1. Functional Analysis of a Carotid Intima-Media Thickness Locus Implicates BCAR1 and Suggests a Causal VariantCLINICAL PERSPECTIVE
by Freya Boardman-Pretty, Andrew J.P. Smith, Jackie Cooper, Jutta Palmen, Lasse Folkersen, Anders Hamsten, Alberico L. Catapano, Olle Melander, Jacqueline F. Price, Meena Kumari, John E. Deanfield, Mika Kivimäki, Karl Gertow, Andrea Baragetti, Giuseppe Danilo Norata, and Steve E. Humphries
Circ Genom Precis Med
Volume 8(5):
October 20, 2015
Copyright © American Heart Association, Inc. All rights reserved.

2. rs4888378 allele is associated with differential expression depending on sequence elements.
rs allele is associated with differential expression depending on sequence elements. Luciferase reporter assay data. Fragments 1 and 2 show no significant differential expression (A and B). Fragments 3 and 4 show a decrease in expression for the A allele, with the decrease in fragment 4 being much more pronounced (C and D). Expression values are normalized to pGL3-promoter control. P values derive from unpaired t test. Error bars show SEM (4 plates of 12 replicates each.)‏
Freya Boardman-Pretty et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

3. Annual change in intima-media thickness (IMT) in Progressione della Lesione Intimale Carotidea by rs genotype.
Annual change in intima-media thickness (IMT) in Progressione della Lesione Intimale Carotidea by rs genotype. A, Overall, despite a trend for lower ΔIMT with each A allele, no significant association was seen with genotype. B, In women only, ΔIMT in the AA genotype was lower than in the GG genotype (P=0.04), although the overall trend was not significant. C, In men only, no trend or significant association was seen.
Freya Boardman-Pretty et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

4. Forest plot showing intima-media thickness (IMT) by rs4888378 allele in (A) men and (B) women.
Forest plot showing intima-media thickness (IMT) by rs allele in (A) men and (B) women. Overall no significant association between the single nucleotide polymorphism and IMT is seen in men (β=−0.0030, P=0.0591), whereas in women, the A allele is associated with a decrease in IMT for each A allele (β=−0.0046, P=1.59×10–4). IMT is here common carotid IMT (the IMT variable common to all cohorts). CI indicates confidence interval; EAS, Edinburgh Artery Study; IMPROVE, IMT and IMT-Progression as Predictors of Vascular Events; MDC, Malmö Diet and Cancer study; PLIC, Progressione della Lesione Intimale Carotidea; and WHII, Whitehall II.
Freya Boardman-Pretty et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

5. Linkage disequilibrium plot for lead single nucleotide polymorphism (SNP) rs4888378.
Linkage disequilibrium plot for lead single nucleotide polymorphism (SNP) rs Data from 1000 Genomes (Pilot 1); graph plotted with data and script from SNP Annotation and Proxy Search.18 Lead SNP rs is shown in red; other SNPs are shown relative to their position at the locus (x axis) and linkage disequilibrium (LD; r2) with rs (y axis). The majority of strong LD SNPs lie within CFDP1 and TMEM170A; they may affect these genes, or lie within enhancer regions which affect expression of other genes. Chromosome coordinates are based on NCBI Build 36.1 (hg18).
Freya Boardman-Pretty et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

6. Single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium selected for further analysis.
Single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium selected for further analysis. The locations of the SNPs are shown with SNP name, alleles, minor allele frequency, and evidence used for selection. Gene coordinate data from UCSC Genome Browser.20
Freya Boardman-Pretty et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

7. Electrophoretic mobility shift assays (EMSAs) show differential protein binding for rs and competition of the protein-binding band.
Electrophoretic mobility shift assays (EMSAs) show differential protein binding for rs and competition of the protein-binding band. Bands show the position of biotin-labeled probe on the gel (direction from top to bottom). Probes unbound by DNA run fastest and are not visible on the picture; bands here are protein-bound probe that migrates more slowly. A and G refer to the respective alleles of rs Competitor probes (unlabelled) are added in excess to nuclear extract 30 minutes before labeled probe; hence, a probe-protein band will disappear when the protein binds first to the competitor. A, Differential protein binding to rs Lane 1 shows binding of the control protein, nuclear factor (NF)-κB, to the control band (NF-κB consensus); lane 2 shows band being competed out by unlabelled NF-κB probe. Lanes 3 and 5 show weak and strong protein binding to the A and G probes, respectively. Lanes 4 and 6 show the binding to be competed out by respective unlabelled probes. B, Multiplex competitor EMSA. Lanes 1 and 2 show NF-κB control as above. Lanes 3 to 14 show protein binding to the G probe is competed out only by the unlabelled G probe, as expected, and by the FOXA competitor.
Freya Boardman-Pretty et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

8. FOXA consensus sequence used in multiplex competitor electrophoretic mobility shift assay compared with genomic sequence around rs
FOXA consensus sequence used in multiplex competitor electrophoretic mobility shift assay compared with genomic sequence around rs The single nucleotide polymorphism G allele matches more closely with the consensus sequence, suggesting FOXA may be the protein binding here.
Freya Boardman-Pretty et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.

9. BCAR1 expression by rs4888378 genotype in (A) aortic artery and (B) tibial artery.
BCAR1 expression by rs genotype in (A) aortic artery and (B) tibial artery. Data and graph from Genotype–Tissue Expression Portal.21 Of the 9 genes analyzed at the locus, only BCAR1 remained significant after correction for multiple testing; expression of BCAR1 increased with each allele of the high-risk G allele. eQTL indicates expression quantitative trait loci.
Freya Boardman-Pretty et al. Circ Cardiovasc Genet. 2015;8:
Copyright © American Heart Association, Inc. All rights reserved.