A Candidate Gene Study: Finding Associations between Recurrent Stroke and Genetic Variants of Five Folate-Mediated One Carbon Metabolism Pathway Genes

Stroke is the second leading cause of death in the world and fifth in the United States. Both in the United States and worldwide, stroke is the leading cause of disability. Stroke is a cerebrovascular event which results from a lack of oxygen being supplied to the brain. This occurs due to either a blockage or rupture of a blood vessel leading to the brain. Worldwide, stroke affects approximately 15 million people annually. In the United States, stroke affects about 795,000 people annually, 185,000 of which will be recurrent events. Stroke and recurrent stroke are influenced by environmental and genetic risk factors. The Folate-Mediated One Carbon Metabolism (FOCM) pathway, through its regulation of homocysteine, may influence stroke and recurrent stroke risk. Genetic data from individuals enrolled in the Vitamin Intervention for Stroke Prevention (VISP) clinical trial were used to investigate associations between FOCM pathway candidate genes and stroke-related phenotypes using candidate gene fine-mapping approaches. 136 single-nucleotide polymorphisms (SNPs) identified from a genome-wide association study and targeted next-generation DNA sequencing of five candidate genes (MTHFR, MTR, GNMT, CBS, and TCN2) were genotyped, followed by single-SNP and gene-based statistical association analyses for 20 stroke-related phenotypes. Notably, the MTHFR variant rs1801133, or C677T, was found to be significantly associated with folate levels in the blood. This polymorphism has been previously associated with a number of fertility and developmental disorders, cancers, and vascular diseases, including stroke. Three novel associations between SNPs and both post-methionine homocysteine and delta post-methionine homocysteine levels in the blood were identified in this research and include rs1129187 and rs2274514, both of which are variants downstream of GNMT, as well as rs466791, a variant downstream of CBS. In total, we identified single-SNP associations for homocysteine (n=16) and folate (n= 1) levels. Gene-based Sequence/SNP-set Kernal Association Test (SKAT) methods identified statistical associations for total cholesterol (CBS), post-methionine homocysteine levels (CBS, GNMT, and PEX6), delta post-methionine homocysteine levels (CBS, GNMT, and PEX6), and VISP recurrent stroke (CBS) through the use of six different SKAT methods. This novel and thorough approach of using multiple SKAT methods yielded significant associations which may not have otherwise been identified. The statistically significant associations suggest a role of genetic variants in FOCM genes and stroke risk. Implications of these findings could pave the way for clinical and functional assays that may lead to more precise therapeutics to alleviate the effects of stroke