2.4. Meta-Analysis

We meta-analyzed the ROS/MAP results with results from two independent datasets. The first independent dataset included participants of the Arizona Study of Aging and Neurodegenerative Disorders, a longitudinal clinical-pathologic study of normal aging, Alzheimer’s disease (AD), and Parkinson’s disease (PD) run by the Banner Sun Health Research Institute [24]. The study population consists of cognitively unimpaired volunteers from retirement communities in northwest greater Phoenix, Arizona with some directed recruitment of participants with AD and PD through neurologists in the metropolitan Phoenix and Tucson areas [24]. Over 90% of participants are of European ancestry. All subjects or their legal representatives sign a Banner Sun Health Research Institute Institutional Review Board-approved informed consent form allowing both clinical assessments during life, several options for brain and/or bodily organ donation after death, and usage of donated biospecimens for approved future research. Circle of Willis atherosclerosis was assessed as the extent of atherosclerotic plaque visible on gross external examination of the circle of Willis. A score of none, mild, moderate, or severe was assigned according to a schematic template [25]. DNA from post-mortem brain tissue was extracted using Qiagen GenePure kit and genotyped using the Affymetrix Precision Medicine Array following the manufacturer’s protocol. Genotypes were imputed and all participants were verified to be of European ancestry and unrelated as described above for the discovery dataset. The SNPs were filtered using the same criteria described above for the discovery dataset. A total of 154 participants with cerebral atherosclerosis assessment and genotype data were available for this analysis.

The second independent dataset included individuals from 31 Alzheimer’s Disease Centers (ADCs) with phenotypes available from the National Alzheimer’s Coordinating Center (NACC) database [26] and genotyping data generated by the Alzheimer’s Genetics Consortium (ADGC) [27]. Each individual ADC received informed consent from their participants and approval from their institutional review board. Atherosclerosis of the circle of Willis was scored as none, mild, moderate, or severe. All ADCs used the same forms for scoring. The autopsies were conducted between 2005 and 2020, and genotyping was performed in 7 batches. A total of 1914 participants with cerebral atherosclerosis assessment and genotype data were available for this analysis.

We calculated association test statistics in these two datasets using the same framework applied to ROS/MAP, with the following covariates: sex, age at death, and 4 genetic PCs (because only the first 4 PCs were significant) for the Banner dataset; and sex, age at death, 10 genetic PCs, and genotyping batch for the ADGC dataset. We then performed inverse variance weighted meta-analysis using METAL [28].