Cindy Boer

124 | Chapter 3.1 and ERP27 , since no SNP in high LD with rs4764133 is present in the coding region. In ASE, the influence of exonic alleles on gene expression in -cis is measured within heterozygote subjects, circumventing strong effects from environmental or trans-acting influences. This property results in ASE analysis to be a more statistically powerful ap- proach, when compared with classical eQTL analysis.[28] Subsequently, we found that the OA risk alleles for three coding variants in high LD with the lead variant, rs4236 ( Supplementary Figure 3a , 39.6%C allele, p-value<5x10 –16 ), rs1049897 ( Supplemen- tary Figure 3b , 44.4% A allele, p-value<5x10 –10 ) and rs1800801 ( Figure 3a , 40.7% T allele, p-value<5x10 –16 ), were significantly correlated with lower expression of MGP , marking imbalanced expression among heterozygotes, independent of the disease sta- tus of the articular cartilage. No ASE was observed between SNPs rs11276, rs3088189 and rs1861698 (residing in C12orf60 and in high LD with the lead SNP, r 2 >0.8, Table 2 ). Technical and biological replication was performed using a custom allele-specific TaqMan assay for rs1800801 in eight additional heterozygous individuals for which we isolated RNA from P cartilage (n=2), OAL (n=2) or both (n=4) from patients with prima- ry knee OA and confirmed the observed imbalance in preserved articular cartilage ( Fig- ure 3b , relative allelic difference=0.92, p-value<1x10 −06 ), as well as in eight knee sub- chondral bone samples ( Figure 3c , relative allelic difference=0.78, p-value<1x10 −04 ). Discussion Here, we show for the first time, that there is a robust genome-wide significant associa- tion between rs4764133, located near MGP , and hand OA. Furthermore, we performed functional validation showing that MGP coding variants in LD with rs4764133 are as- sociated with ASE of MGP , which may increase risk of hand OA by lowering inhibition of articular cartilage calcification, since MGP is an essential inhibitor of cartilage calcifi- cation. [29,30] These findings suggest that MGP could be considered a prioritised drug target for hand OA, since genetically supported drug targets double the success rate of therapeutics in clinical development.[31] MGP is an essential inhibitor of cartilage calcification, and genetic deficiencies of MGP in humans and mice have been linked to abnormal mineralisation of soft tissues, including cartilaginous tissue.[29,32] Furthermore, MGP has been previously implicat- ed in relation to OA. A small candidate study reported marginally significant association between hand OA and genetic variants in MGP (rs1800802 and rs4236).[33] This is consistent with our findings that the minor allele for rs4764133 and related coding variants in high LD (r2>0.8), rs1800802 and rs4236, increase the risk of hand OA and that we found high expression of MGP in both P and OAL articular cartilage. In contrast,