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To Develop and Demonstrate a Technology Enabling the Detection and Quantification of Modified Nucleic Acid Bases from a Mammalian Genome such as Methylation Sites



OBJECTIVE: To develop and demonstrate a technology enabling the detection and quantification of modified nucleic acid bases from a mammalian genome such as methylation sites. The method shall be in an easy-to-use format, not too technically demanding, and require instrumentation with minimal analytics. The method should enable the assessment of DNA methylation targeting a particular region or gene of interest as oppose to discovery of unknown epigenetic changes. 

DESCRIPTION: DNA methylation is the study of chromosomal patterns of DNA or histone modification by methyl groups in vertebrates. The cytosine (C) base in DNA and lysine residue in histone tails can be methylated. These modifications are considered very stable, heritable, and are correlated with locus specific transcriptional status. DNA methylation can also impact gene expression, particularly if the methylation is present in CpG islands, which are found in approximately 50% of promoters. DNA methylation alters gene expression levels primarily through regulating methylation state-dependent interactions with transcriptional activators or repressors, and chromatin remodeling enzymes. There are multiple events that can impact DNA methylation machinery. These biomarkers can be used at any stage of a disease and can be associated with its cause (risk biomarkers), onset (diagnostic biomarkers), clinical course (prognostic biomarkers), or response to treatment (predictive biomarkers). To date vast majority of DNA methylation are reported in cancer research and recently DNA methylation has been known to show a significant role in the pathophysiology of several other diseases such as PTSD (Hammamieh et al 2017), aging ( Hovarth et al 2013) as well as neurodegenerative disorders (Levenson et al 2011). There is a growing body of literature suggesting a role for epigenetic factors as a molecular link between environmental factors and type 2 diabetes. Multiple technologies exist by which these differences can be measured. Most of these methods detect the global DNA methylation or overall changes in DNA methylation status of the sample (1). Bisulphite sequencing that is considered the gold standard method for the detection and quantification of DNA methylation and is similar to genomic sequencing with regards to its prohibitive cost and difficulty in data analysis. To perform a targeted region sequencing, primers are designed that are specific for bisulphite converted DNA; It is a quick method, which could be used for simultaneously profiling of multiple samples/multiple regions (Zymo research, (5)). The obvious drawbacks of the current methods are that they are all time intensive and involve the use of multiple equipment with specialized training. Less common is the detection of methylated bases directly through sequencing of unmodified DNA that could be done without enrichment or bisulfite conversion. Considering the detailed procedure of bisulphite modifications, direct detection of modified bases would be a preferred approach. Another approach for methylated DNA fractions of the genome, usually obtained by immunoprecipitation, could be used for hybridization with microarrays ( 1, 4, 5 ). This is the most popular method which fills that gap between whole genome bisulfite sequencing and cumbersome low throughput methods that can access the methylation of a pre-designed individual CpG sites and can be customized to region of interest. Pyrosequencing is another technology where individual primers are designed to get a short-read pyrosequencing reaction of around 100 bp. The level of methylation for each CpG site within the sequenced region is estimated based on the signal intensities for incorporated dGTP and dATP. The result is quantitative, and the technique is able to detect even small differences in methylation (down to 5%). It is a good technique for heterogeneous samples but requires specialized equipment and training. Advancement in the development of nanopore-based single-molecule real-time sequencing (2-3) technology (Oxford nanopore) can help to detect modified bases directly in short time. Commercialization of each or combination of the unique technique will bring the next generation of assay with even better sensitivity and specificity that would be easy to perform and analyze. The aim of this STTR is to develop a method of choice that should deliver an unbiased answer to the biological question being asked by the researcher. It will be important to consider following factors when choosing a method for targeted DNA methylation analysis: 1) The development of an automated procedure; 2) The investigation is on known methylation sites for specific gene of interest 3) The amount of sample requirements. Considering clinical samples, whole blood would be sample of choice. 4) The sensitivity and specificity of the assay proposed; 5) The robustness and simplicity of the method. 6) The simplicity of software for analysis and interpretation of the data; 7) Effortless use of specialized equipment and reagents; 8) Turn-around time to result 9) Assay cost. 

PHASE I: Given the short duration of Phase I, this phase should not encompass any human use testing that would require formal IRB approval. Phase I should focus on system design for rapid detection of targeted methylation sites using any gene/region of interest. At the end of this phase, a working prototype of the assay (s) should be completed and some demonstration of feasibility, integration, and/or operation of the prototype. In addition, descriptions of data analysis and interpretations concept and concerns should be outlined. Phase I should also include the detailed development of Phase II testing plan. 

PHASE II: During this phase, the integrated system should undergo testing using some targeted genes/regions of interest for evaluation of the operation and effectiveness of utilizing an integrated system and its capability to demonstrate the utility in a diseased condition such as PTSD. Accuracy, reliability, and usability should be assessed. This testing should be controlled and rigorous. Statistical power should be adequate to document initial efficacy and feasibility of the assay. This phase should also demonstrate evidence of commercial viability of the tool. Accompanying the application should be standard protocols and procedures for its use and integration into ongoing programs. These protocols should be presented in multimedia format. 

PHASE III: The ultimate goal of this topic is to develop and demonstrate a technology enabling the direct detection of modified bases such as methylation sites. This assay format should also be seamlessly integrated so that it can be used as monitoring tools for long term health assessment. Once developed and demonstrated, the technology can be used for identification of risk, diagnostic, prognostic, monitoring and/ or predictive biomarkers for diseased state. Development of new technique for methylation analysis will open a multitude of possibilities for biomarker development and might become extremely valuable in clinical practice. 


1. Hammamieh R, Chakraborty N, Gautam A, et al. Whole-genome DNA methylation status associated with clinical PTSD measures of OIF/OEF veterans. Translational Psychiatry. 2017;7(7):e1169-. doi:10.1038/tp.2017.129.; 2. Simpson, J. T., Workman, R. E., Zuzarte, P. C., David, M., Dursi, L. J., & Timp, W. (2017). Detecting DNA cytosine methylation using nanopore sequencing. Nature Methods, 14, 407. doi: 10.1038/nmeth.4184; 3. Wilmot, B, et al. (2015) Methylomic analysis of salivary DNA in childhood ADHD identifies altered DNA methylation in VIPR2. The Journal of Child Psychology and Psychiatry. Doi: 10.1111/jcpp.12457; 4.; 5. Dean et al Multi-omic biomarker identification and validation for diagnosing warzone-related Post-Traumatic Stress Disorder. Submitted to Science Translational Medicine; 6. Levenson VV. DNA methylation as a universal biomarker. Expert Rev Mol Diagn. 2010;10(4):481-8.; 7. Horvath S. DNA methylation age of human tissues and cell types. Genome Biol. 2013;14:R115. doi: 10.1186/gb-2013-14-10-r115

KEYWORDS: Epigenetics, Methylation, Next-generation Sequencing, Technology, Military Health 

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