Differential diagnosis of forms of primary aldosteronism

Abstract

The invention relates to the use of a microRNA or a set of microRNAs in the in vitro differential diagnosis of forms of primary aldosteronism, wherein the microRNA is at least one microRNA selected from the following or wherein the set of microRNAs comprises at least two microRNAs selected from the following: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p. Preferably, the forms of primary aldosteronism are unilateral and bilateral forms, preferably unilateral aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH). The invention also relates to a method for the in vitro differential diagnosis of forms of primary aldosteronism in a subject.

Description

[0001] Differential diagnosis of forms of primary aldosteronism

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the use of a microRNA or a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism. The invention also relates to an in vitro diagnostic method for determining the form of primary aldosteronism in a patient suffering from primary aldosteronism.

[0004] TECHNICAL BACKGROUND

[0005] Primary aldosteronism is the most common cause of secondary hypertension, accounting for 5-10% of all hypertension. It is caused by autonomous overproduction of aldosterone by the adrenal cortex and, if untreated, leads to numerous complications. The disease is underdiagnosed, and its prevalence is probably even higher than reported in the literature [Funder and Carey, 2022], The diagnosis of primary aldosteronism can be made with hormone tests (aldosterone and renin levels) and confirmatory tests.

[0006] The two most common forms of primary aldosteronism are unilateral aldosterone-producing adenoma (APA, 30-40%, classic Conn’s syndrome) and bilateral adrenal hyperplasia (BAH, 60-70%), which are treated differently. APA is to be treated surgically with unilateral adrenalectomy, while BAH is to be treated pharmacologically with mineralocorticoid receptor antagonists [Zennaro et al., 2020; Turcu and Auchus, 2021],

[0007] Differentiating these two forms is therefore essential, but difficult. Imaging can be helpful, but the most reliable method is adrenal venous sampling (AVS), which is an interventional radiological technique requiring great expertise, with limited availability [Rossi et al., 2012; Nanba et al., 2017],

[0008] Imaging (computer tomography, CT; magnetic resonance imaging, MRI) can therefore help in differentiating the forms of primary aldosteronism, buttheir sensitivity varies, and they are not suitable for detecting small adenomas [Nanba et al., 2017], Recognizing aldosterone-producing adenomas with imaging is difficult. The size of the lesion causing the disease may be smaller than the resolution of imaging, and it may not show any differences from the structure of the healthy adrenal cortex on examination. In addition, the frequency of benign adrenal adenomas without function increases with age, as does the frequency of primary aldosteronism, thus laterality determined only by imaging can lead to an incorrect diagnosis in up to 50% of cases.

[0009] The gold standard method accepted in the clinic is adrenal venous sampling (AVS), during which a catheter is inserted into the adrenal vein on both sides, and then the aldosterone and cortisol levels are determined on both sides from the blood obtained from these [Rossi et al., 2012; Rossi, 2019; Turcu and Auchus, 2021] . The ratios of the hormones can be used to determine whether a unilateral or bilateral disease form is present, and if unilateral, which side is affected. However, a serious problem is that this method requires great expertise, has very limited availability, and is often unsuccessful, since the right adrenal vein drains directly into the inferior vena cava and is difficult to cannulate. If both sides cannot be cannulated reliably, the reliability of the method is significantly compromised. The success of the intervention can only be determined retrospectively, after the analysis of the samples, and due to the aforementioned difficulties, repetition is often necessary. Adrenal venous sampling (AVS) is an interventional radiological procedure that requires a high level of expertise and practice, and is performed in centers with high patient turnover. In addition, AVS does not have an internationally accepted clear protocol, some centers administer ACTH (adrenocorticotropic hormone) to patients before the procedure to stimulate the adrenal cortex, while others do not, and there is no internationally accepted and fixed interpretation of the ratios obtained during the analysis, which form the basis of the diagnosis.

[0010] Research is underway to determine the profiles of circulating steroid hormones released by the disease forms, and to use them for differential diagnosis [Wannachalee and Turcu, 2020] .

[0011] Robertson et al. (2013) found that the miRNA expression profile of healthy adrenal tissue and aldosterone-producing adenoma (APA) is significantly different, with significantly lower levels of hsa- miR-24 in the latter. However, hsa-miR-24 was only identified as a therapeutic target, and it was not suggested that this miRNA would be suitable for differentiating the different forms of primary aldosteronism.

[0012] Previously, our research group [Decmann et al., 2019] investigated the differentiation of unilateral and bilateral primary aldosteronism from peripheral blood using circulating microRNAs, but at that time we were unable to find microRNAs showing differences with high sensitivity and specificity. The results at that time showed that miR-30e-5p, miR-30d-5p, miR-223-3p and miR-7-5p were overexpressed in hyperplasia (BAH) compared to APA, and these were also validated by qRT-PCR, with the exception of miR-223-3p. However, their sensitivity and specificity values were not good enough to be reliably used for clinical diagnostic purposes. We examined the three microRNAs individually.

[0013] WO2022 / 171680 discloses a method for identifying combinations of biomarkers for the stratification of hypertensive patients. The disease causing hypertension can be, among others, primary aldosteronism. When classifying patients, biomarkers belonging to at least 3 different groups (e.g. metanephrines, steroids, miRNAs, patient’s age / gender) are considered. In a long list of microRNA biomarkers, they list hsa-miR-199a-5p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-28-3p and hsa- miR-361-5p. However, they do not indicate that these microRNAs would also be suitable for distinguishing forms of a given disease.

[0014] The methods available so far for differentiating the two forms of primary aldosteronism are either not sufficiently accurate or require great expertise and are burdensome for both patients and the institutions providing care for them. For this reason, there is a need for easy-to-implement, minimally invasive, yet highly accurate biomarkers and diagnostic methods.

[0015] Currently, there is no biomarker detectable from blood that can reliably differentiate between unilateral and bilateral forms. None of the above solutions allows for the differentiation of the forms of primary aldosteronism with high accuracy and minimally invasive methods.

[0016] The aim of the solution according to the invention was to find biomarkers that are suitable for the differentiation of the two forms of primary aldosteronism and whose measurement is easy to perform and minimally invasive.

[0017] BRIEF DESCRIPTION OF THE INVENTION

[0018] The invention relates to the use of a microRNA in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the microRNA is at least one microRNA selected from hsa-miR-146a- 5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR- 199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0019] Preferably, the sequences of the microRNAs are as follows:

[0020] Preferably, the microRNA is a single microRNA selected from the above group. Preferably, the microRNA is hsa-miR-146a-5p. Preferably, the microRNA is hsa-miR-361-5p. Preferably, the microRNA is hsa-miR-24-3p. Preferably, the microRNA is hsa-miR-130b-3p. Preferably, the microRNA is hsa-miR-99b-5p. Preferably, the microRNA is hsa-miR-151a-3p. Preferably, the microRNA is hsa-miR-199a-3p. Preferably, the microRNA is hsa-miR-128-3p. Preferably, the microRNA is hsa-miR-28-3p.

[0021] The invention also relates to the use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises at least two microRNAs and wherein the at least two microRNAs are selected from hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24- 3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa- miR-28-3p.

[0022] Preferably, the set of microRNAs comprises at least three, at least four, at least five, at least six, at least seven, at least eight or nine microRNAs selected from the above group. Preferably, the set of microRNAs comprises at least four microRNAs. Preferably, the set of microRNAs comprises at least five microRNAs. Preferably, the set of microRNAs comprises at least six microRNAs. Preferably, the set of microRNAs comprises at least seven microRNAs. Preferably, the set of microRNAs comprises at least eight microRNAs. Preferably, the set of microRNAs comprises nine microRNAs.

[0023] Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-24-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR- 99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0024] Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-199a-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR- 130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0025] Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-361-5p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR- 99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0026] Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-146a-5p and at least one microRNA selected from the group consisting of hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR- 99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0027] Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-130b-3p and at least one microRNA selected from the group consisting ofhsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b- 5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0028] Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-99b-5p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR- 130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0029] Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-151a-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR- 130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0030] Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-128-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR- 130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-28-3p. Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-28-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR- 130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p.

[0031] Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises the microRNA hsa-miR-24-3p and the microRNA hsa-miR- 199a-3p.

[0032] Preferably, the set of microRNAs further comprises at least one additional microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p. Preferably, the set of microRNAs further comprises at least two additional microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0033] The invention also relates to the use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises or consists of the following microRNAs: a) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p and hsa-miR-199a-3p; or b) hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa- miR-199a-3p; or c) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-130b-3p, hsa-miR- 99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p; or d) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR- 15 la-3p and hsa-miR-199a-3p; or e) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-128-3p and hsa- miR-199a-3p; or f) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p, hsa-miR-99b-5p, hsa-miR- 15 la-3p and hsa-miR-199a-3p; or g) hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p and hsa-miR-199a-3p; or h) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa- miR-199a-3p; or i) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-99b-5p, hsa-miR- 15 la-3p and hsa-miR-199a-3p; or j) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-128-3p, hsa-miR- 99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p. Preferably, the set of microRNAs comprises the following six microRNAs: miR-146a-5p, miR- 24-3p, miR-130b-3p, miR-99b-5p, miR-151a-3p and miR-199a-3p.

[0034] Preferably, any use - in the in vitro differential diagnosis of forms of primary aldosteronism - is carried out for a patient suffering from or suspected of suffering from primary aldosteronism. Preferably, the patient has been diagnosed with primary aldosteronism prior to the use of the microRNA or set of microRNAs. Preferably, the patient has been diagnosed with primary aldosteronism based on hormone testing, such as levels of aldosterone and / or renin, and optionally confirmatory tests.

[0035] A patient suspected of suffering from primary aldosteronism is a patient who is suspected of having primary aldosteronism based on at least one symptom characteristic of primary aldosteronism, or who exhibits at least one symptom characteristic of primary aldosteronism, or who can be diagnosed as suffering from primary aldosteronism by at least one alternative diagnostic method.

[0036] The symptom characteristic of primary aldosteronism is preferably selected from hypertension that is difficult to control with medication, low blood potassium levels, and / or cardiovascular consequences that are severe in relation to the degree of hypertension (e.g. stroke).

[0037] The primary aldosteronism is preferably diagnosed by a method selected from hormone testing, aldosterone level testing, renin level testing, followed by a confirmatory test to confirm autonomous aldosterone production.

[0038] Preferably, any use - in the in vitro differential diagnosis of forms of primary aldosteronism - is carried out on a biological sample obtained from a patient suffering from or suspected of suffering from primary aldosteronism.

[0039] The biological sample is preferably a body liquid or body fluid, such as blood, serum, blood plasma, saliva, urine, semen or tears. Preferably, the biological sample is blood, plasma, saliva or urine; more preferably blood. Most preferably, the biological sample is peripheral blood.

[0040] Preferably, the forms of primary aldosteronism are unilateral form or bilateral form. Preferably, the forms of primary aldosteronism are unilateral aldosterone -producing adenoma (APA) and bilateral adrenal hyperplasia (BAH). Preferably, any microRNA or set of microRNAs as defined above can be used to distinguish whether the primary aldosteronism is caused by a unilateral or bilateral process.

[0041] The preferred embodiments or features defined above may also be combined with each other, except where it is clear to a person skilled in the art that they are incompatible.

[0042] Use of a microRNA panel in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the microRNA panel comprises at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR- 151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0043] Use of a marker combination in an in vitro differential diagnosis of forms of primary aldosteronism, said marker combination comprising at least one microRNA selected from hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa- miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p; and comprising at least one clinical parameter selected from gender, imaging result, potassium level and renin activity.

[0044] In one aspect, any of the above-defined microRNAs, sets of microRNAs, microRNA panels or marker combinations are for use in the differential diagnosis of forms of primary aldosteronism in a patient.

[0045] Preferably, the patient is a patient suffering from or suspected of suffering from primary aldosteronism.

[0046] Preferably, the diagnosis is performed on a biological sample of the patient. Preferably, the biological sample is a body liquid or body fluid, such as blood, serum, blood plasma, saliva, urine, semen or tears. Preferably, the biological sample is blood, plasma, saliva or urine; more preferably blood, most preferably peripheral blood.

[0047] Preferably, the forms of primary aldosteronism are unilateral form or bilateral form. Preferably, the forms of primary aldosteronism are unilateral aldosterone -producing adenoma (APA) and bilateral adrenal hyperplasia (BAH). Preferably, any microRNA or set of microRNAs according to the invention can be used to distinguish whether the primary aldosteronism is caused by a unilateral or bilateral process.

[0048] A kit comprising means for determining the expression profile of a set of microRNAs in a biological sample obtained from a subject, wherein the set of microRNAs comprises at least two microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa- miR-28-3p.

[0049] Preferably, the means is a binding molecule, preferably a polynucleotide, for detecting the set of microRNAs, and / or an RT-PCR system, a next-generation sequencing system or a biochip. More preferably, the means is polynucleotides for detecting the set of microRNAs.

[0050] The invention also relates to a kit comprising at least one binding molecule that binds to a microRNA that can be used in the in vitro differential diagnosis of forms of primary aldosteronism, wherein the microRNA is at least one microRNA selected from the group consisting of hsa-miR-146a- 5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR- 199a-3p, hsa-miR-128-3p and hsa-miR-28-3p; and optionally comprising a reagent suitable for detecting the binding. Optionally, the kit comprises at least one binding molecule that binds to a microRNA that can be used in the in vitro differential diagnosis of forms of primary aldosteronism, wherein the microRNA is at least one microRNA selected from the group consisting of hsa-miR-146a- 5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR- 199a-3p, hsa-miR-128-3p and hsa-miR-28-3p, and a means for detecting the binding. Preferably, the kit comprises a pair of primers as binding molecules, which specifically binds to at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p, and is optionally suitable for amplifying it, preferably via reverse transcription PCR.

[0051] Preferably, the kit comprises a plurality of primer pairs, each pair specifically binding to a member of a set of microRNAs selected from any of the microRNA sets defined above.

[0052] A kit comprising a nucleotide matrix, each spot of which comprising a binding molecule that binds to a microRNA that can be used in the in vitro differential diagnosis of forms of primary aldosteronism, preferably a nucleic acid molecule complementary to the microRNA.

[0053] A kit comprising a fluorescent probe that binds to a microRNA that can be used in the in vitro differential diagnosis of forms of primary aldosteronism.

[0054] Use of any of the kits in the differential diagnosis of forms of primary aldosteronism in a biological sample obtained from a subject.

[0055] The invention also relates to a method for the in vitro differential diagnosis of form of primary aldosteronism in a subject, said method comprising:

[0056] (i) determining an expression profile of a set of microRNAs in a biological sample obtained from the subject; and

[0057] (ii) comparing the expression profile determined in step (i) with reference expression profile(s), wherein comparing the expression profile determined in step (i) with the reference expression profile(s) enables the diagnosis of unilateral or bilateral form of primary aldosteronism, and / or applying an algorithm or mathematical function to the expression profile determined in step (i), wherein applying an algorithm or mathematical function to the expression profile determined in step (i) enables the diagnosis of unilateral or bilateral form of primary aldosteronism; wherein the set of microRNAs comprises at least two microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR- 151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0058] Preferably, in the method, the set of microRNAs comprises hsa-miR-24-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b- 3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0059] Preferably, in the method, the set of microRNAs comprises hsa-miR-199a-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0060] Preferably, in the method, the set of microRNAs comprises hsa-miR-361-5p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0061] Preferably, in the method, the set of microRNAs comprises hsa-miR-146a-5p and at least one microRNA selected from the group consisting of hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0062] Preferably, in the method, the set of microRNAs comprises hsa-miR-130b-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0063] Preferably, in the method, the set of microRNAs comprises hsa-miR-99b-5p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0064] Preferably, in the method, the set of microRNAs comprises hsa-miR-151a-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0065] Preferably, in the method, the set of microRNAs comprises the microRNA hsa-miR-128-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-28- 3p.

[0066] Preferably, in the method, the set of microRNAs comprises the microRNA hsa-miR-28-3p and at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128- 3p.

[0067] Preferably, in the method, the set of microRNAs comprises hsa-miR-24-3p and hsa-miR-199a- 3p. Preferably, in the method, the set of microRNAs further comprises at least one additional microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR- 99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p. Preferably, in the method, the set of microRNAs further comprises at least two additional microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR- 128-3p and hsa-miR-28-3p.

[0068] Preferably, in the method, the set of microRNAs comprises at least three microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0069] Preferably, in the method, the set of microRNAs comprises at least four microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0070] Preferably, in the method, the set of microRNAs comprises at least five microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa- miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p. Preferably, in the method, the set of microRNAs comprises at least six microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa- miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0071] Preferably, in the method, the set of microRNAs comprises at least seven microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0072] Preferably, in the method, the set of microRNAs comprises at least eight microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0073] Preferably, in the method, the set of microRNAs comprises the following nine microRNAs: hsa- miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0074] Preferably, in the method, the set of microRNAs comprises or consists of the following microRNAs: a) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p and hsa-miR-199a-3p; or b) hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa- miR-199a-3p; or c) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-130b-3p, hsa-miR- 99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p; or d) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR- 15 la-3p and hsa-miR-199a-3p; or e) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-128-3p and hsa- miR-199a-3p; or f) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p, hsa-miR-99b-5p, hsa-miR- 15 la-3p and hsa-miR-199a-3p; or g) hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p and hsa-miR-199a-3p; or h) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa- miR-199a-3p; or i) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-99b-5p, hsa-miR- 15 la-3p and hsa-miR-199a-3p; or j) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-128-3p, hsa-miR- 99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p.

[0075] Preferably, in the method, the set of microRNAs comprises the following six microRNAs: miR- 146a-5p, miR-24-3p, miR-130b-3p, miR-99b-5p, miR-151a-3p and miR-199a-3p. Preferably, in the method, the subject is a patient suffering from primary aldosteronism or suspected of suffering from primary aldosteronism. Preferably, the patient has been diagnosed with primary aldosteronism prior to the use of the microRNA or the set of microRNAs.

[0076] Preferably, in the method, the biological sample is a body liquid or body fluid, such as blood, serum, blood plasma, saliva, urine, semen or tears. Preferably, the biological sample is blood, plasma, saliva or urine; more preferably blood. Most preferably, the biological sample is peripheral blood.

[0077] Preferably, the form of primary aldosteronism is a unilateral or bilateral form. Preferably, the forms of the primary aldosteronism are unilateral aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH).

[0078] Preferably, in the method, in step (i), the expression profde is determined using a classification algorithm, wherein the classification algorithm is preferably selected from a neural network algorithm, a random forest algorithm, a support vector machine algorithm, a k-nearest neighbours algorithm, a decision tree algorithm, a naive Bayes algorithm, an adaptive Bayesian network algorithm, a logistic regression algorithm, a multinomial logistic regression algorithm, a Fisher’s linear discriminant algorithm, a quadratic classifier algorithm, a perceptron algorithm, and an ensemble learning method combining a plurality of different learning algorithms. Most preferably, the classification algorithm is a neural network model.

[0079] Preferably, the classification algorithm is pre-trained with the expression levels of at least one microRNA selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa- miR-28-3p, measured from a biological sample of patients suffering from unilateral form of primary aldosteronism and / or measured from a biological sample of patients suffering from bilateral form of primary aldosteronism. More preferably, the classification algorithm is pre -trained with the expression levels of at least two microRNAs (i.e. with the expression profile of a set of microRNAs comprising at least two miRNAs) measured from a biological sample of patients suffering from unilateral form of primary aldosteronism and / or measured from a biological sample of patients suffering from bilateral form of primary aldosteronism, said at least two microRNAs being selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR- 151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0080] Preferably, in the method, in step (ii), the reference expression profile(s) is / are an expression profile of the set of microRNAs in a biological sample of a patient suffering from unilateral form of primary aldosteronism, preferably from APA, and / or an expression profile of the set of microRNAs in a biological sample of a patient suffering from bilateral form of primary aldosteronism, preferably from BAH.

[0081] Preferably, in the method, in step (ii), the reference expression profile is an expression profile of the set of microRNAs determined from a biological sample of a patient suffering from unilateral form of primary aldosteronism, preferably from APA. Preferably, in step (ii), the reference expression profile is an expression profile of the set of microRNAs determined from a biological sample of a patient suffering from bilateral form of primary aldosteronism, preferably from BAH. Most preferably, in step (ii), the reference expression profiles are an expression profile of the set of microRNAs determined from a biological sample of a patient suffering from unilateral form of primary aldosteronism, preferably from APA, and an expression profile of the set of microRNAs determined from a biological sample of a patient suffering from bilateral form of primary aldosteronism, preferably from BAH.

[0082] More preferably, the reference expression profile(s) is / are an expression profile determined from biological samples of a group of patients suffering from unilateral form of primary aldosteronism, preferably from APA, and / or an expression profile determined from biological samples of a group of patients suffering from bilateral form of primary aldosteronism, preferably from BAH. Most preferably, the reference expression profiles are an expression profile determined from biological samples of a group of patients suffering from unilateral form of primary aldosteronism, preferably from APA, and an expression profile determined from biological samples of a group of patients suffering from bilateral form of primary aldosteronism, preferably from BAH.

[0083] Preferably, in the method, in step (ii), the algorithm or mathematical function is produced from the reference expression profile, preferably from the reference expression profiles, wherein the reference expression profile(s) is / are defined as above.

[0084] The preferred embodiments or features defined above may also be combined with each other, except where it is clear to a person skilled in the art that they are incompatible.

[0085] The invention also relates to a method for the in vitro differential diagnosis of the form of primary aldosteronism in a subject, said method comprising: a) determining the expression levels of at least two microRNAs in a biological sample obtained from the subject, said at least two microRNAs being selected from the group consisting of hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p; and b) diagnosing the subject as having a unilateral form of primary aldosteronism, preferably APA, or a bilateral form of primary aldosteronism, preferably BAH, based on the determined expression levels.

[0086] Preferably, the at least two microRNAs are any of the sets of microRNAs as defined above.

[0087] Preferably, the subject is any of the subjects as defined above.

[0088] Preferably, the biological sample is any of the biological samples as defined above.

[0089] Preferably, the form of primary aldosteronism is any of the forms as defined above.

[0090] In an embodiment, the expression levels of microRNAs selected from the group consisting of hsa- miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p, are higher in bilateral form, preferably in BAH, than in unilateral form, preferably in APA.

[0091] The preferred embodiments or features defined above may also be combined with each other, except where it is clear to a person skilled in the art that they are incompatible.

[0092] The invention also relates to a method for the in vitro differential diagnosis of the form of primary aldosteronism in a subject, said method comprising: a) determining the expression levels of at least two microRNAs in a biological sample obtained from the subject, said at least two microRNAs being selected from the group consisting of hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p; b) generating an expression profile based on the determined expression levels; and c) diagnosing the subject as having a unilateral form of primary aldosteronism, preferably APA, or a bilateral form of primary aldosteronism, preferably BAH, based on the expression profile.

[0093] Preferably, the at least two microRNAs are any of the sets of microRNAs as defined above.

[0094] Preferably, the subject is any of the subjects as defined above.

[0095] Preferably, the biological sample is any of the biological samples as defined above.

[0096] Preferably, the form of primary aldosteronism is any of the forms as defined above.

[0097] Preferably, the expression profile is defined in any of the ways as described above.

[0098] The preferred embodiments or features defined above may also be combined with each other, except where it is clear to a person skilled in the art that they are incompatible.

[0099] The invention also relates to a method for the in vitro differential diagnosis of the form of primary aldosteronism in a subject, said method comprising:

[0100] - providing a biological sample obtained from the subject,

[0101] - measuring the expression levels of at least two microRNAs in the biological sample, said at least two microRNAs being selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128- 3p and hsa-miR-28-3p,

[0102] - using a score, calculated based on the measured miRNA expression levels, to determine whether the subject has a unilateral form or bilateral form of primary aldosteronism; wherein preferably the score is calculated using a classification algorithm; wherein preferably the classification algorithm is pre -trained with a pattern of expression levels measured from a biological sample of subjects suffering from unilateral form of primary aldosteronism, preferably from APA, and / or a pattern of expression levels measured from a biological sample of subjects suffering from bilateral form of primary aldosteronism, preferably from BAH, more preferably the classification algorithm is pre-trained with a pattern of expression levels measured from a biological sample of subjects suffering from unilateral form of primary aldosteronism, preferably from APA, and a pattern of expression levels measured from a biological sample of subjects suffering from bilateral form of primary aldosteronism, preferably from BAH, and wherein preferably the classification algorithm compares the expression pattern of the subject with the expression pattern of the unilateral (APA) form and / or the bilateral (BAH) form determined during the prior machine learning process, and determines a score that identifies the probability that the subject belongs to the group of unilateral (APA) form and / or the group of bilateral (BAH) form.

[0103] Preferably, the at least two microRNAs are any of the sets of microRNAs as defined above.

[0104] Preferably, the subject is any of the subjects as defined above.

[0105] Preferably, the biological sample is any of the biological samples as defined above.

[0106] Preferably, the form of primary aldosteronism is any of the forms as defined above.

[0107] The preferred embodiments or features defined above may also be combined with each other, except where it is clear to a person skilled in the art that they are incompatible.

[0108] A method for the in vitro differential diagnosis of the form of primary aldosteronism and treatment thereof in a subject, said method comprising:

[0109] (i) determining the expression profile of a set of microRNAs in a biological sample obtained from the subject, wherein the set of microRNAs comprises at least two microRNAs selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b- 5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p; and

[0110] (ii) comparing the expression profile determined in step (i) with reference expression profile(s), wherein comparing the expression profile determined in step (i) with the reference expression profile(s) enables the diagnosis of unilateral or bilateral form of primary aldosteronism, and / or applying an algorithm or mathematical function to the expression profile determined in step (i), wherein applying an algorithm or mathematical function to the expression profile determined in step (i) enables the diagnosis of unilateral or bilateral form of primary aldosteronism; and

[0111] (iii) if the subject is diagnosed with a unilateral form according to point (ii), performing further tests, preferably adrenal venous sampling (AVS), to determine the laterality, i.e. the adrenal gland comprising the adenoma, and then surgically removing the adrenal gland comprising the adenoma, or if the subject is diagnosed with a bilateral form according to point (ii), treating the subject with a drug, preferably with a mineralocorticoid receptor antagonist, such as spironolactone or eplerenone.

[0112] Preferably, the set of microRNAs is any of the sets of microRNAs as defined above.

[0113] Preferably, the subject is any of the subjects as defined above.

[0114] Preferably, the biological sample is any of the biological samples as defined above. Preferably, the form of primary aldosteronism is unilateral or bilateral form. Preferably, the forms of primary aldosteronism are unilateral aldosterone -producing adenoma (APA) and bilateral adrenal hyperplasia (BAH).

[0115] Preferably, the expression profde is defined in any of the ways as described above.

[0116] Preferably, the reference expression profile(s) is / are any of the reference expression profile(s) as defined above.

[0117] The preferred embodiments or features defined above may also be combined with each other, except where it is clear to a person skilled in the art that they are incompatible.

[0118] A method for the in vitro differential diagnosis of the form of primary aldosteronism and treatment thereof in a subject, said method comprising: a) determining the expression levels of at least two microRNAs in a biological sample obtained from the subject, said at least two microRNAs being selected from the group consisting of hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p; b) diagnosing the subject as having a unilateral form of primary aldosteronism, preferably APA, or a bilateral form of primary aldosteronism, preferably BAH, based on the determined expression levels; and c) if the subject is diagnosed with a unilateral form according to point b), performing further tests, preferably adrenal venous sampling (AVS), to determine the laterality, i.e. the adrenal gland comprising the adenoma, and then surgically removing the adrenal gland comprising the adenoma, or if the subject is diagnosed with a bilateral form according to point b), treating the subject with a drug, preferably with a mineralocorticoid receptor antagonist, such as spironolactone or eplerenone.

[0119] Preferably, the at least two microRNAs are any of the sets of microRNAs as defined above.

[0120] Preferably, the subject is any of the subjects as defined above.

[0121] Preferably, the biological sample is any of the biological samples as defined above.

[0122] Preferably, the form of primary aldosteronism is any of the forms as defined above.

[0123] In an embodiment, the expression levels of microRNAs selected from the group consisting of hsa- miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p, are higher in bilateral form, preferably in BAH, than in unilateral form, preferably in APA.

[0124] The preferred embodiments or features defined above may also be combined with each other, except where it is clear to a person skilled in the art that they are incompatible.

[0125] A method for the in vitro differential diagnosis of the form of primary aldosteronism and treatment thereof in a subject, said method comprising: a) determining the expression levels of at least two microRNAs in a biological sample obtained from the subject, said at least two microRNAs being selected from the group consisting of hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p; b) generating an expression profile based on the determined expression levels; c) diagnosing the subject as having a unilateral form of primary aldosteronism, preferably APA, or a bilateral form of primary aldosteronism, preferably BAH, based on the expression profile; and d) if the subject is diagnosed with a unilateral form according to point c), performing further tests, preferably adrenal venous sampling (AVS), to determine the laterality, i.e. the adrenal gland comprising the adenoma, and then surgically removing the adrenal gland comprising the adenoma, or if the subject is diagnosed with a bilateral form according to point c), treating the subject with a drug, preferably with a mineralocorticoid receptor antagonist, such as spironolactone or eplerenone. Preferably, the at least two microRNAs are any of the sets of microRNAs as defined above. Preferably, the subject is any of the subjects as defined above.

[0126] Preferably, the biological sample is any of the biological samples as defined above.

[0127] Preferably, the form of primary aldosteronism is any of the forms as defined above.

[0128] Preferably, the expression profile is defined in any of the ways as described above.

[0129] The preferred embodiments or features defined above may also be combined with each other, except where it is clear to a person skilled in the art that they are incompatible.

[0130] A method for the in vitro differential diagnosis of the form of primary aldosteronism and treatment thereof in a subject, said method comprising:

[0131] - providing a biological sample obtained from the subject,

[0132] - measuring the expression levels of at least two microRNAs in the biological sample, said at least two microRNAs being selected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128- 3p and hsa-miR-28-3p,

[0133] - using a score, calculated based on the measured miRNA expression levels, to determine whether the subject has a unilateral form or bilateral form of primary aldosteronism;

[0134] - if the subject is diagnosed with a unilateral form, performing further tests, preferably adrenal venous sampling (AVS), to determine the laterality, i.e. the adrenal gland comprising the adenoma, and then surgically removing the adrenal gland comprising the adenoma, or if the subject is diagnosed with a bilateral form, treating the subject with a drug, preferably with a mineralocorticoid receptor antagonist, such as spironolactone or eplerenone; wherein preferably the score is calculated using a classification algorithm; wherein preferably the classification algorithm is pre -trained with a pattern of expression levels measured from a biological sample of subjects suffering from unilateral form of primary aldosteronism, preferably from APA, and / or a pattern of expression levels measured from a biological sample of subjects suffering from bilateral form of primary aldosteronism, preferably from BAH, more preferably the classification algorithm is pre-trained with a pattern of expression levels measured from a biological sample of subjects suffering from unilateral form of primary aldosteronism, preferably from APA, and a pattern of expression levels measured from a biological sample of subjects suffering from bilateral form of primary aldosteronism, preferably from BAH, and wherein preferably the classification algorithm compares the expression pattern of the subject with the expression pattern of the unilateral (APA) form and / or the bilateral (BAH) form determined during the prior machine learning process, and determines a score that identifies the probability that the subject belongs to the group of unilateral (APA) form and / or the group of bilateral (BAH) form.

[0135] Preferably, the at least two microRNAs are any of the sets of microRNAs as defined above.

[0136] Preferably, the subject is any of the subjects as defined above.

[0137] Preferably, the biological sample is any of the biological samples as defined above.

[0138] Preferably, the form of primary aldosteronism is any of the forms as defined above.

[0139] The preferred embodiments or features defined above may also be combined with each other, except where it is clear to a person skilled in the art that they are incompatible.

[0140] DEFINITIONS

[0141] “MicroRNA” or “miRNA” or “miR” refers to a short, non-coding ribonucleic acid (RNA) molecule consisting of 18 to 25 nucleotides. miRNAs are involved in RNA silencing and post- transcriptional regulation of gene expression.

[0142] “hsa-miRNA” or “hsa-miR” is a microRNA derived from the human species (“hsa” is short for Homo sapiens).

[0143] “Primary aldosteronism” or also known as “primary hyperaldosteronism” is an endocrine disorder in which the adrenal glands produce too much of the hormone aldosterone. This can cause high blood pressure and other symptoms. High blood pressure caused by primary aldosteronism is often difficult to control with medication and often requires a combination of a plurality of medications. The two most common forms of primary aldosteronism are unilateral aldosterone -producing adenoma (APA) and bilateral adrenal hyperplasia (BAH).

[0144] The division of primary aldosteronism into APA and BAH is essentially a clinical division, i.e. whether a unilateral or bilateral process is involved. Within the unilateral process, several different forms can be distinguished, for example the following: aldosterone-producing adenoma (APA), aldosterone- producing adrenocortical carcinoma (APACC), aldosterone-producing nodule (APN, <10 mm diameter), aldosterone-producing micronodule (APM), multiple aldosterone-producing nodules or micronodules (MAPN, MAPM), and aldosterone-producing diffuse hyperplasia (APDH). The most common unilateral form is APA, the other types are rare. The rare types can only be distinguished from each other by histological examination. The bilateral process is primarily bilateral diffuse hyperplasia, also known as bilateral adrenal hyperplasia (BAH). The sets of microRNAs, uses and methods disclosed herein are aimed at differentiating between unilateral and bilateral processes.

[0145] “Unilateral aldosterone -producing adenoma” or “APA” is a form of primary aldosteronism. Approximately 30% to 40% of patients suffering from primary aldosteronism have this form of the disease. This type is also known as Conn’s syndrome. APA is to be treated surgically by unilateral adrenalectomy, i.e. removal of the affected adrenal gland.

[0146] “Bilateral adrenal hyperplasia” or “BAH” is a form of primary aldosteronism. Approximately 60% to 70% of patients suffering from primary aldosteronism have this form of the disease. BAH is to be treated with medication, with mineralocorticoid receptor antagonists, such as spironolactone or eplerenone.

[0147] A “subject” as used herein refers to an individual of an animal species, preferably a vertebrate, more preferably an individual of a mammalian species, most preferably the individual is a primate, hominid or human.

[0148] A “patient” is a subject who is undergoing or is intended to undergo medical or veterinary observation, monitoring, diagnosis or treatment.

[0149] A “biological sample” refers to any biological material in which miRNAs can be found. A biological sample may be a cell, a group of cells or aggregate of cells, a cell culture, a tissue sample, a biological fluid (such as blood, serum, plasma, saliva, urine, semen or tears) or an organ. The biological sample is preferably a biological fluid.

[0150] The terms “combination” or “panel” or “set” - which are used interchangeably herein, if the context permits - refer to a group comprising more than one element. Preferably, the elements are microRNAs. In an embodiment, the elements are microRNAs and optionally additional parameters (e.g. clinical and laboratory parameters such as gender, renin activity, imaging results). Preferably, the “combination” or “panel” or “set” comprises at least two, at least three, at least four, at least five, at least six, at least seven or at least eight elements. More preferably, the “combination” or “panel” or “set” comprises at least four elements. Preferably, the panel refers to a group or set comprising given elements from which the elements to be examined are to be selected, preferably the set refers to a group of elements actually examined (e.g. measured). The meaning of combination includes both panel and set.

[0151] A “set of microRNAs” or “miRNA set” refers to a plurality of microRNAs. In other words, a set of microRNAs or miRNA set refers to a group of microRNAs that comprises at least two microRNAs. Preferably, the miRNA set comprises at least three, at least four, at least five, at least six, at least seven, or at least eight miRNAs.

[0152] An “expression profile” or “expression pattern” - which are used interchangeably herein, if the context permits - in this specification refers to the expression levels of at least two miRNAs measured in a biological sample or to a value calculated therefrom. Preferably, an expression profile is the expression levels of at least three, at least four, at least five, at least six, at least seven, at least eight or nine miRNAs measured in a biological sample, or a value calculated therefrom.

[0153] “Comparing” two expression profiles is to be understood herein to include a comparison of quantities, expressed in numerical values, characterizing the profiles in order to determine the extent to which the two profiles are similar to or different from each other, optionally complemented with a mathematical procedure or algorithm, as required by a quantification or calculation method.

[0154] A “reference” or “reference expression profile” as used herein is an expression profile of a given set of microRNAs that characterizes a subject or group of subjects (e.g. patients) and that serves as a reference for comparison for patients for whom a determination of the form primary aldosteronism is to be carried out. A “reference” or “reference expression profile” may be an expression profile of a given set of microRNAs that characterizes a patient or group of patients having a given form of primary aldosteronism (i.e. unilateral form, preferably APA, or bilateral form, preferably BAH), wherein the expression profile is preferably determined prior to initiation of treatment specific for the given form. A “unilateral reference expression profile” is an expression profile of a given set of microRNAs that characterizes a patient or group of patients with a unilateral form of primary aldosteronism. An “APA reference” or “APA reference expression profile” is an expression profile of a given set of microRNAs that characterizes a patient or group of patients with the APA form of primary aldosteronism. A “bilateral reference expression profile” is an expression profile of a given set of microRNAs that characterizes a patient or group of patients with a bilateral form of primary aldosteronism. A “BAH reference” or “BAH reference expression profile” is an expression profile of a given set of microRNAs that characterizes a patient or group of patients with the BAH form of primary aldosteronism.

[0155] The term “treatment” refers to any process, action, application, therapy, or the like, wherein the subject or patient is under aid, in particular medical or veterinarian aid, with the object of improving the condition of the subject or patient, either directly or indirectly.

[0156] A “therapy” is understood herein as a method for treatment in which a given medicament or pharmaceutical composition is administered to the patient, preferably administered for a certain period of time, with the object of improving the condition of the subject or patient.

[0157] The articles “a”, “an” and “the”, or at least the indefinite articles “a” and “an”, include plural references unless the context clearly indicates otherwise.

[0158] The term “comprise(s)” or “comprising” or “include(s)” are to be construed herein as having a non-exhaustive meaning and to allow the addition or involvement of further features or method steps or components to anything that comprises the listed features or method steps or components. The term “comprise(s)” can be substituted by the term “include(s)” if the practice of a given language variant so requires, or can be limited to the term “consisting essentially of’ if other features or components are not essential to reduce the invention to practice. The term “consisting essentially of’ or “comprising substantially” is to be understood as consisting of mandatory features or method steps or components listed in a list, e.g. in a claim, while allowing the addition of further features or method steps or components that do not materially affect the essential characteristics of the use, method, composition or other subject matter.

[0159] ABBREVIATIONS

[0160] APA unilateral aldosterone -producing adenoma

[0161] AVS adrenal venous sampling

[0162] BAH bilateral adrenal hyperplasia

[0163] BRIEF DESCRIPTION OF THE FIGURES

[0164] Figure 1. Test method used to select microRNA combinations according to the invention.

[0165] Figure 2. Use of the method according to the invention in practice.

[0166] DETAIEED DESCRIPTION OF THE INVENTION

[0167] Our aim was to find biomarkers that help to distinguish between the two forms of primary aldosteronism and that can be easily tested. We therefore looked for biomarkers that can be obtained from patients using a minimally invasive method (e.g. blood sampling) and that can be easily detected using common laboratory equipment.

[0168] For this purpose, we chose to study microRNAs. The expression of microRNAs is tissue -specific, and they are released from tissues into circulating blood. MicroRNAs are stable molecules, thus they can be ideal diagnostic markers.

[0169] We studied blood samples taken from the adrenal vein of cases of unilateral or bilateral primary aldosteronism confirmed by adrenal venous sampling, and analyzed the expression of microRNAs. We then examined peripheral blood samples and, with the help of artificial intelligence, found microRNA combinations that can be used to determine with high accuracy (with almost 90% sensitivity and specificity) whether a unilateral or bilateral disease process is present.

[0170] After the diagnosis of primary aldosteronism is established, our method helps to differentiate between unilateral and bilateral disease processes, which require different treatments. All this significantly speeds up the diagnostic process, it is easily performed from a peripheral blood sample (such as from a sample from the elbow vein). In the case of the more common BAH diagnosis, medical treatment can be started immediately, while only in the case of the rarer APA diagnosis would it be necessary to perform an AVS intervention on the patient to determine the laterality of the adrenal gland comprising the adenoma, thereby drastically reducing the burden on the centers performing the intervention. All of this has major public health implications, as well.

[0171] In contrast to our previous method (see Decmann et al., 2019), we did not examine the microRNAs individually, but an artificial intelligence algorithm selected the most accurate microRNA combinations, and started the study with blood samples taken from the adrenal vein. For the implementation, we used widely available molecular biology methods. In the method disclosed herein, we first examined the expression of microRNAs in blood samples taken from the adrenal vein by next-generation sequencing, based on the assumption that microRNAs originating directly from the adrenal gland are present in higher quantities in the blood of the adrenal vein than in the periphery. Subsequently, in the validation step, we examined peripheral blood samples in addition to samples from the adrenal vein. Peripheral blood samples are a biological sample type that can be easily used in everyday practice.

[0172] Using artificial intelligence, we identified unique microRNA combinations that can differentiate between unilateral and bilateral disease processes with high sensitivity and specificity. We have previously investigated the microRNA profile of unilateral and bilateral aldosteronism [Decmann et al. , 2019], but compared to previous results, we identified novel microRNAs and microRNA combinations. This result is surprising because the three other microRNAs previously found did not prove to be clinically applicable (their sensitivity and specificity were not high enough).

[0173] MicroRNA combinations or panels (also called sets of microRNAs) are suitable for differentiating the two forms of primary aldosteronism (APA and BAH), with at least the same efficiency as currently used methods.

[0174] The detection of the microRNA or the microRNAs in a microRNA combination is preferably performed by RT-qPCR. The qPCR equipment is part of the clinical genetic laboratory requirements. Furthermore, due to the specific primers and probes, the RT-qPCR method is very sensitive (1 copy can be detected) and specific (due to the perfect match of the TaqMan probe). It is also scalable, meaning that it is easier to create combinations - 1-2 or more assays (miRNAs) can be tested simultaneously.

[0175] The microRNA combination panel facilitates the diagnostic process, allowing determination whether a unilateral or bilateral disease process is present in the patients in a less burdensome way, with a simple blood test. This reduces the burden on patients and also on healthcare institutions, as it would be sufficient to further investigate the rarer, unilateral (APA) form using invasive and technically difficult processes, while the treatment of the bilateral (BAH) form can be started immediately.

[0176] The microRNA combinations, marker combinations and methods expedite the further diagnostic process after the hormonal determination of the presence of primary aldosteronism. Starting from a blood sample, it can be determined with high accuracy whether a unilateral or bilateral disease process is present, the treatment of the latter can begin immediately with the necessary medications, while patients with unilateral disease can be further assessed to determine laterality and location. The solution according to the invention reduces both the radiation, surgical and emotional burden on patients, as well as the burden on the healthcare institution, since with current methods invasive diagnostic procedures must be performed on every patient.

[0177] The analysis of circulating blood-borne microRNAs therefore represents a minimally invasive and potentially reliable method for differentiating between unilateral and bilateral primary aldosteronism. If bilateral primary aldosteronism can be determined using the model (i.e. microRNA combination) according to the invention, then further localization efforts are not needed, thus simplifying the management of patients with primary aldosteronism. This is relevant for both patients and public health.

[0178] Combinations comprising four microRNAs

[0179] In one aspect, the microRNA combination to be used in the in vitro differential diagnosis of forms of primary aldosteronism comprises four microRNAs selected from the group consisting of hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa- miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0180] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p and hsa-miR-99b-5p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-361-5p, hsa-miR-24-3p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-361-5p, hsa-miR-24-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p and hsa-miR-28-3p.

[0181] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p and hsa-miR-99b-5p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p and hsa- miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b- 3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p and hsa-miR-28-3p.

[0182] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p and hsa- miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b- 5p and hsa-miR-28-3p.

[0183] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-151a-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-151a-3p and hsa-miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0184] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0185] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR-99b-5p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa- miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b- 3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR-28-3p.

[0186] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR- 199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a- 5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR- 28-3p.

[0187] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-151a-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-24-3p, hsa-miR-151a-3p and hsa-miR-28-3p.

[0188] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0189] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0190] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa- miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b- 5p and hsa-miR-28-3p.

[0191] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-151a-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-130b-3p, hsa-miR-151a-3p and hsa-miR-28-3p.

[0192] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0193] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0194] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-28-3p.

[0195] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0196] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0197] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0198] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0199] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0200] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR-99b-5p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR- 151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361- 5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR- 128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR-28-3p.

[0201] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR- 199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361- 5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR- 28-3p.

[0202] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p and hsa-miR- 128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0203] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-199a-3p and hsa-miR- 28-3p.

[0204] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0205] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-15 la-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa- miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b- 5p and hsa-miR-28-3p.

[0206] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0207] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa- miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0208] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 361-5p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-28-3p.

[0209] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0210] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0211] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0212] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0213] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0214] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR- 199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR- 28-3p.

[0215] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p and hsa-miR-28-3p.

[0216] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa- miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0217] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR- 128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0218] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR- 28-3p.

[0219] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0220] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0221] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0222] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0223] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0224] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0225] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0226] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa- miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0227] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0228] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0229] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0230] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0231] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0232] Combinations comprising five microRNAs

[0233] In one aspect, the microRNA combination to be used in the in vitro differential diagnosis of forms of primary aldosteronism comprises five microRNAs selected from the group consisting of hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa- miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0234] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR-99b-5p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa- miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p and hsa-miR-28-3p.

[0235] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR-128- 3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p and hsa-miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p and hsa-miR-28- 3p.

[0236] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0237] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0238] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-15 la-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b- 3p, hsa-miR-99b-5p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa- miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-28-3p.

[0239] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b- 3p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0240] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b- 3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0241] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0242] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b- 5p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b- 5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0243] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0244] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-151a- 3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0245] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0246] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0247] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b- 3p, hsa-miR-99b-5p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-28-3p.

[0248] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b- 3p, hsa-miR-151a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p and hsa-miR-28-3p.

[0249] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b- 3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0250] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0251] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-28- 3p.

[0252] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0253] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0254] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-15 la- 3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0255] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0256] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0257] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b- 5p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0258] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b- 5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0259] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0260] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-15 la- 3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0261] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0262] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-15 la- 3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0263] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0264] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0265] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0266] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-151a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-128- 3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-28-3p.

[0267] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa-miR-28- 3p.

[0268] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0269] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0270] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0271] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0272] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0273] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0274] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0275] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0276] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b- 5p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0277] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b- 5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0278] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0279] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la- 3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0280] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0281] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0282] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-15 la- 3p, hsa-miR-199a-3p and hsa-miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0283] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0284] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0285] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-28- 3p.

[0286] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0287] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0288] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-15 la- 3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0289] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0290] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0291] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0292] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0293] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0294] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la- 3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0295] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0296] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0297] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0298] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0299] Combinations comprising six microRNAs

[0300] In one aspect, the microRNA combination to be used in the in vitro differential diagnosis of forms of primary aldosteronism comprises six microRNAs selected from the group consisting of hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa- miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0301] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-15 la-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR- 130b-3p, hsa-miR-99b-5p and hsa-miR-28-3p.

[0302] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0303] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p and hsa-miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0304] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-28-3p.

[0305] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0306] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0307] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0308] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0309] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0310] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0311] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0312] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0313] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-361-5p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0314] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0315] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0316] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-361-5p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0317] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0318] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0319] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0320] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa- miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-28-3p.

[0321] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0322] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0323] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0324] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0325] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0326] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0327] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0328] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0329] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa- miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0330] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0331] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0332] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0333] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0334] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa- miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-28-3p.

[0335] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0336] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR-28-3p.

[0337] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa- miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0338] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0339] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0340] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0341] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0342] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0343] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0344] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa- miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0345] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0346] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0347] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0348] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0349] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR- 130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0350] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0351] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0352] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0353] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0354] Combinations comprising seven microRNAs

[0355] In one aspect, the microRNA combination to be used in the in vitro differential diagnosis of forms of primary aldosteronism comprises seven microRNAs selected from the group consisting of hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa- miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0356] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR- 199a-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a- 5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p and hsa-miR-28-3p.

[0357] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa-miR- 128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a- 5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0358] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0359] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR- 128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a- 5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0360] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR- 28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0361] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR- 128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a- 5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0362] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0363] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0364] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0365] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa- miR-128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR-28-3p.

[0366] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0367] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0368] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa- miR-28-3p.

[0369] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0370] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-199a-3p and hsa-miR- 128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a- 5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa- miR-28-3p.

[0371] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0372] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0373] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0374] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0375] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa- miR-28-3p.

[0376] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR- 128-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p and hsa-miR- 28-3p.

[0377] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0378] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0379] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0380] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

[0381] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa- miR-28-3p. Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128-3p and hsa- miR-28-3p.

[0382] Combinations comprising eight microRNAs

[0383] In one aspect, the microRNA combination to be used in the in vitro differential diagnosis of forms of primary aldosteronism comprises eight microRNAs selected from the group consisting of hsa-miR- 146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa- miR-199a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

[0384] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR- 199a-3p and hsa-miR-128-3p.

[0385] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR- 199a-3p and hsa-miR-28-3p.

[0386] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128- 3p and hsa-miR-28-3p.

[0387] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-199a-3p, hsa-miR-128- 3p and hsa-miR-28-3p.

[0388] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR- 128-3p and hsa-miR-28-3p.

[0389] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR-128- 3p and hsa-miR-28-3p.

[0390] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR- 128-3p and hsa-miR-28-3p.

[0391] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR- 128-3p and hsa-miR-28-3p.

[0392] Preferably, the microRNA combination comprises the following microRNAs: hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR- 128-3p and hsa-miR-28-3p. Combination comprising nine microRNAs

[0393] In one aspect, the microRNA combination to be used in the in vitro differential diagnosis of forms of primary aldosteronism comprises the following nine microRNAs: hsa-miR-146a-5p, hsa-miR-361- 5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-199a-3p, hsa-miR- 128-3p and hsa-miR-28-3p.

[0394] In an embodiment, the method for the in vitro differential diagnosis of a form of primary aldosteronism in a subject comprises the following steps:

[0395] 1. providing a biological sample of a patient suffering from primary aldosteronism,

[0396] 2. isolating RNA from the biological sample, thereby producing an RNA sample,

[0397] 3. examining a microRNA or a set of microRNAs in the RNA sample by RT-qPCR, and

[0398] 4. evaluating the results obtained in point 3, and

[0399] 5. making a diagnosis based on the results obtained in point 4, wherein the diagnosis may be unilateral aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia (BAH).

[0400] EXAMPLES

[0401] Example 1: Materials and Methods

[0402] Our method is based on commercially available molecular biology methods (reverse transcription, TaqMan primers, polymerase chain reaction).

[0403] Samples

[0404] We worked with blood plasma samples taken during bilateral adrenal venous sampling (AVS) and peripheral blood sampling from 84 patients with primary aldosteronism (47 APA, 37 BAH; median age 53 years; 47 women), which were made available to us in the course of an international collaboration (ENS@T, European Network for the Study of Adrenal Tumors), along with the necessary medical documentation. Of these samples, bilateral AVS plasma samples from 18 patients were assigned to the discovery cohort (10 APA, 8 BAH, a total of 36 samples; median age 53.5 years; 10 women), while bilateral AVS samples and peripheral blood plasma samples from 30 patients were included in the validation cohort (15 APA, 15 BAH, a total of 90 samples (3 samples per patient); median age: 54.5 years; 12 women).

[0405] The remaining patient samples were used to independently test our model.

[0406] In a subsequent study, the validation cohort was expanded with 78 additional AVS -validated peripheral blood samples (39 unilateral and 39 BAH samples).

[0407] RNA isolation and concentration measurement

[0408] RNA was isolated from the blood plasma samples (from all 126 blood plasma samples in the first study, i.e. 36 discovery + 90 validation samples) using the same procedure. Thawed on ice from -80 °C, and using the miRNeasy Serum / Plasma Kit (QIAGEN) according to the manufacturer's protocol. As an external control, cel-mir-39 (RNA Spike-in kit, QIAGEN), i.e. a microRNA not found in humans, derived from the nematode C. elegans, was added in equal amounts to all samples.

[0409] After the isolation process, the RNA concentrations of the isolates were measured using a NanoDrop 2000 spectrophotometer.

[0410] Sequencing

[0411] On the 36 samples of the discovery cohort, next-generation microRNA-specific sequencing was performed. For this, the QIASeq miRNA UDI Library kit (QIAGEN) was used, according to the manufacturer's protocol. The DNA concentrations of the produced sequencing libraries were measured using a Qubit 4 spectrophotometer using the Qubit DNA High Sensitivity (HS) Kit (Thermo Fischer Scientific). The final concentration of the pooled library was determined using the NebNext Library Quant Kit (New England Biolabs, Ipswich, MA). Sequencing was performed on the Illumina MiSeq platform with MiSeq Reagent Kit V2 (Illumina, San Diego, CA).

[0412] On the samples of the validation cohort, reverse transcription was performed using the TaqMan microRNA Reverse Transcription Kit (Thermo Fischer Scientific) and specific TaqMan microRNA Assays, according to the manufacturer's protocol. The following assays were used: hsa-miR-146-5p (ID:000468), hsa-miR-361-5p (ID:000554), hsa-miR-24-3p (ID:000402), hsa-miR-28-3p (ID:002446), hsa-miR-128-3p (ID:002216), hsa-miR-130b-3p (ID:000456), hsa-mir-99b-5p (ID:000436), hsa-miR- 151a-5p (ID:002254), hsa-miR-199a-3p (ID:002304). Hsa-mir-16-5p (ID:000391), cel-mir-39 (ID:000200), hsa-miR-139-3p (ID:002313), hsa-miR-142-3p (ID:000464) and hsa-let7d-3p (ID:001178) were applied as controls. After reverse transcription, quantitative real-time polymerase chain reaction (qPCR) was performed using TaqMan Fast Advanced Mastermix 2x (Thermo Fischer Scientific) on a Quantstudio 7 Flex Real-time PCR System, using 3 parallels for each sample.

[0413] Bioinformatics and statistical methods

[0414] The first step in processing the sequencing data obtained from the samples of the discovery cohort was performed on the QIAGEN GeneGlobe web-based platform, which processed and aligned the data, resulting in the establishment of the raw microRNA read counts of the samples. The raw data were normalized to per-million reads, and then, using glmmSeq and DESeq2 algorithms (DESeq2 R package), we obtained the microRNAs that were significantly (p < 0.001 adjusted by the Benjamini- Hochberg method (false discovery rate, FDR)) different in abundance between APA and BAH samples (a 2-based logarithmic fold change, i.e. (Iog2[foldchange])>|±2| were used). This resulted in a total of 47 miRNAs that were significantly differently expressed between BAH and APA samples. From these microRNAs, we excluded those whose read counts were insufficient to be detected by qPCR (miRNAs with a read count of lower than 50).

[0415] Thus, we found 23 significantly different microRNAs present in detectable amounts by qPCR, on which we ran a neural network -based, 90% to 10% random learner-tester cross validation simulation (e.g. nnet algorithm, nnet and caret R packages; single hidden-layer neural network) (1000 iterations per model), which tested the differentiation potential of each microRNA as a standalone marker, as well as constructed combination models comprising 2 to 8 microRNAs (a total of 880946) and examined their accuracy in distinguishing the two forms of the disease. By comparing the groupings predicted from the models and the actual groupings, the sensitivity and specificity values of the models were also obtained. For technical reasons, APA was designated as the “patient” group, while BAH acted as the “control” group. In more detail: the first step of the simulation was the random partitioning of the data into 90% learner and 10% tester subsets. Second, the neural network model was run using the learner subset. Third, using the miRNA expression values of the tester subset, their classification (unilateral primary aldosteronism (UP A) or BAH) was predicted using the obtained model object. Fourth, the predicted and true classifications were compared, resulting in a 2x2 confusion matrix containing true-positive, truenegative, false-positive, and false-negative cases. Then, this whole process was repeated a total of 1000 times per model (1000 iterations). The final output was the average 2x2 confusion matrix of the 1000 iterations. The resulting models were sorted based on their accuracy, and then the 5 most accurate combinations and the 9 microRNAs that made up these combinations were selected for validation.

[0416] In the case of the validation cohort, Ct values obtained after the qPCR reaction were normalized to the Ct value of cel-mir-39, used as control, in the given sample (ddCt method), thus obtaining the ACt value of the sample.

[0417] Statistical analysis of the qPCR data was carried out using GraphPad Prism 10.0 software (Dotmatics Inc, Boston, MA) and STATISTICA 14.0.1 (TIBCO, Palo Alto, CA). Unpaired, two-sided Student t-test was performed to compare miRNA expression between unilateral PA (APA) and BAH samples, with a resulting P<0.05 after Holm’s correction interpreted as significant. To check whether the data sets are normally distributed, Kolmogorov-Smirnov tests were performed, with P<0.05 interpreted as a non-normal distribution. Between peripheral and AVS samples, paired t-tests were performed, as well as a mixed effect model to check between differences. A Spearman correlation was also calculated between microRNA expression values and hormone levels.

[0418] The aforementioned artificial intelligence algorithm was run on the ACt values, first with the right and left AVS plasma samples, then on the peripheral samples, and in order to achieve higher accuracy, 10 000 iterations per model were performed.

[0419] The accuracy of the 10 combination models giving the most accurate classifications obtained in the previous step were also tested with the inclusion of additional clinical parameters. These clinical parameters were the age and sex of the patient, potassium, aldosterone and renin levels, and the diagnosis assumed by imaging results (CT or MRI). When determining renin activity, it is common for different laboratories to use different normal values. For this reason, instead of direct values, we established categories in the models according to the normal values determined by the given laboratory. These were the following: (1) low, (2) between the lowest boundary of the normal range and 25%, (3) between 25% and median, (4) between the median and 75%, (5) between 75% and the upper boundary of the normal range, and (6) elevated renin level. Thus, a total of further 10 x 127 combination models were created. In this case, we also ran the aforementioned artificial intelligence algorithm with 10 000 iterations per model. We also tested our method on an expanded validation cohort, which included peripheral blood samples from an additional 78 patients (39 unilateral and 39 bilateral primary aldosteronism patients), i.e. which contained a total of 108 samples (54 unilateral and 54 bilateral samples).

[0420] For the combination that proved to have the best accuracy, a deep-learning algorithm based on the KERAS application programming interface and TensorFlow library was created, with which all of the 108 peripheral samples of the expanded validation cohort were analyzed. The model included an input layer with 60 neurons, 8 hidden layers with 60 neurons, except for the last hidden layer, which included 30, and an output layer with 2 neurons. LI regularization was used in each layer for both the KERAS kernel as well as the algorithmic bias in a decreasing manner, so the first hidden layer had the strictest regularization and each subsequent layer was less regularized. The algorithm was set to an 80% to 20% learner-tester cross validation and was set to train for 2000 epochs with a built-in early stopping callback, so if during training the accuracy did not improve, or worsened for 200 epochs, the training process stopped. The final output of this model is a binary classification for each sample.

[0421] Example 2: Results

[0422] A) Analysis of the Discovery Cohort

[0423] Sequencing of the 36 samples of the discovery cohort resulted in a median total read count of 970 043 with a median miRNA read count of 103 821. The DESeq2 algorithm, comparing UPA with BAH, found 172 differently expressed miRNAs with P<0.05, 92 miRNAs with P<0.01, and 47 miRNAs with P<0.001. Of the 47 microRNAs, 40 had a logarithmic fold change larger than 2, and of these, only 23 had >50 read counts in all samples, with all of these 23 miRNAs showing higher expression in bilateral PA. Regarding the lateralization in unilateral PA (UPA) between the side of the adenoma and the contralateral healthy side, DESeq2 analysis identified no significantly different miRNAs even with less constraining selection criteria. We analyzed the adenoma and contralateral sides in UPA with gllmSeq as well, wherein some miRNAs had shown statistical significance, but the logarithmic fold changes in all of these cases were no larger than ±1.7, which cannot be considered biologically significant. Sequencing read counts were also under 50 reads per sample for these miRNAs. We then analyzed the two sides of BAH samples with the same 2 algorithms to verify that no inherent biases were present in the previous analyses. With gllmSeq, we found 3 miRNAs that had statistical significance with logarithmic fold change >2 as well, but the sequencing read counts for all these 3 miRNAs were lower than 10 in all samples, so these differences cannot be verified by RT-qPCR, thus we excluded them from further analyses as most probably artificial. The applied neural network machine learning algorithm ran 880 969 combinations in total. The 5 best-performing models contained a total of 9 different miRNAs, which were selected for validation.

[0424] B) Selection of Endogenous Reference miRNAs

[0425] Sequencing (NGS) data were used to select microRNAs with the lowest variability among all samples. Based on these analyses, 4 miRNAs were selected: hsa-miR-16-5p, hsa-miR-139-3p, hsa-miR- 142-3p and hsa-let7d-3p. Hsa-miR-16-5p, hsa-let-7d and hsa-miR-142-3p had already been documented to be useful as endogenous references. Further analysis using the Normfinder algorithm on our qPCR data showed that hsa-let7d-3p is the most stably expressed of these 4, thus we opted to use this as an endogenous control.

[0426] The use of an external control (cel-mir-39) and an internal control or endogenous reference (hsa- let7d-3p) helped to solve the standardization problem: the former was used to monitor the isolation and amplification process, and the latter was used to correct for variability between samples.

[0427] C) Validation of Candidate miRNAs by RT-qPCR

[0428] MiRNAs included in the 5 best-performing models based on the sequencing (NGS) results (9 different miRNAs) were selected for validation by RT-qPCR.

[0429] For the 9 microRNAs selected for validation, the microRNA abundance / expression was not significantly lower in peripheral samples than in AVS samples (both by paired t-tests and the mixed effect model). Thus, our results suggest that AVS plasma samples have no advantage over peripheral samples based on qRT-PCR validation. Therefore, peripheral samples can be reliably used.

[0430] We have found no significant correlation between the expression values of these 9 microRNAs and aldosterone or renin levels.

[0431] When analyzing peripheral plasma samples, out of the different combinations of the 9 microRNAs selected for validation, ten combinations, comprising 4 to 8 microRNAs, had sensitivity and specificity values above the maximum 85% (see Table 1).

[0432] Thus, based on the results of the validation cohort, the 10 combinations shown in Table 1 proved to be the best on peripheral samples.

[0433] The most promising combination comprised 6 microRNAs, which combination had a sensitivity of 86.3%, a specificity of 87.91%, and an AUC value of 87. 1% on the peripheral samples of the subset comprising 30 patients. This most promising combination is the second among the combinations listed in Table 1 (i.e. the combination comprising hsa-miR-146a-5p, miR-24-3p, miR-130b-3p, miR-99b-5p, miR-15 la-3p, and miR-199a-3p). Statistical analysis showed that the 6 miRNAs that made up the bestperforming model (combination) had normal distributions (Kolmogorov-Smirnov test P>0.05) and showed significantly higher expression in BAH than in UPA.

[0434] Using this most promising combination, the original neural network algorithm performed considerably worse on the expanded validation cohort comprised of 108 peripheral samples, with an AUC value of 62.6%. However, our deep-learning model performed with an accuracy of 100% on the peripheral samples of the 30-patient subset, and on the expanded validation cohort comprised of 108 patients, it yielded a result with a sensitivity of 90.9%, a specificity of 81.8%, a positive predictive value of 86.8%, and an AUC value of 86.7%. The inclusion of clinical parameters did not improve the performance of either machine learning method.

[0435] The inclusion of further parameters, such as imaging, potassium and renin levels, did not improve the performance of the model either (see Tables 3 to 4), for either machine learning method. Table 1. The 10 best-performing microRNA combinations

[0436] Table 2 lists the specificity and sensitivity values of each of the 9 microRNAs included in the combinations shown in Table 1 measured individually.

[0437] Table 2. Specificity and sensitivity values of the 9 identified microRNAs measured individually Hsa-miR-24-3p and hsa-miR-199a-3p are found in all combinations listed in Table 1. The dual combination of these two microRNAs has a specificity value of 73.92% and a sensitivity value of 68.91%.

[0438] The best-performing model of the combination comprising hsa-miR-146a-5p, miR-24-3p, miR- 130b-3p, miR-99b-5p, miR-151a-3p and miR-199a-3p was also run with the inclusion of clinical parameters to see if this would further improve the sensitivity-specificity data. However, no significant improvement was achieved, see Table 3.

[0439] Table 3. Specificity and sensitivity values of the best-performing miRNA combination model incorporating various clinical parameters. The 10 best microRNA combinations listed in Table 1 were tested with the inclusion of certain additional clinical parameters, thus obtaining the 10 best marker combinations listed in Table 4.

[0440] Table 4. The 10 best marker combinations. The microRNA combinations are the same as those listed in Table 1 (including their order), but some of them were supplemented with clinical parameters to improve specificity and sensitivity values.

[0441] D) Performance in Gray Zone Samples

[0442] To gauge the performance of our method in a more realistic scenario, we have also studied 6 peripheral blood samples from patients having had AVS with lateralization index (LI) between 0.25 to 0.33 and 3 to 4. The presumed diagnosis based on AVS results was discordant with follow-up data in 3 of these cases. Considering the follow-up data using the Primary Aldosteronism Surgical Outcome criteria, all 6 samples were correctly diagnosed by our model.

[0443] INDUSTRIAL APPLICABILITY OF THE INVENTION The microRNAs and sets of microRNAs (also called microRNA combinations) disclosed herein can be used in the in vitro differential diagnosis of forms of primary aldosteronism. The forms of primary aldosteronism are unilateral form and bilateral form, preferably unilateral aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH).

[0444] The use of the microRNAs and sets of microRNAs, the kits and the methods disclosed herein enable the differentiation between unilateral and bilateral forms in patients suffering from primary aldosteronism in a minimally invasive manner, preferably from a blood sample, more preferably from a peripheral blood sample.

[0445] REFERENCES

[0446] Decmann, A., Nyiro, G., Darvasi, O., Turai, P., Bancos, I., Kaur, R. J., Pezzani, R., lacobone, M., Kraljevic, I., Kastelan, D., Parasiliti-Caprino, M., Maccario, M., Nirschl, N., Heinrich, D., Reincke, M., Patocs, A., & Igaz, P. (2019). Circulating miRNA Expression Profiling in Primary Aldosteronism. Frontiers in endocrinology, 10, 739. https: / / doi.org / 10.3389 / fendo.2019.00739

[0447] Funder, J.W. & R.M. Carey. (2022). Primary Aldosteronism: Where Are We Now? Where to From Here? Hypertension, 79(4), 726-735.

[0448] Nanba AT, Nanba K, Byrd JB et al. (2017). Discordance between imaging and immunohistochemistry in unilateral primary aldosteronism. Clin Endocrinol,' 87, 665-672.

[0449] Robertson et al. (2013). MicroRNA-24 Is a Novel Regulator of Aldosterone and Cortisol Production in the Human Adrenal Cortex. Hypertension 62(3), 572-578.

[0450] Rossi, G. P., Barisa, M., Allolio, B., Auchus, R. J., Amar, L., Cohen, D., Degenhart, C., Deinum, J., Fischer, E., Gordon, R., Kickuth, R., Kline, G., Lacroix, A., Magill, S., Miotto, D., Naruse, M., Nishikawa, T., Omura, M., Pimenta, E., Plouin, P. F., ... Pessina, A. C. (2012). The Adrenal Vein Sampling International Study (AVIS) for identifying the major subtypes of primary aldosteronism. The Journal of clinical endocrinology and metabolism, 97(5), 1606-1614. https: / / doi.org / 10.1210 / jc.2011-2830

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Claims

CLAIMS1. Use of a microRNA in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the microRNA is at least one microRNA selected from hsa-miR-24-3p, hsa-miR-199a-3p, hsa- miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128- 3p and hsa-miR-28-3p.

2. Use of a set of microRNAs in an in vitro differential diagnosis of forms of primary aldosteronism, wherein the set of microRNAs comprises at least two microRNAs and wherein the at least two microRNAs are selected from hsa-miR-24-3p, hsa-miR-199a-3p, hsa-miR-146a-5p, hsa-miR- 361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

3. The use according to claim 2, wherein the set of microRNAs comprises at least three, at least four, at least five, at least six, at least seven or at least eight microRNAs selected from the group consisting of hsa-miR-24-3p, hsa-miR-199a-3p, hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

4. The use according to claim 2 or 3, wherein the set of microRNAs comprises at least four microRNAs selected from the group consisting of hsa-miR-24-3p, hsa-miR-199a-3p, hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR- 28-3p.

5. The use according to any one of claims 2 to 4, wherein the set of microRNAs comprises the microRNA hsa-miR-24-3p and at least one microRNA selected from the group consisting of hsa-miR- 199a-3p, hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa-miR-128-3p and hsa-miR-28-3p.

6. The use according to any one of claims 2 to 4, wherein the set of microRNAs comprises the microRNA hsa-miR-199a-3p and at least one microRNA selected from the group consisting of hsa-miR- 24-3p, hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p, hsa- miR-128-3p and hsa-miR-28-3p.

7. The use according to any one of claims 2 to 6, wherein the set of microRNAs comprises the microRNAs hsa-miR-24-3p and hsa-miR-199a-3p.

8. The use according to any one of claims 2 to 7, wherein the set of microRNAs comprises the microRNAs hsa-miR-24-3p and hsa-miR-199a-3p, and comprises at least one additional microRNAselected from the group consisting of hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR- 99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

9. The use according to any one of claims 2 to 8, wherein the set of microRNAs comprises the following microRNAs: a) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p and hsa-miR-199a-3p; or b) hsa-miR-146a-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa- miR-199a-3p; or c) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-130b-3p, hsa-miR- 99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p; or d) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-130b-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p; or e) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-128-3p and hsa- miR-199a-3p; or f) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p; or g) hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-128-3p and hsa-miR-199a-3p; or h) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa- miR-199a-3p; or i) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p; or j) hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-24-3p, hsa-miR-28-3p, hsa-miR-128-3p, hsa-miR- 99b-5p, hsa-miR-15 la-3p and hsa-miR-199a-3p.

10. The use according to any one of claims 2 to 9, wherein the set of microRNAs comprises the following six microRNAs: miR-146a-5p, miR-24-3p, miR-130b-3p, miR-99b-5p, miR-151a-3p and miR-199a-3p.

11. The use according to any one of claims 1 to 10, wherein the forms of primary aldosteronism are unilateral and bilateral forms.

12. The use according to any one of claims 1 to 11, wherein the forms of primary aldosteronism are unilateral aldosterone -producing adenoma (APA) and bilateral adrenal hyperplasia (BAH).

13. The use according to any one of claims 1 to 12, in the in vitro differential diagnosis of forms of primary aldosteronism in a biological sample obtained from a patient suffering from or suspected of suffering from primary aldosteronism,wherein preferably the biological sample is selected from a body liquid or body fluid, such as blood, serum, blood plasma, saliva, urine, semen and tears; more preferably the biological sample is blood or blood plasma; more preferably the biological sample is peripheral blood.

14. A kit comprising at least one binding molecule that binds to a microRNA that can be used in the in vitro differential diagnosis of forms of primary aldosteronism, wherein the microRNA is at least one microRNA selected from the microRNAs defined in claim 1, and optionally a reagent and / or means suitable for detecting the binding.

15. A method for the in vitro differential diagnosis of forms of primary aldosteronism in a subject, said method comprising:(i) determining an expression profile of a set of microRNAs in a biological sample obtained from the subject; and(ii) comparing the expression profile determined in step (i) with reference expression profile(s), wherein comparing the expression profile determined in step (i) with the reference expression profile(s) enables the diagnosis of unilateral or bilateral form of primary aldosteronism, and / or applying an algorithm or mathematical function to the expression profile determined in step (i), wherein applying an algorithm or mathematical function to the expression profile determined in step (i) enables the diagnosis of unilateral or bilateral form of primary aldosteronism; wherein the set of microRNAs comprises at least two microRNAs selected from the group consisting of hsa-miR-24-3p, hsa-miR-199a-3p, hsa-miR-146a-5p, hsa-miR-361-5p, hsa-miR-130b-3p, hsa-miR- 99b-5p, hsa-miR-151a-3p, hsa-miR-128-3p and hsa-miR-28-3p.

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