

−0.3396

0.2566

h45.1

CDGC

0.2282

0.4661

0.2393

1.9473

0.0519

0.0155

45

p49570delC-rs145376237

125299950

Intron 2

 

W/D

0.2276

0.3121

0.1773

h45.2

CWGC

0.2302

0.6926

0.2376

2.9146

0.0037

 

45

p49690-rs4765615

125299830

Intron 2

 

G/A

0.4426

−0.4646

0.0130

h45.3

TWGC

0.1020

−0.0653

0.3085

−0.2115

0.8325

 

45

p49759-rs146272788

125299761

Intron 2

 

C/T

0.0020

2.5988

0.2219

h45.4 (rare)

****

0.0030

2.0667

2.0848

0.9913

0.3219

 
         

hap.base45

CWAC

0.4366

NA

NA

NA

NA

 

46

p49570delC-rs145376237

125299950

Intron 2

 

W/D

0.2276

0.3121

0.1773

h46.1

DGCG

0.2228

0.4373

0.2413

1.8123

0.0703

0.0278

46

p49690-rs4765615

125299830

Intron 2

 

G/A

0.4426

−0.4646

0.0130

h46.2

WGCG

0.3311

0.4910

0.2105

2.3326

0.0199

 

46

p49759-rs146272788

125299761

Intron 2

 

C/T

0.0020

2.5988

0.2219

h46.3 (rare)

****

0.0080

1.9089

1.0569

1.8061

0.0713

 

46

p49978-rs5891

125299542

Exon 3

Val135lle

G/A

0.0058

1.3374

0.2791

hap.base46

WACG

0.4381

NA

NA

NA

NA

 

47

p49690-rs4765615

125299830

Intron 2

 

G/A

0.4426

−0.4646

0.0130

h47.1

ACGG

0.4346

−0.4701

0.1824

−2.5777

0.0101

0.0079

47

p49759-rs146272788

125299761

Intron 2

 

C/T

0.0020

2.5988

0.2219

h47.2 (rare)

****

0.0101

1.4683

0.9441

1.5552

0.1203

 

47

p49978-rs5891

125299542

Exon 3

Val135lle

G/A

0.0058

1.3374

0.2791

hap.base47

GCGG

0.5553

NA

NA

NA

NA

 

47

p50024-rs368880622

125299496

Intron 3

 

G/T

0.0026

1.6506

0.4362

        

63

p53359-rs112371713

125296161

Intron 5

 

G/A

0.1243

0.4193

0.1651

h63.1

ACGA

0.1237

0.3273

0.3011

1.0871

0.2773

0.0394

63

p53372-rs115604379

125296148

Intron 5

 

C/T

0.0066

3.0372

0.0093

h63.2

GCGG

0.0427

−0.1630

0.4738

−0.3441

0.7309

 

63

p53790-rs4765614

125295730

Intron 5

 

G/A

0.2653

−0.3281

0.1218

h63.3

GCAA

0.2678

−0.2408

0.2194

−1.0975

0.2728

 

63

p54445-rs60910935

125295075

Intron 5

 

A/G

0.0418

−0.1247

0.7963

h63.4 (rare)

****

0.0068

2.9428

1.2559

2.3432

0.0194

 
         

hap.base63

GCGA

0.5591

NA

NA

NA

NA

 

72

p55923-rs838900

125293597

Intron 6

 

G/A

0.3921

0.2787

0.1549

h72.1

ACAG

0.2725

0.4039

0.2520

1.6024

0.1095

0.0315

72

p55963-rs7134858

125293557

Intron 6

 

C/T

0.1560

0.4418

0.0799

h72.2

ACGG

0.1086

−0.1763

0.3929

−0.4486

0.6538

 

72

p56845-rs838902

125292675

Intron 6

 

A/G

0.4249

−0.0786

0.6801

h72.3

GTAG

0.1284

0.3877

0.3170

1.2228

0.2218

 

72

p57004-rs187562853

125292516

Intron 6

 

G/A

0.0098

1.6474

0.0872

h72.4

GTGG

0.0297

0.8722

0.6546

1.3323

0.1832

 
         

h72.5

GCAG

0.1716

−0.4913

0.3344

−1.4690

0.1422

 
         

h72.6 (rare)

****

0.0101

1.7731

0.9506

1.8653

0.0625

 
         

hap.base72

GCGG

0.2791

NA

NA

NA

NA

 

111

p78747-rs2293440

125270773

Intron 11

T/C

0.4112

−0.1684

0.3806

h111.1

CCCG

0.0306

0.7458

0.5599

1.3321

0.1832

0.0040

111

p78791-rs75289200

125270729

Intron 11

T/C

0.0321

0.7037

0.2078

h111.2

CTGC

0.1534

−0.5556

0.2830

−1.9629

0.0500

 

111

p79721-rs838896

125269799

Intron 11

G/C

0.3104

0.3565

0.0817

h111.3

CTCG

0.2269

0.1234

0.2391

0.5162

0.6058

 

111

p79828-rs838895

125269692

Intron 11

C/G

0.3171

0.4961

0.0162

h111.4

TTGG

0.0180

2.3022

0.7617

3.0225

0.0026

 
         

h111.5

TTCG

0.0439

0.5755

0.5317

1.0823

0.2795

 
         

h111.6

TTCC

0.0145

0.9606

0.8068

1.1907

0.2342

 
         

h111.7 (rare)

****

0.0033

0.7755

2.1917

0.3538

0.7236

 
         

hap.base111

TTGC

0.5094

NA

NA

NA

NA

 

112

p78791-rs75289200

125270729

Intron 11

T/C

0.0321

0.7037

0.2078

h112.1

CCGA

0.0311

0.7440

0.5559

1.3384

0.1812

0.0055

112

p79721-rs838896

125269799

Intron 11

G/C

0.3104

0.3565

0.0817

h112.2

TGGA

0.0171

2.3734

0.7506

3.1621

0.0016

 

112

p79828-rs838895

125269692

Intron 11

C/G

0.3171

0.4961

0.0162

h112.3

TGCA

0.0112

−1.2672

0.9074

−1.3964

0.1630

 

112

p80045-rs838893

125269475

Intron 11

G/A

0.3244

0.3127

0.1224

h112.4

TCGA

0.2704

0.2488

0.2164

1.1501

0.2505

 
         

h112.5

TCCG

0.0139

1.1219

0.8186

1.3704

0.1710

 
         

h112.6 (rare)

****

0.0068

1.6244

1.2691

1.2800

0.2009

 
         

hap.base112

TGCG

0.6493

NA

NA

NA

NA

 

113

p79721-rs838896

125269799

Intron 11

G/C

0.3104

0.3565

0.0817

h113.1

GGAG

0.0171

2.3949

0.7509

3.1895

0.0015

0.0048

113

p79828-rs838895

125269692

Intron 11

C/G

0.3171

0.4961

0.0162

h113.2

GCAG

0.0120

−1.1963

0.8784

−1.3619

0.1736

 

113

p80045-rs838893

125269475

Intron 11

G/A

0.3244

0.3127

0.1224

h113.3

CGAG

0.2996

0.3071

0.2067

1.4861

0.1377

 

113

p81863-rs185445624

125267657

Intron 11

G/A

0.0020

−0.9612

0.6510

h113.4

CCGG

0.0139

1.1509

0.8168

1.4090

0.1592

 
         

h113.5 (rare)

****

0.0081

1.1622

1.0896

1.0666

0.2865

 
         

hap.base113

GCGG

0.6493

NA

NA

NA

NA

 

114

p79828-rs838895

125269692

Intron 11

C/G

0.3171

0.4961

0.0162

h114.1

GAGC

0.3173

0.3755

0.2023

1.8559

0.0639

0.0447

114

p80045-rs838893

125269475

Intron 11

G/A

0.3244

0.3127

0.1224

h114.2

CGGT

0.0306

−0.8840

0.5344

−1.6541

0.0985

 

114

p81863-rs185445624

125267657

Intron 11

G/A

0.0020

−0.9612

0.6510

h114.3

CAGC

0.0111

−1.2612

0.9170

−1.3754

0.1694

 

114

p82019-rs838890

125267501

Intron 11

C/T

0.0320

−1.0051

0.0618

h114.4 (rare)

****

0.0086

0.9073

1.0936

0.8296

0.4070

 
         

hap.base114

CGGC

0.6325

NA

NA

NA

NA

 

117

p82019-rs838890

125267501

Intron 11

C/T

0.0320

−1.0051

0.0618

h117.1

CCAG

0.0238

−1.0596

0.6275

−1.6884

0.0917

0.0433

117

p82264-rs141545424

125267256

Exon 12

Gly501Gly

C/A

0.0007

11.5850

0.0016

h117.2

TCGG

0.0311

−0.9657

0.5302

−1.8215

0.0689

 

117

p82340-rs77483223

125267180

Intron 12

G/A

0.0231

−1.0458

0.1012

h117.3 (rare)

****

0.0067

1.6191

1.2946

1.2507

0.2114

 

117

p82369-rs75446635

125267151

Intron 12

G/A

0.0059

0.5896

0.6322

hap.base117

CCGG

0.9383

NA

NA

NA

NA

 

118

p82264-rs141545424

125267256

Exon 12

Gly501Gly

C/A

0.0007

11.5850

0.0016

h118.1

CAGT

0.0238

−1.0621

0.6274

−1.6929

0.0909

0.0375

118

p82340-rs77483223

125267180

Intron 12

G/A

0.0231

−1.0458

0.1012

h118.2

CGGC

0.0307

−1.0134

0.5313

−1.9073

0.0569

 

118

p82369-rs75446635

125267151

Intron 12

G/A

0.0059

0.5896

0.6322

h118.3 (rare)

****

0.0067

1.6189

1.2762

1.2685

0.2050

 

118

p82434-rs838889

125267086

Intron 12

T/C

0.0315

−1.0389

0.0526

hap.base118

CGGT

0.9387

NA

NA

NA

NA

 

123

p83884-rs701106

125265636

Intron 12

C/T

0.2597

0.2471

0.2601

h123.1

TCCT

0.0256

−1.2114

0.6218

−1.9483

0.0518

0.0386

123

p86245-rs188375019

125263275

Intron 12

C/T

0.0341

0.7447

0.1639

h123.2

TCCG

0.2327

0.5306

0.2403

2.2085

0.0275

 

123

p86276-rs747155

125263244

Intron 12

C/T

0.1495

0.2793

0.2980

h123.3

CCTG

0.1476

0.3955

0.2811

1.4071

0.1598

 

123

p86316-rs701104

125263204

Intron 12

G/T

0.0487

−0.9838

0.0286

h123.4

CCCT

0.0233

−0.2329

0.7038

−0.3309

0.7408

 
         

h123.5

CTCG

0.0330

0.8888

0.5458

1.6283

0.1039

 
         

h123.6 (rare)

****

0.0029

1.1191

3.2961

0.3395

0.7343

 
         

hap.base123

CCCG

0.5348

NA

NA

NA

NA

 

124

p86245-rs188375019

125263275

Intron 12

C/T

0.0341

0.7447

0.1639

h124.1

CTGA

0.1476

0.1530

0.2692

0.5683

0.5700

0.0368

124

p86276-rs747155

125263244

Intron 12

C/T

0.1495

0.2793

0.2980

h124.2

CCTG

0.0465

−1.1879

0.4699

−2.5281

0.0117

 

124

p86316-rs701104

125263204

Intron 12

G/T

0.0487

−0.9838

0.0286

h124.3

CCGA

0.0915

0.1086

0.3376

0.3218

0.7477

 

124

p86481-rs701103

125263039

Exon 13-3' UTR

Gly499Arg (isoform 2)

G/A

0.2451

0.1642

0.4492

h124.4

TCGG

0.0337

0.7348

0.5362

1.3702

0.1710

 
         

h124.5 (rare)

****

0.0045

4.0859

2.1131

1.9336

0.0535

 
         

hap.base124

CCGG

0.6761

NA

NA

NA

NA

 

125

p86276-rs747155

125263244

Intron 12

C/T

0.1495

0.2793

0.2980

h125.1

TGAA

0.1476

0.1543

0.2689

0.5737

0.5664

0.0307

125

p86316-rs701104

125263204

Intron 12

G/T

0.0487

−0.9838

0.0286

h125.2

CTGA

0.0465

−1.1980

0.4691

−2.5535

0.0109

 

125

p86481-rs701103

125263039

Exon 13-l3' UTR

Gly499Arg (isoform 2)

G/A

0.2451

0.1642

0.4492

h125.3

CGAA

0.0915

0.1139

0.3375

0.3375

0.7359

 

125

p86967-rs187492239

125262553

Exon 13-3' UTR

 

A/G

0.0355

0.7743

0.1412

h125.4

CGGG

0.0352

0.7974

0.5241

1.5216

0.1285

 
         

h125.5 (rare)

****

0.0045

4.0989

2.1134

1.9394

0.0528

 
         

hap.base125

CGGA

0.6747

NA

NA

NA

NA

 

ApoA-I

               

47

p49690-rs4765615

125299830

Intron 2

 

G/A

0.4426

−0.9139

0.0480

h47.1

ACGG

0.4351

−0.8907

0.4584

−1.9432

0.0524

0.0343

47

p49759-rs146272788

125299761

Intron 2

 

C/T

0.0020

1.5883

0.7630

h47.2 (rare)

****

0.0106

3.5858

2.2998

1.5592

0.1194

 

47

p49978-rs5891

125299542

Exon 3

Val135lle

G/A

0.0058

5.6762

0.0628

hap.base47

GCGG

0.5543

NA

NA

NA

NA

 

47

p50024-rs368880622

125299496

Intron 3

 

G/T

0.0026

1.6012

0.7255

        

48

p49759-rs146272788

125299761

Intron 2

 

C/T

0.0020

1.5883

0.7630

h48.1

CGGT

0.0206

3.3555

1.6564

2.0258

0.0431

0.0293

48

p49978-rs5891

125299542

Exon 3

Val135lle

G/A

0.0058

5.6762

0.0628

h48.2 (rare)

****

0.0106

4.0750

2.3644

1.7235

0.0852

 

48

p50024-rs368880622

125299496

Intron 3

 

G/T

0.0026

1.6012

0.7255

hap.base48

CGGC

0.9688

NA

NA

NA

NA

 

48

p50118-rs58710319

125299402

Intron 3

 

C/T

0.0208

3.1376

0.0571

        

49

p49978-rs5891

125299542

Exon 3

Val135lle

G/A

0.0058

5.6762

0.0628

h49.1

GGTT

0.0213

3.3792

1.6416

2.0584

0.0399

0.0289

49

p50024-rs368880622

125299496

Intron 3

 

G/T

0.0026

1.6012

0.7255

h49.2

GGCC

0.1928

0.8864

0.5841

1.5176

0.1295

 

49

p50118-rs58710319

125299402

Intron 3

 

C/T

0.0208

3.1376

0.0571

h49.3 (rare)

****

0.0086

4.7388

3.1873

1.4868

0.1375

 

49

p50151-rs2278986

125299369

Intron 3

 

T/C

0.1933

0.8568

0.1419

hap.base49

GGCT

0.7774

NA

NA

NA

NA

 

70

p54627-chr12_125294893

125294893

Intron 5

 

G/C

0.0020

3.6910

0.4850

h70.1

GCAC

0.3873

0.8579

0.5090

1.6854

0.0923

0.0140

70

p54856-chr12_125294664

125294664

Intron 6

 

C/T

0.0007

−24.0757

0.0082

h70.2

GCGT

0.1568

2.0940

0.6700

3.1254

0.0018

 

70

p55923-rs838900

125293597

Intron 6

 

G/A

0.3921

0.3606

0.4549

h70.3 (rare)

****

0.0027

−2.5567

5.2200

−0.4898

0.6244

 

70

p55963-rs7134858

125293557

Intron 6

 

C/T

0.1560

1.7537

0.0052

hap.base70

GCGC

0.4532

NA

NA

NA

NA

 

71

p54856-chr12_125294664

125294664

Intron 6

 

C/T

0.0007

−24.0757

0.0082

h71.1

CACA

0.2736

0.7883

0.6210

1.2694

0.2047

0.0488

71

p55923-rs838900

125293597

Intron 6

 

G/A

0.3921

0.3606

0.4549

h71.2

CACG

0.1134

1.1284

0.9724

1.1604

0.2462

 

71

p55963-rs7134858

125293557

Intron 6

 

C/T

0.1560

1.7537

0.0052

h71.3

CGTA

0.1296

2.1103

0.7906

2.6691

0.0078

 

71

p56845-rs838902

125292675

Intron 6

 

A/G

0.4249

−0.3052

0.5129

h71.4

CGTG

0.0300

2.1358

1.6772

1.2734

0.2032

 
         

h71.5

CGCA

0.1706

−0.1013

0.8355

−0.1212

0.9035

 
         

hap.base71

CGCG

0.2822

NA

NA

NA

NA

 

72

p55923-rs838900

125293597

Intron 6

 

G/A

0.3921

0.3606

0.4549

h72.1

ACAG

0.2733

0.7471

0.6218

1.2016

0.2299

0.0463

72

p55963-rs7134858

125293557

Intron 6

 

C/T

0.1560

1.7537

0.0052

h72.2

ACGG

0.1057

0.7094

0.9850

0.7202

0.4716

 

72

p56845-rs838902

125292675

Intron 6

 

A/G

0.4249

−0.3052

0.5129

h72.3

GTAG

0.1297

2.0304

0.7898

2.5707

0.0103

 

72

p57004-rs187562853

125292516

Intron 6

 

G/A

0.0098

3.2853

0.1690

h72.4

GTGG

0.0299

2.1741

1.6857

1.2897

0.1975

 
         

h72.5

GCAG

0.1712

−0.3122

0.8263

−0.3778

0.7057

 
         

h72.6 (rare)

****

0.0100

3.9105

2.4373

1.6044

0.1090

 
         

hap.base72

GCGG

0.2801

NA

NA

NA

NA

 

78

p57592-rs838903

125291928

Intron 7

 

G/A

0.3763

−0.7661

0.1109

h78.1

GCAC

0.0559

1.8913

1.0469

1.8067

0.0712

0.0326

78

p58514-rs838905

125291006

Intron 7

 

T/C

0.4329

−0.4213

0.3646

h78.2

GTAC

0.0367

1.0784

1.2814

0.8415

0.4003

 

78

p58664-rs865716

125290856

Intron 7

 

A/T

0.2708

0.5369

0.3008

h78.3

GTAT

0.2557

0.3365

0.6035

0.5576

0.5773

 

78

p60255-rs3782287

125289265

Intron 7

 

C/T

0.2831

0.3715

0.4856

h78.4

GTTC

0.2463

0.4962

0.5864

0.8462

0.3977

 
         

h78.5

GTTT

0.0238

5.5715

1.6643

3.3477

0.0009

 
         

h78.6 (rare)

****

0.0075

0.6333

2.9303

0.2161

0.8289

 
         

hap.base78

ACAC

0.3740

NA

NA

NA

NA

 

79

p58514-rs838905

125291006

Intron 7

 

T/C

0.4329

−0.4213

0.3646

h79.1

CACT

0.1270

0.3290

0.8318

0.3955

0.6926

0.0256

79

p58664-rs865716

125290856

Intron 7

 

A/T

0.2708

0.5369

0.3008

h79.2

TACC

0.0379

0.6384

1.2921

0.4941

0.6214

 

79

p60255-rs3782287

125289265

Intron 7

 

C/T

0.2831

0.3715

0.4856

h79.3

TATC

0.2563

0.1851

0.6336

0.2921

0.7703

 

79

p61872-rs838909

125287648

Intron 7

 

C/T

0.2199

0.9232

0.1056

h79.4

TTCC

0.1587

−0.6020

0.7769

−0.7749

0.4386

 
         

h79.5

TTCT

0.0880

1.8902

0.8856

2.1342

0.0331

 
         

h79.6

TTTC

0.0238

5.1755

1.6851

3.0714

0.0022

 
         

h79.7 (rare)

****

0.0059

1.2466

3.1079

0.4011

0.6885

 
         

hap.base79

CACC

0.3024

NA

NA

NA

NA

 

80

p58664-rs865716

125290856

Intron 7

 

A/T

0.2708

0.5369

0.3008

h80.1

ACCG

0.0389

−0.3521

1.2793

−0.2753

0.7832

0.0030

80

p60255-rs3782287

125289265

Intron 7

 

C/T

0.2831

0.3715

0.4856

h80.2

ACTG

0.1274

−0.1816

0.7909

−0.2297

0.8184

 

80

p61872-rs838909

125287648

Intron 7

 

C/T

0.2199

0.9232

0.1056

h80.3

ATCG

0.2611

−0.1400

0.6323

−0.2213

0.8249

 

80

p62140-rs838910

125287380

Intron 7

 

G/T

0.3047

−0.0755

0.8821

h80.4

TCCG

0.1549

−1.3614

0.7489

−1.8178

0.0695

 
         

h80.5

TCTG

0.0901

2.0511

0.8921

2.2992

0.0218

 
         

h80.6

TTCG

0.0224

4.7307

1.8842

2.5107

0.0123

 
         

h80.7 (rare)

****

0.0083

3.1429

3.4362

0.9147

0.3607

 
         

hap.base80

ACCT

0.2970

NA

NA

NA

NA

 

81

p60255-rs3782287

125289265

Intron 7

 

C/T

0.2831

0.3715

0.4856

h81.1

CCGC

0.1740

−1.5355

0.7276

−2.1103

0.0352

0.0050

81

p61872-rs838909

125287648

Intron 7

 

C/T

0.2199

0.9232

0.1056

h81.2

CCGT

0.0215

−0.5623

1.6155

−0.3481

0.7279

 

81

p62140-rs838910

125287380

Intron 7

 

G/T

0.3047

−0.0755

0.8821

h81.3

CCTC

0.0352

3.6130

1.4518

2.4886

0.0130

 

81

p62409-rs838911

125287111

Intron 7

 

C/T

0.4211

−0.6245

0.1888

h81.4

CCTT

0.2683

−0.7498

0.6337

−1.1832

0.2371

 
         

h81.5

CTGC

0.0886

1.4787

0.9259

1.5970

0.1107

 
         

h81.6

CTGT

0.1287

−0.2477

0.7967

−0.3109

0.7560

 
         

h81.7 (rare)

****

0.0017

4.9120

8.4190

0.5834

0.5598

 
         

hap.base81

TCGC

0.2819

NA

NA

NA

NA

 

82

p61872-rs838909

125287648

Intron 7

 

C/T

0.2199

0.9232

0.1056

h82.1

CGTT

0.0214

0.3707

1.6055

0.2309

0.8175

0.0137

82

p62140-rs838910

125287380

Intron 7

 

G/T

0.3047

−0.0755

0.8821

h82.2

CTCT

0.0364

3.8641

1.3703

2.8199

0.0049

 

82

p62409-rs838911

125287111

Intron 7

 

C/T

0.4211

−0.6245

0.1888

h82.3

CTTT

0.2692

−0.2007

0.5674

−0.3537

0.7237

 

82

p62615-rs7138386

125286905

Intron 7

 

T/C

0.1137

−0.6495

0.3851

h82.4

TGCT

0.0869

2.1488

0.8777

2.4481

0.0146

 
         

h82.5

TGTT

0.0179

3.0085

1.9599

1.5351

0.1252

 
         

h82.6

TGTC

0.1116

−0.1961

0.7815

−0.2510

0.8019

 
         

h82.7 (rare)

****

0.0020

−4.7635

9.0097

−0.5287

0.5972

 
         

hap.base82

CGCT

0.4546

NA

NA

NA

NA

 

83

p62140-rs838910

125287380

Intron 7

 

G/T

0.3047

−0.0755

0.8821

h83.1

GCTA

0.0854

2.0624

0.8886

2.3211

0.0205

0.0187

83

p62409-rs838911

125287111

Intron 7

 

C/T

0.4211

−0.6245

0.1888

h83.2

GTTG

0.0389

1.3667

1.2527

1.0910

0.2756

 

83

p62615-rs7138386

125286905

Intron 7

 

T/C

0.1137

−0.6495

0.3851

h83.3

GTCG

0.1129

−0.3143

0.7855

−0.4002

0.6891

 

83

p63483-rs838912

125286037

Intron 7

 

G/A

0.0867

1.8700

0.0234

h83.4

TCTG

0.0368

3.8488

1.3757

2.7977

0.0053

 
         

h83.5

TTTG

0.2675

−0.1681

0.5759

−0.2918

0.7705

 
         

h83.6 (rare)

****

0.0031

−0.5696

5.5038

−0.1035

0.9176

 
         

hap.base83

GCTG

0.4554

NA

NA

NA

NA

 

86

p63483-rs838912

125286037

Intron 7

 

G/A

0.0867

1.8700

0.0234

h86.1

ATCG

0.0871

2.5431

0.8550

2.9743

0.0030

0.0290

86

p64772-rs5888

125284748

Exon 8

Ala350Ala

C/T

0.0961

2.0962

0.0080

h86.2

GCAG

0.1457

0.3613

0.6957

0.5194

0.6037

 

86

p64923-rs838915

125284597

Intron 8

 

C/A

0.1435

−0.3684

0.5766

h86.3

GCCA

0.2814

1.0972

0.5782

1.8976

0.0581

 

86

p65999-rs12819677

125283521

Intron 8

 

G/A

0.2813

0.6769

0.2052

h86.4

GTCG

0.0116

1.6563

2.1240

0.7798

0.4357

 
         

hap.base86

GCCG

0.4736

NA

NA

NA

NA

 

95

p71867-rs7954022

125277653

Intron 9

 

C/T

0.1323

0.8502

0.2241

h95.1

TACT

0.1311

0.8202

0.7688

1.0669

0.2864

0.0131

95

p72197-rs838861

125277323

Intron 9

 

A/G

0.3777

−0.1507

0.7464

h95.2

CACC

0.0507

0.3188

1.2809

0.2489

0.8035

 

95

p72777-rs838862

125276743

Intron 9

 

C/T

0.0887

0.7012

0.3938

h95.3

CGCT

0.1846

−0.7832

0.6960

−1.1253

0.2608

 

95

p75766-rs838866

125273754

Intron 9

 

T/C

0.2116

−0.0497

0.9306

h95.4

CGCC

0.1022

0.7176

0.8581

0.8362

0.4033

 
         

h95.5

CGTT

0.0324

4.7525

1.5071

3.1534

0.0017

 
         

h95.6

CGTC

0.0582

−1.3987

1.0854

−1.2887

0.1979

 
         

h95.7 (rare)

****

0.0009

18.2723

NA

NA

NA

 
         

hap.base95

CACT

0.4399

NA

NA

NA

NA

 

96

p72197-rs838861

125277323

Intron 9

 

A/G

0.3777

−0.1507

0.7464

h96.1

ACCT

0.0443

1.0796

1.2832

0.8413

0.4004

0.0484

96

p72777-rs838862

125276743

Intron 9

 

C/T

0.0887

0.7012

0.3938

h96.2

GCTC

0.1849

−0.7979

0.6554

−1.2176

0.2238

 

96

p75766-rs838866

125273754

Intron 9

 

T/C

0.2116

−0.0497

0.9306

h96.3

GCCT

0.0727

−0.3866

0.9478

−0.4079

0.6835

 

96

p75778-rs7301120

125273742

Intron 9

 

C/T

0.1135

0.3767

0.6174

h96.4

GCCC

0.0282

1.9372

1.6107

1.2027

0.2295

 
         

h96.5

GTTC

0.0319

4.2363

1.4400

2.9419

0.0034

 
         

h96.6

GTCC

0.0595

−1.3421

1.0101

−1.3286

0.1844

 
         

h96.7 (rare)

****

0.0058

−3.2342

3.8265

−0.8452

0.3983

 
         

hap.base96

ACTC

0.5728

NA

NA

NA

NA

 

97

p72777-rs838862

125276743

Intron 9

 

C/T

0.0887

0.7012

0.3938

h97.1

CTCT

0.1997

−1.0781

0.6237

−1.7287

0.0843

0.0098

97

p75766-rs838866

125273754

Intron 9

 

T/C

0.2116

−0.0497

0.9306

h97.2

CCTT

0.1141

0.2005

0.7597

0.2639

0.7919

 

97

p75778-rs7301120

125273742

Intron 9

 

C/T

0.1135

0.3767

0.6174

h97.3

CCCT

0.0336

0.7963

1.3894

0.5731

0.5667

 

97

p76757-rs9919713

125272763

Intron 9

 

A/T

0.4390

−0.1860

0.6921

h97.4

TTCT

0.0301

4.3773

1.4494

3.0201

0.0026

 
         

h97.5

TCCT

0.0588

−1.4125

1.0117

−1.3961

0.1631

 
         

h97.6 (rare)

****

0.0050

−6.5869

3.6167

−1.8213

0.0690

 
         

hap.base97

CTCA

0.5587

NA

NA

NA

NA

 

109

p78402-rs838898

125271118

Intron 10

G/A

0.0714

−0.9806

0.2889

h109.1

AGCT

0.0288

−1.4134

1.6436

−0.8600

0.3901

0.0195

109

p78430-rs838897

125271090

Intron 10

C/G

0.3830

−0.1887

0.6887

h109.2

AGTT

0.0451

−1.5093

1.2496

−1.2078

0.2275

 

109

p78747-rs2293440

125270773

Intron 11

T/C

0.4112

−0.2984

0.5352

h109.3

GGCC

0.0317

3.0784

1.3763

2.2366

0.0256

 

109

p78791-rs75289200

125270729

Intron 11

T/C

0.0321

3.6568

0.0086

h109.4

GGCT

0.1633

−0.4126

0.6911

−0.5971

0.5506

 
         

h109.5

GGTT

0.1088

−1.6537

0.8639

−1.9142

0.0560

 
         

h109.6

GCCT

0.1851

−1.8104

0.7168

−2.5256

0.0118

 
         

hap.base109

GCTT

0.4363

NA

NA

NA

NA

 

110

p78430-rs838897

125271090

Intron 10

C/G

0.3830

−0.1887

0.6887

h110.1

GCCC

0.0305

3.0357

1.4224

2.1342

0.0331

0.0012

110

p78747-rs2293440

125270773

Intron 11

T/C

0.4112

−0.2984

0.5352

h110.2

GCTG

0.0189

−3.0973

2.2833

−1.3565

0.1753

 

110

p78791-rs75289200

125270729

Intron 11

T/C

0.0321

3.6568

0.0086

h110.3

GCTC

0.1696

−0.0290

0.6830

−0.0424

0.9662

 

110

p79721-rs838896

125269799

Intron 11

G/C

0.3104

1.1147

0.0278

h110.4

GTTG

0.1400

−2.3158

0.7741

−2.9914

0.0029

 
         

h110.5

GTTC

0.0189

1.3536

2.3385

0.5788

0.5629

 
         

h110.6

CCTG

0.1379

−2.4014

0.7888

−3.0443

0.0024

 
         

h110.7

CCTC

0.0514

−0.8677

1.2628

−0.6871

0.4922

 
         

h110.8

CTTC

0.0398

−0.1892

1.4963

−0.1264

0.8994

 
         

h110.9 (rare)

****

0.0012

7.8235

8.0313

0.9741

0.3303

 
         

hap.base110

CTTG

0.3918

NA

NA

NA

NA

 

111

p78747-rs2293440

125270773

Intron 11

T/C

0.4112

−0.2984

0.5352

h111.1

CCCG

0.0305

3.5704

1.4077

2.5364

0.0114

0.0038

111

p78791-rs75289200

125270729

Intron 11

T/C

0.0321

3.6568

0.0086

h111.2

CTGC

0.1514

−2.1697

0.7058

−3.0742

0.0022

 

111

p79721-rs838896

125269799

Intron 11

G/C

0.3104

1.1147

0.0278

h111.3

CTCG

0.2233

0.3086

0.5985

0.5157

0.6062

 

111

p79828-rs838895

125269692

Intron 11

C/G

0.3171

1.2206

0.0164

h111.4

TTGG

0.0173

1.0502

1.9388

0.5417

0.5882

 
         

h111.5

TTGC

0.0431

0.3464

1.3140

0.2637

0.7921

 
         

h111.6

TTCC

0.0150

0.6429

1.9745

0.3256

0.7448

 
         

h111.7 (rare)

****

0.0047

3.8853

4.0634

0.9562

0.3393

 
         

hap.base111

TTGC

0.5147

NA

NA

NA

NA

 

112

p78791-rs75289200

125270729

Intron 11

T/C

0.0321

3.6568

0.0086

h112.1

CCGA

0.0309

3.7315

1.3947

2.6755

0.0076

0.0412

112

p79721-rs838896

125269799

Intron 11

G/C

0.3104

1.1147

0.0278

h112.2

TGGA

0.0179

1.8646

1.8467

1.0097

0.3130

 

112

p79828-rs838895

125269692

Intron 11

C/G

0.3171

1.2206

0.0164

h112.3

TGCA

0.0109

−3.3720

2.3180

−1.4547

0.1462

 

112

p80045-rs838893

125269475

Intron 11

G/A

0.3244

0.8859

0.0774

h112.4

TCGA

0.2661

0.7087

0.5428

1.3056

0.1921

 
         

h112.5

TCCG

0.0144

1.0316

2.0147

0.5120

0.6088

 
         

h112.6 (rare)

****

0.0068

2.8715

3.2105

0.8944

0.3714

 
         

hap.base112

TGCG

0.6530

NA

NA

NA

NA

 

123

p83884-rs701106

125265636

Intron 12

C/T

0.2597

1.2967

0.0156

h123.1

TCCT

0.0235

−1.7638

1.7393

−1.0141

0.3109

0.0468

123

p86245-rs188375019

125263275

Intron 12

C/T

0.0341

1.8399

0.1674

h123.2

TCCG

0.2351

1.8726

0.6006

3.1179

0.0019

 

123

p86276-rs747155

125263244

Intron 12

C/T

0.1495

−0.2164

0.7433

h123.3

CCTG

0.1485

0.3912

0.6981

0.5604

0.5754

 

123

p86316-rs701104

125263204

Intron 12

G/T

0.0487

−0.6627

0.5579

h123.4

CCCT

0.0238

1.6476

1.7546

0.9390

0.3480

 
         

h123.5

CTCG

0.0328

2.3144

1.3655

1.6949

0.0905

 
         

h123.6 (rare)

****

0.0024

1.2704

8.8153

0.1441

0.8855

 
         

hap.base123

CCCG

0.5340

NA

NA

NA

NA

 

ApoA-I apolipoprotein A-I, Coef coefficient, del/D deletion, HDL-C high-density lipoprotein cholesterol, MAF minor allele frequency, NA not analyzed, SE standard error, SNP single nucleotide polymorphism, UTR untranslated region, W wild type allele for deletion on RefSeq

All alleles on the reverse strand. HDL-C and ApoA-I variables were in mg/dL and Box-Cox transformed

Results were adjusted for covariates: sex, age, waist, current smoking (yes/no), and minutes of daily walking or biking to work (jobmin) for HDL-C; sex and age for ApoA-I

SNP 1-SNP 4 for each window are shown as “SNP name-SNP ID/Chromosome 12 Position (for novel variants)”. All 10 novel variants identified in this study have been submitted to dbSNP database (batch ID: SCARB1_AB): http://www.ncbi.nlm.nih.gov/SNP/snp_viewTable.cgi?handle=KAMBOH.

Nominally significant P-values (P < 0.05) for SNPs with MAF ≥5 % in single-site analysis are shown in bold

Haplotype sequences corresponding to SNP 1-SNP 4 in the 5′ to 3′ direction, respectively

Haplotype association results for all haplotype windows are shown in Additional file 16: Table S11, see haplotype association plots in Fig. 3

a, cRefSeq of SCARB1: hg19, NM_005505 (CHIP Bioinformatics)

bdbSNP build 139: GRCh37.p10

Overall, a total of 21 haplotype windows showed significant associations with ApoA-I, of which 10 contained seven variants associated with ApoA-I in single-site analysis. Haplotype window #110 spanning introns 10–11 showed the best association signal (global P = 0.0012) and contained the rs838896 variant with a nominal evidence of association with ApoA-I (P = 0.0278) in single-site analysis.

A total of 16 haplotype windows yielded significant associations with HDL-C, of which seven contained three HDL-C-associated variants detected in single-site analysis. The most significant association was found with window #111 (global P = 0.0040) spanning intron 11, which contained the rs838895 variant nominally associated with HDL-C (P = 0.0162) in single-site analysis.

We observed nine regions (5 regions for ApoA-I and 4 regions for HDL-C) harboring consecutive significant haplotype windows (global P < 0.05; ranging from 2 to 6 windows per region; Table 8 and Fig. 3). Seven of those regions contained at least one of the six variants that exhibited nominal associations (P < 0.05) with HDL-C and/or ApoA-I (rs4765615, rs7134858, rs838912, rs838896, rs838895, and rs701106) in single-site analysis.
Table 8

Significantly associated haplotype regions (global P < 0.05) with HDL-C and ApoA-I

Region #

Trait

Consecutive Significantly Associated Haplotype Windows (global P < 0.05)

  

Haplotype Windows #

Chr12 Positiona

The Composited Variants in the Region, 5′ to 3′ Direction

Most Relevant Haplotype

(Location)

   

Start (5′)

End (3′)

SNP Nameb-SNP IDc/Chr12 Positiona

Major/Minor Alleles

Haplotype #

Sequence

β (Min-Max)

1

HDL-C

44

125300551

125299542

p48969-rs2343394

C/T

h44.3

CCWGCGG

0.4910–1.0491

 

45

(intron 2)

(exon 3)

p49537-rs7305310

C/T

h45.2

  
 

46

  

p49570delC-rs145376237

W/D

h46.2

  
 

47

  

p49690-rs4765615

G/A

hap.base47

  
    

p49759-rs146272788

C/T

hap.base44

CCWACGG

−0.4701

    

p49978-rs5891 (p.Val135Ile)

G/A

hap.base45

  
    

p50024-rs368880622

G/T

hap.base46

  
      

h47.1

  

2

ApoA-I

47

125299830

125299369

p49690-rs4765615

G/A

h47.1

ACGGTT

(−0.8907)–3.3792

 

48

(intron 2)

(intron 3)

p49759-rs146272788

C/T

h48.1

  
 

49

  

p49978-rs5891 (p.Val135Ile)

G/A

h49.1

  
    

p50024-rs368880622

G/T

   
    

p50118-rs58710319

C/T

   
    

p50151-rs2278986

T/C

   

3

ApoA-I

70

125294893

125292516

p54627-chr12_125294893

G/C

h70.2

GCGTAG

2.0304–2.1103

 

71

(intron 5)

(intron 6)

p54856-chr12_125294664d

C/T

h71.3

  
 

72

  

p55923-rs838900

G/A

h72.3

  
    

p55963-rs7134858

C/T

   
    

p56845-rs838902

A/G

   
    

p57004-rs187562853

G/A

   

4

ApoA-I

78

125291928

125286037

p57592-rs838903

G/A

h78.5

GTTTCGCTG

4.7307–5.5715

 

79

(intron 7)

(intron 7)

p58514-rs838905

T/C

h79.6

  
 

80

  

p58664-rs865716

A/T

h80.6

  
 

81

  

p60255-rs3782287

C/T

hap.base81

  
 

82

  

p61872-rs838909

C/T

hap.base82

  
 

83

  

p62140-rs838910

G/T

hap.base83

  
    

p62409-rs838911

C/T

h78.2

GTACCTCTG

0.6384–3.8641

    

p62615-rs7138386

T/C

h79.2

  
    

p63483-rs838912

G/A

hap.base80

  
      

h81.3

  
      

h82.2

  
      

h83.4

  

5

ApoA-I

95

125277653

125272763

p71867-rs7954022

C/T

h95.5

CGTTCT

4.2363-4.7525

 

96

(intron 9)

(intron 9)

p72197-rs838861

A/G

h96.5

  
 

97

  

p72777-rs838862

C/T

h97.4

  
    

p75766-rs838866

T/C

   
    

p75778-rs7301120

C/T

   
    

p76757-rs9919713

A/T

   

6*

ApoA-I

109

125271118

125269475

p78402-rs838898

G/A

h109.6

GCCTGCA

(−3.3720)─(−1.8104)

 

110

(intron 10)

(intron 11)

p78430-rs838897

C/G

h110.6

  
 

111

  

p78747-rs2293440

T/C

h111.2

  
 

112

  

p78791-rs75289200

T/C

h112.3

  
    

p79721-rs838896

G/C

   
    

p79828-rs838895

C/G

   
    

p80045-rs838893

G/A

   

7*

HDL-C

111

125270773

125267501

p78747-rs2293440

T/C

h111.4

TTGGAGC

0.3755–2.3949

 

112

(intron 11)

(intron 11)

p78791-rs75289200

T/C

h112.2

  
 

113

  

p79721-rs838896

G/C

h113.1

  
 

114

  

p79828-rs838895

C/G

h114.1

  
    

p80045-rs838893

G/A

   
    

p81863-rs185445624

G/A

   
    

p82019-rs838890

C/T

   

8

HDL-C

117

125267501

125267086

p82019-rs838890

C/T

h117.2

TCGGC

(−1.0134)–(−0.9657)

 

118

(intron 11)

(intron 12)

p82264-rs141545424 (p.Gly501Gly)d

C/A

h118.2

  
    

p82340-rs77483223

G/A

   
    

p82369-rs75446635

G/A

   
    

p82434-rs838889

T/C

   

9

HDL-C

123

125265636

125262553

p83884-rs701106

C/T

h123.4

CCCTGA

(−1.180)–(−0.2329)

 

124

(intron 12)

(exon 13-3′ UTR)

p86245-rs188375019

C/T

h124.2

  
 

125

  

p86276-rs747155

C/T

h125.2

  
    

p86316-rs701104

G/T

   
    

p86481-rs701103 (p.Gly499Arg, isoform 2)

G/A

   
    

p86967-rs187492239

A/G

   

ApoA-I apolipoprotein A-I, del/D deletion, HDL-C high-density lipoprotein cholesterol, SNP single nucleotide polymorphism, UTR untranslated region, W wild type allele for deletion on the RefSeq

All alleles on the reverse strand. HDL-C and ApoA-I variables were in mg/dL and Box-Cox transformed

Results were adjusted for covariates: sex, age, waist, current smoking (yes/no), and minutes of daily walking or biking to work (jobmin) for HDL-C; sex and age for ApoA-I

All nine haplotype regions are shown in Fig. 3

Detailed single-site associations are shown in Additional file 14: Table S9 and Additional file 15: Table S10

Detailed haplotype associations are shown in Table 7 and Additional file 16: Table S11

Regions with asterisk (*) indicate regions that included the haplotype window exhibiting the most significant association signal (the smallest global P) for the associated trait

For each region, the most significant associated haplotype window is shown in bold

SNPs with significant evidence of association with the same trait in both single-site and haplotype analyses (single-site P < 0.05 and global P < 0.05) are shown in bold

SNPs with significant evidence of association with different trait in single-site and haplotype analyses (single-site P < 0.05 and global P < 0.05) are shown in italic bold

a, bRefSeq of SCARB1: hg19, NM_005505 (CHIP Bioinformatics)

cdbSNP build 139: GRCh37.p10

dRare variants of interest with potential effects on lipid traits; see details in Table 6

Fig. 3

Haplotype association plots for HDL-C and ApoA-I. Top: The -log10 P-values are presented in the Y-axis. A total of 136 genotyped variants are shown in order on SCARB1 gene (5′ → 3′; RefSeq: hg19, NM_005505) in the X-axis. Middle: gene structure of SCARB1. Marker names are shown as “SNP name-SNP ID/chromosome 12 position (for novel variants)”. Bottom: linkage disequilibrium (LD) plot of 136 variants. SNPs with MAF ≥5 % are shown in bold. SNP ID is based on dbSNP build 139. All 10 novel variants identified in this study have been submitted to dbSNP (batch ID: SCARB1_AB): http://www.ncbi.nlm.nih.gov/SNP/snp_viewTable.cgi?handle=KAMBOH. The dash line indicates the significance threshold (global P = 0.05). Significantly associated haplotype regions are highlighted. The degree of shades and values (r2 × 100) in each square of LD plot represent the pairwise correlations between 136 genotyped variants: black indicating r2 = 1, white indicating r2 = 0, and shade intensity indicating r2 between 0 and 1. ApoA-I, apolipoprotein A-I; HDL-C, high-density lipoprotein cholesterol; LD, linkage disequilibrium; MAF, minor allele frequency; SNP, single nucleotide polymorphism; UTR, untranslated region

Functional evaluation of identified variants

In order to examine the possible regulatory function of all 153 SCARB1 variants (83 variants identified by our sequencing, 68 common HapMap tagSNPs [excluding rs4765180 due to genotyping failure; see Additional file 7: Table S5], and two relevant variants from the literature), we used the RegulomeDB database (version 1.0, Stanford University, http://www.regulomedb.org/) [48]. Although most of 153 variants (n = 132) revealed scores ranging from 1 to 6, only 11 were supported by strong evidence for regulatory function (scores of 1f -2b): one promoter, one 5′ UTR, two coding (rs2070242 [p.Ser4Ser] and rs10396208 [p.Cys21Cys]), five intronic, one 3′ UTR, and one 3′ flanking variants. Summary and detailed regulatory functions are provided in Additional file 17: Table S12 and Additional file 18: Table S13.

Of 10 variants associated with HDL-C and/or ApoA-I, only one ApoA-I associated variant (rs5888 [p.Ala350Ala] in exon 8) showed suggestive evidence of regulatory function with a score of 3a (Table 4).

Of 10 novel variants, one insertion variant (p1048insC/chr12:125348472) located in 5′ UTR-exon 1 had a strong potential for regulatory function with a score of 2a (Additional file 4: Table S4).

Comparison of SCARB1 single-site and haplotype association analysis results between African Blacks (this study) and US Non-Hispanic Whites (previous study [49])

We compared SCARB1 single-site and haplotype association results in African Blacks reported in this study to those in US Non-Hispanic Whites (NHWs) reported in our previously published study [49]. In the sequencing stage, the number of variants identified in African Blacks (n = 83) was greater than that in US NHWs (n = 44). Notably, most (~90 %) of the 22 sequence variants that were shared between the two populations differed in minor alleles and/or MAFs. Although our major findings included the associations with HDL-C and ApoA-I in African Blacks, we also sought to replicate four associations observed with ApoB levels in US NHWs [49] (Table 9); the association between rs11057820 and ApoB (P < 0.05) that we previously reported in US NHWs [49] was also observed in African Blacks (US NHWs [G allele]: β = 0.8700, P = 0.0436; African Blacks [A allele]: β = 1.8661, P = 0.0292). In addition, we observed two variants (rs4765615 and rs701106) exhibiting nominal associations (P < 0.05) in both populations, albeit with different lipid traits (US NHWs| rs4765615 [G allele]: β = 1.2493, P = 0.0059 for ApoB; rs701106 [T allele]: β = 0.0394, P = 0.0066 for HDL-C; African Blacks| rs4765615 [A allele]: β = −0.4646, P = 0.013 for HDL-C and β = −0.9139, P = 0.048 for ApoA-I; rs701106 [T allele]: β = 1.2967, P = 0.0156 for ApoA-I). Moreover, we noticed that two regions associated with HDL-C or ApoA-I (global P < 0.05; Table 10) in African Blacks spanning intron 2 and intron 3 overlapped with the ApoB-associated region (Region I in Fig. 4) previously reported in US NHWs [49]. Three haplotype regions associated with HDL-C (global P < 0.05) spanning intron 11 and exon 13-3′ UTR in African Blacks also overlapped with a large HDL-C-associated region (Region II in Fig. 4) previously reported in US NHWs [49].
Table 9

Results for 7 SCARB1 lipid-associated variants in US Non-Hispanic Whites (previous studya) and in African Blacks (this study)

SNP Nameb

SNP IDc

Chr12 Positiond

Location

RegDB Scoree

Alleles

US Non-Hispanic Whitesa (n = 623)

African Blacks (n = 788)

MA, MAF

β

P

MA, MAF

β

P

Other Assoc Trait(s)f

(SE)

(SE)

HDL-C

            

 p28957

rs11057844

125320563

Intron 1

5

G/A

A, 0.1839

−0.0395

0.0035

A, 0.2362

0.3671

0.1075

 
       

(0.0135)

  

(0.2278)

  

 p83884

rs701106

125265636

Intron 12

5

C/T

T, 0.1527

0.0394

0.0066

T, 0.2597

0.2471

0.2601

ApoA-I

       

(0.0144)

  

(0.2192)

  

 p87927

rs838880

125261593

3′ flanking

5

G/A

G, 0.3237

0.0257

0.0250

A, 0.2414

0.0198

0.9314

 
       

(0.0114)

  

(0.2302)

  

ApoB

            

 p48969

rs2343394

125300551

Intron 2

5

C/T

T, 0.2850

1.2544

0.0082

T, 0.1898

0.0383

0.9544

 
       

(0.4721)

  

(0.6696)

  

 p49690

rs4765615

125299830

Intron 2

5

G/A

G, 0.4497

1.2493

0.0059

A, 0.4426

0.7771

0.1338

HDL-C, ApoA-I

       

(0.4518)

  

(0.5178)

  

 p50151

rs2278986

125299369

Intron 3

5

T/C

C, 0.2890

1.1926

0.0122

C, 0.1933

0.1308

0.8434

 
       

(0.4735)

  

(0.6619)

  

 p52556

rs11057820

125296964

Intron 4

5

G/A

G, 0.4871

0.8700

0.0436

A, 0.1000

1.8661

0.0292

 
       

(0.4300)

  

(0.8542)

  

ApoB apolipoprotein B, HDL-C high-density lipoprotein cholesterol, MA minor allele, MAF minor allele frequency, RegDB RegulomeDB, SE standard error, SNP single nucleotide polymorphism

All alleles on the reverse strand

HDL-C and ApoB values for US Non-Hispanic Whites were in mg/dL, Box-Cox transformed, and adjusted for covariates: sex, age, body mass index, and smoking (past/current/never) for HDL-C; age and smoking for ApoB

HDL-C and ApoB values for African Blacks were in mg/dL, Box-Cox transformed, and adjusted for covariates: sex, age, waist, current smoking (yes/no), and daily walking or biking to work (jobmin) for HDL-C; body mass index and staff status for ApoB

Nominally significant P-values (P < 0.05) are shown in bold

aData from Niemsiri V, et al. Circ Cardiovasc Genet 2014, 7(6):838–847 (Ref [49])

b, dRefSeq of SCARB1: hg19, NM_005505 (CHIP Bioinformatics)

cdbSNP version 139: GRCh37.p10

eThe RegulomeDB (version 1.0) scoring scheme is described at the footnote of Additional file 17: Table S12 or at http://regulome.stanford.edu/help

fEvidence is based on SNPs with MAF ≥5 % exhibiting nominally significant association with either HDL-C or ApoA-I (P < 0.05; Additional file 14: Table S9 and Additional file 15: Table S10) in single-site association results in the current study

Table 10

Significant lipid-associated regions (global P < 0.05) that were observed in US Non-Hispanic Whites (previous studya) and African Blacks (this study)

Region #

Consecutive Haplotype Windows in 623 US Non-Hispanic Whitesa

Consecutive Haplotype Windows in 788 African Blacks

 

Trait

Chr12 Positionb (Location)

Length (bp)

The Composited Variants, 5′ to 3′ Direction

Trait

Chr12 Positionb (Location)

Length (bp)

The Composited Variants, 5′ to 3′ Direction

  

Start (5′)

End (3′)

 

SNP Namec-SNP IDd

Major/Minor Alleles

 

Start (5′)

End (3′)

 

SNP Namec-SNP IDd

Major/Minor Alleles

I

ApoB

125300551

125299369

1183

p48969-rs2343394

C/T

HDL-C

125300551

125299496

1056

p48969-rs2343394

C/T

  

(intron 2)

(intron 3)

 

p49518-rs144194221

G/A

 

(intron 2)

(intron 3)

 

p49537-rs7305310

C/T

     

p49690-rs4765615

A/G

    

p49570delC-rs145376237

W/D

     

p49978-rs5891

G/A

    

p49690-rs4765615

G/A

(p.Val135Ile)

     

p50151-rs2278986

T/C

    

p49759-rs146272788

C/T

           

p49978-rs5891

G/A

(p.Val135Ile)

           

p50024-rs368880622

G/T

       

ApoA-I

125299830

125299369

462

p49690-rs4765615

G/A

        

(intron 2)

(intron 3)

 

p49759-rs146272788

C/T

           

p49978-rs5891

G/A

(p.Val135Ile)

           

p50024-rs368880622

G/T

           

p50118-rs58710319

C/T

           

p50151-rs2278986

T/C

II

HDL-C

125269692

125262516

7177

p79828-rs838895

C/G

HDL-C

125269692

125267501

2192

p79828-rs838895

C/G

  

(intron 11)

(exon 13- 3′ UTR)

 

p80045-rs838893

G/A

 

(intron 11)

(intron 11)

 

p80045-rs838893

G/A

     

p83088-rs797729

A/G

    

p81863-rs185445624

G/A

     

p83884-rs701106

C/T

    

p82019-rs838890

C/T

     

p86436-rs10396214

C/T

HDL-C

125267501

125267086

416

p82019-rs838890

C/T

(p.Arg484Trp, isoform 2)

     

p87004-rs184715678

C/A

 

(intron 11)

(intron 12)

 

p82264-rs141545424

C/A

(p.Gly501Gly)

           

p82340-rs77483223

G/A

           

p82369-rs75446635

G/A

           

p82434-rs838889

T/C

       

HDL-C

125265636

125262553

3084

p83884-rs701106

C/T

        

(intron 12)

(exon 13- 3′ UTR)

 

p86245-rs188375019

C/T

           

p86276-rs747155

C/T

           

p86316-rs701104

G/T

           

p86481-rs701103

G/A

(p.Gly499Arg, isoform 2)

           

p86967-rs187492239

A/G

ApoA-I apolipoprotein A-I, ApoB apolipoprotein B, del/D deletion, HDL-C high-density lipoprotein cholesterol, SNP single nucleotide polymorphism, UTR untranslated region, W wild type allele for deletion on RefSeq

All alleles on the reverse strand

Results for a US Non-Hispanic White sample were Box-Cox transformed, and adjusted for covariates: sex, age, body mass index, and smoking (past/current/never) for HDL-C; age and smoking for ApoB

Results for an African Black sample were Box-Cox transformed, and adjusted for covariates: sex, age, waist, current smoking (yes/no), and minutes of walking or biking to work each day (jobmin) for HDL-C; sex and age for ApoA-I

Location of each region on SCARB1 gene is shown in Fig. 4

SNPs with significant evidence with the same trait in both single-site and haplotype associations (single-site P and global P < 0.05) observed in each population are shown in bold

SNPs with significant evidence with the different trait in single-site and haplotype associations (single-site P and global P < 0.05) in each population are shown in italic bold

aData from Niemsiri V, et al. Circ Cardiovasc Genet 2014, 7(6):838–847 (Ref [49])

b, cRefSeq of SCARB1: hg19, NM_005505 (CHIP Bioinformatics)

ddbSNP version 139: GRCh37.p10

Fig. 4

Lipid-associated SCARB1 common variants and haplotype regions identified in US Non-Hispanic Whites (previous study; Ref [49]) and African Blacks (this study). Lipid-associated variants with MAF ≥5 % with P-values <0.05 and haplotype regions with global P-values < 0.05 that were previously identified in US Non-Hispanic Whites (US NHWs; n = 623) are shown in top panel and those identified in African Blacks (n = 788) are shown in bottom panel (see details in Table 9 and Table 10). SCARB1 variants and haplotype regions are shown on SCARB1 gene (5′ → 3′; RefSeq: hg19, NM_005505). All SNP IDs are based on dbSNP build 139. Regions I and II that are defined based on consecutive haplotype windows with evidence of lipid-association in US NHWs (global P < 0.05; see details in Ref [49]) also show some significant associations in African Blacks (global P < 0.05; see details in Table 7 and Table 8). ApoA-I, apolipoprotein A-I; ApoB, apolipoprotein B; HDL-C, high-density lipoprotein cholesterol; MAF, minor allele frequency; NHW, Non-Hispanic White; SNP, single nucleotide polymorphism; UTR, untranslated region

Discussion

Our sequencing identified 83 variants, of which 78 were selected for follow-up genotyping in the total sample of 788 African Blacks. Additional 69 tagSNPs from the HapMap-YRI data along with two previously reported lipid-associated SCARB1 variants were also genotyped in the total sample. Of 149 genotyped SCARB1 variants, 137 that passed QC were examined for association with major lipid traits (Table 2). The initial gene-based analyses revealed a nominal association with HDL-C (P = 0.0421) as well as a trend for association with ApoA-I (P = 0.1016; Table 3). Consistent with the gene-based results, single-site association analyses also revealed 10 common variants nominally associated (P < 0.05) with HDL-C (n = 5) and/or ApoA-I (n = 8; Table 4 and Fig. 2). The best association signal was between rs11057851 in intron 1 and HDL-C (P = 0.0043, FDR = 0.1465) followed by two associations with ApoA-I including rs7134858 in intron 6 (P = 0.0052, FDR = 0.2918) and rs5888 (p.Ala350Ala) in exon 8 (P = 0.0080, FDR = 0.2918). Moreover, three variants (rs11057851, rs4765615, and rs838895) exhibited evidence of associations (P < 0.05) with both HDL-C and ApoA-I. These findings are supported by the fact that SCARB1 appears to influence ApoA-I in addition to HDL-C [15, 17]. In our data, there was a moderate correlation between ApoA-I and HDL-C levels (r2 = 0.61).

Except for previously reported association of rs5888 (p.Ala350Ala) with lipid traits (HDL-C or LDL-C) in non-African populations [3034, 36, 37, 39], the remaining nine associations observed in this study with the lipid traits (HDL-C and/or ApoA-I levels) in general population are novel and await replication in independent African or African-derived populations. Two of these nine SNPs have previously been shown to have differential effects on cholesterol levels in response to statin (rs4765615) [50] or on HDL-C/TG levels in response to estradiol in post-menopausal women (rs838895) [51]. Another variant (rs838896) was found to be associated with decreased SCARB1 expression in liver [51]. Although the latter SNP was not associated with a low RegulomeDB score (<3), we cannot rule out the possibility that it might be affecting the SCARB1 expression in a tissue-specific manner.

The haplotype analysis revealed evidence of significant association (global P < 0.05) of 32 haplotype windows with HDL-C (n = 16) and/or ApoA-I (n = 21; Table 7) and nine regions harboring consecutive overlapping haplotype windows significantly associated with either HDL-C (4 regions) or ApoA-I (5 regions; Table 8 and Fig. 3). In addition, six variants with nominal association (P < 0.05) in single-site analysis were contained in seven of these nine significantly associated regions, indicating the presence of functional variants in these regions. Our findings demonstrate that haplotype analysis may provide more information than single-site analysis.

Our comparison of the single-site and haplotype association results between in African Blacks (this study) and US NHWs (previous study [49]) has revealed three variants (rs11057820, rs4765615, and rs701106; Table 9) and two regions (Regions I and II; Table 10 and Fig. 4) showing evidence of lipid-associations in both ethnic groups. However, there were differences in associated traits, and/or associated alleles or their directional effects between the two ethnic groups, which reflects the genetic heterogeneity of complex phenotypes like lipid traits among diverse populations. This phenomenon can be explained by different ancestry backgrounds associated with differences in LD structure and genetic architecture, as well as by differences in SNP-SNP, gene-gene, and gene-environment interactions. Nonetheless, the lipid associations observed across different ethnic populations provide convincing evidence that causal/functional variants are present in SCARB1 gene that deserves comprehensive sequencing and functional studies in order to confirm and further characterize the effects of its variants on lipid metabolism.

Rare variant analysis showed significant evidence of association between a group of 23 rare variants (MAF ≤1 %) and HDL-C (P = 0.0478; Table 5). Single-site analysis of these rare variants revealed six (including three novel ones) with effects on HDL-C, of which three also had effects on ApoA-I (Table 6). In addition, four of these six rare variants appeared to be carried by individuals with extreme HDL-C and/or ApoA-I levels (above or under the 3rd percentile). This HDL-C-associated rare variant group also included a novel variant (p70201/chr12:125279319) that was observed in one individual with an unusually high plasma HDL-C level (above the mean + 3.5 SD). Our findings suggest that these rare variants might have functional relevance, thus screening of additional large African samples for these rare variants may help to establish their role in HDL-C and ApoA-I metabolism.

To date, there has been limited information concerning possible functional effects of lipid-associated SCARB1 variants, particularly for those located in non-coding regions. In fact, most of common and rare HDL-C/ApoA-I-associated variants observed in the current study are non-coding and do not show strong evidence of regulatory function based on RegulomeDB database. Nonetheless, three of these HDL-C/ApoA-I-associated SCARB1 variants (rs5888 [p.Ala350Ala], rs838885, and rs838886) have been previously demonstrated to influence the SCARB1 expression [5153]. Therefore, additional functional studies are needed and may help to determine the functional nature of the SCARB1-associated variants and those in LD with them.

Our study has revealed a number of novel findings, although we also acknowledge some limitations. SCARB1 is a large gene and we sequenced only its coding regions and exon-intron junctions and also our sequencing sample size was small. Thus, we may have missed some functional LoF/rare variants due to small sample size and those located in uncovered intronic regions. Moreover, consistent with generally small effect sizes of lipid-associated variants reported in the literature, most of our single-site associations reached nominal significance (P < 0.05) but did not survive multiple testing corrections. Only the top variant (rs11057851) associated with HDL-C yielded an FDR cut-off of <0.20 (FDR = 0.1465; Table 4). Therefore, future larger studies in independent African or African-derived populations are necessary to validate all nominal associations observed in this study.

Conclusions

In conclusion, we report the first comprehensive association study of SCARB1 variants with lipid traits in a native African population, which revealed a number of novel associations in single-site and haplotype analyses. In addition, resequencing allowed us to identify 10 novel rare variants, of which four were in the group of 23 rare variants that has showed association with HDL-C levels. The SCARB1 associated common and rare variants observed in our study explained ~11.09 % of the variation in HDL-C levels and ~8.63 % of the variation in ApoA-I levels. Our findings indicate the genetic contribution of SCARB1, both common and LoF/rare variants, to inter-individual lipid variation in the general African Black population, which warrants further follow-up in independent studies. Insights into the HDL-C and related lipid traits may also lead to new potential targets for CHD treatment.

Notes

Abbreviations

ApoA-I: 

Apolipoprotein A-I

ApoB: 

Apolipoprotein B

BP: 

Base pair

CE: 

Cholesteryl esters

CHD: 

Coronary heart disease

FDR: 

False discovery rate

GWAS: 

Genome-wide association studies

HDL-C: 

High-density lipoprotein cholesterol

HWE: 

Hardy-Weinberg equilibrium

indel: 

Insertion and deletion variation

KB: 

Kilobase pair

LD: 

Linkage disequilibrium

LDL-C: 

Low-density lipoprotein cholesterol

LoF: 

Low-frequency

MAF: 

Minor allele frequency

NHW: 

Non-Hispanic White

PCR: 

Polymerase chain reaction

QC: 

Quality controls

RCT: 

Reverse cholesterol transport

SCARB1: 

Scavenger receptor class B member 1

SD: 

Standard deviation

SKAT-O: 

An optimal sequence kernel association test

SNP: 

Single nucleotide polymorphism

SNV: 

Singlenucleotide variation

TG: 

Triglycerides

UTR: 

Untranslated region

VEGAS: 

Versatile gene-based association study

VLDL-C: 

Very low-density lipoprotein cholesterol

YRI: 

Yoruba people of Ibadan from Nigeria

Declarations

Acknowledgements

This study was supported by the National Heart, Lung and Blood Institute [NHLBI] (grant numbers HL044413 to C.H.B. and HL084613 to M.I.K.).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh
(2)
Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh

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