ES2387292B1 - COMBINATION OF SNPS TO DETERMINE THE RISK OF SUFFERING A NEUROVASCULAR DISEASE - Google Patents

Abstract

Combination of SNPs to determine the risk of suffering a neurovascular disease. # The present invention relates to the detection of a predisposition to neurovascular diseases in an individual, and in particular stroke. The present invention relates to a method and kit for the detection of the risk of suffering a neurovascular disease, in particular strokes, in an individual, comprising the detection of a combination of at least two selected single nucleotide polymorphisms (SNPs) from the group consisting of SNPs rs7956957, rs310585, rs2276109, rs10275136, rs5742912 and rs10947803, as well as the combination itself, the polynucleotides that comprise these SNPs and their uses.

Description

COMBINATION OF SNPS TO DETERMINE THE RISK OF SUFFERING

A NEUROVASCULAR DISEASE

Field of the Invention

5

The present invention relates to the detection of a

predisposition to neurovascular diseases in a

individual, and in particular stroke. The present invention

refers to a method and kit for the detection of

risk of neurovascular disease, in

10

particular stroke, in an individual, comprising the

detection of a combination of at least two polymorphisms

single nucleotide (SNPs), selected from the group that

It consists of the SNPs rs7956957, rs310585,

rs2276109, rs10275136, rs5742912 and rs10947803, as well as a

fifteen

the combination itself, the polynucleotides that comprise

these SNPs and their uses.

Background of the Invention

Neurovascular diseases are a huge burden

for the public health system worldwide. The

twenty

neurovascular pathology, in particular stroke, is the

third cause of death and first cause of disability,

exerting a negative impact not only on the quality of

life of the affected patient, but also in his family,

with a large number of patients suffering from dependency

25

functional more than one year after the event [1]. For other

On the other hand, the cost for health systems is serious because

includes direct costs (hospitalization, rehabilitation,

transportation, medical surveillance, drug use, etc.),

and indirect costs (loss of productivity of the

3 O

patients and caregivers, loss of activity, mortality

premature, etc.). In general, the estimated cost of stroke

in Spain it is € 13,826 the first year, € 8,945 the second

year and € 7,739 the third year per patient [2]. For the

Therefore, there is a need to detect and prevent these

35

strokes.

The present inventors have decided to study the prevention of neurovascular diseases from a genetic point of view. In fact, there is an important genetic implication in neurovascular diseases,

5 discovered in the 1990s in twin studies and family aggregation studies, but the genes responsible for this inheritance remain largely undetermined [3]. For example, classical linkage analysis or gene association studies

10 candidates have demonstrated the association of the PDE4D, ALOX5AP, ApoE, IL6, TNFa or MTHFR genes with ischemic strokes, but the replication of these results has been highly inconsistent [4]. Furthermore, complete genome association studies (GWAS) have revealed the

15 Association of two SNPs with stroke on chromosome 12p13 in the Caucasian population, suggesting a role for the NINJ2 gene [5], an association that has been subsequently contradicted

[6] Additionally, a meta-analysis of GWAS in ischemic stroke in Caucasian populations identified an association of the 20 gene ZFHX3 on chromosome 16q22 with stroke [7]. The PRKCH and CELSR1 genes have also been associated with ischemic stroke in the Japanese population [8-9], but these markers must be validated in new studies. In any case, all studies revealed associations with a small 2 5 effect on the overall prediction of the disease. Similarly, the genetic risk factors for hemorrhagic stroke are not yet defined [3]. However, the genetic basis of all neurovascular diseases is probably identical since all 30 candidate genes identified to date are involved in the pathophysiological processes common to all neurovascular diseases, such as inflammation, angiogenesis, fibrinolysis, coagulation, hypertension, coronary heart disease, angiogenesis, lipid metabolism, or

35 diabetes [3]. In addition, neurovascular diseases

they share the same demographic risk factors and

clinical

how the presence of attacks ischemic

transients,

hypertension, dyslipidemia, diabetes,

fibrillation

handset, disease cardiac, angiopathy

amyloid,

obesity, consumption or abuse of drugs, style of

sedentary life, smoking, medication, alcohol consumption and / or family history [3]. Based on the fact that neurovascular diseases are complex diseases caused by multiple genetic and environmental factors, the inventors thought that the combination of various genetic markers and clinical variables could perhaps reveal stronger relationships with the disease. The inventors have disclosed at some conferences (eg "Predicting the genetic risk of ischemic stroke: A study of 250 polymorphisms in 1080 cases and controls", European Human Genetics Conference, Vienna on 5/23/2009) the possibility that 5 SNPs located in the NOS3, LRP, SCNN1A and MMP12 genes could be associated with an increased risk of suffering ischemic stroke in the Spanish population, although the identity of the SNPs involved in these genes was never revealed, nor is it mentioned or suggested to use these SNPs in combination as a screening tool for stroke risk. Similarly, the use of a combination of genetic markers with risk factors for cerebrovascular disease to detect people at high risk of ischemic stroke was not mentioned or suggested. Furthermore, the inventors published the article "KCNK17 genetic variants in ischemic stroke", in the journal Atherosclerosis (2 OO 9) [1 O], where it was revealed that the A allele of the SNP rs10947803 in the KCNK17 gene could be a risk marker for ischemic stroke, with an OR of 1.48, but it was not disclosed or suggested to use this SNP in association with other SNPs to

predict the risk of stroke. The SNP rs5742912 of the SCNN1A gene was described in article 11 Impact of eNaC Polymorphisms on the Risk of Ischemic Cerebrovascular Events: A Multicenter Case-Control Study ", in the journal Clinical Chemistry (2005) [11], as a risk marker for the ischemic stroke, with an OR of 3.26 in young women homozygous for this mutation, however, no mention was made of the use of this SNP in association with other markers for the diagnosis of ischemic stroke, and no mention of the use of this SNP as a predictive tool in association with other markers to improve stroke detection Several articles have been published on strokes in the Spanish population (for example, 11 Human Vitamin K-Dependent GA86: Gene Structure, Allelic Variation and Association with Stroke ", Human Mutation (2004) [12], or 11Alpha (1) -antichymotrypsin polymorphism: a risk factor for hemorrhagic stroke in normotensive subjects", Stroke (2001) [13 ]). However, there is no disclosure in these articles of the polymorphisms described in the present invention, nor of the possibility of combining various markers and risk factors to create a predictive model of heart attack. Finally, the article 11Association of three-gene interaction among MTHFR, ALOX5AP and NOTCH3 with thrombotic stroke: a multicenter case-control study ", Human Genetics (2 OO 9) [14], describes a genetic study of ischemic strokes in the Chinese population, and suggests that three SNPs in the MTHFR, ALOX5AP and NOTCH3 genes could be combined to improve the prognosis of an ischemic stroke.However, this article only predicts with an OR of 3.16 and does not mention the SNPs or genes described in the present invention In view of the knowledge in the state of the art, it is necessary to give

to know a new tool to determine the risk of suffering neurovascular diseases, in particular strokes. A first objective of the present invention is to disclose new genetic markers for the prevention of neurovascular diseases, in particular strokes. A second objective of the present invention is to disclose a new method and a new kit to determine the probability of suffering a neurovascular accident, in particular strokes. In response to these objectives, the present inventors have surprisingly found that a combination of the SNPs described in the present invention is useful for determining the risk of suffering a neurovascular disease, in particular stroke, and therefore a method and a method can be developed. kit for using this combination.

Summary description of the invention

The present invention relates to a combination of at least two single nucleotide polymorphisms (SNPs), selected from SNPs rs2276109, rs10275136, rs310585, rs7956957, rs10947803, and rs5742912, indicative of an individual's risk of suffering from neurovascular disease, in particular ictus. The present invention also relates to a method for the determination of a genetic predisposition of neurovascular diseases, in particular strokes, in an individual, comprising the detection of alleles of at least two SNPs selected from the group consisting of rs2 2761 O9, rs10275136, rs310585, rs7956957, rs5742912 and rs10947803 in a nucleic acid sample isolated from an individual. The present invention further relates to a kit for determining an individual's risk of suffering a neurovascular disease, in particular stroke. The present invention also relates to the use of the aforementioned combination.

Brief description of the figures Figure l. Model ROC curves with the classic risk factors for stroke (age, male sex, hypertension, smoking, and diabetes) with and without addiction of the 6 genetic variants. Figure 2. Percentage of controls and stroke cases according to the number of genetic variants. Figure 3. Percentage of controls and stroke cases based on the total number of risk factors. Figure 4. Distribution of controls and stroke cases by each score. Figure 5. Percentage of controls and stroke cases in each category proposed by the stroke risk model.

Detailed description of the invention 1.-Definitions and terminology

The following abbreviations and acronyms are used in the present invention: ACM: middle cerebral artery DNA: deoxyribonucleic acid RNA: ribonucleic acid mRNA: messenger ribonucleic acid CADASIL: autosomal dominant cerebral artery disease with subcortical infarcts and leukoencephalopathy (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts Leukoencephalopathy) CeGen: national genotyping center

CI: confidence interval TCD: transcranial Doppler ECG: electrocardiogram EDTA: ethylenediaminetetraacetic acid GWAS: genome-wide association studies or whole genome association study

eNaC: epithelial sodium channel

FDR: False Discovery Rate

HI: hemorrhagic infarction

HT: hemorrhagic transformation

HWE: Hardy-Weinberg equilibrium

IPA: Ingenuity Pathways Analysis

MAF: frequency of minority allele

MELAS: Mitochondrial myopathy, encephalopathy, acidosis

lactic and stroke

MRI: magnetic resonance imaging

mRS: modified Rankin scale

MMP: matrix metalloproteinase

NIHSS: National Institute of Health Stroke Scale

OCSP: Oxfordshire Community Stroke Project

OR: odds ratio

ROC: Operational characteristic of the receiver

PCR: polymerase chain reaction

PH: parenchymal hematoma

RMA: Robust Multiarray Averaging

SNP: single nucleotide polymorphism or polymorphism of

single nucleotide

SPSS: Statistical Package for the Social Sciences

CT: cranial tomography

TOAST: ORG 10172 test in accident treatment

acute cerebrovascular

t-PA: tissue plasminogen activator

Names

of genes:

p38-MAPK:

protein kinase activated by p38 mitogen

ALOX5AP:

Protein activator of Arachidonate 5-

lipoxygenase

ALOX12:

12-lipoxygenase arachidonate

APOE:

apolipoprotein E

C4a:

component of complement 4A

C4b:

component of complement 4B

eDKNlA: cyclin-dependent kinase inhibitor lA (kinase-dependent kinase)

eELSRl: cadherin EGF lag seven-pass G-type receptor 1

eLEe4e: e-type lectin domain family 4, member e

GOS2: GO / Gl switch 2 IL6: interleukin 6 ITGB3: beta 3 integrin

KeNK17: potassium channel, subfamily K, member 17

LRP: lipoprotein receptor related protein

low density

MMP: matrix metalloproteinase

MMP12: matrix metalloproteinase 12

MPL. Myeloproliferative leukemia virus oncogene

MTHFR: 5,10-methylenetetrahydrofolate reductase

NEKl: NIMA (never in mitosis gene a) -related kinase 1

NFkB: nuclear factor kappa-E

NINJ2: nerve injury-induced protein 2

NOS3: nitric oxide synthase 3

PDE4D: phosphodiesterase 4D

PITX2: paired-like homeodomain transcription factor 2

PPIA: cyclophilin A

PRKeH: protein kinase e, eta

signal: sodium channel, non-neuronal (nonvoltage-gated) 1,

alpha subunit

TNFa: tumor necrosis factor alpha ZFHX3: zinc fingers homeobox 3

T: thymine

e: cytosine

A: adenine

G: guanine The term ualelo ", as used here, refers to a variant in the gene sequence.

The terms "polymorphic" and "polymorphism", as used herein, refer to the condition in which two or more variants of a specific genomic sequence, or the encoded amino acid sequence, are found in a population. The terms refer to the nucleic acid sequence or the encoded amino acid sequence, the use is clear from the context. The polymorphic region or polymorphic site refers to a region of the nucleic acid, where the nucleotide difference that distinguishes variants occurs, or, for amino acid sequences, a region of the amino acid sequence where the difference of amino acids that distinguishes variants of the protein. As used herein, a "single nucleotide polymorphism" or "SNP" refers to a polymorphic site consisting of a single nucleotide position.

The term "genotype" refers to the description of the alleles of a gene or genes contained in an individual or a sample. As used here, no distinction is made between the genotype of an individual and the genotype of a sample from the same individual. Although a genotype is normally determined from diploid cell samples, a genotype can be determined from a sample of haploid cells, such as a sperm cell.

The term "odds ratio" or "OR" refers to the ratio of the probability of suffering the disease in people with the marker or polymorphism in relation to the probability of suffering the disease in individuals without the marker or polymorphism.

The term "isolated polynucleotide acid" or "isolated nucleic acid", as used herein, refers to a polynucleotide that is separated and / or recovered from a nucleic acid from nucleotide sequences that normally flank the nucleic acid molecule and / or it has been completely or partially purified from other biological material (eg, proteins) that are normally associated with nucleic acid. Likewise,

said "isolated polynucleotide acid" comprises polynucleotide variants where one or more insertions, deletions and / or substitutions have been carried out.

By "oligonucleotide" is meant a single chain 5 nucleotide polymer composed of more than 2 covalently linked nucleotide subunits.

The term "gene", as used herein, refers not only to the strictly coding part, but also to the non-coding parts.

1 O The term "combination", unless otherwise indicated, refers here to the combination or association of at least two SNPs selected from among the SNPs rs2276109, rs10275136, rs310585, rs7956957, rs10947803 and

rs5742912,

indicative of risk of disease

fifteen

neurovascular, in particular ictus, of a individual, in

where

sayings SNPs is it so included in a sequence of

isolated polynucleotides as defined in this document.

When referring to the SNPs of this invention and the polynucleotides that comprise them, the term "a combination of at least two" and consequently the exact number of "three", "four", "five"

or "six", is not intended to mean a closed group of a maximum of 6 SNPs or corresponding polynucleotides, but may also include combinations with more SNPs or 25 corresponding polynucleotides. In these combinations, one or more SNPs or corresponding polynucleotides may or may not be located in genes associated with neurovascular diseases, in particular strokes, as long as said "at least two",

30 "three", "four", "five" or "six" correspond to the corresponding SNPs and polynucleotides disclosed in the present invention.

Clinical uvariable "means the use of demographic data indicative of neurovascular disease, particularly stroke, such as race or

ethnic origin, age, sex and family history of neurovascular disease, in particular strokes, as well as the

use

of the information clinic, such how the presence of

attacks

ischemic transients,

hypertension,

dyslipidemia, diabetes, fibrillation

atrial, heart disease, amyloid angiopathy, obesity, drug use or abuse, sedentary lifestyle, smoking, medications, alcohol use, and a history of neurovascular disease, particularly stroke ,.

By "genes associated with neurovascular disease" is meant the genes that have been described as being associated with any of the neurovascular diseases defined later in the present invention.

By "stroke-associated genes" or 11 strokes-associated genes "is understood the genes that have been described as being associated with the pathology of strokes and / or processes functionally related to strokes, such as inflammation, fibrinolysis, coagulation, hypertension, heart disease, angiogenesis, lipid metabolism and diabetes.

The term "neurovascular diseases" refers to complex diseases of the brain, spinal cord, and blood vessels, and risk factors associated with complex diseases of the brain, spinal cord, and blood vessels, such as: stroke (also equivalently named in the present invention as cerebrovascular accident (s) "), stroke, arteriovenous malformations, thrombosis, CADASIL, cerebral amyloid angiopathy, aneurysm, dissection, hyper / hypotension, encephalopathies, lacunar syndromes, Fabry, homocystinuria / homocysteinemia, hyper / hypoglycemia, hyper / hypolipidemia, diabetes, MELAS, methylmalonic acidemia, fibromuscular dysplasia, Foix syndrome-

Alajouanine, reperfusion injury, hemorrhagic stroke, ischemic stroke, cardioembolism, atherothrombism, vascular malformations, transient global amnesia, ischemic attacks

transients,

trauma, infections neurological,

seizures,

disorders degenerative, epilepsy,

heart disease,

pain of head and migraines tumors,

inflammation,

demyelination, alterations of the

circulation,

neurodegeneration, dementia, Alzheimer's

Parkinson's, Down syndrome, aphasia, apraxia, psychiatric disorders, substance and drug abuse disorders, cognitive disorders, schizophrenia, sleep disorders.

"Mediterranean population" means persons originating from or residing in any of the following states or territories: Spain, France, Italy, Portugal, Monaco, Malta, Slovenia, Croatia, Bosnia and Herzegovina, Montenegro, Albania, Greece, Turkey, Cyprus , Syria, Lebanon, Israel, Palestine, Egypt, Libya, Tunisia, Algeria, Morocco, Crete, Euboea, Pelagia Islands, Gibraltar, Rhodes, Lesbos, Ceuta, Melilla, Chios, Kefalonia, Corfu, Naxos, Andros, Sardinia, Corsica , Cres, Krk, Brac, Hvar, Pag, Korcula, Akrotiri and Dhekelia, Andorra, Jordan, San Marino, Vatican City, Macedonia and Serbia.

2.-Description

The present invention relates to a combination of at least two SNPs selected from among the SNPs rs2276109, rs10275136, rs310585, rs7956957, rs10947803 and rs5742912, indicative of an individual's risk of suffering a neurovascular disease, in particular stroke.

In a preferred embodiment, said combination consists of three SNPs. In a more preferred embodiment, said combination consists of four SNPs.

In a more preferred embodiment, said combination consists of five SNPs. In a most preferred embodiment, said combination consists of six SNPs.

In a particular embodiment, said at least two, three, four, five or six SNPs are combined with one or more of the SNPs rs405509, rs4934, rs2720723, rs2295778, rs7193343, rs6007897, rs4044210 and rs2200733.

In another embodiment, said single nucleotide polymorphisms according to the previous embodiments are combined with one or more clinical variables associated with neurovascular diseases. Preferably, said clinical variable is selected among age, sex, smoking, hypertension, diabetes and dyslipidemia.

Preferably, said neurovascular disease in the above embodiments is stroke.

Since such SNPs are by nature comprised of an isolated polynucleotide or nucleic acid molecule, the present invention also relates to the

combination

of to the less two polynucleotides isolated

included

in genes associates to diseases

neurovascular,

understanding every one of sayings

polynucleotides

a SNP selected between the SNPs

rs2276109, rs10275136, rs310585, rs7956957, rs10947803 and rs5742912, indicative of an individual's risk of suffering a neurovascular disease, said isolated polynucleotides corresponding to SEQ ID No. 1 to

6. Preferably, said neurovascular disease is stroke.

SEQ ID No. 1 indicates the position of the rs2276109 polymorphism and its adjacent sequence, where nucleotide N is the SNP at position 21 and may be nucleotide A or G, with G being the allele associated with neurovascular disease. rs2276109 is found on human contig NT 033899.7 at chromosomal position 102251001 of

chromosome 11.

SEe ID No. 2 indicates the position of the rs10275136 polymorphism and its adjacent sequence, where nucleotide N is the SNP at position 21 and may be a nucleotide T or e, the allele being associated with neurovascular disease. rs10275136 is found on the human contig NT 007914.14 at chromosome position 150514825 on chromosome 7.

SEe ID No. 3 indicates the position of the rs310585 polymorphism and its adjacent sequence, where nucleotide N is the SNP at position 21 and may be nucleotide G or A, where A is the allele associated with neurovascular disease. rs310585 is found on the human contig NT 007914.14 at chromosome position 150526188 on chromosome 7.

SEe ID No. 4 indicates the position of the rs7956957 polymorphism and its adjacent sequence, where nucleotide N is the SNP at position 21 and may be nucleotide e or G, with G being the allele associated with neurovascular disease. rs7956957 is found on the human contig NT 029419.11 at chromosome position 55889082 on chromosome 12.

or

SEe ID N 5 indicates the position of the rs10947803 polymorphism, also called rs9471058, and its sequence

adjacent, where nucleotide N is the SNP at position 21 and can be nucleotide e or A, where A is the allele associated with neurovascular disease. rs1094 7803 is found on the human contig NT_007592.14 at chromosome position 39378588 on chromosome 6.

SEe ID No. 6 indicates the position of the rs5742912 polymorphism and its adjacent sequence, where nucleotide N is the SNP at position 21 and may be an e or T nucleotide, with the T allele being associated with neurovascular disease. rs5742912 is found on the human contig NT_009759 .15, at chromosomal position 6328611 of the

chromosome 12. Preferably, said neurovascular disease in SEQ ID NOS: 1-6 is stroke.

In a preferred embodiment, said combination consists of three isolated polynucleotides included in genes associated with neurovascular diseases, each of said polynucleotides comprising a SNP selected from SNPs rs2276109, rs10275136, rs310585, rs7956957, rs10947803 and rs5742912, indicative of the risk of an individual suffering from neurovascular disease, said isolated polynucleotides corresponding to SEQ ID NOS: 1 to 6. Preferably, said neurovascular disease is stroke.

In a preferred embodiment, said combination consists of four isolated polynucleotides included in genes associated with neurovascular diseases, each of said polynucleotides comprising a SNP selected among the SNPs rs2276109, rs10275136, rs310585, rs7956957, rs10947803 and rs5742912, indicative of the risk of an individual suffering from neurovascular disease, said isolated polynucleotides corresponding to SEQ ID NOS: 1 to 6. Preferably, said neurovascular disease is stroke.

In a preferred embodiment, said combination consists of five isolated polynucleotides included in genes associated with neurovascular diseases, each of said polynucleotides comprising a SNP selected from SNPs rs2276109, rs10275136, rs310585, rs7956957, rs10947803 and rs5742912, indicative of the risk of an individual suffering from neurovascular disease, said isolated polynucleotides corresponding to SEQ ID NOS: 1 to 6. Preferably, said neurovascular disease is stroke.

In a preferred embodiment, said combination consists of six isolated polynucleotides included in

genes associated with neurovascular diseases, each of said polynucleotides comprising a SNP selected among the SNPs rs2276109, rs10275136, rs310585, rs7956957, rs10947803 and rs5742912, indicative of an individual's risk of suffering a neurovascular disease, said isolated polynucleotides corresponding to SEQ IDs No. 1 to 6. Preferably, said neurovascular disease is stroke.

In another particular embodiment, the alleles of said SNPs are deduced by genetic tests of other markers or by measuring the levels of activity or concentration of the proteins, or the corresponding levels of RNA expression of NOS3, SCNN1A, KCNK17, MMP12 and 1st LRP. Preferably, said neurovascular disease is stroke.

In a particular embodiment, said combination of SNPs or the corresponding isolated polynucleotides are found in an individual belonging to the Mediterranean population. In a more particular embodiment, said individual belongs to the Spanish population. Preferably, said neurovascular disease is stroke.

As will be demonstrated later in the Methods and Results section, such a combination of SNPs or the corresponding isolated polynucleotides can be applied to a new model or method to determine the

risk

genetic of than a individual can suffer a

disease

neurovascular, preferably a ictus, and a

kit for

the application of said method.

2.1.-Methods

The objective of this study was to create a predictive genetic model for stroke or stroke, although perfectly expandable to neurovascular disease as defined in this document,

through

the finding of new markers genetic to

through

of one of the more studies big of Association of

genes

candidates accomplished until now in the accident

cerebrovascular,

including a replica and a analysis

functional

of the results obtained.

2.1.1.-Study population

The study design was a case-control study, where stroke cases and controls collected as described below were used. The sample size was calculated with the Jan 2.0 program to obtain a power of 0.80 with a significance level of 0.05 and a difference of 6% between the frequencies of the alleles of cases and controls.

531 consecutive patients with ischemic stroke in the vascular territory of the basilar cerebral arteries

o mean (ACM) and admitted to the emergency department of a university hospital in the first 3 hours after the onset of symptoms were recruited. Onset of stroke is defined as the last time the patient was determined to be without any neurological deficit. All patients underwent urgent examinations by carotid ultrasound and transcranial Doppler (TCD). They received intravenous t-PA in a standard dose of 0.9-mg / kg (10% bolus, 90% in continuous infusion for 1 hour). Patients with a clinically known inflammatory or malignant disease were excluded from the analysis. To identify the potential mechanism of stroke, a set of diagnostic tests was performed that included an electrocardiogram.

(ECG), chest radiography, complete and differential white blood cell count, blood biochemistry, carotid ultrasound, contrast and cranial tomography (CT) in all patients. When indicated, some patients also underwent special coagulation tests,

magnetic resonance imaging (MRI), transthoracic echocardiography and Holter ECG monitoring. Taking this information into account, previously defined etiological subgroups were determined according to the TOAST criteria [1] and also by criteria of the Oxfordshire Community (OCSP), based on the clinical symptoms, location and extent of cerebral infarction [2]. The clinical examination was performed at admission and at 12, 2, 4 and 4 8 hours after the onset of symptoms and every day until discharge. The severity of stroke was assessed using the NIHSS scale [3]. Neurological improvement was defined as a decrease in score ~ 4 points and neurological deterioration as death or an increase in score of> 4 points at 48 hours [4]. The functional result was defined by the modified Rankin scale (mRS)> 2.3 months after the stroke. TCD examinations were performed in the emergency department before administration of t-PA (<3 hours). Systolic, diastolic blood pressure and heart rate were measured at the time of each TCD examination. The TCD examination was performed with the Multi-Dop X4 TCD device (DWL Elektronische Systeme GmbH), by a hand-held transducer and a pulsed wave mode with a frequency of 2 MHz. The proximal occlusion of ACM was defined as the absence of flow or the presence of a minimum flow signal throughout the MCA at an insonation depth between 45 and 65 mm [5]. Distal ACM occlusion was defined as a mean velocity in the affected area ACM> 21% compared to unaffected ACM [6]. Upon admission, all patients underwent computed tomography in the first 3 hours after the onset of the stroke. No patient with hypodensity> 33% in the MCA territory received t-PA in this study. The CT was repeated later, 24 to 48 hours later (or earlier, when rapid neurological deterioration occurred) to

assess the presence of hemorrhagic transformation

(TH). CT scans were reviewed by a neuroradiologist with extensive experience in acute stroke. Presence and type of HT were defined according to previously published criteria [7-8]. Hemorrhagic infarction (HI) was defined as petechial infarcts without mass effect and parenchymal hematoma (PH) was defined as hemorrhages with mass effect.

54 7 controls were recruited in the stroke unit and in the laboratory of the Vall d 'Hebron hospital in Barcelona between 2 OO 7 and 2 OO 8. All controls were over 65 years of age and declared healthy with dementia, neurovascular disease. and / or cardiovascular, evaluated through an interview before blood collection. Patients with a first or second degree family history of neurovascular disease were excluded from the study.

A detailed history of vascular risk factors has been obtained from each participant. Smoking was defined as smoking or having smoked on occasion. Hypertension was defined as systolic blood pressure ~ 140mmHg and diastolic blood pressure ~ 85mmHg, history of self-reported hypertension and / or treatment for hypertension. Diabetes mellitus was defined by history of self-reported diabetes and / or treatment of type 2 diabetes. Dyslipidemia was defined by elevated lipid levels

(cholesterol> 200 mg / dL or triglycemia> 200 mg / dL), reported history and / or any treatment. Informed consent was obtained from each study participant and all subjects were of European descent. The hospital's ethics committee approved the study.

2.1.2.-Genotyping and SNP associations 221 SNPs in 135 candidate genes chosen in the

literature for its putative functions in diseases

neurovascular and functionally related processes

with these diseases like inflammation, fibrinolysis,

coagulation, hypertension, coronary heart disease,

5

angiogenesis, lipid metabolism or diabetes were

analyzed as follows. Genomic DNA is

extracted 1 mL of peripheral blood from each sample

anticoagulated with EDTA by standard methods. SNPs

were genotyped by SNPlexrn (Applied Biosystems, Inc.,

1 O

Foster City, USA) at the National Genotyping Center

Spanish (CeGen), with a success rate of more than 9O%. Five

genes were analyzed entirely by Tag-SNPs,

defined by HapMap data using pairwise

tagger r2 ~ O, 8 and a minor allele frequency> 0, 1:

fifteen

MMP9, NOS3, VEGF, LRP and IL6. Deviation from the equilibrium of

Hardy-Weinberg (HWE) was evaluated using an X2 test with

1 degree of freedom. An X2 or Fisher test, in your

case, it was used to compare categorical variables between

groups. The Bonferroni correction was used to give

twenty

multiple testing account. Student's tests or

Krustal-Wallis were used to compare variables

continuous, as appropriate. Odds ratios (ORs) with

95% confidence intervals (CI) for the effect on

the risk of ischemic stroke were estimated by

25

Logistic regression. All statistical analyzes are

made with the SPSS 15.0 © program.

The cumulative association of individual SNPs

positively associated was calculated based on the number

of carried risk alleles (from 0 to 6) and the

30

allele combination of the 6 SNPs carried (of the 26 =

64 possible combinations). The analysis was performed under

an additive model, taking into account stage 1, stage 2

and the global database. Then a

logistic regression, including each variable and the

35

predictive probabilities were recorded. I know

performed 2 ROC curves, one with the risk factors and

another with risk factors and genetic variants,

to explore the relationship between sensitivity and

Specificity of a clinical test for the risk of

5

stroke. The

difference between the areas under the curves with the

MedCalc program. Finally, the combination of variants

genetic and clinical performed by each individual is

used to develop a predictive risk model

1 O

of stroke and to determine the

risk categories for strokes.

2.1.3.-Functional analysis

RNA was extracted from healthy controls and the

fifteen

expression by real-time quantitative PCR of the

KCNK17, LRP, SCNN1A and MMP12 genes. Blood cell fraction

blanks were obtained after centrifugation of whole blood

preserved in EDTA for 15 minutes at 35O O rpm, and

preserved in RNAlater ® at -80 ° C. Total RNA was isolated

2 O

by RiboPure-Blood ™ Kit (Ambion ®, Foster City,

USA.). CDNA synthesis was performed using the kit

High-Capacity cDNA Archive Kit (Applied Biosystems Inc.,

Foster City, USA). MRNA levels were determined

by quantitative real-time PCR, using a

25

standard protocol for TaqMan® PCR kit and TaqMan probes

fluorogenic (LRP: Hs00233856_m1, SCNN1A: Hs00168906_m1,

MMP12: Hs001591 78_m1) using 7500 Real Time technology

PCR System (Applied Biosystems Inc., Foster City,

USA). The Cyclophilin A (PPIA) gene was used to

30

normalize the results (Hs99999904_m1). All

Reactions were performed in triplicate on 96 plates

wells and analyzed with the Applied Biosystems SDS

7500 system software (Applied Biosystems Inc., Foster

City, USA) Results are expressed as a percentage in

35

Comparison to a sample calibrator used in

all experiments.

MMP12 protein levels were measured from

controls serum using Fuorokine® MMP12 Analyte

Profiling in 96-well plates. Each sample is

5

concentrated from 500pL to 100uL with Microcon® (MilliporeTM,

Billerica, USA) and were analyzed twice according to the

manufacturer's instructions, samples were measured in

a Luminex analyzer (Human MMP Base Kit, Fluorokine®

MAP, R & D Systems, Minneapolis, USA). The activity of

10

NOS3 protein was analyzed from plasma of

Controls by Nitrate / Nitrite Colorimetric Assay Kit on

96-well plates. Each sample was concentrated from

5O OpL to 1 O OuL with Microcon® (MilliporeTM, Billerica,

USA) and was analyzed twice according to the instructions of the

fifteen

manufacturer (Catalog No. 780001, Cayman ™, Ann Arbor, USA).

2.1.4.-Microarrays and analysis by Ingenuity Pathways

RNA was extracted from 12 subjects. Tubes with EDTA se

centrifuged at 3500RPM for 15 minutes to obtain the

2 O

white blood cell fraction. The RiboPure ™ -Blood kit from

Ambion (Ambion, Woodward st. Austin, USA) was the

used to extract the total RNA following the

manufacturer's instructions. Globin RNA from

erythrocytes from blood samples causing

25

interference in the microarray studies was

removed using Globin-Clear kit (Ambion,

Woodward st. Austin, USA). The images were

processed with Microarray Analysis Suite 5.0

(Affymetrix) All samples showed

30

high quality features and underwent analysis

later. The raw expression values obtained

directly from CEL files were pre-processed by the

RMA method, a three-step process that integrates

correction of background values, normalization and summary of

35

probe values. These normalized values were

the basis for all the following analyzes. The selection of differentially expressed genes was based on an analysis with the Bayes model. P values were used to

sort out

the genes with higher or less changes of

expression.

The values of p I know adjusted using the

correction

by false positive (FDR) and the method of

Benjamini

Hochberg. The genes associates of shape plus

were highlighted graphically using volcano-type graphs with, in horizontal, the amplitude of the change of expression and, in vertical, the values of

obtained. The biological significance of the observed changes was estimated by checking whether the genes that

They turned out to be differentially expressed. They seemed to be concentrated or particularly absent from some categories of Ontology. Finally, the Ingenuity Pathways (IPA) software evaluated the relationships between genes significantly associated with LRP genotypes. The results were recorded in the IPA, and the associations between significant molecules with other molecules, their functions and their participation in molecular networks and in diseases were recorded. The protocol used for the study was as follows:

Scheme 1

Collection of data (questionnaires) and samples (DNA, RNA, serum, plasma, etc.)

+

DNA or RNA extraction

+

Genotyping (PCR and sequencing, real-time PCR, etc.)

+

Statistical analysis of the data

~ ~ Functional studies:

Replication of the. ,

Results in a Protein Level by New ELISA Population Activity of Protein by Calorimetric Assay mRNA Levels by Real-time PCR Microarrays

2.2.-Results

2.2.1.-Genetic analysis Statistical analysis and genotyping of

5 samples were made in two different stages. In total, the cases of ischemic stroke were divided depending on the etiologies into cardioembolic (50%), atherothrombotic (23%) and indeterminate (2 7%). There were no significant differences between stage 1 cases and controls.

10 against stage 2 cases and controls. Established risk factors such as male gender, age, diabetes, hypertension, and smoking were observed with a higher frequency in the stroke group (Table 1)

15 22 SNPs were excluded from the analysis due to the minor allele frequency (MAF) less than 1% and 33 SNPs were excluded due to genotyping failure at any stage of the analysis, so the final statistical analysis included 166 SNPs. Taking into account an additive model, a

20 total of 25 SNPs were associated with strokes in at least one stage of the analysis. Six SNPs located near or within the genes KCNK17 (rs10947803), LRPl (rs7956957), MMP12

(rs2276109), NOS3 (rs10275136 and rs310585) and SCNNlA (rsS 7 42 912) were associated with ischemic stroke, in the two 25 stages of the analysis (Table 2). Table l. Characteristics of the study population.

Total (n = 1062) Controls Cases (n = 535) (n = 527) p-value

Age, years ± SD 71.6 ± 7.1 70.7 ± 12.0 <0.05 Men, n (%) 230 (42.5) 287 (54. 5) <0.001 Smokers, n (%) 68 (12.6) 130 (25.9) <0.001

Hypertension, n (%) 239 (44.2) 308 (59. 2) <0.001 Diabetes mellitus, n (%) 44 (8. 2) 120 (22.9) <0.001

Dyslipidemia, n (%) 155 (28. 7) 173 (33 .1) <0.05

Table 2. SNPs significantly associated with strokes in the

2

stages of analysis

Stage

one

Value of

SNP

Risk Controls Cases p OR (95% IC)

rsl0947803

TO 17.8 25.3 0.003 1.57 (1.16-2.11)

rs7956957

G 63.0 70.0 0.0261 1.37 (1.04-1.80)

rs2276109

G 9.7 17.7 0.0003 2.00 (1.37-2.91)

rsl0275136

and 93.5 9 7. 2 0.0137 2.35 (1.17-4. 73)

rs310585

TO 46.4 58.1 0.0005 1.60 (1.23-2.09)

rs5742912

T 86.8 96.5 3.1E-07 4.19 (2.33-7.54)

Stage 2

Value of

SNP

Risk Controls Cases p OR (95% IC)

rsl0947803

TO 17.8 23.5 0.012 1.42 (1.08-2.12)

rs7956957

G 64.4 70.7 0.0415 1.33 (1.01-1. 76)

rs2276109

G 9.7 14.0 0.0405 1.51 (1.02-2.25)

rsl0275136

and 93.5 9 7. 6 0.0046 2.89 (1.34-6.20)

rs310585

TO 44.9 55.4 0.0038 1.52 (1.14-2.03)

rs5742912

T 93.8 9 7. 2 0.0127 2.31 (1.18-4.55)

Total

Value of

SNP

Risk Controls Cases p OR (95% IC)

rsl0947803

TO 17.8 24.2 0.003 1.48 (1.14-1.91)

rs7956957

G 63.7 70.3 0.0027 1.35 (1.11-1.64)

rs2276109

G 9.7 15.9 4.3E-05 1.76 (1.34-2.31)

rsl0275136

and 93.5 97.4 0.0002 2.59 (1.55-4.34)

rs310585

TO 45.7 56.7 7.9E-06 1.56 (1.28-1.89)

rs5742912

T 91.0 96.8 2.5E-07 3.05 (1.96-4.75)

Interestingly, the combination of six SNPs showed a clear 5 contribution to stroke risk compared to the cumulative risk obtained by risk factors.

Conventional clinical trials, including age, sex, smoking, hypertension, and diabetes, with areas under the ROC curve of 0.687 (95% CI: 0.589-0.669) versus 0.629 (95% CI: O, 649 to O. 725) (Figure 1) . Furthermore, the risk increases with the number of genetic variants presented by the patient (Figure 2), and the risk increases with the number of genetic markers and / or clinical risk variable present, although no one in our population had more than 9 factors of risk (Figure 3). Finally, a predictive risk model was designed to create categories of low to high risk of stroke. The six clinical variables and six replicated genetic variants were entered into the model. All variables are independent of each other. The Hosmer and Lemeshow test showed that our model fits the data very well. A risk score was assigned to each variable and a risk scale of 1 to 45 (Figure 4) was used to classify the subjects into three categories. Stroke risk was defined as low (score: <::; 18), moderate (19 <score: <::; 26) or high (26 <score) (Figure 5). 2.2.2.-Functional analysis

SCNN1A gene expression, measured in 26 healthy controls, revealed that TT carriers had significantly higher mRNA levels than TC and ce (TT: 109 ± 40%, CT: 60 ± 28%, ce: 58 ± 25% , po, 005) However, LRP1 gene expression was determined in 18 healthy controls, but no association was observed between rs7956957 SNP and mRNA levels (GG: 114 ± 73%, CG: 105 ± 60%, CC : 96 ± 47%, p = 0.578). Furthermore, the KCNK1 7 gene mRNA levels were dependent on the rs10947803 SNP alleles (p 0.021). Carriers of the A allele show higher levels than C (114 ± 35%, n = 5 vs. 77 ± 38%, n 8). On the other hand, the activity of the NOS3 protein determined in 80 healthy controls revealed that the two alleles of risk for strokes, the SNPs rs310585 and rs10275136, were associated with lower levels of protein activity (A: 6.8 ± 2, 0mM, G: 8.0 ± 3.4mM, p = 0.005 and

C: 7.2 ± 2.7 mM, T: 8.8 ± 3.6 mM, p = 0.027). On the other hand, MMP12 gene expression could not be detected in 12 healthy controls. However, MMP12 protein levels were determined in 14 healthy controls by Luminex and we were only able to detect very low levels of MMP12 in 6 samples, but no association with rs2276109 SNP genotypes (AA: 22.4 ± 13.3 years , AG: 18.1 ± 12, 8, AP: 11.6 pg 1 mg total protein, p = 0.704). Finally, microarray analysis in 12 subjects revealed that LRP gene rs7956957 SNP genotypes are associated with the expression of several genes after correction for false discovery rate (FDR) and the Ingenuity Pathway Analysis (IPA) program identified various networks associated with LRP genotypes. Notably, the SNP risk allele rs7956957 (allele G) was associated with a lower expression of genes involved in inflammatory processes,

such

how CLEC4C or the accessories 4A and 4B (C4A and C4B),

While

than I know associated with a increase of the expression

of

genes involved in apoptosis how ITGB3, ALOX12, NEK1

or GOS2.

2.3.-Discussion

In this case-control study, the inventors analyzed the association of 221 SNPs functionally related to neurovascular diseases in a Mediterranean population. After multivariate analysis, adjusted for conventional risk factors, the inventors could replicate a statistically significant association between six SNPs.

(rs10947803, rs7956957, rs2276109, rs10275136, rs310585 and rs5 7 42 912) and the risk of neurovascular accidents, or strokes.

The SNP rs5742912 of the SCNNlA gene is an important amino acid change at the structural level of the protein, it is a tryptophan change by arginine

(Trp493 ~ Arg), exchanging a non-polar cyclic amino acid for a positively charged amino acid. The gene encodes a subunit of the ENaC protein, a sodium channel that is

Present

in the cells epithelial, together with others two

subunits,

SCNN1B and SCNN1G [fifteen] The paper of this

protein

in the homeostasis of the sodium, osmolarity

Plasma and blood pressure regulation has made it an ideal candidate to study the development of complex diseases such as stroke, myocardial infarction, or renal failure [16]. In fact, eNaC is important for the limiting step in the rate of sodium reabsorption and is sensitive to amiloride, a diuretic that can block the sodium channel and inhibit its assimilation, used in the treatment of hypertension and congestive heart failure [ 17]. At the genetic level, the SNP rs5742912 from SCNN1A has been associated with ischemic stroke [11]. In our study, the association resisted Bonferroni correction and reached a p-value of 2. SE-O 7 and an OR of 3.59, very low values for a study of candidate genes. The inventors also demonstrate here for the first time that this SNP is associated with SCNN1A gene mRNA levels, and carriers of the risk T allele showed significantly higher levels than carriers of the C allele. Being a non-synonymous SNP, we expect that it could affect the activity of the protein in addition to the mRNA, especially since the SNP is in the extracellular domain, where different ligands and amiloride bind. These results are consistent with other studies showing that other variants of the a subunit are associated with increases in activity of this protein in human epithelial cells [18]. Inventors

observed higher levels of mRNA associated with the risk T allele, which is also the most common allele. The SCNN1A gene is found on chromosome 12p13, where a GWAS recently showed a robust association with the genetic locus of the NINJ2 gene and stroke, indicating that this region could indeed be a major region of susceptibility to stroke.

[5].

Another SNP located on chromosome 12 was associated with ischemic stroke in our study. In fact, the G allele of rs7956957 of the LRP1 gene at the 12q13 locus was identified as a risk factor in all the stages of our study and in all the etiologies of stroke, although it was not associated with mRNA levels. The SNP is in linkage with many other SNPs in the gene, although the only functional SNP described (C766T or rs1799986) is located in a different region of the gene, composed of 8 9 exons, and is not in linkage disequilibrium with the SNP rs7956957. Nothing is known about this SNP (rs7956957), it is an intronic SNP that was selected in this study as a Taq-SNP for LRP1. The effect of rs7956957 on stroke was also examined using microarrays and

we identify

various genes interesting associates to the

genotypes

of the rs7956957 how ALOX12, ITGB3, MPL, C4a and

C4b.

A representation combined of the two nets

Major identified by the IPA software revealed that the genes involved could play an important role in the biological processes of apoptosis, cell signaling, cell death and inflammation. The fusion of these networks also indicates that some genes such as LDL, NFKB, CDKN1A, and p38-MAPK could be important regulators of these networks. The mechanism of the effect of rs7956957 in ischemic stroke is still unclear but it could be due to a modulation of the activity of the protein since it does not affect the expression of LRP1 mRNA, although the

The results indicated that the stroke cases had significantly lower levels of mRNA than the healthy controls.

Two other genes that have been identified in our study, MMP12 and NOS3, are involved in inflammation processes. The MMP12 protein has been linked to ischemic stroke, thanks to its role in the stability of atherosclerotic plaque, while the MMP12 gene, located on chromosome 11q22, has been linked to coronary heart disease and hemorrhagic stroke [21-22] . SNP rs2276109 was associated with ischemic stroke in our cohort. It is a substitution of A for G in the promoter region of the gene, and has been shown to be functional, influencing the binding capacity of transcription factor AP -1 in electromobility assays [23]. The A allele is associated with increased MMP12 promoter activity in vitro in transient transfection studies, and is associated with a lower risk of ischemic stroke in our study. Therefore, it seems that a decrease in MMP12 promoter activity, through the G allele of the SNP rs2276109, could be associated with an increased risk of stroke. Gene expression and MMP12 protein levels were measured in healthy controls, in order to better understand the contribution of the rs2276109 variant, but mRNA and protein levels were barely noticeable and insufficient to draw conclusions. Blood samples were used, while MMP12 manifests itself mainly in activated macrophages and stromal cells, which could explain why it was not detected.

NOS3, on chromosome 7q36, was also associated with stroke in this study. In fact, an association of two SNPs was observed in this gene, rs10275136 and rs310585, the latter reaching p values of 7. 9E-06. Both associations resisted the Bonferroni correction.

These 2 SNPs are not in linkage disequilibrium and,

therefore they are independent of each other and are located in the

5 'region of NOS3. They were selected for this study

like Tag SNPs and their function is unknown. The protein

5

NOS3, also known as eNOS or cNOS participates in

many biological processes, including hypertension and

coronary spasms [2 4 J. It catalyzes the generation of

nitric oxide in blood vessels from L-

arginine, and is particularly important in

10

cell signaling and regulation of function

vascular [25]. At the genetic level, some polymorphisms in

this gene has been associated with stroke, myocardial infarction,

hypertension and coronary heart disease [26]. Curiously,

while the 3 other SNPs were associated with all

fifteen

ischemic stroke subtypes, these two variants are

specific to cardioembolic etiology, indicating

which could be associated with other disorders

cardiovascular. Furthermore, the two risk alleles

were associated with decreased activity of the NOS3 protein and

2O

Stroke cases had lower levels than those

healthy controls, indicating that low NOS3 levels

They could be risk factors for strokes. In fact, this

hypothesis is in agreement with previous studies that

show that NOS3 is an important regulator of f 1 uj o

25

cerebral blood, especially during ischemia

brain, where it has been shown to have effects

neuroprotective through better perfusion and

smaller stroke size [27-28].

The A allele of SNP rs10947803 of the KCNK17 gene is

30

independently associated with ischemic stroke in this

study with an OR of 1.47 (p = 0.010) Little is known about

the KCNK17 gene, but it is widely expressed, especially in

liver, lung, placenta, pancreas, intestine, and aorta

[2 9]. High levels of KCNK1 7 gene mRNA were associated

35

with an increased risk of ischemic stroke. The mechanism of

this association is unknown and the SNP is intronic and therefore does not belong to the promoter region of the gene. The protein encoded by the KCNK17 gene is a member of the K + channel superfamily [29-30]. These channels 5 regulate cellular ion fluxes and consequently cell volume, as well as metabolic acidosis and hypotension caused by HC0-3 secretion [31-34] and taking into account their function and location in many tissues, could play an important role in

10 strokes, being a main trigger common to all stroke subtypes. In summary, it was observed that six SNPs were independent risk factors for stroke in the Spanish population. Four of these SNPs, in the KCNK17 genes,

15 SCNN1A and NOS3 showed a clear influence on the function of the corresponding gene or protein, and a SNP in the MMP12 gene also had a clear effect as described in another study [23]. The cumulative association of

twenty

these variants. The principal objective was establish yes

the

combination of to the less two of these SNP,

preferably

three SNP, best four SNPs, even better

five

SNP, and the optimum six SNPs, could predict the

risk

of accident cerebrovascular best than every SNP of

25

shape individual, and yes the inventors could to get better the

discrimination

between the subjects of risk, in combination

with

factors of risk classics, in contrast  with the

factors

of risk classics alone. The results of this

analysis

they showed than the risk increase gradually with

3O

the number of alleles of the risk carriers. Of done, the

association

accumulated of the six SNPs had a value of

positive prediction

higher to the addition of the OR of every

SNP

of shape individual. The carriers of everybody the

alleles

of susceptibility presented a risk of ictus

35

of almost the 100% comparing it with controls healthy. By other

In addition, the contribution of genetic information with classic risk factors was significant, as indicated in the graph of the ROC curves with clinical or clinical and genetic factors, so the inventors assigned a score to each SNP and each risk factor. conventional, depending on its effect on stroke. The predictive model finally obtained allows the

distinction

of several categories of risk ictus, for

sort out

to the subjects in categories of risk low,

moderate

or high. The inventors believe than this model

could

to be a instrument important in medicine

preventive, since strokes place a huge burden on the health system and especially on patients. They could benefit from specific and individualized primary prevention. In fact, the combination of SNPs presented here could provide important new clinical information at least at the same level as classic risk factors such as hypertension, smoking or diabetes. The results obtained in this study will allow a better classification of individuals who present a moderate risk based on clinical factors, and who could really belong to the high risk group according to this model of genetic and clinical combination.

In this study, the inventors surprisingly found that from a combination of at least two of the SNPs described above, predicting the measured risk was better than any prediction tool described in the literature, and the inventors were able to predict that the Risk of having a stroke increased with the number of SNPs carried, as shown in the adjacent table. In other words, the combination of two of the mentioned SNPs indicated an individual risk of stroke of at least 3,417 times higher than a person who, under the same environmental conditions, does not present any of

these SNPs. As shown in the table, this relationship increases dramatically as the number of SNPs increases, not only through a summation effect, but through a multiplier effect, the model is optimal 5 when the number of SNPs is six. The combination of at least two SNPs is therefore a good model to predict the risk of having a stroke, but the combination of three, four or five SNPs is getting better up to six SNPs which is the optimal combination

10 to predict the risk of stroke.

Table 3. Combinations of SNPs Significantly Associated with Strokes.

Number of SNPs

OR 95% CI

one

one

-

2

3,417 0.670-17.426

3

6,159 1,261-30,089

4

10.26 2,095-50,326

5+

26,056 4,672-145,308

fifteen

Methods

and kits for its application

The

Present invention I know refers too to a method

for

the determination of the predisposition genetics to

2 O

diseases neurovascular, in particular ictus, in a

individual,

than understands the detection of the alleles of to the

less

two SNPs selected of the group than consists in

rs2276109,

rs10275136, rs310585, rs7956957, rs5742912 and

rs10947803

in a a shows of acid nucleic isolated of

25

a individual.

In

a preferred embodiment, saying method I know uses

for

decide the genetic predisposition to diseases

neurovascular,

in particular ictus, in a individual and

it comprises the detection of the alleles of three SNPs selected from the group consisting of rs2 2761 O9, rs10275136, rs310585, rs7956957, rs5742912 and rs10947803 in a nucleic acid sample isolated from an individual.

In a more preferred embodiment, said method is used to determine the genetic predisposition to neurovascular diseases, in particular strokes, in an individual and comprises the detection of the alleles of four SNPs selected from the group consisting of rs2276109, rs10275136, rs310585, rs7956957, rs5742912 and rs10947803 in a nucleic acid sample isolated from an individual.

In a more preferred embodiment, said method is used to determine the genetic predisposition to neurovascular diseases, in particular strokes, in an individual and comprises the detection of the alleles of five SNPs selected from the group consisting of rs2276109, rs10275136, rs310585, rs7956957, rs5742912 and rs10947803 in a nucleic acid sample isolated from an individual.

In the most preferred embodiment, said method is used to determine the genetic predisposition to neurovascular diseases, in particular strokes, in an individual that comprises the detection of the alleles of the six SNPs rs2276109, rs10275136, rs310585, rs7956957, rs5742912 and rs1094 7803 in a nucleic acid sample isolated from an individual.

In a particular embodiment, the detected alleles

of

agreement with anyone  of the realizations previous

would

a allele G of rs7956957, TO of rs310585, G of

r s 2 2 7 61 O 9,

and of r s 1 O 2 7 513 6, T of r s 57 4 2 912 and TO of

rs1094 7803.

The nucleic acid sample isolated from the individual used in any of the embodiments of the method of the present invention may comprise DNA or RNA. On the other hand, said nucleic acid sample can be amplified, generally by a chain reaction of the

polymerase (PCR), in the manner established in the art.

SNP detection can be done by

any of the methods known in the art, for

example without limiting them, by hybridizing a

5

nucleic acid or oligonucleotide probe, or by

radioactive, enzymatic, luminous or

fluorescent [35: page 4183 and 36: pages 246-247].

Said oligonucleotide probe or sequence comprises

a sequence that is completely complementary to a

1O

nucleic acid sequence comprising the SNPs

disclosed in the present invention (SEQ ID No. 1 to SEQ ID

No. 6).

In a preferred embodiment, the present invention

refers to the method according to any of the

fifteen

embodiments described above in which

minus two, three, four, five or six SNPs are combined with

one or more clinical variables associated with a disease

neurovascular, in particular stroke.

Preferably, said clinical variables are

twenty

they select between age, sex, smoking, hypertension,

diabetes and dyslipidemia.

In a particular embodiment, the present invention

refers to the method according to any of the embodiments

described above in which the alleles of said

25

SNPs are deduced using genetic tests.

In another embodiment, the present invention relates

to the method according to any of the described embodiments

above in which the alleles of said SNPs were

infer from activity levels or concentration of

30

proteins, or the corresponding levels of expression

of RNA from NOS3, SCNN1A, KCNK1 7, MMP12 and / or LRP, which are

modulated by the presence of genetic markers,

as shown in the results section.

In a particular embodiment, the present invention

35

refers to the method according to any of the embodiments

described above in which said at least two,

three, four, five or six SNPs are combined with the SNPs

rs405509, rs4934, rs2720723, rs2295778, rs7193343,

rs6007897, rs4044210 and rs2200733.

5

In a particular embodiment, the present invention

refers to the method according to any of the embodiments

described above, which further comprises the

comparison of the combination of alleles obtained for a

individual with the combination of alleles obtained for another

1 O

individual affected by neurovascular disease, in

particular stroke, and the detection of predisposition

genetics to neurovascular disease, in particular

stroke, if the individual to be analyzed presents the same

genetic load than the affected individual. In a

fifteen

more preferred embodiment, said other individual has a

family relationship with the individual to be analyzed.

Preferably, in all the embodiments of the

previous method said neurovascular disease is the

ictus.

twenty

In a particular embodiment, the method according

any of the embodiments described above

to determine an individual's risk of suffering a

neurovascular disease, preferably stroke, is applied

to an individual belonging to the Mediterranean population.

25

In a more particular embodiment, said individual

It belongs to the Spanish population.

The present invention further relates to a kit for

determine an individual's risk of suffering a

30

neurovascular disease, in particular stroke, which

understands:

(A) Two or more probes that allow the alleles of

SNPs rs2276109, rs10275136, rs310585, rs7956957,

rs10947803 and rs5742912.

35

(B) Instructions for using the kit and determining the risk

of an individual suffering from neurovascular disease, in

particular stroke, taking into account the combination of

SNPs filed.

In a preferred embodiment, two SNPs are detected

5

associated with neurovascular disease, in particular

ictus.

In a preferred embodiment, three SNPs are detected

associated with neurovascular disease, in particular

ictus.

10

In a more preferred embodiment, four are detected

SNPs associated with neurovascular disease, in

particular stroke.

In a more preferred embodiment, five are detected

SNPs associated with neurovascular disease, in

fifteen

particular stroke.

In the most preferred embodiment all six are detected

SNPs associated with neurovascular disease, in

particular stroke.

These two or more probes in the kit can be marked. Of

2 O

optionally, the kit may comprise such reagents

such as dilution solutions, reaction buffers, enzymes

polymerization and / or oligonucleotides (primers). To the

use radioactively labeled probes, hybridization

can be detected by autoradiography, scintillation,

25

counter or gamma counter.

In certain kit embodiments, the kit may

further comprise oligonucleotides (primers) for

amplification or sequencing and these can be

specific for sequences SEQ ID NOS 1 to 6.

30

The kit may also comprise reagents

for the labeling of one or more of the oligonucleotides

sequence specific, or you can understand

sequence specific labeled oligonucleotides. The

Useful markers include radioisotopes as well as groups

35

non-radioactive. Isotopic markers include 3H,

35 s, 32p, 125 I, 57Co and 14C. Isotopic markers can

be introduced into the oligonucleotide by techniques

described in the literature. Non-isotopic markers

they can be fluorescent, chemiluminescent, enzymes,

5

cofactors, enzyme substrates, haptens or others

ligands.

In a preferred embodiment, the present invention

refers to the kit according to any of the embodiments

described above in which said at least two,

10

three, four, five or six SNPs are combined with one or more

clinical variables associated with a disease

neurovascular, in particular stroke. Preferably,

These clinical variables are selected between age, sex,

smoking, hypertension, diabetes and dyslipidemia.

fifteen

In a particular embodiment, in the kit according

with the previous embodiments, the alleles of said

SNPs are deduced by genetic testing of others

markers or measuring activity levels or

protein concentration, or the corresponding

twenty

expression levels of NOS3, SCNNlA, KCNK17, MMP12 RNA

and / or LRP.

Preferably, in all embodiments

earlier in the kit, neurovascular disease is the

ictus.

25

In a particular embodiment, said kit for

determine an individual's individual risk of suffering

a neurovascular disease, preferably stroke, is

applies to an individual belonging to the population

Mediterranean.

3O

In a more particular embodiment, said individual

It belongs to the Spanish population.

The present invention further relates to the

use of the combination of at least two SNPs

selected from SNPs rs2276109, rs10275136,

35

rs310585, rs7956957, rs10947803 and rs5742912 and all

combination embodiments described herein

invention in a kit to determine the risk of a

individual suffering from neurovascular disease,

preferably the stroke.

5

In a particular embodiment, said use is applied to

an individual belonging to the Mediterranean population.

In a more particular embodiment, said individual

It belongs to the Spanish population.

The present invention further relates to the

10

use of the combination of at least two SNPs

selected from SNPs rs2276109, rs10275136,

rs310585, rs7956957, rs10947803 and rs5742912, indicative

of an individual's risk of disease

neurovascular, preferably stroke, and all

fifteen

combination embodiments described herein

invention as a genetic testing tool for a

neurovascular disease, preferably stroke.

In a particular embodiment, said use is applied to

an individual belonging to the Mediterranean population.

2 O

In a more particular embodiment, said individual

It belongs to the Spanish population.

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Claims (38)

1.-Combination of single nucleotide polymorphisms (SNPs) comprising rs2276109, rs10275136, rs310585, and rs5742912, indicative of an individual's risk of neurovascular disease. 2. The combination according to claim 1, wherein said combination of SNPs is combined with one or more of the SNPs rs7956957, rs10947803, rs405509, rs4934, rs2720723, rs2295778, rs7193343, rs6007897, rs4044210 and rs2200733. 3. The combination according to any of claims 1 or 2, wherein said combination of single nucleotide polymorphisms is combined with one or more clinical variables associated with neurovascular disease. 4. The combination according to claim 3, wherein said clinical variable is selected from age, sex, smoking, hypertension, diabetes and dyslipidemia. 5.-Combination of isolated polynucleotides included in genes associated with neurovascular diseases that each of said polynucleotides comprise a SNP of the combination of SNPs that includes rs2276109, rs10275136, rs310585, and rs5742912, indicative of an individual's risk of suffering a neurovascular disease , said isolated polynucleotides corresponding to SEQ ID N 1, 2, 3 and 6, respectively. or The combination according to any of claims 1 to 4, in which the alleles of said SNPs are deduced by genetic tests or by measuring the levels of activity or concentration of proteins, or the corresponding levels of expression of NOS3, SCNN1A RNA. , KCNK17, MMP12 or LRP. The combination according to claim S, in which the alleles of said SNPs are deduced by genetic tests or by measuring the levels of activity or concentration of the proteins, or the corresponding levels of RNA expression of NOS3, SCNN1A, KCNK17, MMP12. or LRP. 8. The combination according to any of claims 1-4 or 6, where the neurovascular disease is stroke. 9. The combination according to claim 5 or 7, where the neurovascular disease is stroke. 10.- A method for the determination of a genetic predisposition to an individual's neurovascular diseases, which comprises the detection of alleles of the combination of SNPs comprising rs2276109, rs10275136, rs310585, and rs5742912 in a nucleic acid sample isolated from An individual. The method according to claim 10, wherein an A allele is detected for rs310585, a G allele for rs2276109, a C allele for rs10275136, or a T allele for

rs5742912.

12.-

The method according anyone of the

claims

10 or eleven, where the shows of acid

Nucleic comprises DNA.

13.-

The method according anyone of the

Claims 10-12, where the nucleic acid sample comprises RNA. 14. The method according to any of claims 10-13, wherein said SNPs are detected by hybridization of a nucleic acid probe or oligonucleotide sequence, or by radioactive, enzymatic, light or fluorescent labels. 15. The method according to any of claims 10-14, wherein said combination of SNPs is combined with one or more clinical variables associated with neurovascular diseases. 16.- The method according to claim 15, wherein said clinical variables are selected among age, sex, smoking, hypertension, diabetes and dyslipidemia. 17. -The method according to any of claims 10 to 16, wherein said combination of SNPs

I know

combines with one or plus of the SNPs rs7956957,

rs10947803,

rs405509, rs4934, rs2720723, rs2295778,

rs7193343,

rs6007897, rs4044210 and rs2200733.

18.-

The method according anyone of the

Claims 10 to 17, in which the alleles of said SNPs are deduced by genetic tests or by measuring the levels of activity or concentration of the proteins, or the corresponding levels of RNA expression of NOS3, SCNN1A, KCNK17, MMP12 and / or LRP . 19.- The method according to any of claims 10 to 18, further comprising the comparison of the combination of alleles obtained for one individual with the combination of alleles obtained for another individual affected by neurovascular disease and detection of genetic predisposition to neurovascular disease if the individual to be analyzed has the same genetic load as the affected individual 20.-The method according to claim 19, where the other individual has a family relationship with the

individual

to analyze.

twenty-one.-

The method according anyone of the

claims

10 to twenty, where bliss disease

neurovascular

is the ictus.

22.-A kit to determine the risk of an individual to suffer said neurovascular disease that comprises,

(to)

Probes that allow detecting alleles of the combination of SNPs comprising rs2276109, rs10275136, rs310585, and rs5742912.

(b)

Instructions for using the kit and determining the individual's risk of suffering a neurovascular disease taking into account the presented combination of SNPs.

23. The kit according to claim 22, where one or more probes are marked. 24. The kit according to any of claims 22 or 23, which includes a reagent to detect the marker. 25. The kit according to any of claims 22 to 24, wherein said combination of SNPs is combined with clinical variables associated with neurovascular disease. 26.-The kit according to claim 25, wherein the clinical variables are selected between age, sex, smoking, hypertension, diabetes and dyslipidemia. 27.-The kit according to claims 22 to 26, wherein said combination of SNPs is combined with one or more of the SNPs rs7956957, rs10947803, rs405509, rs4934, rs2720723, rs2295778, rs7193343, rs6007897, rs4044210 and rs2200733. 28.-The kit according to claims 22 to 27, where the alleles of said SNPs are deduced by means of genetic tests or by measuring the levels of activity or concentration of the proteins, or the corresponding levels of RNA expression of NOS3, SCNN1A, KCNK17, MMP12 and / or LRP. 29.-The kit according to any of claims 22 to 28, wherein said neurovascular disease is stroke. 30.-The use of the combination according to any of claims 1 to 9 in a kit to determine the risk of an individual suffering from neurovascular disease. 31.-The use of the combination according to any of claims 1 to 9, as a genetic testing tool for neurovascular diseases. 32.-The use according to claims 30 or 31, wherein said neurovascular disease is stroke. 33.-The combination according to any of claims 1 to 9, wherein said individual belongs to the Mediterranean population. 34.-The combination according to any of claims 1 to 9, wherein said individual belongs to the Spanish population. 35.-The method according to any of claims 10 to 21, wherein said individual belongs to the Mediterranean population. 36.-The method according to any of claims 10 to 21, wherein said individual belongs to the Spanish population. 37.-The kit according to any of claims 22 to 29, wherein said individual belongs to the Mediterranean population. 38-The kit according to any of claims 22 to 29, wherein said individual belongs to the Spanish population. 39.-The use according to any of claims 15 30 to 32, where said individual belongs to the Mediterranean population. 40.-The use according to any of claims 30 to 32, where said individual belongs to the Spanish population.