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

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.

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) [4-6]. 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) [4-8]. 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 [9-11].

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 [15-21].

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 [24-26]. Whereas the overexpression of SCARB1 reduces levels of HDL-C, ApoA-I, very low-density lipoprotein cholesterol (VLDL-C), LDL-C, and ApoB [15–17, 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, 28–39] 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 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 ≥ 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 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, 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) 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

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 × 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

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).

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, 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

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

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

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

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 [30–34, 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 [51-53]. 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.