Effect of disease‐associated variants in TREM2 on splicing and gene dosage

Abstract

AbstractBackgroundLoss‐of‐function variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are responsible for a spectrum of neurodegenerative disorders. In the homozygous state, they cause severe pathologies with early onset dementia, such as Nasu‐Hakola disease (NHD) and behavioral variants of frontotemporal dementia (FTD), while heterozygous variants increase risk of late onset Alzheimer’s disease (AD) and FTD. In over half of TREM2 variants found in families with recessive early onset dementia, the defect occurs at the transcript level, via premature termination codons or via aberrant splicing. The remaining ones are missense variants thought to inactivate the protein; however, the underlying pathogenic mechanism is less clear. In this work we tested whether disease‐associated TREM2 variants may contribute to the pathology via altered splicingMethodSpliceAI prediction algorithm was used to screen 56 disease‐associated/risk‐modifying variants for potential effect on splicing. Variants scored by SpliceAI were tested in the splicing reporter assay with full‐length TREM2. Constructs with the common TREM2 variant or the spliceogenic SNP allele were nucleofected into THP‐1, HMC3 and BV2 cell lines. qRT‐/RT‐PCR and western blots were used to quantify the effect of variants at the transcript and protein levels. Nanostring nCounter analysis was used to measure TREM2 RNA in brains of NHD patients who carried spliceogenic variants.ResultWe demonstrated that multiple variants causative for NHD or associated with AD/FTD affect gene splicing reduced dosage of functional transcript and protein.ConclusionOur findings point to the complex character of pathogenicity of TREM2 variants, in which effects on post‐transcriptional gene regulation and protein function are often combined. This hidden layer of complexity necessitates inclusion of computational and experimental analyses of splicing and mRNA processing for better understanding of genetic variation in disease.