History and Purpose Elevated homocysteine (tHcy) levels are known to be associated with increased risk of ischemic stroke (IS). with IS (P<0.0003) after correcting for multiple testing. For stroke subtypes the sentinel SNP located upstream of was significantly associated with SVD (small vessel disease) (P=0.0022) while one SNP located in was significantly associated with LVD (large vessel disease) (P=0.00019). A genetic risk score including the 18 SNPs did not show significant association with IS or its subtypes. Conclusions This study found several potential associations with IS and its subtypes: an association of an variant with SVD an variant NBI-42902 with LVD and associations of and variants with overall IS. (rs838133 OR 1.04; 95%CI 1.00- 1.07; (rs7422339 OR 0.96; 95%CI 0.92-0.99; (rs9369898 OR 1.12; 95%CI 1.04-1.21; (rs838133 OR 1.07; 95%CI 1.00-1.15; (rs9369898) also passed Bonferroni corrected P-value of 0.0027. The major allele A of NBI-42902 rs9369898 associated with higher tHcy levels was also associated with increased risk of SVD. There was no evidence of between study heterogeneity for rs9369898 (I2=7.4%; P-het=0.37). The GRS including the 18 independent tHcy SNPs did not show an association with SVD risk (OR 1.1; 95%CI 0.85- 1.42; gene was associated at a nominal P-value (P<0.05) but did not pass Bonferroni correction for multiple testing (Supplementary Table V). The GRS of the 18 independent tHcy SNPs did not show an association with LVD risk (OR 1.06; 95%CI 0.82-1.35; were highly correlated (r2=0.766) with each other and moderately correlated with the third SNP rs17271121 located in an intron of (r2[rs9379800 rs17271121]=0.306; r2[rs17271121 rs12664474]=0.545). None of the three SNPs were in LD with the tHcy NBI-42902 associated polymorphism rs548987 (r2<0.035). Table 1 Association with IS and its subtypes of SNPs located ±50kb from the 18 tHcy associated SNPs at a P<0.0003 obtained after adjustment for multiple testing. In addition another SNP rs2287921 located in an intron of gene within 50kb from the polymorphism rs838133 was associated with IS at a P-value of 0.0002 (OR 0.94; 95%CI 0.91-0.97) lower than Bonferroni correction for multiple testing (P<0.0003). This SNP was in moderate LD (r2=0.658) with the sentinel SNP rs838133 which may suggest that this could be a broader risk region spanning the two neighboring genes and gene near the two sentinel SNPs in this gene rs1801133 PPP1R53 and rs12134663 was associated with LVD with a P-value of 1 1.92×10?4 (OR 1.15; 95%CI 1.06-1.23) lower than the Bonferroni correction for multiple testing (P<0.0003). This missense SNP was in low LD with the two tHcy sentinel SNPs (r2[rs1801131 rs1801133]=0.19; r2[rs1801131 rs12134663]=0.268). For SVD one SNP (rs566295) located upstream polymorphism rs9369898 (r2=0.264). For CE no significant associations were observed at a threshold exceeding Bonferroni correction for multiple testing (P<0.0003). Discussion This large study of 12 389 IS cases and 62 4 controls has identified several potential novel associations with IS and its subtypes by testing previously reported NBI-42902 associations with homocysteine levels in stroke. We found evidence of an association of gene with SVD an association of gene with LVD and associations of and with overall IS. Of the 18 tHcy polymorphisms investigated one polymorphism located upstream of gene was significantly associated with SVD while none of the 18 tHcy related SNPs was significantly associated with LVD CE or overall IS. The allele correlated with increased tHcy levels at gene showed to be also associated with increased risk of SVD suggesting a potential small but significant effect on SVD risk. On a closer inspection of this region another SNP located 44kb from the sentinel SNP and in low LD with the sentinel SNP was also associated with SVD. This polymorphism was also significantly associated with homocysteine levels at a genome-wide significance level (P=2.27E-09)11 but conditional analysis has not been conducted to establish if these two polymorphisms were independently influencing homocysteine levels. These two polymorphisms may thus potentially be correlated with either a single or multiple regulatory variants in this region that modulate both tHcy levels and SVD risk. The gene is known to encode the mitochondrial enzyme methylmalonyl Coenzyme A mutase a vitamin B12-dependent enzyme. NBI-42902 Considering that vitamin B12 is an important cofactor in homocysteine metabolism a potential pleiotropic effect of gene on both plasma homocysteine and.