Population-Specific Associations of Deleterious Rare Variants in Coding Region of P2RY1–P2RY12 Purinergic Receptor Genes in Large-Vessel Ischemic Stroke Patients

Abstract

The contribution of low-frequency and damaging genetic variants associated with platelet function to ischemic stroke (IS) susceptibility remains unknown. We employed a deep re-sequencing approach in Polish patients in order to investigate the contribution of rare variants (minor allele frequency, MAF < 1%) to the IS genetic susceptibility in this population. The genes selected for re-sequencing consisted of 26 genes coding for proteins associated with the surface membrane of platelets. Targeted pooled re-sequencing (Illumina HiSeq 2500) was performed on genomic DNA of 500 cases (patients with history of clinically proven diagnosis of large-vessel IS) and 500 controls. After quality control and prioritization based on allele frequency and damaging probability, follow-up individual genotyping of deleterious rare variants was performed in patients from the original cohort. Gene-based analyses identified an association between IS and 6 rare functional and damaging variants in the purinergic genes (P2RY1 and P2RY12 locus). The predicted properties of the most damaging rare variants in P2RY1 and P2RY12 were confirmed by using mouse fibroblast cell cultures transfected with plasmid constructs containing cDNA of mutated variants (FLIPR on FlexStation3). This study identified a putative role for rare variants in P2RY1 and P2RY12 genes involved in platelet reactivity on large-vessel IS susceptibility in a Polish population.

Increased burden of rare deleterious variants of the KCNQ1 gene in patients with large‑vessel ischemic stroke

“…org/packages/AssotesteR/versions/0.1-10. The significance threshold was adjusted to the number of re-sequenced loci, when needed (13,25).…”

“…For the power calculations, instead of using individual rare variants, we decided to use predicted cMAF for all deleterious rare variants in the sequenced loci. We have followed a self-sufficient, closed-form, maximum-likelihood estimator for allele frequencies that accounts for errors associated with sequencing, and a likelihood-ratio test statistic that provides a simple means for evaluating the null hypothesis of monomorphism (13,26,27). Unbiased estimates of allele frequencies 10/N (where N is the number of individuals sampled) appear to be achievable, and near-certain identification of a single nucleotide polymorphism (SNP) requires a cMAF of at least 0.01 (i.e., 10 variants per cohort).…”

“…The fidelity of the mutations for each variant was confirmed by Sanger sequencing. Details about the transfection techniques and cell culture conditions used in our laboratory were published before (13).…”

“…All participants in the study signed the informed consent. Full description of the study cohort, including the inclusion and exclusion criteria were published previously (12,13,(22)(23)(24). Based on the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification we included: i) All patients classified as having ischemic stroke due to large-vessel atherosclerosis and ii) a subset of patients classified as having ischemic stroke of unknown etiology, provided they had at least 50% stenosis of the carotid artery ipsilateral to the infarct side and no evidence or no history of atrial fibrillation.…”

“…In addition, another recent investigation revealed that the rare variants in receptors commonly associated with platelet functions (e.g. purinergic receptors) could be associated with the occurrence of LVIS in the Polish population (13).…”