Supplementary Material for: Contribution of Cystatin C Gene Polymorphisms to Cerebral White Matter Lesions

<i>Background:</i> Vascular remodeling plays an important role in the development of arteriosclerosis and any of the resulting white matter lesions in the brain. An imbalance between cysteine proteases and the cysteine protease inhibitor cystatin C (CST3) may exacerbate vascular remodeling through degradation of extracellular matrix proteins. Therefore, we evaluated the association between functional polymorphisms in the <i>CST3</i> gene and the development of cerebral white matter lesions. <i>Methods:</i> In a total of 2,676 participants, 3<i> CST3</i> genepolymorphisms were genotyped in 92 cases with severe deep white matter hyperintensity (DWMH), and 184 subjects were randomly selected age- and sex-matched controls without any signs of DWMH. The genetic effects of these polymorphisms on DWMH and plasma CST3 levels were examined. CST3 expression vectors were transfected into an astrocytoma cell line and the expression level of CST3 mRNA was analyzed by quantitative RT-PCR. Intracellular and secreted levels of CST3 in the cell culture were quantified by Western blot and ELISA, respectively. <i>Results:</i> A significant association was found between one <i>CST3</i> gene haplotype and DWMH (p = 0.002). This haplotype was also associated with lower plasma CST3 levels (p = 0.01). An in vitro transfection study revealed that the +148A allele, which is included in the risk haplotype, significantly reduced the secretion and increased the intracellular accumulation of CST3; however, it had no effect on the mRNA expression. <i>Conclusions:</i> Our study shows that polymorphisms in the <i>CST3</i> gene are significantly associated with the likelihood of DWMH. Substitution of A for G at +148 of the<i> CST3</i> gene decreased the extracellular availability of CST3 in vitro, which might result in the activation of protease activity.