Updates to the reference genome alter the detection and direction of genes differentially expressed in Alzheimer’s disease

Alzheimer’s Disease gene expression studies often rely on the reproducibility of known disease associations to build confidence in novel discoveries. This approach assumes that true biological variation associated with Alzheimer’s Disease is robust to a range of technical and biological confounders that likely differ across experiments. Here, we systematically assess the impact of an important yet frequently overlooked technical confounder in Alzheimer’s Disease differential expression analysis: changes in gene quantification resulting from ongoing updates to the human genome reference and transcriptome annotations. To investigate this, gene expression in a large brain transcriptomic dataset from Alzheimer’s Disease patients and controls (ROSMAP) was quantified using five transcriptome annotations mapped across a broad range of human genome references, from NCBI36 to CHM13v2.0. Although nearly all genes exhibit significant differences in expression between reference pairs (i.e., were differentially quantified), genome-wide Alzheimer’s Disease differential expression signatures remain highly concordant across references. Notably, while a core set of significantly differentially expressed Alzheimer’s Disease genes is largely conserved across references, reference-specific Alzheimer’s Disease-associated genes are enriched for pathways known to be involved in the disease. Genes whose direction of association with Alzheimer’s Disease significantly differs across references are also identified, highlighting the potential influence of reference choice on Alzheimer’s Disease-relevant genes and pathways. All results were replicated in an independent transcriptomics dataset (MSBB) derived from the same brain region. These findings demonstrate that reference genome choice impacts the identification and interpretation of specific Alzheimer’s Disease-associated genes, despite overall reproducibility of genome-wide differential expression signatures, underscoring the critical importance of reference selection in Alzheimer’s Disease transcriptomic studies.