WO2021097526A1

WO2021097526A1 - Iron status biomarkers

Info

Publication number: WO2021097526A1
Application number: PCT/AU2020/051250
Authority: WO
Inventors: Sherif Boulos
Original assignee: Resonance Health Analysis Services Pty Ltd
Priority date: 2019-11-19
Filing date: 2020-11-19
Publication date: 2021-05-27

Abstract

A method comprising the steps of: (a) assessing an expression level of at least one miRNA selected from SEQ ID No's 1-18 in a sample from a subject, and (b) using the expression level to determine an iron status of the subject.

Description

IRON STATUS BIOMARKERS FIELD OF THE INVENTION

The invention relates to biomarkers associated with iron status and iron status related disorders. The invention also relates to screening, diagnostic and prognostic methods of using the biomarkers. Still further the invention relates to methods of assessing medical interventions for irons status related disorders and methods of identifying drug targets for iron status related disorders.

BACKGROUND TO THE INVENTION

There are a number of pathologies related to iron status (high/low iron levels). Thalassemia, as one example, is a common genetic haemoglobin disorder and is caused by a decrease in the expression or absence of a “globin chain” (commonly beta and alpha globin gene) which leads life threatening anaemia.

Thalassemia severity causes a spectrum of disease outcomes with transfusion dependent thalassemia considered the most severe manifestation as it requires frequent blood transfusions as an ongoing treatment for the management of the disease. Because of regular blood transfusions and/or dysregulation of iron metabolism, Thalassemia patients experience a potentially toxic increase in iron accumulation that can lead to life threatening conditions such as cardiac arrest, liver disease and diabetes.

Various laboratory tests are used to monitor the levels of iron in the body such as serum, transferrin saturation and total blood iron. MRI can be used to accurately determine the liver iron concentration (LIC), but this technique is costly and not always available or offered to patients.

Serum ferritin is often used as a surrogate of total iron stores. Unfortunately, serum ferritin is inaccurate and can be confounded by other non-iron related causes such as inflammation, cancer, metabolic disease and obesity. miRNAs are non coding RNAs that bind to target mRNAs and impact on the expression of the mRNA. miRNAs have been investigated as biomarkers for various disorders with limited success. With the above in mind there is a need for improved iron status biomarkers and associated methods of their use including methods for assessing iron status.

SUMMARY OF THE INVENTION

The present invention provides a method comprising the steps of:

(a) assessing an expression level of at least one miRNA selected from Table 1 in a sample from a subject, and

(b) using the expression level to determine an iron status of the subject.

The present invention also provides for the use of at least one miRNA selected from

Table 1 to determine an iron status of a subject.

The present invention also provides a test comprising:

(a) means for obtaining an expression level of at least one miRNA selected from Table 1 in a sample from a subject; and

(b) means for processing the expression level generated in step (a) to determine an iron status of the subject.

The present invention also provides a method comprising the steps of:

(a) assessing an expression level of at least one miRNA, selected from Table 1 , in a sample from a subject, and

(b) using the expression level to determine whether the subject has an iron status related pathology.

The present invention also provides a test comprising:

(b) means for processing the expression level generated in step (a) to determine whether the subject has an iron status related pathology.

The present invention also provides for the use of at least one miRNA, selected from

Table 1 , as a biomarker for an iron status related pathology. The present invention provides a method of assessing an iron status or iron status related pathology intervention in a subject, the method comprising the steps of:

(a) applying the intervention to the subject;

(b) assessing an expression level of at least one miRNA selected from Table 1 in a sample from the subject; and

(c) using the expression level to determine the effect of the intervention on the subject.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, the present invention provides a method comprising the steps of:

(b) using the expression level to determine an iron status of the subject.

As used herein the term “iron status” includes any measure, including surrogate measures, of the iron concentration, amount of iron, iron levels or total iron stores. Examples of iron status include liver iron concentration (“LIC”), ferritin level and transferrin saturation. Preferably, the iron status corresponds to a clinical threshold. For example, when the iron status is LIC it may be an LIC of less than 3.2, at least 3.2, at least 7, at least 15 and/or at least 25.

As used herein the terms microRNA and miRNA are used interchangeably. Furthermore, when used herein, the term miRNA includes precursors of the miRNA, mature forms of the miRNA and/or complexes including the miRNA. In this regard, as used herein, when reference is made to a “miRNA” it is understood to include all forms of the miRNA such as complexes, precursors and mature forms.

Preferably, the miRNA comprises the mature form of the miRNA. Preferably, the miRNA comprises about 15-30, 17-28, 19-26 or 19-25 nucleotides.

The step (a) of assessing an expression level of at least one miRNA can comprise any suitable method for assessing expression of miRNA. Preferably, step (a) comprises at least one of nucleic acid sequencing such as RNA sequencing, next generation sequencing (NGS), nucleic acid amplification including quantitative PCR, northern blotting, ELISA, aptamer based methods, nucleic acid hybridisation including RNase protection and microarray analysis. When step (a) comprises nucleic acid amplification it may comprise reverse transcription PCR (RT-PCR), quantitative amplification and/or amplification in real time.

Step (a) may also comprise use of (i) techniques that involve the labelling of the miRNA and the subsequent detection of the label including nano-particle, fluorescent and radioactive labels; (ii) biosensors adapted to selectively bind the miRNA; (iii) oligonucleotide templated reactions (OTR) where the miRNA serves as a matrix to catalyse a reaction and the reaction product, such as a fluorogenic reaction product, is then detected; (iv) nanobeads; (v) microfluidic chips adapted to selectively bind the miRNA.

Preferably, the at least one miRNA comprises at least two, three or four miRNAs.

Preferably, the at least one miRNA comprises at least one, two three or four of SEQ ID Nos 1-4.

Preferably, the at least one miRNA comprises at least one, two three or four of SEQ ID Nos 1 -4 in and at least one of SEQ ID Nos 5-18.

Preferably, the at least one miRNA comprises at least one, two three or four of SEQ ID Nos 1 -4 in and at least one to nineteen of SEQ ID Nos 5-18. Preferably, the at least one imiRNA comprises SEQ ID No 1 .

Preferably, the at least one miRNA comprises SEQ ID No 1 and at least one of SEQ ID Nos 2-18.

Preferably, the at least one miRNA comprises: SEQ ID No 1 and SEQ ID No 2; SEQ ID No 1 and SEQ ID No 3; SEQ ID No 1 and SEQ ID No 4; SEQ ID No 2 and SEQ ID No 3; SEQ ID No 2 and SEQ ID No 4; SEQ ID No 3 and SEQ ID No 4.

Preferably, step (a) comprises quantifying the expression level of the at least one miRNA.

Preferably, step (a) comprises forming a nucleic acid sample from the sample wherein the nucleic acid sample comprises the at least one miRNA. Preferably, the nucleic acid sample is isolated from the sample. As used herein, the term “isolated” distinguishes the nucleic acid sample from the its naturally occurring state. Preferably, the nucleic acid sample comprises at least 90-99%, 99.5%, 99.9% or 100% nucleic acid.

Preferably, step (a) comprises the step of contacting the sample with an oligonucleotide capable of selectively binding to the at least one miRNA.

The oligonucleotide may comprise a probe or a primer. When the oligonucleotide is a probe it may comprise a circularised probe. Circularised probes are suitable for use in rolling circle amplification reactions.

Preferably, the oligonucleotide is complimentary to the at least one miRNA. Even more preferably, the oligonucleotide has a nucleotide sequence that is at least about 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or about 100% identical to the nucleotide sequence of the at least one miRNA.

The term "identical," used herein refers to a relationship between the sequences of two or more molecules, as determined by comparing their sequences. "Identical" also means the degree of sequence relatedness between polypeptide or nucleic acid molecule sequences, as the case may be, as determined by the match between strings of nucleotide or amino acid sequences. Sequence identity measures the percent of identical matches between two or more sequences with gap alignments. Various methods and software programs, known to those skilled in the art, and using mathematical models or algorithms can be used to determine the identity or complementarity between two or more nucleic acids.

The oligonucleotide may further comprise a detectable label or tag that facilitates detection or purification. For example, the oligonucleotide may further comprise a radioactive label such as radioactive phosphate or a non-radioactive label such as a fluorophore or biotin or digoxygenin. Nucleic acids can be labelled at the 5' end, the 3' end, or throughout the molecule depending on the application. High specific activity probes can be generated with label distributed throughout the nucleic acid, such as by, for example, nick translation, random priming, by PCR or in vitro transcription using labelled dNTPs or NTPs. Single-stranded or double-stranded nucleic acids can be labelled at either ends of the molecule or randomly throughout the nucleic acid. Non limiting examples of labels include g-³²R rATP, a-³²P dNTP, Biotin-dNTP, FI-dNTP, and FI terminator nucleotide.

Other examples of detectable labels or tags include: fluorescent labels, bioluminescent labels, chemiluminescent labels, isotopic labels, nanoparticles, and metals. A detectable label can refer to a molecule or substance capable of detection, including, but not limited to, fluorescers, chemiluminescers, chromophores, bioluminescent proteins, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, isotopic labels, semiconductor nanoparticles, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens) and the like. A fluorescer can exhibit fluorescence in the detectable range.

The sample may comprise a biological sample and/or sub-samples thereof. Preferably, the biological sample is a body fluid such as blood, serum, plasma, urine, sweat, tears, saliva, sputum, or any combination or fraction thereof. Other non limiting examples of a biological sample include whole blood, peripheral blood, ascites, cerebrospinal fluid, buccal sample, cavity rinse, organ rinse, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, faecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, or other lavage fluids. A biological sample can also include the blastocyl cavity, umbilical cord blood, or maternal circulation which can be of foetal or maternal origin. The biological sample can also be a tissue sample or biopsy. Sub-samples include extracts from the sample including nucleic acid extracts such as RNA extracts.

Preferably, the subject is a mammal such as a human. However, the subject may also be a non-human mammal such as a primate, mouse, rat, dog, cat, horse, or cow. A subject can be one who has been previously diagnosed or identified as having abnormal iron status or an iron status related pathology, and optionally has already undergone, or is undergoing, a therapeutic intervention. Alternatively, a subject can also be one who has not been previously diagnosed or identified as having abnormal iron status or an iron status related pathology. For example, a subject can be one who exhibits one or more risk factors for abnormal iron status or an iron status related pathology, or a subject who does not exhibit any such risk factors or a subject who is asymptomatic for abnormal iron status or an iron status related pathology. A subject can also be one who is suffering from or at risk of developing abnormal iron status or an iron status related pathology.

Step (b) comprises any use of the expression level from step (a) to determine an iron status of the subject.

Preferably, the expression level from step (a) alone determines the iron status. However, the expression level from step (a) may partially determine the iron status. In this regard, the expression level from step (a) may be combined with a second measure to determine the iron status.

Preferably, the second measure comprises ferritin and/or transferrin saturation.

Preferably, step (b) comprises comparing the expression level from step (a) with a reference value indicative of the iron status.

Preferably, the reference value is a miRNA expression level. For example, the reference value may be a reference miRNA expression level from at least one second subject, wherein the reference miRNA expression level is known to correlate with an iron status. The at least one second subject may have a normal iron status or an abnormal iron status.

The at least one second subject can be a cohort or population of subjects.

The reference value may also be a ratio of miRNA expression levels.

Another use, according to step (b) of the expression level from step (a) is comparing it with another expression level from the same subject taken at a different time. Such use allows for the comparison of expression levels, and hence iron status, over time in a subject.

Preferably, the iron status is determined with a sensitivity of at least about 50%, 70%, 77%, 82%, 85%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or about 100%.

Preferably, the iron status is determined with a specificity of at least about 85%, 87%, 89%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or about 100%.

Preferably, the iron status is determined with an accuracy of at least about 85%, 86%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or about 100%.

The method may involve the use of a control to better assess the expression level of the at least on miRNA from Table 1 and use it to determine iron status. Preferably, the control is a control miRNA such as control miRNA that is not differentially expressed with respect to iron status. Even more preferably, the control miRNA is at least one miRNA selected from the list of miRNAs in Table 2.

According to a second aspect the present invention provides for the use of at least one imiRNA selected from Table 1 to determine an iron status of a subject. The other preferred features of the method described above in relation to the first aspect also form preferred features of this aspect of the invention.

According to a third aspect of the present invention there is provided a test comprising:

The other preferred features of the method described above in relation to the first aspect also form preferred features of this aspect of the invention. For example, the means for obtaining the expression level may comprise any suitable method for assessing expression of miRNA. Preferably, the means comprises a nucleic acid sequencing means, a next generation sequencing (NGS) means, a nucleic acid amplification means, a northern blotting means, a nucleic acid hybridisation means and/or a microarray means.

Preferably the test comprises an apparatus.

Preferably, the test comprises a kit. Preferably, the kit comprises an oligonucleotide as described herein such as a probe or primer that is complimentary to the at least one miRNA.

Preferably, the kit comprises a reagent for amplifying the at least one miRNA.

Preferably, the kit comprises written instructions for quantifying an expression level of the miRNA and/or for determining iron status based on the expression level. The written instructions may include instructions for comparing miRNAs and/or a predetermined value (e.g., a value for determining whether the expression level of the miRNA is indicative of iron status.

Preferably, the kit comprises any one or more of the following: a DNA ligase (e.g., T7 ligase or SplintR ligase), a ligation mixture, a buffer, BSA, dNTPs, a DNA polymerase, a denaturing buffer, an RNA, an miRNA, a probe, a DNA, a primer (e.g., forward/reverse primer), hexamers, salts, an oligonucleotide, a padlock probe, and/or a circularized probe.

The iron status may correlate with an iron level related pathology. Thus, according to a fourth aspect, the present invention provides a method comprising the steps of:

Preferably, the iron status related pathology comprises a pathology selected from the list comprising: acquired iron overload disorders (for example caused by repeated blood transfusions and iron transfusions); non-acquired iron overload disorders; bone marrow failure disorders and anaemias (Fanconi anemia (FA), dyskeratosis congenita, aplastic anemia, myelodysplastic syndromes (MDS), Diamond-Blackfan anemia (DBA); haemachromatosis; thalassemia; liver diseases (cirrhosis/fibrosis linked to excess alcohol consumption and metabolic dysfunction, viral and non-viral related hepatitis), atransferrinemia and aceruloplasminaemia. The present invention also provides tests for iron status related pathologies. Thus, according to a fifth aspect of the present invention there is provided a test comprising:

The biomarkers and panels thereof of the present invention can be implemented in a range of test systems. Typically, test systems include a means for obtaining test results from a sample, a means for collecting, storing, processing and/or tracking test results for the sample, usually in a database and a means for reporting test results. The means for obtaining test results can include a module adapted for automatic testing utilising one or more of biochemical, immunological and nucleic acid detection assays. Some test systems can process multiple samples and can run multiple tests on a given sample. The means for collecting, storing, processing and/or tracking test results may comprise a physical and/or electronic data storage device such as a hard drive or flash memory or paper print-outs. The means for reporting test results can include a visible display, a link to a data structure or database, or a printer. In this regard, the reporting means may simply be a data link that is adapted to send results to another device such as a database, visual display, or printer.

Typically, test results from system of the present invention serve as inputs to a computer or microprocessor programmed with a machine code or software that takes the data relating to the expression level of the at least one miRNA described herein and determines the risk of developing or already having abnormal iron status or an iron status related pathology.

The other preferred features of the tests and kits described above in relation to the third aspect also form preferred features of this aspect of the invention. For example, the means for obtaining the expression level may comprise any suitable method for assessing expression of miRNA. Preferably, the means comprises a nucleic acid sequencing means, a next generation sequencing (NGS) means, a nucleic acid amplification means, a northern blotting means, a nucleic acid hybridisation means and/or a microarray means.

The invention provides improved diagnosis and prognosis of an iron status and/or an iron status related pathology. The risk of developing an abnormal iron status and/or an iron status related pathology can be assessed by measuring the expression of one or more of the miRNAs described herein, and comparing the measured values to reference or index values. Such a comparison can be undertaken with mathematical algorithms or formula in order to combine information from results of multiple individual miRNAs and other parameters into a single measurement or index. Subjects identified as having an increased risk of an abnormal iron status and/or an iron status related pathology can optionally be selected to receive treatment regimens, such as administration of prophylactic or therapeutic compounds or implementation of exercise regimens or dietary supplements to prevent, treat or delay disease onset.

The expression level of the at least one miRNA can be measured in the sample and compared to a reference or normal level, utilizing techniques such as reference limits, discrimination limits, or risk defining thresholds to define cut-off points and abnormal values for an iron status and/or an iron status related pathology. The normal control level is the level of one or more miRNAs or combined biomarker indices typically found in a subject not suffering from abnormal levels or a pathology. The normal and abnormal levels and cut-off points may vary based on whether the at least one miRNA is used alone or in a formula combined with other biomarkers into an index. Alternatively, the normal or abnormal level can be a database of biomarker patterns or “signatures” from previously tested subjects who did or did not develop or convert to abnormal iron status or an iron status related pathology over a clinically relevant time horizon.

Thus, the expression levels of the at least one miRNA can be used to generate a profile or signature of subjects: (i) who do not have and are not expected to develop an abnormal iron status or an iron status related pathology and/or (ii) who have or expected to develop such conditions. The profile of a subject can be compared to a predetermined or reference biomarker profile to diagnose or identify subjects at risk for developing an abnormal iron status or an iron status related pathology, to monitor the progression of the pathology, as well as the rate of progression of the pathology, and to monitor the effectiveness of interventions. Profiles of the present invention are preferably contained in a machine-readable medium and are “live” insofar as they can be updated with further data that comes to hand, thus improving the strength and clinical significance of the biomarkers. Data concerning the levels of the at least one miRNA of the present invention can also be combined or correlated with other data or test results, such as, without limitation, measurements of clinical parameters or other algorithms for an iron status or an iron status related pathology. The machine-readable media can also comprise subject information such as medical history and any relevant family history.

The present invention also provides for the use of at least one miRNA, selected from Table 1 , as a biomarker for an iron status related pathology.

The methods of the present disclosure can also include assessing an iron status or iron status related pathology intervention. Thus, according to a sixth aspect the present invention provides a method of assessing an iron status or iron status related pathology intervention in a subject, the method comprising the steps of:

(a) applying the intervention to the subject;

Preferably, the expression level of the at least one miRNA is assessed at least twice. In this regard, changes in the expression levels after the intervention may identify the intervention as an intervention for treating abnormal iron status and/or an iron status related pathology.

Preferably the expression level of the at least one miRNA is assessed before, during and/or after the intervention.

Preferably, the intervention is selected from the list comprising: dietary iron and iron I.V. infusion therapy, iron chelation therapy, agents and drugs affecting iron regulation, mimics or antagonists of biological pathways or processes involved in iron regulation or homeostasis, blood transfusion, folic acid substitutes; stem cell transplants, gene therapy such as haemoglobin gene therapy, haemoglobin based therapies including those designed to increase haemoglobin production.

The present invention also provides for the use of at least one miRNA selected from Table 1 as a target for a therapeutic agent for an iron status or an iron status related pathology. In this regard, the miRNAs described herein may be useful as drug targets.

The various aspects of the present invention can provide, for example, a relatively economical, accurate, non-invasive, and easy to implement test for detection of iron status and/or an iron status related pathology. Methods of the present disclosure can aid early detection of iron status and/or an iron status related pathology. Methods of the present disclosure can be useful for subjects with undiagnosed abnormal iron status and/or an iron status related pathology. Methods of the present disclosure can reduce the rate of false positives and false negatives obtained from other approaches to assessing iron status and/or an iron status related pathology and can improve the accuracy of diagnosis.

General

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. None of the cited material or the information contained in that material should, however be understood to be common general knowledge. The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products and methods are clearly within the scope of the invention as described herein.

The invention described herein may include one or more range of values (e.g. size etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

EXAMPLES

Example 1 - Identification of iron status biomarkers 1. Materials/Methods

Subjects: 29 Normal controls and 30 Patients (with a confirmed diagnosis of Thalassemia Major/Intermedia) were recruited. All necessary ethics approvals were obtained from the respective institutions and government bodies.

MRI imaging: Subjects were imaged (within approximately 1 week of the blood draw) using a 1.5 Tesla Philips Ingenia (Philips, Netherlands) Instrument. Ferriscan MRI image data was acquired according to protocols and instructions provided by Resonance Health Analysis Services. DICOM images were uploaded to a secure web portal for analysis. Ferriscan data was analysed by trained analysts using proprietary in-house software and the similarly analysed using the Ferrismart automated image tool. Ferriscan and Ferrismart are proprietary medical devices owned by Resonance Health Analysis Services that are cleared by the FDA, CE Mark, TGA as tools for the determination of liver iron concentration. Ferriscan is considered the gold standard for the determination of liver iron concentration. Ferrismart is an advanced and automated tool trained on Ferriscan image data using machine learning.

Blood collection and plasma preparation: A total of 10 millilitres of whole blood was collected from each subject (BD Vacutainer® Venous Blood Collection Tubes; cat. no. 367525 with BD Vacutainer Eclipse Blood Collection Needle 22G; cat. No. 368651) with pre-attached holder. Blood collection tubes contained EDTA and were heparin free).

Blood samples were centrifuged at 1900 x g (3000 rpm) in a swinging bucket rotor for 10 minutes at 4 degrees Celsius (4°C). The upper (yellow) plasma phase was transferred to a fresh sterile centrifuge tube (conical bottom), without disturbing the intermediate buffy coat layer (containing white blood cells and platelets). In general, a minimum of 5 millilitres of plasma was obtained from each 10 millilitres of whole blood. The plasma was centrifuged at 4 degrees Celsius (4°C) for 10 minutes at 16,000 x g in a fixed-angle rotor to remove any residual cellular material. Plasma was carefully aliquoted to cryo-storage tubes (Neptune 3102) and stored at -80 C prior to RNA extraction. It should be noted that whole blood was kept at 4°C and processed (as described above) within ONE (1) hour of the blood draw.

Biochemical analysis: performed using standardised methods.

Sample preparation: RNA was isolated using the miRNeasy Serum/Plasma Kit (QIAGEN) according to manufacturers instructions.

Library preparation and Next Generation Sequencing (NGS): Library preparation was done using the QIAseq miRNA Library Kit (QIAGEN). A total of 5ul of RNA was converted into miRNA NGS libraries. Adapters containing UMIs were ligated to the RNA. Then RNA was converted to cDNA. The cDNA was amplified using PCR (22 cycles) and during the PCR indices were added. After PCR the samples were purified. Library preparation QC was performed using either Bioanalyier 2100 (Agilent) or TapeStation 4200 (Agilent). Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar ratios. The library pool(s) were quantified using qPCR. The library pool(s) were then sequenced on a NextSeq500 sequencing instrument (lllumina inc.) according to the manufacturer instructions. The read length was 75 nucleotides with a single-end read (up to 46bp insert + 19bp 3’ linker + 10 UMIs) and a minimum average of 12 million reads/per sample. Raw data was de-multiplexed and FASTQ files were generated for each sample using the bcl2fastq software (lllumina inc.). FASTQ data were checked using the FastQC tool.

Annotation of the obtained sequences was performed using the reference annotation listed as follows: Organism: Flomo_sapiens; Reference genome: GRCh37; Annotation reference: mirbase_20.

Statistical analysis: The correlation coefficients for each miRNA for all subjects was determined for: R2; LIC; Ferritin concentration; Transferrin saturation %; Transferrin receptor expression and; Hepcidin concentration for all subjects, and for the patient group alone. Those showing the best correlation coefficients were further analysed using linear regression to determine the best predictors of LIC for each clinical threshold. In addition to linear regression models, miRNA were also analysed using a decision tree machine learning approach.

2. Results

Table 3 details the correlation coefficients of specific miRNAs (determined by NGS of plasma samples) from 29 NORMAL control subjects and 30 PATIENTS with Thalassemia against R2, LIC, Ferritin concentration, Transferrin saturation %, Transferrin receptor expression and Hepcidin concentration.

^L R2 denotes an MRI parameter calculated from the Ferriscan DICOM image.

^* LIC denotes Liver Iron Concentration as determined by analysing the Ferriscan DICOM image.

Table 4 details the correlation coefficients of specific miRNAs (determined by NGS of plasma samples) 30 PATIENTS with Thalassemia against R2, LIC, Ferritin concentration, Transferrin saturation %, Transferrin receptor expression and Hepcidin concentration.

^L R2 denotes an MRI parameter calculated from the Ferriscan DICOM image.

^* LIC denotes Liver Iron Concentration as determined by analysing the Ferriscan DICOM image. Three additional imiRNAs were also identified through further analysis of data generated in this example: miRNA 151a-5p, miRNA 590-3p and miRNA 144-3p. Further details on these miRNAs are provided elsewhere herein including in Table 1.

Table 5 details the logression analysis of miRNA sp versus LIC as determined by Ferriscan analysis of the DICOM image. LIC (mg/g of dried liver) thresholds of: 3.2; 7; 15 and 25 with and/or without Ferritin in TFIALASSEMIA patients and NORMAL Controls (n=59).

Table 6 details the logression analysis of imiRNA sp versus LIC as determined by Ferrismart analysis of the DICOM image. LIC (mg/g of dried liver) thresholds of: 3.2; 7; 15 and 25 with and/or without Ferritin in TFIALASSEMIA patients and NORMAL Controls (n=59).

Claims

1. A method comprising the steps of:

(b) using the expression level to determine an iron status of the subject.

2. A method according to claim 1 wherein the iron status comprises at least one of liver iron concentration (“LIC”), ferritin level and transferrin saturation.

3. A method according to claim 2 wherein the iron status comprises an LIC selected from the list of: less than 3.2, at least 3.2, at least 7, at least 15 and/or at least 25.

4. A method according to any one of claims 1 to 3 wherein the miRNA comprises a mature form of the miRNA.

5. A method according to claim 4 wherein the mature form of the miRNA comprises about 15-30, 17-28, 19-26 or 19-25 nucleotides.

6. A method according to any one of the preceding claims wherein step (a) comprises at least one of nucleic acid sequencing, next generation sequencing (NGS), nucleic acid amplification, northern blotting, nucleic acid hybridisation and microarray.

7. A method according to claim 6 wherein the nucleic acid amplification comprises reverse transcription PCR (RT-PCR), quantitative amplification and/or amplification in real time.

8. A method according to claim 6 wherein the nucleic acid hybridisation comprises RNase protection.

9. A method according to any one of the preceding claims wherein step (a) comprises labelling the miRNA and detecting the label.

10. A method according to claim 9 wherein the label comprises a label selected from the list comprising: a nano-particle, a fluorescent label and a radioactive label.

11. A method according to any one of the preceding claims wherein step (a) comprises the use of a biosensor adapted to selectively bind the miRNA.

12. A method according to any one of the preceding claims wherein the at least one miRNA comprises at least two, three or four miRNAs.

13. A method according to claim 12 wherein step (a) comprises the assessment of a ratio of at least two of the two, three or four miRNAs.

14. A method according to any one of the preceding claims wherein step (a) comprises quantifying the expression level of the at least one miRNA.

15. A method according to any one of the preceding claims wherein step (a) comprises forming a sub-sample from the sample and wherein the sub-sample comprises the at least one miRNA.

16. A method according to claim 15 wherein the sub-sample comprises at least 90- 99% RNA.

17. A method according to claim 1 wherein step (a) comprises the step of contacting the sample with an oligonucleotide capable of selectively binding to the at least one miRNA.

18. A method according to claim 17 wherein the oligonucleotide is a probe or a primer.

19. A method according to claim 17 or 18 wherein the oligonucleotide has a nucleotide sequence that is at least about 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or about 100% identical to the nucleotide sequence of the at least one miRNA.

20. A method according to any one of claims 17-19 wherein the oligonucleotide further comprises a detectable label or tag.

21. A method according to any one of the preceding claims wherein the biological sample comprise blood or fraction thereof.

22. A method according to any one of the preceding claims wherein the subject is a human.

23. A method according to any one of the preceding claims wherein step (b) comprises comparing the expression level from step (a) with a reference value indicative of the iron status.

24. A method according to claim 23 wherein the reference value is a miRNA expression level.

25. A method according to claim 23 or 24 wherein the reference value is determined from a population.

26. A method according to any one of claims 1 to 22 wherein step (b) comprises comparing the expression level from step (a) with another expression level from the same subject taken at a different time.

27. A method according to any one of the preceding claims wherein the iron status is determined with a sensitivity of at least about 50%, 70%, 77%, 82%, 85%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or about 100%.

28. A method according to any one of the preceding claims wherein the iron status is determined with a specificity of at least about 85%, 87%, 89%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or about 100%.

29. A method according to any one of the preceding claims wherein the iron status is determined with an accuracy of at least about 85%, 86%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or about 100%.

30. A method according to any one of the preceding claims wherein step (b) further comprises using a second measure in combination with the expression level from step (a) to determine the iron status of the subject.

31. A method according to claim 30 wherein the second measure comprises a measure of ferritin and/or transferrin.

32. A test comprising: (a) means for obtaining an expression level of at least one miRNA selected from Table 1 in a sample from a subject; and

33. A test according to claim 32 wherein the means for obtaining the expression level of the at least one miRNA comprises a nucleic acid sequencing means, a next generation sequencing (NGS) means, a nucleic acid amplification means, a northern blotting means, a nucleic acid hybridisation means and/or a microarray.

34. A test according to claim 32 or 33 comprising an apparatus for performing step (a).

35. A kit comprising the test according to any one of claims 32 to 34.

36. A kit according to claim 35 comprising an oligonucleotide capable of selectively binding to the at least one miRNA.

37. A kit according to claim 36 wherein the oligonucleotide is a probe or a primer.

38. A kit according to claim 36 or 37 wherein the oligonucleotide has a nucleotide sequence that is at least about 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or about 100% identical to the nucleotide sequence of the at least one miRNA.

39. A kit according to claim 38 wherein the oligonucleotide is selected from the list comprising:

40. A kit according to any one of claims 36 to 39 wherein the oligonucleotide further comprises a detectable label or tag.

41. A kit according to any one of claims 35 to 40 further comprising a reagent for amplifying the at least one miRNA.

42. A kit according to any one of claims 35 to 41 further comprising instructions for quantifying an expression level of the miRNA and/or for determining iron status based on the expression level.

43. A method comprising the steps of:

44. A method according to claim 43 wherein the iron status related pathology comprises a pathology selected from the list comprising: acquired iron overload disorders (for example caused by repeated blood transfusions and iron transfusions); non-acquired iron overload disorders; bone marrow failure disorders and anaemias (Fanconi anaemia (FA), dyskeratosis congenita, aplastic anaemia, myelodysplastic syndromes (MDS), Diamond-Blackfan anaemia (DBA); haemachromatosis; thalassemia; liver diseases (cirrhosis/fibrosis linked to excess alcohol consumption and metabolic dysfunction, viral and non-viral related hepatitis), atransferrinemia and aceruloplasminaemia.

45. A test comprising:

46. A test according to claim 45 wherein the means for obtaining an expression level of the at least one miRNA comprises a nucleic acid sequencing means, a next generation sequencing (NGS) means, a nucleic acid amplification means, a northern blotting means, a nucleic acid hybridisation means and/or a microarray.

47. A test according to claim 45 or 46 comprising an apparatus for performing step (a).

48. A kit comprising the test according to any one of claims 45 to 47.

49. A kit according to claim 48 comprising an oligonucleotide capable of selectively binding to the at least one miRNA.

50. A kit according to claim 49 wherein the oligonucleotide is a probe or a primer.

51. A kit according to claim 49 or 50 wherein the oligonucleotide has a nucleotide sequence that is at least about 80%, 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or about 100% identical to the nucleotide sequence of the at least one miRNA.

52. A kit according to claim 51 wherein the oligonucleotide is selected from the list comprising:

53. A kit according to any one of claims 48 to 52 wherein the oligonucleotide further comprises a detectable label or tag.

54. A kit according to any one of claims 48 to 53 further comprising a reagent for amplifying the at least one miRNA.

55. A kit according to any one of claims 48 to 54 further comprising instructions for quantifying an expression level of the miRNA and/or for determining iron status based on the expression level.

56. A method of assessing an iron status or iron status related pathology intervention in a subject, the method comprising the steps of:

(a) applying the intervention to the subject;

57. A method according to claim 56 wherein the expression level of the at least one miRNA is assessed at least twice.

58. A method according to claim 56 or 57 wherein the expression level of the at least one miRNA is assessed before, during and/or after the intervention.

59. A method according to any one of claims 56 to 58 wherein the intervention is selected from the list comprising: dietary iron and iron I.V. infusion therapy, iron chelation therapy, agents and drugs affecting iron regulation, mimics or antagonists of biological pathways or processes involved in iron regulation or homeostasis, blood transfusion, folic acid substitutes; stem cell transplants, gene therapy such as haemoglobin gene therapy, haemoglobin based therapies including those designed to increase haemoglobin production.

60. Use of at least one miRNA selected from Table 1 as a target for a therapeutic agent for an iron status or an iron status related pathology.