Previously, we have taken an innovative approach (The Human Brainome; [1-6]) to mapping risk loci for late onset Alzheimer's disease (LOAD). Rather than looking at a single layer of information as in most genome- wide association (GWAS) studies, we have mapped genomic variation in the context of downstream transcriptomic and proteomic expression. This allows for mapping both the crucial variation involved in LOAD, as well as the downstream effects and their directions. Additionally, it allows for building networks of multiple players crucial for disease processes. One shortcoming of the current work is that we have mapped DNA-expression relationships that are subtly changed in Alzheimer's, but we have yet to fully understand why those DNA-expression relationships are altered. We know that expression is altered by specific alleles, but there must be added regulation given our mapped outputs. One target that can alter pathways are natural antisense transcripts (NATs), which can bind to oligonucleotide products and alter their expression and degradation. In our application, we propose to use long read sequencing technology (SMRT; Single Molecule, Real-Time) and fully profile RNA from our human brain bank samples. We will examine where these outputs are located and perform preliminary work to determine if any of these new hits can act on our existing results. We propose to follow these targets through 3 Aims.
Aim 1 will involve following hits from public databases.
Aim 2 will involve collecting additional RNA profiling data. Finally, Aim 3 will seek to validate and order all novel findings from Aims 1 and 2. It is important to use technologies appropriate to our hypothesis for the new data collection. The majority of non-coding RNA belongs to the class of transcripts called long non-coding RNA (lncRNA), which can span from 1000-10,000 bp [7]. Typical short-read RNA sequencing (SRS) technologies are based on the capture of short sequences of ~150 bp, and therefore, SRS has difficulty in capture and alignment of longer products. We are working with Robert Sebra at the Icahn Institute for Genomics and Multicale Biology, who is an expert in SMRT sequencing [8]. This technology offers longer read lengths and will be unique-in-field, since most human RNA profiling involves SRS. By the completion of these Aims, we will have 1. A map of novel long read sequencing in human brain tissues, which will be a significant add-to-field, given most technologies used to date are focused on short read sequencing, and there is limited profiling in pathologically defined human brain tissues, 2. An understanding of how these novel hits are affecting both the direct sense transcript of interest as well as the known LOAD pathologies, 3. Multi-level mapping of rigor and reproducibility of targets through the use of multiple capture techniques and 4. Validation of the effect of hits on the known LOAD pathogenic targets by measuring both expression and protein levels in cell culture.
Our proposal will focus on mapping novel regulators of expression using a large internationally-collected series of human brain tissues. This work will leverage two different sites to perform the necessary experiments for further validation of these targets and obtaining the data necessary. Final outputs will include a map of novel regulators of DNA-transcript expression which act on known Alzheimer's disease pathology.
