[go: up one dir, main page]

US20100129818A1 - Polymorphisms in Genes Affecting CYP2C9-Related Disorders and Uses Thereof - Google Patents

Polymorphisms in Genes Affecting CYP2C9-Related Disorders and Uses Thereof Download PDF

Info

Publication number
US20100129818A1
US20100129818A1 US12/598,268 US59826808A US2010129818A1 US 20100129818 A1 US20100129818 A1 US 20100129818A1 US 59826808 A US59826808 A US 59826808A US 2010129818 A1 US2010129818 A1 US 2010129818A1
Authority
US
United States
Prior art keywords
subject
cyp2c9
polymorphism
snps
allelic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/598,268
Other languages
English (en)
Inventor
Wolfgang Sadee
Danxin Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ohio State University Research Foundation
Original Assignee
Ohio State University Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ohio State University Research Foundation filed Critical Ohio State University Research Foundation
Priority to US12/598,268 priority Critical patent/US20100129818A1/en
Assigned to THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION reassignment THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SADEE, WOLFGANG, WANG, DANXIN
Publication of US20100129818A1 publication Critical patent/US20100129818A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the invention was made with government support from the National Institutes of Health research grants HL 74730, HL 69758 and RR017568. The government may have certain rights in the invention.
  • Single nucleotide polymorphisms are useful as biomarkers for predicting disease susceptibility or progression, or as a guide for individualized therapy, including drug therapy.
  • Angiotensin I-converting enzyme plays a key role in cardiovascular biology. Its functions include formation of angiotensin II and inactivation of bradykinin, resulting in vasoconstriction and increased blood pressure. ACE inhibitors are recommended as first-line treatment of hypertension and heart failure. Expressed in many tissues, ACE further affects a broad spectrum of physiological processes. As a result, the ACE gene has been implicated in susceptibility to hypertension, myocardial infarction, renal pathophysiology, diabetes, and Alzheimer's disease.
  • angiotensin I-converting enzyme ACE1
  • ACE1 angiotensin I-converting enzyme
  • This enzyme hydrolyzes a number of substrates, including conversion of angiotensin I to angiotensin II (as part of the renin-angiotensin system).
  • Angiotensin II (AngII) is a potent vasoconstrictor and pro-hypertrophic factor. Ang II induces production of superoxide free radicals (O 2 ⁇ ) that scavenge available nitric oxide and reduce endothelial vasodilatation.
  • ACE1 has even greater affinity for bradykinin, thus hydrolyzing and inactivating a potent vasodilator. Through these pathways, ACE1 exerts potent physiological influence over salt balance, blood volume and blood pressure levels with significant implications for cardiovascular disease in particular.
  • ACE inhibitors decrease the release of aldosterone and retention of salt and water, significantly lowering blood pressure.
  • Drugs in this class have been shown to reduce mortality in many large clinical trials. These drugs are often administered immediately following myocardial infarction. They currently represent a major pharmaceutical class with millions of prescriptions worldwide, with additional indications in hypertension or renal crisis in relation to scleroderma, and prevention of kidney damage in some diabetics. Furthermore, recent literature indicates that ACE1 may play a role in the degradation of Alzheimer's plaques making it a possible disease factor (26,39).
  • Oxidative stress and damage play a role in the pathogenesis of a number of diseases.
  • mitochondrial-derived oxidants play an important role in the pathogenesis of many human disorders.
  • SOD2 is an antioxidant, the mitochondrial form of SOD and an important defense against oxidative damage.
  • the SOD2 gene is a member of the iron/manganese superoxide dismutase family.
  • the mitochondrial superoxide dismutase protein (SOD2) serves a critical cellular role in protecting from harmful reactive species by reducing these species to hydrogen peroxide (H 2 O 2 ) which is then processed to hydroxide (OH) and then water (H 2 O). This is a normal cellular process that is critical to life and protects the integrity of cellular genomes. Under conditions of stress including disease and environmental conditions (e.g., toxins) reactive species can accumulate to a degree that overwhelms the capacity of endogenous protectors including SOD2. Thus, if common alleles exist that affect SOD2 production these alleles may contribute to many diseases, but may only be important under conditions of accumulated oxidative stress.
  • Dopamine active transporter (SLC6A3, formerly) is a membrane-spanning protein that binds the neurotransmitter dopamine. SLC6A3 provides the primary mechanism through which dopamine is cleared from synapses. SLC6A3 works by transporting dopamine from the synapse into a neuron. SLC6A3 is present in the peri-synaptic area of dopaminergic neurons in areas of the brain where dopamine signaling is common. SLC6A3 terminates the dopamine signal and is implicated in a number of dopamine-related disorders, including alcoholism, attention deficit hyperactivity disorder, bipolar disorder, clinical depression, drug abuse, Parkinson disease, Tourette syndrome and Schizophrenia.
  • dopamine-related disorders including alcoholism, attention deficit hyperactivity disorder, bipolar disorder, clinical depression, drug abuse, Parkinson disease, Tourette syndrome and Schizophrenia.
  • Stimulant medications such as those used to treat ADHD, and drugs of abuse such as amphetamine bind to SLC6A3 and inhibit reuptake of dopamine.
  • Genetic variants of SLC6A3 may influence levels of gene expression and/or ability of drugs to bind to SLC6A3 protein.
  • the gene that encodes the SLC6A3 protein is located on human chromosome 5, consists of 15 coding exons, and is roughly 64 kpb long. It is believed that the associations between SLC6A3 and dopamine related disorders has come from a genetic polymorphism in the SLC6A3 gene, which influences the amount of protein expressed.
  • CYP2C9 (encoding cytochrome P450 2C9) is a liver drug metabolizing enzyme, involved in metabolism of ⁇ 20% of pharmaceuticals.
  • CYP2C9 is a member of the cytochrome P450 mixed-function oxidase system and is involved in the metabolism of xenobiotics in the body.
  • CYP2C9 is involved in the metabolism of several groups of drugs, such as, for example, non-steroidal anti-inflammatory drugs (NSAIDs). Genetic polymorphism exists for CYP2C9 expression and there is a belief that approximately 1-3% of Caucasian populations are poor metabolizers with no CYP2C9 function.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Such tools would likewise enable the identification of new drugs that modulate expression of genes that affect chemosensitivity, particularly new agents that alter expression of these genes to overcome drug resistance or enhance chemosensitivity.
  • the disclosure provides for a method for predicting a subject's risk factors for an ACE-related disorder, such as, but not limited to cardiovascular diseases and/or a subject's responsiveness to a therapeutic agent targeting the subject's renin-angiotension system (for example, ACE inhibitors angiotension receptor blockers (ARBS) and the like).
  • a therapeutic agent targeting the subject's renin-angiotension system for example, ACE inhibitors angiotension receptor blockers (ARBS) and the like.
  • ARBS angiotension receptor blockers
  • the method includes detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is selected from the group of (i) ACE-associated SNPs rs4290, rs7214530, rs7213516, rs4309, rs4343 or combinations thereof; or, (ii) a SNP in linkage disequilibrium with one or more SNPs listed in (i).
  • the allelic status of the polymorphism in the subject is predictive of the subject's risk factors for an ACE-related disorder.
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the subject's risk factors for an ACE-related disorder.
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict whether the subject has risk factors for an ACE-related disorder.
  • the disclosure provides for a method of screening a subject for a prognostic biomarker, comprising detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is one or more of:
  • the disclosure provides for a method of screening a subject for a prognostic biomarker, comprising detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is one or more of:
  • the disclosure provides for a method of screening a subject for a prognostic biomarker, comprising detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject wherein the polymorphism is one or more of:
  • the disclosure provides for a method of screening a subject for a prognostic biomarker, comprising detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is one or more of:
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the prognostic outcome of the subject.
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict whether the subject has a greater or lesser risk factors for an ACE-related disorder.
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the subject's response to treatment.
  • the disorders for which a therapeutic ACE inhibitor may be indicated includes, but is not limited to, one or more of the following: hypertensive treatment for heart disease, lowering blood pressure, myocardial infarction, hypertension or renal crisis in relation to scleroderma, prevention of kidney damage in diabetics, and Alzheimer's disease.
  • SNPs identified herein can be used in combination with additional predictive tests including, but not limited to, additional SNPs, mutations, and clinical tests.
  • the disclosure also provides for a method for finding a functional polymorphism in a target gene implicated a in subject's risk factors for an ACE-related disorder, comprising: (i) providing a sample of a target tissue expressing the target gene; (ii) measuring the target gene's allelic mRNA expression imbalance (AEI) by quantitatively measuring the relative amounts of mRNA generated from each of two alleles in a transcribed region of the target gene and comparing the mRNA expression of one allele against the other allele to obtain an AEI ratio; and (iii) using the AEI ratio as a phenotype to scan the target gene for regions containing polymorphisms. Accordingly, a significant association between the AEI ratio and the polymorphism indicates that the polymorphism is a functional polymorphism that can serve as a biomarker for assessing a subject's risk factors for an ACE-related disorder.
  • AEI allelic mRNA expression imbalance
  • kits comprising useful components for practicing the present method.
  • a useful kit can contain oligonucleotide probes specific for ACE alleles.
  • the kit can also include instructions for correlating the assay results with the subject's responsiveness to a therapeutic agent, the subject's prognostic outcome, or the probability of success or failure of a particular drug treatment in the subject.
  • FIGS. 1 a and 1 b ACE Allelic mRNA expression in left ventricular heart tissues from African-Americans ( FIG. 1 a ) and Caucasian-Americans ( FIG. 1 b ).
  • Allelic mRNA expression ratios (major/minor allele for marker SNPs rs4309 (C/T), rs4343 (A/G)) are averages of results using both markers.
  • AEI was prevalent in African-American ( FIG. 1 a ) but not Caucasian-American ( FIG. 1 b ) heart tissues.
  • Genotypes for the promoter SNPs are indicated above the African-American samples. Data are mean ⁇ SD, ***P ⁇ 0.001 versus pooled DNA ratios.
  • FIGS. 3 a and 3 b Luciferase reporter gene assay of the ACE promoter in bovine aortic endothelial cells (BAEC) FIG. 3 a ) and HEK293 cells ( FIG. 3 b ).
  • BAEC bovine aortic endothelial cells
  • FIG. 3 b HEK293 cells
  • An ACE promoter DNA fragment spanning from ⁇ 4,335 to +1 was cloned into the pGL3-Basic vector, containing various combinations of the promoter SNPs rs7213516 (G/A), rs7214530 (T/G) and rs4290 (C/T).
  • the reference haplotype is G-T-C, while the variant constructs contain 1-3 minor alleles (G-T- T , G- G -C, G- G - T , A - G -C, A - G - T ).
  • BAEC 0.8 ⁇ g plasmids were co-transfected with 40 ng Renilla luciferase plasmid using either Lipofectamine or Fugen reagent, and activity was measured by Dual-Glo luciferase assay kit (Promega). Luciferase activities from fused-pGL3 vector were normalized using Renilla luciferase activity as an internal control.
  • HEK293 cells various amounts of plasmid were transfected using Lipofectamine, with no differences observed between all conditions.
  • FIG. 4 Odds ratios of three polymorphisms for primary outcome in the overall population and within each race/ethnicity group.
  • the three polymorphisms are promoter SNPs rs7213516 and rs4290, and intron 15 SNP rs13447447 (I/D). Odds ratios were adjusted for age, sex, race/ethnicity, BMI, smoking, INVEST treatment strategy, previous myocardial infarction, previous stroke, heart failure, diabetes, renal insufficiency, baseline SBP, diuretic use, and ACE inhibitor use.
  • FIG. 5 ACE gene structure (UCSC genome browser) and location of polymorphisms tested in this study. The boxes indicate the exons coding for the two peptidase domains in the full length ACE isoform.
  • Overviews of HapMap LD in the gene region for individuals from Utah of Northern-European ancestry (CEU) and from Yoruba, Nigeria (YRI) are shown at bottom (Haploview).
  • FIGS. 6 a and 6 b Schematics of the allelic expression imbalance (AEI) assay used to uncover cis-acting functional alleles. Shown here, marker SNP rs4309 (C/T) is used in the SNaPshot reaction for both gDNA and mRNA. Peak area ratios represent allelic ratios in gDNA and mRNA (after conversion to cDNA).
  • AEI allelic expression imbalance
  • FIG. 7 LD structure of polymorphisms in the INVEST-GENES clinical genetic association study. Values for D′ and r 2 are provided and color coded; the light blue boxes indicate very, low allele abundance preventing calculation of D′.
  • FIG. 8 Promoter sequence alignments and TF binding sites.
  • the three promoter SNPs rs7213516 (G/A), rs7214530 (T/G) and rs4290 (C/T) are located ⁇ 2883, ⁇ 2828 and ⁇ 2306 by upstream of the transcription start site (+1).
  • the predicted MEF2A transcription factor binding sites based on the JASPAR database position-weight matrices are shown in detail.
  • Sequence alignments (CLUSTALW) are based on genomic matches identified by BLAST of the human promoter region (* indicates a 1 by insert in rhesus, dog, elephant, and armadillo sequences; ⁇ indicates a 9 by insert in dog and armadillo sequences).
  • FIG. 9 Schematic illustration of ACE gene structure and relevant genetic polyporphism (chromosome 17q.23.3) (not to scale).
  • FIG. 10 Table 1. Unadjusted and adjusted odds ratios and 95% confidence intervals for secondary outcomes by genotype
  • FIGS. 11 a and 11 b Tables 2A and 2B. Polymorphisms analyzed in this study, and minor allele frequencies observed in the 65 heart tissues (Table 2A, FIG. 11 a ) and in the INVEST-GENES cohort (Table 2B, FIG. 11 b ), sorted by race/ethnicity. The P values indicate the level of significance for interethnic differences in minor allele frequencies.
  • FIG. 12 Table 3. Baseline characteristics for the INVEST-GENES case and control patients.
  • FIG. 13 Table 4. Oligonucleotide sequences used in genotyping and allelic expression imbalance (AEI) assays for ACE that employed primer extension technology. Underlined nucleotides were intentionally mismatched against the reference sequence.
  • AEI genotyping and allelic expression imbalance
  • FIG. 14 Table 5. Oligonucleotide sequences employed in genotyping ACE SNPs by the GC-clamp method described in Papp et al.
  • FIG. 15 Table 6. Oligonucleotides sequences employed in ACE Pyrosequencing genotyping.
  • FIG. 16 Table 7. Oligonucleotide primers used in the amplification and direct sequencing of the ACE upstream gene region and cDNA.
  • FIG. 17 Table 8. FAM-labeled oligos and related oligos used in genotyping ACE polymorphisms.
  • FIG. 18 Table 9. Oligonucleotides used in the measurement of ACE expression by RT-PCR, including one that spans cDNA exons.
  • FIG. 19 Table 10. Oligonucleotide sequences used in genotyping and allelic expression imbalance (AEI) assays for SOD2 that employed primer extension technology.
  • AEI genotyping and allelic expression imbalance
  • FIG. 20 Table 11. A list of SNPs used in the SLC6A3 example herein.
  • FIG. 21 Table 12. A list of SNPs used in the CYP2C9 example herein.
  • FIG. 22 Results of AEI analysis for ACE, SOD2, NOS3 and CCL2, in heart left ventricular tissues. Each peak represents a distinct allele measured in genomic DNA or cDNA from a single heterozygous individual.
  • the selected samples (columns, left to right) represent the typical genomic DNA ratio observed, a cDNA showing insignificant deviation from the expected ratio and a cDNA sample showing highly significant deviation from unity. Normalization to the average genomic DNA is used in the calculation of AEI values (cDNA values listed as major:minor allele on a log 2 scale) and accounts for differences in fluorescent dideoxynucleotide incorporation efficiencies and fluorescence yields. See FIG. 26 —Table 13, for a list of genes reported here and FIG. 27 —Table 14 for marker SNPs and genes showing significant AEI results.
  • FIG. 23 Allelic mRNA expression ratios (major allele over minor allele, normalized to the mean allelic ratio in genomic DNA) measured in heart failure samples for 12 cardiovascular candidate genes. Results for individual samples are displayed with the magnitude and direction of AEI indicated on a log 2 scale (y-axis). Potential AEI in individual samples is indicated by ratios >(+0.3) log 2 or ⁇ ( ⁇ 0.3) log 2, a cutoff arrived at by analysis of the extent of variation in genomic DNA ratios. For the present survey study we considered ratios >(+0.5) log 2 or ⁇ ( ⁇ 0.5) log 2 to represent significant AEI.
  • FIG. 24 Lack of correlation between SOD2 allelic mRNA expression ratios and allelic CpG methylation ratios in 34 heart tissue samples. Allelic methylation ratios were determined from triplicate assays using Hpa II digestion of the genomic DNA region containing rs4880 (only non-methylated DNA is cut), followed by SNaPshot analysis of the allelic ratios for uncut genomic DNA.
  • FIG. 25 Computed changes of mRNA folding (minimum free energy conformations) induced by all transitions (SNP generated by C ⁇ >T and G ⁇ >A substitutions) in the transcribed exonic domains of OPRM1 mRNA.
  • the arrow indicates the location of the functional SNP A118G, affecting mRNA levels in human brain (18).
  • the x-axis denotes the nucleotide position in the mature OPRM1 mRNA (cDNA), while the y-axis represent a scale of the extent by which predicted mRNA folding is affected by any given transition.
  • Conformations were calculated for wild-type and mutant sequences using Mfold, and then the sum of the differences in the Mfold single-strandedness count measure at each nucleotide was computed both globally (across the full mRNA structure, each point shown here) and in more regional sliding windows of different sizes. Sliding windows and analysis of both types of transversions at each position (pyrimidine ⁇ >purine), as well as A>G transitions alone all gave very similar results (data not shown).
  • FIG. 26 Table 13. A list of candidate genes tested for the presence of AEI in Example II herein.
  • FIG. 27 Table 14. Gene showing significant allelic mRNA expression ratios (at least one sample showing minimally ⁇ 0.2 0.5 , or ⁇ 40% AEI in either direction.
  • FIG. 28 Table 15. Genotyping of suspected functional polymorphisms compared with AEI data.
  • FIG. 29 Table 16. List of candidate genes analyzed in Example II, grouped by indication (disease or pharmacology). List of candidate genes analyzed in this study, grouped by indication (disease or pharmacology). Marker SNPs are all located in transcribed regions of the mature mRNA, or a splice variant. For some genes more than one marker SNP and tissue were used.
  • FIG. 30 Table 17. List of oligonucleotide primers used for PCT amplification and SNaPshot primer extension reactions.
  • FIG. 31 mRNA sequence of the ACE gene [SEQ ID NO: 261 (DNA) and SEQ ID NO: 287 (protein)].
  • FIG. 32 mRNA sequence of the SOD2 gene[SEQ ID NO: 262 (DNA) and SEQ ID NO: 288 (protein)].
  • FIG. 33 mRNA sequence of the SLC6A3 gene[SEQ ID NO: 263 (DNA) and SEQ ID NO: 289 (protein)].
  • FIG. 34 mRNA sequence of the CYP2C9 gene [SEQ ID NO: 264 (DNA) and SEQ ID NO: 290 (protein)].
  • the disclosure provides diagnostic and prognostic methods, compositions, assays, and kits useful for predicting the phenotype of a subject's risk factors for cardiovascular diseases and/or a subject's responsiveness to therapeutic ACE inhibitors.
  • the methods also include predicting the prognostic outcome of the subject, as well as the subject's responsiveness to drug treatments.
  • the methods and kits include determining the allelic status of polymorphisms in the ACE genes.
  • the disclosure also provides methods for identifying functional polymorphisms using an allele-specific mRNA expression imbalance (AEI) assay combined with SNP scanning of a target gene locus with allelic mRNA ratios as a quantitative phenotype, together with in vitro molecular genetic analysis to identify the functional polymorphisms. Also provided are a number of functional single nucleotide polymorphisms (SNPs) in the ACE gene.
  • AEI allele-specific mRNA expression imbalance
  • allelic expression imbalance Measuring allelic mRNA expression compares one allele against the other in a relevant target tissue of the same individual.
  • the assay quantitatively measures the relative amounts of mRNA generated from each of two alleles in physiologically relevant target tissues (e.g., specific cardiac regions) from subjects that are heterozygous for a marker SNP in the transcribed region of the gene in question.
  • AEI indicates the presence of cis-acting factors in gene regulation and/or mRNA processing.
  • AEI results provide a quantitative measure of the allelic differences in each individual, one allele serving as the control for the other, while canceling out any trans-acting factors.
  • allelic expression ratios are then used as the phenotype to scan a gene locus for regions containing functional polymorphisms. If cis-acting polymorphisms contribute to the measured AEI ratios, significant correlations should be detectable. For this analysis it is helpful to know the phasing of each SNP with, the marker SNPs. As disclosed in the Examples, the inventors conducted a single locus association test between SNP genotype and allelic expression phenotype. The AEI phenotype can be represented either as present/absent; or absent/present low/present high, or as a continuous quantitative trait. Significant associations indicate that a SNP, or one closely linked, contributes to AEI, by affecting mRNA expression levels.
  • polymorphisms as biological biomarkers, used either alone or in combination with each other or with already established biomarkers.
  • For each polymorphism in the candidate genes we have established a link with allelic expression in human biopsy cardiac tissues as the phenotype. Obtained by scanning the entire gene in a number of individuals for polymorphisms that correlate with AEI, these polymorphisms are either directly responsible for altering mRNA expression, or they are in linkage disequilibrium or strong linkage disequilibrium with a functional SNP or SNPs.
  • the listed polymorphisms are frequent (>5%), and have already shown statistically significant associations with clinical phenotypes. These polymorphisms therefore represent biallelic biomarkers associated with functional variants of key genes conveying susceptibility to CNS disorders and treatment outcome.
  • SNPs polymorphisms
  • a polynucleotide includes a plurality of such polynucleotides and reference to “the SNP” includes reference to one or more SNPs known to those skilled in the art, and so forth.
  • alleles are identified with respect to one or more polymorphic positions, with the rest of the gene sequence unspecified.
  • an allele may be defined by the nucleotide present at a single SNP; or by the nucleotides present at a plurality of SNPs, also termed haplotypes.
  • haplotypes also termed haplotypes.
  • a biallelic polymorphism has two forms. Diploid organisms may be homozygous or heterozygous for an allelic form.
  • the allele present at the higher or highest frequency in the population will be referred to as the “main” or “wild-type” allele; less frequent allele(s) will be referred to as “minor” or “variant” allele(s).
  • Assessing the “allelic status” of a polymorphism refers to determining whether a subject is heterozygous (has one minor allele and one main allele), homozygous for the minor allele or homozygous for the main allele.
  • a “gene” refers to a segment of genomic DNA that contains the coding sequence for a protein, wherein the segment may include promoters, exons, introns, and other untranslated regions that control expression.
  • a “genotype” is an unphased 5′ to 3′ sequence of nucleotide pair(s) found at a set of one or more polymorphic sites in a locus on a pair of homologous chromosomes in a subject.
  • genotyping involves determining the specific allele or the specific nucleotide(s) carried by an individual at a biallelic marker.
  • haplotype refers to a combination of alleles present in an individual or a sample on a single chromosome.
  • a haplotype refers to a combination of biallelic marker alleles found in a given individual and which may be associated with a phenotype.
  • Haplotyping is the process for determining one or more haplotypes in a subject and includes use of family pedigrees, molecular techniques and/or statistical inference.
  • polymorphism refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. “Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A “polymorphic site” is the locus at which the variation occurs. A polymorphism may comprise a substitution, deletion or insertion of one or more nucleotides. A single nucleotide polymorphism (SNP) is a single base pair change. Typically, a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site.
  • SNP single nucleotide polymorphism
  • single nucleotide polymorphism refers to a single nucleotide substitution. Typically, between different genomes or between different individuals, the polymorphic site may be occupied by two different nucleotides.
  • biaselic polymorphism refers to a polymorphism having two alleles at a fairly high frequency in the population, sometimes a single nucleotide polymorphism.
  • the frequency of the less common allele of the biallelic polymorphism of the present disclosure has been validated to be greater than 1%, sometimes the frequency is greater than 10%, 20% (i.e. heterozygosity rate of at least 0.32), or 30% (i.e. heterozygosity rate of at least 0.42).
  • mutation refers to a difference in DNA sequence between or among different genomes or individuals that causes a functional change and which can have a frequency below 1%. Sequence variants describe any alteration in DNA sequence regardless of function or frequency.
  • Linkage Disequilibrium refers to alleles at different loci that are not associated at random, i.e., not associated in proportion to their frequencies. If the alleles are in positive linkage disequilibrium, then the alleles occur together more often than expected assuming statistical independence. Conversely, if the alleles are in negative linkage disequilibrium, then the alleles occur together less often than expected assuming statistical independence. As used herein, “strong linkage disequilibrium” is defined by D′ of >0.8.
  • oligonucleotides include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
  • nucleotide as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.
  • nucleotide is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide.
  • purified is used herein to describe a polynucleotide or polynucleotide vector of the disclosure which has been separated from other compounds including, but not limited to other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide), or the separation of covalently closed polynucleotides from linear polynucleotides.
  • isolated requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
  • heterozygosity rate is used herein to refer to the incidence of individuals in a population, which are heterozygous at a particular allele. In a biallelic system, the heterozygosity rate is on average equal to 2 Pa(1-Pa), where Pa is the frequency of the least common allele. In order to be useful in genetic studies, a genetic biomarker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.
  • upstream refers to a location which, is toward the 5′ end of the polynucleotide from a specific reference point.
  • downstream refers to a location which is toward the 3′ end of the polynucleotide from a specific reference point.
  • base paired and “Watson & Crick base paired” are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another be virtue of their sequence identities in a manner like that found in double-helical DNA with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds (See Stryer, L., Biochemistry, 4th edition, 1995; incorporated herein by reference).
  • complementary or “complement thereof” are used herein to refer to the sequences of polynucleotides which are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
  • primer denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.
  • a primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase, or in a single nucleotide extension reaction for the measurement of AEI.
  • probe denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.
  • the primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis.
  • the probes and primers can comprise nucleic acid analogs such as, for example, peptide nucleic acids, locked nucleic acid (LNA) analogs, and morpholino analogs.
  • LNA locked nucleic acid
  • the 3′ end of the probe can be functionalized with a capture or detectable label to assist in detection of a polymorphism.
  • any of the oligonucleotides or nucleic acid of the disclosure can be labeled by incorporating a detectable label measurable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • labels can comprise radioactive substances ( 32 P, 35 S, 3 H, 125 I) fluorescent dyes (5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin), biotin, nanoparticles, and the like.
  • radioactive substances 32 P, 35 S, 3 H, 125 I
  • fluorescent dyes 5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin
  • biotin nanoparticles, and the like.
  • Such oligonucleotides are typically labeled at their 3′ and 5′ ends.
  • Probes can be used to detectably distinguish between target molecules differing in structure. Detection can be accomplished in a variety of different ways depending on the type of probe used and the type of target molecule. Thus, for example, detection may be based on discrimination of activity levels of the target molecule, but typically is based on detection of specific binding. Examples of such specific binding include antibody binding and nucleic acid probe hybridization. Thus, for example, probes can include enzyme substrates, antibodies and antibody fragments, and nucleic acid hybridization probes.
  • the detection of the presence or absence of the at least one variance involves contacting a target polymorphic site with a probe, typically an oligonucleotide probe, where the probe hybridizes with a form of the target nucleic acid containing a complementary base at the variance site as compared to hybridization to a form of the target nucleic acid having a non-complementary base at the variance site, where the hybridization is carried out under selective hybridization conditions.
  • a probe typically an oligonucleotide probe
  • the probe hybridizes with a form of the target nucleic acid containing a complementary base at the variance site as compared to hybridization to a form of the target nucleic acid having a non-complementary base at the variance site, where the hybridization is carried out under selective hybridization conditions.
  • an oligonucleotide probe may span two or more variance sites.
  • an oligonucleotide probe can include one or more nucleic acid analogs, labels or other substituents or moieties so
  • control population refers to a group of subjects or individuals who are predicted to be representative of the genetic variation found in the general population.
  • a “subject” comprises an individual (e.g., a mammalian subject or human) whose genotypes or haplotypes or response to treatment or disease state are to be determined.
  • a “nucleic acid sample” includes blood, serum, plasma, cerebrospinal fluid, urine, saliva, and tissue samples.
  • phenotype refers to any biochemically, anatomically, and clinically distinguishable, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example.
  • phenotype is used herein to refer to symptoms of, or susceptibility to a cardiovascular disorder; or to refer to an individual's response to a therapeutic agent; or to refer to symptoms of, or susceptibility to side effects to a therapeutic agent.
  • a “less severe phenotype” is defined as a less severe form of a cardiovascular disorder, or a form of the cardiovascular disorder that is more responsive to treatment, displays less side effects with treatment, has better prognosis, is not recurrent, or has a combination of these characteristics.
  • a “more severe phenotype” is defined as a more severe form of a cardiovascular disorder, or a form of the disorder that is less responsive to treatment, displays more side effects with treatment, has worse prognosis, is recurrent, or has a combination of these characteristics.
  • the more severe phenotype is a disease state with profound consequences to the patient's life quality and requires more aggressive therapy.
  • a subject who is at risk for “having or developing a cardiovascular disorder” includes a subject with no clinical signs or symptoms of a cardiovascular disorder but with a strong family history of such disorders, a subject who exhibits clinical signs or symptoms associated with a cardiovascular disorder, or a subject who has been clinically diagnosed as having a cardiovascular disorder.
  • prognosis refers to predicting the course or outcome of a condition in a subject. This does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is predictably more or less likely to occur based on the pattern of biomarkers. Instead, the skilled artisan will understand that the term “prognosis” refers to an increased probability that a certain course or outcome will occur.
  • a “diagnostic” biomarker is a biallelic polymorphism, the allelic status of which is indicative of whether or not a subject has, or is at risk for developing, a cardiovascular disorder.
  • a “prognostic” biomarker is a biallelic polymorphism, the allelic status of which is predictive of the severity or prognosis of a cardiovascular disorder.
  • the pattern may signal that the subject is at an increased probability for experiencing a future event in comparison to a similar subject exhibiting a different pattern.
  • a certain pattern of prognostic biomarkers can predict an increased predisposition to an adverse outcome, or the chance of a person responding or not responding to a certain drug.
  • a “prognostic biomarker” can predict the presence of a “prognostic indicator.” For example, the presence of a minor allele of a SNP (prognostic biomarker) is indicative of a lower mRNA expression (prognostic indicator) in a target tissue.
  • ACE-related disorder refers to any ACE-related disorder comprising one or more of the following: cardiovascular diseases, hypertension, myocardial infarction, angioedema, altered kidney function, Alzheimer's, and/or responsiveness to a therapeutic targeting the subject's renin-angiotensin system, including, but not limited to ACE inhibitors, beta blockers, angiotensin receptor blockers (ARBs).
  • cardiovascular diseases hypertension
  • myocardial infarction angioedema
  • angioedema altered kidney function
  • Alzheimer's and/or responsiveness to a therapeutic targeting the subject's renin-angiotensin system
  • ACE inhibitors including, but not limited to ACE inhibitors, beta blockers, angiotensin receptor blockers (ARBs).
  • ARBs angiotensin receptor blockers
  • cardiovascular disorder refers to any disorder in which an increase or decrease in ACE levels, which can lead to hypertension, heart disease, heart failure, myocardial infarction, renal pathophysiology, diabetes, and related pathologies.
  • Treatment means the medical management of a subject, e.g., a human patient, with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement or associated with the cure of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • Treatment also includes symptomatic treatment, that is, treatment directed toward constitutional symptoms of the associated disease, pathological condition, or disorder.
  • Treatment also includes the act of not giving a subject a contra-indicated therapeutic agent.
  • correlating refers to comparing the allelic status of a polymorphism in a subject to the allelic status of the polymorphism in a reference population.
  • the reference population may be persons known to be free of a given condition, i.e., “normal individuals,” or may be persons known to suffer from, or to be at risk of developing, a given mental disorder, persons known to have a form of the mental disorder with better or worse outcome, or persons known to respond to or be resistant to a certain treatment.
  • a SNP pattern in a patient sample can be compared to a SNP pattern known to be associated with response to a certain depression medication.
  • the allelic status of the polymorphism in the subject is predictive of the prognostic outcome.
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the prognostic outcome of the subject.
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict whether the subject has a greater or less severe risk factors for cardiovascular diseases and/or responsiveness to therapeutic agents.
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the subject's response to treatment.
  • the SNPs identified herein can be used in combination with additional predictive tests including, but not limited to, additional SNPs, mutations, and clinical tests.
  • the SNPs can be those provided herein, and discussed in detail in the Examples.
  • the SNPs can also be SNPs in positive linkage disequilibrium with any of the SNPs provided herein.
  • the disclosure provides for a method for predicting a subject's risk factors for cardiovascular diseases and/or a subject's responsiveness to therapeutic ACE inhibitors.
  • the method includes detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is selected from the group: (i) ACE-associated SNPs rs4290, rs7214530, rs7213516 or combinations thereof; or, (ii) a SNP in linkage disequilibrium with one or more SNPs listed in (i).
  • the allelic status of the polymorphism in the subject is predictive of the prognostic outcome of the subject.
  • the allelic status of the polymorphism in the subject is predictive of the subject's risk factors for cardiovascular diseases and/or a subject's responsiveness to therapeutic ACE inhibitors.
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict the subject's risk factors for cardiovascular diseases and/or a subject's responsiveness to therapeutic ACE inhibitors.
  • the method further includes the step of correlating the allelic status of the polymorphism in the subject with the allelic status of the polymorphism in a reference population to predict whether the subject has a more or less severe phenotype for cardiovascular diseases and/or responsiveness to therapeutic ACE inhibitors.
  • the disclosure provides for a method of screening a subject for a prognostic biomarker for determining a subject's risk factors for cardiovascular diseases and/or a subject's responsiveness to therapeutic ACE inhibitors, comprising detecting the allelic status of one or more polymorphisms in a nucleic acid sample of the subject, wherein the polymorphism is one or more of: (i) ACE-associated SNPs rs4290, rs7214530, rs7213516 or combinations thereof; or, (ii) a SNP in linkage disequilibrium with one or more SNPs listed in (i).
  • the allelic status of the polymorphism in the subject is predictive of the prognostic outcome of the subject.
  • AEI Allelic mRNA Expression Imbalance
  • allelic expression imbalance Measuring allelic mRNA expression compares one allele against the other in a relevant target tissue of the same individual. The relative amounts of mRNA generated from each of two alleles in subjects heterozygous for a marker SNP in the transcribed region of the gene in question are quantitatively measured. AEI indicates the presence of cis-acting factors in gene regulation and/or mRNA processing. AEI results provide a quantitative measure of the allelic differences in each individual, one allele serving as the control for the other, while canceling out any trans-acting factors. The allelic expression ratios are used as the phenotype to scan a gene locus for regions containing functional polymorphisms.
  • cis-acting polymorphisms contribute to the measured AEI ratios, significant correlations should be detectable. Also, a single locus association test between SNP genotype and allelic expression phenotype can be conducted. The AEI phenotype is represented either as present/absent; or absent/present low/present high. Significant associations indicate that a SNP, or one closely linked, contributes to AEI, by affecting mRNA expression levels.
  • ACE1 Polymorphisms are Linked to Differences in ACE Expression
  • FIG. 31 contains the mRNA sequence for the ACE gene [SEQ ID NO: 261].
  • the ACE gene consists of 25 exons spanning ⁇ 25 kb and encoding a soluble or a membrane-bound protein variant with two peptidase domains ( FIG. 5 ).
  • FIG. 9 shows a schematic illustration of ACE gene structure and relevant genetic polymorphism (chromosome 17q.23.3) (not to scale).
  • ACE harbors a number of polymorphisms; however, frequent nonsynonymous SNPs that affect the protein sequence are lacking, suggesting that yet to be discovered regulatory polymorphisms may contribute to genetic susceptibility in cardiovascular diseases involving ACE.
  • To search for regulatory polymorphisms we measured allelic mRNA expression of ACE in human cardiac tissues. In contrast to total mRNA levels, allelic mRNA ratios cancel out trans-acting factors, so that any detectable allelic expression imbalance (AEI) is a strong indicator of cis-acting regulatory factors (6-10).
  • AEI detectable allelic expression imbalance
  • rs7213516, rs7214530 and rs4290 were in extensive but incomplete LD, so that we cannot exclude any of the 3 SNPs from contributing to the AEI ratios.
  • allelic mRNA ratios below 1 in the five African-American subjects showing AEI indicated that the less frequent allele had reduced mRNA expression (considering the inferred phasing between the marker SNP alleles and those of the promoter SNPs).
  • overall ACE mRNA levels by RT-PCR. Whereas no association with mRNA levels was observed with the I/D variant ( FIG. 2 a ), carrying the minor allele of the promoter SNPs was associated with decreased ACE mRNA expression (rs4290 T; P ⁇ 0.02 ( FIG. 2 b ), rs7213516 A; P ⁇ 0.04). This result indicates that the minor alleles of the promoter SNPs reduce expression.
  • the expression constructs containing any of the minor alleles of the three promoter SNPs significantly reduced reporter gene expression in BAEC, using two different transfection reagents. While there were differences in the degree of reduction between the various constructs, no single SNP alone could account for all results.
  • FIG. 7 Linkage disequilibrium is shown in FIG. 7 , illustrating the relationships between the genotyped SNPs.
  • allele frequencies in Hispanics were intermediate between Caucasians and African-Americans.
  • Minor allele frequencies of both rs7213516 and rs4290 differed significantly between African-Americans (16%), Hispanics (4%) and Caucasians ( ⁇ 1%).
  • Genotyping quality control checks showed >99.5% concordance between different assays for the same polymorphisms.
  • the ACE I/D polymorphism (rs13447447) was inconsistently associated with outcomes ( FIG. 4 ). Associations were not directionally similar in the different racial/ethnic groups, nor was there a linear trend between I/D heterozygotes and VI homozygotes. Finally, the I/D was not associated with any of the individual components of the composite outcome (FIG. 10 —Table 1). While not wishing to be bound by theory, the inventors herein now believe that there is no meaningful association with clinical outcomes analyzed here, consistent with a lack of effects on ACE mRNA level in heart tissue ( FIG. 2 ). There was also no evidence for association of the primary outcome with polymorphisms rs4291 and rs4366.
  • This study employed allelic mRNA expression analysis of ACE in human heart tissues, followed by SNP scanning, to identify regulatory polymorphisms in the ACE locus, long suspected of conferring genetic risk for cardiovascular disease.
  • This approach revealed strong effects on ACE mRNA expression attributable to three promoter SNPs, rs7213516, rs7214530, and rs4290, located in conserved regions 2-3 kb upstream of the transcription start site.
  • the three ACE promoter SNPs are common in individuals of African-American ancestry ( FIGS. 11 a , 11 b -Tables 2A, 2B), but rare in Caucasians, and intermediate in Hispanics. Consistent with our gene expression results, a clinical association study revealed a robust genetic effect on outcomes in hypertensive patients.
  • FIG. 8 shows the promoter sequence alignments and TF binding sites.
  • the three promoter SNPs rs7213516 (G/A), rs7214530 (T/G) and rs4290 (C/7) are located ⁇ 2883, ⁇ 2828 and ⁇ 2306 by upstream of the transcription start site (+1).
  • the predicted MEF2A transcription factor binding sites based on the JASPAR database position-weight matrices are shown in detail.
  • Sequence alignments (CLUSTALW) are based on genomic matches identified by BLAST of the human promoter region (* indicates a 1 by insert in rhesus, dog, elephant, and armadillo sequences; ⁇ indicates a 9 by insert in dog and armadillo sequences).
  • rs7214530 is part of a predicted recognition site for MEF2, a cardiac transcription factor previously implicated in cardiovascular disease and myocardial infarction (20-22). It is therefore possible that all three SNPs have co-evolved as part of a haplotype block prevalent in subjects of African origin, each contributing to gene regulation, possibly to different extents in different tissues. Reporter genes assays with an ACE promoter fragment, containing various combinations of the three suspected SNPs demonstrated decreased promoter activity for each combination of variant alleles, compared to the reference sequence in endothelial cells (BAEC), but not in HEK293 cells, indicating that these effects can be tissue specific. It is therefore likely that these ACE promoter polymorphisms have different effects in different target tissues, and therefore, could be associated with different pathophysiologies.
  • BAEC endothelial cells
  • the promoter alleles are common in African-Americans, they may partially account for phenotypic variation in ACE levels, blood pressure (e.g., 11) and response to ACE inhibitors (16-18) in individuals of African ancestry. Excessive ACE inhibition in African-Americans carrying the minor alleles of these promoter SNPs could have accounted for the increased susceptibility to angioedema as a main adverse effect of ACE inhibitors (18). While these alleles were found at lower frequency in Hispanics and Caucasians, they could be clinically relevant in the population at large, although we had limited statistical power to address this question.
  • ACE promoter alleles may be relevant in other human pathologies.
  • heart tissues from 12 African-American heart transplant patients only 8 were eligible for AEI analysis.
  • minor allele frequency of rs7213516 and rs4290 (25-27%) were higher than expected (16.0% in INVEST-GENES), with one patient homozygous for the minor alleles, arguing for conducting a larger study of heart failure patients.
  • Approval for use of human subjects was obtained from the OSU IRB.
  • Left ventricle tissue from 65 heart transplant patients was obtained through The Cooperative Human Tissue Network: Midwestern Division at OSU and stored at ⁇ 80° C. until extraction.
  • Genomic DNA and RNA were isolated, and cDNA was prepared from 1.0 ug RNA in three independent preparations, using oligo dT and gene-specific primers close to the two marker SNPs to minimize the effects of mRNA decay in post-extract tissues.
  • allelic mRNA expression was measured by normalizing to the mean ratio of gDNA peaks (SD for gDNA: rs4309 ⁇ 12.4%, rs4343 ⁇ 8.6%).
  • FIG. 6 Examples for assay results are shown in FIG. 6 . Each sample was assayed from three independent cDNA syntheses, each performed at least in duplicate.
  • SNPs selected to represent the major haplotype blocks ( FIGS. 11 a , 11 b -Tables 2A, 2B) in all 65 heart tissues. SNPs were genotyped as described herein. In addition, we sequenced full length cDNAs and the 5′-upstream region over 3 kb in eight African-Americans detecting five SNPs in the upstream region ( FIG. 11 a —Table 2A).
  • AEI allelic mRNA ratios >1.5 or ⁇ 1/1.5 as cutoff.
  • Association between genotype status (heterozygous or homozygous) with AEI was determined using HelixTree (Golden Helix, Inc.).
  • Linkage disequilibrium between SNPs (expressed as D′) and haplotypes were calculated using HelixTree (Golden Helix, Inc.).
  • a promoter fragment ranging from ⁇ 4,335 to +1 (the major transcription start site) in PGL3 basic vector (Promega) was provided by Dr. Melanie Eyries (29).
  • Various combinations of rs7213516/rs7214530/rs4290 haplotypes were obtained via site-directed mutagenesis or restriction digest of amplified genomic DNA with MscI and BstEII and subsequent cloning. All inserts were fully sequenced to verify the intended sequence.
  • the constructs were transfected into HEK-293 and BAEC, cultured in DMEM/F12 media containing 10% fetal bovine serum, penicillin (10 units/ml), and streptomycin (10 ⁇ g/ml), at 37° C. with 5% CO 2 .
  • the INternational VErapamil SR Trandolapril STudy evaluated cardiovascular adverse outcomes in patients randomized to atenolol or verapamil SR hypertension treatment strategy in 22,576 patients with documented coronary artery disease (CAD) and hypertension (19).
  • the primary outcome was the first occurrence of death (all cause), nonfatal MI, or nonfatal stroke. These events were taken separately as secondary outcomes.
  • genomic DNA was collected from 5,979 patients using buccal cells from mouthwash samples (30). All patients provided written informed consent, as approved by the UF IRB.
  • the present case-control study focused on the 258 INVEST-GENES patients who experienced a primary outcome event during study follow-up (cases), frequency matched 3:1 to cases for age, sex, and race/ethnicity with 774 individuals who were event-free during study follow-up (controls).
  • Logistic regression was performed to assess the association of genotypes/haplotypes with the primary and secondary outcomes after adjusting for ancestry and pre-specified confounding factors, namely age (by decades), gender, race/ethnicity, and history of MI and heart failure, and drug treatments.
  • DNA was prepared by a standard salting-out method from heart tissue (34). For RNA isolation, ⁇ 100 mg tissue was pulverized over dry ice and suspended in Trizol reagent, followed by phenol-chloroform extraction, and filtration through an RNAeasy column (Qiagen) after treatment with DNAse I. RNA quantity and quality was confirmed by UV spectrophotometry and nanodrop analysis (Bioanalyzer, Agilent Biotechnologies).
  • cDNA was synthesized following the manufacturer's protocol (Superscript RTII, Invitrogen) from 1.0 ug RNA in three independent preparations using oligo dT and ACE gene-specific reverse primers to increase specific yield. Negative controls (lacking RTII) and positive expression signals for ACE were confirmed by RT-PCR on an ABI7000 cycler followed by gel electrophoresis to confirm correctly sized products. The primers used for RT-PCR verification were the outer primers for the SNaPshot assay.
  • Marker SNPs (rs4309, rs4343) were genotyped at the Ohio State University Pharmacogenomics Core Laboratory in 65 heart failure samples by a melting curve dissociation approach on an ABI7000 real-time PCR instrument in order to determine heterozygotes for allelic expression assays (35). Allelic expression assays in genomic DNA and cDNA for each heterozygote were carried out in triplicate, and analyzed as previously described (6-10). For the rs4343 assay, due to the SNP location near an exon border, separate DNA and cDNA forward primers were used. Outside amplification primers for the assay were as follows (see FIG. 13 —Table 4):
  • rs4309 forward primer TGAGATGGGCCATATACAGTACTAC; [SEQ ID NO: 1] reverse primer: CCCGACGCAGGGAGAC), [SEQ ID NO: 2] and rs4343 DNA forward primer: CCCTTACAAGCAGAGGTGAGCTAA; [SEQ ID NO: 3] cDNA forward primer: ACCACCTACAGCGTGGCC; [SEQ ID NO: 4] common reverse primer: CATGCCCATAACAGGTCTTCATATT. [SEQ ID NO: 5]
  • extension primers for ACE allelic expression assay were as follows:
  • RT-PCR was done with SYBR Green on an ABI7000 (30 cycles, 2 steps: 95° C., 60° C.); values were normalized to ⁇ -actin expression levels. The correct size products were verified by gel electrophoresis.
  • ACE upstream region construct ( ⁇ 4335 to the transcription start site) driving expression of a firefly Luciferase reporter gene (pGL3.Basic, Promega) was kindly provided by M. Eyries (29). Sequencing indicated this construct contained the major allele at all polymorphic sites in the region compared to the reference genome sequence, thus it was labeled (G-T-C). Site-directed mutagenesis (Stratagene) was employed to generate altered constructs with SNP combinations; for
  • rs4290 (G-T-T) sense primer: CTCTGCACCCTTCCTTTGATGAGGTTTTG CCCT [SEQ ID NO: 10];
  • antisense primer AGGGCAAAACCTCATCAAAGGAAGGGTGCAGAG [SEQ ID NO: 11],
  • rs7214530 (G-G-C) sense primer: GAGCATATTTTTAAGGGCTGGTTTTCT CTCCTGTGGTAACT [SEQ ID NO: 12];
  • Genomic DNA isolation and genotyping for rs4290, rs4291 and rs7213516 in INVEST-GENES was performed at the University of Florida Center for Pharmacogenomics. Genomic DNA was isolated from buccal genetic samples using commercially available kits (PureGene, Gentra Systems Inc.) and adjusted to 20 ng/ ⁇ l. Genotyping for rs4290 was performed by polymerase chain reaction (PCR) followed by pyrosequencing using a PSQ HS96A SNP reagent kit according to the manufacturer's protocol (Biotage AB) (36). SNPs rs7213516 and rs4291 were genotyped by Taqman assay. The PCR and sequencing primers used for ACE SNP rs4290 were as follows (see
  • FIG. 15 Table 6
  • the PCR conditions were as follows: 95° C. for 15 min, 40 cycles consisting of denaturation at 94° C. for 30 s, annealing at 59° C. for 30 s, and extension at 72° C. for 1 min, followed by final extension at 72° C. for 7 min.
  • the Applied Biosystems 7900 HT SNP genotyping platform was used for the Taqman assays.
  • the SNP genotyping probes (Applied Biosystems IDs: C — 32160109 — 10 and C — 11942507 — 10) were used for ACE rs7213516 G>A and rs4291 A>T, respectively. Five ⁇ L reactions in 384-well plates were prepared, and the assays were performed and analyzed according to the manufacturer's recommendations.
  • the 287 bp insertion/deletion polymorphism (rs13447447) was genotyped at the Ohio State University Pharmacogenomics Core Laboratory by PCR with
  • FAM-labeled reverse primer (FAM-GTGGCCATCACATTCGTCAG), [SEQ ID NO: 19], and two unlabeled forward primers, one of which was insertion-specific
  • CT 2/3 repeat polymorphism (rs4366) was similarly genotyped by PCR with a FAM-labeled forward primer
  • SNPs rs4291 and rs4292 were genotyped by multiplexed SNaPshot primer extension assay within one amplicon. Extension primers were:
  • rs4291 TGGCT AGAAAGGGCCTCCTCTCTTT
  • rs4292 TTGAG GCGCCGCTG A GGACT
  • FIG. 13 Table 4 presenting the oligonucleotide sequences used in genotyping and allelic expression imbalance (AEI) assays for ACE that employed primer extension technology, showing [SEQ ID NOs: 1-7, 24, 25, 32-33]. Underlined nucleotides were intentionally mismatched against the reference sequence. The original primer sequences [SEQ ID NOs: 1-7, 24, 25, 32-33] are validated assays. In addition, a multiplex assay was developed using the primers [SEQ ID NOs: 265-269].
  • AEI genotyping and allelic expression imbalance
  • the SNPs rs4357 and rs4363 were genotyped by a melting curve dissociation approach as previously described (2) with the following primers:
  • rs4363 forward primer CTGCCCCGCACCCTTG [SEQ ID NO: 26] rs4363 reverse primer G allele CCTTCTGAGCGAGCTGTGC; [SEQ ID NO: 27] rs4363 reverse primer A allele wih GC clamp GGCGGCCGGCCCGCCCCGCCTTCT [SEQ ID NO: 28] GAGCGAGCTGCGT; rs4357 reverse primer TGACTTGAGGGAGGGTCCCT; [SEQ ID NO: 29] rs4357 forward primer C allele GCAGGAGAATGGTTCC; [SEQ ID NO: 30] rs4357 reverse primer T allele with GC clamp CGGGCCGCCGGGCCGCGCGGCAG [SEQ ID NO: 31] GAGAATGGGGTACT.
  • the INternational VErapamil SR Trandolapril STudy evaluated blood pressure and cardiovascular adverse outcomes occurring with either an atenolol or verapamil SR hypertension treatment strategy in 22,576 patients with documented coronary artery disease (CAD) and hypertension (19). Race/ethnicity was based on patient self-report and interaction with the site investigator, choosing all that were applicable among: Caucasian, African-American, Asian, Hispanic, and “other”. Hispanic patients were defined as those who chose only ‘Hispanic’. Patients were seen every six weeks for six months and every six months thereafter until two years after the last patient was enrolled.
  • CAD coronary artery disease
  • AIMs autosomal ancestry informative markers
  • the AIMs were selected to be distributed across the genome and to be distantly interspaced to give independent association with the disease and genetic background.
  • These 87 AIMs were genotyped using either allele-specific PCR with universal energy transfer labeled primers or competitive allele specific PCR at Prevention Genetics (Marshfield, Wis.) (33). Results from this analysis were used in the adjusted genetic association analysis.
  • FIG. 12 shows Table 3 presenting the baseline characteristics for the INVEST-GENES case and control patients.
  • FIG. 14 shows Table 5 presenting the oligonucleotide sequences employed in genotyping ACE SNPs by the GC-clamp method described in Papp et al, showing [SEQ ID NOs: 35-43].
  • FIG. 15 shows Table 6 presenting the oligonucleotides sequences employed in ACE Pyrosequencing genotyping, showing [SEQ ID NOs: 16-18].
  • FIG. 16 shows Table 7 presenting the oligonucleotide primers used in the amplification and direct sequencing of the ACE upstream gene region and cDNA, showing [SEQ ID NOs: 45-69].
  • FIG. 17 shows Table 8 presenting the FAM-labeled oligos and related oligos used in genotyping ACE polymorphisms, ⁇ showing [SEQ ID NOs: 15-16, 20-23, 44].
  • FIG. 18 shows Table 9 presenting the oligonucleotides used in the measurement of ACE expression by RT-PCR, including one that spans cDNA exons, showing [SEQ ID NOs: 8-9].
  • ACE1 renin-angiotensin receptor blockers
  • allelic expression imbalance i.e., a different number or type of mRNAs generated between alleles, is a robust and quantitative phenotype directly linked to cis-acting polymorphisms [3,5,8-21] and epigenetic regulation, including X-inactivation, imprinting, and gene silencing [4,22,23].
  • Also disclosed herein is a method for the rapid detection of regulatory polymorphisms in multiple genes.
  • the method described herein is a robust and fast methodology that is especially applicable to human autopsy tissues.
  • the method described herein fills an important gap between large-scale candidate gene discovery and resolution of the functional variants.
  • the assay relies on PCR/RT-PCR amplification, followed by a primer extension step with fluorescently labeled dideoxynucleotides, and analysis by capillary electrophoresis. Details of the assays applied to single genes have been published previously by us for several genes included in the present survey [9-16].
  • the method described herein includes several steps for obtaining reproducible allelic gDNA and mRNA ratios, including use of multiple gene-specific primers to maximize cDNA yields for the target genes.
  • Assay throughput is ⁇ 150 samples/hour, or higher with multiplexing, with an error rate in the order of 5% (gDNA) and 10-15% (mRNA).
  • AEI analysis was applied to 42 candidate genes in a variety of human tissues (FIG. 26 —Table 13), divided into genes for cardiovascular and CNS disorders, and drug metabolism and transport.
  • FIG. 27 Table 14 contains results for genes meeting the detection threshold in at least one sample, along with information on the marker SNPs, number of replicate analyses, frequency, magnitude and direction of AEI. If a suspected functional polymorphism is in near complete linkage disequilibrium with the marker SNP, most or all AEI ratios are unidirectional (either ⁇ 1 or >1), as observed with SOD2 in heart tissues ( FIG. 26 ).
  • the AEI data confirm previous studies, for example, the modest AEI ratios observed for COMT [17], and a similar frequency and extent of AEI for NQO2 in white blood cells [26] and DTNBP1 in the pons region [29].
  • AEI may be detectable only in certain ethnogeographic populations where regulatory alleles are sufficiently frequent (see ACE below), or in specific tissues, environmental conditions, and diseases. For example, AEI was observed for VKORC1 only in the liver but was undetectable in heart tissues and B-lymphoblasts (CEPH samples) (FIG. 27 —Table 14).
  • AEI analysis was applied to 18 cardiovascular candidate genes that serve as drug targets and have roles in inflammation, coagulation, lipid metabolism, vasomotor tone, and heart contractility (FIG. 26 —Table 13).
  • Target tissues included 65 heart failure explants from transplant recipients, livers, ex vivo monocytes, and peripheral blood monocyte-derived macrophages.
  • AEI was detectable for 15 cardiovascular genes at a 20% imbalance threshold (FIG. 26 —Table 13), while 9 genes displayed AEI when we set our more stringent threshold based on the typical error rates ( ⁇ log 2 0.5).
  • AEI ratios for genes surveyed in heart tissues are shown in FIG. 27 —Table 14.
  • CCL2, PTGDS, and KCNMB 1 showed allelic ratios below and above 1, suggesting multiple functional polymorphisms and/or incomplete linkage disequilibrium between the marker SNPs and functional alleles ( FIG. 23 ).
  • ACE displayed large unidirectional AEI ratios only in African-Americans, suggesting the presence of a cis-acting factor enriched in this population.
  • AEI ratios The frequency and directionality of AEI ratios enables us to investigate whether previously proposed regulatory polymorphisms in NOS3 (rs2070744), CCL2 (rs1024611), SOD2 (rs5746091), PTGDS (rs6926), and ACE (intron 16 VD) contribute to this phenotype.
  • FIG. 32 contains the mRNA sequence for the SOD2 gene[SEQ ID NO: 262]. Allelic mRNA ratios for SOD2 were ⁇ 1.5-fold in 83% of heart tissues heterozygous for marker rs4880, indicating that the ‘major allele’ has ⁇ 50% greater expression (however, since allele frequency is close to 50% assignment of the minor allele is arbitrary).
  • FIG. 19 Table 10 shows the forward PCR primer, the reverse PCR primer and the extension primer for rs4880 and rs5746092, showing [SEQ ID NOs: 79-84].
  • rs5746092 (37% heterozygosity) nor rs4880 (52% heterozygosity) were completely associated with AEI, as several homozygotes or heterozygotes displayed significant or no AEI, respectively.
  • each subject showing discrepancy between AEI and SNP heterozygosity is informative and, thus fails to support a putative functional role for that SNP.
  • SOD2 allelic expression was consistently in a single direction in such a high proportion (>80%) of samples, the inventors now believe that the SOD2 marker SNPs might have a direct, functional effect on expression. Thus, the inventors further analyzed the predicted allelic effects on mRNA folding for the marker SNPs in SOD2 (rs4880, rs5646092).
  • SNPs are in regions that display highly stable structures, with rs5646092 positioned within an 18 bp helix near the transcription and translation initiation sites. These results suggest that one or more of these alleles could affect gene expression through a change in mRNA structure.
  • AEI allelic gene expression
  • the AEI analysis can be scaled up to address multiple genes at a time, and thus, represents an intermediate tool for discovering functional polymorphisms affecting gene regulation (rSNPs) and RNA processing (srSNPs) in candidate genes.
  • rSNPs gene regulation
  • srSNPs RNA processing
  • the effect of rSNPs and srSNPs is expected to vary with the cellular environment, so that studies on human genes in physiologically relevant target tissues are of critical importance, for example the pontine brainstem for SERT and TPH2 mRNA [11,13].
  • Factors other than rSNPs and srSNPs could contribute to AEI, including variable copy number (CNV) in germline DNA or more frequently as somatic mutations in cancer [35].
  • CNV variable copy number
  • complete loss of one allele in germline DNA at the marker SNP locus cannot be assessed with the SNaPshot method as presented because hemizygous carriers would appear as homozygotes, unless the gene dosage is quantitated.
  • the method described herein permits an estimation of the prevalence of cis-acting polymorphisms in multiple (in the example herein, 42) candidate genes in human target tissues, a larger, more diverse sampling than previous studies.
  • FIG. 27 —Table 14 provides information on the magnitude, direction, and frequency of AEI, as guides for more detailed studies.
  • Substantial AEI (>log 2 0.5) in more than one subject was observed for 55% of the surveyed genes (FIG. 27 —Table 14), similar to previous studies [2,3,25]; however, the frequency is higher than estimates from other studies performed with a random selection of genes in cell lines and blood cells [24,26].
  • These differences may be attributable to the selection of strong candidate genes, or differences in methodology, tissue specificity, number of subjects, and stringency of AEI thresholds.
  • the presence of frequent AEI was unexpected for some of the candidate genes that had already been intensely studied for genetic polymorphisms (e.g., SOD2, ACE, TPH2 [13], DRD2 [16]).
  • allelic mRNA expression can be performed after specific amplification of each splice variant, as we have demonstrated for DRD2 (intron 5 and 6 SNPs alter formation of D2S and D2L) [16].
  • AEI patterns provide a means of determining the location of the functional polymorphism by SNP scanning or sequencing the gene locus, followed by molecular genetic analysis of the rSNP or srSNPs, as shown for OPRM1, MDR1, MAOA, SERT, TPH2, and DRD2 [9-13,16].
  • Reporter gene assays in heterologous tissues are commonly used to characterize regulatory polymorphisms. If these polymorphisms are functional in vivo, one expects corresponding changes in the AEI ratios. However, for the five genes tested (FIG. 26 —Table 13) we have failed to detect significant linkage between the observed AEI ratios and the putative regulatory SNPs. Similarly, our genotype scanning with AEI did not support a role for a putative SERT promoter polymorphism (SERT-LPR), although we cannot rule out that this promoter polymorphism might be active in development, or under stress [11]. Previously suggested regulatory polymorphisms in DRD2 also failed to correlate with AEI ratios [16].
  • srSNPs Structural RNA SNPs
  • srSNPs could alter mRNA function through changed folding dynamics [15,16,34].
  • Mfold to predict mRNA structural changes resulting from systematic nucleotide exchanges in opioid receptor mRNAs ( FIG. 25 )
  • SNPs can be detected by a physical method based on ‘single-strand conformational polymorphisms’, with a 95% discovery rate.
  • srSNPs can further affect translation, as suggested for the OPRM1 SNP A118G [16], and COMT haplotypes with altered mRNA folding [38]. Measuring AEI ratios at the protein level with use of nonsynonymous marker SNPs can allow for the determination of quantitative effects of polymorphisms on translation and protein turnover.
  • SOD2 mitochondrial manganese superoxide dismutase
  • SOD2 mitochondrial manganese superoxide dismutase
  • the promoter SNP rs5746091 did not appear to play a main role. Previous studies have implicated structural elements in SOD2 expression, including a GC-rich 5′ region upstream of the transcription start site that also extends into the 5′ end of the transcript [44] and regions in the 3′UTR of the mRNA [45]. Highly favorable RNA structures exist in the region of rs5746092 and rs4880 suggesting multiple structural states in SOD2 mRNA could affect functions. Alternatively, epigenetic regulation of SOD2 expression by CpG methylation [46] could have contributed to AEI, but our initial results argue against this possibility.
  • the measured AEI ratios clearly demonstrate functional variation of SOD2 mRNA expression.
  • FLT1, HIF1A, HMOX1, and LPL did not display common and large AEI.
  • the studied candidate genes all have important physiological roles, even relatively small AEI ratios, as observed for CCL2, NOS3, FLT1, HIF 1A, HMOX1, HMGCR, and LPL, may be of clinical importance [35].
  • Even a small activity change of a critical gene such as HMGCR could affect cholesterol production over an individual's lifetime.
  • pravastatin response was associated with two intronic SNPs in HMGCR, with frequency >5% in the population [47], and a genome-wide association study for LDL cholesterol also revealed an association with an intronic HMGCR SNP [48].
  • the inventors have applied mRNA AEI analysis to the detection of cis-acting variation for many candidate genes, revealing many instances of yet unrecognized functional polymorphisms or other cis-acting factors.
  • the AEI methodology can be applied on a fairly large scale while maintaining high accuracy.
  • EBV-transformed B-lymphoblast cell lines were obtained from the Coriell cell repository, consisting of 30 Caucasian family trios. A majority of the tissues are from normal subjects, while some tissues included subjects diagnosed with schizophrenia, bipolar disorder, Alzheimer's disease, and cancer. Ethnic distributions varied between tissues repositories; no attempt was made to cover ethnic groups evenly. The objective of this study was to detect functional polymorphisms with allele frequencies of 5% or more.
  • Genomic DNA and RNA were prepared from peripheral lymphocytes, or B-lymphocyte pellets, and frozen tissue samples (brain, liver, etc) as described previously [9-16]. Monocytes and monocyte-derived macrophages were cultured as described [49]. For whole blood extractions, the buffy coat was harvested, then red cells were either lysed using ammonium chloride to yield a leukocyte pellet for RNA extraction, or red and white cells were lysed with a sucrose Triton solution, providing a nuclear pellet for DNA purification. Frozen tissue samples were pulverized under liquid nitrogen and portioned into aliquots for DNA and RNA extractions. DNA was prepared by digestion of the pellet or frozen powder with SDS and proteinase K followed by NaCl salting out of proteins.
  • RNA precipitates were dissolved in RNase-free water or Qiagen buffer, and then extracted using Qiagen RNeasy columns.
  • the procedure differs from earlier studies (e.g., [2]) by combining multiple gene-specific primers close to the marker SNP region for cDNA synthesis to compensate for mRNA degradation.
  • Accurate AEI analysis requires robust expression (RT-PCR cycle threshold 27 or less).
  • Selection criteria for a marker SNP were as follows: 1) location in the transcribed region, coding or non-coding, 2) high minor allele frequency (0.15-0.50), 3) position of marker SNPs preferably more than 20 by from exon boundaries so that the same set of primers for PCR amplification can be used in both DNA and RNA.
  • cDNA was generated from total RNA (1 ug) by Superscript II reverse transcriptase (Invitrogen). Because oligo-dT priming often fails in autopsy tissues, we used both oligo-dT and gene-specific oligonucleotide primers targeting a region immediately 3′ of the marker SNP (same oligonucleotide used for PCR). We have multiplexed up to 30 primers to permit 30 different AEI assays per cDNA preparation. Comparisons between single and multiple primers showed no significant differences where tested. cDNA was successfully extracted from autopsy tissues to yield reproducible results between independent cDNA preparations [9-16].
  • RNA levels for each candidate gene in each tissue or cell line were determined using RT-PCR, to assure that expression is sufficient for accurate AEI analysis (cycle thresholds equal to or below 27 cycles).
  • Primers used for RT-PCR were the same as those selected for the AEI analysis, with PCR conditions optimized for each primer pair on an ABI7000 cycler with SYBR-Green. Results were normalized to an internal standard ( ⁇ -actin or GAPDH).
  • SLC6A3 (encoding the dopamine transporter) (newly added gene) is associated with multiple mental disorders such as drug abuse, attention deficit disorder (ADHD/ADD), Parkinson disease, Tourette syndrome and Schizophrenia.
  • FIG. 33 contains the mRNA sequence for the SLC6A3 gene [SEQ ID NO: 263].
  • Stimulant medications such as those used to treat ADHD, and drugs of abuse such as amphetamine bind to SLC6A3 and inhibit reuptake of dopamine.
  • Genetic variants of SLC6A3 may influence levels of gene expression and/or ability of drugs to bind to SLC6A3 protein.
  • rs6347 a synonymous SNP in Exon (rs6347) is associated with higher mRNA expression in both brain tissue and in a heterologous cell culture system. This is the first functional SNP occurring at high frequency in this key gene.
  • Polymorphisms in SLC6A3 are now believed by the inventors herein to be useful as biomarkers in numerous diseases and treatment outcomes including but not restricted to mental disorders and specifically drug addiction.
  • Dopamine transporter is associated with multiple mental disorders such as drug abuse, attention deficit disorder (ADHD/ADD), Parkinson disease, Tourette syndrome and Schizophrenia (for review: see Bannon, 2005 and Sotnikova et al, 2006).
  • Stimulant medications such as those used to treat ADHD, and drugs of abuse such as amphetamine bind to SLC6A3 and inhibit reuptake of dopamine.
  • Genetic variants of SLC6A3 may influence levels of gene expression and/or ability of drugs to bind to SLC6A3 protein.
  • One SNP has been found to be significantly associated with inattention and hyperactivity/impulsivity in children with ADHD.
  • SLC6A3 SNP rs27072 located in the 3′UTR is associated with AEI in brain tissue.
  • An additional synonymous SNP in Exon 9 rs6347 (not linked to rs27072) is associated with AEI in both brain tissue and cell culture. See FIG. 20 Table 11 which shows the sequences for rs27072 and rs6347, showing [SEQ ID NOs: 85-85].
  • FIG. 30 Table 17 shows the forward primer, the reverse primer and the extension primer for rs6437 [SEQ ID NOs: 87-89].
  • the rs6347 biomarker is based on molecular genetics and function. Also the frequency and penetrance are measurable by AEI. In addition, the combined use of two frequent functional polymorphisms can be used to assess disease risk and response to therapy (e.g., SSRIs).
  • CYP2C9 (encoding cytochrome P450 2C9) is a liver drug metabolizing enzyme, involved in metabolism of ⁇ 20% of pharmaceuticals.
  • FIG. 34 contains the mRNA sequence for the CYP2C9 gene [SEQ ID NO: 264].
  • SNPs The most common functional SNPs are CYP2C9*2 (430 C>T) and *3 (1075 A>C). These are non-synonymous SNPs with reduced enzyme activity (*2 50% and *3 25% of wild-type allele).
  • SNP 1425 A>T Described herein is a novel functional SNP, 1425 A>T, which is associated with 20-50% increased in mRNA level in human liver tissues, suggesting a “gain of function”.
  • the frequency of SNP 1425 A>T is ⁇ 4% but may vary significantly in different populations. Because it represents a gain of function (dominant effect), a 4% frequency is pharmacologically relevant.
  • SNP 1425 A>T is in partial linkage disequilbrium with *3 (and hence may affect 8# activity), but is never to link to *2 in >liver tissues.
  • CYP2C9 Polymorphisms in CYP2C9 can be useful as biomarkers in optimizing drug treatment for personalized medicine. It is noted that CYP2C9*2 and *3 already comprise a drug biomarker test, FDA approved and commercialized. See FIG. 21 Table 12 which shows the sequence for sr1057911 [SEQ ID NO: 90].
  • FIG. 30 Table 17 shows the forward primer, the reverse primer and the extension primer for rs9332242 and rs2017319 [SEQ ID NOs: 91-96].

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US12/598,268 2007-04-30 2008-04-30 Polymorphisms in Genes Affecting CYP2C9-Related Disorders and Uses Thereof Abandoned US20100129818A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/598,268 US20100129818A1 (en) 2007-04-30 2008-04-30 Polymorphisms in Genes Affecting CYP2C9-Related Disorders and Uses Thereof

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US92693207P 2007-04-30 2007-04-30
PCT/US2008/005539 WO2008136988A2 (fr) 2007-04-30 2008-04-30 Polymorphismes géniques affectant les troubles associés à cyp2c9 et utilisations
US12/598,268 US20100129818A1 (en) 2007-04-30 2008-04-30 Polymorphisms in Genes Affecting CYP2C9-Related Disorders and Uses Thereof

Publications (1)

Publication Number Publication Date
US20100129818A1 true US20100129818A1 (en) 2010-05-27

Family

ID=39943820

Family Applications (3)

Application Number Title Priority Date Filing Date
US12/598,268 Abandoned US20100129818A1 (en) 2007-04-30 2008-04-30 Polymorphisms in Genes Affecting CYP2C9-Related Disorders and Uses Thereof
US12/598,265 Active 2030-04-30 US9012143B2 (en) 2007-04-30 2008-04-30 Polymorphisms in genes affecting ace-related disorders and uses thereof
US12/598,266 Abandoned US20100143921A1 (en) 2007-04-30 2008-04-30 Polymorphisms in Genes Affecting Dopamine Transporter Disorders and Uses Thereof

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/598,265 Active 2030-04-30 US9012143B2 (en) 2007-04-30 2008-04-30 Polymorphisms in genes affecting ace-related disorders and uses thereof
US12/598,266 Abandoned US20100143921A1 (en) 2007-04-30 2008-04-30 Polymorphisms in Genes Affecting Dopamine Transporter Disorders and Uses Thereof

Country Status (3)

Country Link
US (3) US20100129818A1 (fr)
EP (2) EP2148933A4 (fr)
WO (4) WO2008136988A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100143921A1 (en) * 2007-04-30 2010-06-10 The Ohio State University Research Foundation Polymorphisms in Genes Affecting Dopamine Transporter Disorders and Uses Thereof
US9938576B1 (en) 2012-09-21 2018-04-10 Ohio State Innovation Foundation Materials and methods for determining metabolizer status in humans
US20210158892A1 (en) * 2017-10-02 2021-05-27 Blackthorn Therapeutics, Inc. Methods and tools for detecting, diagnosing, predicting, prognosticating, or treating a neurobehavioral phenotype in a subject
US11213543B2 (en) 2011-08-17 2022-01-04 The Nature's Bounty Co. Human gastrointestinal microbiome modulating composition and methods of use

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ572036A (en) 2008-10-15 2010-03-26 Nikola Kirilov Kasabov Data analysis and predictive systems and related methodologies
AU2010229772A1 (en) * 2009-03-26 2011-10-27 The Ohio State University Research Foundation Polymorphism in CYP3A4 gene affecting drug metabolizing and uses thereof
JP6216486B2 (ja) 2010-08-24 2017-10-18 ザ・チルドレンズ・ホスピタル・オブ・フィラデルフィアＴｈｅ Ｃｈｉｌｄｒｅｎ’Ｓ Ｈｏｓｐｉｔａｌ Ｏｆ Ｐｈｉｌａｄｅｌｐｈｉａ 低頻度反復性遺伝的変異の注意欠陥多動性障害への関連ならびに診断および治療のためのその使用方法
JP5984822B2 (ja) 2010-10-21 2016-09-06 メイヨ・ファウンデーション・フォー・メディカル・エデュケーション・アンド・リサーチ 注意欠陥多動性障害を有する患者を処置するための薬物を選択する方法
WO2012173809A2 (fr) * 2011-06-02 2012-12-20 Ehli Erik Procédé d'identification de variantes du nombre de nouvelles copies (cnv) à l'aide de jumeaux mz discordants pour des problèmes/troubles de l'attention
US11219617B2 (en) 2014-05-30 2022-01-11 The Children's Hospital Of Philadelphia Methods of diagnosing and treating autism
US10395759B2 (en) 2015-05-18 2019-08-27 Regeneron Pharmaceuticals, Inc. Methods and systems for copy number variant detection
EP4023222B1 (fr) 2015-09-08 2024-12-18 The Children's Hospital Of Philadelphia Activateur du récepteur métabotropique du glutamate non sélectif pour le traitement du syndrome 22q
KR101712463B1 (ko) * 2015-12-14 2017-03-06 대한민국 혈압강하제 및 동맥 경화용제 대사효소 관련 유전자 snp 지노타이핑 시약, 이를 포함하는 키트 및 유전자형 분석방법
CN109074426B (zh) 2016-02-12 2022-07-26 瑞泽恩制药公司 用于检测异常核型的方法和系统
US11227690B1 (en) 2020-09-14 2022-01-18 Opendna Ltd. Machine learning prediction of therapy response

Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5882944A (en) * 1993-06-23 1999-03-16 The Regents Of The University Of California Methods for G protein coupled receptor activity screening
US6007986A (en) * 1993-06-23 1999-12-28 The Regents Of The University Of California Methods for anti-addictive narcotic analgesic activity screening
US6228840B1 (en) * 1998-02-27 2001-05-08 Edward T. Wei Melanocortin receptor antagonists and modulations of melanocortin receptor activity
US6270979B1 (en) * 1993-06-23 2001-08-07 The Regents Of The University Of California Methods for anti-addictive narcotic analgesic treatments
US20010034023A1 (en) * 1999-04-26 2001-10-25 Stanton Vincent P. Gene sequence variations with utility in determining the treatment of disease, in genes relating to drug processing
US20010049375A1 (en) * 2000-03-15 2001-12-06 Wolfgang Sadee Neutral antagonists and use thereof in treating drug abuse
US20030059774A1 (en) * 2000-08-30 2003-03-27 Carl Risinger Detection of CYP2C19 polymorphisms
US20030148295A1 (en) * 2001-03-20 2003-08-07 Wan Jackson Shek-Lam Expression profiles and methods of use
US20040115701A1 (en) * 2002-08-30 2004-06-17 Comings David E Method for risk assessment for polygenic disorders
US6812339B1 (en) * 2000-09-08 2004-11-02 Applera Corporation Polymorphisms in known genes associated with human disease, methods of detection and uses thereof
US20050026169A1 (en) * 2002-12-20 2005-02-03 Applera Corporation Genetic polymorphisms associated with myocardial infarction, methods of detection and uses thereof
US20050208512A1 (en) * 2003-10-01 2005-09-22 The Ohio State University Research Foundation Determining the chemosensitivity of cells to cytotoxic agents
US20050272054A1 (en) * 2003-11-26 2005-12-08 Applera Corporation Genetic polymorphisms associated with cardiovascular disorders and drug response, methods of detection and uses thereof
US20060160074A1 (en) * 2001-12-27 2006-07-20 Third Wave Technologies, Inc. Pharmacogenetic DME detection assay methods and kits
US20070065821A1 (en) * 2003-02-21 2007-03-22 Sridhar Kudaravalli Methods for the prediction of suicidality during treatment
US20070197573A1 (en) * 2005-10-04 2007-08-23 Wolfgang Sadee Compositions and methods in the treatment of bone metabolic disorders
US20090111844A1 (en) * 2007-10-18 2009-04-30 Aiko Biotechnology Combination analgesic employing opioid and neutral antagonist
US20100075308A1 (en) * 2006-08-01 2010-03-25 The Ohio State University Research Foundation Polymorphisms in genes affecting cns disorders and uses thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6582908B2 (en) * 1990-12-06 2003-06-24 Affymetrix, Inc. Oligonucleotides
US6197505B1 (en) * 1997-04-04 2001-03-06 Pyrosequencing Ab Methods for assessing cardiovascular status and compositions for use thereof
CA2568729A1 (fr) * 2004-06-04 2005-12-15 Novartis Ag Biomarqueurs de prediction de la reactivite au traitement a la clozapine
WO2008136988A2 (fr) 2007-04-30 2008-11-13 The Ohio State University Research Foundation Polymorphismes géniques affectant les troubles associés à cyp2c9 et utilisations

Patent Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5882944A (en) * 1993-06-23 1999-03-16 The Regents Of The University Of California Methods for G protein coupled receptor activity screening
US6007986A (en) * 1993-06-23 1999-12-28 The Regents Of The University Of California Methods for anti-addictive narcotic analgesic activity screening
US6270979B1 (en) * 1993-06-23 2001-08-07 The Regents Of The University Of California Methods for anti-addictive narcotic analgesic treatments
US6228840B1 (en) * 1998-02-27 2001-05-08 Edward T. Wei Melanocortin receptor antagonists and modulations of melanocortin receptor activity
US6953779B2 (en) * 1998-02-27 2005-10-11 The Regents Of The University Of California Melanocortin receptor antagonists and modulations of melanocortin receptor activity
US20010034023A1 (en) * 1999-04-26 2001-10-25 Stanton Vincent P. Gene sequence variations with utility in determining the treatment of disease, in genes relating to drug processing
US20010049375A1 (en) * 2000-03-15 2001-12-06 Wolfgang Sadee Neutral antagonists and use thereof in treating drug abuse
US6713488B2 (en) * 2000-03-15 2004-03-30 Sadee Wolfgang Neutral antagonists and use thereof in treating drug abuse
US20030059774A1 (en) * 2000-08-30 2003-03-27 Carl Risinger Detection of CYP2C19 polymorphisms
US6812339B1 (en) * 2000-09-08 2004-11-02 Applera Corporation Polymorphisms in known genes associated with human disease, methods of detection and uses thereof
US20030148295A1 (en) * 2001-03-20 2003-08-07 Wan Jackson Shek-Lam Expression profiles and methods of use
US20060160074A1 (en) * 2001-12-27 2006-07-20 Third Wave Technologies, Inc. Pharmacogenetic DME detection assay methods and kits
US20040115701A1 (en) * 2002-08-30 2004-06-17 Comings David E Method for risk assessment for polygenic disorders
US20050026169A1 (en) * 2002-12-20 2005-02-03 Applera Corporation Genetic polymorphisms associated with myocardial infarction, methods of detection and uses thereof
US20070065821A1 (en) * 2003-02-21 2007-03-22 Sridhar Kudaravalli Methods for the prediction of suicidality during treatment
US20050208512A1 (en) * 2003-10-01 2005-09-22 The Ohio State University Research Foundation Determining the chemosensitivity of cells to cytotoxic agents
US20050272054A1 (en) * 2003-11-26 2005-12-08 Applera Corporation Genetic polymorphisms associated with cardiovascular disorders and drug response, methods of detection and uses thereof
US20070197573A1 (en) * 2005-10-04 2007-08-23 Wolfgang Sadee Compositions and methods in the treatment of bone metabolic disorders
US20100075308A1 (en) * 2006-08-01 2010-03-25 The Ohio State University Research Foundation Polymorphisms in genes affecting cns disorders and uses thereof
US20090111844A1 (en) * 2007-10-18 2009-04-30 Aiko Biotechnology Combination analgesic employing opioid and neutral antagonist

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
dbSNP database partial entry for rs2017319, downloaded 10 August 2012 from www.ncbi.nlm.nih.gov/projects/SNP; 2 pages. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100143921A1 (en) * 2007-04-30 2010-06-10 The Ohio State University Research Foundation Polymorphisms in Genes Affecting Dopamine Transporter Disorders and Uses Thereof
US20100167947A1 (en) * 2007-04-30 2010-07-01 The Ohio State University Research Foundation Polymorphisms in Genes Affecting Ace-Related Disorders and Uses Thereof
US9012143B2 (en) 2007-04-30 2015-04-21 The Ohio State University Research Foundation Polymorphisms in genes affecting ace-related disorders and uses thereof
US11213543B2 (en) 2011-08-17 2022-01-04 The Nature's Bounty Co. Human gastrointestinal microbiome modulating composition and methods of use
US9938576B1 (en) 2012-09-21 2018-04-10 Ohio State Innovation Foundation Materials and methods for determining metabolizer status in humans
US10991450B2 (en) 2012-09-21 2021-04-27 Ohio State Innovation Foundation Materials and methods for determining metabolizer status in humans
US20210158892A1 (en) * 2017-10-02 2021-05-27 Blackthorn Therapeutics, Inc. Methods and tools for detecting, diagnosing, predicting, prognosticating, or treating a neurobehavioral phenotype in a subject
US11791016B2 (en) 2017-10-02 2023-10-17 Neumora Therapeutics, Inc. Methods and systems for computer-generated predictive application of neuroimaging and gene expression mapping data
US11842793B2 (en) * 2017-10-02 2023-12-12 Neumora Therapeutics, Inc. Methods and tools for detecting, diagnosing, predicting, prognosticating, or treating a neurobehavioral phenotype in a subject

Also Published As

Publication number Publication date
US20100167947A1 (en) 2010-07-01
EP2149047A4 (fr) 2010-05-19
WO2008136996A2 (fr) 2008-11-13
EP2148933A4 (fr) 2010-05-19
WO2008136989A2 (fr) 2008-11-13
EP2149047A2 (fr) 2010-02-03
WO2008136996A3 (fr) 2009-01-22
WO2008136988A2 (fr) 2008-11-13
WO2008136995A1 (fr) 2008-11-13
WO2008136988A3 (fr) 2009-01-22
US9012143B2 (en) 2015-04-21
US20100143921A1 (en) 2010-06-10
WO2008136989A3 (fr) 2009-02-26
EP2148933A1 (fr) 2010-02-03

Similar Documents

Publication Publication Date Title
US9012143B2 (en) Polymorphisms in genes affecting ace-related disorders and uses thereof
US20200270692A1 (en) Predicting age-related macular degeneration with single nucleotide polymorphisms within or near the genes for complement component c2, factor b, plekha1, htra1, prelp, or loc387715
US20260022420A1 (en) Genomic alterations associated with schizophrenia and methods of use thereof for the diagnosis and treatment of the same
US20100075308A1 (en) Polymorphisms in genes affecting cns disorders and uses thereof
US20210395823A1 (en) Genetic markers associated with asd and other childhood developmental delay disorders
Garcia-Martínez et al. Gene-wide association study reveals RNF122 ubiquitin ligase as a novel susceptibility gene for attention deficit hyperactivity disorder
US8137916B2 (en) Susceptibility gene for alzheimer's disease
US20110035818A1 (en) Diagnostic marker and platform for drug design in myocardial infarction and heart failure
US7935485B2 (en) Genetic polymorphisms for identifying individuals at risk for drug-induced vestibular dysfunction
US20130109589A1 (en) Single nucleotide polymorphisms associated with amyotrophic lateral sclerosis
KR101092580B1 (ko) 위암 감수성 예측용 ｖｃａｎ 다형성 마커 및 이를 이용한위암 감수성 예측 방법
US9909182B1 (en) Methods for identifying subjects susceptible to charcot-marie-tooth neuropathy type 1C
US20120289593A1 (en) Methods for risk assessment, treating, and diagnosing myocardial infarction

Legal Events

Date Code Title Description
AS Assignment

Owner name: THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION, OHI

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SADEE, WOLFGANG;WANG, DANXIN;REEL/FRAME:023725/0227

Effective date: 20091123

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION