FIELD OF THE INVENTION

The present invention relates to a method of diagnosing or aiding diagnosis of pulmonary hypertension (PH), and/or pulmonary arterial hypertension (PAH) or chronic thromboembolic pulmonary hypertension (CTEPH) by detecting the expression of certain panels of microRNAs (miRNAs) in a sample from a subject.

BACKGROUND

Pulmonary hypertension (PH), defined by a resting mean pulmonary artery pressure (PAP) above 20 mmHg, is associated with reduced life expectancy (Maron et al., 2016). There is a diagnostic challenge (Kiely et al., 2019). Patients present with symptoms such as shortness of breath, which are not specific to raised PAP, and the diagnosis is often overlooked and delayed (Khou et al. , 2020). Echocardiography and plasma brain natriuretic peptide (BNP) measurements are used to help triage patients for cardiac catheterisation, the definitive diagnostic test. While echocardiography can offer an estimate of PAP, it is time consuming, and requires specialist interpretation, with significant false positive detection. Measurements of plasma BNP concentration offer a point-of-care option but circulating BNP increases in response to myocardial wall stress, and so elevated levels are not specific for PH.

Once PH is recognised, patients are assigned to one of five groups according to whether their haemodynamic measurements suggest pre-capillary or postcapillary vascular resistance and co-morbidity (Galie et al., 2015); namely, (1 ) pulmonary arterial hypertension (PAH), (2) PH due to left heart disease (PH-LHD), (3) PH due to lung disease (PH-lung), (4) chronic thromboembolic disease (CTEPH) and (5) PH associated with a group of miscellaneous diseases (PH- miscellaneous). PAH and CTEPH are rare subtypes, for which treatment options are available. Especially, diagnosis of PAH requires that physicians have a "suspicion of pulmonary hypertension" for the patient to be referred for echocardiography. It must be determined by a physician that the "symptoms, signs and history are suggestive of PH". As a result, many patients "chum" in the primary and secondary care environment before the diagnosis is made with an average of 2.8-year delay. Furthermore, the next step in the process is echocardiography which has a significant false negative rate. Because of these combined factors, 40% of PAH patients are never diagnosed and there is a substantial delay in the diagnosis of many of those who are ultimately correctly diagnosed. This delay in diagnosis leads to a delay in treatment which is associated with an increase in morbidity & early mortality. Once the patients are referred to a PH center for final diagnosis, after exclusion of other causes of PH, a right heart catheterization is performed for the final confirmatory diagnosis. Given that there is only a 35% five-year survival of untreated patients with PAH, earlier and more complete diagnosis of PAH is predicted to substantially improve mortality and morbidity.

The five subtypes of PH can present as similar manifestations, despite differing in physiologies and aetiologies. Incorrect diagnosis and resultant mismanagement of diseases can have serious consequences (Hoeper et al., 2007). Of particular concern, patients with PH-LHD or PH-lung may be incorrectly managed as PAH and vice versa. T reatments developed for PAH, which target the lung vasculature, have all had negative results in multicentric clinical trials of PH-LHD patients (Fernandez et al., 2019).

Therefore, there exists an urgent need for a more accessible, non-invasive test that can risk-stratify patients to enable early intervention, and distinguish PAH and CTEPH for which there are licensed treatments.

Circulating biomarkers (“liquid biopsies”) have the potential to improve clinical management but to date, only brain natriuretic peptide (BNP) or its prohormone (NT-proBNP) have been adopted by ERS/ESC guidelines and used routinely. But, BNP/NT-pro-BNP measures cardiac strain and is of limited diagnostic value; it does not discriminate between underlying causes of cardiac strain. It also has limited sensitivity, even in heart failure (Verbrugge et al., 2022). microRNAs (miRNAs) are non-protein coding sequences that have a critical role in regulating gene expression. Over 1000 miRNAs can be measured in blood with high confidence. Previous studies have identified miRNAs as dysregulated in PH. Levels change with disease and may offer an alternative to BNP as a blood test to diagnose and risk stratify patients (Rhodes et al., 2013; Rothman et al., 2016; Emington et al., 2021). A recent study has used blood transcriptomics to illuminate the molecular heterogeneity of PAH. In addition to diagnosis, the inventors of the present invention hypothesised that circulating miRNAs may also inform molecular endotypes in a PH cohort.

SUMMARY OF INVENTION

The present invention is based on the surprising discovery that distinct patterns of expression of miRNAs within novel panels detected in a biological sample obtained from a subject can aid in diagnosing and distinguishing between PH, PAH and CTEPH and also aid in identifying subjects who may be at risk of developing PH, PAH and CTEPH. The determination of the pattern of expression of the miRNAs in the panels of the invention can therefore be used to inform treatment protocols for the subject and to ensure the subject receives a suitable course of treatment for their condition.

The present inventors identified panels of circulating miRNAs derived from an unbiased screen of over 600 miRNAs, the most comprehensive screen applied to PH to date. The majority of these miRNAs have not been previously associated with PH in any form. Small panels comprising 9 miRNAs have been identified that can distinguish two treatable subtypes of PH from a population of patients at risk of the condition; namely, pulmonary arterial hypertension (PAH) and chronic thrombolic pulmonary hypertension (CTEPH), alongside a small panel comprising 9 miRNAs that can distinguish PAH from CTEPH. These panels can be used independently or coupled with plasma BNP measurements to enable accurate diagnosis in the patient investigation pathway as illustrated in Figure 1. Additionally, in contrast to current methods for diagnosing PH, the miRNA panels herein described may be used as basis for a rapid and accessible point of care test for earlier diagnosis and distinguishing subtypes of pulmonary hypertension, allowing patients to be aligned with the correct management pathway. It would empower non-specialists, who at present rely on non-specific tools (e.g., ECG, plasma BNP) to identify patients for referral.

Accordingly, the miRNA panels herein disclosed provide basis for an improved method for diagnosing or aiding diagnosis of and distinguishing between PH, PAH and CTEPH, and reducing the risk of mismanaged treatment due to incorrect diagnosis.

In a first aspect, the invention provides a method for diagnosing or aiding diagnosis of pulmonary hypertension (PH) in a subject, determining a subject’s risk of developing PH, and/or determining a suitable course of treatment for a subject with PH, the method comprising the step of detecting expression of microRNAs (miRNAs) comprised in a panel of miRNAs in a sample obtained from the subject, wherein the panel of miRNAs comprises: hsa-miR-210-3p, hsa-miR- 151-5p, hsa-miR-10b-5p, hsa-miR-30a-5p, hsa-miR-145-5p, hsa-miR-193b-3p, hsa-miR-126-3p, hsa-miR-95-3p, and/or hsa-miR-374a-3p.

In a second aspect, the invention provides a method for diagnosing or aiding diagnosis of pulmonary arterial hypertension (PAH) in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH, the method comprising the step of detecting expression of microRNAs (miRNAs) comprised in a panel of miRNAs in a sample obtained from the subject, wherein the panel of miRNAs comprises: hsa-miR- 151a-5p, hsa-miR-210-3p, hsa-miR-193-3p, hsa-miR-30a-5p, hsa-miR-126-3p, hsa-miR-26a-2-3p, hsa-200c-3p, hsa-miR-10b-5p, and/or hsa-miR-1226-3p.

In a third aspect, the invention provides a method for diagnosing or aiding diagnosis of pulmonary arterial hypertension (PAH) in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH, the method comprising the step of detecting expression of microRNAs (miRNAs) comprised in a panel of miRNAs in a sample obtained from the subject, wherein the panel of miRNAs comprises: hsa-miR-361- 3p, hsa-miR-199b-5p, hsa-miR-206, hsa-miR-22-3p, hsa-miR-196b-5p, hsa-miR- 30a-5p, hsa-miR-4306, hsa-miR-150-5p, and/or hsa-miR-135a-5p; preferably wherein the subject has been previously diagnosed with PH.

In a fourth aspect, the invention provides a method for diagnosing or aiding diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH) in a subject, determining a subject’s risk of developing CTEPH, and/or determining a suitable course of treatment for a subject with CTEPH, the method comprising detecting expression of microRNAs (miRNAs) comprised in a panel of miRNAs in a sample obtained from the subject, wherein the panel of miRNAs comprises: hsa- miR-34a-5p, hsa-miR-16-1-3p, hsa-miR-375, hsa-miR-135a-5p, hsa-miR-30d- 5p, hsa-miR-499a-5p, hsa-miR-125b-5p, hsa-miR-885-5p, and/or hsa-miR-99a- 5p, preferably wherein the subject has been previously diagnosed with PH.

In a fifth aspect, the invention provides a kit comprising means for detecting expression of a panel of microRNAs (miRNAs) in a sample, wherein the panel of miRNAs comprises: (i) hsa-miR-210-3p, hsa-miR-151-5p, hsa-miR-10b-5p, hsa- miR-30a-5p, hsa-miR-145-5p, hsa-miR-193b-3p, hsa-miR-126-3p, hsa-miR-95- 3p, and hsa-miR-374a-3p; (ii) hsa-miR-151a-5p, hsa-miR-210-3p, hsa-miR-193- 3p, hsa-miR-30a-5p, hsa-miR-126-3p, hsa-miR-26a-2-3p, hsa-200c-3p, hsa-miR- 10b-5p, and hsa-miR-1226-3p; (iii) hsa-miR-361-3p, hsa-miR-199b-5p, hsa-miR- 206, hsa-miR-22-3p, hsa-miR-196b-5p, hsa-miR-30a-5p, hsa-miR-4306, hsa- miR-150-5p, and hsa-miR-135a-5p; (iv) hsa-miR-34a-5p, hsa-miR-16-1-3p, hsa- miR-375, hsa-miR-135a-5p, hsa-miR-30d-5p, hsa-miR-499a-5p, hsa-miR-125b- 5p, hsa-miR-885-5p, and hsa-miR-99a-5p; or (v) hsa-miR-34a-5p, hsa-miR-135a- 5p, hsa-miR-200b-3p, hsa-miR-885-5p, hsa-miR-16-1-3p, hsa-miR-141-3p, hsa- miR-361-3p, hsa-miR-193b-3p, and/or hsa-miR-651-5p. BRIEF DESCRIPTION OF DRAWINGS

The invention is illustrated with reference to the following drawings, in which:

Figure 1 shows the use of miRNA panels independently or coupled with plasma BNP measurements to enable accurate diagnosis in the patient investigation pathway.

Figure 2 is a Venn diagram showing the six study populations used to identify circulating miRNA biomarkers or panels. These include patients diagnosed with PAH, CTEPH (excluding patients post pulmonary endarterectomy), CTED (chronic thromboembolic pulmonary disease), PH. Controls were healthy controls and disease controls (atypical short of breath (ASOB) patients who have been referred to UK PH clinics). Disease controls can be non-PH, non-PAH or non- CTEPH disease controls (ASOB patients who have been referred to UK PH clinics, excluding those with confirmed PH, PAH or CTEPH), depending on the experimental arm within the study being assessed. Patient classification for pulmonary hypertension was based on the international guidelines.

Figure 3 shows a heat map depicting the variable importance of each miRNA in differentiating clinical classes. Darker values indicate a higher importance score for that miRNA, scaled to between 1-100.

Figures 4A-C show the relative importance of each miRNA in the panel diagnosing PH from disease controls (A). The area under the (receiver-operating characteristic) curve (ROC-AUC) data depicts the performance of the miRNA panel in diagnosing PH from diseases controls in comparison to NT-proBNP (B). PCR cycle threshold box blots comparing the whole cohort expression of each miRNA within the panel for PH and disease controls. A change in one unit represents a two-fold change in expression, such that a lower number indicates a higher abundance of miRNA (C). Figures 5A-C show the relative importance of each miRNA in the panel diagnosing PAH from disease controls (A). The ROC-AUC data depicting the performance of the miRNA panel in diagnosing PAH from diseases controls in comparison to NT-proBNP (B). PCR cycle threshold box blots comparing the whole cohort expression of each miRNA within the panel for PAH and disease controls. A change in one unit represents a two-fold change in expression, such that a lower number indicates a higher abundance of miRNA (C).

Figures 6A-C show the relative importance of each miRNA in the panel diagnosing PAH from other PH subtypes (A). The ROC-AUC data depicts the performance of the miRNA panel in diagnosing PAH from other PH subtypes in comparison to NT-proBNP (B). PCR cycle threshold box blots comparing the whole cohort expression of each miRNA within the panel for PAH and other PH subtypes. A change in one unit represents a two-fold change in expression, such that a lower number indicates a higher abundance of miRNA (C).

Figures 7A-C show the relative importance of each miRNA in the panel diagnosing CTEPH from other PH subtypes (A). The ROC-AUC data depicts the performance of the miRNA panel in diagnosing CTEPH from other PH subtypes in comparison to NT-proBNP (B). PCR cycle threshold box blots comparing the whole cohort expression of each miRNA within the panel for CTEPH and other PH subtypes. A change in one unit represents a two-fold change in expression, such that a lower number indicates a higher abundance of miRNA (C).

Figures 8A-C show the relative importance of each miRNA in the panel diagnosing PAH from CTEPH (A). The ROC-AUC data depicts the performance of the miRNA panel in diagnosing CTEPH from other PH subtypes in comparison to NT-proBNP (B). PCR cycle threshold box blots comparing the whole cohort expression of each miRNA within the panel for PAH and CTEPH. A change in one unit represents a two-fold change in expression, such that a lower number indicates a higher abundance of miRNA (C). DETAILED DESCRIPTION

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The terms "effective amount" or "pharmaceutically effective amount" refer to a sufficient amount of an agent to provide the desired biological or therapeutic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay recurrence. A therapeutically effective amount can be administered in one or more administrations.

The term "treatment" or "therapy" refers to administering an active agent with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a condition (e.g., a disease), the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, biochemical indicia of a disease, or otherwise arrest or inhibit further development of the disease, condition, or disorder in a statistically significant manner.

The term “subject” refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents and the like. In a preferred embodiment, the subject is a human. The subject may be a patient already undergoing care for PH. A suitable subject who may benefit from the invention may be a subject who presents to a medical establishment with unexplained shortness of breath or breathlessness. It is envisaged that the subject may be suspected by a medical practitioner of having PH but may not have been previously diagnosed with PH. Alternatively, the subject may have been diagnosed with PH by another means and the invention may be useful for distinguishing which subtype of PH is present, or for confirming the diagnosis of PH or relevant subtype of PH. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

As used herein, "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" can mean a range of up to 20%. When particular values are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

This invention is predicated on the surprising discovery that distinct patterns of expression of miRNAs within novel panels in a sample from a subject can aid in diagnosing and distinguishing PH, PAH and/or CTEPH in that subject and in identifying subjects at risk of developing PH, PAH and/or CTEPH. The pattern of expression of miRNAs in a panel of miRNAs can therefore be used to ensure the subject receives a suitable course of treatment. The present invention identifies panels of circulating miRNAs derived from an unbiased screen of over 600 miRNAs, the most comprehensive screen applied to PH to date; the majority of these miRNAs have not been previously associated with PH in any form. Small panels comprising nine miRNAs have been identified that can be used to distinguish two treatable subtypes of PH from a population of patients at risk of the condition; namely, PAH and CTEPH, alongside a small panel comprising 9 miRNAs that can be used to distinguish PAH from CTEPH. These panels can be used independently or coupled with plasma BNP measurements to enable accurate diagnosis in the patient investigation pathway as illustrated in Figure 1. Additionally, in contrast to current methods for diagnosing PH, the miRNA panels herein described may be developed and used as basis for a rapid and accessible point of care test for earlier diagnosis and distinguishing of subtypes of pulmonary hypertension, allowing subjects to be aligned with the correct management pathway. It would empower non-specialists, who at present rely on non-specific tools (e.g., ECG, plasma BNP) to identify subjects for referral.

Thus, in a first aspect, the invention provides a method for diagnosing or aiding diagnosis of pulmonary hypertension (PH) in a subject, determining a subject’s risk of developing PH, and/or determining a suitable course of treatment for a subject with PH, the method comprising the step of detecting expression of microRNAs (miRNAs) comprised in a panel of miRNAs in a sample obtained from the subject, wherein the panel of miRNAs comprises: hsa-miR-210-3p, hsa-miR- 151-5p, hsa-miR-10b-5p, hsa-miR-30a-5p, hsa-miR-145-5p, hsa-miR-193b-3p, hsa-miR-126-3p, hsa-miR-95-3p, and/or hsa-miR-374a-3p.

In one embodiment, the expression of two, three, four, five, six, seven, eight or nine of the miRNAs the panel is detected.

In another embodiment, an additional biomarker is detected. In a preferred embodiment, the additional biomarker may be N-terminal pro-brain natriuretic peptide (NT-pro-BNP) or brain natriuretic peptide (BNP).

The methods of the invention may be performed ex vivo on tissue directly obtained from the subject, likely immediately before testing. For the avoidance of doubt, the term “ex vivo" has its usual meaning in the art, referring to methods that are carried out in or on a sample in an artificial environment outside the body of a subject from whom the sample has been obtained. The sample obtained from a subject may also be referred to as a biological sample or a clinical sample.

It is envisaged that the “sample” will be a blood sample but may be any other biological sample (i.e. , any sampling of cells, tissues or bodily fluids or derivation thereof) that may contain circulating miRNA molecules. For example, the sample may be a blood sample, a urine sample, a saliva sample, a lymph sample, a cerebrospinal fluid sample and/or a combination thereof. Other suitable samples will include biopsies and smears. As used herein, the term “blood sample” includes whole blood and blood components (including plasma and/or serum) or any derivation thereof. In one embodiment, the one or more miRNA molecules are extracted from, amplified from, or detected in, the plasma component of a whole blood sample or from the serum component of a whole blood sample. In preferred embodiments, the blood sample is a plasma sample. Samples may be obtained by a variety of techniques which will be well known and understood by those in the art.

As used herein, “plasma” refers to the fluid portion of blood, excluding blood cells and platelets, but including dissolved proteins, glucose, clotting factors, electrolytes and hormones. As used herein, “serum” refers to blood plasma without clotting factors. The skilled person will be familiar with standard phlebotomy techniques, which are suitable for obtaining a blood sample from a subject. The skilled person will also be familiar with routine techniques for obtaining plasma and/or serum from a whole blood sample, e.g., using centrifugation.

The terms “microRNA”, “miRNA” or “miR” are used interchangeably herein and refer to small non-coding RNA molecules. In one instance, the term “miRNA” refers to small, single-stranded nucleotide molecules that have emerged as important regulators of gene expression in almost all eukaryotes; a third of the protein encoding human genome is thought to be regulated by miRNAs. miRNA molecules are composed of uracil (U), adenine (A), guanine (G), and cytosine (C) ribonucleic acid bases. These terms and concepts will be well known to those in the art. miRNA molecules may comprise at least 10 nucleotides, up to 35 nucleotides (i.e., polynucleotides) covalently linked together. In one instance, an miRNA molecule may be approximately 19-25 nucleotides in length, or 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29,30, 31 , 32, 33, 34 or 35 nucleotides in length, not including any optional labels or elongated sequences. miRNAs are non-coding RNAs and function like small-interfering RNA to down- regulate gene expression at the post-transcriptional level. miRNA biogenesis involves a series of steps that lead to gene silencing. Briefly, miRNAs are transcribed in the nucleus as longer primary-miRNAs, which are cleaved to form hair-pin shaped precursor-mi NAs. These precursors are exported from the nucleus and further cleaved to form the mature miRNA which associates with the RNA induced silencing complex to target the 3’-untranslated region of specific mRNAs and inhibit their translation to protein. miRNAs are present in a cell free state in plasma and remain stable and easily measurable. Their potential utility as a biomarker of disease or response to treatment has consequently been widely acknowledged. miRNAs are expressed in a tissue specific manner and have differential expression, both spatially and over time. They remain stable and are easily detectable in blood and are thus suitable for providing a minimally invasive means for diagnosing PH and PH subtypes, enabling early intervention according to the present invention.

The terms “pulmonary hypertension” or “PH” may be used interchangeably, and refer to scenarios in which the pressure in the blood vessels leading from the heart to the lungs is too high. In particular, it refers to scenarios in which a subject has a resting mean pulmonary artery pressure (PAP) above 20 mmHg. Symptoms of PH may include, but are not limited to, shortness of breath, fatigue, dizziness, chest pressure, chest pain, swelling (edema) in the ankles, legs and eventually abdomen (ascites), cyanosis and a pounding heartbeat. The subject may be suffering from angina, dyspnea, cyanosis, fatigue, heart palpitations, swelling of the ankles, legs and/or abdomen, and/or syncope.

The method herein disclosed may further comprise the step of determining a pattern of expression of the miRNAs comprised in the panel and comparing the pattern of expression of the miRNAs with the pattern of expression of miRNAs in a control panel comprising the same miRNAs to determine a relative change in the expression of each of the miRNAs in the sample in comparison with the expression of the control miRNAs. As used herein, the terms “disease control” or “control” may be used interchangeably, and may refer to patients or subjects who present with unexplained breathlessness (atypical short of breath, ASOB). In the context of diagnosing or aiding diagnosis of PH in a subject, determining a subject’s risk of developing PH, and/or determining a suitable course of treatment for a subject with PH, the disease control may be a non-PH disease control, whereby PH has been ruled out in ASOB patients who present with unexplained breathlessness. In one embodiment, the control panel of miRNAs may be from a sample obtained from a non-PH ASOB subject (that is, an ASOB subject who is confirmed as not having PH). In another embodiment, the control panel of miRNAs may be pooled from samples obtained from multiple non-PH ASOB subjects.

The relative change in expression of each miRNA within the panel of the first aspect differs in importance for diagnosing PH in a subject, determining a subject’s risk of developing PH, and/or determining a suitable course of treatment for a subject with PH. Therefore, it is preferred that the panel of the first aspect comprises hsa-miR-210-3p and hsa-miR-151-5p, which appear to be the most significant diagnostic miRNAs in the panel of this aspect. This panel may preferably further comprise hsa-miR-10b-5p and/or hsa-miR-30a-5p, which appear to be the next most significant markers of the condition.

In the context of diagnosing or aiding diagnosis of PH in a subject, determining a subject’s risk of developing PH, and/or determining a suitable course of treatment for a subject with PH according to an embodiment of the first aspect, the relative change may comprise increased expression of one or more of the miRNAs selected from the group comprising: hsa-miR-151-5p, hsa-miR-10b-5p, hsa-miR- 193b-3p, hsa-miR-126-3p, hsa-miR-95-3p, and/or hsa-miR-374a-3p; and decreased expression of one or more of the miRNAs selected from the group comprising hsa-miR-210-3p, hsa-miR-30a-5p, and/or hsa-miR-145-5p.

The terms “decrease” and “increase” in the context herein described, refer to a decrease or increase in the levels of hsa-miR-210-3p, hsa-miR-151-5p, hsa-miR- 10b-5p, hsa-miR-30a-5p, hsa-miR-145-5p, hsa-miR-193b-3p, hsa-miR-126-3p, hsa-miR-95-3p, and/or hsa-miR-374a-3p relative to the level of said miRNAs in a control sample (i.e. , a sample obtained from a non-PH ASOB subject or samples obtained from multiple non-PH ASOB subjects).

If, through the method of the first aspect, the relative change in the expression of each of the miRNAs in the sample is comparable with the expression of the control miRNA, then the subject is diagnosed as having PH, determined to be at high risk of developing PH, and/or directed to a suitable course of treatment for PH. Accordingly, the subject may be referred for further diagnosis in a specialist clinic, and/or medical intervention with a suitable course of treatment. A suitable course of treatment for PH would be known by a person of skill in the art, and may include cardiac catheterisation and/or lung computed tomography scan.

The methods of diagnosis or aiding diagnosis as described herein may further comprise carrying out additional clinical procedures, methods of diagnosis or measuring additional biomarkers. Such additional procedures or analysis may at to confirm or verify the diagnosis. For example, additional procedures may be carried out in addition to the methods of the present invention, such as echocardiogram, electrocardiogram (ECG or EKG), right-heart catheterization, chest X-ray, lung function test, exercise test, ventilation-perfusion scan, computerised tomography scan (CT scan), magnetic resonance imaging scan (MRI scan), and/or blood test. The additional biomarker may be N-terminal probrain natriuretic peptide (NT-pro-BNP) or brain natriuretic peptide (BNP).

In a second aspect, the invention provides a method for diagnosing or aiding diagnosis of pulmonary arterial hypertension (PAH) in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH, the method comprising the step of detecting expression of microRNAs (miRNAs) comprised in a panel of miRNAs in a sample obtained from the subject, wherein the panel of miRNAs comprises: hsa-miR- 151a-5p, hsa-miR-210-3p, hsa-miR-193-3p, hsa-miR-30a-5p, hsa-miR-126-3p, hsa-miR-26a-2-3p, hsa-200c-3p, hsa-miR-10b-5p, and/or hsa-miR-1226-3p. In one embodiment, the expression of two, three, four, five, six, seven, eight or nine of the miRNAs the panel is detected.

In another embodiment, an additional biomarker is detected. In a preferred embodiment, the additional biomarker may be N-terminal pro-brain natriuretic peptide (NT-pro-BNP) or brain natriuretic peptide (BNP).

The terms “pulmonary arterial hypertension” or “PAH” may be used interchangeably, and refer to a subtype of PH, in which the pressure in the blood vessels leading from the heart to the lungs is too high. This high pressure may be caused by obstruction in the small arteries of the lung. PAH is characterised by loss and obstructive remodelling of the pulmonary vascular bed. PAH features precapillary PH, which may be defined as an mPAP of 20 mm Hg or greater. PAH is divided into seven subgroups, namely idiopathic PAH, heritable PAH, drug- induced and toxin-induced PAH, PAH associated with various conditions such as connective tissue diseases, congenital heart diseases, HIV infection and portal hypertension, PAH in long-term responders to calcium channel blockers, PAH with venous/capillary involvement, and persistent PH of the newborn.

In an embodiment of the second aspect, the method may further comprise the step of determining a pattern of expression of the miRNAs comprised in the panel and comparing the pattern of expression of the miRNAs with the pattern of expression of miRNAs in a control panel comprising the same miRNAs to determine a relative change in the expression of each of the miRNAs in the sample in comparison with the expression of the control miRNAs.

In the context of diagnosing or aiding diagnosis of PAH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH according to an embodiment of the second aspect, the disease control may be a non-PAH disease control, whereby PAH has been ruled out in ASOB patients who present with unexplained breathlessness. In one embodiment, the control panel of miRNAs may be from a sample obtained from a non-PAH ASOB subject (that is, an ASOB subject who is confirmed as not having PAH). In another embodiment, the control panel of miRNAs may be pooled from samples obtained from multiple non-PAH ASOB subjects.

The relative change in expression of miRNA within this panel differs in importance for diagnosing or aiding diagnosis of PAH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH. Therefore, it is preferred that the panel of the second aspect comprises hsa-miR-151a-5p and hsa-miR-210-3p, which appear to be the most significant diagnostic miRNAs in the panel of this aspect. In a preferred embodiment, this panel further comprises hsa-miR-193-3p and/or hsa-miR-30a-5p, which appear to be the next most significant markers of the condition.

In the context of diagnosing or aiding diagnosis of PAH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH according to the second aspect, the relative change may comprise increased expression of one or more of the miRNAs selected from the group comprising hsa-miR-151a-5p, hsa-miR-193-3p, hsa-miR- 126-3p, hsa-miR-10b-5p, and/or hsa-miR-1226-3p; and decreased expression of one or more of the miRNAs selected from the group comprising hsa-miR-210-3p, hsa-miR-30a-5p, hsa-miR-26a-2-3p, and/or hsa-200c-3p.

The terms “decrease” and “increase” in the context herein described, refer to a decrease or increase in the levels of hsa-miR-151 a-5p, hsa-miR-210-3p, hsa-miR- 193-3p, hsa-miR-30a-5p, hsa-miR-126-3p, hsa-miR-26a-2-3p, hsa-200c-3p, hsa- miR-10b-5p, and/or hsa-miR-1226-3p relative to the level of said miRNAs in a control sample (i.e. , a sample obtained from a non-PAH ASOB subject or samples obtained from multiple non-PAH ASOB subjects).

For scenarios in which pattern of relative change in expression of the miRNAs comprised in the panel matches, or is similar to the pattern of expression of miRNAs in a control panel comprising the same miRNAs, then the subject is diagnosed as having PAH, determined to be at high risk of developing PAH, and/or directed to a suitable course of treatment for PAH. Accordingly, the subject may be referred for further diagnosis in a specialist clinic, and/or medical intervention with a suitable course of treatment. A suitable course of treatment for PAH would be known within the art, and may include but is not limited to medicaments such as vasodilators, endothelin receptor antagonists (such as bosentan, ambrisentan or macitentan), phosphodiesterase-5 inhibitors (sildenafil and tadalafil), prostaglandins (such as epoprostenol, iloprost and treprostinil), soluble guanylate cyclase stimulators (such as riociguat), calcium channel blockers (such as nifedipine, diltiazem, nicardipine and amlodipine), warfarin, digoxin, diuretics, oxygen therapy, surgeries or combinations thereof. The calcium channel blockers may be high dose calcium channel blockers. Suitable surgeries include pulmonary endarterectomy. Pulmonary endarterectomy is an operation to remove old blood clots from the pulmonary arteries in the lungs in people with chronic thromboembolic pulmonary hypertension. Other surgeries may include balloon pulmonary angioplasty, atrial septostomy or transplant. Pulmonary angioplasty is a new procedure where a small balloon is guided into the arteries and inflated for a few seconds to push the blockage aside and restore blood flow to the lung; it may be considered if pulmonary endarterectomy is not suitable, and has been shown to lower blood pressure in the lung arteries, improve breathing, and increase the ability to exercise. An atrial septostomy is an operation during which a small hole is made in the wall between the left and right atria of the heart using a cardiac catheter (a thin, flexible tube) inserted into the heart's chambers or blood vessels; it reduces the pressure in the right side of the heart, so the heart can pump more efficiently and the blood flow to the lungs can be improved. A lung or heart-lung transplant may be required in severe cases. Transplantation surgeries are rarely used because other effective treatments are available.

The methods of diagnosis or aiding diagnosis as described herein may further comprise carrying out additional clinical procedures, methods of diagnosis or measuring additional biomarkers. For example, additional procedures may be carried out in addition to the methods of the present invention, such as echocardiogram, electrocardiogram (ECG or EKG), right-heart catheterization, chest X-ray, lung function test, exercise test, ventilation-perfusion scan, computerised tomography scan (CT scan), magnetic resonance imaging scan (MRI scan), and/or blood test. The additional biomarker may be N-terminal probrain natriuretic peptide (NT-pro-BNP) or brain natriuretic peptide (BNP).

In a third aspect, the invention provides a method for diagnosing or aiding diagnosis of pulmonary arterial hypertension (PAH) in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH, the method comprising the step of detecting expression of microRNAs (miRNAs) comprised in a panel of miRNAs in a sample obtained from the subject, wherein the panel of miRNAs comprises: hsa-miR-361- 3p, hsa-miR-199b-5p, hsa-miR-206, hsa-miR-22-3p, hsa-miR-196b-5p, hsa-miR- 30a-5p, hsa-miR-4306, hsa-miR-150-5p, and/or hsa-miR-135a-5p; preferably wherein the subject has been previously diagnosed with PH.

In one embodiment, the expression of two, three, four, five, six, seven, eight or nine of the miRNAs the panel is detected.

In another embodiment, an additional biomarker is detected. In a preferred embodiment, the additional biomarker may be N-terminal pro-brain natriuretic peptide (NT-pro-BNP) or brain natriuretic peptide (BNP).

In a preferred embodiment, the panel of miRNAs comprising hsa-miR-361-3p, hsa-miR-199b-5p, hsa-miR-206, hsa-miR-22-3p, hsa-miR-196b-5p, hsa-miR- 30a-5p, hsa-miR-4306, hsa-miR-150-5p, and/or hsa-miR-135a-5p is used in differentiating or diagnosing or aiding diagnosis of PAH from other PH subtypes, i.e., when a subject has confirmed PH, but the subtype is unknown or unclear.

However, in another embodiment, the panel of miRNAs comprising hsa-miR-361- 3p, hsa-miR-199b-5p, hsa-miR-206, hsa-miR-22-3p, hsa-miR-196b-5p, hsa-miR- 30a-5p, hsa-miR-4306, hsa-miR-150-5p, and/or hsa-miR-135a-5p may be used for a subject who has not been diagnosed with PH.

In another embodiment of the third aspect, the method may further comprise the step of determining a pattern of expression of the miRNAs comprised in the panel and comparing the pattern of expression of the miRNAs with the pattern of expression of miRNAs in a control panel comprising the same miRNAs to determine a relative change in the expression of each of the miRNAs in the sample in comparison with the expression of the control miRNAs.

In the context of diagnosing or aiding diagnosis of PAH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH according to the third aspect, if the subject has confirmed PH but the subtype is unknown, the control is a subject who has confirmed PH that is not PAH.

The relative change in expression of miRNA within the panel of the third aspect differs in importance for diagnosing or aiding diagnosis of PAH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH. Therefore, it is preferred that the panel of the third aspect comprises: hsa-miR-361-3p and hsa-miR-199b-5p, which appear to be the most significant diagnostic miRNAs in the panel of this aspect. In a preferred embodiment, this panel further comprises hsa-miR-22-3p and/or hsa-miR-196b-5p, which appear to be the next most significant markers of the condition.

In the context of diagnosing or aiding diagnosis of PAH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH according to the third aspect, if the subject has confirmed PH but the subtype is unknown, the relative change may comprise increased expression of one or more of the miRNAs selected from the group comprising: hsa-miR-135a-5p, hsa-miR-4306, hsa-miR-206, and/or hsa-miR-22- 3p; and decreased expression of one or more of the miRNAs selected from the group comprising: hsa-miR-150-5p, hsa-miR-196b-5p, hsa-miR-199b-5p, hsa- miR-30a-5p, and/or hsa-miR-361-3p.

The terms “decrease” and “increase” in the context herein described, refer to a decrease or increase in the levels of hsa-miR-361 -3p, hsa-miR-199b-5p, hsa-miR- 206, hsa-miR-22-3p, hsa-miR-196b-5p, hsa-miR-30a-5p, hsa-miR-4306, hsa- miR-150-5p, and/or hsa-miR-135a-5p relative to the level of said miRNA’s in a control sample (i.e., a sample obtained from a subject with confirmed PH that is not PAH). For scenarios in which pattern of relative change in expression of the miRNAs comprised in the panel matches, or is similar to the pattern of expression of miRNAs in a control panel comprising the same miRNAs, then the subject is diagnosed as having PAH, determined to be at high risk of developing PAH, and/or directed to a suitable course of treatment for PAH. Accordingly, the subject may be referred for further diagnosis in a specialist clinic, and/or medical intervention with a suitable course of treatment. A suitable course of treatment for PAH would be known within the art, and may include but is not limited to medicaments such as endothelin receptor antagonists (such as bosentan, ambrisentan or macitentan), phosphodiesterase-5 inhibitors (sildenafil and tadalafil), prostaglandins (such as epoprostenol, iloprost and treprostinil), soluble guanylate cyclase stimulators (such as riociguat), or calcium channel blockers (such as nifedipine, diltiazem, nicardipine and amlodipine), or surgeries such as pulmonary endarterectomy. Pulmonary endarterectomy is an operation to remove old blood clots from the pulmonary arteries in the lungs in people with chronic thromboembolic pulmonary hypertension. Other surgeries may include balloon pulmonary angioplasty, atrial septostomy or transplant. Pulmonary angioplasty is a new procedure where a small balloon is guided into the arteries and inflated for a few seconds to push the blockage aside and restore blood flow to the lung; it may be considered if pulmonary endarterectomy is not suitable, and has been shown to lower blood pressure in the lung arteries, improve breathing, and increase the ability to exercise. An atrial septostomy is an operation during which a small hole is made in the wall between the left and right atria of the heart using a cardiac catheter (a thin, flexible tube) inserted into the heart's chambers or blood vessels; it reduces the pressure in the right side of the heart, so the heart can pump more efficiently and the blood flow to the lungs can be improved. A lung or heart-lung transplant may be required in severe cases. Transplantation surgeries are rarely used because other effective treatments are available. The methods of diagnosis or aiding diagnosis as described herein may further comprise carrying out additional clinical procedures, methods of diagnosis or measuring additional biomarkers. For example, additional procedures may be carried out in addition to the methods of the present invention, such as echocardiogram, electrocardiogram (ECG or EKG), right-heart catheterization, chest X-ray, lung function test, exercise test, ventilation-perfusion scan, computerised tomography scan (CT scan), magnetic resonance imaging scan (MRI scan), and/or blood test. The additional biomarker may be N-terminal probrain natriuretic peptide (NT-pro-BNP) or brain natriuretic peptide (BNP).

In a fourth aspect, the invention provides a method for diagnosing or aiding diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH) in a subject, determining a subject’s risk of developing CTEPH, and/or determining a suitable course of treatment for a subject with CTEPH, the method comprising detecting expression of microRNAs (miRNAs) comprised in a panel of miRNAs in a sample obtained from the subject, wherein the panel of miRNAs comprises: hsa- miR-34a-5p, hsa-miR-16-1-3p, hsa-miR-375, hsa-miR-135a-5p, hsa-miR-30d- 5p, hsa-miR-499a-5p, hsa-miR-125b-5p, hsa-miR-885-5p, and/or hsa-miR-99a- 5p, preferably wherein the subject has been previously diagnosed with PH.

In one embodiment, the expression of two, three, four, five, six, seven, eight or nine of the miRNAs the panel is detected.

In another embodiment, an additional biomarker is detected. In a preferred embodiment, the additional biomarker may be N-terminal pro-brain natriuretic peptide (NT-pro-BNP) or brain natriuretic peptide (BNP).

The terms “chronic thromboembolic pulmonary hypertension” or “CTEPH” may be used interchangeably, and refer to a subtype of PH in which there is pulmonary hypertension defined by resting mean pulmonary artery pressure (PAP) above 20 mmHg, due to obstructive thrombus in the pulmonary circulation. In a preferred embodiment, the panel of miRNAs comprising hsa-miR-34a-5p, hsa-miR-16-1-3p, hsa-miR-375, hsa-miR-135a-5p, hsa-miR-30d-5p, hsa-miR- 499a-5p, hsa-miR-125b-5p, hsa-miR-885-5p, and/or hsa-miR-99a-5p is used in differentiating or diagnosing or aiding diagnosis of the CTEPH from other PH subtypes, i.e. , when a subject has confirmed PH, but the subtype is unknown or unclear.

However, in another embodiment, the panel of miRNAs comprising hsa-miR-34a- 5p, hsa-miR-16-1-3p, hsa-miR-375, hsa-miR-135a-5p, hsa-miR-30d-5p, hsa-miR- 499a-5p, hsa-miR-125b-5p, hsa-miR-885-5p, and/or hsa-miR-99a-5p may be used for a subject who has not been diagnosed with PH.

In another embodiment of the fourth aspect, the method may further comprise the step of determining a pattern of expression of the miRNAs comprised in the panel and comparing the pattern of expression of the miRNAs with the pattern of expression of miRNAs in a control panel comprising the same miRNAs to determine a relative change in the expression of each of the miRNAs in the sample in comparison with the expression of the control miRNAs.

In the context of diagnosing or aiding diagnosis of CTEPH in a subject, determining a subject’s risk of developing CTEPH, and/or determining a suitable course of treatment for a subject with CTEPH, if the subject has confirmed PH but the subtype is unknown, the control is a subject who has confirmed PH that is not CTEPH.

The relative change in expression of miRNA within the panel of the fourth aspect differs in importance for diagnosing or aiding diagnosis of CTEPH in a subject, determining a subject’s risk of developing CTEPH, and/or determining a suitable course of treatment for a subject with CTEPH. Therefore, it is preferred that the panel of the fourth aspect comprises: hsa-miR-34a-5p and hsa-miR-16-1-3p, which appear to be the most significant diagnostic miRNAs in the panel of this aspect. In a preferred embodiment, this panel further comprises: hsa-miR-375 and/or hsa-miR-135a-5p, which appear to be the next most significant markers of the condition.

In the context of diagnosing or aiding diagnosis of CTEPH in a subject, determining a subject’s risk of developing CTEPH, and/or determining a suitable course of treatment for a subject with CTEPH according to the fourth aspect, if the subject has confirmed PH but the subtype is unknown, the relative change may comprise increased expression of one or more of the miRNAs selected from the group comprising: hsa-miR-34a-5p and/or hsa-miR-375; and decreased expression of one or more of the miRNAs selected from the group comprising: hsa-miR-16-1-3p, hsa-miR-135a-5p, hsa-miR-30d-5p, hsa-miR-499a-5p, hsa- miR-125b-5p, hsa-miR-885-5p, and/or hsa-miR-99a-5p.

The terms “decrease” and “increase” in the context herein described, refer to a decrease or increase in the levels of hsa-miR-34a-5p, hsa-miR-16-1 -3p, hsa-miR- 375, hsa-miR-135a-5p, hsa-miR-30d-5p, hsa-miR-499a-5p, hsa-miR-125b-5p, hsa-miR-885-5p, and/or hsa-miR-99a-5p, relative to the level of said miRNAs in a control sample (i.e. , a sample obtained from a subject who has confirmed PH that is not CTEPH).

For scenarios in which pattern of relative change in expression of the miRNAs comprised in the panel matches, or is similar to the pattern of expression of miRNAs in a control panel comprising the same miRNAs, then the subject is diagnosed as having CTEPH, determined to be at high risk of developing CTEPH, and/or directed to a suitable course of treatment for CTEPH. Accordingly, the subject may be referred for further diagnosis in a specialist clinic, and/or medical intervention with a suitable course of treatment. A suitable course of treatment for CTEPH would be known within the art, and may include surgical intervention, in which the subject is treated with a thrombo-embolectomy. Thrombo-embolectomy refers to a procedure designed to remove blood clots of foreign bodies from a blood vessel (vein or artery), as would be understood by a skilled person. The present invention further provides a panel of miRNAs that may be used for differentiating between PAH and CTEPH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH. This method may comprise detecting expression of microRNAs (miRNAs), comprised in a panel of miRNAs, in a sample obtained from the subject, wherein the panel of miRNAs comprises: hsa-miR-34a-5p, hsa-miR-135a-5p, hsa- miR-200b-3p, hsa-miR-885-5p, hsa-miR-16-1-3p, hsa-miR-141-3p, hsa-miR-361- 3p, hsa-miR-193-3p, and/or hsa-miR-651-5p.

In a preferred embodiment, the panel of miRNAs comprising hsa-miR-34a-5p, hsa-miR-135a-5p, hsa-miR-200b-3p, hsa-miR-885-5p, hsa-miR-16-1-3p, hsa- miR-141-3p, hsa-miR-361-3p, hsa-miR-193-3p, and/or hsa-miR-651-5p is used in differentiating or diagnosing or aiding diagnosis of PAH from CTEPH, i.e. , when a subject has confirmed PH, but the subtype is unknown or unclear.

However, in another embodiment, the panel of miRNAs comprising hsa-miR-34a- 5p, hsa-miR-135a-5p, hsa-miR-200b-3p, hsa-miR-885-5p, hsa-miR-16-1-3p, hsa- miR-141-3p, hsa-miR-361-3p, hsa-miR-193-3p, and/or hsa-miR-651-5p may be used for a subject who has not been diagnosed with PH.

In another embodiment, the method further comprises the step of determining a pattern of expression of the miRNAs comprised in the panel and comparing the pattern of expression of the miRNAs with the pattern of expression of miRNAs in a control panel comprising the same miRNAs to determine a relative change in the expression of each of the miRNAs in the sample in comparison with the expression of the control miRNAs.

In the context of differentiating between PAH and CTEPH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH, the control may be a subject with confirmed PH that is not PAH or CTEPH. The relative change in expression of miRNA within this panel differs in importance for differentiating between PAH and CTEPH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH. Therefore, it is preferred that the panel comprises: hsa-miR- 34a-5p and hsa-miR-135a-5p. Preferably the panel further comprises: hsa-miR- 200b-3p and/or hsa-miR-885-5p.

In the context of differentiating between PAH and CTEPH in a subject, determining a subject’s risk of developing PAH, and/or determining a suitable course of treatment for a subject with PAH, the relative change may comprise increased expression of one or more of the miRNAs selected from the group comprising: hsa-miR-135a-5p, hsa-miR-885-5p and/or hsa-miR-193-3p; and decreased expression of one or more of the miRNAs selected from the group comprising: hsa-miR-34a-5p, hsa-miR-200b-3p, hsa-miR-16-1-3p, hsa-miR-141-3p, hsa-miR- 361 -3p, and/or hsa-miR-651-5p.

The terms “decrease” and “increase” in the context herein described, refer to a decrease or increase in the levels of hsa-miR-34a-5p, hsa-miR-135a-5p, hsa-miR- 200b-3p, hsa-miR-885-5p, hsa-miR-16-1-3p, hsa-miR-141-3p, hsa-miR-361-3p, hsa-miR-193-3p, and/or hsa-miR-651-5p, relative to the level of said miRNAs in a control sample (i.e., a sample obtained from a subject with confirmed PH that is not PAH or CTEPH).

For scenarios in which pattern of relative change in expression of the miRNAs comprised in the panel matches, or is similar to the pattern of expression of miRNAs in a control panel comprising the same miRNAs, then the subject is diagnosed as having PAH, determined to be at high risk of developing PAH, and/or directed to a suitable course of treatment for PAH. Accordingly, the subject may be referred for further diagnosis in a specialist clinic, and/or medical intervention with a suitable course of treatment. A suitable course of treatment for PAH would be known within the art, and may include but is not limited to medicaments such as endothelin receptor antagonists (such as bosentan, ambrisentan or macitentan), phosphodiesterase-5 inhibitors (sildenafil and tadalafil), prostaglandins (such as epoprostenol, iloprost and treprostinil), soluble guanylate cyclase stimulators (such as riociguat), or calcium channel blockers (such as nifedipine, diltiazem, nicardipine and amlodipine), or surgeries such as pulmonary endarterectomy. Pulmonary endarterectomy is an operation to remove old blood clots from the pulmonary arteries in the lungs in people with chronic thromboembolic pulmonary hypertension. Other surgeries may include balloon pulmonary angioplasty, atrial septostomy or transplant. Pulmonary angioplasty is a new procedure where a small balloon is guided into the arteries and inflated for a few seconds to push the blockage aside and restore blood flow to the lung; it may be considered if pulmonary endarterectomy is not suitable, and has been shown to lower blood pressure in the lung arteries, improve breathing, and increase the ability to exercise. An atrial septostomy is an operation during which a small hole is made in the wall between the left and right atria of the heart using a cardiac catheter (a thin, flexible tube) inserted into the heart's chambers or blood vessels; it reduces the pressure in the right side of the heart, so the heart can pump more efficiently and the blood flow to the lungs can be improved. A lung or heart-lung transplant may be required in severe cases. Transplantation surgeries are rarely used because other effective treatments are available.

The methods of diagnosis or aiding diagnosis as described herein may further comprise carrying out additional clinical procedures, methods of diagnosis or measuring additional biomarkers. For example, additional procedures may be carried out in addition to the methods of the present invention, such as echocardiogram, electrocardiogram (ECG or EKG), right-heart catheterization, chest X-ray, lung function test, exercise test, ventilation-perfusion scan, computerised tomography scan (CT scan), magnetic resonance imaging scan (MRI scan), and/or blood test. The additional biomarker may be N-terminal probrain natriuretic peptide (NT-pro-BNP) or brain natriuretic peptide (BNP).

For the avoidance of doubt, “hsa-” refers to Homo sapiens, and is a standard abbreviation to differentiate the miRNAs from those of other species. The suffixes “3p” and “5p” denote 3 prime and 5 prime, respectively. These suffixes are used to distinguish two miRNAs originating from opposite arms of the same pre-miRNA.

All miRNAs are identified herein using standard nomenclature.

The method of the present invention also provides for, prior to detecting expression of miRNAs comprised in a panel of miRNAs in a sample, the step of amplifying RNA present in the sample. Amplification of total RNA in a sample may be achieved by various techniques which will be known in the art. Amplification of RNA enables the expression of miRNAs comprised in a panel of miRNAs in a sample to be detected, for example using quantitative polymerase chain reaction (qPCR).

The expression of miRNAs comprised in a panel of miRNAs in a subject’s sample and/or a control sample can be detected using any convenient means for detecting a level of a nucleic acid sequence, e.g., qPCR, quantitative nucleic acid hybridisation of miRNA, labelled miRNA, and/or nucleic acid amplification techniques which are routinely used in the art and which the skilled person will be familiar with.

Preferred techniques for determining the expression of miRNAs include: qPCR, real time PCR (RT-PCR), a technique suitable for large scale/multiple analysis and useful for screening large populations; microarray, comprising a 2D array on a solid substrate; next generation sequencing platforms for example RNAseq or miRNA-seq, in which the advantages of next generation sequencing include its high throughput, speed and low cost per base; and in situ hybridisation.

In a fifth aspect, the invention provides a kit comprising means for detecting expression of a panel of microRNAs (miRNAs) in a sample, wherein the panel of miRNAs comprises: (i) hsa-miR-210-3p, hsa-miR-151-5p, hsa-miR-10b-5p, hsa- miR-30a-5p, hsa-miR-145-5p, hsa-miR-193b-3p, hsa-miR-126-3p, hsa-miR-95- 3p, and hsa-miR-374a-3p; (ii) hsa-miR-151a-5p, hsa-miR-210-3p, hsa-miR-193- 3p, hsa-miR-30a-5p, hsa-miR-126-3p, hsa-miR-26a-2-3p, hsa-200c-3p, hsa-miR- 10b-5p, and hsa-miR-1226-3p; (iii) hsa-miR-361-3p, hsa-miR-199b-5p, hsa-miR- 206, hsa-miR-22-3p, hsa-miR-196b-5p, hsa-miR-30a-5p, hsa-miR-4306, hsa- miR-150-5p, and hsa-miR-135a-5p; (iv) hsa-miR-34a-5p, hsa-miR-16-1-3p, hsa- miR-375, hsa-miR-135a-5p, hsa-miR-30d-5p, hsa-miR-499a-5p, hsa-miR-125b- 5p, hsa-miR-885-5p, and hsa-miR-99a-5p; or (v) hsa-miR-34a-5p, hsa-miR-135a- 5p, hsa-miR-200b-3p, hsa-miR-885-5p, hsa-miR-16-1-3p, hsa-miR-141-3p, hsa- miR-361-3p, hsa-miR-193b-3p, and/or hsa-miR-651-5p.

As used herein, the term “means for detection” refers to any molecule or agent capable of selectively recognising the target biomolecule. A target biomolecule may be the microRNA(s) of the panels described herein. The means for detection may be a probe. Suitable probes may be synthesised by one of skill in the art, and may be appropriately labelled. For example, the probe may comprise a nucleic acid probe with a suitable degree of complementarity with the microRNA(s) of the panels described herein. Such a probe designed to recognise microRNAs may be directed to the target region, complementary nucleic acid sequence on the reverse strand, or copies of these regions or sequences generated through an amplification technique. In other embodiments, the probe may comprise a DNA or RNA molecule (such as a primer), an aptamer, an antibody, an affibody, a peptide, a protein, or an organic molecule.

In one embodiment, the kit may comprise an isolated set of probes for detecting expression of one or more of the panels of microRNAs (miRNAs) herein disclosed in a sample. The isolated set of probes may comprise at least two of the miRNAs from any one or more of the panels described herein ((i), (ii), (iii), (iv) or (v)). This embodiment is applicable to each panel described herein. In a preferred embodiment, the isolated set of probes for detecting expression of a panel of microRNAs (miRNAs) in a sample may comprise two, three, four, five, six, seven, eight or nine of the miRNAs from each of the panels described herein ((i), (ii), (iii), (iv) or (v)). This embodiment is applicable to each panel described herein.

In another embodiment, the kit further comprises a means for detection of at least one additional biomarker. In a preferred embodiment, the additional biomarker may be N-terminal pro-brain natriuretic peptide (NT-pro-BNP) or brain natriuretic peptide (BNP). In one embodiment, the kit comprises nucleic acid primer pairs that specifically hybridise to the miRNAs contained in the panel following treatment of the miRNAs with reverse transcriptase. As would be understood by a person of skill in the art, specifically hybridise in this context is intended to mean that the primer pairs have complementary nucleotide sequences to opposing portions of the either strand of the double stranded transcribed target and are thus configured to enable amplification of the target in the presence of suitable DNA polymerase and deoxyribonucleotides under thermocycling conditions.

The kit of the present invention may further comprise instructions for determining a pattern of expression of the miRNAs comprised in the panel and comparing the pattern of expression of the miRNAs with a the pattern of expression of miRNAs in a control panel comprising the same miRNAs to determine a relative change in the expression of each of the miRNAs in the sample in comparison with the expression of the control miRNAs; preferably wherein the relative change comprises: (i) increased expression of miRNAs selected from the group comprising: hsa-miR-151-5p, hsa-miR-10b-5p, hsa-miR-193b-3p, hsa-miR-126- 3p, hsa-miR-95-3p, and hsa-miR-374a-3p; and decreased expression of one or more of the miRNAs selected from the group comprising: hsa-miR-210-3p, hsa- miR-30a-5p, and hsa-miR-145-5p; (ii) increased expression of miRNAs selected from the group comprising: hsa-miR-151a-5p, hsa-miR-193-3p, hsa-miR-126-3p, hsa-miR-10b-5p, and hsa-miR-1226-3p; and decreased expression of miRNAs selected from the group comprising: hsa-miR-210-3p, hsa-miR-30a-5p, hsa-miR- 26a-2-3p, and hsa-200c-3p; (iii) increased expression of miRNAs selected from the group comprising: hsa-miR-135a-5p, hsa-miR-4306, hsa-miR-206, and hsa- miR-22-3p; and decreased expression of miRNAs selected from the group comprising: hsa-miR-150-5p, hsa-miR-196b-5p, hsa-miR-199b-5p, hsa-miR-30a- 5p, and hsa-miR-361-3p; (iv) increased expression of miRNAs selected from the group comprising: hsa-miR-34a-5p and hsa-miR-375; and decreased expression of miRNAs selected from the group comprising: hsa-miR-16-1-3p, hsa-miR-135a- 5p, hsa-miR-30d-5p, hsa-miR-499a-5p, hsa-miR-125b-5p, hsa-miR-885-5p, and hsa-miR-99a-5p; and/or (v) increased expression of miRNAs selected from the group comprising hsa-miR-135a-5p, hsa-miR-885-5p, and hsa-miR-193b-3p; and decreased expression of miRNAs selected from the group comprising: hsa-miR- 34a-5p, hsa-miR-200b-3p, hsa-miR-16-1-3p, hsa-miR-141-3p, hsa-miR-361-3p, and hsa-miR-651-5p.

The invention therefore provides an isolated set of probes as a means for detection of the microRNA(s) of the panels described herein. It is envisaged that the isolated set of probes will be capable of detecting the expression of at least two miRNAs of any of the panels described herein. The expression of the at least two miRNAs may be determined by several techniques which will be appreciated and understood by one of skill in the art, and may include, but are not limited to, sequencing, microarray, quantitative polymerase chain reaction (qPCR), northern blot, reverse transcription-polymerase chain reaction (RT-PCR), quantitative nucleic acid hybridisation of miRNA, labelled miRNA, and/or any other nucleic acid amplification techniques which are routinely used in the art and which the skilled person will be familiar with may be used. Other suitable techniques will include, but are not limited to, non-sequencing-based techniques, such as isothermal amplification-based techniques, including nicking endonuclease amplification reaction assay, transcription mediated amplification assay, loop-mediated isothermal amplification assay, helicase-dependent amplification assay, clustered regularly interspaced short palindromic repeat assay, or strand displacement assay.

The kit of the present invention provides for the expression of miRNAs comprised in a panel of miRNAs in a subject’s sample and/or a control sample to be detected using any convenient means for detecting a level of nucleic acid sequence as described herein, for example, using quantitative polymerase chain reaction (qPCR).

Preferred techniques for determining the expression of miRNAs include: qPCR, reverse-transcriptase PCR (RT-PCR), a technique suitable for large scale/multiple analysis and useful for screening large populations; microarray, comprising a 2D array on a solid substrate; next generation sequencing platforms for example RNAseq, in which the advantages of next generation sequencing include its high throughput, speed and low cost per base; and in situ hybridisation.

It will be apparent to the person skilled in the art that the methods of the invention 5 disclosed herein can be used in conjunction with other methods for diagnosing or aiding diagnosis of PH and PH subtypes, namely PAH and CTEPH, that are well known in the art.

SEQUENCES

10

The following table comprises the sequences forming part of the present application and each corresponding SEQ ID NO:

Table 1: SEQ ID NOs, sequence name, accession number, and sequence of each miRNA within each miRNA panel.

The invention will be further described with reference to the following non-limiting examples. The contents of any document mentioned herein are hereby incorporated by reference in its entirety.

EXAMPLES

The inventors have surprisingly found that distinct patterns of expression of miRNAs within novel panels in a sample from a subject can diagnose PH, PAH and CTEPH and identify subjects at high risk of PH, PAH and CTEPH. The pattern of expression of miRNAs in a panel of miRNAs can therefore be used to ensure the subject receives a suitable course of treatment.

Methodology

Example 1 - Study population and clinical data

The study population comprised 1150 patients with PH and 334 disease controls as summarised in Table 2. Patients were recruited from three UK national PH referral centres at the Hammersmith Hospital (Imperial), Royal Hallamshire Hospital (Sheffield) and Royal Papworth Hospital (Cambridge) (Table 3). All cases were diagnosed between 2008 and 2019 using diagnostic guidelines (Galie et al., 20150. All samples were obtained following informed consent to either the Imperial College Prospective study of patients with pulmonary vascular disease (PPVD, UK REC Ref 17/LO/0563), the Sheffield Teaching Hospitals observational study of pulmonary hypertension, cardiovascular and other respiratory diseases (STH- ObS, UK REC Ref 18/YH/0441) or Papworth (Cambridgeshire East Research Ethics Committee reference 08/H0304/56). All samples were collected as per local standard operating procedures and stored at -80°C until assayed. All cases/samples were pre-processed into training, interim and validation datasets to balance age, sex, PH classification and recruitment site.

Normally distributed variables reported as mean (standard deviation), not normally distributed variables reported as median [IQR], Categorical variables reported as number (% from reported total of column).

Hammersmith Hospital (Imperial), Royal Hallamshire Hospital (Sheffield) and Royal Papworth Hospital (Cambridge). Example 2 - Quantification of serum NT-proBNP and miRNAs

Total RNAwas extracted from 200 pl of serum or plasma using the Maxwell® RSC miRNA Plasma and Serum Kit (Promega, Madison, USA) as per the manufacturer’s recommendations with the following modifications: (a) a set of three proprietary spike-in controls (MiRXES, Singapore) was added, representing high, medium, and low levels of RNA, into the lysis buffer C prior to sample RNA isolation. The spike-in controls are 20-nucleotide RNAs with unique sequences (distinct from any of the 2588 annotated mature human miRNAs in miRBase version 21.0, RRID:SCR_003152) and are used to monitor RNA isolation efficiency and normalise for technical variations during RNA isolation; (b) bacteriophage MS2 RNA (Roche, Basel, Switzerland) was added at 0.4ng per sample isolation to improve RNA isolation yield. For biomarker discovery, a highly- controlled RT-qPCR workflow was used to quantify the expression of miRNA in each sample. Isolated RNA was reverse transcribed using miRNA-specific reverse transcription (RT) primers according to manufacturer’s instructions (ID3EAL Customized Individual miRNA RT Primer, MiRXES) on QuantStudio™ 5 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

Example 3 - Pre-processing of miRNA expression data

590 miRNAs were measured in the discovery and interim cohorts and 359 highly expressed miRNAs were measured in the validation cohort. Data from 326 miRNAs that are detected in no less than 90% of samples in both phases were combined. Missing values were imputed separately in the combined discovery and interim, and validation sets, by replacing missing values with miRNA mean - 4 standard deviations. miRNA data were further global normalized (a novel and universal method for microRNA RT-qPCR data normalisation). Samples from Cambridge showed higher total miRNA counts than the other centres. To correct for this batch effect, total miRNA counts were modelled with a Least absolute shrinkage and selection operator (LASSO) model composed of 11 miRNAs (Table 4). A linear regression using this model was then used to adjust the counts, retaining the mean miRNA levels. hsa.miR.222.3p 18.635122

Table 4: LASSO coefficients for calculating total miRNA counts.

Example 4 - Training classification models The Boruta package (v.7.0.0) was utilised, using default settings with 300 iterations of the normalised permutation importance function using random forest to identify potentially relevant features within the classification framework. Once the 300 runs were completed, miRNAs not classified as important by the algorithm were rejected. This process was repeated 100 times, with miRNAs selected on at least 99 occasions carried forwards into a random forest model, using the randomForest package (v4.6-14). The caret package was used to identify the optimal number of trees, from 1000, 1500, 2000 or 2500. Then, the number of variables available for splitting at each tree node was optimised across the range 1 to 15 (Table 5). Once a model was created, if the original number of variables selected was greater than 10, the model was re-tuned by removing the variables with the lowest contributed importance to the model.

Table 5: Final Random forest model parameters for each comparison. *Random forest was not the final model for these comparisons.

Recursive partition trees were computed using Rpart (v4.1-15)(Therneau and Atkinson 2018) and caret in R. Trees were generated from the root of the tree downwards, using a greedy feature selection algorithm and recursive binary splitting to return features in order. The tree construction was controlled by setting the number of minimum number of observations in a terminal node to 5, and the minimum number of observations in a node for a split to occur set to 4. The Gini index was minimised to create each split. A variable was deemed to be selected if it appeared in the final model.

LASSO using the glmnet package in R (v4.1-2) was used to choose pertinent miRNAs by eliminating variables with a coefficient shrunk to 0. The regularisation parameter, A, was chosen using 10-fold cross-validation with binomial deviance as the criterion. The value of A with minimum binomial deviance was selected and used to fit the final model.

XGBoost (v1 .4.1.1) parameters were tuned over previously described optimisation ranges for miRNAs (Emington et al. 2021). Once models had been trained, the variables were ranked in terms of their importance. The top 9 miRNAs were then selected (as summarised in Figures 3-8), and the model re-trained over the same parameter range using only the 9 selected miRNAs. The optimal values for these parameters for each model can be seen in Table 6.

Table 6: Final XGBoost model parameters for each comparison. *XGBoost not the final model for this comparison.

Example 5 - Performance of NT-proBNP as a PH Classifier

The performance of NT-proBNP as a standalone variable for classification was also investigated. The glmnet package in R was used to build binary logistic regression models for each comparison. Example 6 - Multivariable miRNA selection

All statistical analyses were carried out using R (v4.0.3). Following the approach described in (Emington et al. 2021), we used four different machine learning feature selection methods, and each fed forward to binary classifiers. For each method, parameters were tuned using 10-fold cross validation (repeated 10 times). Each classifier was then assessed for ROC AUC using the interim set. The models with the best cross-validated AUC on the discovery set, and highest AUC on the interim set were refined, using the combined discovery and interim sets, with the best performing method (assessed as the highest mean cross-validated AUC on the combined sets) was selected as the final model. Final performance was then assessed on the validation set.

Example 7 - Pathway Enrichment Analysis

An over representation analysis was carried out for each miRNA panel using the GeneTrail 3.2 miRNomics platform. The miRPathDB 2.0 collection was used to select strongly validated miRTarBase REACTOME and WIKIPATHWAYS. A significance level of 0.05 was used for FDR adjusted results (Benjamini and Yetutieli 2001).

Results

Example 8 - miRNAs can identify PH clinical classes as well as, or better, than NT-proBNP

The performance of miRNA signatures to identify PH assigned to internationally adopted clinical classification groups was measured by receiver operator characteristics (ROC) area under the curve (AUC). For each comparison, we used the performance of miRNA to compare to NT-proBNP at distinguishing PH from disease controls (DC), which included all symptomatic patients (including chronic thromboembolic disease, CTED and connective tissue disease, CTD) in which PH was excluded by cardiac catheterization. Signatures were first derived in the training and interim dataset, and the final performance then independently evaluated and reported in the validation samples (Table 7).

There was no significant difference in the overall performance between NT- proBNP and the miRNA signature in distinguishing patients with PH or PAH from symptomatic disease controls (DC) (All PH vs DC, PAH vs DC) (Table 7). However, miRNA signatures did offer improved performance over NT pro-BNP at classifying both PAH and CTEPH from other forms of PH, with an AUC for of 0.69 and 0.7 respectively using miRNAs (compared to 0.51 and 0.58 for NT-proBNP) (Table 7). Furthermore, miRNAs could also better distinguish PAH from CTEPH than NT-proBNP (0.77 vs 0.55). No performance was analysed on samples from patients with PH-miscellaneous due to the small sample size. ROC curves for miRNA and NT-proBNP classification of PAH vs PH, CTEPH vs PH and PAH v CTEPH are shown in Figures 4B, 5B, 6B, 7B and 8B.

Table 7: Performance of miRNA signatures in training and validation datasets.

Mean cross-validated AUCs on discovery and interim datasets, and AUC on validation set for the best performing models trained on miRNAs and NT-proBNP across five clinically defined classes (Pulmonary Hypertension groups 1-4 and DC).

Example 9 - Shared miRNAs may show overlapping pathology

While the precise miRNA composition of each of the panels used to discriminate the clinical classes is different, some miRNAs repeat across the panels, albeit with varying importance as a contributor to the overall signature (Figure 3). The signals that separate PH and PAH from disease controls share six miRNAs (miR-RNA- 151a-5p, miR-210-3p, miR-30a-5p, miR-193b-3p, miR-126-3p and miR-10b-5p). The relative abundance of miR-34a-5p features in discriminating PH-LHD, PH- lung and CTEPH from PH, and CTEPH from PAH.

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