ES2984299A1 - ACUTE CORONARY SYNDROME BIOMARKER (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Biomarker for acute coronary syndrome. The present invention is based on the detection of single nucleotide genetic polymorphisms that are associated with acute coronary syndrome (ACS). In particular, the present invention relates to nucleic acid molecules containing the polymorphisms, reagents for detecting the polymorphic nucleic acid molecules and proteins, and methods of using the nucleic acid and proteins, as well as methods of using reagents for their detection. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

ACUTE CORONARY SYNDROME BIOMARKER

Field of Technology

The present invention relates to genetic polymorphisms that are associated with myocardial infarction.

Background

Cardiovascular diseases are the leading cause of death in developed countries. Among them, acute coronary syndrome (ACS) is the most prevalent manifestation, and is associated with high morbidity and mortality. Although the ACS mortality rate has decreased in the last four decades, it is still the cause of approximately one third of all deaths in subjects aged >35 years.

The pathogenesis of ACS involves at least three interrelated processes: atherosclerosis, inflammation and thrombosis. It is well established that the different clinical presentations of ACS share a common pathophysiological mechanism, rupture or erosion of the atherosclerotic plaque. Even so, 20% of these events in young adults are not associated with arterial atherosclerosis and it has been described that 8% of hospital admissions for ACS in Spain are under 45 years of age. What is remarkable is that this group has significantly higher readmission rates than those patients who suffer the first event at older ages. At these ages, instead of atherosclerosis, the common triggers of ACS are coronary artery embolisms, thrombosis abnormalities and inflammation or spasms in the vessels. For all these reasons, young adults represent a unique and distinct subgroup of ACS. These differences in etiology and risk factors can lead to a different prognosis, as well as different evolution and treatment. This is why early detection of subclinical disease is crucial to prevent the occurrence of coronary events.

miRNAs (micro Ribonucleic Acids) are small non-coding RNAs, with a length of ~22 nucleotides (nt) in their mature form and a total of 2654 of these sequences have been characterized in humans. miRNAs regulate gene expression through the specific recognition of the sequence of their targets mainly (but not always) within the 3'UTR region (untranslated region). In this way, gene silencing is achieved either by blocking translation or by degrading the target mRNA.

Functional genetic polymorphisms and/or mutations affecting miRNA (miRNA-SNPs) can directly regulate the thrombotic response and indirectly influence the inflammatory response. Furthermore, genetic alterations are of particular interest in young subjects, due to the absence or lower incidence of risk factors in these individuals acquired throughout life, which allows for a clearer cause/effect association. To date, we do not know the frequency of miRNA-SNPs in miRNA genes potentially involved in inflammatory and atherothrombotic processes, nor their functional consequences. Thus, the role of genetic variants, specifically in miRNA genes, has not been studied in depth to date.

In the state of the art there are disclosures of methods for detecting SCA biomarkers based on genetic polymorphisms in miRNA:

The scientific article Ghaffarzadeh M, Ghaedi H, Alipoor B, et al.Association of MiR-149 (RS2292832) Variant with the Risk of Coronary Artery Disease.J Med Biochem.

2017;36(3):251-258. Published 2017 Jul 14. doi:10.1515/jomb-2017-0005 reports the rs2292832 polymorphism in miRNA-149 in patients with coronary artery disease vs. healthy controls.

The scientific article Goretti E, Wagner DR, Devaux Y.miRNAs as biomarkers of myocardial infarction: a step forward towards personalized medicine?.Trends Mol Med.

2014;20(12):716-725. doi:10.1016/j.molmed.2014.10.006 is a review that discloses the usefulness of SNPs located in miRNAs as biomarkers in myocardial infarction. This document mentions miRNA-296 and miRNA-199 but as part of different studies in which the expression levels have been analyzed (in the presence or not of the pathology) without taking into account the relevance of SNPs in said miRNAs.

Patent application WO2012094366A1 discloses a method for detecting coronary artery disease comprising determining the expression level of a series of miRNAs (Table 8). This document does not disclose any specific SNP associated with said miRNAs.

Patent EP2151507B1 discloses a method for determining an individual's risk of having a myocardial infarction that comprises detecting the presence of a SNP in a nucleotide sequence.

However, none of these documents focuses on young populations nor disclose the relevance of the SNPs of the present invention located in non-coding regions of the genome.

Description

In this specification, “individual,” “subject,” and “patient” are synonyms and are used interchangeably.

In this report, the term "young people" refers to individuals aged 45 years or younger.

In this specification the terms “miRNA” and “miR” are synonyms and are used interchangeably.

In this specification the term "non-coding nucleic acid" means "nucleic acid not coding for a protein"

The term "biological sample" includes any biological material that can be obtained from a subject, that can be preserved and that can store information about the subject's characteristic genetic composition. The sample of the invention can be of cellular, tissue or fluid type. In a particular embodiment of the invention, the sample is a blood and/or saliva sample.

The terms “polymorphism” and “single nucleotide polymorphism” (i.e. SNP) are used interchangeably and refer to a nucleotide sequence variation that occurs when a single nucleotide in the genome or other shared sequence differs between members of species or between paired chromosomes in an individual. A SNP may also be designated as a mutation with a low allele frequency of greater than about 1% in a defined population. Single nucleotide polymorphism according to the present application may only fall within coding sequences for miRNAs, non-coding regions of genes, or the intronic regions between genes.

A first object of the invention relates to an in vitro method for identifying a young individual having an altered risk of developing an acute coronary syndrome (ACS), comprising detecting the single nucleotide polymorphism (SNP) in the nucleotide sequence of SEQ ID NO: 1 and/or SEQ ID NO: 2 in the nucleic acids of said individual, wherein said individual has an increased risk of developing an acute coronary syndrome (ACS) due to:

- the presence of G at position 26 of SEQ ID NO: 1 or the presence of A at position 26 of its complement or

- the presence of C at position 26 of SEQ ID NO: 2 or the presence of T at position 26 of its complement

Clinical presentations of ACS covered by this invention include ST segment elevation myocardial infarction (STEMI), non-ST segment elevation myocardial infarction (NSTEMI), and unstable angina, with STEMI being the most common form of ACS.

SEQ ID NO: 1; GCGCTCTTCAGAGCCCTTCAGGAGAGCCTCCACCCAACCCTCTGA ATTAGG where the SNP of the invention is the G in position 26, which in some individuals there is an A instead, this change is associated with an increased risk of developing myocardial infarction. The code for this SNP is rs117258475 and is located in the miR-296, whose position in the genome is chr20:58817631 (GRCh38.p13).

SEQ ID NO: 3, shows the same sequence as SEQ ID NO 1, but with more flanking nucleotides at both ends and the [G/A] variation of the SNP

SEQ ID NO: 2; CGGCGGCTGCCCACATCCCCAGTCCCCATTGACCCCCCCCATCCTC TCAGTC where the SNP of the invention is the C at position 26, which in some individuals there is a T instead, this change is associated with an increased risk of developing myocardial infarction. The code for this SNP is rs557283175, whose position in the genome is chr9:128244522 (GRCh38.p13), located in intron 14 of dynamin 1, near miRNA-199b and miRNA-3154

SEQ ID NO: 4, shows the same sequence as SEQ ID NO 2, but with more flanking nucleotides at both ends and the [C/T] variation of the SNP

The rs117258475 has been studied as a possible marker for other diseases such as susceptibility to childhood acute lymphoblastic leukemia, a disease very far removed from cardiac pathologies, but it was found not to be determinant in these models. Doctoral ThesisGenetic variants involved in ChildhoodAcute Lymphoblastic Leukemia Susceptibilityby Ángela Gutiérrez Camino. University of the Basque Country, Faculty of Medicine and Dentistry. Leioa 2016.

The use of genetic biomarkers, such as SNPs, has the advantage that they are constant and do not vary throughout the individual's life, which allows for early diagnosis and therefore it is not necessary to observe the evolution of the individual over the years.

In a particular embodiment, the young individual is a human being up to 45 years old. The term does not denote a specific sex. Thus, it is intended to cover adult and newborn subjects, as well as fetuses, whether male or female.

The term "increased risk" refers to increases above baseline levels, e.g., compared to a control, e.g., the overall risk of the global population that does not possess any of the SNPs of the invention.

In another particular embodiment, the nucleic acid is obtained from cells of a biological sample of said individual.

In another particular embodiment, the nucleic acid is a non-coding nucleic acid for a protein, preferably it is a nucleic acid that codes for a miRNA or a non-coding region adjacent to the coding region for a miRNA. Adjacent region means that it is located at least 100 base pairs from the 3' or 5' end of the coding region for a miRNA, preferably at least 80 base pairs, more preferably at least 60 base pairs, even more preferably at least 40 base pairs and even more preferably at least 20 base pairs from the 3' or 5' end.

In another particular embodiment, the biological sample is blood, saliva or buccal swabs, preferably peripheral blood. As these techniques are non-invasive, the patient is not subjected to risks inherent to surgical procedures.

In another particular embodiment, the method of the invention comprises isolating said nucleic acid from said biological sample before said step of detecting one or more of the SNPs.

Those skilled in the art will readily recognize that the analysis of the nucleotides present according to the method of the invention in the nucleic acid of an individual can be performed by any method or technique capable of determining the nucleotides present at a polymorphic site. As is obvious in the art, the nucleotides present at the polymorphic markers can be determined either from either nucleic acid chain or from both chains.

Virtually any method known to those skilled in the art can be used. Perhaps the most direct method is to actually determine the sequence of either the genomic DNA or the cDNA and compare these sequences to the SNPs of known alleles of the gene. This can be a fairly expensive and time-consuming procedure. However, this technology is quite common and well understood.

Any of a variety of methods that exist to detect sequence variations can be used in the methods of the invention. The particular detection method used is not important in estimating the risk of SCA.

There are other commercially available methods for high-throughput SNP identification without using direct sequencing technologies. For example, Illumina's Veracode Technology, Taqman® SNP genotyping chemistry, and KASPar SNP genotyping chemistry.

In another particular embodiment, the detection comprises the amplification of nucleic acids, preferably carried out by polymerase chain reaction.

SNP detection is performed by allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, sequencing, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism analysis, or denaturing gradient gel electrophoresis (DGGE).

The SNPs of the invention can be detected by means of the primers (polynucleotide sequences) of Table 2 or a variant of said sequences having at least 80% identity, more preferably 85% identity, even more preferably at least 90% identity, and even more preferably 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or even up to 99% identity with respect to the polynucleotide sequences of Table 2. A person skilled in the art will understand that to detect the SNP of SEQ ID NO: 1, primers SEQ ID NO: 5, 6 and 7 should be used as well as variations thereof, and to detect the SNP of SEQ ID NO: 2, primers SEQ ID NO: 8, 9 and 10 should be used as well as variations thereof.

As used herein, an "amplified polynucleotide" is a nucleic acid molecule containing the SNP of the invention that has been increased in amount at least two-fold by any nucleic acid amplification method performed in vitro compared to its initial amount in a test sample. In other preferred embodiments, an amplified polynucleotide results from an increase of at least ten-fold, fifty-fold, one hundred-fold, one thousand-fold, or even ten thousand-fold compared to its initial amount in a test sample. In a typical PCR amplification, a polynucleotide of interest is typically amplified at least fifty thousand-fold in amount relative to unamplified genomic DNA, but the precise amount of amplification needed for an assay depends on the sensitivity of the downstream detection method used.

In another particular embodiment, the method comprises contacting the nucleic acids of the individual with a detection reagent, and determining which nucleotide is present or absent at position 26 of SEQ ID NO: 1 and/or position 26 of SEQ ID NO: 2.

A SNP detection reagent is an isolated or synthetic DNA or RNA polynucleotide probe or primer or an RNAP oligomer, or a combination of DNA, RNA, and/or RNAP, that hybridizes to a segment of a target nucleic acid molecule containing the SNP of SEQ ID NO:1 and/or 2. A detection reagent in polynucleotide form may optionally contain modified base analogs, intercalators, or minor groove binders. A detection reagent in polynucleotide form may optionally contain modified base analogs, intercalators, or minor groove binders. Multiplex detection reagents, such as probes, may, for example, be fixed to a solid support (e.g., arrays or beads) or supplied in solution (e.g., probe/primer sets for enzymatic reactions such as PCR, RT-PCR, TaqMan assays, or primer-extension reactions) to form a SNP detection kit.

In a preferred embodiment, the detection reagent is a polynucleotide probe.

A probe or primer is typically a substantially purified oligonucleotide or RNA oligomer. Such an oligonucleotide typically comprises a region of complementary nucleotide sequence that hybridizes under stringent conditions to at least about 8, 10, 12, 16, 18, 20, 22, 25, 30, 40, 50, 60, 100 (or any number in between) or more consecutive nucleotides in a target nucleic acid molecule. Depending on the particular assay, the consecutive nucleotides may include the target SNP position, or be a specific region sufficiently proximal 5' and/or 3' to the SNP position to perform the desired assay.

It will be apparent to one skilled in the art that such primers and probes are directly useful as reagents for genotyping the SNPs of the present invention, and can be incorporated into any kit/system format.

The 3' end of the probe can hybridize to the SNP in the nucleic acid, optionally the probe is labeled with a reporter dye.

In another particular embodiment, the detection is carried out using:

- at least one allele-specific probe or primer comprising at least 8 contiguous nucleotides of SEQ ID NO: 1, wherein said at least 8 contiguous nucleotides of SEQ ID NO: 1 include position 26 of SEQ ID NO: 1, or the complement thereof, in the method according to any of claims 1-10 and/or

- at least one allele-specific probe or primer comprising at least 8 contiguous nucleotides of SEQ ID NO: 2, wherein said at least 8 contiguous nucleotides of SEQ ID NO: 2 include position 26 of SEQ ID NO: 2, or the complement thereof, in the method according to any of claims 1-10.

Another additional object of the invention relates to the use of an amplified polynucleotide containing the single nucleotide polymorphism (SNP) at position 26 of SEQ ID 1, or the complement thereof, in the method according to any of claims 1-10, wherein the amplified polynucleotide is between about 16 and about 1,000 nucleotides in length.

In a particular embodiment, the amplified polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1.

Another additional object of the invention relates to the use of an amplified polynucleotide containing the single nucleotide polymorphism (SNP) at position 26 of SEQ ID 2, or the complement thereof, in the method according to any of claims 1-10, wherein the amplified polynucleotide is between about 16 and about 1,000 nucleotides in length.

In a particular embodiment, the amplified polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2.

Another additional object of the invention relates to the use of an isolated polynucleotide that specifically hybridizes with a nucleic acid molecule containing:

the single nucleotide polymorphism (SNP) at position 26 of SEQ ID NO: 1, or the complement thereof, in the method according to any of claims 1-10 and/or

the single nucleotide polymorphism (SNP) at position 26 of SEQ ID NO: 2, or the complement thereof, in the method according to any of claims 1-10.

In a particular embodiment, the isolated polynucleotide has a length of 8-70 nucleotides or wherein the isolated polynucleotide is an allele-specific probe or an allele-specific primer.

One of skill in the art will understand that, based on the SNP and associated sequence information disclosed herein, detection reagents can be developed and used to analyze any SNP described herein individually or in combination, and such detection reagents can be readily incorporated into one of the established kit or system formats that are well known in the art. The terms "kits" and "systems" as used herein in the context of SNP detection reagents are intended to refer to such things as combinations of multiple SNP detection reagents, or one or more SNP detection reagents in combination with one or more types of elements or components (e.g., other types of biochemical reagents, containers, packaging such as packaging intended for commercial sale, substrates to which the SNP detection reagents are affixed, electronic hardware components, etc.). Accordingly, the present invention further provides the use of SNP detection kits and systems, including but not limited to, packaged primer and probe sets (e.g., TaqMan primer/probe sets), nucleic acid molecule arrays/microarrays, and beads containing one or more probes, primers, or other detection reagents in the method of the present invention. The kits/systems may optionally include various electronic hardware components; for example, arrays ("DNA chips") and microfluidic systems ("lab-on-a-chip" systems) provided by various manufacturers often include hardware components. Other kits/systems (e.g., primer/probe sets) may not include electronic hardware components, but may be comprised of, for example, one or more SNP detection reagents (along with, optionally, other biochemical reagents) packaged in one or more containers.

Another additional object of the invention relates to the use of a kit comprising reagents for detecting the identity of the nucleotide at position 26 within a nucleic acid sequence selected from the group of SEQ ID NO: 1 and 2, or the equivalent position in SEQ ID NO: 3 and 4.

In some embodiments, a SNP detection kit typically contains one or more detection reagents and other components (e.g., a buffer, enzymes such as DNA polymerases or ligases, chain extension nucleotides such as deoxynucleotide triphosphates, and in the case of Sanger-type DNA sequencing reactions, chain terminating nucleotides, positive control sequences, negative control sequences, and the like) necessary to carry out an assay or reaction, such as amplification and/or detection of a SNP-containing nucleic acid molecule. A kit may further contain means for determining the amount of a target nucleic acid, and means for comparing the amount to a standard, and may comprise instructions for using the kit to detect the SNP-containing nucleic acid molecule of interest. In one embodiment of the present invention, the use of kits containing the reagents necessary to carry out one or more assays to detect the SNP disclosed herein in the method of the present invention is provided. In a preferred embodiment of the present invention, SNP detection kits/systems are in the form of nucleic acid arrays, or compartmentalized kits, including microfluidic/lab-on-a-chip systems.

SNP detection kits/systems may contain, for example, one or more probes, or probe pairs, that hybridize to a nucleic acid molecule at or near each target SNP position. Multiple allele-specific probe pairs may be included in the kit/system to simultaneously assay a large number of SNPs, at least one of which is a SNP of those described herein. In some kits/systems, the allele-specific probes are immobilized to a substrate such as an array or bead. For example, the same substrate may comprise allele-specific probes to detect at least 1; 10; 100; 1000; 10,000; 100,000 (or any other number in between) or substantially all of the SNPs described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims, the word "comprises" and variations thereof are not intended to exclude other technical features, additives, components or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention.

Examples of implementation

In order to identify new SNPs that could have pathological consequences in acute coronary syndrome, using massive sequencing, we collected a first cohort of 150 patients and 150 healthy controls matched in age and sex with the patients, that is, for each patient a healthy control of the same age and sex as similar as possible was sought, Table 1.

Table 1: Characteristics of 150 patients and 150 controls in the study

All included patients met the criteria of having suffered a primary ACS event before the age of 45 years. In patients with a recurrent event, the inclusion criterion was that the first ACS event had occurred before the age of 45 years. The presence of congenital vascular malformation or traumatic ACS was ruled out in patients who suffered a secondary ACS event. To reflect the "real life" of clinical practice, no other specific exclusion criteria were established. Participating healthy volunteers were selected from regular blood donors, matched in age and sex to the patients.

We performed blood extractions from these subjects and purified the genomic DNA. We made 60 pools of 5 samples, that is, 30 pools from 5 patients each and 30 pools from 5 healthy controls. We obtained whole blood from all subjects and centrifuged it (2500xg, 15min) to purify the gDNA from the buffy coat using the QIAmpR DNA Blood Mini kit (Qiagen, Madrid, Spain) manually. The samples were stored at -20°C until use. An expert in the field would know how to use another standard protocol in the technical field to perform the extraction.

We made a selection of miRNA genes that were expressed in disease-related cells, such as platelets, leukocytes, cardiomyocytes and endothelial cells. Specifically, two criteria were used:

1. That they are expressed in one or more of the following cells or tissues: platelets, leukocytes, liver, cardiomyocytes and endothelial cells.

2. That their published expression levels (quantified in expression arrays) were above 100 copies (equivalent to the number of RNA molecules) in the case of platelets, leukocytes and liver; for cardiomyocytes and endothelial cells, expression or non-expression was considered.

Using this analysis, 365 miRNAs were selected for the sequencing assay (Next generation sequencing, NGS). Since miRNA coding regions are very small, the first primers for NGS were designed to cover more genomic sequence at both ends, with the idea of covering the region of interest without technical problems. For this reason, some of the SNPs identified are outside, but in a region adjacent to, the coding region of a miRNA. These SNPs may have regulatory functions on the miRNA in question. It is important to note that when choosing these 365 miRNAs, it was not taken into account whether they had been related in the state of the art with acute coronary syndrome.

Massive sequencing was then carried out on the genomic DNA regions encoding miRNA using the Ion PGM system from Ion Torrent (Life Technologies) as is routinely used in the technical field. Once these results were obtained, various statistical tests were performed to ensure the reliability of the results obtained. The first test performed was a Fisher Test, which verified that the same number of readings appeared from the two DNA strands, in both possible directions (forward and reverse). The second test was a Binomial Test, which verified that the variant readings exceeded a minimum established percentage. As we were working with pools of 5 samples, 10 alleles were studied in each pool at a time. This means that it is necessary for the variants to appear in at least 10% of the readings.

After discarding the underrepresented or non-real variants, we found a total of 1175 SNPs and we selected only those variants represented at least 4 times more in patient pools compared to pools of healthy controls in order to eliminate the most frequent SNPs in the general population, which had a frequency described in databases. With this criterion, we obtained 10 SNPs of interest.

To validate these 10 SNPs of interest (see Table 3), we expanded the cohort with another 150 patients with acute coronary syndrome and 150 healthy controls. We performed individual genotyping of these 10 SNPs in a total of 300 patients and 300 age- and sex-matched healthy controls. Genotyping was performed using KASP probes (allele-specific competitive PCR) designed and validated by the company (LGC Biosearch Technologies). Table 2 shows the probes used for each SNP. The sequence listing indicates the SEQ ID corresponding to each of the sequences in Table 2.

Table 2. Probes used in competitive allele-specific PCR (KASP)

Table 3 shows the results of individual genotyping of controls and patients for the SNPs of interest.

Table 3: Genotyping of SNPs of interest

EMAF: (European major allied frequency), and is the data that indicates in the databases the frequency of the alteration in the European population.

The previous results led us to select 2 SNPs that were found in a higher percentage of patients, in both cases without being expressed in any control: rs117258475 and rs557283175.

To validate the interest of these SNPs and their relationship with acute coronary syndrome, we expanded our control group with a third cohort of 300 healthy controls, blood donors, the characteristics of these patients are found in Table 4. In this new batch of 300 subjects, 2 carriers of rs117258475 and one carrier of rs557283175 were found.

Table 4: Characteristics of the 300 patients and 300 healthy controls

Thus, the results obtained in 600 healthy subjects vs. 300 patients are those shown in Table 5. As previously mentioned, 2/600 healthy controls versus 11/300 patients (0.33% vs. 3.7%) and 1/600 control versus 5/300 patients (0.17% vs. 1.6%) are significant figures that relate these variants with ACS. These results demonstrate that the frequency of SNPs associated with a higher risk of suffering an acute myocardial infarction is around 10 times higher in patients who have suffered from this disease compared to healthy individuals.

Table 5 Presence of SNPs of interest.

Functional tests

It has been determined that the expression in cells that present the rs117258475 variant of miRNA-296, presents an affectation in said miRNA and, this change, influences genes involved in inflammation, the generation of atherosclerotic plaque and/or other processes involved in the early development of acute coronary syndrome.

Claims (20)

1. An in vitro method for identifying a young individual who has an altered risk of developing an acute coronary syndrome (ACS), comprising detecting the single nucleotide polymorphism (SNP) in the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in the nucleic acids of said individual, wherein said individual has an increased risk of developing ACS due to the presence of G at position 26 of SEQ ID NO: 1 or the presence of A at position 26 of its complement or the presence of C at position 26 of SEQ ID NO: 2 or the presence of T at position 26 of its complement. 2. The method of the preceding claim, wherein SCA comprises ST-segment elevation myocardial infarction, non-ST-segment elevation myocardial infarction, and unstable angina. 3. The method according to any one of the preceding claims, wherein said nucleic acid has been isolated from cells of a biological sample of said individual. 4. The method according to any one of the preceding claims, wherein the nucleic acid is a non-coding nucleic acid, preferably a miRNA. 5. The method according to any one of the preceding claims 1 to 3, wherein the nucleic acid is a non-coding region located at least 100 base pairs from the 3' or 5' end of the coding region for a miRNA. 6. The method according to any one of the preceding claims, wherein said biological sample is blood, saliva or buccal swabs. 7. The method according to any one of the preceding claims, further comprising isolating said nucleic acid from said biological sample prior to detecting at least one SNP. 8. The method according to any one of the preceding claims, wherein the detection comprises the amplification of nucleic acids, preferably carried out by polymerase chain reaction. 9. The method according to any one of claims 1 to 8, comprising contacting the nucleic acids of the individual with a detection reagent, and determining which nucleotide is present or absent at position 26 of SEQ ID NO: 1 and/or position 26 of SEQ ID NO: 2. 10. The method of claim 6, wherein the detection reagent is a polynucleotide probe. 11. The method according to any one of claims 9-10, wherein the 3' end of the probe hybridizes to the SNP in the nucleic acid. 12. The method according to any one of claims 9-11, wherein the probe is labeled with a reporter dye. 13. The method of claim 1, wherein said detection step is carried out using at least one allele-specific probe or primer comprising at least 8 contiguous nucleotides of SEQ ID NO: 1, wherein said at least 8 contiguous nucleotides of SEQ ID NO: 1 include position 26 of SEQ ID NO: 1, or the complement thereof, in the method according to any of claims 1-12 and/or at least one allele-specific probe or primer comprising at least 8 contiguous nucleotides of SEQ ID NO: 2, wherein said at least 8 contiguous nucleotides of SEQ ID NO: 2 include position 26 of SEQ ID NO: 2, or the complement thereof, in the method according to any of claims 1-12. 14. Use of an amplified polynucleotide containing the single nucleotide polymorphism (SNP) at position 26 of SEQ ID 1, or the complement thereof, in the method according to any of claims 1-12, wherein the amplified polynucleotide is between about 16 and about 1,000 nucleotides in length. 15. Use according to the preceding claim, wherein the amplified polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1. 16. Use of an amplified polynucleotide containing the single nucleotide polymorphism (SNP) at position 26 of SEQ ID 2, or the complement thereof, in the method according to any of claims 1-12, wherein the amplified polynucleotide is between about 16 and about 1,000 nucleotides in length. 17. Use according to the preceding claim, wherein the amplified polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2. 18. Use of an isolated polynucleotide that specifically hybridizes to a nucleic acid molecule containing: the single nucleotide polymorphism (SNP) at position 26 of SEQ ID NO: 1, or the complement thereof, in the method according to any of claims 1-10 and/or the single nucleotide polymorphism (SNP) at position 26 of SEQ ID NO: 2, or the complement thereof, in the method according to any of claims 1-12. 19. Use according to the preceding claim, wherein the isolated polynucleotide has a length of 8-70 nucleotides or wherein the isolated polynucleotide is an allele-specific probe or an allele-specific primer. 20. Use of an assay kit comprising the polynucleotide as defined in any one of claims 11-16, a buffer, and an enzyme in the method according to any one of claims 1-12.