WO2015168252A1 - Mitochondrial dna copy number as a predictor of frailty, cardiovascular disease, diabetes, and all-cause mortality - Google Patents

Abstract

The present invention provides methods and compositions related to the use of mitochondrial DNA copy number as a predictor of frailty, cardiovascular disease, diabetes and all-cause mortality. In one embodiment, a method for predicting cardiovascular disease in a subject comprises the steps of (a) obtaining a biological sample from the subject; (b) performing an assay to measure the mitochondrial DNA (mtDNA) copy number in the biological sample; and (c) identifying the subject as likely to develop cardiovascular disease by comparison of the mitochondrial DNA copy number to a control.

Description

MITOCHONDRIAL DNA COPY NUMBER AS A PREDICTOR OF FRAILTY, CARDIOVASCULAR DISEASE, DIABETES, AND ALL-CAUSE MORTALITY

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61/985,764, filed April 29, 2014, which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no. P30-AG021334, awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE FNVENTION

The present invention provides methods and compositions related to the use of mitochondrial DNA copy number as a predictor of frailty, cardiovascular disease, diabetes, and all-cause mortality.

BACKGROUND OF THE FNVENTION

Age-related declines in mitochondrial function have long been hypothesized to underlie multiple biological changes that increase vulnerability to multiple disease states, functional and cognitive decline, and ultimately, mortality. The mechanisms contributing to age-related mitochondrial functional change encompass multiple domains, including declines in energy (ATP) production, increased free radical production, altered rates of apoptosis and mitophagy, and altered fusion/fission. Alterations in these crucial intracellular processes lead to dysfunctional cells, altered tissues, and increased risk of disease states. The evidence for the age-related changes in mitochondrial function leading to altered phenotypes and disease states is bolstered by the observation that mice with deficiency of the proofreading mechanism of the mitochondrial polymerase display a premature aging phenotype and that mitochondrial dysfunction is a core component of several neurodegenerative disorders in humans.

The role of mitochondrial DNA (mtDNA) in aging and late life decline has also been studied, with evidence to support the fact that mtDNA variants modulate risk of several age- associated diseases. Along these lines, we have previously implicated a specific

mitochondrial D loop variant in frailty, a clinical syndrome prevalent in older individuals, characterized by broad decline in resilience, and increased risk for disability and all-cause mortality. Given the role of the D-loop in mitochondrial replication, and hence, its possibility of affecting the levels of mitochondrial DNA, we hypothesized that mtDNA copy number is likely to play an important role in the aging process.

While the role of mitochondrial depletion in severe disorders, such as MDS (mtDNA depletion syndrome) is well established, the extent of normal variation in mitochondrial copy number and its effect on aging and mortality is less understood. Several studies have examined the correlation between age and mtDNA copy number with often ambiguous and conflicting results. To address this gap in the literature, we examined mtDNA copy number in two large multi-center prospective studies-the Cardiovascular Health Study (CHS) and the Atherosclerosis Risk in Communities (ARIC) study-in a total of 16,401 samples of European and African descent. We demonstrate that mtDNA copy number, ascertained from DNA isolated from lymphocytes, is associated with frailty, and is a significant predictor of all- cause mortality and cardiovascular disease (CVD).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery that mitochondrial DNA (mtDNA) copy number is associated with frailty, cardiovascular disease, diabetes, and all-cause mortality.

Mitochondrial function is altered with age and variants in mitochondrial DNA (mtDNA) have been shown to modulate risk for several age-related disease states. However, the effect of mtDNA copy number, which reflects energy reserves and oxidative stress, on aging and mortality in the general population has not been addressed.

The present inventors determined mtDNA copy number using a qPCR-based method in 4,892 participants from the Cardiovascular Health Study (CHS), and an array-based method in 11,509 participants from the Atherosclerosis Risk in Communities Study (ARIC). We examined the association between mtDNA copy number and two primary phenotypes- prevalent frailty and all-cause mortality.

MtDNA copy number was significantly associated with prevalent frailty in self- identified white participants from CHS (P<0.001). Additionally, mtDNA copy number was a strong independent predictor of all-cause mortality in an age and sex-adjusted, race-stratified analysis of 16,401 participants from both cohorts with a pooled hazard ratio of 1.47 (95% CI, 1.33-1.62, P<0.001) for the lowest quintile of mtDNA copy number relative to the highest quintile.

MtDNA copy number was strongly associated with prevalent frailty and was an independent predictor of all-cause mortality. Accordingly, in one aspect, the present invention provides methods for predicting mtDNA copy number with certain outcomes. In one embodiment, a method for predicting cardiovascular disease in a subject comprises the steps of (a) obtaining a biological sample from the subject; (b) performing an assay to measure the mitochondrial DNA (mtDNA) copy number in the biological sample; and (c) identifying the subject as likely to develop cardiovascular disease by comparison of the mitochondrial DNA copy number to a control. In certain embodiments, the assay comprises quantitative polymerase chain reaction (qPCR). In a specific embodiment, the assay comprises PCR amplifying a mitochondrial gene and a nuclear gene and determining the mtDNA copy number relative to nuclear DNA copy number. The mitochondrial gene can be one or more of NDI, ND2, ND3, ND4L, ND5, ND6, CYB COl C02, C03, ATP6, ATP8 and RNR2. The nuclear gene is one or more of RPPH1, tubulin, RPS18, HGB, B2M, and actin.

In another embodiment, a method for predicting diabetes in a subject comprises the steps of (a) obtaining a biological sample from the subject; (b) performing an assay to measure the mitochondrial DNA (mtDNA) copy number in the biological sample; and (c) identifying the subject as likely to develop diabetes by comparison of the mitochondrial DNA copy number to a control. In a specific embodiment, the assay comprises PCR amplifying a mitochondrial gene and a nuclear gene and determining the mtDNA copy number relative to nuclear DNA copy number. The mitochondrial gene can be one or more of NDI, ND2, ND3, ND4L, ND5, ND6, CYB COl C02, C03, ATP6, ATP8 and RNR2. The nuclear gene is one or more of RPPH1, tubulin, RPS18, HGB, B2M, and actin.

In particular embodiments, the methods of the present invention also predict frailty and all-cause mortality.

In other embodiments, mtDNA copy number can be calculated by extracting information from microarrays, in particular, genotyping arrays. In a specific embodiment, a method for predicting cardiovascular disease in a subject comprises the steps of (a) obtaining a biological sample from the subject; (b) extracting genomic DNA from the biological sample; (c) performing a genotyping assay using a microarray; (d) calculating mtDNA copy number based on the probe intensity of one or more mitochondrial SNPs; and (e) identifying the subject as likely to develop cardiovascular disease by comparison of the mitochondrial DNA copy number to a control. In another specific embodiment, a method for predicting diabetes in a subject comprises the steps of (a) obtaining a biological sample from the subject; (b) extracting genomic DNA from the biological sample; (c) performing a genotyping assay using a microarray; (d) calculating mtDNA copy number based on the probe intensity of one or more mitochondrial SNPs; and (e) identifying the subject as likely to develop diabetes by comparison of the mitochondrial DNA copy number to a control. In other embodiments, the calculation of mtDNA copy number and prediction of CVD, diabetes or other outcome described herein is performed on information previously extracted from microarray or sequencing analyses.

In another aspect, the present invention provides methods for managing treatment. In one embodiment, a method for managing treatment of a diabetes patient comprising the steps of (a) ordering a diagnostic test that measures mtDNA copy number from a DNA sample obtained from the diabetes patient; and (b) adjusting treatment for the diabetes patient based on a comparison of the measured mtDNA copy number against a control value. The present invention also provides a method for managing treatment of a CVD patient comprising the steps of (a) ordering a diagnostic test that measures mtDNA copy number from a DNA sample obtained from the CVD patient; and (b) adjusting treatment for the CVD patient based on a comparison of the measured mtDNA copy number against a control value.

In a further embodiment, a method for managing treatment of an elderly patient comprising the steps of (a) ordering a diagnostic test that measures mtDNA copy number from a DNA sample obtained from the patient; and (b) recommending, prescribing or administering a treatment regimen designed to prevent or treat frailty or symptoms thereof to the patient based on a comparison of the measured mtDNA copy number against a control value. In particular embodiments, the treatment regimen comprises one or more of medical treatment, nutritional supplementation and exercise.

In certain embodiments, the measured mtDNA copy number is statistically significantly lower than the control. The methods of the present invention can further comprise the steps of periodically measuring mtDNA copy number during the course of treatment and adjusting treatment based on the measured mtDNA copy number as compared to a control.

The present invention also provides a method comprising the step of recommending, prescribing or administering a cardiovascular disease (CVD) treatment to a patient having a mitochondrial DNA copy number that is statistically significantly lower than mtDNA copy number of a control. In another embodiment, a method comprises the step of recommending, prescribing or administering a diabetes treatment to a patient having a mitochondrial DNA copy number that is statistically significantly lower than mtDNA copy number of a control. In another specific embodiment, a method comprises the step of recommending, prescribing or administering a frailty treatment regimen to a patient having a mitochondrial DNA copy number that is statistically significantly lower than mtDNA copy number of a control. The methods of the present invention can further comprise monitoring mtDNA copy number levels during treatment to assess effectiveness and adjusting treatment accordingly.

In particular embodiments, the frailty treatment regimen can be a combination of interventions including medical, nutritional supplementation and exercise. Medical intervention can include, but it not limited to, HMG-CoA reductase inhibitors, angiotensin converting enzyme inhibitors (ACE) inhibitors, sex steroids (e.g., testosterone, estrogen or precursors thereof), growth hormones (replacement and/or supplementation), insulin like growth factor- 1, and the like. Nutritional supplementation can include, but is not limited to, vitamin D, carotenoids, creatine, dehydroepiandrosterone (DHEA), and beta-hydroxy-beta- methylbutyrate. Nutritional supplementation can also include increasing protein and/or omega-3 intake. Exercise intervention can include recommending or prescribing physical therapy, tai chi, walking, and resistance exercises.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Frailty components in CHS. Association between age, sex and collection site adjusted mitochondrial copy number and frailty components in study participants who self- identify as "white" (top panel) and "black" (bottom panel) from CHS. MtDNA copy number is expressed in terms of standard deviation units. Participants were scored as being at risk (1) or not at risk (0) for each characteristic of frailty. Overall frailty was scored in terms of number of characteristics that each participant was at risk for—robust 0 characteristics, pre- frail 1-2 characteristics and frail >2 characteristics.

FIG. 2. Survival curves. Kaplan-Meier estimates for all-cause mortality by quintile of mtDNA copy number were calculated for both race groups in CHS and ARIC. Inset table indicates the total number of people in the model at each time point.

FIG. 3. Meta-analysis of effects mtDNA copy number on mortality. Effects of highest copy quintile of copy number relative to lowest quintile from race stratified analyses in each cohort were meta-analyzed using an inverse-variance weighted approach.

FIG. 4. Effects of mtDNA copy number on cause-specific mortality. Hazards ratio reflect effect of lowest quintile of mtDNA relative to highest quintile on survival. Baseline models were adjusted for age, sex, and collection site. Heterogeneity between estimates of HR for subgroups of cause of death was evaluated using a random effects model. Diseases of the circulatory system were defined by ICD9 codes 390-459, neoplasms by 140-239 and diseases of the respiratory system by 460-519. FIG. 5. Ten-year cardiovascular disease (CVD) risk in white men, white women, black men and black women in the ARIC study.

FIG. 6A-6D. Model (mtDNA copy number+ACRS) classifies a significant proportion of people from high risk (>7.5% 10 year risk) to lower risk categories.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to the particular methods and components, etc., described herein, as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to a "protein" is a reference to one or more proteins, and includes equivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

All publications cited herein are hereby incorporated by reference including all journal articles, books, manuals, published patent applications, and issued patents. In addition, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present invention.

I. Definitions

Ranges may be expressed herein as from "about" one particular value, and/or to

"about" another particular value. The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term "about." "Complementary" refers to sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In certain embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In other embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

The "copy number of a gene" or the "copy number of a marker" refers to the number of DNA sequences in a cell encoding a particular gene product. Generally, for a given gene, a mammal has two copies of each gene. Copy numbers can be determined for either or both of a mitochondrial gene(s) and a nuclear gene(s).

The "normal" copy number of a marker is copy number of the marker in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, saliva, cerebrospinal fluid, urine, stool, bile, from a subject, e.g., a human, not afflicted with a disease or condition described herein including frailty, cardiovascular disease, and diabetes.

A "significantly lower copy number" of a mitochondrial DNA marker refers to a copy number in a test sample that is lower than the standard error of the assay employed to assess copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times lower than the copy number of the marker in a control sample (e.g., sample from a healthy subject not afflicted with a disease or condition described herein) and preferably, the average expression level or copy number of the marker in several control samples.

A "significantly higher copy number" of a mitochondrial DNA marker refers to a copy number in a test sample that is greater than the standard error of the assay employed to assess copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the copy number of the marker in a control sample (e.g., sample from a healthy subject not afflicted with a disease or condition described herein) and preferably, the average expression level or copy number of the marker in several control samples.

As used herein, "frailty" refers to an adverse, primarily gerontologic, health condition, which can include low functional reserve, accelerated osteoporosis, easy tiring, decreased muscle strength, high susceptibility to disease and decreased libido (e.g., see Bandeen-Roche et ah, The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61 : 262-266 (2006)). Frailty can be characterized by meeting three of the following five attributes: unintentional weight loss, muscle weakness, slow walking speed, exhaustion, and low physical activity.

The phrase "nucleic acid" as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single- stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.

Optional" or "optionally" means that the subsequently described event or

circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The terms "patient," "individual," or "subject" are used interchangeably herein, and refer to a mammal, particularly, a human. The patient may have a mild, intermediate or severe disease or condition. The patient may be an individual in need of treatment or in need of diagnosis based on particular symptoms or family history. In some cases, the terms may refer to treatment in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates. In particular, the term also includes mammals diagnosed with a cardiovascular disease and/or diabetes.

"Polypeptide" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules. In addition, as used herein, the term "polypeptide" refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc., and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer- R A mediated addition of amino acids to protein such as arginylation. See Proteins- Structure and Molecular Properties 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

By "probe," "primer," or oligonucleotide is meant a single-stranded DNA or RNA molecule of defined sequence that can base-pair to a second DNA or RNA molecule that contains a complementary sequence (the "target"). The stability of the resulting hybrid depends upon the extent of the base-pairing that occurs. The extent of base-pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions. The degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art. Probes or primers specific for nucleic acids (for example, genes and/or mRNAs) have at least 80%-90% sequence complementarity, preferably at least 91%-95% sequence complementarity, more preferably at least 96%-99% sequence complementarity, and most preferably 100% sequence complementarity to the region of the nucleic acid to which they hybridize. Probes, primers, and oligonucleotides may be detectably-labeled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art. Probes, primers, and oligonucleotides are used for methods involving nucleic acid hybridization, such as: nucleic acid sequencing, reverse transcription and/or nucleic acid amplification by the polymerase chain reaction, single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, electrophoretic mobility shift assay (EMS A).

The terms "sample," "patient sample," "biological sample," and the like, encompass a variety of sample types obtained from a patient, individual, or subject and can be used in a diagnostic or monitoring assay. The patient sample may be obtained from a healthy subject or a patient having symptoms associated with or is suspected of having or likely to have or develop a disease or condition described herein. Moreover, a sample obtained from a patient can be divided and only a portion may be used for diagnosis. Further, the sample, or a portion thereof, can be stored under conditions to maintain sample for later analysis. The definition specifically encompasses blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, serum, plasma, cord blood, amniotic fluid, cerebrospinal fluid, urine, saliva, stool and synovial fluid), solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. In certain embodiments, a sample comprises blood. In other embodiments, a sample comprises serum. In a specific embodiment, a sample comprises plasma.

The definition of "sample" also includes samples that have been manipulated in any way after their procurement, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washed, or enriched for certain cell populations. The terms further encompass a clinical sample, and also include cells in culture, cell supernatants, tissue samples, organs, and the like. Samples may also comprise fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or study by

immunohistochemistry.

"Statistically significant" means that the alteration is greater than what might be expected to happen by chance alone. Statistical significance can be determined by any method known in the art. For example, statistical significance can be determined by p-value. The p- value is a measure of probability that a difference between groups during an experiment happened by chance. For example, a P-value of 0.01 means that there is a 1 in 100 chance the result occurred by chance. The lower the P-value, the more likely it is that the difference between groups was caused by, e.g., treatment. An alteration is considered to be statistically significant if the P-value is at least 0.05. Preferably, the P-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 or less. Various methodologies of the instant invention include a step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control," referred to interchangeably herein as an "appropriate control," a "control sample" or a "reference." A "suitable control," "appropriate control," "control sample" or a "reference" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc., determined in a cell, organ, or patient, e.g., a control cell, organ, or patient, exhibiting, for example, a normal phenotype. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, ratio, etc. determined prior to performing a therapy (e.g., diabetes treatment) on a patient. In yet another embodiment, a mitochondrial copy number profile can be determined prior to, during, or after administering a therapy into a cell, organ, or patient. In a further

embodiment, a "suitable control," "appropriate control" or a "reference" is a predefined value, level, feature, characteristic, property, ratio, etc. A "suitable control" can be a profile or pattern of levels/ratios of mitochondrial DNA copy number that correlates to a disease or condition described herein, to which a patient sample can be compared. The patient sample can also be compared to a negative control.

As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease. In a specific embodiment, the disease or condition is cardiovascular disease. In particular embodiments, the disease or condition is diabetes. In further embodiments, the disease or condition is frailty.

As used herein, the term "effective," means adequate to accomplish a desired, expected, or intended result. More particularly, a "therapeutically effective amount" as provided herein refers to an amount of a treatment necessary to provide the desired therapeutic effect, e.g., an amount that is effective to prevent, alleviate, or ameliorate symptoms of disease or condition or prolong the survival of the subject being treated. In a particular embodiment, the disease or condition is cardiovascular disease. In particular embodiments, the disease or condition is diabetes. In further embodiments, the disease or condition is frailty. As would be appreciated by one of ordinary skill in the art, the exact amount required will vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, the particular compound and/or composition administered, and the like. An appropriate "therapeutically effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

The terms "specifically binds to," "specific for," and related grammatical variants refer to that binding which occurs between such paired species as antibody/antigen, enzyme/substrate, receptor/agonist, and lectin/carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. Thus, for example, an antibody typically binds to a single epitope and to no other epitope within the family of proteins. In some embodiments, specific binding between an antigen and an antibody will have a binding affinity of at least 10^~6 M. In other embodiments, the antigen and antibody will bind with affinities of at least 10^"7 M, 10^"8 M to 10^"9 M, 10^"10 M, 10^"11 M, or 10^"12 M.

By "specifically hybridizes" is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid under high stringency conditions, and does not substantially base pair with other nucleic acids.

By "high stringency conditions" is meant conditions that allow hybridization comparable with that resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHP0₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65°C, or a buffer containing 48% formamide, 4.8XSSC, 0.2 M Tris-Cl, pH 7.6, lXDenhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42°C Other conditions for high stringency hybridization, such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well-known by those skilled in the art of molecular biology. (See, for example, F. Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998).

In certain embodiments, DNA can be isolated from a biological sample taken from a subject. DNA can be extracted and purified from biological samples using any suitable technique. A number of techniques for DNA extraction and/or purification are known in the art, and several are commercially available (e.g., ChargeS witch®, MELT™ total nucleic acid isolation system, MagMAX™ FFPE total nucleic acid isolation kit, MagMAX™ total nucleic acid isolation kit, QIAamp DNA kit, Omni-Pure™ genomic DNA purification system, WaterMaster™ DNA purification kit). Reagents such as DNAzoI® and TR1 Reagent® can also be used to extract and/or purify DNA. DNA can be further purified using Proteinase K and/or RNAse.

In further embodiments, primers can be used to amplify a region of a mitochondrial DNA gene. In one embodiment, a mitochondrial gene that is used to determine

mitochondrial DNA (mtDNA) copy number is a single copy gene with low frequency of mutations and/or polymorphisms. MtDNA genes can include NADH dehydrogenase 1 (ND1), ND2, or ND6. Other genes can include ND3, ND4L, ND5, CYB (cytochrome B), COl (cytochrome x oxidase I), C02, C03, ATP 6 (ATP synthase F0 subunit 6), ATP8 and RNR2 (16S rRNA).

Primers can also be designed and used to amplify a region of a nuclear gene. In particular embodiments, the nuclear gene is non-repetitive with no known alternative splicing events. In a specific embodiment, the gene is ribonuclease PRNA component HI (RPPH1). Other nuclear genes can include tubulin, 40S ribosomal protein S18 (RPS18), human globulin (HGB), beta-2 -microglobulin (B2M), and actin.

In particular embodiments, a primer is contacted with isolated DNA from the subject under conditions such that the primer specifically hybridizes with the target gene

(mitochondrial or nuclear). The primer and DNA thus form a primer:DNA complex. In certain embodiments, the primer:DNA complex is amplified using polymerase chain reaction.

As described herein, in certain embodiments, the primers can used to support DNA amplification reactions. Typically the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription.

Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, such as PCR. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner.

The size of the primers for interaction with the mitochondrial and nuclear gene sequences in certain embodiments can be any size that supports the desired enzymatic manipulation of the primer, such as DNA amplification. . A typical primer or probe would be at least 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 nucleotides long or any length in-between.

The primers of the present invention can be prepared by conventional techniques well-known to those skilled in the art. For example, the primers can be prepared using solid- phase synthesis using commercially available equipment. Modified oligonucleotides can also be readily prepared by similar methods. The probes can also be synthesized directly on a solid support according to methods standard in the art. This method of synthesizing polynucleotides is particularly useful when the polynucleotide probes are part of a nucleic acid array.

The present invention therefore also provides predictive, diagnostic, and prognostic kits comprising degenerate primers to amplify a target nucleic acid (e.g., mitochondrial and optionally nuclear reference genes) and instructions comprising amplification protocol and analysis of the results. The kit may alternatively also comprise buffers, enzymes, and containers for performing the amplification and analysis of the amplification products. In some embodiments, the kit also provides one or more control templates, such as nucleic acids isolated from normal tissue sample.

In certain embodiments, a patient can be identified as likely to develop, for example,

CVD or diabetes by adding a biological sample (e.g., blood or blood serum) obtained from the patient to the kit and measuring the mtDNA copy number, for example, by a method which comprises the steps of: (i) collecting blood or blood serum from the patient; (ii) separating DNA from the patient's blood; (iii) adding the DNA from patient to a diagnostic kit; and, (iv) measuring mtDNA copy number. In this exemplary method, primers are brought into contact with the patient's DNA. The formation of the primer:DNA complex (a complex between a primer and a mitochondrial gene; and a complex between a primer and a nuclear gene) can, for example, be PCR amplified and the mtDNA copy number determined by comparison to a control. In other kit and diagnostic embodiments, blood or blood serum need not be collected from the patient (i.e., it is already collected). Moreover, in other embodiments, the sample may comprise a tissue sample, urine or a clinical sample.

Without further elaboration, it is believed that one skilled in the art, using the preceding description, can utilize the present invention to the fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices, and/or methods described and claimed herein are made and evaluated, and are intended to be purely illustrative and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for herein. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component

concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

EXAMPLE 1 : Association of Mitochondrial DNA Levels with Frailty and All-Cause Mortality. Mitochondrial function is altered with age and variants in mitochondrial DNA (mtDNA) have been shown to modulate risk for several age-related disease states. However, the effect of mtDNA copy number, which reflects energy reserves and oxidative stress, on aging and mortality in the general population has not been addressed.

We determined mtDNA copy number using a qPCR-based method in 4,892 participants from the Cardiovascular Health Study (CHS), and an array-based method in 11,509 participants from the Atherosclerosis Risk in Communities Study (ARIC). We examined the association between mtDNA copy number and two primary phenotypes- prevalent frailty and all-cause mortality.

MtDNA copy number was significantly associated with prevalent frailty in self- identified white participants from CHS (P<0.001). Additionally, mtDNA copy number was a strong independent predictor of all-cause mortality in an age and sex-adjusted, race-stratified analysis of 16,401 participants from both cohorts with a pooled hazard ratio of 1.47 (95% CI, 1.33-1.62, P<0.001) for the lowest quintile of mtDNA copy number relative to the highest quintile.

MtDNA copy number was strongly associated with prevalent frailty and was an independent predictor of all-cause mortality.

Materials and Methods

Cohorts.

CHS: The Cardiovascular Health Study (CHS) is a prospective multi-center study comprising of 5,888 older individuals aged 65 years and above (15.69% African American, 42.37% female), drawn from 4 US communities with initial enrollment in 1989-90, and follow-up recruitment of a minority cohort comprising 687 participants in 1992-93.

Participants were followed by annual telephone interviews and clinic visits through 1998-99 and semi-annual telephone interviews subsequently. Mortality information was obtained via contact with next of kin, death certificates, autopsy and coroner's reports. DNA was extracted by salt precipitation following proteinase K digestion of the buffy coat from whole blood. Only participants self-identifying as white or black were included in this analysis. Participants were included only if they consented to use of their DNA for studies of cardiovascular disease outcomes.

ARIC: The Atherosclerosis Risk In Communities (ARIC) study was established in 1986 as a prospective study of 15,792 individuals, 45-65 years of age, from 4 different US communities. Investigators, T.A., 129 AM. J. EPIDEMIOL. 687-702 (1989). The first visit was carried out in 1987-89, with four subsequent in-person visits and annual telephone interviews after initial visit. DNA was isolated from whole blood using the Gentra Puregene Blood Kit (Qiagen). Mortality was tracked via telephone follow-ups, hospitalization records, state records, and the National Death Index. Cause of death was determined using cause of death on the death certificate (ICD-9 code). Only samples with a self-reported race of white or black were included in this analysis.

Frailty in CHS. We operationalized frailty in CHS participants as detailed previously by Fried et al, 56 J. GERONTOL. A. BIOL. SCI. MED. SCI. 146-56 (2001). Briefly, participants were scored on a 0-1 scale (1 being at risk and 0 being not at risk for frailty) for 5 characteristics-slowness, exhaustion, shrinking, weakness, and low activity, and classified as robust (0 characteristics), pre-frail (1 or 2 characteristics), or frail (>=3 characteristics).

MtDNA Copy Number qPCR Assay. mtDNA copy number in the CHS samples was determined using a multiplexed real time quantitative polymerase chain reaction (qPCR) utilizing ABI TaqMan chemistry (Applied Biosystems). Each well consisted of a VIC labeled, primer-limited assay specific to a mitochondrial target (ND1) (Assay ID

Hs02596873_sl), and a FAM labeled assay specific to a region of the nuclear genome selected for being non-repetitive with no known alternative splicing events (RPPH1) (Assay ID Hs03297761_sl). Each sample was run in triplicate on a 384 well plate in a ΙΟμΙ_^ reaction containing 20ng of DNA. The cycle threshold (Ct) value was determined from the amplification curve for each target by the ABI Viia7 software. A ACt value was computed for each well as the difference between the Ct for the RPPH1 target and the Ct for the ND1 target, as a measure of mtDNA copy number relative to nuclear DNA copy number. For samples with standard deviation of ACt values of the three replicates > 0.5, an outlier replicate was detected and excluded from analysis. If sample ACt standard deviation remained >0.5 post replicate exclusion, the sample was excluded completely from further analyses. Replicates with values of Ct for ND1 >28, Ct for RPPH1 >5 standard deviations from the mean, or ACt value >3 standard deviations from the mean, were removed from each plate. Additionally, we observed a linear increase in ACt value by order in which the replicate was pipetted onto the plate. This effect was adjusted for using a linear regression, and ACt values corrected for pipetting order were used for all subsequent analyses.

MtDNA Copy Number from Microarray Intensities. 13,444 ARIC samples were genotyped on the Affymetrix Genome- Wide Human SNP Array 6.0. Sample exclusions based on sample quality and relatedness have been previously described. See Arking et al, 5 PLoS ONE e9879 (2010). Genotypes were called using Birdseed (version 2) as implemented in the Affymetrix Power Tools software. In addition to determining genotype calls, the software was used to compute probe intensities for each of the two alleles at every SNP (A and B alleles).

To determine mtDNA copy number, data for 119 mitochondrial SNPs were collected across all samples. For mitochondrial SNPs, the software assumes haploidy and hence all genotype calls are homozygous. At a SNP with genotype call AA, probe intensity corresponding to the A allele is considered the true signal, and probe intensity for B allele is considered background. At each SNP, the overall signal intensity was calculated as the absolute difference of the probe intensities of the two alleles (|A-B|). The median probe intensity difference across all mitochondrial SNPs was taken as a measure of the relative mtDNA copy number for each sample. Additionally, we generated principal components (PC) on probe intensities for both alleles of a randomly chosen subset of 1,000 autosomal SNPs. PCs generated from these data allow for correction of both technical artifacts (plate and batch effects) and population substructure. The mtDNA copy number was adjusted for the first 20 PCs, age, sex, and collection site using a linear model. Residuals generated from this model were used for all subsequent analyses.

Statistical Analysis. All statistical analyses were performed using R version 3.0.1. For the qPCR based assay, across plate normalization was performed using quantile normalization as implemented in the R package 'qpcrNorm'. Plate layouts used were non- random with respect to race, requiring all analyses post-normalization and post-removal of plate effects to be stratified by race. Mean ACt value was calculated per sample and adjusted for age, sex and collection site using a linear regression model. Standardized residuals were used as the measure of mtDNA copy number. Effect estimates for age and sex on mtDNA copy number were obtained by linear regression with mtDNA copy number as the outcome (standardized mean ACt in CHS; PC adjusted median mitochondrial probe intensity in ARIC). Effect estimates are expressed in terms of standard deviation units (sd) of mtDNA copy number. The frailty characteristics were treated as binary variables and overall frailty was treated as an ordered variable (0, 1 , 2). The association with mtDNA copy number was determined using a logistic regression model for the individual frailty characteristics, and a proportional odds model for overall frailty.

To assess the effect of mtDNA copy number on mortality, a Cox proportional-hazards model was used, adjusting for age, sex, and collection site, as the baseline model. A secondary multivariate mortality analysis was run including age, sex, collection site, body mass index (BMI), high-density lipoprotein (HDL), total cholesterol, and smoking status with respect to whether the participant had ever smoked as covariates, and excluding participants with prevalent coronary heart disease (CHD), diabetes, or history of myocardial infarction (MI).

For our analyses, baseline was defined as time at which the blood sample that was used to determine mtDNA copy number was collected. Age, follow-up time, and other variables were adjusted accordingly. Samples for which time of DNA extraction was unavailable were excluded. Quintiles were calculated using residuals from age, sex, collection site (for both cohorts), and PCs (for ARIC) adjusted mtDNA copy number. The hazard ratios from both cohorts were pooled using a random effect, inverse-variance weighted meta-analysis, as implemented by the 'metagen' function in R package 'meta' (version 3.1-2).

The extent to which association between sex and mortality may be attributable to sex differences in mtDNA was estimated by comparing the variance explained by each term (mtDNA and sex) on mortality in separate univariate models, with the variance explained by the terms together in a bivariate model.

Results

Sample characteristics.

CHS: Initial analysis was carried out on samples from CHS. The baseline characteristics of the 4,892 participants (4108 whites, 784 blacks) from the cohort included in the current analysis after sample exclusions, are detailed in Table 1. We observed an inverse association between mtDNA copy number and age in both racial groups-a reduction of 0.14 (95% CI, 0.08-0.19, PO.001) and 0.19 (95% CI, 0.06-0.31, P=0.002) standard deviation units (sd) over 10 years in whites and blacks, respectively. Additionally, we noted a higher mtDNA copy number in women relative to men, (OR=l .21 for women relative to men, 95% CI, 1.14-1.28, P<0.001) in whites, with a consistent, but not statistically significant effect in blacks (OR=1.14 for women relative to men, 95% CI, 0.99-1.31, P=0.08).

Table 1. Sample characteristics by quintiles

yrs)

No. of deaths— no

(%) 96 (61.5) 79 (50.3) 88 (56.4) 81 (51.6) 85 (54.1) 0.47

Mean age at death

(in yrs) 81.97 ± 6.02 82.13 ± 5.84 81.81 ± 5.79 81.86 ± 6.21 82.08 ± 6.12 0.87

ARIC-Whites

Quintile 1 Quintile 2 Quintile 3 Quintile 4 Quintile 5 Pval

No. of samples 1804 1804 1805 1804 1805

Age (in yrs) 58.41 ± 5.93 57.97 ± 5.85 58.05 ± 5.88 58.19 ± 5.96 58.13 ± 6.09

Number of males- no (%) 863 (47.8) 844 (46.8) 837 (46.4) 829 (46) 869 (48.1)

Follow up time (in

yrs) 15.82 ± 4.50 16.69 ± 4.46 16.59 ± 4.84 17 ± 4.83 16.95 ± 5.62 O.001

No. of deaths— no

(%) 628 (34.8) 468 (25.9) 496 (27.5) 423 (23.4) 419 (23.2) O.001

Mean age at death

(in yrs) 71.71 ± 6.54 72.44 ± 6.54 72.95 ± 6.47 72.32 ± 6.64 72.78 ± 7.05 0.01

ARIC-Blacks

Quintile 1 Quintile 2 Quintile 3 Quintile 4 Quintile 5 Pval

No. of samples 496 496 497 496 497

Age (in yrs) 57.52 ± 5.91 57.15 ± 6.00 57.22 ± 5.83 57.65 ± 5.94 57.15 ± 6.09

Number of males- no (%) 182 (36.7) 181 (36.5) 191 (38.4) 190 (38.3) 177 (35.6)

Follow up time (in

yrs) 14.67 ± 5.16 15.46 ± 5.22 15.66 ± 5.43 15.84 ± 5.63 16.12 ± 6.15 O.001

No. of deaths— no

(%) 213 (42.9) 193 (38.9) 188 (37.8) 174 (35.1) 158 (31.8) O.001

Mean age at death

(in yrs) 68.87 ± 6.48 69.64 ± 6.82 70.37 ± 6.30 70.54 ± 6.66 70.35 ± 7.45 0.1

Data are presented as Mean±SD. Quintiles were calculated from age, sex, collection site adjusted mtDNA copy number (details in Methods). 'Pval for trend' is the pvalue for effect of trait on age, sex, collection site standardized mtDNA copy number as a continuous variable.

APvIC. We used 11,509 samples (9,025 whites, 2,484 blacks) from ARIC to validate our initial findings from CHS (Table 1). As in CHS, we observed an inverse association of mtDNA copy number with age with a reduction of 0.11 sd (95% CI, 0.07-0.14, P<0.001) in whites and 0.11 sd (95% CI, 0.04-0.17, P=0.001) in blacks, over a 10 year period, and a significantly higher mtDNA copy number in women relative to men (whites OR=l .52 for women relative to men, 95% CI 1.46- 1.59, P<0.001; blacks OR=1.42 for women relative to men, 95% CI 1.31-1.54, PO.001). Frailty. In a race-stratified analysis of samples from CHS, we observe a statistically significant association between lower mtDNA copy number and frailty, adjusted for age and sex in whites (P=0.005). Furthermore, this association was not driven by any single component of the frailty phenotype, with three out of five frailty characteristics showing statistically significant association with lower mtDNA copy number in whites (FIG. 1), and a similar trend of association for the remaining characteristics.

Mortality.

CHS: A total of 2,961 deaths (60.4% samples) were observed in the CHS participants during 26,770 person-years of follow-up. In an age, sex, and collection site adjusted, race- stratified analysis, we observed a statistically significant association between lower mtDNA copy number and mortality, with overall hazard ratio of 1.39 (95% CI, 1.23-1.58, P<0.001) for the lowest quintile of copy number relative to the highest quintile in whites (FIG. 2, Table 2 ). A more stringent multivariate model adjusted for age, sex, collection center, BMI, HDL, total cholesterol and smoking status, and excluding all samples with prevalent CHD, diabetes or previous history of MI, yielded a hazard ratio of 1.42 (95% CI, 1.17-1.74, P<0.001) (Table 2). When stratified by sex, we observed no significant difference in the inverse association between mtDNA copy number and mortality in men and women (P for interaction=0.80). Effect estimates were similar, albeit not statistically significant, in the blacks (Table 2).

Table 2 Lower mtDNA copy number is associated with increased risk for all-cause mortality

Model 1 was the baseline model adjusted for age, sex and collection site. Model 2 was more stringent model that included age, sex, collection site, BMI, HDL, total cholesterol and smoking status as covariates, and excluded samples with prevalent CHD, diabetes or previous history of MI.

dARIC: We observed a similar inverse association of mtDNA copy number with mortality in ARIC, as seen in CHS, with a hazard ratio of 1.63 (95% CI, 1.44-1.84, P<0.001) for the lowest quintile of mtDNA copy number relative to the highest quintile, in whites in an age, sex, and center adjusted analysis (Table 2). We also observed a significantly higher risk of mortality in blacks with hazard ratio of 1.47 (95% CI, 1.19-1.81, PO.001) for the lowest quintile of copy number relative to the highest quintile. In the subsequent multivariate analyses, low copy number remained strongly associated with increased risk for mortality in whites (hazard ratio=l .42, 95% CI, 1.21- 1.68, P<0.001). We observe a similar, albeit not statistically significant, inverse association in blacks (hazard ratio=1.27, 95% CI 0.93-1.73, P=0.06).

Combined e ffect on mortality. A meta-analysis of race-stratified results from both cohorts for the effect of the lowest quintile relative to the highest quintile on mortality, yielded an overall hazard ratio of 1.47 (95% CI, 1.33-1.62, P<0.001), with no significant heterogeneity between the subgroups (P=0.26) (FIG. 3). Additionally, we evaluated the effects of mtDNA copy number on cause-specific mortality, and observe a consistent detrimental effect of low mtDNA copy number in death due to diseases of the circulatory system, respiratory system or neoplasms (FIG. 4). Heterogeneity between effect estimates from cause of death subgroups was determined to be non- significant (P=0.21) using a random-effects model.

Cardiovascular Disease. Lower mtDNA copy number was associated with an increased prevalence of coronary heart disease (CHD) (combined OR from both

cohorts=1.23, 95% CI, 1.17-1.30, P=2.25xl0^"13).

Discussion

We demonstrate that low mtDNA copy number is strongly associated with age, sex, and frailty, and an independent predictor of mortality in 16,401 samples from two large multi-ethnic cohorts, even after adjustment for traditional mortality risk factors and exclusion of prevalent disease states associated with high risk of mortality.

Our results demonstrating a strong inverse correlation between age and mtDNA copy number are in line with previous studies that have shown decreased mtDNA copy number with age in different tissue types. Additionally, our data indicating a higher mtDNA copy number in women relative to men across all the subgroups might suggest that a mito- protective effect may account for the disparity in life expectancy between men and women. We estimate that mtDNA copy number accounts for 3-10% of the effect of sex on mortality (see Methods), suggesting that while mtDNA copy number is not the primary driver of differences in mortality between men and women, it is one of the many mechanisms contributing to the disparity of the phenotype. Frailty has been previously shown to be predictive of both incident disability and mortality. While there has been considerable debate about what drives the onset of frailty, our findings add to the evidence of a role for mitochondria in this process. Given that energy utilization forms a core feature of the phenotype, and low copy number is associated with many of the energy related components of frailty it is not surprising that mtDNA levels might form part of the biological component of the phenotype.

Several limitations to the study should be noted. First, the mtDNA copy number used in this study is derived from a single time-point, and thus does not take into account the dynamic nature of mtDNA copy number during the life of an individual. Second, while mtDNA copy number has been associated with ATP production rate, it is an indirect measure, and further, does not account for acquired mutational burden-a mechanism that forms a critical part of the mitochondrial theory of aging. Third, while we are able to comment on differences between men and women with respect to mtDNA copy number, we cannot do so for race due to technical limitations of study design (see Methods Statistical analysis). This is an important issue, given the significant disparities in health outcomes in the U.S. between whites and blacks. Finally, we were measuring mtDNA copy number in DNA derived from whole blood, which is not necessarily the relevant tissue with respect to many aging-related diseases. Nevertheless, it is noteworthy that a single, easily implemented, measure of mtDNA copy number, isolated from whole blood decades before the event of interest (death), is strongly predictive of all-cause mortality.

Mitochondrial DNA Copy Number and Cardiovascular Disease Risk Prediction As described above, the present inventors hypothesize that mitochondrial stability and copy number of the mitochondrial genome influence the frailty phenotype and successful aging. Furthermore, lower mtDNA copy number was associated with an increased prevalence of coronary heart disease (CHD) (OR=1.23, 95% CI, 1.17-1.30, P=2.25xl0-13). Given the associations with overall mortality and prevalent CHD, we hypothesize that decreased mtDNA copy number is a significant predictor of incident CHD, serving as an important biomarker for CHD.

mtDNA copy number and CHD. Given the robust association we observed between mtDNA copy number and overall mortality, we explored the association of mtDNA copy number, adjusted for age, sex, and collection site, with prevalent risk factors for mortality in both ARIC and CHS. We found that lower mtDNA copy was associated with increased prevalence of CHD, diabetes, and hypertension, with significant results observed for whites in both cohorts, and blacks showing the same direction of effects in both cohorts, with significant associations in ARIC blacks for CHD and diabetes (Table 3).

Table 3. Quartiles of age-,sex-, collection-site adjusted mtDNA copy number in CHS (top panel) and ARIC (bottom panel).

CHD=coronary heart disease; HTN=hypertension; Ql=quartile 1.

We further demonstrate that mitochondrial DNA (mtDNA) copy number is a strong predictor of incident coronary heart disease (CHD) in 11,478 samples from the

Atherosclerosis Risk in Communities (ARIC) study. Additionally, we show that including mtDNA copy number in traditional CHD risk prediction models, that adjust for effects of age, sex, lipid (HDL and total cholesterol) levels, smoking status, diabetes status, systolic blood pressure and the use of hypertensive medications (ACRS score according to 2013 AHA CVD Risk Prediction Guidelines), improves 10 year CHD risk prediction as seen by difference in AUC (area under the ROC curve). See FIG. 5.

Another prediction of the utility of a biomarker is the ability to reclassify subjects more accurately between risk categories. Reclassification is formalized primarily by 2 statistics-the net reclassification index (NRI) and the integrated discrimination improvement (IDI). In FIG. 6, data is included to show that an updated model (mtDNA copy

number+ACRS) classifies a significant proportion of people from high risk (>7.5% 10 year risk) to lower risk categories. Risk cutoffs are consistent with 2013 AHA/ACC Guidelines on Assessment of Cardiovascular Risk4.

EXAMPLE 2: Determining mtDNA copy number using Digital Droplet PCR. In particular embodiments, a Digital Droplet PCR system to determine mitochondrial DNA (mtDNA) copy number in DNA isolated from a biological sample, for example, whole blood. ddPCR is based on using an oil-based master mix to create 20,000 emulsion drops that act as independent measures of copy number for a single sample. The reaction scores each droplet as PCR positive or negative after 30 cycles. In certain embodiments, a Poisson distribution is used to estimate the number of copies of each target from this information. To determine the ideal reaction conditions, 8 samples were tested with the variables of DNA concentration, Taqman probe used and addition of restriction enzyme.

DNA concentration. The sensitivity of ddPCR assay depends on the software detecting both positive, as well as negative PCR droplets for all probe sets in the reaction. 5 Because mitochondrial DNA copy number is highly variable and can be present in hundreds to thousands of copies, it is essential that we determine the right concentration of input DNA that will not saturate the reaction. We tested all reaction conditions with lOng, lng and O. lng of input DNA and determine lng to be the optimal DNA concentration that will allow us to detect mtDNA copy number in most samples while staying within the dynamic range of the 10 ddPCR instrument.

Taqman probe used. Given the differences in the chemistries of the ddPCR and regular qPCR reactions, we tested the same samples using the ABI Mt-NDl and RPPH1 Taqman probes that were previously used in our qPCR assay, as well as new BioRad Mt-NDl and RPP30 probes that are optimized for ddPCR use. Data from our samples suggests no 15 difference between the two probesets with variability between the copy number estimates being well within the 20% range of measurement error.

Addition of restriction enzyme. mtDNA exists in the cell as a supercoiled circular structure that is often hard to denature by heat alone. In order to allow for more uniform access of the primers to mtDNA fragments, we hypothesized that performing a restriction 20 digest within our PCR reaction with enzyme BamHI would increase accuracy of the mtDNA copy number measurement. However, the data from our pilot experiment suggest no additional benefit of the digestion step and suggest that heat denaturation is sufficient for accurate measurement of copy number using ddPCR.

Table 4.

BioRai I Assay Taqman Assay

Reagent Volume for 1 reaction (uL) Reagent Volume for 1 reaction (uL)

2X ddPCR Supermix 10 2X ddPCR Supermix 10

OX BioRad Mt-NDl assay 1 60X Taqman Mt-NDl assay 0.335

0X BioRad RPPH30 assay 1 60X Taqman RPPHl assay 0.335

Water 7 Water 8.33

DNA at Ing/uL 1 DNA at Ing/uL 1

Total 20 Total 20

Detailed reaction setup volumes are as follows.

Claims

We claim:

1. A method for predicting cardiovascular disease in a subject comprising the steps of:

(a) obtaining a biological sample from the subject;

(b) performing an assay to measure the mitochondrial DNA (mtDNA) copy number in the biological sample; and

(c) identifying the subject as likely to develop cardiovascular disease by comparison of the mitochondrial DNA copy number to a control.

2. The method of claim 1, wherein the assay comprises quantitative polymerase chain reaction (qPCR).

3. The method of claim 1, wherein the assay comprises PCR amplifying a mitochondrial gene and a nuclear gene and determining the mtDNA copy number relative to nuclear DNA copy number.

4. The method of claim 3, wherein the mitochondrial gene is one or more of NDl, ND2, ND3, ND4L, ND5, ND6, CYB COl C02, C03, ATP6, ATP8 and RNR2.

5. The method of claim 3, wherein the nuclear gene is one or more of RPPH1, tubulin, RPS18, HGB, B2M, and actin.

6. A method for predicting diabetes in a subject comprising the steps of:

(a) obtaining a biological sample from the subject;

(b) performing an assay to measure the mitochondrial DNA (mtDNA) copy number in the biological sample; and

(c) identifying the subject as likely to develop diabetes by comparison of the mitochondrial DNA copy number to a control.

7. The method of claim 1, wherein the assay comprises quantitative polymerase chain reaction (qPCR).

8. The method of claim 1, wherein the assay comprises PCR amplifying a mitochondrial gene and a nuclear gene and determining the mtDNA copy number relative to nuclear DNA copy number.

9. The method of claim 3, wherein the mitochondrial gene is one or more of NDl, ND2, ND3, ND4L, ND5, ND6, CYB COl C02, C03, ATP6, ATP8 and RNR2.

10. The method of claim 3, wherein the nuclear gene is one or more of RPPH1, tubulin, RPS18, HGB, B2M, and actin.

11. The method of claim 1 or 6, wherein the method also predicts frailty and all-cause mortality.

12. A method for predicting cardiovascular disease in a subject comprising the steps of:

(a) obtaining a biological sample from the subject;

(b) extracting genomic DNA from the biological sample;

(c) performing a genotyping assay using a microarray;

(d) calculating mtDNA copy number based on the probe intensity of one or more mitochondrial SNPs; and

(e) identifying the subject as likely to develop cardiovascular disease by comparison of the mitochondrial DNA copy number to a control.

13. A method for predicting diabetes in a subject comprising the steps of:

(a) obtaining a biological sample from the subject;

(b) extracting genomic DNA from the biological sample;

(c) performing a genotyping assay using a microarray;

(d) calculating mtDNA copy number based on the probe intensity of one or more mitochondrial SNPs; and

(e) identifying the subject as likely to develop diabetes by comparison of the mitochondrial DNA copy number to a control.

14. A method for managing treatment of a diabetes patient comprising the steps of:

(a) ordering a diagnostic test that measures mtDNA copy number from a DNA sample obtained from the diabetes patient; and (b) adjusting treatment for the diabetes patient based on a comparison of the measured mtDNA copy number against a control value.

15. A method for managing treatment of a CVD patient comprising the steps of:

(c) ordering a diagnostic test that measures mtDNA copy number from a DNA sample obtained from the CVD patient; and

(d) adjusting treatment for the CVD patient based on a comparison of the measured mtDNA copy number against a control value.

16. A method for managing treatment of an elderly patient comprising the steps of:

(e) ordering a diagnostic test that measures mtDNA copy number from a DNA sample obtained from the patient; and

(f) recommending, prescribing or administering a treatment regimen designed to prevent or treat frailty or symptoms thereof to the patient based on a comparison of the measured mtDNA copy number against a control value.

17. The method of any one of claims 14-16, wherein the treatment regimen comprises one or more of medical treatment, nutritional supplementation and exercise.

18. A method comprising the step of recommending, prescribing or administering a cardiovascular disease (CVD) treatment to a patient having a mitochondrial DNA copy number that is statistically significantly lower than mtDNA copy number of a control.

19. A method comprising the step of recommending, prescribing or administering a diabetes treatment to a patient having a mitochondrial DNA copy number that is statistically significantly lower than mtDNA copy number of a control.

20. A method comprising the step of recommending, prescribing or administering a frailty treatment regimen to a patient having a mitochondrial DNA copy number that is statistically significantly lower than mtDNA copy number of a control.