WO2014067965A1

WO2014067965A1 - Methods of predicting or diagnosing a pulmonary arterial hypertension

Info

Publication number: WO2014067965A1
Application number: PCT/EP2013/072640
Authority: WO
Inventors: Florent Soubrier; Marc Humbert; David Alexandre TREGOUËT; Mélanie EYRIES
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm); Universite Paris-Sud Xi; Assistance Publique - Hopitaux De Paris
Priority date: 2012-10-29
Filing date: 2013-10-29
Publication date: 2014-05-08

Abstract

The invention relates to methods of predicting or diagnosing pulmonary arterial hypertension and to kits for performing these methods.

Description

METHODS OF PREDICTING OR DIAGNOSING A PULMONARY ARTERIAL

HYPERTENSION

Field of the invention

The invention relates to methods of predicting or diagnosing pulmonary arterial hypertension in patients. The invention also relates to kits for performing these methods.

Background to the invention

Pulmonary arterial hypertension (PAH) is a rare and devastating disease, resulting from progressive obliteration of small caliber pulmonary arteries by proliferating vascular cells, and leading to cardiac failure, with untreated mean survival less than three years (Rabinovitch, M. Molecular pathogenesis of pulmonary arterial hypertension. J Clin Invest 1 18, 2372-9 (2008); Rubin, L.J. Primary pulmonary hypertension. N Engl J Med 336, 1 1 1 - 7 (1997)).

PAH can complicate well identified pathological conditions, or occur in the context of genetic mutations causing heritable PAH, or is considered as idiopathic (iPAH), representing around 70% of PAH in the absence of an identified predisposing factor (Humbert, M. et al. Pulmonary arterial hypertension in France: results from a national registry. Am J Respir Crit Care Med 173, 1023-30 (2006); Simonneau, G. et al. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 54, S43-54 (2009)).

Currently, the diagnosis is suspected on the basis of unexplained respiratory symptoms, secondary to right-heart failure, such as exercise dyspnea. The diagnosis is firmly established by hemodynamic measurements (right-heart catheterization) showing an increase in mean pulmonary artery pressure above 25 mmHg with a normal pulmonary artery capillary wedge pressure of less than 15mmHg (pre-capillary pulmonary hypertension). When symptomatic, the disease has already reached an advanced stage, where drugs are less efficacious. Therefore finding predisposing markers to the disease, either in the idiopathic form, or in subjects carrying a predisposing mutation, would allow a better prediction of the individual risk for developing the disease.

There is today a real challenge in discovering novel genetic factors involved in the pathogenesis of PAH as well as new genetic markers to predict and diagnose major risks of PAH in patients.

Description of the invention

The invention provides methods of predicting or diagnosing PAH.

According to an embodiment of the invention, the method of predicting or diagnosing pulmonary arterial hypertension in a patient comprises: a) determining the presence or absence of a nucleotide polymorphism (SNP) of the cerebellin-2 gene in a sample obtained from said patient; and optionally

b) deducing therefrom if the patient is at risk of having or has PAH. According to another embodiment of the invention, the method of predicting or diagnosing PAH in a patient comprises:

a) measuring the level of expression of the cerebellin-2 gene in a sample from said patient; optionally

b) comparing the level measured in step a) with a reference level for said gene; and optionally

c) deducing therefrom if the patient is at risk of having or has PAH. According to another embodiment, the invention relates to a method of diagnosing genetic predisposition to pulmonary arterial hypertension in a patient comprising:

a) determining the presence or absence of a nucleotide polymorphism (SNP) of the cerebellin-2 gene in a sample obtained from said patient; and optionally

b) deducing therefrom if the patient has a genetic predisposition to PAH.

The invention further relates to kits for performing these methods.

The term PAH refers to an increase in blood pressure in the pulmonary artery mean pulmonary arterial pressure above 25mmHg at rest, with a normal pulmonary artery capillary wedge pressure, leading to shortness of breath, dizziness, fainting, and other symptoms, all of which are exacerbated by exertion. PAH can be a severe disease with a markedly decreased exercise tolerance and heart failure. PAH can be considered as idiopathic (iPAH) or familial (fPAH).

The term "idiopathic pulmonary arterial hypertension "(iPAH) refers to the case where no identified predisposing factor for the occurrence of PAH has been determined.

The term "familial pulmonary arterial hypertension "(fPAH) refers to the case where

PAH occurs in a familial context, where mutations are found in 70% of cases on the

BMPR2 gene. In this latter case, PAH is considered as "heritable" since it occurs in the context of heritable genetic mutations. However, BMPR2 mutations are found in approximately 15% of sporadic forms, and are also called in this case "heritable" forms.

The terms "biomarker" or "marker" can be used herein interchangeably. They refer to a substance that is a distinctive indicator of a biological process, biological event, and/or pathologic condition.

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to PAH. A "sample" in the context of the present invention is a biological sample isolated from a subject and can include, by way of example, bodily fluids. Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof. In a preferred embodiment, the sample to be tested is blood. As used herein "blood" includes whole blood, plasma, serum, circulating epithelial cells, constituents, or any derivative of blood.

The term "Single nucleotide polymorphism" or "SIMP" means a single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population. There are millions of SNPs in the human genome. Most commonly, these variations are found in the DNA between genes. When SNPs occur within a gene or in a regulatory region near a gene, they may play a direct role in disease by affecting the gene's function or expression. According to an embodiment of the present invention, SNPs are SNPs of the cerebellin-2 gene, in particular those selected from the group consisting of rs1432071 , rs10514048, rs12955878, rs99601 17, rs79135430, rs59675169, rs7228776, rs7226690, rs1432070, rs1432069, rs1432067, rs2217560 and rs9916909, more particularly from the group consisting of rs2217560 and rs9916909, even more particularly from the group consisting of allele A or G of rs2217560 and allele C or A of rs9916909, and preferably the SNP of the cerebellin-2 gene is the allele G of the rs2217560.

The term "Allele" has the meaning which is commonly known in the art, that is, an alternative form of a gene or a genetic locus that is located at the same specific position on a specific chromosome and that differs by its DNA sequence.

By "SNP of the cerebellin-2 gene", it is meant a SNP that occurs within the cerebellin-2 gene or its flanking region and that is in complete or strong (r²>0.8) linkage disequilibrium with the SNPs rs2217560 and rs9916909.

By "complete or strong (r²>0.8) linkage disequilibrium", it is meant a complete or nearly complete association between the alleles of the two SNPs.

The term "genetic predisposition" refers to a genetic status which influences the phenotype of an individual organism within a specie or population, that is, in the context of the present invention, a genetic status in a patient that would lead to an increased risk of developing a pulmonary arterial hypertension.

The term "the expression level of a gene" refers to an amount or a concentration of a transcription product, for instance mRNA, or a translation product, for instance a protein or polypeptide. Typically, a level of mRNA expression can be expressed in units such as transcripts per cell or nanograms per microgram of tissue. A level of a polypeptide can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe an expression level.

The expression "measuring the level of expression of a gene" encompasses the step of measuring the quantity of a transcription product, preferably mRNA obtained through transcription of said gene, and/or of translation product, preferably the protein obtained through translation of said gene.

As used herein, the term "patient" refers to a human that may or may not have a pulmonary arterial hypertension.

The term "control patient" refers to a patient that has not shown any pulmonary arterial hypertension symptoms and has not been diagnosed for this disease.

The term "reference level" denotes a level of expression of the cerebellin-2 gene in a control patient or group of control patients, or in a patient or a group of patients diagnosed for a PAH.

The reference level(s) may be determined as a single value or a range of values which is determined based on the level of expression of the cerebellin-2 gene measured in a population of control patients.

Typically, the analysed population could be divided into quantiles based on the measured level of expression of the cerebellin-2 gene. The reference level could be defined as the median, or the second tertile, or the second or third quartile, or the third or fourth quintile etc.

Comparison with a reference level may also be performed by comparing the level of expression of the cerebellin-2 gene with the level of expression of the cerebellin-2 gene, as appropriate, measured in a standard sample obtained from patients having PAH.

The reference level for the cerebellin-2 gene may vary depending on the method used for measuring.

By "measuring" is meant measuring the level of expression of the cerebellin-2 gene, or detecting a decrease or increase of the level of expression of the cerebellin-2 gene.

By "decrease in the level of expression" is meant a decrease of expression level of the cerebellin-2 gene in comparison to a reference or to a predetermined threshold value, for example a decrease of expression level of the cerebellin-2 gene of 5% or 10% in comparison to a reference or to a predetermined threshold value.

By "increase in the level of expression" is meant an increase of expression level of the cerebellin-2 gene in comparison to a reference or to a predetermined threshold value, for example an increase of expression level of the cerebellin-2 gene of 5% or 10% in comparison to a reference or to a predetermined threshold value.

The term "predetermined threshold" for one protein may refer to the median value of the expression level of the cerebellin-2 gene in biological samples of a control patient, or to the median value of the expression level of the cerebellin-2 gene in patients having a PAH. The skilled person can easily determine such a predetermined threshold using methods well-known in the art.

Detailed description of the invention

The inventors have conducted pioneering studies to identify novel genetic factors associated with PAH.

For the first time, the inventors have found a completely novel genome wide significant association at the cerebellin-2 locus with respect to PAH pathogenesis.

The invention thus relates inter alia to a method of diagnosing genetic predisposition to pulmonary arterial hypertension in a patient, said method comprising:

a) determining the presence or absence of a nucleotide polymorphism (SNP) of the cerebellin-2 gene in a sample obtained from said patient; b) deducing therefrom if the patient has a genetic predisposition to PAH.

According to an embodiment, the invention also relates to a method of predicting or diagnosing pulmonary arterial hypertension in a patient comprises:

b) deducing therefrom if the patient is at risk of having or has PAH. According to a further embodiment, the invention also relates to a method of treating pulmonary arterial hypertension in a patient comprising the steps of:

b) deducing therefrom if the patient is at risk of having or has PAH; and optionally

c) treating the patient at risk of having or having a pulmonary arterial hypertension.

The cerebellin-2 gene (CBLN2) belongs to the cerebellin gene family, a group of secreted neuronal glycoproteins (Cbln1 -4) and encodes the precursor of CBLN2, a hexadecapeptide with sequence homology of 94% and 44% to the cerebellin 1 and 3 peptides, respectively (Yiangou, Y., Burnet, P., Nikou, G., Chrysanthou, B.J. & Bloom, S.R. Purification and characterisation of cerebellins from human and porcine cerebellum, J Neurochem 53, 886-9 (1989)). CBLN2 has been previously reported to be expressed mainly in various regions of the brain (Miura, E., lijima, T., Yuzaki, M. & Watanabe, M. Distinct expression of Cbln family mRNAs in developing and adult mouse brains, Eur J Neurosci 24, 750-60 (2006)). The CBLN2 gene is further described by its gene sequence SEQ ID N°1 and its corresponding protein sequence SEQ ID N°2.

The inventors have further demonstrated that the presence of a SNP of the cerebellin-2 gene indicates that the patient has a genetic predisposition to pulmonary arterial hypertension.

In particular, the inventors shown in the context of the present invention that when the genetic predisposition to arterial hypertension is diagnosed, it indicates that the patient is at risk of having or has pulmonary arterial hypertension.

According to an embodiment of the present invention, the SNP of the cerebellin-2 gene is selected from the group consisting of rs1432071 , rs10514048, rs12955878, rs99601 17, rs79135430, rs59675169, rs7228776, rs7226690, rs1432070, rs1432069, rs1432067, rs2217560 and rs9916909, more particularly from the group consisting of rs2217560 and rs9916909, and even more particularly from the group consisting of allele A or G of rs2217560 and allele C or A of rs9916909.

According to a preferred embodiment, the SNP of the cerebellin-2 gene is the allele G of rs2217560.

The above cited SNPs are listed in the dbSNP international basis which can be found on the NIH server at the following web address www.ncbi.nlm.nih.gov/snp/ and where the sequences of these SNPs are provided as well as their exact position in the genome.

These sequences as well as their HGVS (human genome variation society) names can be found in the Table 1 below:

Table 1

SNP HGVS names SEQ Sequences

ID N°

rs 1432071 NC_000018.9:g.70162305A>G 3 TTTAGTAGAGAGCGTTTAATGTTTTCRTAGCTGAC

AAAAATATATATAAAGT NT_025028.14:g.17953169A>G

rs10514048 NC_000018.9:g.70165505T>C 4 ATTAAGTCAACAGTTACTTGAAGATAYTTACTTAA

AGACAAGACATGTTGAT NT_025028.14:g.17956369T>C

rs12955878 NC_000018.9:g.70165553G>A 5 TGATCATTATTTGCTTTATTTTTGGCRCAGTGCTC

TATCCTCTAAG CAATAC NT_025028.14:g.17956417G>A

rs99601 17 NC_000018.9:g.70148236T>C 6 TGCAGGGGTGGGGCTCTCATGGAGAAYGTCTGC

CAGGGCAGTGCAGAAGGGA NT_025028.14:g.179391 OOT>C

rs79135430 NC_000018.9:g.70162857C>T 7 AATATCTAAGAATAATAAAGAAGAAGYACCATTAG

AATAAATGGGGGGTTAA NT_025028.14:g.17953721 OT

rs59675169 NC_000018.9:g.70162875G>A 8 AG AAG AAGCACCATTAG AATAAATG G RGG GTTAA

TACAGTGTTTCCATCGGT NT_025028.14:g.17953739G>A

rs7228776 NC_000018.9:g.70149733C>T 9 AGGATTTCTTTGTATTAGTCCACTCTYTCATTGCT

GTGAAGAAATACCTGAG NT_025028.14:g.17940597C>T

rs7226690 NC_000018.9:g.70161540G>A 10 AAAG AG CT AG TG ATG AATC AAT AATT RT AACTTAA

CATAAAATATACTCATA NT_025028.14:g.17952404G>A

rs 1432070 NC_000018.9:g.70162291 C>T 11 TACAGGACATTTTATTTAGTAGAGAGYGTTTAATG

I I I I CATAGCTGACAAA NT_025028.14:g.17953155C>T

rs 1432069 NC_000018.9:g.70162160C>T 12 GGCAAATATCTTCTTTCTCCCAAAAGYGAAATGC

NT_025028.14:g.17953024C>T AGTCCATGATAAAATAAT

rs 1432067 NC_000018.9:g.70157358T>C 13 CCTGGGAGGTCAATGCTGCAGTCAGCYGTGATC

TTGCTGCTGTACCCACACT NT_025028.14:g.17948222T>C

rs2217560 NC_000018.9:g.70150939G>A 14 GTGCACATTAAAATATACACTGCAAARTCAGGCA

GAGTTCTGTAGAGAGAAT NT_025028.14:g.17941803G>A

rs9916909 NC_000018.9:g.70156123A>C 15 TCTGAAATCATTGTCCTCACATTGAAMAAGGAAA

GCCTTATTTTTGACCAAG NT_025028.14:g.17946987A>C

The SNPs rs2217560 and rs9916909 are further described in the Table 2 below:

Table 2

It has notably been shown that the presence of a SNP of the cerebellin-2 gene, as defined above, indicates an increased relative risk of having PAH, more particularly a twofold increased relative risk.

By "relative risk" is meant the ratio of the probability of an event occurring in an exposed group, that is in the context of the present invention the risk of developing or having PAH for patients having a genetic predisposition to PAH, to the probability of the event occurring in a comparison non-exposed group, here for patients not having a genetic predisposition to PAH. In the methods according to the present the invention, the presence or absence of a SNP of the cerebellin-2 gene can be determined by nucleic acid sequencing, PCR analysis or any genotyping method known in the art. Examples of such methods include, but are not limited to, chemical assays such as allele specific hybridation, pyrosequencing, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry.

For example, the presence or absence of said polymorphism may be detected in a DNA sample, preferably after amplification. For instance, the isolated DNA may be subjected to couple reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for the polymorphism or that enable amplification of a region containing the polymorphism. According to a first alternative, conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of the polymorphism according to the invention. Otherwise, DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.

Currently numerous strategies for genotype analysis are available (Antonarakis et al., 1989; Cooper et al., 1991 ; Grompe, 1993). Briefly, the nucleic acid molecule may be tested for the presence or absence of a restriction site. When a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype the polymorphism. Further strategies include, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele- specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al., 1997). Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method; by enzymatic sequencing, using the Sanger method; mass spectrometry sequencing; pyrosequencing; sequencing using a chip-based technology and real-time quantitative PCR. Preferably, DNA from a patient is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers. However several other methods are available, allowing DNA to be studied independently of PCR, such as the rolling circle amplification (RCA), the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may be used for revealing base polymorphisms. According to this method, two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the polymorphism. DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized to one of the allele.

Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.

For the first time, the inventors also found that the cerebellin-2 gene is differentially expressed in patients suffering from PAH.

The present invention thus also relates to a method of predicting or diagnosing pulmonary arterial hypertension in a patient, said method comprising:

a) measuring the level of expression of the cerebellin-2 gene in a sample from said patient;

b) comparing the level measured in step a) with a reference level for said gene;

c) deducing therefrom if the patient is at risk of having or has pulmonary arterial hypertension.

The inventors have further reported the first evidence that when the level of expression of the cerebellin-2 gene is increased as compared with the reference level of expression of said gene, it indicates that the patient is at risk of having or has PAH.

In one embodiment according to the invention, the sample is obtained from endothelial cells of lung tissue or endothelial cells from vessels, or from a blood sample.

In the methods according to the present invention, the level of expression of the cerebellin-2 gene can be determined by any known method allowing the level of expression to be determined. Examples of such methods include methods for measuring the quantity of cerebellin-2 protein. It can be performed by immunoassay or immunoblots or by analytical methods, like for example mass spectrometry (MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC-MS, LC-MS/MS), quantitative methods with isotopic labeling (stable isotope labeling by amino acids in cell culture (SILAC), isotope coded affinity tags (ICAT), isobaric tag for relative and absolute quantitation (ITRAQ), label-free methods like selective reaction monitoring (SRM) or multiple reaction monitoring (MRM) assays, or bio- molecular interaction analysis/surface plasmon resonance (BIA/SPR) technologies encompassing methods with calibration and without calibration as calibration free concentration analysis for example.

The term "immunoassay" as used according to the present invention includes competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, agglutination test, enzyme-labelled and mediated immunoassays, such as ELISA, biotin/avidin type assay, radioimmunoassay, Immunoelectrophoresis, and immunoprecipitation.

Mass spectrometry (MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC-MS/MS), stable isotope labeling by amino acids in cell culture (SILAC), isotope coded affinity tags(ICAT), isobaric tag for relative and absolute quantitation (ITRAQ), selective reaction monitoring (SRM) assays, multiple reaction monitoring (MRM) assays, bio-molecular interaction analysis/surface plasmon resonance (BIA/SPR) technologies, calibration free concentration analysis, are all analytical methods very well know by the man skilled in the art which are suitable to carry out the measure of the cerebellin-2 protein level according to the invention.

Such methods to determine the level of expression of the cerebellin-2 gene also include methods for measuring the quantity of transcription products of the cerebellin-2 gene, preferably mRNA. Methods for measuring the quantity of mRNA are well known in the art. Typically, the nucleic acid contained in the biological sample may be extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA may be then detected by hybridization (e. g., Northern blot analysis). Alternatively, the extracted mRNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that enable amplification of a region in said genes. Quantitative or semi- quantitative RT-PCR can be used. Extracted mRNA may be reverse-transcribed and amplified, after which amplified sequences may be detected by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. Other methods of amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

According to an embodiment of the present invention, PAH can be idiopathic or familial. It can also be a BMPR2 mutation negative pulmonary arterial hypertension.

Bone morphogenetic protein receptor type II or BMPR2 is a serine/threonine receptor kinase. It binds bone morphogenetic proteins, members of the TGF beta superfamily of ligands. BMPR2 mutations have been observed in PAH. Low penetrance dominant BMPR2 mutations are found in around 70% of f PAH and in around 15% of iPAH (Sztrymf, B. et al. Clinical outcomes of pulmonary arterial hypertension in carriers of BMPR2 mutation. Am J Respir Crit Care Med 177, 1377-83 (2008); Girerd, B. et al. Absence of influence of gender and BMPR2 mutation type on clinical phenotypes of pulmonary arterial hypertension. Respir Res 1 1 , 73 (2010)).

The present invention also relates to kit for performing methods herein described. Said kit comprises means for detecting SNPs of the cerebellin-2 gene or means for measuring the level of expression of the cerebellin-2 gene and optionally instructions for use in the prediction or diagnosis of PAH.

According to an embodiment of the present invention, the kit comprises means for detecting SNPs of the cerebellin-2 gene, particularly those selected from the group consisting of rs1432071 , rs10514048, rs12955878, rs99601 17, rs79135430, rs59675169, rs7228776, rs7226690, rs1432070, rs1432069, rs1432067, rs2217560 and rs9916909, more particularly from the group consisting of rs2217560 and rs9916909, and even more particularly from the group consisting of allele A or G of rs2217560 and allele C or A of rs9916909, and in a preferred embodiment the allele G of the rs2217560.

In one embodiment of the invention, the kit for identifying whether a patient has a genetic predisposition to PAH or has or is at risk of having pulmonary arterial hypertension, comprises at least one primer and/or at least one probe for amplification of a sequence comprising a SNP selected from the group consisting of rs1432071 , rs10514048, rs12955878, rs99601 17, rs79135430, rs59675169, rs7228776, rs7226690, rs1432070, rs1432069, rs1432067, rs2217560 and rs9916909, more particularly from the group consisting of rs2217560 and rs9916909, and optionally instructions for use.

In one embodiment of the invention, the primer or probe may be labeled with a suitable marker. In another embodiment of the invention, the primer or probe may be coated on an array.

According to a further embodiment of the present invention, the kit comprises means for measuring the level of expression of the cerebellin-2 gene.

As described above, the level of expression of the cerebellin-2 gene can be determined by measuring the quantity of cerebellin-2 protein or the quantity of transcription products of the cerebellin-2 gene, preferably mRNA.

According to an embodiment of the present invention, said means can be a specific antibody directed against the cerebellin-2 protein.

Means for measuring the level of expression of the cerebellin-2 protein include antibodies specifically binding to the cerebellin-2 protein. Such means can be labeled with detectable compound such as fluorophores or radioactive compounds. For example, the antibody specifically binding to the cerebellin-2 protein may be labeled with a detectable compound. Alternatively, the kit may further comprise a secondary antibody, labeled with a detectable compound, which binds to an unlabelled antibody specifically binding to the cerebellin-2 protein.

The antibody may be polyclonal or monoclonal, preferably monoclonal.

Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.

Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV- hybridoma technique (Cole et al. 1985).

Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies directed against biomarkers of the invention. Antibodies useful in practicing the present invention also include anti-biomarkers fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to biomarkers of the invention.

For example, phage display of antibodies may be used. In such a method, single- chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e.g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay. Examples of commercially available monoclonal antibodies are human cerebellin-2 antibody from R&D system (monoclonal mouse antibodies catalog numbers MAB7044 and MAB70441 ), and from Abgent (polyclonal antibody against C-terminal peptide AP1 1835b).

Instructions for using the kit according to methods of the invention may comprise instructions for processing the biological sample obtained from the patient and/or for performing the test, or instructions for interpreting the results. A kit may also contain a notice in the form prescribed by a governmental agency regu lating the man ufacture, use or sale of pharmaceuticals or biological products.

In addition, a kit of the invention generally also comprises at least one reagent for the detection of a complex between the means for measuring the level of expression of the cerebellin-2 gene included in the kit and the cerebellin-2 protein or mRNA cited above, or between means for detecting SNPs of the cerebellin-2 gene included in the kit and said SNPs of the cerebellin-2 gene cited above.

Depending on the procedure, the kit may further comprise one or more of: extraction buffer and/or reagents, western blotting buffer and/or reagents, and detection means.

Protocols for using these buffers and reagents for performing different steps of the procedure may be included in the kit.

The different reagents included in a kit of the invention may be supplied in a solid (e.g. lyophilized) or liquid form.

The kits of the present invention may optionally comprise different containers

(e.g., vial, ampoule, test tube, flask or bottle) for each individual buffer and/or reagent. Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Other containers suitable for conducting certain steps of the disclosed methods may also be provided. The individual containers of the kit are preferably maintained in close confinement for commercial sale.

The invention will be further illustrated by the following figures and examples.

FIGURES

Figure 1 : Figure 1 represents the testing of the association of SNPs with i/fPAH with the application of the Eigenstrat program.

Figure 2: Figure 2 illustrates CBLN2 mRNA levels in lungs from PAH patients. CBLN2 mRNA levels were measured by real time RT-PCR in lungs from control (CTL, white bars) and PAH patients (PAH, black bars) and normalized to GAPDH mRNA levels. ^*** p<0.001 vs controls, n=8 to 9. Figure 3: Figure 3 illustrates CBLN2 mRNA levels in PA-EC and PA-SMC (human pulmonary artery endothelial cells and human pulmonary artery smooth muscle cells) from PAH patients. CBLN2 mRNA levels were measured by real time RT-PCR in cultured PA- EC and PA-SMC from control (CTL, white bars) and PAH patients (PAH, black bars) and normalized to GAPDH mRNA levels. ^* p<0.05 vs controls, n=7 to 1 1.

EXAMPLES

Example 1 : two-stage genome-wide association study (GWAS) of patients with iPAH and fPAH (i/fPAH) without detectable BMPR2 mutations

Methods

Study population

Patients and controls:

The diagnosis of PAH was defined by hemodynamic measurement during right- heart catheterization for all patients included in the study (Discovery stage and Replication stage), including those seen in the French PAH Network between January 1 , 2003 and April 1 , 2010. For all patients, PAH was defined as a mean pulmonary arterial pressure equal to or exceeding 25 mm Hg associated with a normal pulmonary capillary wedge pressure. Hemodynamic evaluation by right-heart catheterization was performed at baseline in all subjects according to previously described protocols (Sitbon, O. et al. Long- term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation 1 1 1 , 3105-1 1 (2005); Loyd, J.E., Primm, R.K. & Newman, J.H. Familial primary pulmonary hypertension: clinical patterns. Am Rev Respir Dis 129, 194-7 (1984)).

PAH was considered to be idiopathic (iPAH) after clinical and biological investigation allowing elimination of all known causes. Patients with iPAH were tested for BMPR2 mutations, and patients with a family history of PAH, were tested for BMPR2 and ACVRL1 mutations. Screening for point mutations and large rearrangements was performed as previously reported (Sztrymf, B. et al. Clinical outcomes of pulmonary arterial hypertension in carriers of BMPR2 mutation. Am J Respir Crit Care Med 177, 1377-83 (2008), Eyries, M. et al. ACVRL1 germinal mosaic with two mutant alleles in hereditary hemorrhagic telangiectasia associated with pulmonary arterial hypertension. Clin Genet (201 1 )). Patients carrying a mutation of either of these genes were excluded. When patients had a family history of PAH without evidence of either BMPR2 or ACVRL1 mutation, a single index case from the family was included in the GWAS analysis.

Discovery stage 378 patients meeting these criteria were included. All clinical characteristics at PAH diagnosis and follow-up were stored in the Registry of the French PAH Network (Humbert, M. et al. Pulmonary arterial hypertension in France: results from a national registry. Am J Respir Crit Care Med 173, 1023-30 (2006)). This registry was set up in agreement with French bioethics laws (French Commission Nationale de Nnformatique et des Liberies), and patients gave their consent to be included (Sztrymf, B. et al. Clinical outcomes of pulmonary arterial hypertension in carriers of BMPR2 mutation. Am J Respir Crit Care Med 177, 1377-83 (2008)).

The control group was composed of a random sample of 1 ,140 subjects free of any chronic diseases selected from the 3C study (Vascular factors and risk of dementia: design of the Three-City Study and baseline characteristics of the study population, Neuroepidemiology 22, 316-25 (2003)). The 3C Study is a population-based prospective cohort with a 4-years follow up carried out in three French cities: Bordeaux (southwest France), Montpellier (southeast France) and Dijon (central eastern France). This study has served as a control population for several French GWAS projects (Allanore, Y. et al. Genome-wide scan identifies TNIP1 , PSORS1 C1 , and RHOB as novel risk loci for systemic sclerosis. PLoS Genet 7, e1002091 (201 1 ); Germain, M. et al. Genetics of venous thrombosis: insights from a new genome wide association study. PLoS One 6, e25581 (201 1 ); Hollingworth, P. et al. Common variants at ABCA7, MS4A6A/MS4A4E, EPHA1 , CD33 and CD2AP are associated with Alzheimer's disease. Nat Genet 43, 429- 35 (201 1 ), Saad, M. et al. Genome-wide association study confirms BST1 and suggests a locus on 12q24 as the risk loci for Parkinson's disease in the European population, Hum Mol Genet 20, 615-27 (201 1 )).

Replication stage

Specimens (315 PAH patients and 500 healthy controls) from participants in the

Vanderbilt Prospective Pulmonary Hypertension Research Cohort study, and from the Columbia University Pulmonary Hypertension Center, were used. These subjects were recruited via the Vanderbilt and Columbia Pulmonary Hypertension Centers, the NIH Clinical Trials website (http://clinicaltrials.gov), and in collaboration at the Pulmonary Hypertension Association Conference Research Recruitment Room (2010 Conference, Anaheim, California). The Vanderbilt University Medical Center and Columbia University Institutional Review Boards approved all study protocols. All participants gave informed written consent to participate in genetic and clinical studies and underwent genetic counseling when appropriate, in accordance with the guidelines of the American College of Chest Physicians (McGoon, M. et al. Screening, early detection, and diagnosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 126, 14S-34S (2004)).

As with the Discovery stage, PAH was defined as a mean pulmonary arterial pressure equal to or exceeding 25 mm Hg associated with a normal pulmonary capillary wedge pressure. Hemodynamic evaluation by right-heart catheterization was performed at baseline in all subjects according to previously described protocols (Sitbon, O. et al. Long- term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation 1 1 1 , 3105-1 1 (2005), Loyd, J.E., Primm, R.K. & Newman, J.H. Familial primary pulmonary hypertension: clinical patterns. Am Rev Respir Dis 129, 194-7 (1984)). PAH was considered to be idiopathic (iPAH) after clinical and biological investigation allowing elimination of all known causes, or familial as appropriate. Genotypic evaluation for mutations in BMPR2 and ACVRL1 mutations was the same as in the Discovery stage cohort. Genotyping

Genotyping

Discovery stage

The sample of 378 i/fPAH patients and 1 ,140 healthy controls were typed with the lllumina Human 610-Quad Beadchip. Individuals with genotyping success lower than 95% were excluded from the analyses as were individuals demonstrating close relatedness. The latter was assessed by pairwise clustering of identity by state distance (IBS) and multi-dimensional scaling (MDS) using the PLINK software (Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81 , 559-75 (2007)). The Eigenstrat program (Price, A.L. et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38, 904- 9 (2006)) was further used to detect individuals of non-European ancestry. SNPs showing significant (p < 10^"5) deviation from Hardy-Weinberg Equilibrium (HWE) in controls, with minor allele frequency (MAF) less than 1 % in controls, less than 5% in cases or genotyping call rate < 99% were filtered out. This resulted in the final analysis of 462,499 autosomal SNPs in a sample of 340 i/fPAH patients and 1 ,068 healthy individuals.

Replication stage

The 384 SNPs showing the strongest association with i/fPAH in the GWAS and assigned to an lllumina ScoreDesign greater than 0.4 were selected for genotying in an independent sample of 297 i/fPAH patients and 479 controls using a lllumina GoldenGate assay. 34 individuals (12 patients and 22 controls) were discarded due to low genotype calling (< 80%). SNPs showing significant deviation from HWE at p< 10^"5 in controls and call rate <99% were filtered out from the statistical analysis, resulting into the statistical analysis of 319 SNPs in a sample of 285 patients and 457 controls. Statistical analysis

At the first (Discovery) stage, the genome-wide association analysis of autosomal SNPs was conducted using the Eigenstrat program that corrects for any uncontrolled population stratification (Price, A.L. et al. Principal components analysis corrects for stratification in genome-wide association studies, Nat Genet 38, 904-9 (2006)). The genomic control (GC) inflation factor was also computed according to the median test statistic (Devlin, B. & Roeder, K. Genomic control for association studies. Biometrics 55, 997-1004 (1999). At the second (Replication) stage, the association of SNPs with i/fPAH was assessed by use of the Cochran-Armitage trend test (Sasieni, P.D. From genotypes to genes: doubling the sample size. Biometrics 53, 1253-61 (1997)). Homogeneity of associations across the two stages was tested using the Mantel-Haenszel method (Mantel, N. & Haenszel, W. Statistical aspects of the analysis of data from retrospective studies of disease, J Natl Cancer Inst 22, 719-48 (1959)).

The imputation of 1 1 ,572,501 autosomal SNPs was conducted using the MACH (http://www.sph.umich.edu/csg/abecasis/mach/) and minimac (http://genome.sph. umich. edu/wiki/Minimac) according the 1000G 2010-08 release CEU reference dataset. The association of each imputed SNP with i/fPAH was tested by use of a logistic regression analysis in which allele dosage (from 0 to 2 copies of the minor allele) of imputed SNPs was used. Analyses were adjusted for the first four principal components and were performed using the mach2dat (v 1 .08.18) software (http://www.sph. umich. edu/csg/abecasis/MACH/download/).

Results

A two-stage genome-wide association study (GWAS) of patients with iPAH and fPAH (i/fPAH) without detectable BMPR2 mutations was conducted to identify novel predisposing genes to idiopathic and familial pulmonary hypertension (iPAH and fPAH, respectively). At the first stage of the analysis, 462,499 autosomal SNPs typed by the lllumina 610-Quad DNA beadchip and checked for quality controls were tested for association with i/fPAH in a sample of 340 i/fPAH patients for whom no BMPR2 mutation was identified and 1 ,068 controls of French origin (see Methods). The application of the Eigenstrat program for testing the association of SNP with i/fPAH (Price, A.L. et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38, 904-9 (2006)) did not reveal any evidence for population stratification, the genomic inflation coefficient being 1.02 (Figure 1 ). The 384 most significant SNPs with corresponding association p-values ranging from 1.82 x 10^"6 to 6.87 x 10^"4 were then selected for further validation of association with i/fPAH in an independent sample of 285 patients and 457 controls. Among these 384 SNPs genotyped by a dedicated lllumina GoldenGate assay (see Methods), 319 SNPs passed the quality controls filtering. Of these 319 SNPs, two showed significant association with i/fPAH after Bonferroni correction for the number of tested SNPs, rs2217560 (P = 1.63 x 10^"5) and rs9916909 (P = 3.50 x 10^"5). These two SNPs were in complete linkage association (r² = 0.99) and located around 52 kb in the downstream region of the CBLN2 gene on locus 18q22.3.

As indicated in Table 3 below, the pattern of association of the rs2217560 with i/fPAH was very homogeneous in the discovery and replication phases. In the discovery GWAS, the rs2217560-G allele was more frequent in cases than in controls (0.123 vs 0.070) and was associated with an increased risk for i/fPAH of 1.87 [1.41 - 2.48] (Pcochran = 1 .56 x 10^"5' PEIGENSTRAT =3.88 x 10^"4 ).

Table 3. Association of CBLN2 rs2217560 with i/fPAH in two independent case-control studies

First-Stage Cohort Second-Stage Cohort

Controls i/fPAH Controls i/fPAH

rs2217560 N = 1 ,068 N = 340 N = 456 N = 284

AA 925 (87%) 262 (77%) 400 (88%) 21 1 (74%)

AG 136 (13%) 72 (21 %) 52 (1 1 %) 71 (25%)

GG 7 (<1 %) 6 (2%) 4 (1 %) 2 (1 %)

MAF (G) 0.070 0.123 0.066 0.132

pd⁾ P = 1.56 x 10^"5 P = 1.63 x 10^"5

1.866 [1.407 - 2.475] 2.160 [1.51 1 - 3.089]

Allelic

Odds Ratio 1.973 [1.587

p = 7.47 10^"10

MAF: Minor Allele Frequency

⁽ ' P-value of the Cochran-Armitage trend test. The p-value of the association test in the discovery cohort after controlling for any underlying population stratification (EIGENSTRAT software) was p = 3.9 10^"4

The Mantel-Haenszel test for homogeneity of the Odds Ratio across the two cohorts was not significant (P = 0.81 1 ). The resulting Mantel-Haenszel allelic odds ratio estimate was 1.977 [1.584 - 2.467], P = 1.649 x 10^"9 At the second stage, the rs2217560-G allele was also at increased frequency in cases (0.132 vs 0.066) and the corresponding odds ratio was 2.16 [1 .51 - 3.09] (P_Coc_hran= 1 -63 10^"5). In the combined sample, the rs2217560-G allele was associated with an increased risk for i/fPAH of 1.97 [1.59 - 2.45] with an overall statistical evidence reaching P = 7.47 x 10^"10 (Table 2). No evidence for a sex-specific association was observed as shown in Table 4.

Table 4. Association of CBLN2 rs2217560 with iPAH according to sex

Male Female

First-Stage Second-Stage First-Stage Second-Stage

Cohort Cohort Cohort Cohort

Controls iPAH Controls iPAH Controls iPAH Controls iPAH rs2217560 N = 333 N = 103 N = 122 N = 91 N = 735 N =237 N = 334 N = 193

285 77 640 185 136

AA 106 (87%) 75 (82%) 294 (88%)

(86%) (75%) (87%) (78%) (71 %)

25

AG 45 (13%) 13 (1 1 %) 16 (18%) 91 (12%) 47 (20%) 39 (12%) 55 (29%)

(24%)

GG 3 (<1 %) 1 (1 %) 3 (2%) 0 (-) 4 (<1 %) 5 (2%) 1 (<1 %) 2 (<1 %)

MAF (G) 0.077 0.131 0.078 0.088 0.067 0.120 0.061 0.153

MAF: Minor AlleleFrequency

It was further examined whether the observed association could be explained by untyped SNP(s) located in the vicinity of the rs2217560. Using the 1000 Genomes 2010-08 release as a reference, it was conducted an imputation analysis in the GWAS discovery cohort (see Methods). This analysis did not reveal any single imputed SNP at the 18q22.3 locus that showed stronger evidence of association than the rs2217560.

The rs2217560 identified through this two-stage GWAS strategy lies about 50 kb upstream from the CBLN2 gene.

Example 2: study of the cerebellin-2 gene expression in pulmonary vascular cells.

Methods

Tissues and cells

Lung specimens were obtained at the time of lung transplantation from patients with idiopathic PAH, at the Marie Lannelongue Hospital, Le Plessis-Robinson, France. Control-lung specimens were obtained from patients without any evidence of pulmonary vascular disease who underwent lobectomy or pneumonectomy for localized lung cancer, with the normal tissue collected at a distance from the tumors. This study was approved by the local ethics committee (Comite de Protection des Personnes lle-de-France, Le Kremlin-Bicetre, France). The patients gave their informed consent before the study.

For RNA extraction, lungs were washed in PBS, immediately frozen in liquid nitrogen and stored at -80°.

Human pulmonary artery endothelial cells (PA-EC) and human pulmonary artery smooth muscle cells (PA-SMC) were isolated and cultured as previously described (Guignabert, C. et al. Dichloroacetate treatment partially regresses established pulmonary hypertension in mice with SM22{alpha}-targeted overexpression of the serotonin transporter. Faseb J (2009); Tu, L. et al. Autocrine fibroblast growth factor-2 signaling contributes to altered endothelial phenotype in pulmonary hypertension. Am J Respir Cell Mol Biol 45, 31 1-22 (201 1 )). Cells were used between passages 3-6. Real-time RT-PCR assay

Real-time RT PCR assay was performed as previously described (Dupuis, M. et al. Profiling of aortic smooth muscle cell gene expression in response to chronic inhibition of nitric oxide synthase in rats, Circulation 1 10, 867-73 (2004)) using primers described in Table 5 below:

Table 5. Primers used in Real-time RT PCR assays

Data are expressed as mean fold change ± standard deviation (SD) of at least three independent experiments. Statistical analysis was performed using a non parametric Mann and Whitney test for single comparisons. P values less than 0.05 were considered to be significant.

Results

Expression of the cerebellin-2 gene (CBLN2) in the lung and other cell types was studied. It was found that CBLN2 mRNA is expressed in the whole lung and in circulating cells, such as lymphocytes, but at lower levels. CBLN2 mRNA levels were significantly higher in lungs explanted from PAH patients than in histologically normal lungs from subjects having lung surgery for cancer (control lungs) (Figure 2).

In cultured pulmonary vascular cells, it was found that CBLN2 mRNA was expressed in endothelial cells (EC) isolated from pulmonary artery (PA-EC), in contrast with pulmonary artery vascular smooth muscle cells (PA-SMC) where expression was detected at very low levels (Figure 3). The levels of expression in primary cultured EC explanted from PAH lungs in the course of lung transplantation were higher than in EC from control lungs (Figure 3).

CONCLUSION

Altogether, these results strongly support the role of the CBLN2 gene as a novel contributor to the physiopathology of i/fPAH.

Claims

Method of diagnosing genetic predisposition to pulmonary arterial hypertension in a patient, said method comprising:

a) determining the presence or absence of a nucleotide polymorphism (SNP) of the cerebellin-2 gene in a sample obtained from said patient;

b) deducing therefrom if the patient has a genetic predisposition to pulmonary arterial hypertension.

Method according to claim 1 , wherein the presence of a SNP of the cerebellin-2 gene indicates that the patient has a genetic predisposition to pulmonary arterial hypertension.

Method according to claim 2, wherein the genetic predisposition to arterial hypertension indicates that the patient is at risk of having or has pulmonary arterial hypertension.

Method according to claims any one of claims 1 to 3, wherein the SNP of the cerebellin-2 gene is selected from the group consisting of rs1432071 , rs10514048, rs12955878, rs99601 17, rs79135430, rs59675169, rs7228776, rs7226690, rs1432070, rs1432069, rs1432067, rs2217560 and rs9916909.

Method according to any one of the preceding claims, wherein the SNP of the cerebellin-2 gene is selected from the group consisting of rs2217560 and rs9916909.

Method according to claim 5, wherein the SNP of the cerebellin-2 gene is selected from the group consisting of allele A or G of rs2217560 and allele C or A of rs9916909.

Method according to claim 6, wherein the SNP of the cerebellin-2 gene is the allele G of the rs2217560.

Method according to any one of the preceding claims, wherein the presence of a SNP of the cerebellin-2 gene indicates a two-fold increased relative risk of having pulmonary arterial hypertension.

9. Method according to any one of the preceding claims, wherein the presence or absence of a SNP of the cerebellin-2 gene is determined by DNA sequencing or PCR analysis.

10. Method of predicting or diagnosing pulmonary arterial hypertension in a patient, said method comprising:

1 1. Method according to claim 10, wherein a level of expression of said gene which is increased as compared with the reference level of expression of said gene indicates that the patient is at risk of having or has pulmonary arterial hypertension.

12. Method according to claim 10 or 1 1 , wherein the sample is obtained from endothelial cells of lung tissue, endothelial cells from vessels or from a blood sample.

13. Method according to any one of claims 10 to 12, wherein the level of expression of the cerebellin-2 gene is determined by measuring the level of expression of the cerebellin-2 protein.

14. Method according to claim 13, wherein the measure of the level of expression of the cerebellin-2 protein is performed by a method chosen from immunoassay, immunoblot, mass spectrometry (MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC- MS/MS), stable isotope labeling by amino acids in cell culture (SI LAC), isotope coded affinity tags (ICAT), isobaric tag for relative and absolute quantitation (ITRAQ), selective reaction monitoring (SRM) assays, multiple reaction monitoring (MRM) assays, bio-molecular interaction analysis/surface plasmon resonance

(BIA/SPR) technologies, calibration free concentration analysis.

15. Method according to any one of the preceding claims, wherein said pulmonary arterial hypertension is idiopathic pulmonary arterial hypertension or familial pulmonary arterial hypertension.

16. Kit for performing a method according to any one of claims 1 to 15, wherein said kit comprises:

a) means for detecting SNPs of the cerebellin-2 gene for performing a method according to any one of claims 1 to 9 and 15 or means for measuring the level of expression of the cerebellin-2 gene for performing a method according to any one of claims 10 to 15 ; and

b) instructions for use in the prediction and diagnosis of pulmonary arterial hypertension.