A PATIENT-DERIVED IPSC MODEL OF A RARE TTC3 MUTATION

Abstract

While 20+ loci have been associated with late onset Alzheimer's disease (LOAD), a large number of genetic factors still have yet to be revealed. We recently performed whole exome sequencing on a non-Hispanic white LOAD family with 11 AD individuals with a range of onset between 70–85 years old (average 75.7 years) that was negative for alterations in known AD candidate genes (Kohli, et al, 2016). A single rare, nonsynonymous variant was identified in all affected individuals in the tetratricopeptide repeat domain 3 (TTC3) gene. This missense alteration, rs377155188 (p.S1038C), is predicted to be deleterious by five in silico algorithms and extremely rare in the ExAC database (1.96x10-5). Studies have reported that TTC3 expression is reduced in LOAD patients and negatively correlated with AD neuropathology, making this gene an interesting LOAD candidate to further investigate. To understand the mechanism by which the TTC3 alteration may be acting to contribute to LOAD risk, induced pluripotent stem cells (iPSC) lines were developed to examine cellular consequences in neuronal tissue, which can otherwise only be collected postmortem. Peripheral blood mononuclear cells (PBMCs) were extracted from the whole blood of two LOAD individuals bearing the TTC3 change, as well as two aged, and ethnically matched control individuals. iPSC reprogramming was performed using Sendai virus. Each iPSC line generated was validated for pluripotency through immunocytochemical staining and shown to be negative for any large scale chromosomal abnormalities via karyotyping. These iPSC lines will be differentiated into forebrain neurons to generate a disease relevant tissue to assess. There is evidence that modulation of TTC3 affects neurite growth (Berto, et al, 2007); therefore, morphological measures of axon and synapse formation will be assessed in differentiating neuronal cultures using live cell imaging. Alzheimer-specific phenotypes such as the levels of secreted beta amyloid and intercellular whole and phosphorylated tau will also be measured. Utilizing patient-specific iPSC lines will permit assessing the cellular and molecular consequences of genetic alterations which may contribute to LOAD risk. We will present the morphological and cellular phenotypes of these lines carrying a potential risk factor for AD.