Identification and analysis of a SMAD3 cis-acting eQTL operating in primary osteoarthritis and in the aneurysms and osteoarthritis syndrome  E.V.A. Raine, L.N. Reynard, I.M.B.H. van de Laar, A.M. Bertoli-Avella, J. Loughlin  Osteoarthritis and Cartilage  Volume 22, Issue 5, Pages 698-705 (May 2014) DOI: 10.1016/j.joca.2014.02.931 Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Columnar scatter plots of the quantitative expression of SMAD3 in knee OA vs hip OA cartilage. n is the number of patients studied. The horizontal lines in each plot represent the mean and the 95% confidence interval. P-value was calculated using a Mann–Whitney U Test. Osteoarthritis and Cartilage 2014 22, 698-705DOI: (10.1016/j.joca.2014.02.931) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Allelic expression analysis of the 3'UTR transcript SNP rs8031440 using cDNA from cartilage. The genotypes of the patients for the associated SNP rs12901499 are shown. The cDNA allelic ratio in each patient was compared to the 1:1 allelic ratio calculated from the DNA of that patient. Osteoarthritis and Cartilage 2014 22, 698-705DOI: (10.1016/j.joca.2014.02.931) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Columnar scatter plots of the quantitative expression data of SMAD3. (A) shows OA cartilage, (B) shows OA knee cartilage, and (C) shows OA hip cartilage, with patients stratified by genotype at the 3′UTR transcript SNP rs8031440. Due to the low frequency of AA homozygotes at rs8031440 the analysis was performed between GG homozygotes and A-allele carriers (GA and AA combined). n is the number of patients studied. The horizontal lines in each plot represent the mean and the 95% confidence interval. P-values were calculated using a Mann–Whitney U Test. Osteoarthritis and Cartilage 2014 22, 698-705DOI: (10.1016/j.joca.2014.02.931) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Functional analysis of the six SMAD3 3'UTR SNPs in SW1353 cells. (A–C) show the results of the luciferase reporter assays for amplicons A, B and C, respectively. (D) shows the results of the luciferase reporter assay for the four possible haplotypes (CC, CT, TT, and TC) for rs12595334 and rs3743342. Data are the fold expression relative to Renilla activity and are shown as the mean and 95% confidence intervals from three biological repeats each with six technical replicates. P-values were calculated using a Mann–Whitney U Test. Osteoarthritis and Cartilage 2014 22, 698-705DOI: (10.1016/j.joca.2014.02.931) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Functional analysis of the six SMAD3 3'UTR SNPs in MG63 cells. (A–C) show the results of the luciferase reporter assays for amplicons A, B, and C, respectively. (D) shows the results of the luciferase reporter assay for the four possible haplotypes (CC, CT, TT, and TC) for rs12595334 and rs3743342. Data are the fold expression relative to Renilla activity and are shown as the mean and 95% confidence intervals from three biological repeats each with six technical replicates. P-values were calculated using a Mann–Whitney U Test. Osteoarthritis and Cartilage 2014 22, 698-705DOI: (10.1016/j.joca.2014.02.931) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Allelic expression analysis of the 3'UTR SNP rs8031440 using cDNA from vascular smooth muscle cells. The cDNA allelic ratio in each individual was compared to the 1:1 allelic ratio calculated from the DNA of that individual. Osteoarthritis and Cartilage 2014 22, 698-705DOI: (10.1016/j.joca.2014.02.931) Copyright © 2014 Osteoarthritis Research Society International Terms and Conditions