Patent Grant
11344121
Preeclampsia (PE) is responsible for 76,000 maternal and 500,000 infant deaths worldwide each year. Adverse maternal events include stroke, organ dysfunction and disseminated intravascular coagulation; whereas, adverse fetal complications include intrauterine growth restriction, premature birth, and stillbirth. PE is also associated with increased risk of chronic diseases in the mother and child later in life. The cause of PE is unknown; however, it is often diagnosed in the third trimester and there is no known prevention or cure.
Most of the suggested biomarkers for PE focus on late gestation and lack sufficient sensitivity and specificity. Successful intervention of PE requires a better understanding of disease progression and development of accurate and early biomarkers that appear before the appearance of clinical symptoms. Placental DNA methylation and/or microRNA (miRNA) regulation, particularly, the presence of certain miRNAs in a mother's blood in the second or third trimester have been implicated in PE.
The invention provides biomarkers which can be analyzed during the first trimester of pregnancy for identifying a subject as having high risk of the development PE later in the pregnancy. These noninvasive biomarkers presented herein include miRNAs, post-translational modification of histone proteins, amount, expression and/or activity of histone or DNA modifying enzymes and methylation of certain sites in the genomic DNA of certain cells in the mother.
Accordingly, in one embodiment, the levels of certain miRNAs in a body fluid, for example, blood, serum or plasma, of a subject are used to predict the development of PE. In another embodiment, the levels post-translational modifications of histone proteins in the cells, for example, blood cells, of a subject are used to predict the development of PE. In a further embodiment, the amounts, expression and/or activities of certain enzymes capable of modifying histone proteins or sites in the genomic DNA of cells, for example, blood cells, of a subject are used to predict the development of PE. In an even further embodiment, the levels of methylation of certain sites in the genomic DNA of cells, for example, blood cells, of a subject are used to predict the development of PE.
In an embodiment, increased miR-17 (SEQ ID NO: 77) in blood, serum or plasma of a subject compared to a control subject is used to predict the development of PE in the subject. In another embodiment, increased acetylation of H4 histone protein on N-terminus, lysine 12 and/or lysine 16 and/or methylation/demethylation on lysine 20 or a combination thereof compared to that of a control subject is used to predict the development of PE in the subject. In a further embodiment, decreased amount, expression and/or activity of HDAC5 protein or mRNA compared to that of a control subject is used to predict the development of PE in the subject. In an even further embodiment, hypermethylation of DNA at the genomic site CYP19A1 (SEQ ID NO: 46) compared to that of a control subject is used to predict the development of PE in the subject. In certain embodiment, increased mirR-17; increased acetylation of histone H4 on N-terminal, lysine 12 and/or lysine 16 and/or methylation/demethylation of lysine 20 or a combination thereof; decreased amount, expression and/or activity of HDAC5 protein or mRNA; and increased methylation of DNA in the genomic site CYP19A1 compared to that of a control sample are used to predict development of PE in the subject.
The invention also provides the methods of treating and/or managing PE in a subject identified as having a high risk of the development of PE.
The invention further provides kits and reagents to conduct assays to quantify biomarkers described herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, with color drawing(s), will be provided by the Office upon request and payment of the necessary fee.
SEQ ID NO: 1: Sequence of Histone H4 protein lacking the first methionine residue. The first methionine is removed from this sequence to correctly indicate the position for lysine residues, e.g., lysine 12, lysine 16 and lysine 20.
SEQ ID NOs: 2-45: Sequences of genomic DNA sites that are hypomethylated in PE patients.
SEQ ID NOs: 46-55: Sequences of genomic DNA sites that are hypermethylated in PE patients.
SEQ ID NOs: 56 to 115 and 191: Sequences of pre-miRNAs and mature miRNAs that are differentially expressed in PE patients.
SEQ ID NOs: 116-190: Sequences of the probes for determining methylation of the genomic sites that are differentially methylated in PE patients.
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising”. The transitional terms/phrases (and any grammatical variations thereof) “comprising”, “comprises”, “comprise”, “consisting essentially of”, “consists essentially of”, “consisting” and “consists” can be used interchangeably.
“Treatment”, “treating”, “palliating” and “ameliorating” (and grammatical variants of these terms), as used herein, are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. A therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with PE such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with PE.
“Subject” refers to an animal, such as a mammal, for example a human. The methods described herein can be useful in both humans and non-human animals. In some embodiments, the subject is a mammal (such as an animal model of disease), and in some embodiments, the subject is human. The terms “subject” and “patient” can be used interchangeably.
Epigenetic dysregulation during early pregnancy may lead to PE. DNA methylation, histone modification, and miRNA are all inter-related and may work in concert to regulate gene expression leading to PE. The current invention provides that alterations in epigenetic features and miRNA could presage PE and be reflected in tissues of the pregnant mother, for example, during the first trimester in the blood of a pregnant mother who later developed PE.
To discover early noninvasive novel biomarkers of PE, epigenetic (DNA methylation, histone modification, and epigenetic modifying enzyme) and miRNA profiling was conducted in a case-controlled study in the first trimester in tissues of pregnant mothers, for example, in blood of pregnant mothers. Altered expressions of certain miRNA (e.g., upregulated miR-17); altered post-translational modifications of certain histone proteins (e.g., hyperacetylation of H4); altered expression, amount and/or activity of certain histone or DNA modifying enzymes (e.g., decreased HDAC5 protein and/or mRNA); and/or altered methylation of certain genomic DNA sites (e.g., hypermethylated CYP19A1) were found to be interrelated and associated with the development of PE.
Accordingly, an embodiment of the invention provides a method of predicting the development of PE in a subject, the method comprising:
(a) determining the level of one or more miRNAs in:
(b) optionally obtaining one or more reference values corresponding to levels of one or miRNAs,
wherein the presence of the one or more miRNAs:
(c) identifying the subject as having high risk of developing PE based on the level of one or more miRNAs in the test sample and optionally, administering a therapy to the subject to treat and/or manage PE, or
(d) identifying the subject as not having high risk of developing PE based on the level of one or more miRNAs in the test sample and withholding the therapy to the subject to treat and/or manage PE.
Various techniques are well known to a person of ordinary skill in the art to determine the level of miRNA in a sample. Non-limiting examples of such techniques include microarray analysis, real-time polymerase chain reaction (PCR), Northern blot, in situ hybridization, solution hybridization, or quantitative reverse transcription PCR (qRT-PCR). Methods of carrying out these techniques are routine in the art. Additional methods of determining the level of miRNA in a sample are also well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
The reference values corresponding to levels of one or miRNAs indicate the level of miRNA associated with no risk or low risk of the development of PE or high risk of development of PE. As such, the reference values corresponding to levels of one or miRNAs may be indicative of the absence or presence of high risk of the development of PE. A reference value associated with no risk or low risk of the development of PE may be obtained based on samples obtained from subjects known to be free of PE. A reference value associated with high risk of the development of PE may be obtained based on samples obtained from subjects known to have PE.
For example, tissues from a group of pregnant women can obtained during the first trimester and the levels of one or more miRNAs can be determined. The group of women can then be monitored for the development of PE. Reference values corresponding to levels of one or more miRNAs that are associated with low risk or no risk of the development of PE or high risk of the development of PE can be determined based on the presence of absence of PE in various women whose samples were analyzed. Additional examples of determining references values associated with no risk or low risk or high risk of the development of PE are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
The step of identifying the subject as having high risk or not having high risk of developing PE depends on the level of one or more miRNAs in the test sample. For example, if the levels of certain miRNAs in the test sample are significant higher or lower than the levels of corresponding miRNAs in the control sample, the subject is identified as having high risk of development of PE. For example, if the levels of one or more of miR-7a, miR-7c, miR-93, mir-106a, mir-126, miR-128a, miR-130b, miR-140-3p, miR-142-3p, miR-146b, miR-15a-5p, miR-17, miR-191, miR-196, miR-19b-1, miR-20a, miR-331-5p, miR-886-5p, miR-26a, miR-29a, miR-517a and miR-296 miRNAs are higher in the test sample compared to control sample, the subject is identified as having high risk of the development of PE (See Table 1). Thus, a woman is identified as having high risk of the development of PE if the levels of one or more of miR-7a, miR-7c, miR-93, mir-106a, mir-126, miR-128a, miR-130b, miR-140-3p, miR-142-3p, miR-146b, miR-15a-5p, miR-17, miR-191, miR-196, miR-19b-1, miR-20a, miR-331-5p, miR-886-5p, miR-26a, miR-29a, miR-517a and miR-296, are higher in a blood, serum or plasma sample of the woman compared to a control sample.
A further embodiment of the invention provides a kit comprising reagents to carry out the methods of the current invention. In one embodiment, the kit comprises primers or probes specific for miRNAs of interest. Reagents for treating the samples, for example, deproteination, degradation of DNA, or removal of other impurities can also be provided in the kit.
An aspect of the invention provides a kit, for example, a point-of-care (POC) diagnostic device for assaying one or more miRNAs which can be used to identify the subject as having high risk of the development PE. In another embodiment, the kit comprises an oligonucleotide chip and reagents to conduct the assay to determine the levels of miRNAs corresponding to the oligonucleotides on the oligonucleotide chip. The oligonucleotide chip according to the invention contains oligonucleotides corresponding to a group of miRNAs that are present at different levels in a sample of an individual having a high risk of the development of PE as compared to the corresponding sample of an individual having no risk or low risk of the development of PE.
In one embodiment, the oligonucleotide chip essentially consists of oligonucleotides corresponding to one or more miRNAs selected from miR-7c, miR-93, miR-128a, miR-140-3p, miR-142-3p, miR-146b, miR-15a, miR-196b, miR-331-5p, miR-886-5p, miR-17, miR-26a, miR-130b, miR-7a, miR-29a, miR-517a, miR-191, miR-296, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-92a-1, miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2, and miR-363 and optionally, one or more control oligonucleotides.
For the purposes of the invention, the term “oligonucleotide chip essentially consists of oligonucleotides” indicates that the oligonucleotide chip contains oligonucleotides corresponding to only those miRNAs that present at different levels in a sample of an individual having a high risk of the development of PE as compared to the corresponding sample of an individual having no risk or low risk of the development of PE and optionally, contain one or more control oligonucleotides.
The control oligonucleotides are oligonucleotides corresponding to an miRNA or messenger RNAs (mRNA) known to be present in the equal amount in a sample of an individual having a high risk of the development of PE as compared to the corresponding sample of an individual having no risk or low risk of the development of PE. Non-limiting examples of control oligonucleotides include oligonucleotides corresponding to mRNAs of 18S, U6 form microRNA, β-actin, β-glucoronidase and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Additional examples of control miRNAs or mRNAs depend on the tissue under examination. A person of ordinary skill in the art can determine control oligonucleotides appropriate for a particular assay and such embodiments are within the purview of the invention.
Epigenetic biomarkers of PE according to the invention include post-translational modification of one or more histone proteins. Accordingly, an embodiment of the invention also provides a method of predicting the development PE in a subject, the method comprising:
(a) determining the levels of post-translational modifications of one or more histone proteins in:
(b) optionally obtaining one or more reference values corresponding to the levels of post-translational modifications of the one or more histone proteins,
wherein the presence of the post-translational modifications in the one or more histone proteins:
at different levels in the test sample as compared to the control sample, or
relative to the reference values indicates high risk of development of PE in the subject; and
(c) identifying the subject as having high risk of developing PE based the levels of post-translational modifications in the one or more histone proteins in the test sample and optionally, administering a therapy to the subject to treat and/or manage PE, or
(d) identifying the subject as having not having high risk of developing PE based on the levels of post-translational modifications in the one or more histone proteins in the test sample and withholding the therapy to the subject to treat and/or manage PE.
Non-limiting examples of post-translational modifications of histone proteins include methylation, acetylation, ADP-ribosylation, ubiquitination, citrullination, and phosphorylation. The one or more histones can be selected from H1, H2A, H2B, H3, H4 and H5. In one embodiment, hyperacetylation of H3 is indicative of the development of PE.
Various techniques are well known to a person of ordinary skill in the art to determine the level of post-translational modifications of one or more histone proteins in a sample. Non-limiting examples of such techniques include protein mass-spectrometry and antibody based analysis.
Determination of post-translation modification of a histone protein by protein spectrometry in a sample involves analyzing protein lysates or purified histone protein from a sample and analyze them by mass spectrometry to identify specific peptides within the histone protein which have different spectrometric behavior based on the presence or absence of post-translational modifications, for example, acetylation, methylation, demethylation. Certain techniques of spectrometric analysis of post-translational modification of proteins are described in Harvey (2005), which is herein incorporated by reference in its entirety.
In a further embodiment, post-translational modification of a histone protein is determined in an antibody based assay using antibody specific for a post-translational modification. For example, acetylation of H4 histone protein on one or more of: N-terminus, Lysine 12, Lysine 16 and methylation/dimethylation on Lysine 20 is determined in an antibody based assay using antibody specific for the recited modification. In another embodiment, two or more antibodies specific for different post-translational modification are used to determine post-translational modification of a histone protein. Non-limiting examples of the antibody based assays include western blot analysis, enzyme immunoassay (EIA), enzyme linked immunosorbent assay (ELISA), radioimmune assay (MA) and antigen-antibody precipitation assay. Additional examples of antibody-based assays are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
Methods of carrying out these techniques are routine in the art. Additional methods of determining the level post-translational modifications of histone proteins in a sample are also well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
The reference value corresponding to levels of post-translational modifications of one or more histone proteins indicate the level of post-translational modifications associated with no risk or low risk of the development of PE or high risk of development of PE. As such, the reference values corresponding to levels of post-translational modifications of certain histone proteins may be indicative of the absence or presence of high risk of the development of PE. A reference value associated with no risk or low risk of the development of PE may be obtained based on samples obtained from subjects known to be free of PE. A reference value associated with high risk of the development of PE may be obtained based on samples obtained from subjects known to have PE. For example, tissues from a group of pregnant women can obtained during the first trimester and the levels of post-translational modifications of one or more histone proteins can be determined. The group of women can then be monitored for the development of PE. Reference values corresponding to levels of post-translational modifications of one or more histone proteins that are associated with low risk or no risk of the development of PE or high risk of the development of PE can be determined based on the presence of absence of PE in various women whose samples were analyzed. Additional examples of determining references values associated with no risk or low risk or high risk of the development of PE are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
The step of identifying the subject as having high risk or not having high risk of developing PE depends on the level of post-translational modifications of one or more histone proteins in the test sample. For example, if the levels of certain post-translational modifications of certain histone proteins in the test sample are significant higher or lower than the levels of corresponding post-translational modifications of certain histone proteins in the control sample, the subject is identified as having high risk of development of PE.
For example, a subject is identified as having high risk of the development of PE if H4 histone in a sample from the subject has one or more of:
In another example, a woman is identified as having high risk of the development of PE if H4 histone protein (SGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRL ARRGGVKRISGLIYEETRGVLKVFLENVIRDAVTYTEHAKRKTVTAMDVVYALKRQ GRTLYGFGG, SEQ ID NO: 1) in a buffy coat sample of blood from the woman has one or more of:
A further embodiment of the invention provides a kit comprising reagents to carry out the methods of the current invention, for example, identifying a subject as having high risk of the development of PE and optionally, administering therapy to treat and/or manage PE in the subject. The kit comprises reagents to conduct the assay to determine the levels of certain post-translational modifications of certain histones, for example, an antibody chip containing specific antibodies.
An aspect of the invention provides a kit, for example, POC diagnostic device for assaying one or more post-translational modifications of histone proteins which can be used to identify the subject as having high risk of the development PE. The antibody chip according to the invention comprises or essentially consists of antibodies against histone proteins post-translationally modified on certain residues, wherein the histone proteins are post-translationally modified on certain residues at different levels in a sample of an individual having a high risk of the development of PE as compared to the corresponding sample of an individual having no risk or low risk of the development of PE.
In one embodiment, the antibody chip essentially consists of one or more of antibodies against:
Epigenetic biomarkers of PE according to the invention also include expression, amount and/or activity of histone and DNA modifying enzymes, i.e., one or more enzymes that mediate post-translational modification of histone proteins or modification of DNA, for example, methylation. Accordingly, an embodiment of the invention also provides a method of predicting the development of PE in a subject, the method comprising:
(a) determining the levels of expression, activity and/or amount of one or more histone or DNA modifying enzymes in:
(b) optionally obtaining one or more reference values corresponding to the levels of expression, activity and/or amount histone proteins or DNA modifying enzymes,
wherein the presence of expression, activity and/or amount of one or more histone or DNA modifying enzymes:
at different levels in the test sample as compared to the control sample, or
relative to the reference values indicates high risk of development of PE in the subject; and
(c) identifying the subject as having high risk of developing PE based on the levels of one or more histone or DNA modifying enzymes in the test sample and optionally, administering a therapy to the subject to treat and/or manage PE, or
(d) identifying the subject as not having high risk of developing PE based on the levels of one or more histone or DNA modifying enzymes in the test sample and withholding the therapy to the subject to treat and/or manage PE if the subject.
Non-limiting examples of modifications of histone proteins include methylation, acetylation, ADP-ribosylation, ubiquitination, citrullination, and phosphorylation. Non-limiting examples of histone modifying enzymes include histone acetyl transferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT) and histone demethylase. Non-limiting examples of DNA modifying enzymes include DNA methyl transferase (DNMT). Additional examples of enzymes involved in modifying histone proteins or DNA are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
The activity of histone or DNA modifying enzymes in the test sample and optionally, the control sample can be determined by assays to determine the activity of histone or DNA modifying activity, expression and/or amount of histone or DNA modifying enzyme, expression and/or amount of mRNA encoding histone or DNA modifying enzyme. Various techniques are well known to a person of ordinary skill in the art to determine the level of expression, amount and/or activity of one or more histone or DNA modifying enzymes or the corresponding mRNAs. Non-limiting examples of techniques used to determine the activity histone or DNA modifying enzymes include fluorometric and colorimetric assays; whereas, techniques used to determine histone or DNA modifying enzyme amount include mass spectrometry or antibody based assays. Example of techniques used to determine activity of histone or DNA modifying enzymes are well known to a person of ordinary skill in the art and such methods are within the purview of the invention.
Determination of the amount of an enzyme by protein spectrometry in a sample involves analyzing protein lysates or purified enzymes of interest from a sample by mass spectrometry to identify the amounts of specific peptides within the histone protein. The amounts of enzymes within a sample can be determined based on the amount of peptides originating from the enzyme in the sample.
Non-limiting examples of the antibody based assays which can be used to determine the amount of histone or DNA modifying enzymes in a sample include western blot analysis, EIA, ELISA, RIA and antigen-antibody precipitation assay. Additional examples of antibody-based assays are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
Methods of carrying out these techniques are routine in the art. Additional methods of determining the level amount or activity of histone or DNA modifying enzymes in a sample are also well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
The reference value corresponding to levels of expression, amount and/or activity of histone or DNA modifying enzymes indicate the levels associated with no risk or low risk of the development of PE or high risk of development of PE. As such, the reference values corresponding to levels of expression, amount and/or activity of histone or DNA modifying enzymes may be indicative of the absence or presence of high risk of the development of PE. A reference value associated with no risk or low risk of the development of PE may be obtained based on samples obtained from subjects known to be free of PE. A reference value associated with high risk of the development of PE may be obtained based on samples obtained from subjects known to have PE. For example, tissues from a group of pregnant women can obtained during the first trimester and the levels of expression, amount and/or activity of histone or DNA modifying enzymes can be determined. The group of women can then be monitored for the development of PE. Reference values corresponding to levels of expression, amount and/or activity of histone or DNA modifying enzymes that are associated with low risk or no risk of the development of PE or high risk of the development of PE can be determined based on the presence of absence of PE in various women whose samples were analyzed. Additional examples of determining references values associated with no risk or low risk or high risk of the development of PE are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
The step of identifying the subject as having high risk or not having high risk of developing PE depends on the level of expression, amount and/or activity of histone or DNA modifying enzymes in the test sample. For example, if the levels of expression, amount and/or activity certain of histone or DNA modifying enzymes in the test sample are significant higher or lower than the levels of corresponding enzymes in the control sample, the subject is identified as having high risk of development of PE.
In one embodiment, a subject is identified as having high risk of the development of PE if histone deacetylase 1 (HDAC1) protein, mRNA or activity is increased in a test sample as compared to a control sample or histone deacetylase 5 (HDAC5) protein, mRNA or activity is decreased in a test sample as compared to a control sample. In another example, a woman is identified as having high risk of the development of PE if the HDAC1 protein, mRNA or activity is increased in a buffy coat sample of blood from the woman as compared to a control sample or HDAC5 protein, mRNA or activity is decreased in a buffy coat sample of blood from the woman as compared to a control sample.
Alternately, if the level of Aurora Kinase C (AURKC) protein, mRNA or activity is higher in the test sample compared to the control sample, the subject is identified as having high risk of developing PE (
Further, if the level of Aurora Kinase A (AURKA) or protein arginine N-methyltransferase 8 (PRMT8) protein, mRNA or activity is lower in the test sample compared to the control sample, the subject is identified as having high risk of developing PE (
A further embodiment of the invention provides a kit comprising reagents to carry out the methods of the current invention, for example, identifying a subject as having high risk of the development of PE and optionally, administering therapy to treat and/or manage PE in the subject. The kit comprises reagents to conduct the assay to determine the levels of expression, amount and/or activity of certain histone or DNA modifying enzymes, for example, an antibody chip containing antibodies against certain histone or DNA modifying enzymes or oligonucleotide chips containing mRNAs corresponding to certain histone or DNA modifying enzymes.
The antibody chip or oligonucleotide chip according to the invention contains antibodies or oligonucleotides corresponding to certain histone or DNA modifying enzymes, wherein the histone or DNA modifying enzymes have different amount, expression and/or activity in a sample of an individual having a high risk of the development of PE as compared to the corresponding sample of an individual having no risk or low risk of the development of PE and optionally, contain one or more control antibodies or control oligonucleotides.
In one embodiment, the antibody chip essentially consists of an antibody against HDAC1 and an antibody against HDAC5; whereas, the oligonucleotide chip essentially consists of an oligonucleotide corresponding to HDAC1 mRNA and an oligonucleotide corresponding to HDAC5 mRNA.
For the purposes of the invention, the term “chip essentially consists of antibodies or oligonucleotides” indicates that the antibody or oligonucleotide chip contains antibodies or oligonucleotides corresponding only those histone or DNA modifying enzymes that are present at different levels in a sample of an individual having a high risk of the development of PE as compared to the corresponding sample of an individual having no risk or low risk of the development of PE and optionally, contains one or more control antibodies or oligonucleotides. The control oligonucleotides or antibodies correspond to mRNA or proteins known to be present in the equal amount in a sample of an individual having a high risk of the development of PE as compared to the corresponding sample of an individual having no risk or low risk of the development of PE. Non-limiting examples of control oligonucleotides or antibodies include oligonucleotides or antibodies corresponding to β-actin, β-glucoronidase and GAPDH. Additional examples of control miRNAs or mRNAs depend on the tissue under examination. A person of ordinary skill in the art can determine control oligonucleotides appropriate for a particular assay and such embodiments are within the purview of the invention.
An aspect of the invention provides a kit, for example, a POC diagnostic device for assaying one or more histone or DNA modifying enzymes which can be used to identify the subject as having high risk of the development PE.
Epigenetic biomarkers of PE according to the invention also include the level of methylation of certain DNA loci in the genomic DNA of certain cells. Accordingly, an embodiment of the invention also provides a method of predicting the development of PE in a subject, the method comprising:
(a) determining the levels of methylation of one or more sites in the genomic DNA in:
(b) optionally obtaining one or more reference values corresponding to levels of methylation of the one or more sites,
wherein the presence methylation of one or more sites in the genomic DNA:
at different levels in the test sample as compared to the control sample, or
relative to the reference values indicates high risk of development of PE in the subject; and
(c) identifying the subject as having high risk of developing PE based the levels of methylation of the one or more sites in the genomic DNA in the test sample and optionally, administering a therapy to the subject to treat and/or manage PE, or
(d) identifying the subject as not having high risk of developing PE based the levels methylation of the one or more sites in the genomic DNA in the test sample and withholding the therapy to the subject to treat and/or manage PE.
As used herein, the term “level of methylation” as applied to a genomic site refers to whether one or more cytosine residues present in a CpG context have or do not have a methylation group. The level of methylation may also refer to the fraction of cells in a sample that do or do not have a methylation group on such cytosines. These cytosines are typically in the promoter region of the gene, though may also be found in the body of the gene, including introns and exons. The Beta-value is a ratio between methylated probe intensity and total probe intensities (sum of methylated and demethylated probe intensities). It is in the range of 0 and 1, which can also be interpreted as the percentage of methylation.
Various techniques are well known to a person of ordinary skill in the art to determine the level of methylation of one or more sites in the genomic DNA in a sample. Non-limiting examples of such techniques include bisulfite conversion, digestion by restriction enzymes followed by polymerase chain reaction (Combined Bisulfite Restriction Analysis, COBRA), direct sequencing, cloning and sequencing, pyrosequencing, mass spectrometry analysis or probe/microarray based assay. Certain techniques of determining methylation of genomic sites are described in Eads et al., Xiong et al., Paul et al., Warnecke et al., Tost et al., and Ehrich et al., the contents of which are herein incorporated in their entirety. Additional techniques for determining DNA methylation of one or more sites in the genomic DNA of a sample are well known to a person of ordinary skill in the art and such techniques are within the purview of the invention.
The reference value corresponding to levels methylation of one or more sites in the genomic DNA indicate the levels associated with no risk or low risk of the development of PE or high risk of the development of PE. As such, the reference values corresponding to levels of methylation of one or more sites in the genomic DNA may be indicative of the absence or presence of high risk of the development of PE. A reference value associated with no risk or low risk of the development of PE may be obtained based on samples obtained from women known to be free of PE. A reference value associated with high risk of the development of PE may be obtained based on samples obtained from women known to have PE. For example, tissues from a group of pregnant women can obtained during the first trimester and the levels methylation of one or more sites in the genomic DNA can be determined. The group of women can then be monitored for the development of PE. Reference values corresponding to the levels of methylation of one or more sites in the genomic DNA that are associated with low risk or no risk of the development of PE or high risk of the development of PE can be determined based on the presence of absence of PE in various women whose samples were analyzed. Additional examples of determining references values associated with no risk or low risk or high risk of the development of PE are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
The step of identifying the subject as having high risk or not having high risk of developing PE depends on the levels methylation of one or more sites in the genomic DNA in the test sample. For example, if the levels of methylation of one or more sites in the genomic DNA in the test sample are significant higher or lower than corresponding levels in the control sample, the subject is identified as having high risk of development of PE. Table 2 provides the Illumina ID, the corresponding sequences of the genomic sites and the level of methylation of the genomic sites in PE patients.
In one embodiment, a subject is identified as having high risk of the development of PE if the methylation of one or more genomic sites selected from SEQ ID NOs: 46-55 is increased in the genomic DNA of a test sample as compared to a control sample. In a specific embodiment, a woman is identified as having high risk of the development of PE if methylation of genomic site CYP19A1 (SEQ ID NO: 46) is increased in a sample, for example, buffy coat sample of blood, from the woman as compared to a control sample.
In another embodiment, a subject is identified as having high risk of the development of PE the methylation of one or more genomic sites selected from SEQ ID NOs: 2-45 is decreased in the genomic DNA of a test sample as compared to a control sample. A further embodiment of the invention provides a kit comprising reagents to carry out the methods of the current invention. The kit comprises reagents to conduct the assay to determine the levels methylation of certain sites in the genomic DNA in certain cells of a subject. The kit can include reagents for isolation of genomic DNA from a sample, reagents to treat the genomic DNA, for example, bisulfite treatment, specific primers to analyze the genomic sites of interests and reagents for PCR amplification of the sites of interest.
An aspect of the invention provides a kit, for example, POC diagnostic device for assaying methylation of one or more sites in the genomic DNA which can be used to identify the subject as having high risk of the development PE. PE arises from a complex interplay among several factors. Epigenetic mechanisms and miRNAs closely interact with each other, thereby creating reciprocal regulatory circuits which lead to gene regulation. The invention identifies novel interactive sets of epigenetic and miRNA biomarkers in the first trimester which can be used to predict the development of PE. Accordingly, one embodiment of the invention provides the methods of predicting, treating and/or managing PE in a subject; the method comprises determining two or more, for example, three, four, five, six, seven, eight, nine or ten biomarkers described herein to identify a subject as having a high risk of the development of PE. The multiple biomarkers can belong to the same class, for example, multiple miRNAs or multiple post-translational modifications of histone proteins; or the multiple biomarkers can be chosen from different classes, for example, a combination of miRNAs, post-translational modification of histones, histone or DNA modifying enzymes or methylation of certain genomic DNA sites. In an embodiment, the multiple biomarkers do not contain any biomarker from one or more classes described herein, e.g., the multiple biomarkers may not contain an miRNA, a post-translational modification of histone, a histone or DNA modifying enzyme or a methylation of a genomic DNA site.
Accordingly, an embodiment of the invention also provides a method of predicting the development of PE in a subject, the method comprising:
(a) determining the levels of two or more biomarkers selected from one or more of miRNA, post-translational modification of histones, histone or DNA modifying enzymes, methylation of certain genomic DNA sites in:
(b) optionally obtaining one or more reference values corresponding to levels of one or more biomarkers,
wherein the presence of two or more biomarkers:
at different levels in the test sample as compared to the control sample, or
relative to the reference values indicates high risk of development of PE in the subject; and
(c) identifying the subject as having high risk of developing PE based on the levels of two or more biomarkers in the test sample and optionally, administering a therapy to the subject to treat and/or manage PE, or
(d) identifying the subject as not having high risk of developing PE based on the levels of two or more biomarkers in the test sample and withholding the therapy to the subject to treat and/or manage PE.
The combination of two or more biomarkers can be selected from the miRNAs, post-translational modification of histones, histone or DNA modifying enzymes, methylation of certain genomic DNA sites described earlier in this disclosure. In one embodiment, a subject is identified as having high risk of the development of PE if all of the analyzed biomarkers are significant different between the test sample and the control sample. In another embodiment, a subject is identified as having high risk of the development of PE if a pre-determined number of biomarkers out of the analyzed biomarkers are significant different between the test sample and the control sample. For example, if five biomarkers are analyzed, a subject can be identified as having high risk of the development of PE if more than three biomarkers are significant different between the test sample and the control sample.
In another embodiment, the comparison in the levels of two or more biomarkers between the test sample and the control sample is performed by as a combination of the two or more biomarkers, for example, by multivariable analysis. An example of multivariable analysis is multiple regression analysis. When the levels of two or more biomarkers are compared between the test sample and the control sample as a combination, the two or more biomarkers as a combination can be identified as significantly different between the two samples despite one or more of the multiple biomarkers not being different when considered individually.
In an embodiment, levels of four biomarkers, namely, miR-17, post-translational modification of H4 histone protein, amount of HDAC5 mRNA and/or protein and methylation of CYP19A1 site are determined. A subject is identified as having a high risk of the development of PE if the subject has increased miR-17, hyperacetylated H4 histone protein, decreased HDAC5 mRNA and/or protein and hypermethylated CYP19A1.
A further embodiment of the invention provides a kit, for example, POC diagnostic device, for identifying a subject as having high risk of PE based on the levels of two or more biomarkers. The POC device of the invention provides low-tech and cost-effective tool that still produces an accurate measurement, is portable, physically strong (compared to chip/sensing device), and simple to use. The kit can be used by virtually anyone, anywhere.
3-D printing technique can be used to manufacture the housing of the kit. Recycled materials, for example, recycled thermoplastic with added fibrous reinforcement, can be used to reduce the material cost and produce a light weight and unbreakable biomarker tool.
The invention provides a POC device capable of assaying miR-17, methylated CYP19A1, HDAC5 mRNA and acetylation of histone H4 in a sample, for example, a blood sample obtained from a subject. The sample can be treated before subject the sample to the analysis using the POC device.
The POC device can comprise of one or more locations for the introduction of the treated or untreated sample, which can be directed to two or more compartments, wherein each compartments is designed to assay different biomarker. For example, the POC device comprises of four compartments: one for assaying miR-17, one for assaying acetylation of H4 histone protein, one for assaying HDAC5 mRNA, and one for assaying methylation of CYP19A1.
Accordingly, a POC for reliable and rapid detection of biomarkers described herein is provided. In one embodiment, the POC utilizes an opto-fluidics-based platform for use as a biosensor.
In a certain embodiment, the POC incorporates functionalized colloidal nanoparticles trapped at the entrance to a nanofluidic channel providing a robust means for analyte detection at trace levels using surface enhanced Raman spectroscopy. The POC device can be used for sensitive detection of epigenetic modification in either blood or urine, is small and inexpensive, and can provide results in less than 15 minutes. Briefly, following the introduction of blood or urine, small molecules in the sample would compete with competing probes or aptamers depending on the molecule of interest. These probes will be already pre-bound to small molecule derivatives, and Raman reporter molecules attached on nanoparticles. The competition releases the gold particles which then aggregate at a nanochannel constriction downstream. To demonstrate the diagnostic potential of the system, a “gate” can be imposed; i.e., the lowest and highest value obtained from a healthy sample considered the healthy range. All values beyond this range can be assumed to be indicative of a change from normal conditions. As such, a low-cost, rapid, sensitive epigenetic diagnostic and prognostic tool for early detection of pre-eclampsia is provided.
To practice the methods described herein for identifying a subject as having high risk of the development of PE, control samples can be obtained from one or more of the following:
a) an individual belonging to the same species as the subject and not having PE,
b) an individual belonging to the same species as the subject and known to have a low risk or no risk of developing PE, or
c) the subject prior to becoming pregnant.
Additional examples of control samples are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.
In certain embodiments, the control sample and the test sample are obtained from the same type of an organ or tissue. Non-limiting examples of the organ or tissue which can be used as samples are placenta, brain, eyes, pineal gland, pituitary gland, thyroid gland, parathyroid glands, thorax, heart, lung, esophagus, thymus gland, pleura, adrenal glands, appendix, gall bladder, urinary bladder, large intestine, small intestine, kidneys, liver, pancreas, spleen, stoma, ovaries, uterus, skin, blood or buffy coat sample of blood. Additional examples of organs and tissues are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
In certain other embodiments, the control sample and the test sample are obtained from the same type of a body fluid. Non-limiting examples of the body fluids which can be used as samples include amniotic fluid, aqueous humor, vitreous humor, bile, blood, cerebrospinal fluid, chyle, endolymph, perilymph, female ejaculate, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sputum, synovial fluid, vaginal secretion, blood, serum or plasma. Additional examples of body fluids are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.
The methods described herein can be used to identify a subject as having high risk of the development of PE. In certain embodiments, the subject is a mammal. Non-limiting examples of mammals include human, ape, canine, pig, bovine, rodent, or feline.
In one embodiment, the methods described herein are used to identify a pregnant woman as having high risk of the development of PE. In another embodiment, the methods described herein are performed during the first trimester of pregnancy of a woman to identify the woman as having high risk of the development of PE.
Once a subject is identified as having high risk of the development of PE based on the methods described herein, the step of treating and/or managing PE includes one or more of:
a) administering medications to lower blood pressure: these medications, called antihypertensives, are used to lower blood pressure,
b) administering corticosteroids,
c) administering anticonvulsant medications, for example, magnesium sulfate,
d) bed rest for the patient,
e) hospitalization to perform regular non-stress tests or biophysical profiles to monitor the fetus' well-being and measure the volume of amniotic fluid,
f) administering low-dose aspirin,
g) administering calcium supplements,
h) inducing delivery before natural labor is initiated.
As such, the invention provides that epigenetics and miRNA regulation provides very early manifestation of PE pathogenesis—one that presages the clinical onset of PE by a few months, for example, four to five months. In addition, the invention indicates a new paradigm of discovering interactive epigenetic biomarkers for prediction of PE at a very early stage, for example, during the first trimester. This study also paves new avenues to look for biomarkers in a unique perspective for other diseases. These epigenetic changes happen prior to gene expression, and they are often reversible, making them good candidates for therapeutic interventions.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
First trimester blood samples and uterine artery Doppler ultrasonography were obtained from 1007 women between 11 and 13 6/7 weeks of gestation. Epigenetic and miRNA profiling was performed on the serum or buffy coat samples from total of 51 controls and 17 severe PE cases. Cases and controls were closely matched with respect to age, sex, body mass index (BMI) and other relevant parameters (Table 3).
To compare PE women with controls, 381 miRNAs in 28 patients (C=14, PE=14) were examined. Expression profiles revealed 22 significantly upregulated miRNAs which are shown to be involved in reproductive system disease (p<0.02) (
Upregulation of certain miRNAs, namely, 17, 26a, 130b, 7a, 29a, 517a, 191 & 296 in the third trimester in the serum or in the placenta is reported. Conversely, Wang et al. (2008) and Hong et al. (2014) showed that miR-126 functions as a pro-angiogenic factor in rat placenta and is decreased in endothelial progenitor cells in term placenta of PE patients. However, evidence showed that miR-126 functions as a pro-angiogenic factor in rat placenta and is decreased in endothelial progenitor cells in term placenta of PE patients.
Several groups showed that the miR-17 cluster (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1) and its paralog, the miR-106a cluster (miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2, and miR-363) are significantly increased in term placentas of PE women. This is consistent with the results at 11-13 weeks (
Post-translationally modified (PTM) histones in buffy coat samples were characterized using reverse-phase liquid chromatography mass spectrometry. The profiles showed several species that corresponded in mass to core and linker histones variants and their PTM isoforms (data not shown, Su et al. (2007)). Multiple PTMs were measured; however, the core histones (H3, H2A, H2B) showed complicated spectra due to the presence of multiple variants and a high degree of PTMs, in particular acetylation and methylation. ELISA study showed no changes in H2A in PE serum. Histone H4 showed unique spectra as H4 does not have sequence variants (uncomplicated by multiple variants, unlike other histones).
In H4 spectra, the most abundant species was observed at 11,306 Da which correspond in mass with dimethylation (DiMe) and N-terminal acetylation (N-Ac) of H4 (Su et al. (2007)). The next most abundant peaks, 11,348 Da and 11,390 Da, correspond to additional H4 acetylation (Su et al. (2007)). Ratio of relative abundance of 11349/11307 peak area demonstrated the H4 acetylation levels in patients with and without PE.
The statistical power was limited by small sample size (PE=8, C=8); however, more H4 acetylation was observed in PE patients (p=0.09) compared to controls (
The evidence suggests that histone acetylation may mediate development of chronic inflammation by modulating the expression of pro-inflammatory cytokine TNF-α and interleukins, and activation of the transcription factor NF-κB. These molecules are constitutively produced by a variety of cells under chronic inflammatory conditions, which in turn leads to the development of major diseases such as PE. The invention provides that H4 acetylation is associated with PE.
The expression levels of 81 epigenetic genes which are involved in histone modifications and DNA methylation was measured. Two of the eleven HDACs were significantly altered with no changes in HAT expression (p<0.05) (
In addition to the HDACs, aurora kinase A (AURKA), aurora kinase C (AURKC), and protein arginine methyltransferase 8 (PRMT8) were significantly altered (p<0.05).
DNA methylation was determined using Comprehensive High-throughput Arrays for Relative Methylation (CHARM) [6 Control (C) and 6 PE patients]. Significant (p<0.05) hypomethylation was associated with 81 genes in PE patients with an average methylation difference of 37.5% (min: 30%, max: 45%).
Using Infinium HumanMethylation450 assay in 36 samples (PE=12, C=24), 5904 significant CpG islands (p<0.05) were identified to be associated with PE. Out of 5904 islands, 86 CpG islands were significantly methylated (adjusted p-values, corrected for multiplicity-q=0.01). Of the 86 sites, 54 were associated with genes (10 hypermethylated & 44 hypomethylated, Table 2). Repetition of the same genes was not observed when the results of CHARM and Infinium assays were compared; however, repetitions were observed in the same families (e.g., solute carrier (SLC) family, zinc finger protein (ZNF) family), related upstream and downstream regulators, and pathways (Table 5).
A large portion of these gene sets do not have a known function but several have been reported in the context of PE or a function related to PE (e.g. angiogenesis, invasion, migration etc.). Using TARGETSCAN, mirBase and IPA, several of these genes are discovered as targets of the significantly upregulated miRNAs. In epigenetic gene expression assay changes in DNA methyl transferase were not observed. This lack of correlation may indicate that DNA methyltransferase (DNMT) activity might be the primary route, or frequency of DNA methylation may result from other mechanisms besides expression of DNMT since both hyper and hypo methylation are observed.
Hypermethylated CYP19A1 is provided as a first trimester PE biomarker. The synthesis of estrogens from C19 steroids is catalyzed by aromatase P450 (P450arom, product of the hCYP19A1 gene) and the ability of the human placenta to synthesize estrogens is vastly increased after the ninth week of gestation. Placental aromatase deficiency has been found in PE. In addition, biologically active estrogens and their metabolites formed by placental aromatase may also enhance angiogenesis and uteroplacental blood flow and reduce systemic vascular resistance. The scenario is mostly hampered in PE. Since the blood was collected after nine weeks of gestation, decreased CYP19A1 was expected in the patients who develop PE. Epigenetic modifications presage any gene expression and pathophysiology. A hypermethylated gene leads to decreased gene expression which is expected to be evident at later gestation. Upregulated mir-17 and -106a cluster was associated with decreased expression of CYP19A1 in term placenta (correspondence of decreased estrogen levels with increasing severity of PE). These miRNA clusters inhibited trophoblast differentiation by repressing CYP19A1. miR-17 is also predicted to target HDAC5 and subsequently a decrease in HDAC5 has been shown to increase H4 acetylation. In addition, target analysis has shown that miR-17 and other miRNAs target several well-known PE markers, PAPPA, VEGF, MMP, etc. Evidence also supports a role to these four markers in other PE pathologies (e.g. hypoxia, oxidative stress, inflammatory response, invasion, placental insufficiency). Therefore, these four interacting biomarkers underline the robustness of this analysis and also strengthen the previously published work and might serve as novel predictors of PE.
PE arises from a complex interplay among several factors. The invention shows that epigenetic mechanisms and miRNAs closely interact with each other, thereby creating reciprocal regulatory circuits which lead to gene regulation. The invention identifies novel interactive sets of noninvasive epigenetic and miRNA biomarkers in the first trimester which have a strong potential to predict the future development of PE.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.
This application is the U.S. national stage application of International Patent Application No. PCT/US2017/027593, filed Apr. 14, 2017, which claims the benefit of U.S. Provisional Application Ser. No. 62/322,422, filed Apr. 14, 2016, the disclosures of which are hereby incorporated by reference in their entirety, including all figures, tables and amino acid or nucleic acid sequences. The Sequence Listing for this application is labeled “Seq-List.txt” which was created on Apr. 14, 2017 and is 32 KB. The entire content of the sequence listing is incorporated herein by reference in its entirety.
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| WO2017/180984 | 10/19/2017 | WO | A |
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