SUPPLEMENTAL MATERIAL

Size: px

Start display at page:

Download "SUPPLEMENTAL MATERIAL"

Gyles Perkins
6 years ago
Views:

1 SUPPLEMENTAL MATERIAL Supplementary Table 1: RT-qPCR primer sequences. Sequences are shown from 5 to 3 direction; all primers are designed using mouse genome as reference. 36B4-F; TGAAGCAAAGGAAGAGTCGGAGGA LIPA-F; TGACTGACTAGCAAGCGTCCACAA 36B4-R; AAGCAGGCTGACTTGGTTGCTTTG L14Rik-F: TGGATACTGGTTTGAAGGGAGGCA L14Rik-R: AGCCTTTGTCCCAGCTAAAGTCCT ABCG8-F: TTGGACAACCTGTGGATAGTGCCT ABCG8-R: GGTGAAGTTGCCGATTTGTGTGGT ABO-F: ACAGACACTGAACCATCCTGGGTT ABO-R: AGACAAACACTGCGAAGGGAAGGA ADAMTS7-F; TCATGAACATGGTGGCTGGACTCT ADAMTS7-R; AGTCTCTTCGGCATGGTGTGTGAT ApoA1-F: TCACCCACACCCTTCAGGATGAAA ApoA1-R: ACACATAGTCTCTGCCGCTGTCTT APOA4-F: TTCTGACTCCGGGAAACATCCAGT APOA4-R: CTTGGCATTGTTGCTTAGCTGGGT APOA5-F: GCTTTGCAAACCCAGCTTCTGGTA APOA5-R: TTAACAGGAGCCCAACTCCAGGTT APOC3-F: TACAGGGCTACATGGAACAAGCCT APOC3-R: AGAATCCCAGAAGCCGGTGAACTT ATP5G1-F; ACGGGAATTCCAGACCAGTGTCAT ATP5G1-R; AGCTACCAAACACTGTGCCAATGC B2M-F; GCAGGTTCAAATGAATCTTCAGAGCATC B2M-R; TACGTAACACAGTTCCACCCGCCTC CD31-F; TGGAGTGGTCATCGCCACCTTAAT CD31-R; AACAGAAACCCGTGGAGATGTCCA CDKN2A-F: ACATCGTGCGATATTTGCGTTCCG CDKN2A-R: TTTAGCTCTGCTCTTGGGATTGGC CDKN2B-F: TGTTTGACATTTGGGTGGGTGCAG CDKN2B-R: ATTTCCTTGTCGAGCTGGAGGTGA CELSR2-F: ATGAGTGTGCTGGTGTCAGATGGT CELSR2-R: TGAATGAAGAGTCCCAGCAGTGGT CNNM2-F; GCCCACCGTGAAAGAAAGCAAGAT CNNM2-R; ATCTGGGATGGGCTAAACGCTTCT COL4A1-F; AGGATGCAACGGTACAAAGGGAGA COL4A1-R; TGGCCGAGAATTTCACCAGGATCT COL4A2-F: GCCAAACGCACTTCCTGGAATCAA COL4A2-R: CGGTGTTGCCCATGAATCCTTGTT CXCL12-F; TGCACGGCTGAAGAACAACAACAG LIPA-R; ATTACCTCCACACAAGGGCCAGAA MAPKAPK5-F; GCACCAAGCCAAAGGACGGTATTT MAPKAPK5-R; GAGCTTGCATTCGCGGTTGTACTT MIA3-F; TGAGTGCCATCGGGTTCAAGAAGA MIA3-R; TCGGCATGCGATTTACTCCACTCT MORF4L1-F; AATGGAGATGGTGGCAGTACCAGT MORF4L1-R; AATGGAGATGGTGGCAGTACCAGT MRAS-F: TCAGTGGGCCATCTTGGATGTTCT MRAS-R: TTGGGAATGACTCCCTGTCCTTGA MRPS6-F; TTGCTGCAGGAGGTAGAAACTGCT MRPS6-R; AGGCTTGCAAGACAACCAAGTGTG NT5C2-F: AAGTCAAAGAAACGGCAAGGGTGG NT5C2-R: TGCTGTCCAGGTGCTTGTAGAGTT PCSK9-F; ACAAGGACAGTCAACTCACAGCCA PCSK9-R; TGCAGCAGATGAGGACATCTGGAA PDGFD-F: ACTCCGCAGAGAGCATCCATCAAA PDGFD-R: GGCCATTGCTTGTCACCTGAATGT PEMT-F; TATGATGAGCCAGCCCAAGATGGA PEMT-R; GAAGCTGGACAGCACAAACACGAA PHACTR1-F; AAATCAAGAGGAGGCTGACTCGCA PHACTR1-R; TGCCACTTCCACGTAGTCACTGAA PIK3CG-F; TTTGTGGAAGCGAACATCCAGCAC PIK3CG-R; ATGCCAAACTCCAGCCACACATTC PPBP-F-F: TAACCTCCAGATCTTGCTGCTGCT PPBP-R-R: ACTCCTGGCCTGTACACATTCACA PSRC1-F; TCCTCAGCTAGCAACTCCCAATGT PSRC1-R; AGAATCTGGAGCTAGGGCACCTTT RASD1-F: AAGAGGTGCAAAGGCTCAAACAGC RASD1-R: TTTGTTACCGCAAATGACCAGCGG SH2B3-F: AGCCACTTTCTGCAGCTCTTCGAT SH2B3-R: AGTTCAGCTGCTGTTCATCTCCCA SLC22A3-F; ATCACCCGGAAGCAAGGAGAGAAA SLC22A3-R; GCGCTCGTGAACCAAGCAAACATA SLC5A3-F; TATTGTGGAGATGCAAGGAGGCCA SLC5A3-R; AAGAACAAAGCCTGCCATTCCACC SMG6-F; TATCGGGAGCAAGCCAACGATACA

2 CXCL12-R; TCACACCTCTCACATCTTGAGCCT CYP17A1-F: AAGCATATCCTTGTCACGGTGGGA CYP17A1-R: ACGGTGTTCGACTGAAGCCTACAT GALNT4-F; GATTGGTGGGTTTGACTGGCGTTT GALNT4-R; ATTCTTGACGTTCGCCTGTCCCTT GIP-F: TGGCTTTGAAGACCTGCTCTCTGT GIP-R: GGGCCAGCAAATTTAGCATGGGAT HHIPL1-F; AAGCTGGGTAAATCAGTCACCGGA HHIPL1-R; ACCTCGCTATACTTCCATTGGCCT HNF1A-F;TGTCTGCGGCCTTACACCAAGTAT HNF1A-R; AGGTAGCGAGGCCATGATAAGGTT ICAM1-F; AGATCACATTCACGGTGCTGGCTA ICAM1-R; AGCTTTGGGATGGTAGCTGGAAGA IL5-F: TCCCATGAGCACAGTGGTGAAAGA IL5-R : AGGAAGCCTCATCGTCTCATTGCT KCNE2-F: AGGGCTTACAGGTCTCAAGCTGAA KCNE2-R: ATTGGCTAATGTGGCCATGCTTCC KIAA1462-F;AGAATCGCGTCTTGGTGTTCCTGA KIAA1462-R; AGGGAAGACTGAGTGCAAAGCTGA KLHDC10-F; ACGGACTGGGTCGCTGTTTAAGAT KLHDC10-R; AACGCTCGATGAGTTCCTGTGTGA SMG6-R; AGGATAGGGTTGCTGGCTGCTAAA SNF8-F; AGAAGAAACTCGCAGAGGCCAAGT SNF8-R; TCTTCCGGATCTCTTGCTTGTGCT SORT1-F; ACCTGTTAGCTCTCAGCACCGAAA SORT1-R; CAGCTTTGCAGGAGCCATTCACAT SRR-F; AAGGAACAATTGCCCTGGAAGTGC SRR-R; ATTCCAGCAACCATTCCTCCTCCT TCF21-F; AGATCCCACCTCAAACCCAACACA TCF21-R; TGTTGGAGTCCACTTTCAGGGAGT TCTN1-F; TGCTGGTGCTCCTAAACTGCTACA TCTN1-R; TGGCCTTGGTGGAATTGAGATCCT TRIB1-F: CTTCAAGCAGATTGTTTCCGCCGT TRIB1-R: AGGCTTTCCAGTCTAAGCTGGGTT UBE2Z-F: AGTCTGTGACATGATGGAGGGCAA UBE2Z-R TTGCAGGCCACCTCATAGAAGTCA WDR12-F; AACGTTTGGCATCTGGAAGCACAG WDR12-R; AGAGACAGCGACACCAAAGAACCA ZC3HC1-F; AGTGAGTTCCTGGACCGGTTTCAA ZC3HC1-R; AATCAAGCTCATCCTCCAGCAGGT ZFP259-F; TGATGATCCCTCGGGAAACAGCTT ZFP259-R; TGCACTCTGGGCAGTTAGTGTTGA LDLR-F; CAACAATGGTGGCTGTTCCCACAT LDLR-R; ACTCACACTTGTAGCTGCCTTCCA

3 Supplementary Table 2: Fold-change values of GWAS CAD candidate genes exhibiting differential expression from healthy to diseased cell types.

4 Supplementary Table 3: SNPs that had the most significant association in published GWAS studied were interrogated for their association with nearby gene expression in human aortic endothelial cells. Proxies for SNPs that were not genotyped were used to determine significant association. NA indicates no information was available for that SNP. GWAS SNP Chrom osome Proxy SNP Pairwise LD (r2) Distance from GWAS SNP (bp) Candidate gene(s) reported mouse homologs rs rs PPAP2B Ppap2b rs rs IL5 Il5 rs rs PIK3CG Pik3cg rs rs TRIB1 Trib1 rs rs ABO Abo rs rs PDGFD Pdgfd rs rs SH2B3 Sh2b3 rs rs ADAMTS7,MORF4L1 Adamts7, Morf4l1 rs rs RASD1, SMCR3, PEMT Rasd1, --, Pemt rs rs SMG6-SRR Smg6-Srr rs rs SLC5A3-MRPS6-KCNE2 Slc5a3-Mrps6-Kcne rs genotyped PCSK9 Pcsk9 rs genotyped MIA3 Mia3 rs genotyped SORT1-CELSR2-PSRC1 Sort1-Celsr2-Psrc1 rs genotyped MRAS Mras rs genotyped TCF21 Tcf21 rs genotyped PHACTR1 Phactr1 rs genotyped ANKS1A Anks1a rs genotyped ZC3HC1 Zc3hc1 rs genotyped ANRIL/CDKN2BAS Cdkn2a-Cdkn2b rs genotyped CYP17A1-NT5C2- CNNM2 rs genotyped LIPA Lipa rs genotyped CXCL12 Cxcl12 rs genotyped KIAA1462 Kiaa1462 Cyp17a1-Nt5c2- Cnnm2 rs genotyped ZNF259, APOA1-C3- A4-A5 Zfp259, ApoA1-C3- A4-A5 rs genotyped COL4A1-A2 Col4a1-Col4a2 rs genotyped UBE2Z-GIP-ATP5G1- SNF8 Ube2z-Gip-Atp5g1- Snf8 rs genotyped LDLR Ldlr rs NA LPA -- rs NA ABCG8 Abcg8 rs NA WDR12 Wdr12 rs NA C6orf L14Rik rs NA SLC22A3-LPAL2-LPA Slc22a3 rs NA HHIPL1 Hhipl1 rs NA APOE ApoE

5 Supplementary Table 4: Differential expression of genes at CAD GWAS loci with multiple candidate genes reported.

6 Supplementary Table 5: Differential expression of genes at CAD GWAS loci across all four cell types as determined by multivariate analysis comparing healthy to diseased state. Hotelling s T 2 test was performed for each gene using all samples; significance threshold is set at Gene Symbol Hotelling s T 2 test P-value Gene Symbol Hotelling s T 2 test P-value Col4a2 6.99E-03 Apoa1 1.00E+00 Cxcl E-03 Apoc3 1.00E+00 Nt5c2 2.60E-02 Cdkn2a 1.00E+00 Icam1 2.65E-02 Celsr2 1.00E+00 Mia3 2.67E-02 Cyp17a1 1.00E+00 Ppap2b 3.88E-02 Gip 1.00E+00 Pik3cg 4.41E-02 Hhipl1 1.00E+00 Mapkapk5 5.65E-02 Il5 1.00E+00 Smg6 5.80E-02 Klhdc E+00 Trib1 6.01E-02 Ldlr 1.00E+00 Adamts7 6.15E-02 Morf4l1 1.00E+00 Mrps6 1.08E-01 Pdgfd 1.00E+00 Cdkn2b 1.17E-01 Pemt 1.00E+00 Mras 1.22E-01 Phactr1 1.00E+00 Anks1a 1.33E-01 Rasd1 1.00E+00 Ube2z 1.37E-01 Slc22a3 1.00E+00 Snf8 1.43E-01 Slc5a3 1.00E+00 Srr 1.54E-01 Sort1 1.00E+00 Pcsk9 1.57E-01 Tcf E+00 KIAA E-01 Tctn1 1.00E+00 Col4a1 1.78E-01 Wdr E+00 Galnt4 1.94E-01 Zc3hc1 1.00E+00 Psrc1 2.63E-01 Zfp E+00 Cnnm2 2.87E-01 Abcg8 Not expressed Lipa 3.77E-01 Abo Not expressed Sh2b3 4.28E-01 Apoa4 Not expressed L14Rik 5.11E-01 Apoa5 Not expressed Atp5g1 6.28E-01 Kcne Not expressed

SUPPLEMENTARY FIGURES Supplementary Figure 1: ApoE -/- BL6 mice at 4 weeks of age do not have the visible lipid deposits that are present in 24 week ApoE -/- BL6 mice, while 24 week wild-type BL6

7 SUPPLEMENTARY FIGURES Supplementary Figure 1: ApoE -/- BL6 mice at 4 weeks of age do not have the visible lipid deposits that are present in 24 week ApoE -/- BL6 mice, while 24 week wild-type BL6 mice do not develop atherosclerosis. Histology slides of sectioned aortic arch stained with oil red o, magnification 20x. A: 4 week, BL6 ApoE -/-; no lipid deposits observed B: 24 week BL6 ApoE -/-; intimal lesions visible C: 24 week BL6 wild-type; no lipid deposits observed

8 Supplementary Figure 2: Clustering of donors used in the study based on their genotypes with HapMap3 populations. HAEC: Human Aortic Endothelial Cell Donors; ASW: African ancestry in Southwest USA; CEU: Utah residents with Northern and Western European ancestry from the CEPH collection; CHB: Han Chinese in Beijing, China; CHD: Chinese in Metropolitan Denver, Colorado; GIH: Gujarati Indians in Houston, Texas; JPT: Japanese in Tokyo, Japan; LWK: Luhya in Webuye, Kenya; MXL: Mexican ancestry in Los Angeles, California; MKK: Maasai in Kinyawa, Kenya; TSI: Toscani in Italia; YRI: Yoruba in Ibadan, Nigeria.

9 Supplementary Figure 3. Genome-wide association of gene expression in HAECs under control and oxpapc conditions. The diagonal line with strong association depicts the local eqtls and each off-diagonal dot depicts the location of distant eqtls. Color of the dot is proportional to the association significance p-value. 1,502 and 1,504 genes had a significant cis association with nearby SNPs in control and oxpapc-treated cells, respectively. 1,239 of the transcripts overlapped between the two datasets. 185 and 164 genes showed evidence for trans associations in control and oxpapc-treated cells, respectively. 22 of the transcripts overlapped between two datasets

10 Supplementary Figure 4. Regional association plots of SNF8 transcript levels. Left panels show the regional association of SNF8 gene expression in (A) control and (B) oxpapc conditions. Association of the genotyped SNPs and the transcript expression as indicated by log 10 (P) values are plotted. The color of the SNPs indicates the strength of linkage disequilibrium with the peak SNP, which is in purple. Recombination rates are based on 1000 genomes European populations (1). Right panels show the expression level of SNF8 in individuals based on their SNP rs46522 genotypes.

11 Supplementary Figure 5. Regional association plots of SRR transcript levels. Left panels show the regional association of SRR gene expression in (A) control and (B) oxpapc conditions. Association of the genotyped SNPs and the transcript expression as indicated by log 10 (P) values are plotted. The color of the SNPs indicates the strength of linkage disequilibrium with the peak SNP, which is in purple. Recombination rates are based on 1000 genomes European populations (1). Right panels show the expression level of SRR in individuals based on their SNP rs (proxy for GWAS peak SNP rs216172, r 2 =1) genotypes.

12 Supplementary Figure 6. Regional association plots of MAPKAPK5 transcript levels. Left panels show the regional association of MAPKAPK5 gene expression in (A) control and (B) oxpapc conditions. Association of the genotyped SNPs and the transcript expression as indicated by log 10 (P) values are plotted. The color of the SNPs indicates the strength of linkage disequilibrium with the peak SNP, which is in purple. Recombination rates are based on 1000 genomes European populations (1). Right panels show the expression level of MAPKAPK5 in individuals based on their SNP rs (proxy for GWAS peak SNP rs , r 2 =0.873) genotypes.

13 Supplementary Figure 7. Regional association plots of TCTN1 transcript levels. Left panels show the regional association of TCTN1 gene expression in (A) control and (B) oxpapc conditions. Association of the genotyped SNPs and the transcript expression as indicated by log 10 (P) values are plotted. The color of the SNPs indicates the strength of linkage disequilibrium with the peak SNP, which is in purple. Recombination rates are based on 1000 genomes European populations (1). Right panels show the expression level of TCTN1 in individuals based on their SNP rs (proxy for GWAS peak SNP rs , r 2 =0.873) genotypes.

14 Supplementary Figure 8. Regional association plots of GALNT4 transcript levels. Left panels show the regional association of GALNT4 gene expression in (A) control and (B) oxpapc conditions. Association of the genotyped SNPs and the transcript expression as indicated by log 10 (P) values are plotted. The color of the SNPs indicates the strength of linkage disequilibrium with the peak SNP, which is in purple. Recombination rates are based on 1000 genomes European populations (1). Right panels show the expression level of GALNT4 in individuals based on their SNP rs genotypes.

15 Supplementary Figure 9. Regional association plots of PPAP2B transcript levels. Left panels show the regional association of PPAP2B gene expression in (A) control and (B) oxpapc conditions. Association of the genotyped SNPs and the transcript expression as indicated by log 10 (P) values are plotted. The color of the SNPs indicates the strength of linkage disequilibrium with the peak SNP, which is in purple. Recombination rates are based on 1000 genomes European populations (1). Right panels show the expression level of GALNT4 in individuals based on their SNP rs (proxy for GWAS peak SNP rs , r 2 =0.831) genotypes.

16 Supplementary Figure 10. Regional association plots of ICAM1 transcript levels. Left panels show the regional association of ICAM1 gene expression in (A) control and (B) oxpapc conditions. Association of the genotyped SNPs and the transcript expression as indicated by log 10 (P) values are plotted. The color of the SNPs indicates the strength of linkage disequilibrium with the peak SNP, which is in purple. Recombination rates are based on 1000 genomes European populations (1). Right panels show the expression level of ICAM1 in individuals based on their SNP rs genotypes. SUPPLEMENTARY REFERENCE 1. Pruim, R. J., R. P. Welch, S. Sanna, T. M. Teslovich, P. S. Chines, T. P. Gliedt, M. Boehnke, G. R. Abecasis, and C. J. Willer LocusZoom: regional visualization of genome-wide association scan results. Bioinformatics 26:

Genotyping Technology How to Analyze Your Own Genome Fall 2013

Genotyping Technology How to Analyze Your Own Genome Fall 2013 Genotyping Technology 02-223 How to nalyze Your Own Genome Fall 2013 HapMap Project Phase 1 Phase 2 Phase 3 Samples & POP panels Genotyping centers Unique QC+ SNPs 269 samples (4 populations) HapMap International