Abstract

Coronary artery disease (CAD) is a complex disorder involving both genetic and non-genetic factors. Genome-wide association studies (GWAS) have identified hundreds of single nucleotides polymorphisms (SNPs) tagging over > 40 CAD risk loci. We hypothesized that some non-coding variants might directly regulate the gene expression rather than tagging a nearby locus. We used RegulomeDB to examine regulatory functions of 58 SNPs identified in two GWAS and those SNPs in linkage disequilibrium (LD) (r2 ≥ 0.80) with the GWAS SNPs. Of the tested 1200 SNPs, 858 returned scores of 1–6 by RegulomeDB. Of these 858 SNPs, 97 were predicted to have regulatory functions with RegulomeDB score of < 3. Notably, only 8 of the 97 predicted regulatory variants were genome-wide significant SNPs (LIPA/rs2246833, RegulomeDB score = 1b; ZC3HC1/rs11556924, CYP17A1-CNNM2-NT5C2/rs12413409, APOE-APOC1/rs2075650 and UBE2Z/rs46522, each with a RegulomeDB score = 1f; ZNF259-APOA5-APOA1/rs964184, SMG6/rs2281727 and COL4A1-COL4A2/rs4773144, each with a RegulomeDB score = 2b). The remainder 89 functional SNPs were in linkage disequilibrium with GWAS SNPs. This study supports the hypothesis that some of the non-coding variants are true signals via regulation of gene expression at transcription level. Our study indicates that RegulomeDB is a useful database to examine the putative functions of large number of genetic variants and it may help to identify a true association among multiple tagged SNPs in a complex disease, such as CAD.

Background

Most human DNA sequence is non-coding (98%) and hence only small portion (2%) of human genome encodes proteins (1). Although the pathogenesis of monogenic disorders is largely explained, it has been difficult to determine the underlying mechanisms of complex disorders like coronary artery disease (CAD). Before the development of genome-wide association studies (GWAS), only the APOE*4 allele showed consistent association with the risk of CAD across many populations (2–5).

The hypothesis-free GWAS approach was designed with the assumption that common DNA variants explain the bulk of the variation in common diseases (6). About 90% of GWAS-implicated variants, exert only minimal to modest effect sizes on disease phenotypes, and they are present in non-coding rather than coding regions (7). Highly sensitive molecular and computational techniques have identified different regulatory elements (DNAse hypersensitive regions, sequences affecting the binding of transcription factors and promoters or enhancers) in intergenic regions (8). Common variants located in one of these regulatory elements may affect gene expression. To predict the role of these variants in gene regulation and to differentiate between physically tagged and functional single nucleotides polymorphism (SNPs), many databases have been created (9). RegulomeDB is one of such databases that describes the role of these variants in transcriptional regulation.

Similar to many other complex diseases, GWAS have identified hundreds of risk variants associated with CAD that need to be analyzed for their functional role in gene expression (10). Recently, we have used SNAP Webportal and Regulome DB to identify potential regulatory function of variants in associated risk loci for Alzheimer’s disease (11). In this study, we have applied the same approach to identify the regulatory nature of GWAS-implicated variants with CAD and those that are in linkage disequilibrium (LD) with these variants.

Objective

The objective of our study was to assess the GWAS-implicated CAD variants and those variants in LD with GWAS variants for their potential regulatory effects on gene transcription using bioinformatics tools.

Materials and methods

SNPs selection

A total of 58 SNPs within 54 CAD loci was selected, including 52 with accepted genome-wide significant threshold (P < 5 × 10−8) and 6 with suggestive associations (P > 5 × 10−8) identified in two GWAS (12, 13). Detailed information on the selected 58 SNPs is provided in Supplementary Table S1.

Linkage disequilibrium

For the LD assessment of the selected 58 SNPs, we used SNAP web portal (https://www.broadinstitute.org/mpg/snap/, accessed 13 July 2016) (14) (Supplementary Table S2). SNAP contains data from the Northern European from Utah (CEU) population derived from the 1000 Genomes Pilot Project 1 and three different releases of the International-Hap Map Project. We used data from both the 1000 Genomes Project and HapMap 3 (release 2) to identify SNPs in strong LD (r2 ≥ 0.80) with our SNPs of interest. We did not select an array bound search, and query SNPs were included in the output. We performed the search at three thresholds—r2 ≥ 0.80, r2 ≥ 0.90 and r2 ≥ 1.0—for both SNP datasets and identified a total of 1,200 SNPs in LD with the 58 published GWAS SNPs, including the GWAS SNPs themselves. As shown in Table 1, the number of proxy SNPs decreased with the increased level of r2.

Table 1.

Number of SNPs in LD for all published GWAS SNPs for HapMap3 and 1000 genomes populations at tested r2 threshold

LD
r2threshold0.800.901.0
1000 Genomes1176928480
Hap Map321015774
Total (overlaps removed)1200934485
LD
r2threshold0.800.901.0
1000 Genomes1176928480
Hap Map321015774
Total (overlaps removed)1200934485
Table 1.

Number of SNPs in LD for all published GWAS SNPs for HapMap3 and 1000 genomes populations at tested r2 threshold

LD
r2threshold0.800.901.0
1000 Genomes1176928480
Hap Map321015774
Total (overlaps removed)1200934485
LD
r2threshold0.800.901.0
1000 Genomes1176928480
Hap Map321015774
Total (overlaps removed)1200934485

Functional assessment of CAD-associated SNPs

We used RegulomeDB to identify potentially functional SNPs among the 1200 SNPs of interest. Regulome DB is a database that scores SNPs functionality based upon experimental data, such as its existence in a DNAase hypersensitive site or transcription factor binding site. These regions have been characterized biochemically, and data are drawn from published literature, Gene Expression Omnibus and ENCODE project that include a total of 962 experimental datasets, covering over 100 tissues and cell lines and representing nearly 60 million annotations. The output data can be mapped to Human genome version 19. It is a user friendly and freely accessible database (http://www.regulomedb.org/accessed 17 July 2016) (15). The functional Grades 1–6 of RegulomeDB are given in Table 2. SNPs showing the strongest evidence of being regulatory (affecting the binding of transcription factor) are given a score of 1 and SNPs demonstrating the least evidence of being functional are given a score of 6.

Table 2.

RegulomeDB category summaries (15)

CategoryDescription
Likely to affect binding and linked to expression of a gene target
1beQTL + TF binding + any motif + DNase footprint + DNase peak
1ceQTL + TF binding + matched TF motif + DNase peak
1deQTL + TF binding + any motif + DNase peak
1eeQTL + TF binding + matched TF motif
1feQTL + TF binding/DNase peak
Likely to affect binding
2aTF binding + matched TF motif + matched DNase footprint + DNase peak
2bTF binding + any motif + DNase footprint + DNase peak
2cTF binding + matched TF motif + DNase peak
Less likely to affect binding
3aTF binding + any motif + DNase peak
3bTF binding + matched TF motif
Minimal binding evidence
4TF binding + DNase peak
5TF binding or DNase peak
6Motif hit
CategoryDescription
Likely to affect binding and linked to expression of a gene target
1beQTL + TF binding + any motif + DNase footprint + DNase peak
1ceQTL + TF binding + matched TF motif + DNase peak
1deQTL + TF binding + any motif + DNase peak
1eeQTL + TF binding + matched TF motif
1feQTL + TF binding/DNase peak
Likely to affect binding
2aTF binding + matched TF motif + matched DNase footprint + DNase peak
2bTF binding + any motif + DNase footprint + DNase peak
2cTF binding + matched TF motif + DNase peak
Less likely to affect binding
3aTF binding + any motif + DNase peak
3bTF binding + matched TF motif
Minimal binding evidence
4TF binding + DNase peak
5TF binding or DNase peak
6Motif hit
Table 2.

RegulomeDB category summaries (15)

CategoryDescription
Likely to affect binding and linked to expression of a gene target
1beQTL + TF binding + any motif + DNase footprint + DNase peak
1ceQTL + TF binding + matched TF motif + DNase peak
1deQTL + TF binding + any motif + DNase peak
1eeQTL + TF binding + matched TF motif
1feQTL + TF binding/DNase peak
Likely to affect binding
2aTF binding + matched TF motif + matched DNase footprint + DNase peak
2bTF binding + any motif + DNase footprint + DNase peak
2cTF binding + matched TF motif + DNase peak
Less likely to affect binding
3aTF binding + any motif + DNase peak
3bTF binding + matched TF motif
Minimal binding evidence
4TF binding + DNase peak
5TF binding or DNase peak
6Motif hit
CategoryDescription
Likely to affect binding and linked to expression of a gene target
1beQTL + TF binding + any motif + DNase footprint + DNase peak
1ceQTL + TF binding + matched TF motif + DNase peak
1deQTL + TF binding + any motif + DNase peak
1eeQTL + TF binding + matched TF motif
1feQTL + TF binding/DNase peak
Likely to affect binding
2aTF binding + matched TF motif + matched DNase footprint + DNase peak
2bTF binding + any motif + DNase footprint + DNase peak
2cTF binding + matched TF motif + DNase peak
Less likely to affect binding
3aTF binding + any motif + DNase peak
3bTF binding + matched TF motif
Minimal binding evidence
4TF binding + DNase peak
5TF binding or DNase peak
6Motif hit

Results

Among the 1200 SNPs evaluated with RegulomeDB, 342 had no data (Supplementary Table S3). Of the 858 SNPs for which RegulomeDB provided a score, 97 had a score of <3 (likely to affect the binding) and among these only 8 SNPs were genome-wide significant, including LIPA/rs2246833 (RegulomeDB score = 1b; eQTL in monocytes), ZC3HC1/rs11556924 (RegulomeDB score = 1f; eQTL in monocytes), CYP17A1-CNNM2-NT5C2/rs12413409 (RegulomeDB score = 1f; eQTL in monocytes and lymphoblasts), APOE-APOC1/rs2075650, and UBE2Z/rs46522 (RegulomeDB score = 1f; eQTL in monocytes), ZNF259-APOA5-APOA1/rs964184, UBE2Z/rs46522, SMG6/rs2281727, COL4A1-COL4A2/rs4773144 (RegulomeDB score =2b; eQTLs in monocytes and lymphoblasts). A flow chart summarizes these results (Figure 1) . The remaining 89 SNPs with RegulomeDB scores < 3 were not identified in GWAS but they were in LD (r2 ≥ 0.80) with the 29 GWAS reported SNPs. A summary of the regulatory SNPs in LD with GWAS SNPs is provided in Table 3.

Table 3.

Functional SNPs (RegluomeDB Score < 3) in LD (r2 ≥ 0.80) with published GWAS SNPs

GWAS SNPsFunctional proxy SNPsRegulome DB score
LIPA/rs2246833LIPA/rs13323272b
LIPA/rs13323282b
LIPA/rs14124441d
LIPA/rs2246833a1b
LIPA/rs22506442b
ZC3HC1/rs11556924ZC3HC1/rs11556924a1f
CYP17A1-CNNM2-NT5C2/rs12413409AS3MT/rs111914541f
BORCS7-ASMT/rs44097661f
CNNM2/rs108838081f
MAT2A/rs14466682a
NT5C2/rs108838321f
CNNM2/rs111914791f
NT5C2/rs111915571f
CNNM2/rs111914991f
NT5C2/rs111915581f
CNNM2/rs111915141f
NT5C2/rs111915801f
CNNM2/rs111915151f
NT5C2/rs111915821f
CNNM2/rs122210642b
NT5C2/rs124120381f
CNNM2/rs124118861f
NT5C2/rs124130461f
CNNM2/rs12413409a1f
NT5C2/rs96337121e
CNNM2/rs171152131f
NT5C2/rs111915481f
CNNM2/rs22977872a
CNNM2/rs37812851f
CNNM2/rs9430371f
CNNM2/rs122199012b
APOE-APOC1/TOMM40/rs2075650APOE-APOC1/rs2075650a1f
UBE2Z/rs46522GIP/rs22917251f
GIP/rs47940041d
SNF8/rs19949701f
SNF8/rs47939921f
UBE2Z/rs126016722b
UBE2Z/rs155631f
UBE2Z/rs37446082a
UBE2Z/rs38484601f
UBE2Z/rs46522a1f
UBE2Z/rs110798441f
ZNF259-APOA5-APOA1/rs964184ZNF259-APOA5-APOA1/rs9641841f
SMG6/rs2281727SMG6/rs2281727a2b
SMG6/rs72176872b
SMG6/rs99088882b
COL4A1-COL4A2/rs4773144COL4A1-COL4A2/rs4773144a2b
ABO/rs579459ABO/rs6491292b
ADMTS7/rs7173743LOC105370915/rs50299042b
PHACTR1/rs47731432b
CXCL12/rs501120CXCL12/rs5185942b
CXCL12/rs17460522b
FURIN-FES/rs17514846FES/rs18944011b
HHIPL1/rs2895811HHIPL1/rs283915272b
HHIPL1/rs46241072b
HHIPL1/rs71452622b
IL6R/rs4845625IL6R/rs75492502b
IL6R/rs75493382b
IL6R/rs75537962b
KCNE2/rs9982601KCNE2/rs285914152b
KIAA1462/rs2505083KIAA1462/rs37399982b
LPL/rs264LPL/rs2711f
LPL/rs37797882b
MIA3/rs17465637MIA3/rs171633012b
PLG/rs4252120PLG/rs42521261f
PLG/rs42521351f
PPAP2B/rs17114036LOC101929929/rs726643042a
PLPP3/rs46349321f
SLC22A4-SLC22A5/rs273909SLC22A5/rs176895501f
SMG6/rs72176872b
SMG6/rs99088882b
SORT1/rs602633CELSR2/rs127403742b
CELSR2/rs6293011f
CELSR2/rs6467761f
TRIB1/rs2954029LOC105375745/rs29808532b
LOC105375745/rs20018442b
LOC105375745/rs69826362b
TRIB1/rs29808562b
VAMP5-VAMP8-GGCX/rs1561198GGCX/rs67386451f
GGCX/rs101874241f
VAMP8/rs10091b
GGCX/rs65476211f
VAMP8/rs13488181f
GGCX/rs28867221f
VAMP8/rs37700981f
VAMP8/rs67572631f
WDR12/rs6725887ICA1L/rs729347152b
NBEAL1/rs23515241f
WDR12/rs729368522b
NBEAL1/rs46753101f
NBEAL1/rs729345122b
REST-NOA1/rs17087335REST/rs22279011f
REST-NOA1/rs76877671d
SWAP70/rs10840293SWAP70/rs931381f
SWAP70/rs3601361f
SMAD3/rs56062135SMAD3/rs172936322a
SMAD3/rs18663162b
MTERF1/rs80327392b
CDKN2BAS1/rs1333049CDKN2BAS1/rs49775742c
GWAS SNPsFunctional proxy SNPsRegulome DB score
LIPA/rs2246833LIPA/rs13323272b
LIPA/rs13323282b
LIPA/rs14124441d
LIPA/rs2246833a1b
LIPA/rs22506442b
ZC3HC1/rs11556924ZC3HC1/rs11556924a1f
CYP17A1-CNNM2-NT5C2/rs12413409AS3MT/rs111914541f
BORCS7-ASMT/rs44097661f
CNNM2/rs108838081f
MAT2A/rs14466682a
NT5C2/rs108838321f
CNNM2/rs111914791f
NT5C2/rs111915571f
CNNM2/rs111914991f
NT5C2/rs111915581f
CNNM2/rs111915141f
NT5C2/rs111915801f
CNNM2/rs111915151f
NT5C2/rs111915821f
CNNM2/rs122210642b
NT5C2/rs124120381f
CNNM2/rs124118861f
NT5C2/rs124130461f
CNNM2/rs12413409a1f
NT5C2/rs96337121e
CNNM2/rs171152131f
NT5C2/rs111915481f
CNNM2/rs22977872a
CNNM2/rs37812851f
CNNM2/rs9430371f
CNNM2/rs122199012b
APOE-APOC1/TOMM40/rs2075650APOE-APOC1/rs2075650a1f
UBE2Z/rs46522GIP/rs22917251f
GIP/rs47940041d
SNF8/rs19949701f
SNF8/rs47939921f
UBE2Z/rs126016722b
UBE2Z/rs155631f
UBE2Z/rs37446082a
UBE2Z/rs38484601f
UBE2Z/rs46522a1f
UBE2Z/rs110798441f
ZNF259-APOA5-APOA1/rs964184ZNF259-APOA5-APOA1/rs9641841f
SMG6/rs2281727SMG6/rs2281727a2b
SMG6/rs72176872b
SMG6/rs99088882b
COL4A1-COL4A2/rs4773144COL4A1-COL4A2/rs4773144a2b
ABO/rs579459ABO/rs6491292b
ADMTS7/rs7173743LOC105370915/rs50299042b
PHACTR1/rs47731432b
CXCL12/rs501120CXCL12/rs5185942b
CXCL12/rs17460522b
FURIN-FES/rs17514846FES/rs18944011b
HHIPL1/rs2895811HHIPL1/rs283915272b
HHIPL1/rs46241072b
HHIPL1/rs71452622b
IL6R/rs4845625IL6R/rs75492502b
IL6R/rs75493382b
IL6R/rs75537962b
KCNE2/rs9982601KCNE2/rs285914152b
KIAA1462/rs2505083KIAA1462/rs37399982b
LPL/rs264LPL/rs2711f
LPL/rs37797882b
MIA3/rs17465637MIA3/rs171633012b
PLG/rs4252120PLG/rs42521261f
PLG/rs42521351f
PPAP2B/rs17114036LOC101929929/rs726643042a
PLPP3/rs46349321f
SLC22A4-SLC22A5/rs273909SLC22A5/rs176895501f
SMG6/rs72176872b
SMG6/rs99088882b
SORT1/rs602633CELSR2/rs127403742b
CELSR2/rs6293011f
CELSR2/rs6467761f
TRIB1/rs2954029LOC105375745/rs29808532b
LOC105375745/rs20018442b
LOC105375745/rs69826362b
TRIB1/rs29808562b
VAMP5-VAMP8-GGCX/rs1561198GGCX/rs67386451f
GGCX/rs101874241f
VAMP8/rs10091b
GGCX/rs65476211f
VAMP8/rs13488181f
GGCX/rs28867221f
VAMP8/rs37700981f
VAMP8/rs67572631f
WDR12/rs6725887ICA1L/rs729347152b
NBEAL1/rs23515241f
WDR12/rs729368522b
NBEAL1/rs46753101f
NBEAL1/rs729345122b
REST-NOA1/rs17087335REST/rs22279011f
REST-NOA1/rs76877671d
SWAP70/rs10840293SWAP70/rs931381f
SWAP70/rs3601361f
SMAD3/rs56062135SMAD3/rs172936322a
SMAD3/rs18663162b
MTERF1/rs80327392b
CDKN2BAS1/rs1333049CDKN2BAS1/rs49775742c
a

GWAS significant SNPs with functional evidence (RegulomeDB score < 3) are bolded.

Table 3.

Functional SNPs (RegluomeDB Score < 3) in LD (r2 ≥ 0.80) with published GWAS SNPs

GWAS SNPsFunctional proxy SNPsRegulome DB score
LIPA/rs2246833LIPA/rs13323272b
LIPA/rs13323282b
LIPA/rs14124441d
LIPA/rs2246833a1b
LIPA/rs22506442b
ZC3HC1/rs11556924ZC3HC1/rs11556924a1f
CYP17A1-CNNM2-NT5C2/rs12413409AS3MT/rs111914541f
BORCS7-ASMT/rs44097661f
CNNM2/rs108838081f
MAT2A/rs14466682a
NT5C2/rs108838321f
CNNM2/rs111914791f
NT5C2/rs111915571f
CNNM2/rs111914991f
NT5C2/rs111915581f
CNNM2/rs111915141f
NT5C2/rs111915801f
CNNM2/rs111915151f
NT5C2/rs111915821f
CNNM2/rs122210642b
NT5C2/rs124120381f
CNNM2/rs124118861f
NT5C2/rs124130461f
CNNM2/rs12413409a1f
NT5C2/rs96337121e
CNNM2/rs171152131f
NT5C2/rs111915481f
CNNM2/rs22977872a
CNNM2/rs37812851f
CNNM2/rs9430371f
CNNM2/rs122199012b
APOE-APOC1/TOMM40/rs2075650APOE-APOC1/rs2075650a1f
UBE2Z/rs46522GIP/rs22917251f
GIP/rs47940041d
SNF8/rs19949701f
SNF8/rs47939921f
UBE2Z/rs126016722b
UBE2Z/rs155631f
UBE2Z/rs37446082a
UBE2Z/rs38484601f
UBE2Z/rs46522a1f
UBE2Z/rs110798441f
ZNF259-APOA5-APOA1/rs964184ZNF259-APOA5-APOA1/rs9641841f
SMG6/rs2281727SMG6/rs2281727a2b
SMG6/rs72176872b
SMG6/rs99088882b
COL4A1-COL4A2/rs4773144COL4A1-COL4A2/rs4773144a2b
ABO/rs579459ABO/rs6491292b
ADMTS7/rs7173743LOC105370915/rs50299042b
PHACTR1/rs47731432b
CXCL12/rs501120CXCL12/rs5185942b
CXCL12/rs17460522b
FURIN-FES/rs17514846FES/rs18944011b
HHIPL1/rs2895811HHIPL1/rs283915272b
HHIPL1/rs46241072b
HHIPL1/rs71452622b
IL6R/rs4845625IL6R/rs75492502b
IL6R/rs75493382b
IL6R/rs75537962b
KCNE2/rs9982601KCNE2/rs285914152b
KIAA1462/rs2505083KIAA1462/rs37399982b
LPL/rs264LPL/rs2711f
LPL/rs37797882b
MIA3/rs17465637MIA3/rs171633012b
PLG/rs4252120PLG/rs42521261f
PLG/rs42521351f
PPAP2B/rs17114036LOC101929929/rs726643042a
PLPP3/rs46349321f
SLC22A4-SLC22A5/rs273909SLC22A5/rs176895501f
SMG6/rs72176872b
SMG6/rs99088882b
SORT1/rs602633CELSR2/rs127403742b
CELSR2/rs6293011f
CELSR2/rs6467761f
TRIB1/rs2954029LOC105375745/rs29808532b
LOC105375745/rs20018442b
LOC105375745/rs69826362b
TRIB1/rs29808562b
VAMP5-VAMP8-GGCX/rs1561198GGCX/rs67386451f
GGCX/rs101874241f
VAMP8/rs10091b
GGCX/rs65476211f
VAMP8/rs13488181f
GGCX/rs28867221f
VAMP8/rs37700981f
VAMP8/rs67572631f
WDR12/rs6725887ICA1L/rs729347152b
NBEAL1/rs23515241f
WDR12/rs729368522b
NBEAL1/rs46753101f
NBEAL1/rs729345122b
REST-NOA1/rs17087335REST/rs22279011f
REST-NOA1/rs76877671d
SWAP70/rs10840293SWAP70/rs931381f
SWAP70/rs3601361f
SMAD3/rs56062135SMAD3/rs172936322a
SMAD3/rs18663162b
MTERF1/rs80327392b
CDKN2BAS1/rs1333049CDKN2BAS1/rs49775742c
GWAS SNPsFunctional proxy SNPsRegulome DB score
LIPA/rs2246833LIPA/rs13323272b
LIPA/rs13323282b
LIPA/rs14124441d
LIPA/rs2246833a1b
LIPA/rs22506442b
ZC3HC1/rs11556924ZC3HC1/rs11556924a1f
CYP17A1-CNNM2-NT5C2/rs12413409AS3MT/rs111914541f
BORCS7-ASMT/rs44097661f
CNNM2/rs108838081f
MAT2A/rs14466682a
NT5C2/rs108838321f
CNNM2/rs111914791f
NT5C2/rs111915571f
CNNM2/rs111914991f
NT5C2/rs111915581f
CNNM2/rs111915141f
NT5C2/rs111915801f
CNNM2/rs111915151f
NT5C2/rs111915821f
CNNM2/rs122210642b
NT5C2/rs124120381f
CNNM2/rs124118861f
NT5C2/rs124130461f
CNNM2/rs12413409a1f
NT5C2/rs96337121e
CNNM2/rs171152131f
NT5C2/rs111915481f
CNNM2/rs22977872a
CNNM2/rs37812851f
CNNM2/rs9430371f
CNNM2/rs122199012b
APOE-APOC1/TOMM40/rs2075650APOE-APOC1/rs2075650a1f
UBE2Z/rs46522GIP/rs22917251f
GIP/rs47940041d
SNF8/rs19949701f
SNF8/rs47939921f
UBE2Z/rs126016722b
UBE2Z/rs155631f
UBE2Z/rs37446082a
UBE2Z/rs38484601f
UBE2Z/rs46522a1f
UBE2Z/rs110798441f
ZNF259-APOA5-APOA1/rs964184ZNF259-APOA5-APOA1/rs9641841f
SMG6/rs2281727SMG6/rs2281727a2b
SMG6/rs72176872b
SMG6/rs99088882b
COL4A1-COL4A2/rs4773144COL4A1-COL4A2/rs4773144a2b
ABO/rs579459ABO/rs6491292b
ADMTS7/rs7173743LOC105370915/rs50299042b
PHACTR1/rs47731432b
CXCL12/rs501120CXCL12/rs5185942b
CXCL12/rs17460522b
FURIN-FES/rs17514846FES/rs18944011b
HHIPL1/rs2895811HHIPL1/rs283915272b
HHIPL1/rs46241072b
HHIPL1/rs71452622b
IL6R/rs4845625IL6R/rs75492502b
IL6R/rs75493382b
IL6R/rs75537962b
KCNE2/rs9982601KCNE2/rs285914152b
KIAA1462/rs2505083KIAA1462/rs37399982b
LPL/rs264LPL/rs2711f
LPL/rs37797882b
MIA3/rs17465637MIA3/rs171633012b
PLG/rs4252120PLG/rs42521261f
PLG/rs42521351f
PPAP2B/rs17114036LOC101929929/rs726643042a
PLPP3/rs46349321f
SLC22A4-SLC22A5/rs273909SLC22A5/rs176895501f
SMG6/rs72176872b
SMG6/rs99088882b
SORT1/rs602633CELSR2/rs127403742b
CELSR2/rs6293011f
CELSR2/rs6467761f
TRIB1/rs2954029LOC105375745/rs29808532b
LOC105375745/rs20018442b
LOC105375745/rs69826362b
TRIB1/rs29808562b
VAMP5-VAMP8-GGCX/rs1561198GGCX/rs67386451f
GGCX/rs101874241f
VAMP8/rs10091b
GGCX/rs65476211f
VAMP8/rs13488181f
GGCX/rs28867221f
VAMP8/rs37700981f
VAMP8/rs67572631f
WDR12/rs6725887ICA1L/rs729347152b
NBEAL1/rs23515241f
WDR12/rs729368522b
NBEAL1/rs46753101f
NBEAL1/rs729345122b
REST-NOA1/rs17087335REST/rs22279011f
REST-NOA1/rs76877671d
SWAP70/rs10840293SWAP70/rs931381f
SWAP70/rs3601361f
SMAD3/rs56062135SMAD3/rs172936322a
SMAD3/rs18663162b
MTERF1/rs80327392b
CDKN2BAS1/rs1333049CDKN2BAS1/rs49775742c
a

GWAS significant SNPs with functional evidence (RegulomeDB score < 3) are bolded.

58 GWAS ANPs in LD with 1200 SNPs. We used SNAP webportal to determine LD SNPs. These 1200 SNPs were further evaluated by RegulomeDB to identify their functional role. RegulomeDB did not provide data for 342 SNPs. A total of 858 SNPs returned the scores of 1–6 by RegulomeDB. Of those 858 SNPs, 97 returned the scores of < 3. Among 97 functional SNPs, only 8 were GWAS SNPs. Lower the RegulomeDB score, more evidence of functionality.
Figure 1.

58 GWAS ANPs in LD with 1200 SNPs. We used SNAP webportal to determine LD SNPs. These 1200 SNPs were further evaluated by RegulomeDB to identify their functional role. RegulomeDB did not provide data for 342 SNPs. A total of 858 SNPs returned the scores of 1–6 by RegulomeDB. Of those 858 SNPs, 97 returned the scores of < 3. Among 97 functional SNPs, only 8 were GWAS SNPs. Lower the RegulomeDB score, more evidence of functionality.

Overall, we had 97 functional SNPs (RegulomeDB < 3). Eight of these were GWAS SNPs, and the remaining 89 were in LD (r2 ≥ 0.80) with the GWAS SNPs.

Three variants, FES/rs1894401, LIPA/rs2246833 and VAMP8/rs1009, were strongly predicted to be functional with score of 1b. FES/rs1894401 is an intronic SNP that is an eQTL for FES in thyroid and transformed lymphoblasts, is present in the binding motif of Pax5, and affects the binding of eleven transcription factors. LIPA/rs2246833 (RegulomeDB score = 1b), located in Intron 6 of LIPA, in the DNA motif of EWSRCFLI1, is a GWAS reported SNP along with 4 other functional SNPs (of 12 tested) in this region and it and it affects the binding of CTCF. It is an eQTL in the whole blood. VAMP5-VAMP8-GGCX/rs1009 is in exon 3 of VAMP8 and affects the binding of CTCF and HSF1. rs1009 of VAMP8 is an eQTL in lymhoblasts, skeletal muscles, adipose tissue and thyroid. Of 42 SNPs analyzed in this locus, we found 8 other SNPs with RegulomeDB score < 3 (Table 3).

There were 33 functional SNPs within 15 GWAS identified CAD loci: ABO (1of 10 assessed), ADAMTS7 (1 of 15 assessed), CXCL12 (2 of 36 assessed), HHIPL1 (3 of 17 assessed), KCNE2(2 of 18 assessed), KIAA1462 (1 of 9 assessed), MIA3 (1 of 27 assessed), PPAP2B (2 of 22 assessed), SORT1 (3 of 9 assessed), WDR12 (5 of 214 assessed), IL6R (3 of 14 assessed), LPL (2 of 6 assessed), PLG (2 of 41 assessed), SLC22A4-SLC22A5 (1 of 2 assessed) and TRIB1 (4 of 16 assessed).

Of 97 SNPs with RegulomeDB score < 3, 25 were in the CYP17A1-CNNM2-NT5C2 region, and one of them was a GWAS reported SNP (rs12413409). The regional LD plot of this SNP is given in Supplementary Figure S1. rs9633712 (RegulomeDB score = 1e) is located in Intron 3 of NT5C2 and is an eQTL for USMG5 in monocytes. This SNP was also found in the motifs of the following transcription factors: PU1, ELF1, Sfpil, PU.1 and c-Ets-1. It appears to affect the binding of SPI1. Twenty SNPs returned a score of 1f (likely to affect the binding), and 18 of them were in intronic regions. NT5C2/rs11191558 lies in HOXC series of DNA motifs, and CNNM2/rs3781285 lies between NF-kappaB and P50:50. NT5C2/rs2297787 returned a score of 2a, affecting the binding motifs of FOXI1, HNF3-alpha and FOXP1 and the binding of FOXA1. SNP rs12412038 is located in Intron 10 of NT5C2 and is in the binding motif of Irx. The remaining two SNPs, rs12219901 and rs12221064, lie in the CNNM2-NT5C2 intergenic region and upstream of CNNM2, respectively. They are located in DNA motifs of SRF and MAZR and affect the binding of POLRA2 and CTCF/ETS. Interestingly, rs943037 resides in exon 7 of CNNM2. Nineteen of the 25 SNPs in the region of CYP17A1-CNNM2-NT5C2 are eQTLs for USMG5 (Table 4).

Table 4.

Putative functional SNPs and corresponding motifs, eQTL and related transcription factors (Regulome DB score < 3)

Coordinate 0-basedSNP IDRegulomeDB scoreGene/LocusPositioneQTLMotifProtein-binding
chr15:91429041rs18944011bFESIntron 2FESPax5SPI1
USF1
POLR2A
GABPA
BHLHE40
CEBPB
CTCF
MAX
RFX5
RUNX3
STAT5A
chr10:91005853rs22468331bLIPAIntron 6LIPAEWSR-FLI1CTCF
znf143
chr2:85808736rs10091bVAMP8Exon 3VAMP8CTCF
LOC388969HSF1
chr17:47038470rs47940041dGIPIntron 4ATP5G1Gata5NR3C1
UBE2ZIN3AK20
CREB1
TAF1
TCF12
CTCF
POLR2A
USF1
FOXA1
FOXA2
RBBP5
chr10:91002926rs14124441dLIPAIntron3LIPASAP1aATF2
ELK1FOXM1
ELK3SP1
ELK4SPI1
MECP2MTA3
ERFRUNX3
ERG
ETS1
ETV1
ETV2
ETV3
Gabpa
chr10:104873760rs96337121eNT5C2Intron 3UMG5PU1SP11
ELF-1
Sfpil
PU.1
c-Ets-1
chr11:116648916rs9641841fZPR1Downstream ZRP1TAGLNFOXJ2
chr1:109818529rs6467761fCELSR2Upstream CELSR2PSMA5CTCF
HEY1
REST
POLR2A
ZBTB7A
TAF7
chr10:104616662rs44097661fBORCS7-ASMTIntron 1C10orf77Tcf3BACH1
USMG5MAFF
MAFK
chr17:47008206rs47939921fSNF8Intron 7ATP5G1POLR2A
UBE2ZTEAD4
chr6:161152293rs42521261fPLGIntron 11PLGCTCF
RUNX3
TEAD4
RAD21
chr6:161154231rs42521351fPLGIntron 12PLGCTCF
FOXA1
NFKB1
RAD21
ZNF263
SMC3
ZNF143
FOXA2
chr10:104846177rs111915481fNT5C2 gene regionDownstream NT5C2USMG5TEAD1
TEAD3
chr10:104864613rs111915571fNT5C2Intron 5USMG5
chr10:104864677rs111915581fNT5C2Intron 5USMG5HOXC13
Hoxa13
Hoxc13
Hoxd12
HOXA13
HOXD9
HOXC11
chr10:104871203rs124130461fNT5C2Intron 3USMG5NR3C1
TRIM28
CTCF
ATF2
IKZF1
TCF7L2
ZNF263
chr10:104871278rs108838321fNT5C2Intron 3USMG5TRIM28
TCF7L2
chr10:104913652rs111915821fNT5C2Intron 2USMG5EP300
NFIC
TCF12
TEAD4
STAT1
ARID3A
EP300
JUN
RCOR1
chr10:104906210rs111915801fNT5C2Intron 2USMG5TRIM28
SETDB1
GATA1
GTF2F1
CEBPB
FOS
JUND
ZNF263
chr10:104856161rs124120381fNT5C2Intron 10USMG5Irx-3
Irx-2
Irx-4
Irx-6
chr2:85807081rs13488181fVAMP8Intron 2GGCXHMGIYEBF1,
Mtf1
Srf
Zfp105
HMGIY
chr2:85805366rs37700981fVAMP8Intron1VAMP8POLR2A
LOC388969BHLHE40
E2F6
KDM5B
MAX
MXI1
MYC
NFIC
WRNIP1
chr2:85803541rs67572631fVAMP8Upstream VAMP8GGCXSP1
VAMP8EP300
LOC388969NFIC
chr17:46988596rs465221fUBE2ZIntron 2ATP5G1NFKB
UBE2ZNFYB
RUNX3
chr19:45395618rs20756501fTOMM40Intron 2TOMM40RREB1
chr1:56996190rs46349321fPLPP3Intron 2PPAP2BPOLR2A
chr2:203880833rs46753101fNBEAL1Intron 1ALS2CR13
chr10:104681142rs171152131fCNNM2Intron 1USMG5
chr10:104721125rs108838081fCNNM2Intron 1USMG5
chr10:104723619rs111914791fCNNM2Intron 1USMG5GATA1
TAL1
CEBPB
chr10:104773363rs111915141fCNNM2Intron 1USMG5PAX5
chr10:104776526rs111915151fCNNM2Intron 1USMG5
chr10:104825664rs37812851fCNNM2Intron 4USMG5NF-kappaBIKZF1
P50:50
chr10:104835918rs9430371fCNNM2Exon 7USMG5TBX20
Foxj1
chr8:19813701rs2711fLPLIntron 6LPL
chr17:47039131rs22917251fGIPExon 4GIPGATA2
TCF4
FOSL2
EGR1
ELF1
FOS
NR3C1
EP300
RXRA
CHD2
JUND
POLR2A
RAD21
FOSL1
REST
chr2:203880991rs23515241fNBEAL15' UTRALS2CR13
chr1:109818305rs6293011fCELSR23' UTRPSRC1CTCF
POLR2A
chr2:85774675rs65476211f3' UTRGGCXELK4
POLR2A
chr10:104660003rs111914541fAS3MTIntron10USMG5
chr10:104685298rs124118861fCNNM2Intron1USMG5Zec
chr10:104719095rs124134091fCNNM2Intron1USMG5POLR3A
chr10:104764270rs111914991fCNNM2Intron1USMG5
chr17:47014126rs19949701fSNF8Intron4ATP5G1TFII-I
UBE2Z
chr2:85742296rs28867221fPseudogeneLOC388969TCF7L2
chr2:85783127rs67386451fGGCXIntron5Evi-1POLR2A
chr2:85794296rs101874241fPseudogeneGGCX
LOC388969
chr5:131723064rs176895501fRAPGEF6
chr7:129663495rs115569241fZC3HC1Exon8KIAA0265
chr17:4702833rs110798441fPseudogeneATP5G1
chr17: 47005192rs155631fUBE2ZExon7ATP5G1PRDM1
chr17:47047113rs38484601fUBE2ZATP5G1CEBPB
chr10:104680136rs22977872aCNNM2Intron 1Freac-7FOXA1
HFH3(FOXI1)SIN3A
HNF3alphaZNF263
FOXP1HNF4G
Elf3FOXA1
Foxl1
Srf
Tcf3
Tcfap2e
Zfp105
HFH(FOXl1)
chr1:56948289rs726643042aC8BIntron 6FOXA1FOXA1
Foxa2FOXA2
TCF4
SP1
HNF4G
HNF4A
HDAC2
JUND
EP300
chr17:46993232rs37446082aUBE2ZIntron 3Zfp740SPI1
MZF1POLR2A
MAZRIKZF1
SP1MAX
SP1:SP3TFAP2A
WT1ZNF21TFAP2C
Zfp281SP1
ZFp740CEBPB
WT1NR3C1
ZNF219BATF
ZNF740BCL11A
SP4MEF2A
NFKB1
JUND
EP300
STAT3
IRF4
EBF1
FOSL2
BATF
NR3C1
RUNX3
MYC
STAT3
chr2:85764959rs14466682aMAT2Anc transcriptCTCFCTCF
Upstream MAT2APOLR2A
TAF1
RFX5
RAD21
HEY1
CDX2
HNF4A
ZNF263
NR3C1
CTCF
MYC
AR
MYBL2
TEAD4
MAZ
CHD2
SMC3
TBP
ZNF143
CDX2
E2F6
MAX
NR3C1
SIN3A
YY1
REST
HMGN3
chr17:47006492rs126016722bUBE2ZDownstream UBE2ZZfxPOLR2A
EGR1
SPI1
ELF1
chr10:30316071rs37399982bKIAA1462Exon 2RELACTCF
MYC
PAX5
ZNF143
chr8:126476378rs29808562bTRIB1 geneIntergenic regionpax-8JUND
regionDownstream TRIB1Sox17POLR2A
TFAP2C
MXI1
CEBPB
chr9:136154303rs6491292bABO gene regionIntergenic regionIRFNFYA
Upstream ABOPOLR2A
FOS
IRF1
NFYB
PML
chr10:104840966rs122199012bCNNM2 geneIntergenic regionSRFPOLR2A
regionDownstream CXCL12
chr10:44778545rs17460522bCXCL12 geneIntergenic regionGATA1TAL1
regionDownstream CXCL12
chr21:35593826rs284510642bLINC00310 geneIntergenic regionPPARSP1
regionDownstreamFOXA2
LINC00310
chr17:2098271rs72176872bSMG6Intron 13-NF-1SIN3A
TCF12
MAX
YY1
ZNF263
EP300
TEAD4
chr13:110960711rs47731442bCOL4A2Intron 3STAT3:STAT3POLR2A
EZH2
chr14:100116251rs283915272bHHIPL1Intron 3MyoDBHLHE40
SCRT1USF1
FIGLAFOXA1
MAX
chr1:154404335rs75492502bIL6RIntron 3TBX15MXI1
FOS
JUNB
MAX
JUND
JUN
STAT3
FOSL1
MAFK
RCOR1
MYC
USF2
TEAD4
RCOR1
YY1
chr1:154404379rs75493382bIL6RIntron 3GRFOS
ARJUNB
JUND
JUN
STAT3
chr1:154404405rs75537962bIL6RIntron 3NF-kappaB,FOS
JUND
JUN
STAT3
chr14:100127439rs46241072bHHIPL1Intron 7Pax5JUND
chr10:91011457rs13323282bLIPAIntron 9UF1H3BETACREBBP
ZNF263
CDX2
ELF1
ZEB1
TBP
TFAPC2
TBP
POLR2A
ETS1
GABPA
HEY1
chr17:2117944rs22817272bSMG6Intron 13SRYCREBBP
SrfEP300
Zfp105STAT3
TRIM28
MYC
RBBP5
chr8:126479314rs69826362bLOC105375745Intron1MAFSMARCC1
RFX3
POLR2A
GATA2
CHD2
GTF2F1
chr14:100125720rs71452622bHHIPL1Intron4ESR2SMARC4
ZBTB7A
SMARB1
POLR2A
EZH2
RAD21
BACH1
chr10:104677125rs122210642bCNNM2Upstream CNNM2MAZR,CTCF, ETS1
ETS1
chr8:126478349rs29808532bLOC105375745UpstreamPit-1,RFX3
LOC105375745
chr15:79152421rs50299042bLOC105370915UpstreamNeuroDUSF1
LOC105370915POLR2A
YY1
FOXA1
E2F4
MAX
TAF7
TAF1
MXI1
chr8:126478744rs20018442bLOC105375745UpstreamHSF1RFX3
LOC105375745
chr10:91011680rs13323272bLIPA5' UTRAP-4,CREBBP
CDX2
ELF1
TBP
SPI1
NRF1
ELF1
ETS1
GABPA
SPI1
PAX5
SREBF1
chr17:2102452rs99088882bSMG6Intron10GRCEBPB
AR
chr10:91008878rs22506442bLIPAIntron1Oct-1RUNX3
XBP-1
MAfb
Mafk
MAFB
MAFK
NRL
chr1:109817589rs127403742bCELSR2Exon34HNF1EBF1
HNF1A
DUXA
chr1:222794090rs171633012bMIA3Intron1HNF1EBF1
HNF1A
HNF1B
DUXA
chr21:35644028rs285914152bpseudogenePPAREP300
FOXA1
HDAC2
NFIC
SP1
chr2:203713279rs729347152bICA1LIntron2HMGIYATF2
NFIC
EBF1
EP300
NFKB1
PAX5
chr2:203775474rs729368522bWDR12Intron1ARMAFF
chr2:203926270rs729345122bNBEAL1Intron6TEAD1TEAD4
TEAD3
chr8:19803092rs37797882bLPLIntron1TGIFCEBPB
chr10:44757106rs5185942bCXCL12Downstream intergenicE2AFOXM1
NRSENFIC
NRSFMAX
EBF1
TBL1XR1
chr4:57824931rs76877671dCECR6Sox8FOXA1
chr11:5759712rs931381fSWAP70Intron8
rs3601361fSWAP70Exon13
chr:457798188rs22279011fREST-NOA1Exon6SPI1
chr:1567442595rs172936322aSMAD3Intron4Bach1SIN3A
AP-1TCF7L2
JundM2TFAP2A
Pou1f1TFAP2C
Pou3f1ZNF217
Sox5POLR2A
JDP2STAT1
MXI1
TAF1
E2F1
POLR2A
HDAC1
TCF7L2
MYC
POLR2A
ESR1
EP300
CDX2
HNF4A
CEBPB
EP300
FOS
FOSL1
GATA3
JUNB
JUN
MYC
NR2F2
NR3C1
RAD21
RCOR1
TCF12
JUND
MAFK
EGR1
MAX
chr15:67441996rs188663162bSMAD3Exon4AIREESR1
Elf3NR3C1
SrfPOLR2A
Tcf3
Tcfap2e
chr15:67448898rs80327392bMTERFIntron4SRFCEBPB
FOS
MYC
STAT3
chr9:22098573rs49775742cCDKN2BAS1Intron16ARAR
NR3C1
NR3C2
Ar
Elk-1
c-Ets-1(p54)
Coordinate 0-basedSNP IDRegulomeDB scoreGene/LocusPositioneQTLMotifProtein-binding
chr15:91429041rs18944011bFESIntron 2FESPax5SPI1
USF1
POLR2A
GABPA
BHLHE40
CEBPB
CTCF
MAX
RFX5
RUNX3
STAT5A
chr10:91005853rs22468331bLIPAIntron 6LIPAEWSR-FLI1CTCF
znf143
chr2:85808736rs10091bVAMP8Exon 3VAMP8CTCF
LOC388969HSF1
chr17:47038470rs47940041dGIPIntron 4ATP5G1Gata5NR3C1
UBE2ZIN3AK20
CREB1
TAF1
TCF12
CTCF
POLR2A
USF1
FOXA1
FOXA2
RBBP5
chr10:91002926rs14124441dLIPAIntron3LIPASAP1aATF2
ELK1FOXM1
ELK3SP1
ELK4SPI1
MECP2MTA3
ERFRUNX3
ERG
ETS1
ETV1
ETV2
ETV3
Gabpa
chr10:104873760rs96337121eNT5C2Intron 3UMG5PU1SP11
ELF-1
Sfpil
PU.1
c-Ets-1
chr11:116648916rs9641841fZPR1Downstream ZRP1TAGLNFOXJ2
chr1:109818529rs6467761fCELSR2Upstream CELSR2PSMA5CTCF
HEY1
REST
POLR2A
ZBTB7A
TAF7
chr10:104616662rs44097661fBORCS7-ASMTIntron 1C10orf77Tcf3BACH1
USMG5MAFF
MAFK
chr17:47008206rs47939921fSNF8Intron 7ATP5G1POLR2A
UBE2ZTEAD4
chr6:161152293rs42521261fPLGIntron 11PLGCTCF
RUNX3
TEAD4
RAD21
chr6:161154231rs42521351fPLGIntron 12PLGCTCF
FOXA1
NFKB1
RAD21
ZNF263
SMC3
ZNF143
FOXA2
chr10:104846177rs111915481fNT5C2 gene regionDownstream NT5C2USMG5TEAD1
TEAD3
chr10:104864613rs111915571fNT5C2Intron 5USMG5
chr10:104864677rs111915581fNT5C2Intron 5USMG5HOXC13
Hoxa13
Hoxc13
Hoxd12
HOXA13
HOXD9
HOXC11
chr10:104871203rs124130461fNT5C2Intron 3USMG5NR3C1
TRIM28
CTCF
ATF2
IKZF1
TCF7L2
ZNF263
chr10:104871278rs108838321fNT5C2Intron 3USMG5TRIM28
TCF7L2
chr10:104913652rs111915821fNT5C2Intron 2USMG5EP300
NFIC
TCF12
TEAD4
STAT1
ARID3A
EP300
JUN
RCOR1
chr10:104906210rs111915801fNT5C2Intron 2USMG5TRIM28
SETDB1
GATA1
GTF2F1
CEBPB
FOS
JUND
ZNF263
chr10:104856161rs124120381fNT5C2Intron 10USMG5Irx-3
Irx-2
Irx-4
Irx-6
chr2:85807081rs13488181fVAMP8Intron 2GGCXHMGIYEBF1,
Mtf1
Srf
Zfp105
HMGIY
chr2:85805366rs37700981fVAMP8Intron1VAMP8POLR2A
LOC388969BHLHE40
E2F6
KDM5B
MAX
MXI1
MYC
NFIC
WRNIP1
chr2:85803541rs67572631fVAMP8Upstream VAMP8GGCXSP1
VAMP8EP300
LOC388969NFIC
chr17:46988596rs465221fUBE2ZIntron 2ATP5G1NFKB
UBE2ZNFYB
RUNX3
chr19:45395618rs20756501fTOMM40Intron 2TOMM40RREB1
chr1:56996190rs46349321fPLPP3Intron 2PPAP2BPOLR2A
chr2:203880833rs46753101fNBEAL1Intron 1ALS2CR13
chr10:104681142rs171152131fCNNM2Intron 1USMG5
chr10:104721125rs108838081fCNNM2Intron 1USMG5
chr10:104723619rs111914791fCNNM2Intron 1USMG5GATA1
TAL1
CEBPB
chr10:104773363rs111915141fCNNM2Intron 1USMG5PAX5
chr10:104776526rs111915151fCNNM2Intron 1USMG5
chr10:104825664rs37812851fCNNM2Intron 4USMG5NF-kappaBIKZF1
P50:50
chr10:104835918rs9430371fCNNM2Exon 7USMG5TBX20
Foxj1
chr8:19813701rs2711fLPLIntron 6LPL
chr17:47039131rs22917251fGIPExon 4GIPGATA2
TCF4
FOSL2
EGR1
ELF1
FOS
NR3C1
EP300
RXRA
CHD2
JUND
POLR2A
RAD21
FOSL1
REST
chr2:203880991rs23515241fNBEAL15' UTRALS2CR13
chr1:109818305rs6293011fCELSR23' UTRPSRC1CTCF
POLR2A
chr2:85774675rs65476211f3' UTRGGCXELK4
POLR2A
chr10:104660003rs111914541fAS3MTIntron10USMG5
chr10:104685298rs124118861fCNNM2Intron1USMG5Zec
chr10:104719095rs124134091fCNNM2Intron1USMG5POLR3A
chr10:104764270rs111914991fCNNM2Intron1USMG5
chr17:47014126rs19949701fSNF8Intron4ATP5G1TFII-I
UBE2Z
chr2:85742296rs28867221fPseudogeneLOC388969TCF7L2
chr2:85783127rs67386451fGGCXIntron5Evi-1POLR2A
chr2:85794296rs101874241fPseudogeneGGCX
LOC388969
chr5:131723064rs176895501fRAPGEF6
chr7:129663495rs115569241fZC3HC1Exon8KIAA0265
chr17:4702833rs110798441fPseudogeneATP5G1
chr17: 47005192rs155631fUBE2ZExon7ATP5G1PRDM1
chr17:47047113rs38484601fUBE2ZATP5G1CEBPB
chr10:104680136rs22977872aCNNM2Intron 1Freac-7FOXA1
HFH3(FOXI1)SIN3A
HNF3alphaZNF263
FOXP1HNF4G
Elf3FOXA1
Foxl1
Srf
Tcf3
Tcfap2e
Zfp105
HFH(FOXl1)
chr1:56948289rs726643042aC8BIntron 6FOXA1FOXA1
Foxa2FOXA2
TCF4
SP1
HNF4G
HNF4A
HDAC2
JUND
EP300
chr17:46993232rs37446082aUBE2ZIntron 3Zfp740SPI1
MZF1POLR2A
MAZRIKZF1
SP1MAX
SP1:SP3TFAP2A
WT1ZNF21TFAP2C
Zfp281SP1
ZFp740CEBPB
WT1NR3C1
ZNF219BATF
ZNF740BCL11A
SP4MEF2A
NFKB1
JUND
EP300
STAT3
IRF4
EBF1
FOSL2
BATF
NR3C1
RUNX3
MYC
STAT3
chr2:85764959rs14466682aMAT2Anc transcriptCTCFCTCF
Upstream MAT2APOLR2A
TAF1
RFX5
RAD21
HEY1
CDX2
HNF4A
ZNF263
NR3C1
CTCF
MYC
AR
MYBL2
TEAD4
MAZ
CHD2
SMC3
TBP
ZNF143
CDX2
E2F6
MAX
NR3C1
SIN3A
YY1
REST
HMGN3
chr17:47006492rs126016722bUBE2ZDownstream UBE2ZZfxPOLR2A
EGR1
SPI1
ELF1
chr10:30316071rs37399982bKIAA1462Exon 2RELACTCF
MYC
PAX5
ZNF143
chr8:126476378rs29808562bTRIB1 geneIntergenic regionpax-8JUND
regionDownstream TRIB1Sox17POLR2A
TFAP2C
MXI1
CEBPB
chr9:136154303rs6491292bABO gene regionIntergenic regionIRFNFYA
Upstream ABOPOLR2A
FOS
IRF1
NFYB
PML
chr10:104840966rs122199012bCNNM2 geneIntergenic regionSRFPOLR2A
regionDownstream CXCL12
chr10:44778545rs17460522bCXCL12 geneIntergenic regionGATA1TAL1
regionDownstream CXCL12
chr21:35593826rs284510642bLINC00310 geneIntergenic regionPPARSP1
regionDownstreamFOXA2
LINC00310
chr17:2098271rs72176872bSMG6Intron 13-NF-1SIN3A
TCF12
MAX
YY1
ZNF263
EP300
TEAD4
chr13:110960711rs47731442bCOL4A2Intron 3STAT3:STAT3POLR2A
EZH2
chr14:100116251rs283915272bHHIPL1Intron 3MyoDBHLHE40
SCRT1USF1
FIGLAFOXA1
MAX
chr1:154404335rs75492502bIL6RIntron 3TBX15MXI1
FOS
JUNB
MAX
JUND
JUN
STAT3
FOSL1
MAFK
RCOR1
MYC
USF2
TEAD4
RCOR1
YY1
chr1:154404379rs75493382bIL6RIntron 3GRFOS
ARJUNB
JUND
JUN
STAT3
chr1:154404405rs75537962bIL6RIntron 3NF-kappaB,FOS
JUND
JUN
STAT3
chr14:100127439rs46241072bHHIPL1Intron 7Pax5JUND
chr10:91011457rs13323282bLIPAIntron 9UF1H3BETACREBBP
ZNF263
CDX2
ELF1
ZEB1
TBP
TFAPC2
TBP
POLR2A
ETS1
GABPA
HEY1
chr17:2117944rs22817272bSMG6Intron 13SRYCREBBP
SrfEP300
Zfp105STAT3
TRIM28
MYC
RBBP5
chr8:126479314rs69826362bLOC105375745Intron1MAFSMARCC1
RFX3
POLR2A
GATA2
CHD2
GTF2F1
chr14:100125720rs71452622bHHIPL1Intron4ESR2SMARC4
ZBTB7A
SMARB1
POLR2A
EZH2
RAD21
BACH1
chr10:104677125rs122210642bCNNM2Upstream CNNM2MAZR,CTCF, ETS1
ETS1
chr8:126478349rs29808532bLOC105375745UpstreamPit-1,RFX3
LOC105375745
chr15:79152421rs50299042bLOC105370915UpstreamNeuroDUSF1
LOC105370915POLR2A
YY1
FOXA1
E2F4
MAX
TAF7
TAF1
MXI1
chr8:126478744rs20018442bLOC105375745UpstreamHSF1RFX3
LOC105375745
chr10:91011680rs13323272bLIPA5' UTRAP-4,CREBBP
CDX2
ELF1
TBP
SPI1
NRF1
ELF1
ETS1
GABPA
SPI1
PAX5
SREBF1
chr17:2102452rs99088882bSMG6Intron10GRCEBPB
AR
chr10:91008878rs22506442bLIPAIntron1Oct-1RUNX3
XBP-1
MAfb
Mafk
MAFB
MAFK
NRL
chr1:109817589rs127403742bCELSR2Exon34HNF1EBF1
HNF1A
DUXA
chr1:222794090rs171633012bMIA3Intron1HNF1EBF1
HNF1A
HNF1B
DUXA
chr21:35644028rs285914152bpseudogenePPAREP300
FOXA1
HDAC2
NFIC
SP1
chr2:203713279rs729347152bICA1LIntron2HMGIYATF2
NFIC
EBF1
EP300
NFKB1
PAX5
chr2:203775474rs729368522bWDR12Intron1ARMAFF
chr2:203926270rs729345122bNBEAL1Intron6TEAD1TEAD4
TEAD3
chr8:19803092rs37797882bLPLIntron1TGIFCEBPB
chr10:44757106rs5185942bCXCL12Downstream intergenicE2AFOXM1
NRSENFIC
NRSFMAX
EBF1
TBL1XR1
chr4:57824931rs76877671dCECR6Sox8FOXA1
chr11:5759712rs931381fSWAP70Intron8
rs3601361fSWAP70Exon13
chr:457798188rs22279011fREST-NOA1Exon6SPI1
chr:1567442595rs172936322aSMAD3Intron4Bach1SIN3A
AP-1TCF7L2
JundM2TFAP2A
Pou1f1TFAP2C
Pou3f1ZNF217
Sox5POLR2A
JDP2STAT1
MXI1
TAF1
E2F1
POLR2A
HDAC1
TCF7L2
MYC
POLR2A
ESR1
EP300
CDX2
HNF4A
CEBPB
EP300
FOS
FOSL1
GATA3
JUNB
JUN
MYC
NR2F2
NR3C1
RAD21
RCOR1
TCF12
JUND
MAFK
EGR1
MAX
chr15:67441996rs188663162bSMAD3Exon4AIREESR1
Elf3NR3C1
SrfPOLR2A
Tcf3
Tcfap2e
chr15:67448898rs80327392bMTERFIntron4SRFCEBPB
FOS
MYC
STAT3
chr9:22098573rs49775742cCDKN2BAS1Intron16ARAR
NR3C1
NR3C2
Ar
Elk-1
c-Ets-1(p54)
Table 4.

Putative functional SNPs and corresponding motifs, eQTL and related transcription factors (Regulome DB score < 3)

Coordinate 0-basedSNP IDRegulomeDB scoreGene/LocusPositioneQTLMotifProtein-binding
chr15:91429041rs18944011bFESIntron 2FESPax5SPI1
USF1
POLR2A
GABPA
BHLHE40
CEBPB
CTCF
MAX
RFX5
RUNX3
STAT5A
chr10:91005853rs22468331bLIPAIntron 6LIPAEWSR-FLI1CTCF
znf143
chr2:85808736rs10091bVAMP8Exon 3VAMP8CTCF
LOC388969HSF1
chr17:47038470rs47940041dGIPIntron 4ATP5G1Gata5NR3C1
UBE2ZIN3AK20
CREB1
TAF1
TCF12
CTCF
POLR2A
USF1
FOXA1
FOXA2
RBBP5
chr10:91002926rs14124441dLIPAIntron3LIPASAP1aATF2
ELK1FOXM1
ELK3SP1
ELK4SPI1
MECP2MTA3
ERFRUNX3
ERG
ETS1
ETV1
ETV2
ETV3
Gabpa
chr10:104873760rs96337121eNT5C2Intron 3UMG5PU1SP11
ELF-1
Sfpil
PU.1
c-Ets-1
chr11:116648916rs9641841fZPR1Downstream ZRP1TAGLNFOXJ2
chr1:109818529rs6467761fCELSR2Upstream CELSR2PSMA5CTCF
HEY1
REST
POLR2A
ZBTB7A
TAF7
chr10:104616662rs44097661fBORCS7-ASMTIntron 1C10orf77Tcf3BACH1
USMG5MAFF
MAFK
chr17:47008206rs47939921fSNF8Intron 7ATP5G1POLR2A
UBE2ZTEAD4
chr6:161152293rs42521261fPLGIntron 11PLGCTCF
RUNX3
TEAD4
RAD21
chr6:161154231rs42521351fPLGIntron 12PLGCTCF
FOXA1
NFKB1
RAD21
ZNF263
SMC3
ZNF143
FOXA2
chr10:104846177rs111915481fNT5C2 gene regionDownstream NT5C2USMG5TEAD1
TEAD3
chr10:104864613rs111915571fNT5C2Intron 5USMG5
chr10:104864677rs111915581fNT5C2Intron 5USMG5HOXC13
Hoxa13
Hoxc13
Hoxd12
HOXA13
HOXD9
HOXC11
chr10:104871203rs124130461fNT5C2Intron 3USMG5NR3C1
TRIM28
CTCF
ATF2
IKZF1
TCF7L2
ZNF263
chr10:104871278rs108838321fNT5C2Intron 3USMG5TRIM28
TCF7L2
chr10:104913652rs111915821fNT5C2Intron 2USMG5EP300
NFIC
TCF12
TEAD4
STAT1
ARID3A
EP300
JUN
RCOR1
chr10:104906210rs111915801fNT5C2Intron 2USMG5TRIM28
SETDB1
GATA1
GTF2F1
CEBPB
FOS
JUND
ZNF263
chr10:104856161rs124120381fNT5C2Intron 10USMG5Irx-3
Irx-2
Irx-4
Irx-6
chr2:85807081rs13488181fVAMP8Intron 2GGCXHMGIYEBF1,
Mtf1
Srf
Zfp105
HMGIY
chr2:85805366rs37700981fVAMP8Intron1VAMP8POLR2A
LOC388969BHLHE40
E2F6
KDM5B
MAX
MXI1
MYC
NFIC
WRNIP1
chr2:85803541rs67572631fVAMP8Upstream VAMP8GGCXSP1
VAMP8EP300
LOC388969NFIC
chr17:46988596rs465221fUBE2ZIntron 2ATP5G1NFKB
UBE2ZNFYB
RUNX3
chr19:45395618rs20756501fTOMM40Intron 2TOMM40RREB1
chr1:56996190rs46349321fPLPP3Intron 2PPAP2BPOLR2A
chr2:203880833rs46753101fNBEAL1Intron 1ALS2CR13
chr10:104681142rs171152131fCNNM2Intron 1USMG5
chr10:104721125rs108838081fCNNM2Intron 1USMG5
chr10:104723619rs111914791fCNNM2Intron 1USMG5GATA1
TAL1
CEBPB
chr10:104773363rs111915141fCNNM2Intron 1USMG5PAX5
chr10:104776526rs111915151fCNNM2Intron 1USMG5
chr10:104825664rs37812851fCNNM2Intron 4USMG5NF-kappaBIKZF1
P50:50
chr10:104835918rs9430371fCNNM2Exon 7USMG5TBX20
Foxj1
chr8:19813701rs2711fLPLIntron 6LPL
chr17:47039131rs22917251fGIPExon 4GIPGATA2
TCF4
FOSL2
EGR1
ELF1
FOS
NR3C1
EP300
RXRA
CHD2
JUND
POLR2A
RAD21
FOSL1
REST
chr2:203880991rs23515241fNBEAL15' UTRALS2CR13
chr1:109818305rs6293011fCELSR23' UTRPSRC1CTCF
POLR2A
chr2:85774675rs65476211f3' UTRGGCXELK4
POLR2A
chr10:104660003rs111914541fAS3MTIntron10USMG5
chr10:104685298rs124118861fCNNM2Intron1USMG5Zec
chr10:104719095rs124134091fCNNM2Intron1USMG5POLR3A
chr10:104764270rs111914991fCNNM2Intron1USMG5
chr17:47014126rs19949701fSNF8Intron4ATP5G1TFII-I
UBE2Z
chr2:85742296rs28867221fPseudogeneLOC388969TCF7L2
chr2:85783127rs67386451fGGCXIntron5Evi-1POLR2A
chr2:85794296rs101874241fPseudogeneGGCX
LOC388969
chr5:131723064rs176895501fRAPGEF6
chr7:129663495rs115569241fZC3HC1Exon8KIAA0265
chr17:4702833rs110798441fPseudogeneATP5G1
chr17: 47005192rs155631fUBE2ZExon7ATP5G1PRDM1
chr17:47047113rs38484601fUBE2ZATP5G1CEBPB
chr10:104680136rs22977872aCNNM2Intron 1Freac-7FOXA1
HFH3(FOXI1)SIN3A
HNF3alphaZNF263
FOXP1HNF4G
Elf3FOXA1
Foxl1
Srf
Tcf3
Tcfap2e
Zfp105
HFH(FOXl1)
chr1:56948289rs726643042aC8BIntron 6FOXA1FOXA1
Foxa2FOXA2
TCF4
SP1
HNF4G
HNF4A
HDAC2
JUND
EP300
chr17:46993232rs37446082aUBE2ZIntron 3Zfp740SPI1
MZF1POLR2A
MAZRIKZF1
SP1MAX
SP1:SP3TFAP2A
WT1ZNF21TFAP2C
Zfp281SP1
ZFp740CEBPB
WT1NR3C1
ZNF219BATF
ZNF740BCL11A
SP4MEF2A
NFKB1
JUND
EP300
STAT3
IRF4
EBF1
FOSL2
BATF
NR3C1
RUNX3
MYC
STAT3
chr2:85764959rs14466682aMAT2Anc transcriptCTCFCTCF
Upstream MAT2APOLR2A
TAF1
RFX5
RAD21
HEY1
CDX2
HNF4A
ZNF263
NR3C1
CTCF
MYC
AR
MYBL2
TEAD4
MAZ
CHD2
SMC3
TBP
ZNF143
CDX2
E2F6
MAX
NR3C1
SIN3A
YY1
REST
HMGN3
chr17:47006492rs126016722bUBE2ZDownstream UBE2ZZfxPOLR2A
EGR1
SPI1
ELF1
chr10:30316071rs37399982bKIAA1462Exon 2RELACTCF
MYC
PAX5
ZNF143
chr8:126476378rs29808562bTRIB1 geneIntergenic regionpax-8JUND
regionDownstream TRIB1Sox17POLR2A
TFAP2C
MXI1
CEBPB
chr9:136154303rs6491292bABO gene regionIntergenic regionIRFNFYA
Upstream ABOPOLR2A
FOS
IRF1
NFYB
PML
chr10:104840966rs122199012bCNNM2 geneIntergenic regionSRFPOLR2A
regionDownstream CXCL12
chr10:44778545rs17460522bCXCL12 geneIntergenic regionGATA1TAL1
regionDownstream CXCL12
chr21:35593826rs284510642bLINC00310 geneIntergenic regionPPARSP1
regionDownstreamFOXA2
LINC00310
chr17:2098271rs72176872bSMG6Intron 13-NF-1SIN3A
TCF12
MAX
YY1
ZNF263
EP300
TEAD4
chr13:110960711rs47731442bCOL4A2Intron 3STAT3:STAT3POLR2A
EZH2
chr14:100116251rs283915272bHHIPL1Intron 3MyoDBHLHE40
SCRT1USF1
FIGLAFOXA1
MAX
chr1:154404335rs75492502bIL6RIntron 3TBX15MXI1
FOS
JUNB
MAX
JUND
JUN
STAT3
FOSL1
MAFK
RCOR1
MYC
USF2
TEAD4
RCOR1
YY1
chr1:154404379rs75493382bIL6RIntron 3GRFOS
ARJUNB
JUND
JUN
STAT3
chr1:154404405rs75537962bIL6RIntron 3NF-kappaB,FOS
JUND
JUN
STAT3
chr14:100127439rs46241072bHHIPL1Intron 7Pax5JUND
chr10:91011457rs13323282bLIPAIntron 9UF1H3BETACREBBP
ZNF263
CDX2
ELF1
ZEB1
TBP
TFAPC2
TBP
POLR2A
ETS1
GABPA
HEY1
chr17:2117944rs22817272bSMG6Intron 13SRYCREBBP
SrfEP300
Zfp105STAT3
TRIM28
MYC
RBBP5
chr8:126479314rs69826362bLOC105375745Intron1MAFSMARCC1
RFX3
POLR2A
GATA2
CHD2
GTF2F1
chr14:100125720rs71452622bHHIPL1Intron4ESR2SMARC4
ZBTB7A
SMARB1
POLR2A
EZH2
RAD21
BACH1
chr10:104677125rs122210642bCNNM2Upstream CNNM2MAZR,CTCF, ETS1
ETS1
chr8:126478349rs29808532bLOC105375745UpstreamPit-1,RFX3
LOC105375745
chr15:79152421rs50299042bLOC105370915UpstreamNeuroDUSF1
LOC105370915POLR2A
YY1
FOXA1
E2F4
MAX
TAF7
TAF1
MXI1
chr8:126478744rs20018442bLOC105375745UpstreamHSF1RFX3
LOC105375745
chr10:91011680rs13323272bLIPA5' UTRAP-4,CREBBP
CDX2
ELF1
TBP
SPI1
NRF1
ELF1
ETS1
GABPA
SPI1
PAX5
SREBF1
chr17:2102452rs99088882bSMG6Intron10GRCEBPB
AR
chr10:91008878rs22506442bLIPAIntron1Oct-1RUNX3
XBP-1
MAfb
Mafk
MAFB
MAFK
NRL
chr1:109817589rs127403742bCELSR2Exon34HNF1EBF1
HNF1A
DUXA
chr1:222794090rs171633012bMIA3Intron1HNF1EBF1
HNF1A
HNF1B
DUXA
chr21:35644028rs285914152bpseudogenePPAREP300
FOXA1
HDAC2
NFIC
SP1
chr2:203713279rs729347152bICA1LIntron2HMGIYATF2
NFIC
EBF1
EP300
NFKB1
PAX5
chr2:203775474rs729368522bWDR12Intron1ARMAFF
chr2:203926270rs729345122bNBEAL1Intron6TEAD1TEAD4
TEAD3
chr8:19803092rs37797882bLPLIntron1TGIFCEBPB
chr10:44757106rs5185942bCXCL12Downstream intergenicE2AFOXM1
NRSENFIC
NRSFMAX
EBF1
TBL1XR1
chr4:57824931rs76877671dCECR6Sox8FOXA1
chr11:5759712rs931381fSWAP70Intron8
rs3601361fSWAP70Exon13
chr:457798188rs22279011fREST-NOA1Exon6SPI1
chr:1567442595rs172936322aSMAD3Intron4Bach1SIN3A
AP-1TCF7L2
JundM2TFAP2A
Pou1f1TFAP2C
Pou3f1ZNF217
Sox5POLR2A
JDP2STAT1
MXI1
TAF1
E2F1
POLR2A
HDAC1
TCF7L2
MYC
POLR2A
ESR1
EP300
CDX2
HNF4A
CEBPB
EP300
FOS
FOSL1
GATA3
JUNB
JUN
MYC
NR2F2
NR3C1
RAD21
RCOR1
TCF12
JUND
MAFK
EGR1
MAX
chr15:67441996rs188663162bSMAD3Exon4AIREESR1
Elf3NR3C1
SrfPOLR2A
Tcf3
Tcfap2e
chr15:67448898rs80327392bMTERFIntron4SRFCEBPB
FOS
MYC
STAT3
chr9:22098573rs49775742cCDKN2BAS1Intron16ARAR
NR3C1
NR3C2
Ar
Elk-1
c-Ets-1(p54)
Coordinate 0-basedSNP IDRegulomeDB scoreGene/LocusPositioneQTLMotifProtein-binding
chr15:91429041rs18944011bFESIntron 2FESPax5SPI1
USF1
POLR2A
GABPA
BHLHE40
CEBPB
CTCF
MAX
RFX5
RUNX3
STAT5A
chr10:91005853rs22468331bLIPAIntron 6LIPAEWSR-FLI1CTCF
znf143
chr2:85808736rs10091bVAMP8Exon 3VAMP8CTCF
LOC388969HSF1
chr17:47038470rs47940041dGIPIntron 4ATP5G1Gata5NR3C1
UBE2ZIN3AK20
CREB1
TAF1
TCF12
CTCF
POLR2A
USF1
FOXA1
FOXA2
RBBP5
chr10:91002926rs14124441dLIPAIntron3LIPASAP1aATF2
ELK1FOXM1
ELK3SP1
ELK4SPI1
MECP2MTA3
ERFRUNX3
ERG
ETS1
ETV1
ETV2
ETV3
Gabpa
chr10:104873760rs96337121eNT5C2Intron 3UMG5PU1SP11
ELF-1
Sfpil
PU.1
c-Ets-1
chr11:116648916rs9641841fZPR1Downstream ZRP1TAGLNFOXJ2
chr1:109818529rs6467761fCELSR2Upstream CELSR2PSMA5CTCF
HEY1
REST
POLR2A
ZBTB7A
TAF7
chr10:104616662rs44097661fBORCS7-ASMTIntron 1C10orf77Tcf3BACH1
USMG5MAFF
MAFK
chr17:47008206rs47939921fSNF8Intron 7ATP5G1POLR2A
UBE2ZTEAD4
chr6:161152293rs42521261fPLGIntron 11PLGCTCF
RUNX3
TEAD4
RAD21
chr6:161154231rs42521351fPLGIntron 12PLGCTCF
FOXA1
NFKB1
RAD21
ZNF263
SMC3
ZNF143
FOXA2
chr10:104846177rs111915481fNT5C2 gene regionDownstream NT5C2USMG5TEAD1
TEAD3
chr10:104864613rs111915571fNT5C2Intron 5USMG5
chr10:104864677rs111915581fNT5C2Intron 5USMG5HOXC13
Hoxa13
Hoxc13
Hoxd12
HOXA13
HOXD9
HOXC11
chr10:104871203rs124130461fNT5C2Intron 3USMG5NR3C1
TRIM28
CTCF
ATF2
IKZF1
TCF7L2
ZNF263
chr10:104871278rs108838321fNT5C2Intron 3USMG5TRIM28
TCF7L2
chr10:104913652rs111915821fNT5C2Intron 2USMG5EP300
NFIC
TCF12
TEAD4
STAT1
ARID3A
EP300
JUN
RCOR1
chr10:104906210rs111915801fNT5C2Intron 2USMG5TRIM28
SETDB1
GATA1
GTF2F1
CEBPB
FOS
JUND
ZNF263
chr10:104856161rs124120381fNT5C2Intron 10USMG5Irx-3
Irx-2
Irx-4
Irx-6
chr2:85807081rs13488181fVAMP8Intron 2GGCXHMGIYEBF1,
Mtf1
Srf
Zfp105
HMGIY
chr2:85805366rs37700981fVAMP8Intron1VAMP8POLR2A
LOC388969BHLHE40
E2F6
KDM5B
MAX
MXI1
MYC
NFIC
WRNIP1
chr2:85803541rs67572631fVAMP8Upstream VAMP8GGCXSP1
VAMP8EP300
LOC388969NFIC
chr17:46988596rs465221fUBE2ZIntron 2ATP5G1NFKB
UBE2ZNFYB
RUNX3
chr19:45395618rs20756501fTOMM40Intron 2TOMM40RREB1
chr1:56996190rs46349321fPLPP3Intron 2PPAP2BPOLR2A
chr2:203880833rs46753101fNBEAL1Intron 1ALS2CR13
chr10:104681142rs171152131fCNNM2Intron 1USMG5
chr10:104721125rs108838081fCNNM2Intron 1USMG5
chr10:104723619rs111914791fCNNM2Intron 1USMG5GATA1
TAL1
CEBPB
chr10:104773363rs111915141fCNNM2Intron 1USMG5PAX5
chr10:104776526rs111915151fCNNM2Intron 1USMG5
chr10:104825664rs37812851fCNNM2Intron 4USMG5NF-kappaBIKZF1
P50:50
chr10:104835918rs9430371fCNNM2Exon 7USMG5TBX20
Foxj1
chr8:19813701rs2711fLPLIntron 6LPL
chr17:47039131rs22917251fGIPExon 4GIPGATA2
TCF4
FOSL2
EGR1
ELF1
FOS
NR3C1
EP300
RXRA
CHD2
JUND
POLR2A
RAD21
FOSL1
REST
chr2:203880991rs23515241fNBEAL15' UTRALS2CR13
chr1:109818305rs6293011fCELSR23' UTRPSRC1CTCF
POLR2A
chr2:85774675rs65476211f3' UTRGGCXELK4
POLR2A
chr10:104660003rs111914541fAS3MTIntron10USMG5
chr10:104685298rs124118861fCNNM2Intron1USMG5Zec
chr10:104719095rs124134091fCNNM2Intron1USMG5POLR3A
chr10:104764270rs111914991fCNNM2Intron1USMG5
chr17:47014126rs19949701fSNF8Intron4ATP5G1TFII-I
UBE2Z
chr2:85742296rs28867221fPseudogeneLOC388969TCF7L2
chr2:85783127rs67386451fGGCXIntron5Evi-1POLR2A
chr2:85794296rs101874241fPseudogeneGGCX
LOC388969
chr5:131723064rs176895501fRAPGEF6
chr7:129663495rs115569241fZC3HC1Exon8KIAA0265
chr17:4702833rs110798441fPseudogeneATP5G1
chr17: 47005192rs155631fUBE2ZExon7ATP5G1PRDM1
chr17:47047113rs38484601fUBE2ZATP5G1CEBPB
chr10:104680136rs22977872aCNNM2Intron 1Freac-7FOXA1
HFH3(FOXI1)SIN3A
HNF3alphaZNF263
FOXP1HNF4G
Elf3FOXA1
Foxl1
Srf
Tcf3
Tcfap2e
Zfp105
HFH(FOXl1)
chr1:56948289rs726643042aC8BIntron 6FOXA1FOXA1
Foxa2FOXA2
TCF4
SP1
HNF4G
HNF4A
HDAC2
JUND
EP300
chr17:46993232rs37446082aUBE2ZIntron 3Zfp740SPI1
MZF1POLR2A
MAZRIKZF1
SP1MAX
SP1:SP3TFAP2A
WT1ZNF21TFAP2C
Zfp281SP1
ZFp740CEBPB
WT1NR3C1
ZNF219BATF
ZNF740BCL11A
SP4MEF2A
NFKB1
JUND
EP300
STAT3
IRF4
EBF1
FOSL2
BATF
NR3C1
RUNX3
MYC
STAT3
chr2:85764959rs14466682aMAT2Anc transcriptCTCFCTCF
Upstream MAT2APOLR2A
TAF1
RFX5
RAD21
HEY1
CDX2
HNF4A
ZNF263
NR3C1
CTCF
MYC
AR
MYBL2
TEAD4
MAZ
CHD2
SMC3
TBP
ZNF143
CDX2
E2F6
MAX
NR3C1
SIN3A
YY1
REST
HMGN3
chr17:47006492rs126016722bUBE2ZDownstream UBE2ZZfxPOLR2A
EGR1
SPI1
ELF1
chr10:30316071rs37399982bKIAA1462Exon 2RELACTCF
MYC
PAX5
ZNF143
chr8:126476378rs29808562bTRIB1 geneIntergenic regionpax-8JUND
regionDownstream TRIB1Sox17POLR2A
TFAP2C
MXI1
CEBPB
chr9:136154303rs6491292bABO gene regionIntergenic regionIRFNFYA
Upstream ABOPOLR2A
FOS
IRF1
NFYB
PML
chr10:104840966rs122199012bCNNM2 geneIntergenic regionSRFPOLR2A
regionDownstream CXCL12
chr10:44778545rs17460522bCXCL12 geneIntergenic regionGATA1TAL1
regionDownstream CXCL12
chr21:35593826rs284510642bLINC00310 geneIntergenic regionPPARSP1
regionDownstreamFOXA2
LINC00310
chr17:2098271rs72176872bSMG6Intron 13-NF-1SIN3A
TCF12
MAX
YY1
ZNF263
EP300
TEAD4
chr13:110960711rs47731442bCOL4A2Intron 3STAT3:STAT3POLR2A
EZH2
chr14:100116251rs283915272bHHIPL1Intron 3MyoDBHLHE40
SCRT1USF1
FIGLAFOXA1
MAX
chr1:154404335rs75492502bIL6RIntron 3TBX15MXI1
FOS
JUNB
MAX
JUND
JUN
STAT3
FOSL1
MAFK
RCOR1
MYC
USF2
TEAD4
RCOR1
YY1
chr1:154404379rs75493382bIL6RIntron 3GRFOS
ARJUNB
JUND
JUN
STAT3
chr1:154404405rs75537962bIL6RIntron 3NF-kappaB,FOS
JUND
JUN
STAT3
chr14:100127439rs46241072bHHIPL1Intron 7Pax5JUND
chr10:91011457rs13323282bLIPAIntron 9UF1H3BETACREBBP
ZNF263
CDX2
ELF1
ZEB1
TBP
TFAPC2
TBP
POLR2A
ETS1
GABPA
HEY1
chr17:2117944rs22817272bSMG6Intron 13SRYCREBBP
SrfEP300
Zfp105STAT3
TRIM28
MYC
RBBP5
chr8:126479314rs69826362bLOC105375745Intron1MAFSMARCC1
RFX3
POLR2A
GATA2
CHD2
GTF2F1
chr14:100125720rs71452622bHHIPL1Intron4ESR2SMARC4
ZBTB7A
SMARB1
POLR2A
EZH2
RAD21
BACH1
chr10:104677125rs122210642bCNNM2Upstream CNNM2MAZR,CTCF, ETS1
ETS1
chr8:126478349rs29808532bLOC105375745UpstreamPit-1,RFX3
LOC105375745
chr15:79152421rs50299042bLOC105370915UpstreamNeuroDUSF1
LOC105370915POLR2A
YY1
FOXA1
E2F4
MAX
TAF7
TAF1
MXI1
chr8:126478744rs20018442bLOC105375745UpstreamHSF1RFX3
LOC105375745
chr10:91011680rs13323272bLIPA5' UTRAP-4,CREBBP
CDX2
ELF1
TBP
SPI1
NRF1
ELF1
ETS1
GABPA
SPI1
PAX5
SREBF1
chr17:2102452rs99088882bSMG6Intron10GRCEBPB
AR
chr10:91008878rs22506442bLIPAIntron1Oct-1RUNX3
XBP-1
MAfb
Mafk
MAFB
MAFK
NRL
chr1:109817589rs127403742bCELSR2Exon34HNF1EBF1
HNF1A
DUXA
chr1:222794090rs171633012bMIA3Intron1HNF1EBF1
HNF1A
HNF1B
DUXA
chr21:35644028rs285914152bpseudogenePPAREP300
FOXA1
HDAC2
NFIC
SP1
chr2:203713279rs729347152bICA1LIntron2HMGIYATF2
NFIC
EBF1
EP300
NFKB1
PAX5
chr2:203775474rs729368522bWDR12Intron1ARMAFF
chr2:203926270rs729345122bNBEAL1Intron6TEAD1TEAD4
TEAD3
chr8:19803092rs37797882bLPLIntron1TGIFCEBPB
chr10:44757106rs5185942bCXCL12Downstream intergenicE2AFOXM1
NRSENFIC
NRSFMAX
EBF1
TBL1XR1
chr4:57824931rs76877671dCECR6Sox8FOXA1
chr11:5759712rs931381fSWAP70Intron8
rs3601361fSWAP70Exon13
chr:457798188rs22279011fREST-NOA1Exon6SPI1
chr:1567442595rs172936322aSMAD3Intron4Bach1SIN3A
AP-1TCF7L2
JundM2TFAP2A
Pou1f1TFAP2C
Pou3f1ZNF217
Sox5POLR2A
JDP2STAT1
MXI1
TAF1
E2F1
POLR2A
HDAC1
TCF7L2
MYC
POLR2A
ESR1
EP300
CDX2
HNF4A
CEBPB
EP300
FOS
FOSL1
GATA3
JUNB
JUN
MYC
NR2F2
NR3C1
RAD21
RCOR1
TCF12
JUND
MAFK
EGR1
MAX
chr15:67441996rs188663162bSMAD3Exon4AIREESR1
Elf3NR3C1
SrfPOLR2A
Tcf3
Tcfap2e
chr15:67448898rs80327392bMTERFIntron4SRFCEBPB
FOS
MYC
STAT3
chr9:22098573rs49775742cCDKN2BAS1Intron16ARAR
NR3C1
NR3C2
Ar
Elk-1
c-Ets-1(p54)

One SNP rs2075650 lies in Intron 2 of ApoEApoC1/TOMM40 with a RegulomeDB score of 1f. It is located in RREB1 DNA motif and is an eQTL for TOMM40 (Table 4).

In total 3 of 107 SMG6 associated SNPs, rs2281727, rs7217687 and rs9908888 had a score of 2b and they affect the binding of EP300. rs2281727 is a genome-wide significant SNP located in Intron 9 of SMG6. It is in binding motifs of SRY, Srf and Zfp105 and affects the binding of CREBBP, EP300, STAT3, TRIM28, MYC and RBBP5 (Table 4).

The UBE2Z region had 10 functional SNPs, including a GWAS reported SNP, UBE2Z/rs46522 (RegulomeDB score of 1f). The SNP with the most evidence of regulatory function in this locus is rs4794004 with a score of 1d. It is in DNA motif of Gata5 that alters the expression of UBE2Z and ATP5G1and affects the binding of NR3C1, IN3AK20, CREB1, TAF12, CTCF, POLR2A, USF1, FOXA1, FOXA2 and RBBP5. The other 5 SNPs in this region have a score of 1f. The remaining two regulatory SNPs, rs3744608 and rs12601672, have scores of 2a and 2b, respectively. rs3744608 is located in Intron 3 of UBE2Z and it affects the binding of large number of transcription factors (Table 4).

COL4A1-COL4A2/rs473144 is a GWAS reported SNP, achieving a RegulomeDB score of 2b. This SNP lies in Intron 3 of COL4A2 between STST3:STAT3 DNA motif and affects the binding of POLR2A and EZH2 (Table 4). ZNF259-APOA5-APOA1/rs964184 is a GWAS significant SNP with a score of 1f and is an eQTL for TAGLN. This SNP is located downstream of this gene region and is present in FOXJ2 DNA motif. Another GWAS significant SNP, ZC3HC1/rs11556924 is an exonic variant and the only functional SNP (score = 1f) in this locus; it is also an eQTL for ZC3HC1 (Table 4).

REST-NOA1/rs17087335 is in LD with two functional SNPs (rs2227901 and rs7687767 with RegulomeDB scores of 1f and 1d, respectively). rs768776 lies in DNA motif of Sox8 and affects the binding of FOXA1. SWAP70 has two functional SNPs, rs93138 and rs360136, each with a RegulomeDB score of 1f. SWAP70/rs93138 is an eQTL as evidenced in monocytes.

SMAD3 has three functional SNPs, SMAD3/rs17293632 and SMAD3/rs1866316 and MTERF1/rs8032739 with RegulomeDB scores of 2a, 2a and 2b, respectively. Both are in LD with a lead GWAS SNP (SMAD3/rs56062135). CDKN2BAS1/rs1333049 has one functional SNP(rs4977574) only with RegulomeDB score of 3c. It is a part of a gene cluster on chromosome 9p21 and it maps to Intron 16 of cyclin dependent kinase, an important regulator of cell cycle.

Discussion

Following the sequencing of human genome, a large number of SNPs have been identified that affect disease phenotypes, but their exact roles remain unclear (16). One possible explanation is that some variation affects disease expression at the transcriptional level other than at the protein level. For example, a base pair change in a transcription factor binding site may affect the binding affinity of transcription factors that consequently may alter the transcription of the related genes. These effects are indirect and may seem subtle, but their interactions with other genetic or environmental factors may result in the pathogenesis of common diseases.

Like other complex disorders, a large number of CAD associated risk variants have been discovered by multiple GWAS (12, 13, 17). ENCODE provides information regarding the functionality of human genome (18). This data requires careful interpretation and helps to define the biological function of previously termed ‘junk DNA’. Using bioinformatics tools, we may generate new hypotheses about the gene regulation of complex disorders. In this study, we have used two bioinformatics tools, SNAP and RegulomeDB, in order to identify the putative roles of CAD-associated SNPs.

We examined a total 1,200 SNPs in 54 loci implicated by GWAS, including 58 genome-wide significant SNPs. Ninety-seven SNPs were predicted to have regulatory functions with a RegulomeDB score of <3, but only 8 of them were genome-wide significant. Interestingly, all 8 genome-wide significant SNPs with suggested regulatory function are located either in intronic or intergenic regions, suggesting that these are true associations that regulate gene expression at the transcriptional level.

Among these eight GWAS reported functional SNPs, the SNP with the top RegulomeDB score was LIPA/rs2246833 (Regulome DB score = 1b). This variant is located in Intron 6 of lipase A (LIPA) and is an eQTL for the same gene which catalyzes intracellular triglyceride and hydrolyses cholesterol ester (19).

ZC3HC1/rs11556924 is a GWAS significant CAD associated SNP that returned a score of 1f. rs11556924 is a coding SNP located in the ZC3HC1 gene region encoding NIPA (Nuclear Interaction Partner of ALK) protein. This polymorphism is responsible for arginine-histidine amino acid alteration at position 363 (R363H). The SNP has been associated with essential hypertension in Finnish population (20, 21).

The CYP17A1-CNNM2-NT5C2 gene region has the highest number of regulatory SNPs, including one GWAS significant SNP, rs12413409. This locus affects diastolic blood pressure, systolic blood pressure and body mass index. All three measures are important risk factors for CAD (22). There are 25 putative regulatory SNPs in LD with rs12413409 that are located across four genes on chromosome 10 (CNNM2, NT5C2, AS3MT and BORCS7/ASMT) but affect the expression of same protein USMG5. These findings suggest that USMG5 should be investigated as an important player for CAD pathogenesis. USMG5 (upregulated during skeletal muscle growth protein 5) is also known as diabetes-associated protein in insulin-sensitive tissues that plays a crucial role in the maintenance of ATP synthase structure in mitochondria (23). Chen et al. (24) have purified this protein from bovine heart mitochondria and suggested its role in cell energy metabolism.

APOE-APOC1/TOMM40/rs2075650 is present in the TOMM40 gene region near the APOE-C1 cluster. TOMM40 encodes TOMM40 protein, which is an important subunit 40 of outer mitochondrial membrane protein complex. rs2075650 risk allele has shown an association with low levels of CRP in CAD patients (25).

rs46522, an intronic SNP in Ubiquitin-conjugating enzyme E2Z (UBE2Z) gene region returns a RegulomeDB score of 1f. This SNP is associated with CAD in Iranian and Han Chinese populations (26, 27). The exact mechanism by which genetic alteration in UBE2Z can attribute to the CAD risk is not yet clear; however, rs46522 is in strong LD with the causal SNPs in gastric inhibitory peptide (GIP) gene that encodes GIP protein, a protein that modifies the glucose and lipid metabolism potentially mediating known CAD risk factors.

ZNF259-APOA5APOA1/rs964184 is also an important regulatory SNP. ZNF259 protein polymorphism has been associated with metabolic syndrome in Chinese population. Aung et al. (28) have also shown its association with lipid levels. ZNF259 is located close to APOA5. Overexpression of APOA5 in mice reduces plasma triglyceride levels and mice lacking APOA5 have hypertriglyceridemia (29).

COL4A2/rs4773144 has been identified as functional lead SNP by RegulomeDB (score = 2b). This gene controls collagen proliferation, indicating a potential functional role in atherosclerotic plaque strengthening (30).

SMG6/rs2281727 is an intronic SNP. The potential function of SMG6 in CAD is not yet established. This gene promotes the endonuclease activity and is responsible for protection of telomere ends of chromosomes (16).

Although regulatory elements are most often found in non-coding regions of the genome, we found 5 loci with exonic regulatory SNPs (VAMP8/rs1009, CNNM2/rs943037, GIP/rs2291725, KIAA1462/rs3739998 and UBE2Z/rs15563), indicating the presence of regulatory signals inside the coding sequences as well.

USF1 is an upstream transcription factor whose binding is affected by three SNPs (GIP/rs4794004, FES/rs1894401, HHIPL1/rs28391527), suggests a potential functional link between FES, GIP and HHIPL1 (31).

RegulomeDB identified three important functional SNPs affecting CAD phenotype. Among these, REST-NOA1/rs17087335 is the lead GWAS SNP that encodes a transcription factor which suppresses the voltage gated sodium and potassium channels and it has shown to maintain vascular smooth muscle cells in non-proliferative phase (32). SWAP70/rs10840293 encodes a signaling molecule that is implicated in cell adhesion and migration and it appears to be a potential regulator of leukocyte migration and their adhesion to endothelial cells (33). SMAD3 is a major regulator of TGF-ß. A study on mice has shown that mutations in this gene lead to decreased connective tissue deposition in response to vascular injury (34).

It should be noted that 342 SNPs had returned ‘No Data’ when queried by RegulomeDB. This suggests that current evidence does not support a functional role for those variants. Our results also showed that some loci harbor markedly more regulatory SNPs as compared with other regions. We caution against interpreting this finding to meant that one region is more functionally relevant, as regions with ‘fewer’ functional SNPs may have yet to be interrogated as thoroughly and thus have fewer annotations.

Since these loci are mostly in Europeans, and only 5 of them are replicated in South Asians (35), the findings may not be as relevant to other populations as they are to Europeans as genetic effects can differ across populations. The cause of this varying association with disease phenotype may be the ethnic admixture resulting in population stratification. It is also noteworthy that robust associations of variants with different diseases have been reported in Europeans while other populations (Africans, Asians and Hispanics) failed to demonstrate those associations (36, 37).

Though the cellular mechanisms underlying CAD pathogenesis are established, the molecular basis is not yet agreed upon. Comprehending the molecular basis of disease is crucial before pathogenesis is completely described. The study has identified 97 regulatory SNPs associated with CAD. In summary, our results highlight the importance of considering both disease-associated SNPs and those SNPs in LD, as well as the regulatory function of these SNPs to help identify the causal genetic mechanisms of CAD. The methods which we have implemented here can inform planning of more complete and better directed functional genomic studies.

Supplementary data

Supplementary data are available at Database Online.

Funding

This study was partially supported by Higher Education Commission of Pakistan and the US National Institutes of Health grants (AG030653 and AG041718).

Conflict of interest. None declared.

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Author notes

*

Citation details: Cheema,A.N., Rosenthal,S.L., and Kamboh,M.I. Proficiency of data interpretation: Identification of signaling SNPs/specific loci for coronary artery disease. Database (2017) Vol. 2017: article ID bax078; doi:10.1093/database/bax078

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