EP-4741515-A2 - CELL FREE DNA DECONVOLUTION AND USE THEREOF

Abstract

Methods of determining the origin of cell free DNA (cfDNA) and for detecting death of a cell type or tissue in a subject by determining the origin of cfDNA in the subject are provided. Computer program products, computerized methods and computerized systems for doing same, as well as methods for constructing a methylome atlas, are also provided.

Inventors

• DOR, Yuval
• SHEMER, Ruth
• GLASER, BENJAMIN
• KAPLAN, Tomer
• MOSS, Joshua

Assignees

• Yissum Research Development Company of the Hebrew University of Jerusalem Ltd.
• Hadasit Medical Research Services and Development Ltd.

Dates

Publication Date

20260513

Application Date

20190218

Claims (15)

1. A method for determining a cell type or tissue of origin of cell-free DNA (cfDNA), comprising: (a) obtaining cfDNA from a biological sample; (b) determining DNA methylation values at a plurality of CpG sites in the cfDNA; and (c) determining a relative contribution of a plurality of cell types or tissues to the cfDNA by comparing the DNA methylation values with a methylome atlas, wherein the methylome atlas comprises DNA methylation data derived from purified cell populations.
2. The method of claim 1, wherein the purified cell populations are obtained from tissues by cell separation.
3. The method of claim 1 or 2, wherein the methylome atlas comprises DNA methylation data from at least 5 different cell types or tissues.
4. The method of any one of claims 1-3, wherein the purified cell populations comprise non-blood-derived cell populations.
5. The method of any one of claims 1-4, wherein a contribution of a cell type or tissue representing 1% or less of total cfDNA is detected.
6. The method of any one of claims 1-5, wherein determining the relative contribution comprises solving a deconvolution problem.
7. The method of claim 6, wherein the deconvolution comprises non-negative least squares analysis.
8. The method of any one of claims 1-7, wherein determining DNA methylation values comprises bisulfite conversion of the cfDNA.
9. The method of claim 8, wherein determining DNA methylation values further comprises sequencing or array-based analysis.
10. The method of any one of claims 1-9, wherein the biological sample comprises blood or plasma.
11. The method of any one of claims 1-10, wherein the plurality of CpG sites comprises CpG sites selected based on differential methylation between the plurality of cell types or tissues.
12. The method of claim 11, wherein the CpG sites are selected such that methylation levels distinguish one cell type or tissue from at least one other cell type or tissue.
13. The method of any one of claims 1-12, wherein determining the cell type or tissue of origin indicates cell death in said cell type or tissue.
14. The method of claim 13, wherein the method is used for detecting or monitoring a disease selected from cancer, organ transplantation, or sepsis.
15. A computer-implemented method comprising: (a) receiving DNA methylation data from a cfDNA sample; (b) comparing the DNA methylation data with a methylome atlas comprising DNA methylation data derived from purified cell populations; and (c) determining a relative contribution of a plurality of cell types or tissues to the cfDNA based on the comparison.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/631,791, filed February 18, 2018, and U.S. Provisional Patent Application No. 62/661,179, filed April 23, 2018, the contents of which are all incorporated herein by reference in their entirety. FIELD OF INVENTION The present invention is in the field of cell free DNA methylome analysis. BACKGROUND OF THE INVENTION Small fragments of DNA circulate freely in the peripheral blood of healthy and diseased individuals. These cell-free DNA (cfDNA) molecules are thought to derive from dying cells and are thus a reflection of ongoing cell death taking place in the body. In recent years, this understanding has led to the emergence of diagnostic tools which are impacting multiple areas of medicine. Specifically, next generation sequencing of fetal DNA circulating in maternal blood has allowed non-invasive prenatal testing of fetal chromosomal abnormalities; detection of donor-derived DNA in the circulation of organ transplant recipients can be used for early identification of graft rejection; and the evaluation of mutated DNA in the circulation can be used to detect, genotype and monitor cancer. These technologies are powerful at identifying genetic anomalies in circulating DNA yet are not informative when cfDNA does not carry mutations. A key limitation is that sequencing does not reveal the tissue origins of cfDNA, precluding the identification of tissue-specific cell death. The latter is critical in many settings such as neurodegenerative, inflammatory or ischemic diseases, not involving DNA mutations. Even in oncology, it is often important to determine the tissue origins of the tumor in addition to determining its mutational profile, for example in cancers of unknown primary (CUP) and in the setting of early cancer diagnosis. Finally, identification of collateral tissue damage (e.g. toxicity of drugs in genetically normal tissues) is a key element in drug development and monitoring of response to therapy. Several approaches have been proposed recently for tracing the tissue sources of cfDNA, all based on tissue-specific epigenetic signatures. Nucleosome positioning in different tissues has been used to infer the origins of cfDNA, based on the idea that nucleosome-free regions are more likely to be degraded upon cell death and hence will be under-represented in cfDNA. An alternative approach is based on DNA methylation patterns. Methylation of cytosine adjacent to guanine (CpG sites) is an essential component of cell type-specific gene regulation, and hence is a fundamental mark of cell identity. It was recently shown that the detection of cfDNA molecules with a methylation pattern unique to a certain tissue can be used to identify cell death derived from that tissue. Others have taken a genome-wide approach to the problem and used the plasma methylome to assess the origins of cfDNA. It has been showed that the deconvolution of cfDNA methylation profiles can be used to infer relative contributions of four different tissues, using a low-depth whole genome bisulfite sequencing (WGBS). It was further demonstrated that the potential exists of using cfDNA methylation in detecting cancer DNA in circulation, as well as identifying its tissue of origin in two cancer types, using a reduced representation bisulfite sequencing (RRBS) approach. Nevertheless, a method of reliably determining the tissue of origin of all cfDNA found in a subject, and not just the most abundant is greatly in need. Such a method would allow for early diagnosis of conditions that may be unknown to the physician or patient, and not just a directed assessment of an already predicted condition. SUMMARY OF THE INVENTION The present invention provides methods of determining the origin of cell free DNA (cfDNA) and for detecting death of a cell type or tissue in a subject by determining the origin of cfDNA in the subject are provided. Computer program products for doing same and methods of constructing a methylome atlas are also provided. According to a first aspect, there is provided a method of determining the cell type or tissue of origin of cell free DNA (cfDNA) comprising: a. providing cfDNA;b. measuring DNA methylation of the cfDNA; andc. assigning a cfDNA molecule from the cfDNA to a cell type or tissue of origin by comparing the methylation of the molecule to a methylome atlas of at least l cell type or tissue, wherein the atlas comprises at least 25 of the 100 most uniquely methylated sites and at least 25 of the 100 most uniquely unmethylated sites in each of the at least 1 cell type or tissue; thereby determining the cell or tissue of origin of cfDNA. According to another aspect, there is provided a method of detecting death of a cell type or tissue in a subject comprising: a. providing cfDNA from the subject;b. measuring DNA methylation of the cfDNA; andc. assigning a cfDNA molecule from the cfDNA to a cell type