Cell-Free DNA Hydroxymethylation Profiling for Tissue-of-Origin Assignment
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Solution Overview
Problem
Current methods for analyzing cell-free DNA, particularly for epigenetic analysis, face challenges in detecting rare DNA modifications like 5-hydroxymethylcytosine (5hmC) with high efficiency and selectivity, especially in small sample volumes, which are crucial for determining the tissue or organ of origin.
Innovation Solution
A method utilizing hydroxymethylation profiles to probabilistically assign a tissue of origin to nucleic acids by comparing sample profiles with a reference data set of hydroxymethylation patterns at specific genomic loci associated with tissue-specific genes, allowing for accurate tissue determination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If cell-free DNA is used for epigenetic analysis to enable noninvasive evaluation, then patient sampling becomes noninvasive, but the small sample volume and low DNA concentration make detection of rare modifications like 5hmC difficult
Solution Approach 1:
The patent segments the DNA modification detection process into distinct enzymatic steps: first converting 5hmC to 5mC using TET enzyme, then detecting 5mC using bisulfite sequencing. This segmentation allows the rare 5hmC modification to be converted into a more abundant and detectable form (5mC), overcoming the limitation of low concentration in cell-free DNA samples.
Solution Approach 2:
The patent introduces TET enzyme as an intermediary that converts 5hmC to 5mC, making the rare modification detectable through existing 5mC detection methods. This intermediary step bridges the gap between detecting rare 5hmC in cell-free DNA and using well-established 5mC detection techniques.
2Measurement precision
If bisulfite sequencing is used to detect DNA methylation, then 5mC can be detected, but it cannot distinguish between 5mC and 5hmC modifications
Solution Approach 1:
The patent performs preliminary conversion of 5hmC to 5mC using TET enzyme before applying bisulfite sequencing. This preliminary action ensures that both original 5mC and converted 5hmC are detected as 5mC, allowing the method to distinguish between the two modifications based on their different conversion behaviors rather than relying solely on post-conversion detection.
Solution Approach 2:
The patent changes the chemical state of 5hmC to 5mC through enzymatic conversion, altering the parameter of DNA modification type. This parameter change enables the use of bisulfite sequencing to detect both modifications, as the converted 5hmC behaves like 5mC in the sequencing process, while the ability to reverse the conversion (using fidelity enzymes) allows for distinction between the two original modifications.
3Measurement precision
If genome-wide bisulfite sequencing is performed to generate methylation profiles, then tissue of origin can be identified, but the process is time-consuming and not suitable for rapid diagnosis
Solution Approach 1:
The patent segments the genome-wide sequencing approach by focusing on specific loci known to be tissue-specific for hydroxymethylation. Instead of sequencing the entire genome, the method targets particular genomic regions where 5hmC patterns are characteristic of specific tissues, dramatically reducing analysis time while maintaining tissue identification accuracy.
Solution Approach 2:
The patent applies local quality analysis by examining hydroxymethylation patterns at specific tissue-specific loci rather than performing uniform analysis across the entire genome. This localized approach concentrates resources on the most informative regions, enabling rapid tissue identification without the time cost of whole-genome sequencing.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables precise and efficient identification of the tissue or organ of origin for DNA fragments in cell-free samples, providing high sensitivity and specificity in epigenetic analysis.
Implementation Method 1
5hmC is a stable DNA modification, formed from the catalytic oxidation of 5mC by a Ten-Eleven Translocation (TET) enzyme such as TET1
Data Source
AI summary
A method is provided for probabilistically assigning a tissue of origin to a nucleic acid in a sample, e.g., DNA in a cell-free fluid sample obtained from a human subject. A hydroxymethylation profile is generated for the sample DNA and then compared across a reference data set of hydroxymethylation profile vectors, where each hydroxymethylation profile vector identifies the hydroxymethylation profile at a specific reference locus, the tissue-specific gene associated with the reference locus, and the tissue with which the gene and reference locus are associated. A tissue of origin can be probabilistically assigned to the sample nucleic acid using the results of the comparison. Other methods of use are also provided.