Patent Application
20120225427
The invention is in the field of cardiovascular disease/event diagnosis and therapy. In particular, the invention relates to specific single nucleotide polymorphisms (SNPs) in the human genome and their association with cardiovascular disease.
A key problem in treating vascular diseases is proper diagnosis. Often the first sign of the disease is sudden death. For example, approximately half of all individuals who die of coronary artery disease die suddenly, Furthermore, for 40-60% of the patients who are eventually diagnosed as having coronary artery disease, myocardial infarction is the first presentation of the disease. Unfortunately, approximately 40% of those initial events go unnoticed by the patient. Because of our limited ability to provide early and accurate diagnosis followed by aggressive treatment, cardiovascular diseases (CD) remain the primary cause of morbidity and mortality worldwide. Patients with CD represent a heterogeneous group of individuals, with a disease that progresses at different rates and in distinctly different patterns. Despite appropriate evidence-based treatments for patients with CD, recurrence and mortality rates remain high. Also, the full benefits of primary prevention are unrealized due to our inability to accurately identify those patients who would benefit from aggressive risk reduction.
Whereas certain disease markers have been shown to predict outcome or response to therapy at a population level, they are not sufficiently sensitive or specific to provide adequate clinical utility in an individual patient. As a result, the first clinical presentation for more than half of the patients with coronary artery disease is either myocardial infarction or death.
Physical examination and current diagnostic tools cannot accurately determine an individual's risk for suffering a complication of CD. Known risk factors such as hypertension, hyperlipidemia, diabetes, family history, and smoking do not establish the diagnosis of atherosclerosis disease. Diagnostic modalities which rely on anatomical data (such as coronary angiography, coronary calcium score, CT or MRI angiography) lack information on the biological activity of the disease process and can be poor predictors of future cardiac events. Functional assessment of endothelial function can be non-specific and unrelated to the presence of atherosclerotic disease process, although some data has demonstrated the prognostic value of these measurements.
Individual biomarkers, such as the lipid and inflammatory markers, have been shown to predict outcome and response to therapy in patients with CD and some are utilized as important risk factors for developing atherosclerotic disease.
Nonetheless, up to this point, no single biomarker is sufficiently specific to provide adequate clinical utility for the diagnosis of CD in an individual patient. Therefore, there is a need for identifying other factors that provide a more accurate diagnosis/prognosis of CD.
The development of molecular biological techniques and the primary completion of the human genome project have enabled the detection of genetic variations that may be directly or indirectly related to a cardiovascular disease/event. Thus, the invention aims to provide a novel method for the diagnosis/prognosis of CD using a genetic factor.
One object of the invention is a method of identifying a subject having or at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising determining, in a sample obtained from said subject, the presence or absence of a variant allele of nucleotide polymorphism (SNP) of the sPLA2 type IIA nucleic acid, wherein the SNP is selected from the group consisting of rs11573156 C>G and rs2236771 G>C, wherein:
Another object of the invention is a method of identifying a subject having or at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising determining, in a sample obtained from said subject, the presence or absence of the sPLA2 type IIA haplotype comprising variant alleles in rs11573156 or rs2236771 SNPs wherein:
Another object of the invention is a kit for identifying whether a subject has or is at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising:
Another object of the invention is a method for predicting the responsiveness of a subject at risk of having or developing a cardiovascular disease and/or a cardiovascular event, to a drug decreasing the quantity and/or inhibiting the activity of sPLA2 type IIA, said method comprising a step of determining if the minor allele (G) of SNP rs 11573156 is present or if the haplotype wherein the variant allele (G) in SNP rs 11573156 is present, wherein said presence of the minor allele (G) of SNP rs 11573156 or said presence of the haplotype wherein the variant allele (G) in SNP rs 11573156 is present are indicative of responsiveness of the patient to said drug.
“Cardiovascular disease” or “arteriovascular disease” as defined herein is a general term used to classify numerous conditions affecting the heart, heart valves, blood, and vasculature of the body and encompasses any disease affecting the heart or blood vessels, including, but not limited to, Metabolic Syndrome, Syndrome X, atherosclerosis, atherothrombosis, coronary artery disease, stable and unstable angina pectoris, stroke, diseases of the aorta and its branches (such as aortic stenosis, thrombosis or aortic aneurysm), peripheral artery disease, peripheral vascular disease, cerebrovascular disease, and including, without limitation, any acute ischemic arteriovascular event. Arteriovascular disease as used herein is meant to most commonly refer to the ischemic or pro-ischemic disease, rather than generally to-non-ischemic disease.
“Cardiovascular event” is used interchangeably herein with the term “cardiac event”, “acute arteriovascular event”, or “Arteriovascular event” and refers to sudden cardiac death, acute coronary syndromes such as, but not limited to, plaque rupture, myocardial infarction, unstable angina, as well as non-cardiac acute arteriovascular events such as blood clots of the leg, aneurysms, stroke and other arteriovascular ischemic events where arteriovascular blood flow and oxygenation is interrupted.
As used herein, “atherosclerosis” and “atherothrombosis” refer to systemic inflammatory disease states associated with complex inflammatory responses to multifaceted vascular pathologies involving inflammatory activation of the endothelium, inflammatory leukocytes as a source of thrombogenic stimuli, smooth muscle cells as a source of procoagulants and amplifier of the inflammatory response during thrombosis, and platelets as mediators of inflammation and thrombosis. Arteries harden and narrow due to build up of a material called “plaque” on their inner walls. As the plaque develops and increases in size, the insides of the arteries get narrower (“stenosis”) and less blood can flow through them. Stenosis or plaque rupture may cause partial or complete occlusion of the affected vasculature. Tissues supplied by the vasculature are thus deprived of their source of oxygenation (ischemia) and cell death (necrosis) can occur.
“CAD” or “coronary artery disease” is an arteriovascular disease which occurs when the arteries that supply blood to the heart muscle (the coronary arteries) become atherosclerotic, calcified and/or narrowed. Eventually, blood flow to the heart muscle is reduced, and, because blood carries much-needed oxygen, the heart muscle is not able to receive the amount of oxygen it needs, and often undergoes necrosis. CAD encompasses disease states such as acute coronary syndromes (ACS), myocardial infarction (heart attack), angina (stable and unstable), and atherosclerosis and atherothrombosis that occurs in the blood vessels that supply the heart with oxygen-rich blood. An estimated 13 million Americans are currently diagnosed with CAD, with approximately 7 million being the survivors of past acute events. Over 1 million new acute CAD events occur each year, many resulting in death. The lifetime risk of CAD after age 40 is 49 percent for men and 32 percent for women. Subjects who are deemed clinically to be at low risk or no risk for developing arteriovascular disease such as CAD often exhibit none or few of the traditional risk factors for the arteriovascular disease, but nevertheless may still be at risk for an acute arteriovascular event. Approximately 20% of all acute CAD events occur in subjects with none of the traditional risk factors, and the majority of all acute CAD occur in subjects who have not been previously diagnosed with CAD. Often these subjects do not exhibit the symptoms of an acute CAD event, i.e. shortness of breath and/or chest pain, until the actual occurrence of such an acute event. A substantial detection gap remains for those who are at risk for an acute CAD event yet are asymptomatic, without traditional risk factors, or are currently deemed clinically to be at low risk and have not yet been diagnosed with CAD.
“CVD” or “cerebrovascular disease” is an arteriovascular disease in the blood vessels that feed oxygen-rich blood to the face and brain, such as atherosclerosis and atherothrombosis. This term is often used to describe “hardening” of the carotid arteries, which supply the brain with blood. It is a common comorbid disease with CAD and/or PAD. It is also referred to as an ischemic disease, or a disease that causes a lack of blood flow. CVD encompasses disease states such as cerebrovascular ischemia, acute cerebral infarction, stroke, ischemic stroke, hemorrhagic stroke, aneurysm, mild cognitive impairment (MCI) and transient ischemic attacks (TIA). Ischemic CVD is believed to closely relate to CAD and PAD; non-ischemic CVD may have multiple pathophysiologies. An estimated 5 million Americans are the survivors of past diagnosed acute CVD events, with an estimated 700 thousand acute CVD events occurring each year. As disclosed herein, subjects deemed to be at low risk or no risk of CVD based on clinical assessments of traditional arteriovascular disease risk factors, or without symptoms such as TIAs, MCI or severe headache, may still be at risk for an acute CVD event.
“PAD” or “peripheral artery disease” encompasses disease states such as atherosclerosis and atherothrombosis that occur outside the heart and brain. It is a common comorbid disease with CAD. Subjects who are deemed to be at low risk or no risk of PAD based upon an assessment of traditional risk factors of PAD (or arteriovascular disease), or who are asymptomatic for PAD or an arteriovascular disease may nevertheless be at risk for an arteriovascular event, even in the absence of claudication. Claudication can be defined as pain or discomfort in the muscles of the legs occurring due to a decreased amount of blood flowing to a muscle from narrowing of the peripheral arteries, producing ischemia and often arterial occlusion, causing skeletal muscle and limb necrosis. The pain or discomfort often occurs when walking and dissipates under resting conditions (intermittent claudication). Pain, tightness, cramping, tiredness or weakness is often experienced as a result of claudication. PAD not only causes the hemodynamic alterations common in CAD, but also results in metabolic changes in skeletal muscle. When PAD has progressed to severe chronic and acute peripheral arterial occlusion, surgery and limb amputation often become the sole therapeutic options. PAD is widely considered to be an underdiagnosed disease, with the majority of confirmed diagnoses occurring only after symptoms are manifested, or only with other arteriovascular disease, and irreversible arteriovascular damage due to such ischemic events has already occurred.
“Cardiovascular Risk Factor” encompasses one or more biomarker whose level is changed in subjects having a cardiovascular disease or is predisposed to developing a cardiovascular disease, or at risk of a cardiovascular event.
“Risk” in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to arteriovascular events, and can mean a subject's “absolute” risk or “relative” risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(1-p) where p is the probability of event and (1-p) is the probability of no event) to no-conversion. Alternative continuous measures which may be assessed in the context of the present invention include time to arteriovascular disease conversion and therapeutic arteriovascular disease conversion risk reduction ratios. “Risk evaluation,” or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to an arteriovascular condition or to one at risk of developing an arteriovascular event, or from at risk of an arteriovascular event to a more stable arteriovascular condition. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of arteriovascular disease, such as coronary calcium scores, other imaging or treadmill scores, passive or provocative testing results, arteriovasculature percentage stenosis or occlusion and other measurements of plaque burden and activity, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to arteriovascular disease and events, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for an arteriovascular event. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for arteriovascular events. In other embodiments, the present invention may be used so as to discriminate those at risk for developing an arteriovascular event from those having arteriovascular disease, or those having arteriovascular disease from normal.
A “sample” in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof. As used herein “blood” includes whole blood, plasma, serum, circulating epithelial cells, constituents, or any derivative of blood.
“Clinical parameters or indicia” encompasses all non-sample or non-analyte biomarkers of subject health status or other characteristics, such as, without limitation, age (Age), ethnicity (RACE), gender (Sex), diastolic blood pressure (DBP) and systolic blood pressure (SBP), family history (FamHX), height (HT), weight (WT), waist (Waist) and hip (Hip) circumference, body-mass index (BMI), as well as others such as Type I or Type II Diabetes Mellitus or Gestational Diabetes Mellitus (DM or GDM, collectively referred to here as Diabetes), and resting heart rate.
A “subject” in the context of the present invention is preferably a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of arteriovascular disease or arteriovascular events. A subject can be male or female. A subject can be one who has been previously diagnosed or identified as having arteriovascular disease or an arteriovascular event, and optionally has already undergone, or is undergoing, a therapeutic intervention for the arteriovascular disease or arteriovascular event. Alternatively, a subject can also be one who has not been previously diagnosed as having arteriovascular disease. For example, a subject can be one who exhibits one or more risk factors for arteriovascular disease, or a subject who does not exhibit arteriovascular risk factors, or a subject who is asymptomatic for arteriovascular disease or arteriovascular events. A subject can also be one who is suffering from or at risk of developing arteriovascular disease or an arteriovascular event.
The term “Allele” has the meaning which is commonly known in the art, that is, an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome which, when translated result in functional or dysfunctional (including non-existent) gene products.
The term “polymorphism” or “allelic variant” means an alteration in the normal sequence of a gene. Allelic variants can be found in the exons, introns, or the coding region of the gene, or in the sequences that control expression of the gene. Complete gene sequencing often identifies numerous allelic variants (sometimes hundreds) for a given gene. The significance of allelic variants is often unclear until further study of the genotype and corresponding phenotype occurs in a sufficiently large population.
The term “Single nucleotide polymorphism” or “SNP” means a single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population. There are millions of SNPs in the human genome. Most commonly, these variations are found in the DNA between genes. When SNPs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting the gene's function.
The term “linkage disequilibrium” (LD) refers to a population association among alleles at two or more loci. It is a measure of co-segregation of alleles in a population. Linkage disequilibrium or allelic association is the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population.
The term “haplotype” means a 5′ to 3′ sequence of nucleotides found at a set of one or more polymorphic sites in a locus on a single chromosome from a single individual. The term “polymorphic site” refers to a region in a nucleic acid at which two or more alternative nucleotide sequences are observed, often in a significant number of nucleic acid samples from a population of individuals.
The term “locus” as it is used herein refers to any specific region of DNA, a gene, a group of genes or any group of nucleotides defining a DNA region of interest. The term “sPLA2 type IIA” refers to the member IIA of the secreted phospholipases A2 (sPLA2) family of enzymes that hydrolyze phospholipids at the sn-2 position to release free fatty acids and lysophospholipid. In humans, nine catalytically-active sPLA2s (IB, IIA, IID, IIE, IIF, III, V, X and XIIA) and two catalytically-inactive sPLA2-like proteins (XIIB and otoconin-95) have been identified. sPLA2 type IIA has been the most characterized enzyme.
As used herein, the expression “sPLA2 type IIA activity” refers to the ability of the sPLA2 type IIA of metabolizing oxidized phospholipids (OxPL) by cleaving the oxidized fatty acid side chain at the sn2 position of OxPL to generate lysophosphatidylcholine and an oxidized free fatty acid.
According to the invention, the measurement of the quantity (or mass) of sPLA2 type IIA can be performed following routine techniques, well known by the person skilled in the art. For example, said measurement may be performed by an immunoassay allowing the measurement of sPLA2 type IIA concentrations in cell culture supernatant, serum and plasma. Said immunoassay is based on a double-antibody “sandwich” technique. Commercial kits for carrying out this immunoassay are available on the market. For instance, the quantity of sPLA2 type IIA may be performed with sPLA2 EIA Kit commercialised by Cayman. This immunoassay is based on a double-antibody “sandwich” technique. An antibody specific for sPLA2 has been pre-coated onto a microplate. Standards and samples are pipetted into wells and any sPLA2 type IIA present is bound by the immobilized antibody. An enzyme-linked antibody specific for sPLA2 is also added to the wells. The “sandwiches” are immobilized on the plate so that the excess reagents may be washed away. Following this step of washing, a substrate solution is added to the wells and color develops in proportion to the amount of sPLA2 bound in the initial step. The color development is stopped and the intensity of the color is measured.
According to the invention, the measurement of sPLA2 type IIA activity can be performed by a fluorimetric assay according to Radvanyi et al. (1989 Anal Biochem 177: 103-9) as modified by Pernas et al. (1991 Biochem Biophys Res Commun 178: 1298-1305), all incorporated by reference. In particular, the following assay is used. The 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphomethanol sodium salt (Interchim, Montlucon, France) is used as a substrate for sPLA2. The hydrolysis of this substrate by sPLA2 yields 1-pyrenedecanoic acid, which emits fluorescence at 397 nm. A volume (E) of 0.03 ml of the aliquoted plasmas is mixed with 5 nmol of substrate in presence of a 10 mM Tris-HCL pH 8.7, 0.1% albumin, 10 mM CaCl2 in a total volume of 2.5 ml, and fluorescence (F) is measured at 397 nm after one minute. 100% hydrolysis of the substrate is obtained with 0.1 U of bee venom PLA2 (Sigma Chemical Co., France) during one minute, the value of the fluorescence at the end of the one minute reaction (Fmax) thus corresponds to an activity of 2 nmoles/min/ml (Vmax). The activity (A) of the sample (expressed in nmol/ml/min) is given by the following formula: A=(Vmax*F)/(E*Fmax). The samples are diluted when substrate hydrolysis is above 50%. The hydrolysis of substrate in the absence of plasma is used as negative control and deduced from sPLA2 activity. All samples are tested in duplicate. The minimum detectable activity and detection limit is 0.10 nmole/min/ml and the intra and inter assay coefficient of variation is lower than 10%.
According to the invention, the measurement of sPLA2 activity can be performed by an improved fluorimetric assay, using an automated fluorimetric measurement, with a small sample volume, a modified substrate/enzyme ratio (10 nmoles/U instead of 50 nmoles/U) and a thermostat ruled at 30° C., providing a higher precision and sensitivity (2.7%<within batch coefficient of variation (CV)<3.2% and between batch CV=5.7%) than the previous method (within batch CV<10% and between batch CV<10%) and a substantially shorter time to complete the assay. In particular, the following assay is used for automated measurement. The 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphomethanol sodium salt (Interchim, Montlucon, France) is used as a substrate for sPLA2. The hydrolysis of this substrate by sPLA2 yields 1-pyrenedecanoic acid, which emits fluorescence at 405 nm. Briefly, 1 nmol of fluorescent substrate in 0.2 ml of buffer substrate (10 mM Tris-HCL pH 8.7, 0.1% albumin, 10 mM CaCl2) was automatically distributed in Black Maxisorp microtitration plate (96 wells). Because the self-quenching properties of the substrate, a low fluorescence is firstly recorded (Fmin) in a Fluostar Optima fluorimeter equipped with a stirring device and thermostat ruled at 30° C. The addition of 30 μl (100 U/mL) of bee venom PLA2 (Sigma Chemical Co., France) leads to a rapid hydrolysis of all substrate (100% of hydrolysis) and an increase in fluorescence to a maximal value (Fmax), corresponding to an activity of 5 nmol/ml. To determine the sPLA2 activity in unknown blood samples, 30 μl of sera (E) were automatically distributed and added to the substrate mixture and the fluorescence was recorded at one minute (F). A two-point procedure was used to measure the corrected fluorescence intensity of each sample and to evaluate the enzymatic activity (expressed in nmol/min/ml). All samples were tested in duplicate. The activity (A) of the sample (expressed in nmol/ml/min) is given by the following formula: A=(Vmax*F)/(E*Fmax). The hydrolysis of the substrate in the absence of serum is used as negative control and deduced from PLA2 activity. All samples are tested in duplicate. Unless otherwise mentioned, all the numerical values given herein for serum sPLA2 activity are measured according to the above defined assay for automated measurement. The phospholipase that can be used to perform the assay is a secretory phospholipase or a phospholipase with a known activity and preferably a bee venom phospholipase.
In another embodiment, sPLA2 activity may be determined by a process based on a fluorimetric assay comprising contacting a biological sample containing said sPLA2 and taken from said patient, with a substrate at a concentration from 1 nM to 15 nM, the serum sample volume being from 50 to 50 μl and the substrate volume being from 1000 to 300 μl, at a temperature range from about 15° C. to about 40° C. and preferably 30° C. The phospholipase used could be a phospholipase from bee venom or snake venom like Naja venom, preferably bee venom. It could be a recombinant phospholipase from any species. This assay is described in example 2 of WO2008/015546, which is incorporated by reference. The advantage of this method is the small sample volume of substrate used and the thermostating, providing a higher precision and sensitivity.
Alternatively, a variant of the automated fluorimetric measurement as defined above can be used, which enables to alleviate imprecision which might result from a non-specific increase in fluorescence intensity due to other factors in the sample, thus interfering with the measure of sPLA2 activity. This method only differs from the above-defined automated fluorimetric measurement method in that the following formula is used for determining sPLA2 activity:
A=F*s/[(Fmax−Fmin)*V]
wherein:
Secretory phospholipase A2 (sPLA2) enzymes, including type IIA, V and X, are believed to play important roles in several atherogenesis-related pathways, ranging from lipoprotein modification and foam-cell formation to the production of proinflammatory bioactive lipids that perpetuate vascular inflammation1-4.
Elevated circulating levels of sPLA2 type IIA mass and sPLA2 activity, which result from several types of circulating sPLA2 enzymes including type IIA, are associated with an increased incidence of future coronary artery disease in apparently healthy individuals5,6 and with adverse outcomes in smaller populations of patients with stable7 or unstable coronary artery disease8.
The sPLA2 type IIA gene comprises several SNPs. While studying the association between five SNPs (rs11573156, rs3753827, rs2236771, rs876018 and rs3767221) of PLA2 type IIA gene polymorphism, sPLA2 activity and the occurrence of cardiovascular events, the inventors surprisingly found that specific genetic variations in the sPLA2 type IIA gene are associated with sPLA2 activity and the diagnosis/prognosis of a cardiovascular disease/event.
This finding suggests a causal role of sPLA2 activity in cardiovascular disease/event. In addition, this finding also strongly supports the clinical testing of sPLA2 inhibitors to limit the progression and complications of ischaemic cardiovascular disease.
The five SNPs studied in the sPLA2 type IIA gene are described here after:
A first object of the invention is a method of identifying a subject having or at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising determining, in a sample obtained from said subject, the presence or absence of allelic variant in a single nucleotide polymorphism (SNP) of the sPLA2 type IIA nucleic acid, wherein the SNP is selected from the group consisting of rs11573156 and rs2236771,
wherein
According to the invention, the presence of the minor allele (G) of SNP rs11573156 indicates an increased risk of having or being at risk of having or developing a cardiovascular disease and/or cardiovascular event. As shown in the examples, the presence of the minor allele SNP rs11573156 is significantly associated with an increase in sPLA2 activity compared to subjects that do not exhibit said SNP.
According to the invention, the presence of minor allele (C) in SNP rs2236771 indicates a decreased risk of having or being at risk of having or developing a cardiovascular disease and/or cardiovascular event. As shown in the examples, the presence of minor allele SNP rs2236771 is significantly associated to a decrease in sPLA2 activity compared to subjects that do not exhibit said SNP.
Of the 30 potential haplotypes defined by the five SNPs present in the sPLA2 type IIA gene, the inventors also found that
i) haplotype in which the variant allele (minor allele G) in SNP rs11573156 is present is significantly associated with an increase in sPLA2 activity compared to the most frequent haplotype, and
ii) haplotype in which the variant allele (minor allele C) in SNP rs2236771 is present is significantly associated with a decrease in sPLA2 activity compared to the most frequent haplotype.
Thus, a second object of the invention is a method of identifying a subject having or at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising determining, in a sample obtained from said subject, the presence or absence of the sPLA2 type IIA haplotype comprising variant alleles in rs11573156 or rs2236771 SNPs,
wherein
According to the invention, the presence of haplotype in which the variant allele in SNP rs11573156 is present indicates an increased risk of having or being at risk of having or developing a cardiovascular disease and/or cardiovascular event. As shown in the examples, the presence of the GCGAA haplotype, wherein G represents the variant allele of rs11573156, is significantly associated with an increase in sPLA2 activity and with an increased risk of development of cardiovascular disease/event, independently of classic cardiovascular risk factors, compared to subjects that do not exhibit said haplotype.
According to the invention, the presence of the haplotype in which the variant allele in SNP rs2236771 is present indicates a decreased risk of having or being at risk of having or developing a cardiovascular disease and/or cardiovascular event. As shown in the examples, the presence of the CCCAC haplotype, wherein C represents the variant allele of SNP rs2236771, is significantly associated to a decrease in sPLA2 activity and is associated with a decreased risk of development of cardiovascular disease/event, compared to subjects that do not exhibit said SNP.
In one embodiment of the invention, the subject having or being at risk of having or developing a cardiovascular disease/event may be a substantially healthy subject, which means that the subject has not been previously diagnosed or identified as having or suffering from a cardiovascular disease, or that has not developed a cardiovascular event.
In another embodiment, said subject may also be one that is asymptomatic for the cardiovascular disease. As used herein, an “asymptomatic” subject refers to a subject that do not exhibit the traditional symptoms of a cardiovascular disease or event, including, but not limited to, chest pain and shortness of breath for CAD, claudication for PAD, and TIAS, MCI and severe headache for CVD.
In another embodiment of the invention, said subject may be one that is at risk of having or developing a cardiovascular disease or cardiovascular event, as defined by clinical indicia such as for example: age, gender, LDL concentration, HDL concentration, triglyceride concentration, blood pressure, body mass index, CRP concentration, coronary calcium score, waist circumference, tobacco smoking status, previous history of cardiovascular disease, family history of cardiovascular disease, heart rate, fasting insulin concentration, fasting glucose concentration, diabetes status, and use of high blood pressure medication.
In another embodiment of the invention, said subject may be one that has been previously diagnosed or identified for a cardiovascular disease or cardiovascular event, such as for example chronic ischemic disorders without myocardial necrosis (for example stable or effort angina pectoris), acute ischemic disorders without myocardial necrosis (for example unstable angina pectoris), ischemic disorders with myocardial necrosis (for example ST segment evaluation myocardial infarction or non-ST segment elevation myocardial infarction).
Tissue ischemia is often defined in relative terms and occurs when the needs in oxygen exceed the delivery of oxygen to tissues. There is an imbalance between tissue (myocardial for example) oxygen demands and supply. This condition of oxygen deprivation may be accompanied by inadequate removal of metabolites consequent to reduced perfusion. Myocardial ischemia can be diagnosed clinically (chest pain for example), biologically (increase in myeloperoxidase activity for example), metabolically, using scintigraphy, by analyzing regional wall motion disorders or by use of an electrocardiogram (typical modifications of the ST segment, upper or lower ST segment deviation, typical changes in T wave such as T wave inversion or steep symmetric or high amplitude positive T waves). Silent ischemia is typically diagnosed using scintigraphy or a 24 h electrocardiogram recording.
Stable and effort angina is typically manifested by a chest pain during exercise and slowly recovers at rest. It usually reflects tissue ischemia during exercise. Unstable angina is a recent increase in the frequency and/or severity of stable angina, a first episode of angina, or an angina at rest.
Myocardial necrosis is typically diagnosed by an increase in myocardial enzymes (for example troponin I, troponin T, CPK) in the circulating blood.
In another embodiment of the invention, said subject may be one who shows an improvement in cardiovascular risk factors as a result of treatments and/or therapies for cardiovascular diseases. Such improvements include a reduction in body mass index, a reduction in total cholesterol, a reduction in LDL levels, an increase in HDLC levels, a reduction in systolic and/or diastolic blood pressure, or other aforementioned risk factor or combinations thereof.
In one embodiment of the invention, no onset of ischemic symptom has been diagnosed in the subject. Myocardial ischemia can be diagnosed clinically (chest pain for example), biologically (increase in myeloperoxidase activity for example), metabolically using scintigraphy, by analysing regional wall motion disorders or by use of an electrocardiogram (typical modifications of the ST segment, upper or lower ST segment deviation, typical changes in T wave such as T wave insertion or steep symmetric or high amplitude positive T waves).
In another embodiment, an onset of ischemic symptoms has been diagnosed in the subject.
In one embodiment of the invention, the subject is a mammal, preferably a human.
In another embodiment of the invention, the sample obtained from the subject comprises bodily fluids (such as blood, saliva or any other bodily secretion or derivative thereof), and/or tissue extracts such as homogenates or solubilized tissue obtained from the subject. In a preferred embodiment, the sample to be tested is blood.
According to the invention, the sample comprises sPLA2 type IIA nucleic acid, wherein the sPLA2 type IIA nucleic acid may be genomic DNA, heterogenous nuclear RNA (hnRNA, also referred as incompletely processed single strand of ribonucleic acid) and/or cDNA.
According to the invention, the determination of the presence or absence of said SNPs may be determined by nucleic acid sequencing, PCR analysis or any genotyping method known in the art. Examples of such methods include, but are not limited to, chemical assays such as allele specific hybridation, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry.
A third object of the invention is a kit for identifying whether a subject has or is at risk of having or developing a cardiovascular disease and/or a cardiovascular event, comprising:
In one embodiment of the invention, the primer or probe may be labelled with a suitable marker. In another embodiment of the invention, the primer or probe may be coated on an array.
In a further aspect of the invention is a method for predicting the medical response of a subject having or developing a cardiovascular disease and/or a cardiovascular event, to a drug decreasing the quantity and/or inhibiting the activity of sPLA2 type IIA. Indeed, the inventors have shown that the presence of the minor allele SNP rs11573156 is associated with an increased risk of having or developing a cardiovascular disease and/or a cardiovascular event. Accordingly, the invention provides a mean by which a physicist may predict the reaction of a patient if subjected to a treatment which decreases the quantity and/or inhibits the activity of sPLA2 type IIA. Preferably, the invention provides a mean by which a physicist may predicts the reaction of a patient subjected to a treatment which inhibits the activity of sPLA2 type IIA.
The invention thus provides a method for predicting the responsiveness of a subject at risk of having or developing a cardiovascular disease and/or a cardiovascular event, to a drug decreasing the quantity and/or inhibiting the activity of sPLA2 type IIA, said method comprising a step of determining if the minor allele (G) of SNP rs 11573156 is present or if the haplotype wherein the variant allele (G) in SNP rs 11573156 is present, wherein said presence of the minor allele (G) of SNP rs 11573156 or said presence of the haplotype wherein the variant allele (G) in SNP rs 11573156 is present are indicative of responsiveness of the patient to said drug.
(B) HAPLOVIEW LD (D′) display of the five SNPs. The darker the box the stronger the LD. The D′ LD for any two SNPs is presented in the box representing their intersection. No number denotes complete LD.
The population and methods of the French registry of Acute ST-elevation and non-ST-elevation Myocardial Infarction (FAST-MI) have been described in detail elsewhere.11 Briefly, the objective of the FAST-MI registry was to gather complete and representative data on the management and outcome of patients admitted to intensive care units for definite acute MI, irrespective of the type of institution to which they were admitted (i.e., university hospitals, public hospitals, or private clinics). All consecutive adult patients (≧18 years) were included in the registry if they had elevated serum markers of myocardial necrosis higher than twice the upper limit of normal for creatine kinase, creatine kinase-MB or elevated troponins, and either symptoms compatible with acute MI and/or electrocardiographic changes on at least two contiguous leads with pathologic Q waves (≧0.04 sec) and/or persisting ST elevation or depression >0.1 mV. The time from symptom onset to intensive care unit admission had to be <48 h. Patients were managed according to usual practice; treatment was not affected by participation in the registry. At the time of admission, when the first routine blood sample was drawn, an additional 50 mL were taken to create DNA and serum banks. The duration of recruitment was 1 month (31 days) per centre for all patients and 2 months for diabetic patients, ranging from 1 Oct. to 24 Dec., 2005. Of the 374 centres in France that treated patients with acute MI at that time, 223 (60%) participated in the registry. Among these, 100 centres recruited 1029 patients who contributed to both serum and DNA banks. Written informed consent was provided by each patient. Follow-up was collected through contacts with the patients' physicians, the patients themselves or their family, and registry offices of their birthplace. One-year follow-up was >99% complete.
The study was reviewed by the Committee for the Protection of Human Subjects in Biomedical Research of Saint Antoine University Hospital and the data file was declared to the Commission Nationale Informatique et Liberté.
Blood samples were stored at −80° C. at the Department of Clinical Pharmacology, University of Pierre et Marie Curie. All samples were identified by number only and were analysed in random order. Serum concentrations of sPLA2 type IIA were measured by a quantitative and specific time-resolved fluoroimmunoassay (TR-FIA) as previously described.12 sPLA2 activity was measured using a selective fluorimetric assay as previously described.6 All samples were tested in duplicate. Serum levels of sPLA2 activity are expressed as nmol/min/mL and sPLA2 IIA mass as ng/mL.
Genomic DNA was extracted from whole blood with the MagNA Pure Compact Instrument according to the manufacturer's recommendations (www.roche-applied-science.com). Five non-synonymous polymorphisms (rs11573156, rs3753827, rs2236771, rs876018, rs3767221) were selected for PLA2GA genotyping. All single nucleotide polymorphisms (SNPs) were genotyped with the use of an oligoligation assay (SNPlex, Applied Biosystems, Foster City, Calif.) after initial polymerase-chain reaction amplification as described previously.13
Outcome events, a composite of all-cause death and non-fatal myocardial infarction (MI), were adjudicated by a committee whose members were unaware of patients' medications, blood measurements, and genotypes.
Baseline demographic and clinical characteristics, treatment factors, and therapeutic management during hospitalisation were compared among the tertile ranges of sPLA2 activity, with the use of a univariate Cox proportional hazards model. We used a multivariable Cox proportional-hazards model to assess the independent prognostic value of variables with the outcome events during the 1-year follow-up period. The multivariable model comprised sex, age, previous or current smoking, family history of coronary disease, history of hypertension, acute MI, heart failure, renal failure, diabetes, blood pressure at admission, heart rate at admission, Killip class, left ventricular ejection fraction, prior angioplasty or coronary artery bypass surgery, hospital management (including reperfusion therapy, statins, beta-blockers, aspirin, clopidogrel, diuretics, digitalis glycosides, heparin), tertiles of sPLA2 activity, and sPLA2 IIA mass. Serum levels of sPLA2 activity and sPLA2 IIA mass were log-transformed to remove positive skewness. Results are expressed as hazard ratios for Cox models with 95% confidence intervals (CIs). All statistical tests were two-sided and performed using SAS software version 9.1.
The Hardy-Weinberg equilibrium and the linkage disequilibrium (D′) of the different SNPs were assessed using the Haploview program.14 Haplotype analyses were performed using the THESIAS software as described previously.15 Haplotype frequencies were estimated assuming Hardy-Weinberg equilibrium at the haplotypic level. Haplotypes with frequencies <0.05 were not used for further analysis. Haplotypic association effects, expressed as mean quantitative effects for sPLA2 activity and sPLA2 IIA levels or as odds ratios (ORs) for clinical outcomes, were estimated by comparison with a reference haplotype chosen as the most frequent one.
The relationship between the phenotype and haplotypes was modelled using a regression linear model for sPLA2 activity and sPLA IIA levels and a regression model for cardiovascular outcome. Models assumed additive effects of haplotypes on phenotype. A global test of association between haplotypes and the phenotype was performed by a likelihood ratio test. Because of multiple testing, the significance level was taken as P<0.01.16,17
The sponsors had no involvement in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.
Patients who had an outcome event during 1 year follow-up were older (75±12 vs 65±13 years) than those without an outcome event and a smaller proportion were male (60% vs 72%) (table 1). They also had a higher rates of hypertension (80% vs 60%), diabetes (50% vs 29%), prior myocardial infarction (25% vs 16%), and heart or renal failure (both 15% vs 4%). In addition, patients who had an outcome were less likely to be treated during the first 48 hours of admission with statins (67% vs 81%), beta-blockers (49% vs 75%), clopidogrel (79% vs 90%), or unfractionated heparin (76% vs 84%) but more often received diuretics (63% vs 30%) and digitalis treatment (5% vs 2%) (table 1).
The baseline characteristics of patients also varied according to sPLA2 activity, as shown in table 2. Patients in the highest tertile were older, with a higher frequency of previous hypertension, diabetes, myocardial infarction, stroke or transient ischaemic attack, heart failure, and chronic renal failure. In-hospital, they less frequently received reperfusion therapy (percutaneous coronary intervention or thrombolysis), statins, beta-blockers, clopidogrel, and heparin treatments, but more frequently received diuretics and digitalis.
sPLA2 Activity and Clinical Outcomes at 1 year
Of the 1029 patients enrolled, 136 (13.2%) patients died or had a myocardial infarction during the 1-year follow-up period. The probability of outcome events as a function of baseline sPLA2 activity is presented in
After adjustment for known cardiovascular risk factors, C-reactive protein, sPLA2 type IIA levels, and treatments including statins, sPLA2 activity remained an independent correlate of the risk of death or MI. The hazards of death or recurrent acute MI with increasing tertile of sPLA2 activity were 1.62 (95% CI 0.96-2.72) and 1.92 (95% CI 1.17-3.17) compared with the first tertile (p=0.037).
PLA2G2A Haplotypes and sPLA2 Activity
The genotype distribution of the five SNPs for the PLA2G2A gene (rs11573156, rs3753827, rs2236771, rs876018, rs3767221) was consistent with the Hardy-Weinberg equilibrium (observed allele frequencies are listed in table 3). Their location is presented in
A/C
C/G
By single locus analysis, rs11573156 C/G, rs3753827 A/C, rs2236771 C/G, and rs3767221 A/C polymorphisms were significantly associated with concentration levels of sPLA2 IIA and sPLA2 activity. Among them, the rs11573156 G allele was associated with elevated sPLA2 activity, whereas the rs3753827 A, rs2236771 C, and rs3767221 C alleles were linked with a decrease in sPLA2 activity (p<10−6, p=0.005, p=0.00008, p<10−6, respectively).
Of the 30 potential haplotypes defined by the five SNPs, 16 inferred haplotypes were observed in the sample. Seven of these occurred at frequencies ≧5% and accounted for 94% of the observed haplotypes. Table 4 shows the association of these haplotypes with sPLA2 activity, giving the mean value for one copy of each haplotype as determined by THESIAS.15 Overall, haplotypic variation in PLA2G2A was associated with a significant effect on sPLA2 activity (p<0.0001). After applying correction for multiple testing, the GCGAA haplotype was the single haplotype that remained significantly associated with variations in sPLA2 activity. This haplotype, the only one carrying the rs11573156 G allele, was associated with 34.5% increase in sPLA2 activity compared with the most frequent CAGAA haplotype chosen as the reference (2.22[2.1-2.36] vs 1.65 nmol/mL/min [1.55-1.75], p<0.0001). The CCCAC haplotype, the only haplotype carrying the rs2236771 C allele, was associated with a trend towards a decrease (15.8%) in sPLA2 activity compared with the CAGAA haplotype (1.39 [1.24-1.54] vs 1.65 nmol/mL/min [1.55-1.75], p=0.01).
CAGAC
The GCGAA haplotype was the only haplotype associated with the risk of death and recurrent MI during the first year of follow-up. After adjustment for age, sex, diabetes, hypertension, C-reactive protein, and early use of statins, the OR for outcome events as a function of the GCGAA haplotype remained significant (1.84, 95% CI 1.14-2.95, p=0.01). As expected from a causal association, additional adjustment for serum levels of sPLA2 type IIA mass and sPLA2 activity abolished the association between GCGAA haplotype and risk of death and recurrent MI (OR 1.33, 95% CI 0.81-2.18, p=0.25).
The OR of death and recurrent MI was reduced for the CCCAC haplotype, the only haplotype associated with a trend towards a decrease in sPLA2 activity, but the reduction was not significant compared with the reference CAGAA haplotype (OR 0.61, 95% CI 0.26-1.48, p=0.27).
All the cited references are incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2010/062144 | 8/20/2010 | WO | 00 | 4/30/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61235825 | Aug 2009 | US |
