WO2019122340A1 - In vitro method for predicting a predisposition to acute coronary syndrome - Google Patents

Abstract

The invention relates to the field of molecular diagnosis. The invention provides in vitro methods for detecting a genetic predisposition to acute coronary syndrome in a subject. More specifically, the invention relates to an in vitro method for detecting a genetic predisposition to acute coronary syndrome in a human subject, said method comprising determining the presence of a PLIN1 disease associated variant in a biological sample from said subject, wherein said PLIN1 disease associated variant is an allele of the PLIN1 gene comprising at least a genetic mutation in the open reading frame of the PLINI gene, and the detection of said PLIN1 disease associated variant is indicative of a genetic predisposition to acute coronary syndrome in said subject.

Description

IN VITRO METHOD FOR PREDICTING A PREDISPOSITION TO ACUTE

CORONARY SYNDROME

TECHNICAL FIELD The invention relates to the field of molecular diagnosis. The invention provides in vitro methods for detecting a genetic predisposition to acute coronary syndrome in a human subject. More specifically, this method comprises determining the presence of a PLIN1 disease associated variant in a biological sample from said subject, wherein said PLIN1 disease associated variant is an allele of the PLIN1 gene comprising at least a genetic mutation in the open reading frame of the PLIN1 gene. The detection of said PLIN1 disease associated variant is indicative of a genetic predisposition to acute coronary syndrome in said subject.

BACKGROUND

Acute coronary syndrome (ACS) is the result of atheroma plaque rupture or erosion. It is the second cause of death in developed countries and the first cause of premature death (INSEE). Among the risk factors for coronary artery disease (CAD), heredity is well documented (Bachmann 2012; Vinkhuyzen AA 2013). Although a family history of CAD is associated with increased risk of ACS, the mechanisms involved remain not fully elucidated. A network of proteins were suggested to participate in atheroma plaque development and rupture such as proteins involved in lipid metabolism but to date, trials have failed to identify molecular anomalies responsible for premature ACS (Nikpay et al, 2015)

Genetic partial lipodystrophies refer to a group of rare diseases characterized by the abnormal distribution of adipose tissue and metabolic anomalies including insulin resistance or type 2 diabetes and hypertriglyceridemia. These anomalies result in atherosclerosis development ultimately leading to CAD (Vantyghem et al 2012, Fardet et al, 2013). Molecular causes of partial lipodystrophies include a broad spectrum of proteins which can be classified in 2 categories. The first group of proteins is key to adipogenesis (or adipocytes differentiation) and includes proteins involved in premature aging syndromes. The second group includes proteins involved in the adipocyte lipid droplet formation and regulation (Vigouroux 2011).

Mazzali et al (2015) have studied heart fat infiltration in patients with or without coronary artery diseases. They reported an accumulation of perilipin-l and -2 in patients with coronary artery diseases. Meirhaeghe et al (2006) further studied the impact of perilipin polymorphisms in a French population. In particular, they evaluated whether SNP rs8179070 is associated to obesity or type 2 diabetes. However, the study concluded that this SNP, as well as two others, are not associated to an increased risk in obesity or type 2 diabetes.

Therefore, to the knowledge of the inventors, there is still no simple and sensitive in vitro method for detecting ACS predisposition.

Clinically, in genetic partial lipodystrophies, the fat loss is predominant on the limbs but can be present on abdomen and chest in the most severe forms. In parallel, adipose tissue accumulates on the face and neck and on organs which are not supposed to store fat including the liver and the heart. The severity of these syndromes varies ranging from early onset right after puberty to later onset with an expression similar to the metabolic syndrome (Hussain I, 2016).

The inventors hypothesized that minor forms of lipodystrophy could be involved in premature CAD responsible for ACS. In an attempt to diagnose lipodystrophy in patients with ACS, the inventors investigated a series of 46 individuals who suffered a premature ACS by high throughput sequencing of a panel of genes involved in genetic partial lipodystrophies, diabetes and/or obesity.

They surprisingly observed a high rate of mutation in the PLIN1 gene encoding for perilipin- 1 in patients who suffered a premature ACS compared to controls. Indeed, four missense mutations in the PLIN1 gene were identified with a high frequency compared to the observed prevalence in the match population. In particular, the inventors showed that not only mutations in PLIN1 were significantly more frequent than in the control group, but also that the presence of any mutation among the identified missense mutations in the PLIN1 gene significantly increases the risk (at least eight times higher) to develop an acute coronary syndrome. The inventors also surprisingly identified that these four mutations locate close to phosphorylation sites of the Perilipin 1 protein and are thus predicted to be deleterious to the phosphorylation level of the protein.

SUMMARY

The present disclosure relates to an in vitro method for detecting a genetic predisposition to acute coronary syndrome in a subject. The method comprises determining the presence of a PLIN1 disease associated variant in a biological sample from said subject, wherein said PLIN1 disease associated variant is an allele of the PLIN1 gene comprising at least a genetic mutation in the open reading frame of the PLIN1 gene, and the detection of said PLIN1 disease associated variant is indicative of a genetic predisposition to acute coronary syndrome in said subject.

In specific embodiments, said genetic mutation is one or more single nucleotide polymorphism (SNP) non-synonymous mutation in the PLIN1 coding sequence.

Typically, said PLIN1 disease associated variant may be a genetic mutation in the PLIN1 gene altering the predicted amino acid sequence encoded by the PLIN1 gene.

In a specific embodiment, said genetic mutation is a non-synonymous mutation in the coding sequence of PLIN1 gene that alters the phosphorylation level of the Perilipin-l protein in said subject. For example, said PLIN1 mutation may be a non-synonymous SNP resulting in a single amino acid substitution in a phosphorylation site of Perilipin-l protein selected from the group consisting of SEQ ID NOs: 3-7.

Preferred examples of said PLIN1 disease associated variant include SNPs selected from the group consisting of rsl50004289 as shown in SEQ ID NO:9, rsl3927l800 as shown in SEQ ID NO:l l, rs8l79070 as shown in SEQ ID NO:l3, and/or rsl49989895 as shown in SEQ ID NO:l5.

In specific embodiments, the presence of a PLIN1 disease associated variant is determined by a SNP detection method selected from the group consisting of sequencing methods including Sanger sequencing, next generation sequencing, pyrosequencing, sequencing by ligation; PCR-based methods including PCR, real-time PCR, quantitative PCR and high- resolution melting analysis; and/or any other SNP genotyping techniques such as amplification refractory mutation system (ARMS), restriction fragment length polymorphism (RFLP) analysis, denaturing gradient gel electrophoresis (DGGE), single strand conformation polymorphism (SSCP), and allele discrimination methods including allele-specific hybridization, molecular beacons, allele-specific single base primer extension, Flap endonuclease discrimination, 5’nuclease, oligonucleotide ligation and micro-array analysis of genomic DNA.

The present disclosure also provides a kit for performing the in vitro method as described above, said kit comprising: probes or primers for specifically detecting one or more SNPs in PLIN1 gene coding sequence; optionally, instructions for use of the kit. In preferred embodiments of the kit, the kit comprises probes having nucleic acids for the specific detection of one or more of the following SNPs: rsl50004289 (SEQ ID NO:8 or 9), rsl3927l800 (SEQ ID NO:lO or 11) , rs8l79070 (SEQ ID NO:l2 or 13) and/or rsl49989895 (SEQ ID NO: 14 or 15). In other specific embodiments, the kit includes at least a probe that binds specifically to a nucleic acid comprising one SNP allele of the preferred SNPs as defined above, and/or primers that are capable of amplifying a nucleic acid comprising one preferred SNP allele as defined above.

Typically, said probe may be selected from the probes comprising any one of SEQ ID NOs 24-31 or their fragments including the SNP allele with corresponding 5’ and 3’ flanking regions of at least 5 nucleotides. Alternatively, said primers may be selected among the following groups of pair of primers:

SEQ ID NO: 16 and SEQ ID NO: 17 for rs 150004289;

SEQ ID NO:l8 and SEQ ID NO:l9 for rsl3927l800;

SEQ ID NO:20 and SEQ ID NO:2l for rs8179070 and/or;

SEQ ID NO: 22 and SEQ ID NO:23 for rs 149989895.

Another aspect of the disclosure concerns a method for preventing a subject from developing acute coronary syndrome, comprising

a) identifying whether said subject is at increased risk of developing acute coronary syndrome according to the above-described in vitro method, b) treating said subject identified at step a) with a suitable treatment for preventing acute coronary syndrome.

DETAILED DESCRIPTION

The present disclosure relates to an in vitro method for detecting a genetic predisposition to acute coronary syndrome in a subject, said method comprising determining the presence of a PLIN1 disease associated variant in a biological sample from said subject, wherein said PLIN1 disease associated variant is an allele of the PLIN1 gene comprising at least a genetic mutation in the open reading frame of the PLIN1 gene, and the detection of said PLIN1 disease associated variant is indicative of a genetic predisposition to acute coronary syndrome in said subject.

As used herein, a subject has a genetic predisposition to a disease when this subject has a higher risk to develop such disease, compared to the average risk in a population to develop such disease. Of course, a predisposition does not mean that the subject will develop the disease. The method may not give a precise probability for such risk but may give a relative risk assessment as compared to the average risk in a given population.

The subject according to the present method can be a man or a woman. In some embodiments the subject is preferably a man. Typically the subject is less than 55 yo. In particular, the subject is a man of less than 50 yo or a woman of less than 55 yo. In some embodiments, the subject is more than 35 yo, typically, the subject is at least 38 yo, notably at least 40 yo, or at least 45 yo.

In some embodiment also the subject is an active smoking subject and/or has a smoking history of at least 20 years.

As used herein, “Detecting a predisposition to acute coronary syndrome” therefore includes detecting a higher risk of developing the disease, or determining the susceptibility of that subject to develop the disease or to have an increased risk in developing premature acute coronary syndrome.

As used herein, the term“acute coronary syndrome” or“ACS” refers to any group of clinical symptoms compatible with acute myocardial ischemia and includes unstable angina (UA), non— ST-segment elevation myocardial infarction (NSTEMI), and ST- segment elevation myocardial infarction (STEMI). ACS is more specifically defined in the guidelines (Grundy et al. 2004; Philips et al. 2007; Nasir et al. 2007; Roffi et al. 2016). In specific embodiments, this excludes ACS related to non-atherosclerosis cause including trauma, dissection, septic embol, toxic or coronary artery dissection.

As used herein,“premature ACS” refers to age of onset (<50yo for men and <55yo for women).

Providing a biological sample

The method of the disclosure can be carried out on any appropriate biological sample obtained from a subject.

As used herein, the term“biological sample” refers to a sample that contains either nucleic acid or protein materials reflecting the genomic information of cells, tissue or organs of the subject. In preferred embodiments, it refers to a sample containing nucleic acid materials reflecting the genomic information of cells, tissue or organs of the subject.

In one specific embodiment, said sample is obtained from a mammal, for example from rodents, cats, dogs, horses, primates or human. In one preferred embodiment, said sample is obtained from a human subject. For example, said biological sample may be obtained from urine, blood including without limitation peripheral blood or plasma, stool, sputum, bronchoalveolar fluid, endotracheal aspirates, wounds, cerebrospinal fluid, lymph node, exsudate and more generally any human biopsy tissue or body fluids, tissues or materials.

Genomic DNA of individuals can readily be purified from individual blood sample by conventional methods well known in the art. Therefore, in a preferred embodiment, said biological sample is blood, more preferably human blood sample.

PLIN1 disease associated variant

As used herein, the term“disease associated variant” means any genotypic biomarker, such as a genetic mutation, that is associated with an increased risk of developing the disease.

The inventors have identified specific single nucleotide polymorphisms of PLIN1 that are associated to an increased risk of developing acute coronary syndrome.

The inventors have further shown that some of such SNPs are associated to alteration of the amino acid sequence of Perilipin-l protein and/or may be associated to a change in the phosphorylation status of the corresponding Perilipin-l protein.

Therefore, in a specific embodiment, predisposition to acute coronary syndrome may be assessed by detecting a genetic mutation in the PLIN1 gene, which genetic mutations alters the predicted amino acid sequence encoded by PLIN1 gene.

The human wild-type PLIN1 gene is the gene comprising the nucleotide sequence as shown in SEQ ID NO:l, and coding for the polypeptide Perilipin-l of SEQ ID NO:2.

By alteration, it is meant that at least one amino acid is substituted, deleted or inserted as compared to the wild-type sequence of Perilipin-l protein (for example SEQ ID NO:2).

In one specific embodiment, predisposition to acute coronary syndrome may also be detected by detecting a genetic mutation in the coding sequence of PLIN1 altering the phosphorylation level of the Perilipin-l protein in a subject.

“Alteration of the phosphorylation level” includes in particular that the corresponding Perilipin-l protein cannot be phosphorylated at one or more of the phosphorylation sites in vivo, or is less phosphorylated as compared to the corresponding wild-type Perilipin-l protein which do not have the genetic mutation in PLIN1 gene. Such alteration can be assessed for example in an in vitro assay for determination the phosphorylation level of the perlipin-l protein. A“genetic mutation” refers to one or several nucleotide change(s) in the wild type sequence of the corresponding gene. Said genetic mutation refers to a germline mutation that can be considered as a causal mutation (present only in patients at increased risk of developing the disease, with a direct causal link with the disease). For example, mutations can occur within a gene or chromosome, including specific changes in non-coding regions of a chromosome, for instance changes in or near regulatory regions of genes. Types of mutations include, but are not limited to, base substitution point mutations (which are either transitions or transversions), deletions, and insertions.

In specific embodiments of the methods of the invention, a disease associated variant is one or more single nucleotide polymorphism (SNP) in the open reading frame of the PLIN1 gene. Preferably, said PLIN1 disease associated variant is a SNP non-synonymous mutation in the PLIN1 coding region.

As used herein, a "single nucleotide polymorphism (SNP)" is a single base (nucleotide) polymorphism in a DNA sequence among individuals in a population. Typically in the literature, a single nucleotide polymorphism (SNP) may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. SNPs within a coding sequence will not necessarily change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code. A SNP in which both forms lead to the same polypeptide sequence is termed "synonymous" (sometimes called a silent or isosemantic mutation)-if a different polypeptide sequence is produced they are "nonsynonymous". A nonsynonymous change may either be missense or "nonsense", where a missense change results in a different amino acid, while a nonsense change results in a premature stop codon. The exact sequence of a SNP can be determined from the database of SNPs available at the NCBI website (Entrez SNP, dbSNP build 128, Jan. 28, 2009, https://www.ncbi.nlm.nih.gov/snp/). The "position" of the nucleotide of interest gives the location in the genome of the SNP, referring to the nucleotide position from the p-terminus of the chromosome in the human genome, see the NCBI SNP website (dbSNP), available on the internet.

In specific embodiments, a PLIN1 disease associated variant is a non-synonymous SNP resulting in a single amino acid substitution in a phosphorylation site of Perilipin-l selected from the group consisting of SEQ ID NOs: 3-7, preferably SEQ ID NO:3 or SEQ ID NO:5. In a specific embodiment, a PLIN1 disease associated variant is a non- synonymous SNP resulting in one of the following single amino acid substitution in Perilipin-l protein: Thr82Ile, Leu90Pro, Arg274Trp and Arg280Gln.

Typically, in preferred embodiments, said non-synonymous SNP is a SNP selected from the group consisting of rsl50004289 as shown in SEQ ID NO:9, rsl3927l800 as shown in SEQ ID NO:l l; rs8l79070 as shown in SEQ ID NO:l3 and/or rsl49989895 as shown in SEQ ID NO:l5.

The above-mentioned disease associated variants for PLIN1 may be detected in the biological sample either at the nucleic acid level or at the polypeptide level and includes methods to detect a genetic mutation in the genomic DNA or RNA transcripts and method to detect abnormal expression or phosphorylation of the gene product or mutation in the gene product (RNA transcripts or polypeptide).

The identification of the genetic mutation(s) (e.g. one or more specific SNPs as described above) on at least one chromosome indicates that the subject has an increased risk of developing acute coronary syndrome. Determining the presence or absence of PLIN1 disease associated variant

The presence of PLIN1 disease associated variant may be determined by any suitable techniques for detecting genetic mutations within a sample.

For example, specific genetic mutations of PLIN1 gene associated to predisposition to acute coronary syndrome as described above may be readily detected on genomic DNA or transcript RNA or cDNA obtained from the biological sample.

A variety of techniques are known in the art for detecting a genetic mutation within a sample.

In one specific embodiment, determining the presence of PLIN1 disease associated variant may be carried out by sequencing a nucleic acid comprising a fragment of PLIN1 open reading frame and analysing the sequence for detecting the presence or the absence of said genetic mutation. Sequencing methods include without limitation, Sanger sequencing, next generation sequencing, pyrosequencing, sequencing by ligation. Prior to sequencing, amplification of said genomic fragment including the SNP of interest may be carried out from the genomic DNA of the biological sample. In other specific embodiments, determining the presence of PLIN1 disease associated variant comprises the step of amplifying a nucleic acid fragment containing one of the PLIN1 disease associated variant, such as the specific SNPs as described above, using PCR-based methods. PCR-based methods useful for SNP detection include without limitation real-time PCR, quantification PCR, high-resolution melting analysis and amplification refractory mutation system PCR (ARMS-PCR). The amplified nucleic acid fragment may be a fragment of at least 20, 30, 40, 50, 100, 200, 300 or at least 500 consecutive nucleotides, for example comprised between 20 and 1000 consecutive nucleotides, preferably between 30 and 200 consecutive nucleotides. The skilled person in the art will adapt the size of the fragments according to the method used for determining the presence of the SNPs.

PCR amplification may be performed on genomic DNA from said biological sample allowing amplification of the relevant fragments.

Thus, primers which span one or more fragments that comprise the putative location of a PLIN1 genetic mutation (e.g specific SNPs as described in the above paragraphs) may be used to detect said genetic mutations.

Other available methods for SNPs genotyping may be used in the methods of the invention. The traditional gel-based approach uses standard molecular techniques, such as amplification refractory mutation system (ARMS), restriction digests and various forms of gel electrophoresis (e.g., RFLP), denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP). High throughput methods include allele discrimination methods (Allele-Specific Hybridization, Molecular Beacons, Allele- Specific Single-Base Primer Extension, 5’ nuclease), High-throughput assay chemistry (Flap endonuclease discrimination, Oligonucleotide ligation), microarray analysis of genomic DNA, pyrosequencing and light cycler.

In other embodiments, the genetic mutations are detected by specific hybridization of nucleic acid probes, such as oligonucleotide probes to genomic DNA or RNA transcripts or corresponding cDNA, containing the PLIN1 genetic mutations, for example containing the specific PLIN1 SNPs as described in the above paragraphs. In a specific embodiment, dynamic allele specific hybridization method (DASH) is used to detect the presence of a PLIN1 disease associated variant. Dynamic allele-specific hybridization (DASH) genotyping takes advantage of the differences in the melting temperature in DNA that results from the instability of mismatched base pairs. The process can be vastly automated and encompasses a few simple principles. Typically, the target genomic segment is amplified and separated from non-target sequence, e.g., through use of a biotinylated primer and chromatography. A probe that is specific for the particular allele is added to the amplification product. The probe can be designed to hybridize specifically to the PLIN1 disease associated variant (such as corresponding identified SNPs). The probe can be either labeled with or added in the presence of a molecule that fluoresces when bound to double stranded-DNA. The signal intensity is then measured as temperature is increased until the Tm can be determined. A non-matching sequence (either PLIN1 disease associated variant, depending on probe design) will result in a lower than expected Tm. Thus, in a number of the above methods, primers and probes which span one or more fragments that comprise the PLIN1 disease associated variant (e.g specific SNP as described in the above paragraphs) may be used to detect said PLIN1 disease associated variant.

As used herein, the term“probe” or“primer” refer to one or more nucleic acid fragments whose specific hybridization to a sample can be detected. A probe or primer can be of any length depending on the particular technique it will be used for.

Such probes or primers which may be used in the methods of the invention may typically be short nucleic acid molecules, for instance DNA oligonucleotides of 10 nucleotides or more in length, which can be annealed to the complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer or probe and the target nucleic acid strand.

The probes or primers can be unlabelled or labelled so that its binding to a target sequence can be detected (e.g. with a FRET donor or acceptor label).

A primer can be extended along the target nucleic acid molecule by a polymerase enzyme. Therefore, primers can be used to amplify the target nucleic acid molecule, such as fragments including any of the SNPs described herein associated to predisposition to acute coronary syndrome.

The specificity of a probe or a primer increases with its length. Thus, for example, a probe or primer that includes 30 consecutive nucleotides will anneal to a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, to obtain greater specificity, probes and primers can be selected that include at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more consecutive nucleotides.

In particular examples, a primer is at least 10 nucleotides in length, such as at least 10 contiguous nucleotides complementary to a target nucleic acid molecule. Particular lengths of primers that can be used to practice the methods of the present disclosure include primers having at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, at least 55, at least

60, at least 65, at least 70, or more contiguous nucleotides complementary to the target nucleic acid molecule to be amplified, such as a primer of 10-70 nucleotides, 10-60 nucleotides, 10-50 nucleotides, 10-30 nucleotides or 10-20 nucleotides.

An "upstream" or "forward" primer is a primer 5' to a reference point on a nucleic acid sequence. A "downstream" or "reverse" primer is a primer 3' to a reference point on a nucleic acid sequence. In general, at least one forward and one reverse primer are included in an amplification reaction.

Nucleic acid probes and primers can be readily prepared based on the nucleic acid sequence flanking the SNPs of interest for use in the methods of the invention, and for example the genomic sequence.

PCR primer pairs can be derived from a known sequence by using computer programs intended for that purpose such as Primer 3 (v. 0.4.0 Whitehead Institute for Biomedical Research, Steve Rozen, and Helen Skaletsky). In other embodiments, the SNPs of interest are detected by specific hybridization of nucleic acid probes, such as oligonucleotide probes to genomic DNA or RNA transcripts or corresponding cDNA, potentially containing the PLIN1 disease associated variant, for example containing any one of the SNPs as described in the above paragraphs.

Such probes can also be immobilized on a solid surface (such as nitrocellulose, glass, quartz, fused silica slide) as in an array or microarray or DNA chip. One of skill in the art will recognize that the precise sequence of particular probes and primers can be modified from the target sequence to a certain degree to produce probes that are“substantially identical” or“substantially complementary” to a target sequence, while retaining the ability to specifically bind to (i.e. hybridize specifically to) the same targets from which they are derived.

In the context of the present invention, the terms“capable of hybridizing to” and“binds specifically to”, which are used interchangeably, refer to a polynucleotide sequence that forms Watson-Crick bonds with a complementary sequence. One of skill will understand that the percent complementary need not be 100% for hybridization or specific binding to occur, depending on the length of the polynucleotides, length of the complementary region and stringency of the conditions. For example, a primer or probe is at least 60%, 70%, 80%, 90%, 95%, 99% or 100% complementary over the stretch of the complementary region.

Examples of primers that may be used for amplifying PLIN1 exons prior to sequencing are shown in the following table 1. Typical PCR amplification conditions that may be used are: 95°C 5 min followed by 35 cycles :[95°C-l5sec / Tm - 30sec / Elongation Temp - between 15 sec to 30sec]

Table 1: Examples of PCR primers for specific detection of PLIN1 SNPs

SNP primers (5’>3’) _ ld _

1 SEQ 1D NO :l6 rsl50004289

_ SEQ 1D NO :17 _

2 SEQ 1D NO :l8 rsl3927l800

_ SEQ 1D NO :19 _

3 SEQ 1D NO :20 rs8179070

_ SEQ 1D NO :2l _ 4 SEQ ID NO :22 rsl49989895

SEQ ID NO :23

Typical examples of probes useful for detecting the SNPs of PLIN1 disease associated variants are shown in the following Table 2: Table 2: Probes

In another embodiment, a polypeptide disease associated variant is detected using a binding agent that specifically binds to a polypeptide disease associated variant. For example, an agent that specifically binds to a mutant variant of Perlipin-l corresponding to the altered Perilipin-l polypeptide but not to wild-type Perilipin-l may be used. Said binding agent may be for example an antibody or antibody fragments comprising antigen binding regions. Specific antibodies, or antibody fragments, reactive against particular disease associated variant, for example, a particular Perilipin-l mutant polypeptide may be selected by screening expression libraries encoding immunoglobulin genes using phage display technologies. In one embodiment, antibodies are used to detect mutant Perilipin-l protein, in particular the mutant Perilipin-l protein as encoded by a PLIN1 coding sequence including one or more of the SNPs associated to predisposition to acute coronary syndrome as described above, such as rsl50004289, rsl3927l800, rs8l79070, and rsl49989895. Said antibodies or fragments thereof bind to mutant Perilipin-l protein but not to wild type Perilipin-l protein. Alternatively, the antibodies may recognize specifically the phosphorylated perilipin-l protein (or non-phosphorylated perilipin-l protein) and may be used to assess abnormal level of phosphorylation of the perilipin-l protein.

A person skilled in the art will understand that a number of methods can be used to detect and/or quantify specific polypeptide, including immunoassays such as Western Blots, ELISA, and immunoprecipitation followed by SDS-PAGE, as well as immunocytochemistry or immunohistochemistry.

Kits for performing the method

Kits may be prepared for carrying out one of the above mentioned detection methods. Thus, the invention further relates to a kit for carrying out the above-described method, said kit comprising: a. probes and/or primers for detecting a genetic mutation in PLIN1 gene coding sequence, and, b. optionally, instructions for use of the kit.

Said probes or primers for detecting a genetic mutation in PLIN1 gene coding sequence may therefore comprise specific primers or oligonucleotides probes as described above, or specific binding agents, such as antibody or antibody fragments as described above.

The kits can include one or more isolated primers or primer pairs for amplifying a target nucleic acid in PLIN1 gene coding sequence, such as a region comprising a SNP associated to predisposition to acute coronary syndrome as described above. For example, the kit can include primers for amplifying a haplotype including one, two, three or four SNPs in PLIN1 gene coding sequence, wherein the amplified sequence includes the SNP associated with predisposition to acute coronary syndrome.

The kit can further include one or more of a buffer solution, a conjugating solution for developing the signal of interest, or a detection reagent for detecting the signal of interest, each in separate packaging, such as a container.

In another example, the kit includes binding reagent to disease associated polypeptide variant, such as antibodies or fragment thereof.

The kit can also include instructions in a tangible form, such as written instructions or in a computer-readable format. In one preferred embodiment, said kit comprises probes, wherein said probes comprise nucleic acids for the specific detection of one or more of the following SNPs: rsl50004289 (SEQ ID NO:8 or 9), rsl3927l800 (SEQ ID NO:lO orl l); rs8l79070 (SEQ ID NO:l2 or 13) and/or rs 149989895 (SEQ ID NO: 14 or 15).

For example, the kit includes at least a probe that binds specifically to a nucleic acid comprising one SNP allele of the SNPs rsl50004289, rsl3927l800, rs8l79070, and/or rsl49989895 and/or primers that are capable of amplifying a nucleic acid comprising one SNP allele of the SNPs rsl50004289, rsl3927l800, rs8l79070 and/or rs 149989895.

In specific embodiments, the kit include a probe selected from nucleic acids comprising any one of SEQ ID NOs 24-31 or their fragments including the SNP allele with corresponding 5’ and 3’ flanking regions of at least 5 nucleotides.

In other specific embodiments said kit include primers selected among the following groups of pair of primers:

- SEQ ID NO: 16 and SEQ ID NO: 17 for rs 150004289;

- SEQ ID NO: 18 and SEQ ID NO:l9 for rsl3927l800 ;

- SEQ ID NO:20 and SEQ ID NO:2l for rs8179070 and/or;

- SEQ ID NO:22 and SEQ ID NO:23 for rs 149989895.

Prophylactic methods

The invention further relates to methods for preventing a subject from developing acute coronary syndrome or reducing the risk of a subject to develop acute coronary syndrome, said method comprising a. identifying whether said subject is at increased risk of developing acute coronary syndrome as described in the previous sections, b. treating said subject identified at step (a) at risk with a suitable treatment to reduce the risk of developing acute coronary syndrome.

As used herein, the term“suitable treatment” refers to measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder or slow down or relieve one or more of the symptoms of the disorder. Such measures encompass not only a particular preventive pharmaceutical treatment, but also changes of lifestyle and/or recommendations in care, and/or special care. The invention will now be further illustrated by the following examples. However, these examples should not be interpreted in any way as limiting the scope of the present invention.

EXAMPLES

METHODS

The inventors prospectively included 122 patients who had been admitted in the cardiology department of the Hopital Nord of Marseille for an ACS treated by percutaneous coronary intervention (PCI) before 55 years old for women and before 50 years old for men. ACS was defined according to the guidelines and includes both ST elevation (STEMI) and non ST elevation (NSTEMI) ACS (Erbel et al, 2014). Only patients with at least one significant athero -thrombotic stenosis requiring PCI were included.

The inventors excluded patients whose ACS was due to non-atherosclerosis cause including trauma, dissection, septic embolus, toxic or coronary artery dissection.These patients were compared to a healthy control group constituted of 120 individuals matched for gender and close geographical origin. These subjects were part of a larger cohort of 6154 individuals recruited as healthy controls from French origin with no cardiovascular events before the age of 60 (Mazoyer et al. 2009).

The present study was approved by the institution review board and agrees with the declaration of Helsinki. All patients gave an informed consent before inclusion.

Clinical endpoint

The inventors recorded clinical ischemic and bleeding events during a 1 year follow-up period. Ischemic events are composed of major adverse cardiovascular events (MACE): cardiovascular death, myocardial infarction, urgent revascularization, and stroke. Bleedings were measured using the TIMI classification (Merhan et al, 2011).

Genetic analysis

DNA was extracted from peripheral blood using standard procedures. DNAs were prepared and stored at the certified Biological Resource Center (CRB TAC component (NF S96-900 & ISO 9001 v20l5 Certification)). All DNAs belong to biological sample collections declared to the French ministry of Health (declaration numbers DC-2008-429 and rajouter n° FITENAT) whose use for research purposes was authorized by the French ministry of Health (authorization AC-2011-1312 and AC-2017-2986). NGS study:

The targeted regions corresponded to the coding regions and intron-exon boundaries of the following genes: AGPAT2, BSCL2, CAV1, LMNA, PLIN1, PPARG, PTRF, and ZMPSTE24. This panel focuses on genes associated with lipodystrophy and nucleopathy. The capture was performed with reagents from a custom design HaloPlex Target Enrichment kit (Agilent Technologies, USA), according to the HaloPlex Target Enrichment for Ion Torrent Sequencing v D4. Libraries were quantified and qualified using the Qubit Fluorometer (Thermo Fisher Scientific Inc. USA) and the Agilent 2100 Bioanalyzer instrument (High Sensitivity DNA Kit) to enable equi-molar pooling of barcoded samples. Template preparation, emulsion PCR, and Ion Sphere Particles (ISP) enrichment were carried out using the Ion PI™ Template OT2 200 Kit v2 on the Ion OneTouch™ 2 System (Thermo Fisher Scientific Inc. USA). The quality of the ISPs was assessed using a Qubit 2.0 Fluorometer, and the ISPs were loaded and sequenced on a Ion PI™ Chip Kit v2 using Ion PI™ Sequencing 200 Kit v2 on the Ion Proton™ Sequencer (Thermo Fisher Scientific Inc. USA). Raw data were first aligned with the provided software suite included with the Ion Proton system to generate BAM files. The coverage and sequencing depth analysis were computed using the BEDtools suite v2T7 and in- house scripts. Variants were identified using the Torrent Browser Variant caller (version 4.0.2), annotated and prioritized with the in-house “VarAFT” system that includes Annovar (JP Desvignes & al). All mutations in the PLIN1 gene were confirmed using Sanger sequencing according to standard procedures on ABI3500XL (Life Technologies, Carlsbad, C.A, U.S.A.). Mutations are numbered according to the GenBank reference sequence NM 002666 and the Human Genome Variation Society recommendations (http ://vamomen.hgvs.org/T Transcript study:

RNA was extracted by conventional method: trizol / chloroform (Thermo fischer) as described above. Reverse transcriptase was performed according to standard procedure using Superscrit III Reverse Transcrpitase (Thermofisher). And then PCR and Sanger sequencing was done according to standard procedures on ABI3500XL (Life Technologies, Carlsbad, C.A, U.S.A.).

Statistical analysis

Statistical analyses were performed using PASW Statistics version 17.0. Continuous variables are reported as means and standard deviation or as medians and range (according to their distribution), and categorical variables are reported as count and percentages. Univariate and multivariate analyses were performed using a logistic regression model to estimate the risk of ACS associated with the presence of PLIN1 mutated alleles. Odds ratios (OR) were estimated with a 95% confidence interval. Calibration of the logistic model was assessed using the Hosmer-Lemeshow goodnessof-fit test. A classification table was used to evaluate the predictive accuracy of the logistic regression model.

RESULTS

Clinical data

The cohort was composed by a majority of men (81.6%). The mean age at the time of first ACS was 43.6 ±7 years old.

Among cardiovascular risk factors, a family history of CAD was present in 37.5% of patients, obesity in 67.8%, active smoking in 80.8% and diabetes in 20.5%. Hypercholesterolemia was present in 13%. Hypertriglyceridemia was present in 35%.

Coronary angiography revealed a multi-vessel disease in 67% of patients. All patients received PCI of the culprit lesion with drug-eluting stents. PCI was successful in all patients.

Among the 120 patients included, a recurrent event of ACS was observed in 30.8% over the one year follow-up.

Molecular findings

Next-Generation-Sequencing was performed on a panel of 38 genes on 62 patients.

The inventors collected 85 nonsynonymous variants out of 8 genes passing the quality controls. Among those, 16 passed the frequency filter set up at less than 0.15% in GnomAD database. The variants were located in 6 different genes. Those predicted as having a benign effect by bioinformatics software prediction tools were eliminated, leading to an end-up of 12 variants in 5 genes. No causative mutation described previously was found in genes associated with lipodystrophy phenotype as AGPAT2, BSCL2, CAV1, PTRF, PPARG, ZMPSTE24 and LMNA. Then, they selected genes mutated in at least 2 patients. They found 11 variants in 4 genes (LMNA, PLIN1, PPARG and PTRF). The gene which was found mutated in the higher number of patients was PLIN1 (n=6). Four individuals were found to share the same variant at heterozygous condition in PLIN1 : c.245C>T (p.Thr82Ile) and two other patients have a different variant in this same gene: c.269T>C (p.Leu90Pro) and c.820C>T (p.Arg274Trp) also in heterozygous condition. All 3 mutations were independently confirmed by Sanger sequencing (see primers sequences on supplementary data). The inventors then tested a second cohort of 60 patients with a premature SCA by direct Sanger sequencing of the coding regions of PLIN1. In this second cohort, two more patients were found carrying a mutation in this gene, one carrying the recurrent mutation previously identified: c.245C>T (p.Thr82Ile), the second one carrying a novel mutation c.839G>A (p.Arg280Trp), both in heterozygous conditions. These variants were found mostly in men (7/8).

All the mutations locates in or close to two of the five phosphorylation sites of the N- terminal domain of Perilipin 1, Ser8l and Ser276 (Zhang et al. 2003). All of them are described as deleterious in at least two of the following prediction tools: MutationT aster, Sift, Polyphen2 and UMDpredictor. Severity prediction scores and frequencies in general population are shown in Table 3.

Table 3: bioinformatics software prediction and allelic frequencies in GnomAd for the 4 mutations identified in PLIN1.

GnomAD: http :// gnomad.broadinstitute.org/

Transcriptional study was performed for mutations c.245C>T (p.Thr82Ile) and c.269T>C (p.Leu90Pro) because of prediction by bioinformatics tools of splicing alteration (http://www.umd.be/HSF3/HSF.shtml). But these analyses did not confirm splicing alteration and showed the transcript with the mutation at heterozygous condition also as in genomic level.

Link between PLIN 1 mutations and premature ACS Allele frequencies and OR are summarized below in Table 4.

Univariate analysis confirmed the significantly higher prevalence of each mutation of PLIN1 in patients with ACS compared to the control group extracted from the GnomAD database. The risk for ACS was significantly increased with the mutations: c.245C>T (r<10⁴) and c.839G>A (r<0.014). For the 2 other mutations, a tendency was observed (c.269T>C: p=0.73 and C.820OT: p=0.l).

To investigate a potential bias linked to the region of recruitment, the inventors tested a local control cohort of 120 voluntary persons by Sanger sequencing of all PLIN1 coding regions. They calculated the risk to develop an ACS conferred by the presence of any of the 4 mutations in PLIN l . The frequency of the mutations were again significantly higher in the ACS cohort compared to controls (OR, 8.2946, 95% Cl: 1.0834; 372.9476, P = 0.035).

_ _ _

Table 4: Odd Ratios counted in the SCA cohort versus control population.

Relationship between mutations condition and clinical profile or recurrence events When comparing patients with a mutation to the non-mutated ones, the inventors observed no significant difference regarding baseline clinical characteristics or biological parameters. In particular there was no difference in disease diffusion or rate of recurrent events (p respectively 0.6 and 1). At the contrary, a clear tendency for one risk factor, tobacco, in the group SCA vs. control group (p-value=0.l43) was observed.

Functional study of mutational events

The four mutations described in the study are located in close proximity of the phosphorylation sites 1 and 2 of perilipinl (p.Ser8l and p.Ser277 respectively). We thus hypothesized a phosphorylation defect of the mutant proteins, indeed p.Arg274Trp is predicted in silico to fully disrupt perilipin phospho-site 2 using GPS software

(http://gps.biocuckoo.org/online.php). These analyses are done using the high specificity cutoff to minimize false positive results; however, under this condition, it is known that the GPS software has a high level of false negative rate (“GPS' 2.0, a Tool to Predict Kinase- specific Phosphorylation Sites in Hierarchy”, Mol Cell Proteomic, 2008), therefore the functional consequences of the four mutations: c.245C>T and c.269T>C on perilipin phospho-site 1 and c.820C>T and c.839G>A on perilipin phospho-site 2 are also investigated by an experimental approach.

To do so, Perilipinl variants are purified in vitro and differential PKA phosphorylation kinetics are measured by western blotting. As no anti-phospho perilipin antibody exists, a non-specific anti phospho-serine antibody can be used. All perilipin phospho sites are abolished by in vitro mutagenesis except the phospho site 1 or 2 to prevent any false positive phospho-band on the western blot.

The following different perilipins are for example produced:

- p.[Ser277Ala; Ser436Ala; Ser497Ala; Ser522Ala]

- p.[Thr82Iso; Ser277Ala; Ser436Ala; Ser497Ala; Ser522Ala]

- p.[Leu90Pro; Ser277Ala; Ser436Ala; Ser497Aia; Ser522A!a]

- p.[Ser81Ala; Ser436Ala; Ser497Ala; Ser522A!a]

- p.[ Ser81Aia; Arg274Trp; Ser436Aia; Ser497Ala; Ser522Aia]

- p.[ Ser81Aia; Arg280Gln; Ser436Ala; Ser497Ala; Ser522Ala]

- p.[ Ser81Ala; Ser277A!a; Ser436Ala; Ser497Ala; Ser522Ala] as a negative control. Typically, from an adipocyte enriched tissue sample, the PLIN1 transcript ENST00000300055.9 is amplified and inserted it in the pDONR22l vector using BP reaction of the gateway technology. After sequencing, the transcript is transferred in a bacterial expression vector (such as the bacterial expression vector pDESTl7). The cDNA is then placed in frame downstream a 6xHis tag allowing efficient purification of the corresponding recombinant protein.

Finally, to evaluate the effect of the mutations on the perilipin phosphorylation rate, the mutant protein stability is assessed by nano Differential Scanning Fluorimetry (nano DSF). Briefly, purified proteins are submitted to linear temperature ramp and intrinsic tryptophan or tyrosine fluorescence is measured. The gradual heating makes the Trp and Tyr residues accessible and the fluorescence increases as the protein unfolds. The evolution of the fluorescence is assessed to compare the unfolding of the WT perilipin v.v. the mutant perilipins. The same cloning strategy canl be conducted as previously described and different perilipin forms can thus be purified: WT, p.Thr82Iso, p.Leu90Pro, Arg274Trp and Arg280Gln.

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USEFUL SEQUENCES FOR PRACTICING THE INVENTION

Claims

1. An in vitro method for detecting a genetic predisposition to acute coronary syndrome in a subject, said method comprising determining the presence of a PLIN1 disease associated variant in a biological sample from said subject, wherein said PLIN1 disease associated variant is an allele of the PLIN1 gene comprising at least a genetic mutation in the open reading frame of the PLIN1 gene, and the detection of said PLIN1 disease associated variant is indicative of a genetic predisposition to acute coronary syndrome in said subject.

2. The method according to Claim 1, wherein said genetic mutation is one or more single nucleotide polymorphism (SNP) non-synonymous mutation(s) in the open-reading frame of the PLIN1 gene.

3. The diagnosis method according to Claim 1 or 2, wherein said PLIN1 disease associated variant is a genetic mutation in the PLIN1 gene altering the predicted amino acid sequence encoded by the PLIN1 gene.

4. The method according to any one of Claims 1-3, wherein said genetic mutation is a non-synonymous mutation in the coding sequence of PLIN1 gene that alters the phosphorylation level of the Perilipin-l protein in said subject.

5. The method according to any one of Claims 1-4, wherein said PLIN1 disease associated variant is a non-synonymous SNP resulting in a single amino acid substitution in a phosphorylation site of Perilipin-l protein selected from the group consisting of SEQ ID NOs: 3-8.

6. The method according to any one of Claims 1-5, wherein said PLIN1 disease associated variant is a SNP selected from the group consisting of rsl50004289 as shown in SEQ ID NO:9, rsl3927l800 as shown in SEQ ID NO:l l, rs8l79070 as shown in SEQ ID NO: 13 and/or rs 149989895 as shown in SEQ ID NO: 15.

7. The method according to any one of Claims 1 to 6, wherein the presence of a PLIN1 disease associated variant is determined by a SNP detection method selected from the group consisting of sequencing methods including Sanger sequencing, next generation sequencing, pyrosequencing, sequencing by ligation; PCR-based methods including PCR, real-time PCR, quantitative PCR and high-resolution melting analysis; and/or any other SNP genotyping techniques such as amplification refractory mutation system (ARMS), restriction fragment length polymorphism (RFLP) analysis, denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP), and allele discrimination methods including allele-specific hybridization, molecular beacons, allele-specific single base primer extension, Flap endonuclease discrimination, 5’nuclease, oligonucleotide ligation and micro-array analysis of genomic DNA.

8. The method according to any one of Claims 1-7, wherein said biological sample is a blood sample.

9. A kit for performing the in vitro method according to any one of Claims 1-8, comprising: a. probes or primers for specifically detecting one or more SNPs in PLIN1 gene coding sequence; b. optionally, instructions for use of the kit.

10. The kit according to Claim 9, wherein the probes comprise nucleic acids for the specific detection of one or more of the following SNPs: rsl50004289 (SEQ ID NO:8 or 9), rsl3927l800 (SEQ ID NO:lO or 11), rs8l79070 (SEQ ID NO:l2 or 13) and/or rsl49989895 (SEQ ID NO:l4or 15).

11. The kit according to Claim 9, wherein the kit includes at least a probe that binds specifically to a nucleic acid comprising one SNP allele of the SNPs as defined in Claim 10, and/or primers that are capable of amplifying a nucleic acid comprising one SNP allele as defined in Claim 10.

12. The kit according to Claim 11, wherein said probe is selected from the probes comprising any one of SEQ ID NOs 24-31 or their fragments including the SNP allele with corresponding 5’ and 3’ flanking regions of at least 5 nucleotides.

13. The kit according to Claim 11, wherein said primers are selected among the following groups of pair of primers:

- SEQ ID NO: 16 and SEQ ID NO: 17 for rs 150004289;

- SEQ ID NO:l8 and SEQ ID NO:l9 for rsl3927l800 and/or;

- SEQ ID NO:20 and SEQ ID NO:2l for rs8179070;

- SEQ ID NO:22 and SEQ ID NO:23 for rs 149989895.

14. A method for preventing a subject from developing acute coronary syndrome, comprising

a. identifying whether said subject is at increased risk of developing acute coronary syndrome according to a method of any one of claims 1-8, b. treating said subject identified at step a at risk with a suitable treatment for preventing acute coronary syndrome.