Methods using DNA methylation for identifying a cell or a mixture of cells for prognosis and diagnosis of diseases, and for cell remediation therapies
AssigneesThe Regents of the University of California, Brown University
InventorsKarl Kelsey, Eugene Andres Houseman, John Wiencke, William P. Accomando, JR., Carmen Marsit
Life of this patent
Estimating the term…
Abstract
Methods using DNA Methylation arrays are provided for identifying a cell or mixture of cells and for quantification of alterations in distribution of cells in blood or in tissues, and for diagnosing, prognosing and treating disease conditions, particularly cancer. The methods use fresh and archival samples.
Claims34
A method for assessing a disease condition in a subject, comprising:
- measuring a CD3Z positive T lymphocyte cell number in a sample from the subject by analyzing methylation in the sample of at least one CpG dinucleotide (CpG) in gene CD3Z or in an orthologous or a paralogous gene thereof, wherein an amount of a demethylated C of the at least one CpG in the sample is a measure of CD3+ T lymphocyte cell number;
- and
- comparing the amount of the demethylated C in the sample from the subject with that in positive control samples from patients with the disease condition, and with that in negative control samples from healthy subjects, wherein the disease condition is selected from:
- an autoimmune disease, an allergy, a transplant rejection, obesity, an inherited disease, immunosuppression and a cancer.
3-13. (canceled)
A kit for measuring CD3+ T lymphocyte and FOXP3+ T regulatory cell numbers, by analyzing methylation of CpG positions in CD3Z and FOXP3 genes, the kit comprising sequencing and PCR primers specific for the CD3Z and the FOXP3 gene DMRs and instructions for analyzing and comparing methylation of the CpG positions of a subject in need of diagnosis of a disease with that of control subjects.
A method for assessing a disease condition by estimating an alteration in proportions of types of leukocytes in a sample from a subject, the method comprising:
- measuring a DNA methylation profile for each type of leukocyte and for unfractionated cells, wherein DNA methylation profiles are obtained for a plurality of CpG loci, and obtaining the status of an individual CpG locus by amplifying DNA from each of the types of leukocyte and from the unfractionated cells, wherein amplifying comprises hybridizing methylation sensitive locus-specific DNA oligomers corresponding to each CpG locus;
- ordering CpG loci by ability to distinguish types of leukocytes, wherein the ordering of the CpG loci determines differentially methylated DNA regions (DMRs), wherein obtaining DMRs comprises statistically minimizing introduction of bias in amount of total methylation status of a large number of CpG loci obtained from the unfractionated cells by employing a Bayesian treatment utilizing prior probabilities of the methylation status at each individual locus, thereby identifying a plurality of CpG loci to include in the measurement, wherein an amount of CpG loci distinguishes DMR signatures among the types of leukocytes and minimizes bias;
- obtaining DNA methylation profiles comprising DMRs from the types of leukocytes, wherein the DNA methylation profiles comprise validating measures of relative amounts of the types of leukocytes, and obtaining DNA methylation profiles of the unfractionated cells as surrogate measures of relative amounts of each type of leukocyte in the unfractionated cells;
- employing an analog of a measurement error model wherein a DNA methylation surrogate y is reverse formulated with respect to the disease outcome z, as
- y=f(z),
- wherein y denotes a multivariate random variable representing a methylation profile, z denotes a disease outcome or state, and f denotes a probability distribution;
- y, z, and leukocyte distribution, ω are related by the estimator equations,
- E(y|ω)=g(ω), and
- under an assumption E=(z|ω,y)=E(z|ω), wherein E denotes an expectation of a random variable and ω denotes a subject specific distribution of leukocytes;
- and,
- comparing relative amounts of each type of leukocyte in the sample from the subject with those in a control sample, thereby providing an assessment of the disease condition.
17-26. (canceled)
A method of predicting a methylation class membership in a bodily fluid sample of a subject for assessing disease status of the subject, wherein the methylation class membership corresponds to an epigenetic signature of a plurality of leukocyte types, the method comprising:
- measuring amounts of DNA methylation in each of a plurality of leukocyte type populations to determine differentially methylated regions (DMRs);
- ranking leukocyte DMRs for each leukocyte type according to statistical strength of association of the DMR with each leukocyte type;
- randomly dividing a data set of control subjects and subjects with a disease into groups having substantially the same numbers of control subjects and subjects with the disease to obtain a training set and a testing set;
- clustering samples in the training set using a defined number of highest ranked leukocyte DMRs to determine clustering solutions, wherein a clustering solution corresponds to the methylation class membership;
- and
- predicting the methylation class membership for subjects within the testing set by applying the clustering solutions obtained from the training set to the highest ranked leukocyte DMRs in the testing set, wherein clinical utility of the predicted methylation class membership is determined by testing association of the predicted methylation class membership with the disease status of the subject.
29-36. (canceled)
An array for estimating proportions of leukocyte types in a sample from a mammal for assessing a disease condition of the mammal by analyzing differential methylation of CpG dinucleotides in a plurality of genes of the sample, the array comprising:
- a plurality of DNA probes attached to a plurality of surfaces at known addressable locations on the array, wherein the surface at each location is attached to a DNA probe having a specific nucleotide sequence, wherein the DNA probe having the specific nucleotide sequence hybridizes to a DNA sequence of a methylated form or an ummethylated form of a CpG dinucleotide in a sequence of a gene of the plurality of genes in the sample, wherein the array is selected from having:
- at least 16 probes, at least 64 probes, at least 96 probes, and at least 384 probes.
40-46. (canceled)
A method for estimating proportions of types of leukocytes in a sample from a subject for assessing a disease condition of the subject by analyzing differential methylation of CpG dinucleotides in a plurality of genes of the sample, the method comprising:
- providing an array having a plurality of DNA probes attached to a plurality of surfaces at known addressable locations on the array, wherein the surface at each location is attached to a DNA probe having a specific nucleotide sequence;
- reacting genomic DNA in the sample with a bisulfite reagent to convert unmethylated cytosine residues to uracil;
- hybridizing resulting bisulfite treated genomic DNA with the array to obtain resulting hybridized probes on the array, wherein the DNA probes hybridize to a DNA sequence of each of a methylated form and an ummethylated form of a sequence having a CpG dinucleotide in a gene for each of the plurality of genes;
- and
- detecting the methylation status of each of the CpG dinucleotides in each sequence, thereby estimating proportions of types of leukocyte in the sample from the subject for assessing the disease condition of the subject.
The method according to claim 48, wherein amplifying by PCR further comprises:
- using primers pairs having a 5′ primer specific to each of the methylated or the unmethylated form of the CpG dinucleotide containing gene, and a 3′ primer specific to the gene containing the CpG dinucleotide, thereby obtaining a first PCR product;
- amplifying the first PCR product with differentially labeled 5′ primers specific for each of the methylated and the unmethylated form of the CpG dinucleotide sequence containing gene, and a common 3′ primer, thereby obtaining a differentially labeled second PCR product, and hybridizing the second PCR product to the CpG dinucleotide containing gene for measuring amount of the second PCR product, thereby detecting the methylation status of the CpG dinucleotide sequence.
50-51. (canceled)
55-58. (canceled)
A kit for estimating proportions of leukocyte types in a sample from a subject by analyzing differential methylation of CpG dinucleotides in a plurality of genes of the sample, the kit comprising:
- an array comprising:
- a plurality of DNA probes attached to a plurality of surfaces at known addressable locations on the array, wherein the surface at each location is attached to a DNA probe having a specific nucleotide sequence, wherein the DNA probe having the specific nucleotide sequence hybridizes to a DNA sequence of a methylated form or an ummethylated form of a CpG dinucleotide in a sequence of a gene of the plurality of genes in the sample, wherein the array is selected from having:
- at least 16 probes, at least 64 probes, at least 96 probes, and at least 384 probes;
- primers and reagents for detecting the hybridized probes and for detecting the reaction products derived from the hybridized probes;
- and
- instructions for using the array with a bisulfate reagent, thereby providing an estimation of proportions of leukocyte types in the sample.
(canceled)
62-65. (canceled)
A method of treating a subject for a disease condition, wherein the subject is a human patient and wherein the disease condition is a cancer, the method comprising:
- obtaining signatures comprising differentially methylated regions (DMRs) from types of leukocytes in a blood sample of the patient, the types of leukocytes comprising at least one selected from:
- CD19+ B lymphocyte, CD15+ granulocyte, CD14+ monocyte, CD56dim Natural Killer cell, CD56bright Natural Killer cell, and CD3+ T lymphocyte;
- and from a healthy control human subject not having the cancer;
- comparing a signature for a specific type of leukocyte in the patient with that in the healthy subject, wherein the signature for the specific type of leukocyte is an indication of amount of cells of the specific type of leukocyte circulating in blood, and wherein a decreased amount of the cells of the specific type of leukocyte circulating in the blood of the patient compared to the healthy subject is an indicium of the cancer;
- and,
- administering a composition comprising the cells of the type of leukocyte to the patient, thereby increasing the amount of the cells of the type of leukocyte in the patient and treating the cancer.
68-69. (canceled)
72-73. (canceled)
Dependencies are inferred from the claim text.