Method and kit for diagnosing muscle atrophy

Abstract

The present invention relates to compositions and methods for detecting muscle atrophy in a subject. The present invention also provides novel biomarkers that allow for in vitro or ex vivo detection of muscle mass loss in a subject. The present invention also relates to the use of muscle atrophy as an indicator of the onset or risk of onset of a disease or disorder in a subject. Furthermore, the present invention relates to kits and reagents for use in these methods.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to compositions and methods for detecting muscle atrophy in a blood sample of a subject. The present invention also provides novel biomarkers that allow for in vitro or ex vivo detection of muscle mass loss in a blood sample of a subject. The present invention also relates to the use of muscle atrophy as an indicator of the onset or risk of onset of a disease or disorder in a subject, including addressing potential adverse effects of some treatments and the efficacy of potential countermeasures. Furthermore, the present invention relates to reagents, such as kits and oligonucleotides, for use in these methods. [Background technology]

[0002] 2. Background of the Invention Skeletal muscle accounts for nearly half of the mass of the human body. In addition to its role in movement, metabolism, homeostasis, and energy and nitrogen storage, skeletal muscle (containing 50-75% of the total protein in the human body) is an essential supplier of amino acids used by vital organs during pathology.

[0003] Malnutrition, muscle disuse, aging, disease or injury can cause skeletal muscle fibers to atrophy, ultimately resulting in loss of muscle mass. This process, called skeletal muscle atrophy, is associated with reduced quality of life, reduced tolerance and response to treatment, and increased morbidity and mortality, regardless of the underlying disease (Kalantar-Zadeh et al., J Cachexia Sarcopenia Muscle (2013) 4:89-94). Once skeletal muscle atrophy has been established for a long time, it is very difficult to reverse. Therefore, it seems essential to predict early in the disease that muscle atrophy is becoming established.

[0004] Skeletal muscle homeostasis is controlled by numerous signaling pathways. Loss of muscle mass is due to an imbalance in protein homeostasis that favors protein degradation. Comparison of gene expression profiles of muscles from different catabolic states allowed the identification of a common panel of genes, called atrogenes, that appear to be important for protein loss. Several atrogenes belong to two major cellular degradation systems, the ubiquitin-proteasome and the autophagy-lysosome. In atrophying rodent muscles, two ubiquitin ligases, muscle ring finger-1 (MuRF1 / TRIM63) and muscle atrophy F-box (MAFbx / atrogin-1), are considered master genes for muscle atrophy (Bodine et al., Science (2001) 294(5547):1704-1708), whereas TRIM32 is considered a master regulator of muscle regeneration by initiating autophagy (Overa et al., J Cell Sci. (2019) 132(23):jcs236596).

[0005] Following the discovery of atrogenes, the concept that transcription factors promote muscle atrophy is now well established (Sartori et al., Nature Communications (2021) 12:330). FoxO1, 3, and 4 are downstream transcription factors of the IGF1 / insulin-Akt pathway, and their inhibition completely avoids muscle loss and strength decline in fasting, hindlimb suspension, immobilization, diabetes, and glucocorticoid treatment. NF-κB and Stat3 transcription factors mediate the action of TNF-α and IL-6 on muscle wasting, particularly by upregulating either MuRF1 or MAFbx. The unfolded protein response (UPR) and endoplasmic reticulum (ER) stress-related pathways have been found to regulate muscle atrophy via ATF4 and XBP1 transcription factors.

[0006] Muscle atrophy may also be associated with muscle-specific diseases. Although less severe than disuse muscle atrophy, disease-related muscle atrophy may result from either diseases affecting the nerves supplying individual muscles (i.e., neurogenic atrophy) or diseases intrinsic to muscle tissue (i.e., myopathy). In neurogenic atrophy, the nerve supply to the muscle may be interrupted or impaired by compression, damage, or disease in the nerve cells, resulting in temporary or permanent nerve deficits. Diseases in nerve cells that may interrupt or impair the nerve supply to the muscle include, for example, multiple sclerosis, amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease), Guillain-Barre syndrome, stroke, and viral infection of nerve cells (e.g., poliomyelitis). Muscle diseases may be intrinsic to muscle tissue (e.g., muscular dystrophy, polymyositis, or myotonia) or may occur as a response to a systemic illness (e.g., hypo- or hyperthyroidism, adrenal deficiency, diabetes, or autoimmune disease). Sarcopenia is a debilitating disease that afflicts older adults and is characterized by age-related loss of muscle mass and function.

[0007] Generalized muscle wasting (cachexia) may also occur as a secondary consequence of diseases such as advanced cancer, acquired immune deficiency syndrome (AIDS), chronic obstructive pulmonary disease, congestive heart failure, cardiomyopathy, chronic liver disease, kidney disease, emphysema, tuberculosis, osteomalacia, hormone deficiency, anorexia nervosa, generalized malnutrition, and drug abuse (e.g., alcohol, opiate, or steroid abuse). Skeletal muscle atrophy in cachexia cannot be reversed by increasing food intake in the absence of treatment of the underlying pathology (Konishi et al., J Cachexia Sarcopenia (2016) 7:107-109). Mechanical stress and neurohormonal mediators also play a role in the control of muscle mass in cachexia. Most studies of the mechanisms of muscle wasting have been performed in animal models, such as young growing rodents. Although some abnormalities described in animal models have been confirmed in humans, it remains to be established whether the observed alterations are directly related to the atrophy program or are specific to the disease itself. Furthermore, muscle wasting is generally detected within 1-2 weeks after a catabolic stimulus in young rodents, whereas in human disease it is generally detected within longer time periods and in adults. Additionally, rodent physiology is not strictly equivalent to human physiology.

[0008] Attempts to identify biomarkers of muscle atrophy have been performed in muscle biopsies of patients suffering from certain pathologies such as chronic obstructive pulmonary disease (COPD) (Debigare et al., Journal of chronic obstructive pulmonary disease (2008) 5(2):75-84). However, it is not clear whether the identified markers are related to muscle atrophy or COPD. Other studies carried out on human muscle biopsies have allowed the identification of biomarkers involved in muscle atrophy induced in specific situations such as immobilization (US Pat. No. 11,090,313 B2) or denervation (US Pat. Pub. No. 2006 / 003959 A1). The mechanisms of muscle atrophy have also been studied in vitro (KR2020 0001068 A) or in vivo in rodent muscle biopsies (US Pat. Pub. No. 2006 / 069049 A1), but it is well known that 90% of the results obtained in rodents are not transposable to humans. Furthermore, the use of muscle biopsy for routine diagnosis is not clinically feasible.

[0009] Recently, we performed a study on atrophied skeletal muscle biopsies from lung cancer (LC) and chronic hemodialysis (HD) patients (Aniort et al., J Cachexia Sarcopenia Muscle (2019) 10(2):323-337). Both the ubiquitin proteasome and autophagy systems were activated in LC and HD patients, and several already known E3 ligases were confirmed as strong markers of muscle atrophy in humans. The Wnt-β-catenin and ATF4 signaling pathways were also activated in patients. A panel of more than 230 proteins is differentially expressed in LC and HD patients when compared to the healthy human proteome. However, protein variations were not addressed at the transcriptome level. Furthermore, mass spectrometry of proteins is of little use for direct clinical use. In addition, muscle biopsies are painful, difficult to access, and not suitable for monitoring muscle atrophy over time.

[0010] Therefore, there is a strong need to identify biomarkers of muscle atrophy from easily accessible samples with good sensitivity and specificity independent of disease. Such biomarkers can be very useful for diagnosing muscle atrophy in subjects, in particular for monitoring the loss of muscle mass in subjects suffering from a disease or disorder, and for assisting in the management of treatment. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention is based on the identification of novel biomarkers detectable in blood samples that allow the diagnosis of muscle atrophy, especially skeletal muscle atrophy. By identifying a restricted panel of genes in blood samples from diseased subjects suffering from different types of pathologies and suffering from loss of muscle mass, the inventors have developed a simple and reliable method for diagnosing and monitoring muscle atrophy. The expression of these genes is highly correlated with the atrophy program of any disease. The present invention therefore allows for adjusting and improving the treatment of subjects by treating or reducing muscle atrophy in disease management. In particular, the detection and treatment of muscle atrophy in diseased subjects in a clinically feasible manner can increase the effectiveness of therapeutic treatment. [Means for solving the problem]

[0012] The present invention relates to the use of at least one gene or its gene product selected from ACTR6 (SEQ ID NO: 1), GIMAP2 (SEQ ID NO: 2), RCN2 (SEQ ID NO: 3), RPL22L1 (SEQ ID NO: 4), TRIAP1 (SEQ ID NO: 5), NIFK (SEQ ID NO: 6), APIP (SEQ ID NO: 7), GEMIN6 (SEQ ID NO: 8), RWDD1 (SEQ ID NO: 9), ZNF613 (SEQ ID NO: 10), BPNT1 (SEQ ID NO: 11), CCT2 (SEQ ID NO: 12) and LYRM2 (SEQ ID NO: 13) as a biomarker for the in vitro or ex vivo detection in a blood sample of muscle atrophy, in particular skeletal muscle atrophy.

[0013] Another object of the present invention relates to an in vitro or ex vivo method for diagnosing muscle atrophy, in particular skeletal muscle atrophy, in a subject, which typically comprises determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in a blood sample obtained from the subject, wherein a decrease in the expression level of said at least one biomarker compared to a reference value is indicative of muscle atrophy, in particular skeletal muscle atrophy, in the subject. Effect of the Invention

[0014] The present invention also relates to an in vitro or ex vivo method for assessing the efficacy of a treatment to reverse muscle atrophy, in particular skeletal muscle atrophy, in a blood sample from a subject, the method typically comprising: (i) determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in a first blood sample from the subject obtained prior to said treatment or at an initial stage of said treatment; and (ii) determining the expression level of the same at least one biomarker in a second blood sample from the subject obtained at a later stage during or after said treatment, wherein an expression level of the at least one biomarker measured in step (ii) that is greater than the expression level of the same at least one biomarker measured in step (i) indicates that the treatment is efficacious. Includes.

[0015] The present invention further relates to an in vitro or ex vivo method for assessing the onset or risk of onset of a disease in a subject comprising assessing the presence of muscle atrophy, in particular skeletal muscle atrophy, by a method described herein; wherein the presence of muscle atrophy, in particular skeletal muscle atrophy, is indicative of the onset or risk of onset of a disease in the subject.

[0016] In another aspect, the present invention relates to an in vitro or ex vivo method for assisting in selecting, adapting or altering a treatment in a subject suffering from a disease, the method typically comprising: (i) assessing the presence of muscle atrophy, particularly skeletal muscle atrophy, in a subject by the methods described herein; and (iii) taking into account the presence of muscle atrophy, in particular skeletal muscle atrophy, to select, adapt or modify a treatment in a subject suffering from a disease Includes.

[0017] In a preferred embodiment, the in vitro or ex vivo method for assisting in selecting, adapting or altering a treatment in a subject suffering from a disease further comprises the step (iii) of selecting a treatment for muscle atrophy, in particular skeletal muscle atrophy, said treatment typically comprising a drug (pharmacological agent such as glucocorticoids, anti-inflammatory drugs, etc.), a nutritional agent (nutraceuticals, ursolic acid, tomatidine, etc.), exercise, or a combination thereof.

[0018] The present invention may be used in mammals, preferably human subjects, and even more preferably adult human subjects.

[0019] In various embodiments of the methods of the invention, the measurement of the expression level of the biomarkers may be determined at the level of nucleic acids, in particular RNA or cDNA, using well-known techniques such as, for example, real-time reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), digital PCR, RNAseq, microarrays, gene chips, nCounter gene expression assays, serial analysis of gene expression (SAGE), rapid analysis of gene expression (RAGE), nuclease protection assays, Northern blotting, or any other equivalent gene expression detection technique. In a preferred embodiment, the expression level of at least one biomarker is determined using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) or digital PCR.

[0020] In other embodiments of the methods of the present invention, measurement of the expression level of the biomarker may be determined at the protein level, for example using immunoassays, immunosorbent assays (ELISA), radioimmunoassays (RIA), mass spectrometry, Western blotting, flow cytometry, or any other equivalent protein detection technique.

[0021] The present invention may be used on blood samples, preferably selected from peripheral blood, plasma and serum samples. In a preferred embodiment, the sample is a peripheral blood sample.

[0022] In another aspect, the present invention relates to an oligonucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14 to 39. The oligonucleotides may be used as primers, in particular primer pairs, each pair comprising specific forward and reverse primers for amplifying one of the target biomarkers.

[0023] For carrying out the methods of the invention, the oligonucleotide primer pairs described above can be part of a device, such as, for example, a column, an array or a microarray; or a kit.

[0024] In a further aspect, the present invention relates to a kit comprising at least two oligonucleotide primers as described herein for amplifying at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2, and optionally reagents and / or instructions for performing the amplification reaction.

[0025] The present invention also relates to the use of the device or kit with a blood sample obtained from a subject for diagnosing muscle atrophy, in particular skeletal muscle atrophy; for assessing the effectiveness of a treatment to reverse muscle atrophy, in particular skeletal muscle atrophy; for assessing the onset or risk of onset of a disease in a subject and / or to assist in selecting, adapting or modifying a treatment in a subject suffering from a disease.

[0026] The present invention also relates to a method for treating muscle atrophy, comprising: (i) determining an expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in a blood sample obtained from the subject; wherein a decrease in the expression level of the at least one biomarker compared to a reference value is indicative of muscle atrophy, in particular skeletal muscle atrophy, in the subject; and (ii) treating the subject identified in step (i) as having muscle atrophy, particularly skeletal muscle atrophy, with drugs (pharmacological agents such as glucocorticoids, anti-inflammatory drugs, etc.), nutritional agents (nutraceuticals, ursolic acid, tomatidine, etc.), exercise, or a combination thereof. The present invention relates to a method for diagnosing and treating muscle atrophy, particularly skeletal muscle atrophy, in a subject, comprising: [Brief description of the drawings]

[0027] [Figure 1A] Figure 1A: sPLS-DA was performed using qRT-PCR data from both PROMETHE and PHD patients, including four outliers. Healthy volunteers and patients with muscle atrophy (hemodialysis or lung cancer) are located in the space defined by the two first components of the sPLS-DA model. Patients with catabolic states appear to be a distinct population from healthy volunteers. [Figure 1B] Figure 1B: Using the data used in Figure 1A, sPLS-DA was performed using qRT-PCR data from both PROMETHE and PHD patients but without the four outliers. [Figure 2A] Figure 2A: ROC analysis was performed using qRT-PCR data from both PROMETHE and PHD patients using a one-component model. ACTR6 and GIMAP2 best predicted the one-component model. Area under the ROC curve (AUC) = 0.9736. [Figure 2B]Figure 2B: ROC analysis was performed using qRT-PCR data from both PROMETHE and PHD patients using a two-component model. NIFK, RPL22L1, CCT2, GEMIN6 and RCN2 predicted the two-component model. Area under the ROC curve (AUC) = 0.9934. [Diagram 3] Figure 3: Biomarker levels determined by RT-qPCR for the different cohorts. *, significantly different from control (CT, n=13), p<0.05. CKD, chronic kidney disease, n=57; lung cancer, n=14; abdominal cancer (pancreatic or colon cancer), n=7. [Figure 4] Figure 4: Standard curves obtained by qRT-PCR for biomarkers and housekeeping genes. [Diagram 5] Figure 5: Absolute quantification of biomarkers by qRT-PCR with standard curves. The copy number per ng of RNA used for reverse transcription was calculated using the corresponding standard curve. Data are expressed as mean ± SEM of n=3–5. Significantly different from control (CTL), *p<0.05; **p<0.01. The same patients were used for all tests. CKD, chronic kidney disease. Absolute quantification makes it possible to identify catabolic patients by determining the number of RNA molecules of the biomarker per ng of total RNA. [Figure 6] Figure 6: Comparison of absolute quantification of biomarkers obtained by RT-qPCR and digital PCR (dPCR). Biomarker concentrations were calculated using digital PCR from Bio-Rad and compared to data obtained using standard curves for RT-qPCR absolute quantification. Cancer and CKD patients were pooled because no differences were observed between the groups in previous experiments ( [ka] ), healthy volunteers (CTL, [ka] ) compared with CTL. * p < 0.05; ** p<0.01, ***p<0.001. Digital PCR is effective in measuring biomarker levels, allowing for more accurate quantification than traditional absolute RT-qPCR analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description of the Invention Skeletal muscle atrophy occurs in response to a variety of conditions, including sepsis, glucocorticoid administration, fasting, muscle disuse, chronic kidney disease, diabetes, renal failure, cancer, HIV / AIDS, uremia, and many other chronic or systemic diseases.

[0029] Understanding the diversity of genes and proteins that influence muscle mass loss makes it difficult to identify and select relevant biomarkers that allow diagnosis in subjects with muscle atrophy, especially skeletal muscle atrophy. Identifying specific biomarkers that are directly linked to the atrophy program and independent of the disease or disorder itself is even more complex.

[0030] The present invention is based on the identification of specific and reliable biomarkers of muscle atrophy, preferably skeletal muscle atrophy. These markers are ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2, LYRM2 and / or variants thereof. These markers are detectable in blood samples of patients and can reliably predict muscle atrophy, regardless of the disease or disorder affecting said patient. These markers are specifically identified in blood samples and indirectly reflect the mechanisms present in muscle during the muscle atrophy program. The identified markers are independent of the markers identified in muscle biopsies of the prior art. First, there is no reason to find the same markers (blood markers vs. muscle biopsy), since the samples are different. In addition, the inventors have developed a new strategy. Indeed, to ensure the identification of markers completely independent of the pathology, patients with different diseases at different stages of progression (e.g. early diagnosis vs. late disease course [2 years]; pre-treatment vs. post-chemotherapy) were used.

[0031] In a first aspect, the present invention relates to the use of at least one gene or its gene product selected from ACTR6 (SEQ ID NO: 1), GIMAP2 (SEQ ID NO: 2), RCN2 (SEQ ID NO: 3), RPL22L1 (SEQ ID NO: 4), TRIAP1 (SEQ ID NO: 5), NIFK (SEQ ID NO: 6), APIP (SEQ ID NO: 7), GEMIN6 (SEQ ID NO: 8), RWDD1 (SEQ ID NO: 9), ZNF613 (SEQ ID NO: 10), BPNT1 (SEQ ID NO: 11), CCT2 (SEQ ID NO: 12) and LYRM2 (SEQ ID NO: 13) as a biomarker for the in vitro or ex vivo detection of muscle atrophy, in particular skeletal muscle atrophy, in a blood sample.

[0032] The present invention provides a method for determining a biomarker useful for diagnosing muscle atrophy, particularly skeletal muscle atrophy, by using the change in the expression level of at least one of the target genes listed above in a blood sample from a subject.The change in the expression level of at least one biomarker is generally obtained by comparing with a reference value.

[0033] definition The term "gene" refers to a DNA sequence (whose component segments are not necessarily physically contiguous) that specifies one or more sequence-related RNA / proteins. As intended in the present invention, a gene may be in the form of a sequence of only the protein-coding portion of the sequence of the respective gene (cDNA) or may include sequences (DNA) other than the protein-coding portion. In all humans, the variability of the genetic material leads to genetic polymorphisms. A "genetic polymorphism" is an individual difference in the base sequence of the DNA that constitutes a gene. In general, it is defined as occurring with a collective frequency of 1% or higher. Single nucleotide polymorphisms (SNPs), defined as one possible change per 1000 base pairs, are common in the human genome and can be located in different regions of a gene, for example in the promoter, coding or non-coding regions and also in intergenic regions.

[0034] The term "gene product" refers to what is transcribed or translated from the gene in question, such as mRNA or protein. Different isoforms or variants of the mRNA and the resulting protein isoforms or variants are contemplated within the term gene product. Fragments of the gene product are also contemplated, so long as they are functionally active.

[0035] As used herein, the term "biomarker" refers to a biological molecule whose presence and / or concentration can be detected and typically correlated with a condition of interest. A biomarker can be a polypeptide or a nucleic acid (e.g., mRNA) encoding it. In the context of the present invention, a biomarker can be used to classify a sample from a subject as a "muscle atrophy" sample, to assess the severity of muscle atrophy, to assess the effectiveness of treatment in an affected subject, and / or to assess the onset or risk of onset of a disease in a subject suffering from muscle atrophy.

[0036] The term "atrophy" defines a decrease in size of a tissue or organ due to cell shrinkage and / or a decrease in cell number. The decrease in cell size is caused by a loss of organelles, proteins and cytoplasm.

[0037] The expression "muscle atrophy" refers to the loss of muscle mass, including the loss of muscle fibers and / or the progressive weakening and / or degeneration of muscle. The loss of muscle mass and / or the progressive weakening and / or degeneration of muscle can occur, for example, due to an abnormally high protein degradation rate, an abnormally low protein synthesis rate, or a combination of both. An abnormally high muscle protein degradation rate can also occur due to muscle protein catabolism (i.e., the breakdown of muscle protein to use amino acids as substrates for gluconeogenesis). Muscle atrophy can also include a significant loss of muscle strength. "Significant loss of muscle strength" refers to the reduction in strength in diseased, damaged, or unused muscle tissue in a subject, relative to the same muscle tissue in a control subject.

[0038] Muscle atrophy can have a genetic, organic or systemic origin. The main categories of genetic disorders that primarily affect skeletal muscle tissue are muscular dystrophies and myopathies.

[0039] Muscle atrophy is a debilitating response not only to activity but also to many systemic diseases, such as hyperuremia, chronic obstructive pulmonary disease, diabetes, sepsis, obesity, AIDS, cancer and heart failure. In general, loss of muscle mass is accompanied by loss of muscle function and quality (i.e., the force generated by each volume unit of muscle tissue). As defined herein, "skeletal muscle atrophy" refers to the loss of muscle mass caused by excessive protein degradation and / or the progressive weakening and degeneration of skeletal muscle.

[0040] The term "skeletal muscle atrophy" refers to the loss of skeletal muscle mass and / or the progressive weakening and / or degeneration of skeletal muscle.

[0041] Muscles are soft tissues found in most animals that contain muscle cells. Muscle cells contain protein filaments that slide past each other, resulting in contractions that change both the length and shape of the muscle cells. Muscles function to produce force and movement. There are three types of muscles in the body: a) skeletal muscles (muscles involved in moving the limbs and external areas of the body); b) cardiac muscles (muscles of the heart); and c) smooth muscles (muscles found in the walls of arteries and intestines).

[0042] Skeletal muscles, or voluntary muscles, are generally anchored to bones by tendons and are generally used to effect skeletal movement, such as walking, or to maintain posture. Although some control of skeletal muscles is generally maintained as an involuntary reflex (e.g., postural muscles or the diaphragm), skeletal muscles are responsive to conscious control. Skeletal muscles are striated and contain sarcomeres packed into highly regular bundle arrangements.

[0043] Skeletal muscles are further divided into two major types: type I (or "slow-twitch") and type II (or "fast-twitch"). Type I muscle fibers are densely packed with capillaries and rich in mitochondria and myoglobin, which give type I muscle tissue its characteristic red color. Type I muscle fibers can carry more oxygen and sustain aerobic activity using fat or carbohydrates for fuel. Type I muscle fibers contract for long periods of time but exert little force. In large mammals, including humans, type II muscle fibers can be subdivided into two main subtypes (IIa and IIx) that differ in both the speed of contraction and the force they produce. Type II muscle fibers contract quickly and powerfully, but fatigue very quickly and therefore produce only brief anaerobic bursts of activity before muscle contractions become painful.

[0044] The term "subject" or "patient" refers to an individual who requires detection and / or evaluation of muscle atrophy, particularly skeletal muscle atrophy. The subject is at risk of or suffers from (skeletal) muscle atrophy. The subject may be asymptomatic or suffer from symptoms unrelated to (skeletal) muscle atrophy. Typically, the subject may have one of the systemic diseases mentioned above. The term "subject" or "patient" includes, but is not limited to, a mammal, either a human or a non-human mammal. Preferably, the subject is an adult.

[0045] The term "biological sample" includes any biological sample from a subject that can be used for diagnostic, prognostic or monitoring purposes. The biological sample may be a body fluid sample. A typical example of a biological fluid sample that can be used in the context of the present invention is a blood sample. Optionally, the biological sample is a plasma, peripheral blood or serum sample. In the method of the present invention, the sample may be used pure or diluted. Dilution of the sample may be useful to remove inhibitors that may interfere with the measurement of the expression level of the biomarker of interest. Preconcentration of the sample may be performed to concentrate the biomarkers. The sample may be processed before use and / or frozen or lyophilized and / or used immediately.

[0046] The expression "determining the expression level" of a biomarker in a sample, control or reference material as described herein refers to the detection and quantification of the presence of said biomarker in the sample to be tested. For example, the concentration of a biomarker in said sample can be directly quantified by measuring the amount of protein / polypeptide present in the sample to be tested. However, it is also possible to indirectly quantify the amount of a biomarker by evaluating the genetic expression of the coding gene of the biomarker, for example by quantification of the expressed mRNA encoding the respective biomarker. The present invention is not limited to any particular method for determining the level of a given biomarker, but is intended to encompass all means that allow, directly or indirectly, to quantify or estimate the expression level of said biomarker. "Level" in the context of the present invention is therefore a parameter that describes the absolute amount of a biomarker in a given sample, for example as an absolute weight, volume or molar amount; or alternatively "level" refers to a relative amount, for example and preferably the concentration of said biomarker in a test sample, for example mol / l, g / l, g / mol, etc. When analyzing gene expression using qRT-PCR, the "expression level" of a target gene is expressed as a ΔΔCt value, as number of mRNA molecules per μl, or as copy number per ng of RNA. When analyzing gene expression using digital PCR, the "expression level" of a target gene is expressed as copies of target per μl, copies of target per droplet (CPD), or copies per ng of RNA.

[0047] To quantify the difference in the expression level of a biomarker, an expression ratio can be calculated. This differential expression refers to the quantitative and qualitative difference in the expression level of a biomarker between a subject with muscle atrophy and a reference value. The term "reduction" in the expression level of a biomarker refers to an expression value for at least one biomarker in a blood sample of a subject being tested that is lower than the reference value for the same biomarker. Typically, a reduction in the expression level of a biomarker can correspond to a decrease of 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of the expression level of the reference value for the same biomarker. Alternatively, a reduction in the expression level of a biomarker can correspond to a 2-10-fold decrease compared to the reference value.

[0048] The term "reference value" may refer to a basal value corresponding to the median or mean of the values ​​(measured levels, amounts or concentrations) obtained using blood sample(s) of a reference subject or population, usually healthy subjects or a population or cohort of healthy subjects, i.e. subjects not suffering from muscle atrophy, in particular skeletal muscle atrophy. The reference value may be a statistical value / threshold or a discrimination value / threshold, i.e. a value determined by measuring the parameter both in a healthy control population and in a population suffering from (skeletal) muscle atrophy and / or any other disease, disorder or dysfunction state having skeletal muscle atrophy as a secondary consequence. The threshold between "normal" and "atrophy" is defined as the border between healthy subjects not suffering from muscle atrophy, in particular skeletal muscle atrophy, and disease patients experiencing muscle atrophy, in particular skeletal muscle atrophy. The discrimination value identifies the diseased population with a specificity and / or sensitivity, both of which are pre-determined by a skilled person, based on an analysis of the relationship between the value of the parameter and known clinical data of healthy control subjects / populations and of diseased patient subjects / populations. The discrimination value determined in this way is valid for the same experimental setting in future individual tests. The reference value can also be a value measured in samples from control subjects. The "reference value" can also be replaced by a calibration curve with known amounts (μmoles or copies) of the target biomarker(s).

[0049] In a particular embodiment, the reference value is a threshold amount of the marker in the sample that allows to distinguish between subjects with muscle atrophy and subjects without muscle atrophy.

[0050] The terms "diagnosis" and "diagnosing" refer to the detection or identification of a medical condition, disease, disorder or dysfunctional state, usually the presence, stage, severity, progression, etc. of (skeletal) muscle atrophy, or the assessment / determination (prescribing, comparing) of the severity or stage of such muscle atrophy in a subject. Diagnosis may allow early detection of muscle atrophy, i.e. before the appearance of clinical signs, or intervention to confirm clinical findings by a medical practitioner.

[0051] The term "about" as used herein in connection with any and all values ​​(including the lower and upper limits of a numerical range) means any value with a tolerance of deviation of up to ±10% (e.g., ±0.5%, ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±7.5%, ±8%, ±8.5%, ±9%, ±9.5%). Use of the term "about" at the beginning of a value string modifies the respective value (i.e., "about 1, 2 and 3" refers to about 1, about 2 and about 3). Additionally, when a list of values ​​is provided herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%), the list includes all intermediate and fractional values ​​therein (e.g., 54%, 85.4%).

[0052] Biomarkers The present invention discloses the identification of biomarkers that allow efficient diagnosis of muscle atrophy, in particular skeletal muscle atrophy. These biomarkers were selected from a set of about 1,500 mRNAs identified by RNAseq approaches in patients with lung cancer or renal failure. A second round of selection based on criteria such as high amplitude of variation between healthy and diseased subjects, good reproducibility from one subject to another, resulted in the identification of a subset of 30 mRNAs. Among this subset, 12 mRNAs are sufficient, alone and / or in combination(s), to provide a strong and specific diagnostic result of muscle atrophy, in particular skeletal muscle atrophy.

[0053] The sequence information for these biomarkers is publicly available and can be queried in the known NCBI Entrez gene or protein databases. The identity and nucleic acid and protein sequences for each of these biomarkers are provided below and in Table A.

[0054] Table A: List of biomarkers [Table 1]

[0055] In the context of the present invention, the expression "ACTR6 gene" refers preferably to a nucleic acid molecule or sequence comprising the human ACTR6 gene, in particular (i) the sequence of gene #64431 or a sequence complementary thereto; (ii) a natural variant of the sequence of (i), such as a polymorphism; or (iii) a sequence having at least 90% identity, preferably at least 95, 96, 97, 98 or 99% identity, with the sequence of (i) or (ii). In a particular embodiment, the method comprises determining the presence, absence or amount of at least one sequence encoded by an exon of the ACTR6 gene.

[0056] The same definition applies to any other genes cited in this application by reference to the corresponding reference sequence in Table A. If the subject is a mammal other than a human, the corresponding ortholog is used.

[0057] Preferred biomarkers for use in the present invention are ACTR6, GIMAP2 and RCN2 or any combination thereof. As disclosed in the Examples, measuring the expression level of one of these biomarkers is sufficient to provide a reliable diagnosis of muscle atrophy, particularly skeletal muscle atrophy.

[0058] With respect to ACTR6, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 1 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number Q9GZN1.

[0059] With respect to GIMAP2, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 2 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number Q9UG22.

[0060] With respect to RCN2, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 3 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number Q14257.

[0061] The present invention shows that the above genes or their alternative isoforms are deregulated in subjects with muscle atrophy, particularly skeletal muscle atrophy, such that any sequence of the expression products encoded by these genes can be measured and correlated with muscle atrophy.

[0062] It is believed that some combination of the aforementioned biomarkers will show very high sensitivity and specificity for diagnosing muscle atrophy, especially skeletal muscle atrophy.

[0063] In a preferred embodiment, the invention comprises measuring the expression level of a combination of at least two biomarkers selected from ACTR6, GIMAP2 and RCN2.

[0064] In a more preferred embodiment, the present invention includes measuring the expression levels of two biomarkers, ACTR6 and GIMAP2. The results shown in the experimental section demonstrate that by measuring the expression of ACTR6 and GIMAP2, the area under the ROC curve is about 0.9736. The prediction accuracy of "skeletal muscle atrophy" is 95%.

[0065] In another preferred embodiment, the invention comprises measuring the expression levels of two biomarkers, ACTR6 and RCN2.

[0066] In another preferred embodiment, the invention involves measuring the expression levels of two biomarkers, GIMAP2 and RCN2.

[0067] In another preferred embodiment, the invention involves measuring the expression levels of a combination of three biomarkers: ACTR6, GIMAP2 and RCN2.

[0068] Furthermore, although the above three biomarkers provide a reliable test, the method can be further improved by combining the above markers with further biomarkers identified by the inventors. Valid (secondary) diagnostic biomarkers suitable for use in the present invention are selected from RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2, in addition to ACTR6, GIMAP2 and RCN2.

[0069] In one embodiment of the invention, the number of biomarkers includes any one of the following numbers of biomarkers: 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 13. Generally, the optimal panel size for the biomarkers of the present invention is 1-5, preferably 3-5.

[0070] With respect to RPL22L1, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 4 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number Q6P5R6.

[0071] With respect to TRIAP1, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 5 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number O43715.

[0072] With respect to NIFK, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 6 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number Q9BYG3.

[0073] With respect to APIP, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 7 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number Q96GX9.

[0074] With respect to GEMIN6, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 8 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by accession number Q8WXD5.

[0075] With respect to RWDD1, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 9 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number Q9H446.

[0076] With respect to ZNF613, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 10 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number Q6PF04.

[0077] With respect to BPNT1, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 11 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number O95861.

[0078] With respect to CCT2, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 12 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number P78371.

[0079] With respect to LYRM2, in a particular embodiment, the method preferably comprises measuring a target sequence consisting of or contained in SEQ ID NO: 13 or its complement. In another particular embodiment, the method preferably comprises measuring the protein identified by Accession Number Q9NU23.

[0080] Methods for determining the expression level of a biomarker According to a preferred embodiment, determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 may be performed at the nucleic acid and / or protein level.

[0081] If an mRNA is selected as the (or one of) gene products whose levels are determined, this may be the sum of all mRNA isoforms of the gene(s) under study, or one or more specific mRNAs. If a protein is selected as the (or one of) gene products whose levels are determined, the same considerations apply: total protein levels may be determined, or protein levels of only specific isoforms may be determined (e.g., using antibodies against different C-termini). Most particularly, all protein isoforms may be detected (e.g., using antibodies against a common epitope). Of note, it is also contemplated that both mRNA and protein are determined. In this case, the isoforms detected may be all isoforms, identical isoforms (completely overlapping), or different isoforms (partially overlapping or not overlapping), both of the mRNA and the protein, depending on the experimental setup. By identical isoforms, it is meant that the mRNA isoforms code for the corresponding protein isoforms. However, it is known that for some genes, the number of protein isoforms detected is generally less than the number of possible mRNA transcripts (and thus the number of protein isoforms). If the target gene exists in several isoforms, one can choose to amplify the majority, some or all isoforms of this gene.

[0082] According to a preferred embodiment, the method comprises determining, preferably by selective amplification, the expression level of RNA encoded by a target gene as defined above.

[0083] Measurement by selective amplification Selective amplification of a target gene is preferably performed using a primer, a primer pair, or multiple primer pairs to amplify all or a portion of the target gene(s), i.e., the target nucleic acid(s), in a sample from a subject.

[0084] The term "primer" refers to a nucleic acid molecule that can base-pair with a complementary template and serve to initiate amplification of the template in the presence of reagents (polymerase or reverse transcriptase) and four different nucleoside triphosphates (NTPs) at a suitable buffer solution and temperature.

[0085] The primer pairs of the present invention are carefully designed to maximize the sensitivity and specificity of the detection of the target gene. The primers are usually single-stranded nucleic acids comprising 10-30 bases, preferably 15-30 bases, more preferably 15-25 bases in length. The primers can perfectly match the target sequence in the target gene or can have one or more mismatches with the target sequence, provided that the mismatch(es) do not impair the specific amplification of the target sequence, in cases where several isoforms of the target gene are present. The primers of the present invention can incorporate additional features / modifications known in the art that do not alter their basic properties. Examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more nucleotides, and uncharged bonds such as phosphonates, phosphotriesters, phosphoramidates or carbamates, phosphorothioates or phosphorodithioates. The nucleic acid sequences of the primers used in the present invention may also be modified with a label capable of providing a detectable signal directly or indirectly.

[0086] As used herein, the terms "amplification" and "amplifying" encompass all methods for copying or reproducing a target nucleic acid sequence, thereby increasing the copy number or amount of the nucleic acid sequence in a sample. Amplification can be exponential or linear, and the target nucleic acid can be DNA, cDNA, or RNA. The sequence thus amplified is referred to herein as an "amplicon."

[0087] The method of the invention may comprise amplifying at least one target sequence from at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in a blood sample obtained from a subject. In certain embodiments, the target sequence to be amplified (i.e., the amplicon) is 70-250 nt in length, preferably 80-230 nt, more preferably 90-210 nt in length. Specific examples of primers suitable for carrying out the amplification(s) of the target biomarker are provided in Table B below. These primers are designed to bind the target sequences of SEQ ID NOs: 1-13. Additional primers can be designed to bind and amplify one or more of the target sequences of SEQ ID NOs: 1-13.

[0088] Table B: List of primers for amplifying target sequences [Table 2]

[0089] In preferred embodiments, the expression level of the target biomarker is determined at the nucleic acid level, e.g., by detecting and / or quantifying nucleic acid, such as RNA or cDNA, encoding the target gene in a blood sample of the subject. In certain embodiments, the method comprises extracting RNA from the blood sample and measuring the nucleic acid level of the marker.

[0090] In this particular embodiment, determining the expression level of the at least one biomarker comprises performing real-time reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), digital PCR, RNAseq, microarray, gene chip, nCounter gene expression assay, serial analysis of gene expression (SAGE), rapid analysis of gene expression (RAGE), nuclease protection assay, northern blotting, or any other equivalent gene expression detection technique. In a preferred embodiment, determining the expression level of the at least one biomarker comprises performing quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) or digital PCR.

[0091] In some embodiments, sequences from two or more genes of interest are amplified in the same reaction vessel. In this case, the amplicon(s) can be detected by first size-separating the amplicons and then detecting the size-separated amplicons. Separation of amplicons of different sizes can be achieved, for example, by gel electrophoresis, column chromatography, denaturing HPLC, capillary electrophoresis, hybridization with a probe, or sequencing. These and other separation methods are well known in the art. In one example, amplicons of about 10 to about 150 base pairs that differ in size by 10 or more base pairs can be separated, for example, on a 4% to 5% agarose gel (2% to 3% agarose gel for amplicons of about 150 to about 300 base pairs) or a 6% to 10% polyacrylamide gel. The separated nucleic acids can then be stained with a dye such as ethidium bromide, and the size of the resulting stained band or bands can be compared to a standard DNA ladder.

[0092] Alternatively, the amplicon may be detected by hybridization with a specific probe. As used herein, the term "probe" refers to a nucleic acid molecule that can specifically bind to a target sequence and is complementary to a portion of the amplified target sequence (i.e., amplicon) consisting of several to several tens of bases, and may be labeled to identify the presence of the specifically amplified target sequence.

[0093] Useful labels include, for example, fluorescent dyes (e.g., Cy5, Cy3, FITC, rhodamine, lanthamide phosphor, Texas Red), 32P, 35S, 3H, 14C, 125I, 131I, electron-dense reagents (e.g., gold), enzymes, such as those commonly used in ELISAs (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g., colloidal gold), magnetic labels (e.g., Dynabcads™), biotin, dioxygenin, or haptens and proteins for which antisera or monoclonal antibodies are available. Other labels include ligands or oligonucleotides capable of forming complexes with the corresponding oligonucleotides. Labels can be directly incorporated into the nucleic acid to be detected or can be attached to a probe (e.g., an oligonucleotide) or antibody that hybridizes or binds to the nucleic acid to be detected.

[0094] Real-time quantitative RT-PCR (qRT-PCR) measures the accumulation of PCR products via dual-labeled fluorogenic probes. The probes employed in qRT-PCR are based on the principle of fluorescence quenching and contain a donor fluorescent dye (also called a reporter dye) at the 5' end and a quenching moiety (also called a quencher dye) at the 3' end. As used herein, the term "quencher moiety" refers to a molecule in close proximity to a donor fluorescent dye that captures the luminescence energy generated by the donor and dissipates the energy as heat or emits light at a wavelength longer than the emission wavelength of the donor. Fluorophores can be, but are not limited to, FAM, TAMRA, VIC, JOE, TET, HEX, ROX, RED610, RED670, NED, Cy3, Cy5, and Texas Red. Quenchers can be, but are not limited to, 6-TAMRA, BHQ-1,2,3, and MGB-NFQ. The fluorescent dye-quencher pair can be selected so that the excitation spectrum of the quencher overlaps with the emission spectrum of the fluorescent dye. Examples are pairs such as FAM-TAMRA, FAM-MGB, VIC-MGB, etc. The skilled artisan will know how to recognize other suitable pairs.

[0095] The probe may be prepared in the form of single-stranded DNA, double-stranded DNA, RNA, or hybrid DNA-RNA. The typical length of the probe is 10 to 60 nt, preferably 15 to 55 nt, more preferably 20 to 50 nt, more preferably 30 to 45 nt, and even more preferably 10 to 30 nt.

[0096] A variety of probe formats can be used to perform real-time RT-PCR, including TaqMan™, Molecular Beacon™, Scorpion™, and LUX™ probes.

[0097] Another object of the invention therefore relates to nucleic acids having any one of the nucleotide sequences set out in Table B, compositions comprising them, as well as the use of these nucleic acids for diagnosing muscle atrophy, in particular skeletal muscle atrophy.

[0098] In particular, the present invention relates to an oligonucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14-39.

[0099] Oligonucleotides can be used as primers, particularly primer pairs, for amplifying one or more specific amplicons of the biomarkers identified above. Typically, a primer pair consists of a forward primer and a reverse primer, both of which hybridize to a target sequence. In the context of the present invention, an oligonucleotide primer pair comprises a forward oligonucleotide primer and a reverse oligonucleotide primer capable of binding and amplifying at least one target sequence of SEQ ID NO: 1-13, where the oligonucleotides have the nucleic acid sequences of SEQ ID NO: 14-39.

[0100] In a particular embodiment, the present invention relates to a set of oligonucleotide primer pairs specific for at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2. As used herein, the term "specific" means that the primers bind only to one of the biomarkers of the present invention and have negligible binding to other biomarkers of the present invention or other analytes in the blood sample being analyzed. This ensures that the integrity of the diagnostic assay using the biomarkers of the present invention and its results are not compromised by additional binding events. Examples of oligonucleotide primer pairs suitable for amplifying each of the target biomarkers are listed in Table B. As an example, a set of oligonucleotide primers comprising a forward primer of SEQ ID NO: 14 and a reverse primer of SEQ ID NO: 15 allows the amplification of ACTR6.

[0101] In some embodiments of the methods of the invention described herein, the probes can be used to detect at least one of the biomarkers mentioned above or to quantify the obtained amplicons.

[0102] The amplified nucleic acid for each of the target sequences may be detected simultaneously (i.e., in the same reaction vessel) or individually (i.e., in separate reaction vessels). In some embodiments, the amplified DNA is detected simultaneously in a multiplex format using distinguishably labeled gene-specific oligonucleotide probes, each hybridizing to a different target sequence. If the assays are performed in parallel in separate reaction vessels, the gene-specific oligonucleotide probes may have the same label. To minimize interactions between the biomarker(s) and the reaction vessel (tubes, microplates, etc.), it is preferable to use vessels (tubes, microplates, etc.) that are free of any surface coating or have low binding properties. In a preferred embodiment, the vessel is a low-binding / low-retention plastic vessel or tube, such as an anti-adherent pipette tip, a polypropylene tube, such as a PCR tube or a 1.5-2 ml tube. To avoid amplification bias, it may be desirable to block non-specific binding of the target biomarker during the amplification reaction. Therefore, in a preferred embodiment, the amplification is performed in the presence of a carrier, preferably an RNA carrier, such as yeast tRNA and MS2 RNA, or a DNA carrier, such as salmon sperm DNA.

[0103] To correct (normalize) for both differences in the amount of RNA assayed and variability in the quality of the RNA used, the assay may optionally incorporate analysis of the expression of certain reference genes (or "normalization genes"), including well-known housekeeping genes such as GAPDH (Entrez Gene ID#P04406, SEQ ID NO:40), 36B4 / RPLP0 (Entrez Gene ID#P05388, SEQ ID NO:41), ACTB (Entrez Gene ID#p60709, SEQ ID NO:42). Specific examples of primers suitable for amplifying(s) the reference genes are provided in Table C below. These primers are designed to bind the target sequences of SEQ ID NOs:40-42. Additional primers can be designed to bind and amplify one or more of the target sequences of SEQ ID NOs:40-42.

[0104] Table C: List of primers for amplification of reference genes [Table 3]

[0105] Alternatively, absolute quantification of target biomarkers may be obtained by calibration standards (calibration curves) or digital PCR (dPCR). The amount of RNA or DNA may be determined by comparing the results with a calibration curve made by real-time PCR of serial dilutions of known amounts of RNA or DNA. The absolute or relative copy number of target molecules in a polymerase chain reaction (PCR) amplification may be determined by comparing the cycle threshold (Ct) value with a calibration curve, with the cycle threshold (Ct) value of a reference nucleic acid, or with an absolute quantification standard nucleic acid. In the case of absolute quantification, the range of expression of the same at least one biomarker is expressed as the number of molecules present in a blood sample.

[0106] Measurement of polypeptides According to another preferred embodiment, the method comprises determining the expression level of a polypeptide encoded by a target gene as defined above.

[0107] In this particular embodiment, the expression level of the target biomarker is determined at the protein level in the subject's blood sample. In other words, the expression level of the target biomarker is determined by detecting the polypeptides of ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in the subject's blood sample.

[0108] Measuring or assaying a polypeptide in a sample can be performed by any known technique, most notably by a specific ligand that binds a polypeptide encoded by a target gene, such as an antibody, antibody fragment, or derivative thereof. As used herein, the term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments and F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The term "epitope" refers to the site on an antigen to which an antibody binds. An epitope can be formed from contiguous or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. As used herein, the term "antibody" also refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds an antigen. The immunoglobulin molecules of the present disclosure can be of any class (e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.

[0109] Non-limiting examples of antibody fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked using recombinant methods by synthetic or natural linkers that allow them to be produced as a single protein chain (known as single-chain Fv (scFv)) in which the VL and VH domains pair to form a monovalent molecule. The VH and VL sequences of specific single chain antibodies can be linked to human immunoglobulin constant region cDNA or genomic sequences to generate expression vectors encoding complete IgG molecules or other isotypes. The VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies, are also encompassed.

[0110] The "F(ab')2" and "Fab'" portions include antibody fragments that can be generated by treating immunoglobulins (monoclonal antibodies) with proteases such as pepsin and papain, and are generated by digesting the immunoglobulins near the disulfide bond present between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond present between the hinge regions in each of the two H chains to generate two homologous antibody fragments in which the L chain composed of VL (L chain variable region) and CL (L chain constant region) and the H chain fragment composed of VH (H chain variable region) and CHγ1 (γ1 region in the constant region of the H chain) are linked at their C-terminal regions via a disulfide bond. Each of these two homologous antibody fragments is called Fab'. Pepsin also cleaves IgG downstream of the disulfide bond present between the hinge regions in each of the two H chains to generate antibody fragments that are slightly larger than the fragment in which the two Fab's are linked at the hinge regions described above. This antibody fragment is called F(ab')2.

[0111] Fab fragments also contain the constant domain of the light chain; the first constant domain (CH1) of the heavy chain Fab' fragment differs from that of the Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteine(s) from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0112] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, these six hypervariable regions confer antigen binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only the three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0113] "Single-chain Fv" or "sFv" antibody fragments comprise the VH domain, the VL domain, or both the VH and VL domains of an antibody, where both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.

[0114] Preferably, the ligand is a specific antibody, antibody fragment or derivative thereof that binds to an epitope of an ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 or LYRM2 polypeptide.

[0115] Commercially available antibodies include, for example, mouse monoclonal antibodies sc-514988 (anti-ACTR6 antibody), ABIN525619 (anti-GIMAP2 antibody), SAB1404305 (anti-RCN2 antibody), ABIN2586231 (anti-NIFK antibody), sc-376666 (anti-APIP antibody), sc-514919 (anti-GEMIN6 antibody), sc-514496 (anti-RWDD1 antibody), sc-393185 (anti-BPNT1 antibody), and sc-374152 (anti-CCT2 antibody); and rabbit polyclonal antibodies HPA038587 (anti-ACTR6 antibody), HPA013 589 (anti-GIMAP2 antibody), AB104516 (anti-RCN2 antibody), HPA056207 (anti-RPL22L1 antibody), HPA043640 (anti-TRIAP1 antibody), HPA021188 (anti-APIP antibody), HPA035727 (anti-GEMIN6 antibody), HPA028712 (anti-RWDD1 antibody), HPA026833 (anti-ZNF613 antibody), HPA048461 (anti-BPNT1 antibody), HPA003198 (anti-CCT2 antibody) and HPA063932 (anti-LYRM2 antibody).

[0116] Other specific antibodies of the target polypeptide can be produced by conventional techniques, in particular by immunization of a non-human mammal with an immunogen comprising part or all of the target polypeptide, and recovery of antibody (polyclonal) or monoclonal antibody producing cells. The immunogen can be produced synthetically or by expression in a suitable host of a nucleic acid target as previously defined. In the general monoclonal antibody method, the synthetic antibody genes can be expressed and purified by inserting them into a viral-based or non-viral expression vector for antibody expression. Both prokaryotic cells, such as E. coli, Bacillus subtilis, Salmonella, Serratia or Pseudomonas, and eukaryotic cells, such as HEK293, CHO or CHO-DG44, are suitable for producing antibodies. Other microorganisms, such as yeast, can be used to express the antibodies of the invention, and insect cells in combination with baculovirus vectors may also be used. Such monoclonal or polyclonal antibodies, as well as their fragments or derivatives having the same antigen specificity, constitute further objects of the present invention, as well as their use for diagnosing muscle atrophy, in particular skeletal muscle atrophy.

[0117] In this particular aspect of the invention, determining the expression level of at least one biomarker comprises performing an immunoassay, an immunosorbent assay (ELISA), a radioimmunoassay (RIA), mass spectrometry, Western blotting, flow cytometry, or any other equivalent protein detection technique.

[0118] method The expression level of the biomarkers described herein is highly correlated with the muscle atrophy program that occurs in affected subjects. Provided herein is an efficient method for assessing muscle atrophy status in subjects. The present invention can be used, for example, for one or more of: (i) diagnosing, prognosing and / or evaluating subjects with muscle atrophy, particularly skeletal muscle atrophy; (ii) evaluating the effectiveness of treatments for reversing (skeletal) muscle atrophy; (iii) evaluating the onset or risk of onset of disease in patients diagnosed with (skeletal) muscle atrophy; and / or (iv) assisting in selecting, adapting or changing treatments in affected subjects.

[0119] In another aspect, the present invention relates to an in vitro or ex vivo method for diagnosing muscle atrophy, in particular skeletal muscle atrophy, in a subject, comprising determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in a blood sample obtained from the subject; wherein a decrease in the expression level of said at least one biomarker compared to a reference value indicates muscle atrophy, in particular skeletal muscle atrophy, in the subject. Optionally, the blood sample is selected from the group consisting of peripheral blood, plasma and serum samples.

[0120] In certain embodiments, the subject is a mammal, preferably a human subject, and even more preferably an adult human subject, such as, but not limited to, 18 years of age or older.

[0121] The subject may be tested regardless of gender. The subject may be apparently healthy or may suffer from any disease or disorder, such as hyperuremia, sepsis, stroke, spinal cord injury, cancer, diabetes, obesity, neuromuscular disease, chronic inflammatory disease, chronic kidney disease, heart failure (such as congestive heart failure or congestive cardiac failure), lung disease (such as chronic obstructive pulmonary disease), and acute critical illness chronic renal failure, or any other disease. In a particular embodiment, the subject suffers from cancer, particularly lung cancer or abdominal cancer, such as small intestine cancer, colon cancer, pancreatic cancer or gastric cancer. In another particular embodiment, the subject suffers from a chronic disease, particularly chronic renal failure.

[0122] In particular aspects, the blood sample is from peripheral blood, plasma and serum, in a more particular aspect, the blood sample is peripheral blood.

[0123] The expression levels of the biomarkers identified by the present inventors may be decreased in subjects suffering from muscle atrophy, in particular skeletal muscle atrophy, compared to healthy subjects, which may correspond to a 2- to 10-fold decrease compared to reference values ​​in healthy subjects.

[0124] Alternatively, the method may comprise determining the absolute amount of marker in sample, for example the copy number per amount of RNA extracted from the sample, such as the copy number per ng of RNA.Based on the analysis of a healthy population and a population of subjects with muscle atrophy (particularly whatever the disease), it is possible to determine the threshold value for distinguishing healthy subjects from subjects with muscle atrophy for one or several markers of the present invention.The threshold value may be different for each marker.

[0125] For example, and more precisely, when the amplification is carried out by qRT-PCR, the threshold value is: - for ACTR6, the threshold may be that subjects with less than about 12 copies per ng of RNA are likely to have muscle atrophy while subjects with more than about 12 copies per ng of RNA are likely to not have muscle atrophy; and / or -For RCN2, the threshold may be that subjects with less than about 10 copies per ng of RNA are likely to have muscle atrophy, while subjects with more than about 10 copies per ng of RNA are likely to not have muscle atrophy; and / or For GIMAP2, the threshold may be that subjects with less than about 205-220 copies per ng of RNA may have muscle atrophy, while subjects with more than about 205-220 copies per ng of RNA may not have muscle atrophy (e.g., the threshold may be 205, 210, 215, or 220 copies per ng of RNA, or any range defined by these values). It could be.

[0126] Threshold values ​​can also be determined for other markers of the invention.

[0127] The absolute amounts or thresholds of several markers may be combined to improve the performance of the discrimination between subjects with and without muscle atrophy. For example, the combination of absolute amounts or thresholds may lead to the calculation of a score that allows to discriminate between subjects with and without muscle atrophy.

[0128] For example, and more precisely, when the amplification is performed by digital PCR, the threshold value is: - for ACTR6, the threshold may be that subjects having less than about 12-15 copies per ng of RNA are likely to have muscle atrophy while subjects having more than about 12-15 copies per ng of RNA are likely to not have muscle atrophy (e.g., the threshold may be 12, 13, 14, or 15 copies per ng of RNA, or any range defined by these values); and / or -For RCN2, the threshold may be that subjects having less than about 40-70 copies per ng of RNA are likely to have muscle atrophy, while subjects having about 40-70 copies per ng of RNA are likely to not have muscle atrophy (e.g., the threshold may be 40, 45, 50, 55, 60, 65, or 70 copies per ng of RNA, or any range defined by these values); and / or For GIMAP2, the threshold may be that subjects with less than about 220-280 copies per ng of RNA have muscle atrophy, while subjects with more than about 220-280 copies per ng of RNA may not have muscle atrophy (e.g., the threshold may be 220, 230, 240, 250, 260, 270, or 280 copies per ng of RNA, or any range defined by these values). It could be.

[0129] Threshold values ​​can also be determined for other markers of the invention.

[0130] The absolute amounts or thresholds of several markers may be combined to improve the performance of the discrimination between subjects with and without muscle atrophy. For example, the combination of absolute amounts or thresholds may lead to the calculation of a score that allows to discriminate between subjects with and without muscle atrophy.

[0131] The expression levels of the individual biomarkers (e.g., the three biomarkers ACTR6, GIMAP2 and RCN2) can be combined to obtain a score, e.g., a single value that can be called an "atrophy score". The expression levels obtained with the different cohorts (i.e., absolute RT-qPCR or dPCR) can be analyzed by partial least squares discriminant analysis (PLS-DA, Bereton and Lloyd, Chemometrics, 2014; 28, 213-225). This analysis allows prediction of the absolute data of the individual on the covariance space of the presence / absence of the muscle atrophy factors for all the retained quantification measures of the biomarkers. As in standard PCA, the principal components (by PLS-DA) based on the maximum covariance can be obtained, and only the first components can be retained to create an "atrophy score". The threshold for atrophy detection can then be easily estimated and used to distinguish between normal (no atrophy), intermediate (atrophy / recovery process not stabilized) and atrophy status. A macro containing this analysis can be created in an Excel file (or any other software) to provide a detection kit for use in medical laboratories. The absolute values ​​of the biomarkers (copy numbers / μg of RNA) obtained by dPCR or absolute RT-qPCR can be stored in the Excel file at the indicated locations to calculate the "atrophy score" of any future patient. The atrophy score is automatically displayed, for example with a color-coded highlight (green=no atrophy; orange=moderate [no atrophy or recovery stable], red=atrophy).

[0132] In certain embodiments, an in vitro or ex vivo method for diagnosing muscle atrophy, in particular skeletal muscle atrophy, in a subject comprises determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, or a combination thereof in a blood sample obtained from the subject; wherein a decrease in the expression level of the at least one biomarker compared to a reference value is indicative of muscle atrophy in the subject.

[0133] The combination of ACTR6, GIMAP2 and RCN2 biomarkers contemplated by the present invention may be a combination of two biomarkers: ACTR6 and GIMAP2, ACTR6 and RCN2, GIMAP2 and RCN2, or the three biomarkers ACTR6, GIMAP2 and RCN2.

[0134] Although the ACTR6, GIMAP2 and RCN2 biomarkers provide a reliable test, the method can be further improved by combining said biomarkers with further biomarkers identified by the inventors.Suitable (secondary) diagnostic biomarkers suitable for use in the present invention are selected from RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in addition to ACTR6, GIMAP2 and RCN2.

[0135] In another particular embodiment, the in vitro or ex vivo method for diagnosing muscle atrophy, in particular skeletal muscle atrophy, in a subject further comprises determining the expression level of at least one further biomarker selected from the group consisting of RPL22L1 (SEQ ID NO: 4), TRIAP1 (SEQ ID NO: 5), NIFK (SEQ ID NO: 6), APIP (SEQ ID NO: 7), GEMIN6 (SEQ ID NO: 8), RWDD1 (SEQ ID NO: 9), ZNF613 (SEQ ID NO: 10), BPNT1 (SEQ ID NO: 11), CCT2 (SEQ ID NO: 12) and LYRM2 (SEQ ID NO: 13).

[0136] The accuracy of a method aimed at diagnosing an event is best described by its receiver operating characteristics (ROC). Each point on the ROC plot represents a sensitivity / specificity pair corresponding to a certain decision threshold. Depending on the desired confidence interval, a threshold can be derived from the ROC curve, allowing a diagnosis or prediction for a given event, respectively, with a suitable balance of sensitivity and specificity. Thus, the reference used in the method of the present invention, i.e. the threshold that allows distinguishing subjects with and without muscle atrophy, can preferably be generated by establishing a ROC analysis for the cohort as described above and deriving one or more threshold amounts or concentrations therefrom. Depending on the sensitivity and specificity desired for the diagnostic method, the ROC plot allows the derivation of a suitable threshold value that can be adjusted. It will be understood that an optimal sensitivity is desired to exclude (i.e. exclude) muscle atrophy, while an optimal specificity is assumed for subjects identified as having (i.e. likely to have) muscle atrophy.

[0137] For the three preferred biomarkers, absolute quantification of at least one additional biomarker in the subject's blood sample described herein is obtained by calibration standard (calibration curve) or digital PCR. Calibration curves are generally made using a 10-fold serial dilution series or any other dilution factor, for example 2-fold or 3-fold dilution. The interval between the upper and lower limits of the concentration of the biomarker determined to be suitable for the method corresponds to the practical range. Each biomarker has its own practical range. In the case of absolute quantification, the range of expression of the same at least one biomarker is expressed by the number of molecules present in the blood sample. When a calibration curve is used, the expression level of at least one additional biomarker in the subject's blood sample described herein is compared with a statistical value / threshold or a discrimination value / threshold that allows the diagnosis of muscle atrophy, in particular skeletal muscle atrophy.

[0138] Once a diagnosis of muscle atrophy has been made, the subject may be treated to restore all or part of the loss of muscle mass. Various approaches, referred to as "countermeasures" or "treatments", may be implemented in an attempt to reverse (skeletal) muscle atrophy and loss of function (Chopard et al., J. Cell. Mol. Med., (2009) 13(9b):3032-3050). These approaches can be grouped into three separate classes: exercise and physical training, nutritional supplements and the use of drugs. Dynamic and maximal resistance exercise is associated with a decrease in the induction of MAFbx and MURF1 (i.e., two muscle-specific atrophy-related genes), thus allowing the maintenance of muscle mass by counteracting the decrease in contractile, cytoskeletal and membrane-associated proteins. Amino acid supplementation can also promote protein synthesis. Among the essential amino acids, leucine is thought to mediate most of the effects of protein / amino acid intake on protein metabolism, and its potential anabolic effects have been recognized. Growth factors such as IGF, FGF and TGF-β are also known to have a significant impact on muscle gene expression by promoting the proliferation of satellite cells in muscle. Compounds with antioxidant properties, such as soy protein, vitamin C, vitamin E, N-acetylcysteine ​​and curcumin, exert their effects by regulating the expression of muscle protein degradation-related genes. More recently, ursolic acid and tomatidine were found to reduce skeletal muscle atrophy in mouse models by stimulating skeletal muscle hypertrophy (Ebert et al., Physiology (2019), 34: 232-239).

[0139] Diagnosis of muscle atrophy, particularly skeletal muscle atrophy, may allow follow-up of recovery of muscle mass and / or strength.

[0140] In another aspect, the invention relates to an in vitro or ex vivo method for assessing the efficacy of a treatment to reverse muscle atrophy in a subject, the method comprising: (i) determining in a first blood sample from the subject obtained prior to said treatment or at an initial stage of said treatment, the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2; and (ii) determining the expression level of the same at least one biomarker in a second blood sample obtained from the subject during said treatment, at a later stage, or after said treatment, wherein an expression level of the at least one biomarker measured in step (ii) that is greater than the expression level of the same at least one biomarker measured in step (i) indicates that the treatment is efficacious. Includes.

[0141] The expression "evaluate the effectiveness of a treatment to reverse muscle atrophy" for the purposes of the present invention means observing the progression or regression of muscle atrophy, particularly skeletal muscle atrophy, in a subject receiving measures (i.e., treatment) to reverse muscle atrophy. In other words, the subject undergoing treatment is monitored periodically for the effectiveness of the applied treatment, which allows the practitioner to estimate at an early stage during treatment whether the prescribed treatment is effective to reverse muscle atrophy and adjust the treatment accordingly. Muscle atrophy, particularly skeletal muscle atrophy, is reversed if at least one symptom of muscle atrophy is alleviated, alleviated, terminated, slowed down or prevented. Muscle mass can be determined by the following approaches: DXA (dual energy X-ray densitometer), skeletal muscle index, skeletal muscle area, masseter cross-sectional area (CSA), psoas CSA, total psoas area, L4 spine index, CT scan, etc. (For details, see Xiao-Ming Zhang, BMC Geriatrics, 2021).

[0142] In another preferred aspect, the present invention relates to an in vitro or ex vivo method for assessing the efficacy of a treatment to reverse muscle atrophy in a subject, the method comprising: (i) determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, or a combination thereof in a first blood sample from the subject obtained prior to said treatment or at an initial stage of said treatment; and (ii) determining the expression level of the same at least one biomarker, or combination thereof, in a second blood sample obtained from the subject during, at a later stage, or after said treatment, wherein an expression level of the at least one biomarker, or combination thereof, measured in step (ii) that is greater than the expression level of the same at least one biomarker measured in step (i) indicates that the treatment is efficacious. Includes.

[0143] All possible combinations of the three biomarkers are described herein.

[0144] In yet another preferred embodiment, the method further comprises in step (i) determining the expression level of at least one further biomarker selected from RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2.

[0145] Many diseases or disorders are associated with skeletal muscle atrophy, muscle weakness, and general muscle fatigue. Skeletal muscle wasting (i.e., atrophy) is associated with increased morbidity and mortality, and reduced quality of life. In addition to muscle wasting and the associated weakness, muscle fatigue is one of the most common and debilitating symptoms experienced by subjects suffering from cancer. It is believed that chronic inflammatory diseases have a direct detrimental effect on the skeletal muscles of the trunk and limbs, leading to reduced skills and therefore fatigue. However, in neurological disorders, fatigue often appears before changes in muscle performance are observed. Diagnosis of muscle atrophy, particularly skeletal muscle atrophy, can also be useful in determining whether a subject facing muscle atrophy has, is suspected of having, or is at risk of having a disease or disorder.

[0146] In another aspect, the present invention relates to an in vitro or ex vivo method for assessing the onset or risk of onset of a disease in a subject comprising assessing the presence of muscle atrophy in a subject by a method described herein; wherein the presence of muscle atrophy is indicative of the onset or risk of onset of a disease in the subject.

[0147] In a particular aspect of this method, the presence of muscle atrophy, in particular skeletal muscle atrophy, is established by determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2.

[0148] In another particular embodiment of this method, the presence of muscle atrophy, in particular skeletal muscle atrophy, is established by determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2 or a combination thereof. In this particular embodiment, determining the expression level of at least one further biomarker selected from RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 can be used to improve the diagnosis of muscle atrophy, in particular skeletal muscle atrophy.

[0149] Fatigue caused by or preceding muscle atrophy may limit treatment options for disease or disorder.In fact, perceived fatigue is recognized to have more negative impact on daily activities and quality of life than other symptoms associated with disease or disorder.Fatigue occurs as a result of both disease or disorder itself and the side effects of disease or disorder treatment.Therefore, it may be necessary to adjust treatment with therapeutic drugs or treatment regimens in subjects suffering from disease or disorder.

[0150] In another aspect, the invention relates to an in vitro or ex vivo method for assisting in selecting, adapting or altering a treatment in a subject suffering from a disease, the method comprising: (i) assessing the presence of muscle atrophy, particularly skeletal muscle atrophy, in a subject by the methods described herein; and (ii) taking into account the presence of muscle atrophy, in particular skeletal muscle atrophy, to select, adapt or modify a treatment in a subject suffering from a disease; Includes.

[0151] In a particular aspect of this method, the presence of muscle atrophy, in particular skeletal muscle atrophy, is established by determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2.

[0152] In another particular embodiment of this method, the presence of muscle atrophy, in particular skeletal muscle atrophy, is established by determining the expression level of at least one biomarker selected from ACTR6, GIMAP2 and RCN2, or a combination thereof. In this particular embodiment, determining the expression level of at least one further biomarker selected from RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 can be used to improve the diagnosis of muscle atrophy, in particular skeletal muscle atrophy.

[0153] In another further particular aspect, the in vitro or ex vivo method for assisting in selecting, adapting or altering a treatment in a subject suffering from a disease further comprises the step (iii) of selecting a treatment for muscle atrophy, wherein said treatment comprises a drug (pharmacological agent such as glucocorticoid, anti-inflammatory drug, etc.), a nutritional agent (nutraceutical, ursolic acid, tomatidine, etc.), exercise, or a combination thereof.

[0154] In yet another aspect, the present invention also relates to a method for diagnosing and treating muscle atrophy, particularly skeletal muscle atrophy, in a subject, the method comprising: (i) determining an expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in a blood sample obtained from the subject; wherein a decrease in the expression level of the at least one biomarker compared to a reference value is indicative of muscle atrophy, in particular skeletal muscle atrophy, in the subject; and (ii) treating the subject identified in step (i) as having muscle atrophy, particularly skeletal muscle atrophy, with a drug (pharmacologic agent such as a glucocorticoid, an anti-inflammatory drug, etc.), a nutritional agent (nutraceutical, ursolic acid, tomatidine, etc.), exercise, or a combination thereof; Includes.

[0155] "Method of treating" refers to a process intended to bring about a beneficial change in the condition of an individual, e.g., a mammal, particularly a human. The beneficial change may include one or more of restoring muscle mass and / or strength, limiting or delaying muscle atrophy, or preventing, limiting or delaying the deterioration of a patient's condition, disease or disorder. In particular, as used herein, the term "treatment" (also "treat" or "treating") refers to any measure that partially or completely alleviates, improves, alleviates, inhibits, delays the onset, reduces the severity, and / or reduces the incidence of muscle atrophy caused by a particular disease, disorder, and / or condition. Such treatment may be for subjects who do not show signs of muscle atrophy and / or who show only early signs of muscle atrophy. Alternatively or additionally, such treatment may be for subjects who show one or more established signs of muscle atrophy.

[0156] Step (i) of the method may also be carried out using a calibration curve used as a calibrator. When a calibration curve is used, the expression level of at least one biomarker described herein in a blood sample of a subject is compared with a statistical value / threshold or discrimination value / threshold that allows the diagnosis of muscle atrophy, in particular skeletal muscle atrophy.

[0157] In a particular embodiment, the presence of muscle atrophy, particularly skeletal muscle atrophy, in a subject is established in step (i) by determining the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, or a combination thereof. In this particular embodiment, determining the expression level of at least one further biomarker selected from RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 can be used to improve the diagnosis of muscle atrophy, particularly skeletal muscle atrophy, in a subject.

[0158] kit In another aspect, the present invention relates to a suitable kit comprising suitable means for measuring the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in a blood sample of a subject. The kit typically comprises reagents, e.g., nucleic acid extraction / purification reagents, amplification reagents; containers; and optionally, an instruction leaflet. The reagents may be present in the same container or in separate containers. The user may use any part of the reagents to carry out a reaction according to the instructions.

[0159] In a particular aspect, a kit for measuring the expression level of at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in a blood sample from a subject comprises at least two oligonucleotides having nucleic acid sequences selected from the group consisting of SEQ ID NOs: 14-39, and optionally reagents and / or instructions for performing an amplification reaction.

[0160] Specific examples of oligonucleotide pairs selected to amplify at least one biomarker selected from ACTR6, GIMAP2, RCN2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 correspond to the forward and reverse primers listed in Table B.

[0161] In another particular embodiment, the kit comprises an oligonucleotide primer pair suitable for measuring expression of at least one biomarker selected from ACTR6, GIMAP2 and RCN2, or a combination thereof. In this particular embodiment, the oligonucleotides have the nucleic acid sequences of SEQ ID NOs: 14-19, where the oligonucleotides of SEQ ID NOs: 14 and 15 allow amplification of ACTR6, the oligonucleotides of SEQ ID NOs: 16 and 17 allow amplification of GIMAP2, and the oligonucleotides of SEQ ID NOs: 18 and 19 allow amplification of the RCN2 biomarker.

[0162] In another particular embodiment, the kit may further comprise oligonucleotides suitable for measuring the expression level of at least one additional biomarker selected from RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2. In this particular embodiment, the kit comprises oligonucleotides having the base sequences of SEQ ID NOs: 20 to 39, wherein the oligonucleotides of SEQ ID NOs: 20 and 21 enable amplification of RPL22L1, the oligonucleotides of SEQ ID NOs: 22 and 23 enable amplification of TRIAP1, the oligonucleotides of SEQ ID NOs: 24 and 25 enable amplification of NIFK, the oligonucleotides of SEQ ID NOs: 26 and 27 enable amplification of APIP, the oligonucleotides of SEQ ID NOs: 28 and 29 enable amplification of GEMIN6, the oligonucleotides of SEQ ID NOs: 30 and 31 enable amplification of RWDD1, the oligonucleotides of SEQ ID NOs: 32 and 33 enable amplification of ZNF613, the oligonucleotides of SEQ ID NOs: 34 and 35 enable amplification of BPNT1, the oligonucleotides of SEQ ID NOs: 36 and 37 enable amplification of CCT2, and the oligonucleotides of SEQ ID NOs: 38 and 39 enable amplification of LYRM2 biomarker.

[0163] The present invention also relates to the use of the above kit with a blood sample obtained from a subject for diagnosing muscle atrophy, preferably skeletal muscle atrophy, in vitro or ex vivo; for assessing the effectiveness of a treatment to reverse muscle atrophy, preferably skeletal muscle atrophy; for assessing the onset or risk of onset of the disease in a subject diagnosed with (skeletal) muscle atrophy and / or for assisting in selecting, adapting or modifying a treatment in a subject suffering from a disease.

[0164] The following examples are provided to demonstrate and further illustrate certain preferred embodiments of the present invention and are not to be construed as limiting the scope of the invention.

[0165] Working Example Materials and Methods 1- Biological samples Whole blood samples were collected from patients in the PROMETHE and PHD cohorts for a total of 92 subjects. 53 patients had chronic renal failure, 14 patients had lung cancer, 7 patients had colon or pancreatic cancer, and 13 subjects were used as controls or healthy subjects. Table 1 below provides the main parameters of the subjects enrolled in this study.

[0166] Table 1: Patient characteristics [Table 4]

[0167] 2.5 ml blood samples were collected into PAXgene® Blood RNA tubes (PreAnalytiX, Catalog number / ID: 762165, distributed by Qiagen) and stored upright at room temperature (18-25°C) for a minimum of 2 hours and a maximum of 72 hours before processing or placed in a refrigerator (2-8°C) or freezer (-20°C or -70 / -80°C).

[0168] 2- Blood RNA extraction, purification and concentration PAXgene® Blood RNA tubes containing blood samples were thawed at room temperature (18-25°C) for approximately 2 hours, the time required for complete lysis of blood cells. Total RNA was extracted using the PAXgene Blood miRNA kit (PreAnalytiX, Catalog No. / ID: 763134, distributed by Qiagen) according to the manufacturer's procedure, except for replacing buffer BM5 with RNase-free water in the final RNA elution step. Briefly, the PAXgene Blood RNA tubes were first centrifuged to pellet the sample, which was then washed with water and resuspended in buffer BM1. After digestion in buffer BM2 with proteinase K, the sample was homogenized by centrifugation through a PAXgene Shredder spin column. Isopropanol was added to the sample to optimize binding conditions, and the sample was then centrifuged through a PAXgene RNA spin column, where total RNA >18 nucleotides (including miRNA) binds to the PAXgene silica membrane. The bound RNA is subjected to DNase digestion to remove contaminating genomic DNA and washed with Buffer BM3 followed by Buffer BM4. The pure RNA is then eluted in RNAse-free water. The extracted RNA (80 μl) is denatured in a water bath at 65° C. for 5 min, immediately placed on ice and then stored at −80° C.

[0169] The extracted RNA was then further purified and concentrated using the RNeasy® MinElute® Cleanup Kit (Cat. No. / ID: 74204, Qiagen) according to the manufacturer's procedures.

[0170] 3-RNAseq Gene expression analysis (mRNA analysis) was performed on human blood samples. The Illumina Ribo-Zero rRNA removal kit was used initially for rRNA removal. Globin removal was also performed as recommended for RNA samples. PolyA mRNA was purified using oligo-dT linked to magnetic beads, fragmented and random primed. cDNA synthesis was followed by end repair, phosphorylation (5' end) and A-tailing (3' end). Unique adapters were ligated and cDNA fragments were amplified by PCR and sequenced (2 x 150 bp) using the Illumina platform (13-21 million reads per sample).

[0171] 4-qRT-PCR Purified and concentrated RNA obtained with the RNeasy® MinElute® Cleanup Kit was denatured in a water bath at 65°C for 5 min and then placed on ice to prevent renaturation. Removal of genomic DNA and reverse transcription of RNA to cDNA was performed using the QuantiTect® Reverse Transcription Kit (Cat. No. / ID: 205311 or 205313, Qiagen) according to the manufacturer's procedure. Briefly, purified RNA samples were incubated in gDNA Wipeout buffer at 42°C for 2 min to effectively remove contaminating genomic DNA. After removal of genomic DNA, the RNA samples were ready for reverse transcription using a master mix prepared from Quantiscrip reverse transcriptase, Quantiscript RT buffer, and RT primer mix. The entire reaction was performed at 42°C for 30 min and then inactivated at 95°C for 3 min. cDNA samples were stored at -20°C until qPCR.

[0172] Semi-quantitative qPCR is performed on the cDNA samples using the SsoADV Univer SYBR® Green Supermix Kit (Cat. No. / ID: 1725274, Bio-Rad) according to the manufacturer's procedure. First, the cDNA samples are diluted 1 / 50. For each sample (1 well), the volume of the reaction mixture is 10 μl. It contains 0.7 μl of 10 μM sense primer, 0.7 μl of 10 μM antisense primer, 7 μl of Mix iQ and 1.6 μl of RNase-free water. In a 96-well PCR plate, 4 μl of the cDNA sample diluted 1 / 16 is distributed into each well with 10 μl of the reaction mixture. Each cDNA sample is distributed in duplicate. The semi-quantitative qPCR reaction is performed in a CFX96 Bio-Rad Laboratories thermal cycler. Calculations are performed using the comparative method ΔΔCt with a housekeeping gene.

[0173] 5- Absolute RT-qPCR Gene cloning and standard curve The different genes (including housekeeping genes) were cloned into pBluescript SK- (ThermoFisher) plasmids using HEK293 cells as starting material and E. coli Novablue (Novagen) for amplification of the plasmids. Plasmids were purified using QIAprep Spin Miniprep (QIAgen®) and quantified using a Nanodrop spectrometer (Thermofisher Scientific). We created standard curves by serial dilution for the different biomarkers (300,000 to 30 copies per μl) using the following formula:

[0174] Based on the size of the ACTR6 construct (4173 base pairs) and the average mass of one base pair (1,096 x 10^(-21) g), the mass (m) of 300,000 copies of the ACTR6 plasmid is: m=4173×(1,096×10^(-21))×300000 m=1,37208×10^(-12)g It is.

[0175] Using the spectrophotometric assay, standard curves were generated according to this calculation using the exact size of each plasmid construct.

[0176] The reliability of a PCR is defined by two criteria: a slope of -3.6 to -3.1 and an efficiency of 90 to 110%, with a correlation coefficient >99% (for example, see the Thermofisher website [https: / / www.thermofisher.com / fr / fr / home / life-science / pcr / real-time-pcr / real-time-pcr-learning-center / real-time-pcr-basics / efficiency-real-time-pcr-qpcr.html] for more information).

[0177] To obtain reliable and reproducible calibration curves corresponding to these criteria, we performed several assays, which included different plastic suppliers, the use of carrier nucleic acids and freeze-thaw cycles.

[0178] Randomly selected RNA samples from healthy volunteers (n=5) and atrophy patients (n=12, including CKD, lung cancer, and colon cancer) were analyzed by RT-qPCR using 200 ng of RNA from each sample, and absolute quantification was calculated using a previously established standard curve. Data were calculated as copy number per ng of starting total RNA.

[0179] 6-Digital PCR Absolute quantification of key biomarkers and housekeeping genes was performed using digital PCR (dPCR) technology (QX600 Droplet Digital PCR System, Bio-Rad). The exact same RNA samples used for absolute quantification by RT-qPCR were also used for dPCR analysis using QX200 ddPCR Evagreen Supermix® according to the manufacturer's instructions. The same primers designed for relative RT-qPCR (see Tables B and C) were used at a final concentration of 100 nM, and 200 ng of total RNA was used for each sample.

[0180] 7- Bioinformatics analysis of mRNA expression data Sequence reads were trimmed to remove possible adapter sequences and low quality nucleotides using Trimmomatic v.0.36. Trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using Star aligner v.2.5.2b. STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align all reads. BAM files were generated as a result of this process. Below are the statistics of mapping reads to the reference genome. Unique gene hits were calculated using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exonic regions were counted. Reads were counted strand-specifically since strand-specific library preparation was performed.

[0181] 8-Statistical analysis Statistical analysis was performed using R software (https: / / www.R-project.org). Tests were two-sided and type I error was set at α=0.05. p-values ​​were adjusted using the Benjamini-Hoschberg method. Differential expression analysis was performed using linear models with the Limma Voom R package. Genes whose expression exceeded 0.5 CPM in at least all samples of the same condition were removed. Normalization was performed using the TMM method. RNAs that were concordantly significantly increased or decreased in lung cancer and hemodialysis patients compared to healthy controls were selected. A PLS model was then constructed using the mixomic R package to predict the expression matrix from the proteomic analysis of muscle biopsies using the RNA expression matrix of whole blood. The first 30 RNAs selected to construct the model were retained. RNA expression data measured by qRT-PCR were then used to model patient group (disease or healthy) membership using PLS-DA. The ability of the model to predict group membership was assessed using the area under the receiver operating characteristic curve.

[0182] Absolute RT-qPCR data were analyzed using the Mann & Whitney test and dPCR analysis was performed using the Welch t test.

[0183] result Some mRNAs present in the whole blood of patients have expression levels directly related to the presence of muscle atrophy, regardless of the pathology. Approximately 1,500 mRNAs have been identified by RNAseq in patients with lung cancer or renal failure. The RNAseq approach identifies all mRNAs in one sample, but is too expensive to be routinely applied in hospitals. Therefore, the 30 best candidates (with high amplitude of variation between healthy and diseased patients, good reproducibility from one patient to another, etc.) were selected and verified by RT-qPCR testing. In this way, we selected the ones that are most able to discriminate between healthy and diseased patients from the 30 preselected mRNAs identified.

[0184] We first identified whole blood RNA whose expression levels best correlated with common proteomic changes observed in muscle biopsies from humans with two different catabolic states (hemodialysis n = 7 and lung cancer n = 7 vs. controls n = 7). Differential analysis of the blood transcriptome by RNAseq was combined with skeletal muscle proteomic analysis by mass spectrometry to identify a set of candidate genes (PLS sparse regression algorithm in the MixOmic R package).

[0185] qRT-PCR was performed to validate the RNAseq results in the PROMETHE cohort (renal failure n = 7 and lung cancer n = 14 vs. control n = 13) and in a hemodialysis patient cohort n = 38 (PHD cohort). Logistic regression was used to evaluate the ability of potential biomarkers to identify the catabolic state in derivation cohorts (renal failure n = 7 patients, lung cancer n = 7 patients and control n = 7 patients from the PROMETHE cohort) and validated in different validation cohorts (hemodialysis n = 38 patients from the PHD cohort, lung cancer n = 7 patients from the PROMETHE cohort, control n = 7 patients). The obtained results are summarized in Table 2.

[0186] Table 2: ROC parameters [Table 5]

[0187] Using qRT-PCR data, partial least squares discriminant analysis (PLS-DA) was performed using all patients (PROMETHE+PHD) for prediction of having muscle wasting due to a pathological condition (either cancer or renal failure). Patients were first randomly divided into a derivation cohort (n=21) and a validation cohort (n=52).

[0188] To select the most predictive or discriminatory features to help classify samples, we performed sparse partial least squares discriminant analysis (sPLS-DA) using qRT-PCR data from both PROMETHE and PHD patients with (Figure 1A) or without (Figure 1B) the four outliers.

[0189] We found that one-component analysis including ACTR6 and GIMAP2 was sufficient to predict that patients belonged to the muscle atrophy / pathology group. The AUC of the ROC was 0.9736 (Figure 2A). The prediction accuracy of "skeletal muscle atrophy" in the validation cohort was 95%. In addition, RCN2 behaved like ACTR6.

[0190] In the two-component model, NIFK, RPL22L1, CCT2, GEMIN6 and RCN2 were selected. The AUC of the ROC is 0.9934 (Figure 2B). The prediction accuracy of "skeletal muscle atrophy" in the validation cohort is 95%.

[0191] Overall, the mRNAs encoding the following genes were kept in the main list: ACTR6, GIMAP2, RCN2. The mRNAs encoding the following genes were kept in the complementary list: RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2, LYRM2. All these genes can be used in combination for a blood test as proposed by the present invention, but three mRNAs used individually or in combination are sufficient to predict muscle atrophy (sPLS-DA statistical test), thus limiting the cost of the test. Although some mRNAs can be used individually, a combination of several mRNAs provides significant additional value, since it reduces the number of false negatives and can be used for the largest feasible population. A maximum of 10-12 complementary mRNAs can nevertheless be kept, since additional experiments will eventually allow the inclusion of other markers based on the number of patients responding, the amplitude of variation, the number of disease states responding to each mRNA, etc.

[0192] Following these initial determinations, the biomarkers were tested in a small cohort of patients with abdominal cancer (colon or pancreatic) and compared to expression measured in patients from the PROMETHE and PHD cohorts (Figure 3). The data showed that abdominal cancer decreased the relative expression of mRNAs encoding the 13 biomarkers, thus demonstrating their applicability for detecting muscle wasting in patients with colon or pancreatic cancer.

[0193] It should be noted that these biomarkers are only effective in blood, even though they reflect muscle atrophy. Indeed, in skeletal muscle, genes that are systematically up- or down-regulated at the mRNA level are called "atrogens". We recently compiled data from 259 papers that analyzed the transcriptome of skeletal muscle in a total of 30 different catabolic and / or disease states in rodents and humans (Taillandier & Polge, Biochimie, 2019, https: / / doi.org / 10.1016 / j.biochi.2019.07.014). Only genes whose expression was similarly altered in at least two catabolic situations were selected. None of the 13 genes described herein as blood biomarkers were found in skeletal muscle, thus demonstrating that these biomarkers are only effective when analyzed in blood.

[0194] Absolute quantification by RT-qPCR was first used to obtain a valid standard curve (Figure 4). We first observed that the use of low-binding plastics allowed for the acquisition of highly reproducible standard curves (r 2 >0.99, Figure 4). In addition, the slopes were in the range of -3.2 / -3.5, and the efficiencies were 93-104%. In contrast, the use of standard pipette tips and tubes did not improve the linearity of the standard curve (r 2= 0.85-0.95%, not shown), with slopes ranging from -3.8 to -4.6 (Figure 4) and efficiencies ranging from 64 to 86%. We also found that the use of carrier RNA (yeast tRNA, 10 µg per sample) allowed the acquisition of a reliable calibration curve (Figure 4). Up to five freeze-thaw cycles did not noticeably alter the efficiency and slope of the calibration curve (Figure 4).

[0195] Using the optimal standard curves, we confirmed the differences in expression of the main biomarkers between healthy volunteers and patients and were now able to quantify the exact number of copies of each mRNA present in the samples (Figure 5). For ACTR6, 11-25 copies / ng of RNA were present in healthy volunteers and 0.5-12 copies / ng of RNA for patients. Expression of GIMAP2 was 230-380 and 45-205 copies / ng of RNA for healthy volunteers and patients, respectively. For RCN2, it was 105-170 and 20-105 copies / ng of RNA for healthy volunteers and patients, respectively. For APIP, it was 38-57 and 9-35 copies / ng of RNA for healthy volunteers and patients, respectively. In the case of dPCR, we did not detect differences between the different groups, so we mixed the data obtained for atrophy patients suffering from different pathologies. For easier comparison, qPCR and dPCR data obtained from the same samples were placed side by side (Figure 6). We found that although dPCR is perfectly suitable for absolute quantification of biomarkers, the values ​​obtained are slightly different but within the same range as those obtained when using a standard curve (Figure 6 and Table 3). This is known in the field of PCR, and this means that the values ​​obtained for healthy volunteers and patients are technique dependent. Each technique is effective to separate healthy volunteers and patients with muscle atrophy. However, a more precise threshold between patient and control values ​​can be determined for each approach. It should be noted that although dPCR is currently considered the gold standard for absolute quantification of mRNA (Williams et al., Front Oncol 2022, doi:10.3389 / fonc.2022.864820), very few biology laboratories have already installed dPCR equipment.

[0196] Table 3: Biomarker concentrations expressed as copies per ng of total RNA. [Table 6]

Claims

1. Use of at least one gene or gene product thereof selected from RCN2 (SEQ ID NO: 3), ACTR6 (SEQ ID NO: 1), GIMAP2 (SEQ ID NO: 2), RPL22L1 (SEQ ID NO: 4), TRIAP1 (SEQ ID NO: 5), NIFK (SEQ ID NO: 6), APIP (SEQ ID NO: 7), GEMIN6 (SEQ ID NO: 8), RWDD1 (SEQ ID NO: 9), ZNF613 (SEQ ID NO: 10), BPNT1 (SEQ ID NO: 11), CCT2 (SEQ ID NO: 12) and LYRM2 (SEQ ID NO: 13) as a biomarker for the in vitro or ex vivo detection of muscle atrophy in blood samples.

2. 1. An in vitro or ex vivo method for diagnosing muscle atrophy in a subject, comprising determining the expression level of at least one biomarker selected from RCN2 (SEQ ID NO: 3), ACTR6 (SEQ ID NO: 1), GIMAP2 (SEQ ID NO: 2), RPL22L1 (SEQ ID NO: 4), TRIAP1 (SEQ ID NO: 5), NIFK (SEQ ID NO: 6), APIP (SEQ ID NO: 7), GEMIN6 (SEQ ID NO: 8), RWDD1 (SEQ ID NO: 9), ZNF613 (SEQ ID NO: 10), BPNT1 (SEQ ID NO: 11), CCT2 (SEQ ID NO: 12) and LYRM2 (SEQ ID NO: 13) in a blood sample obtained from the subject, wherein a decrease in the expression level of the at least one biomarker compared to a reference value is indicative of muscle atrophy in the subject.

3. 3. The method of claim 2, wherein the at least one biomarker is selected from RCN2, ACTR6 and GIMAP2, or a combination thereof, and a decrease in the expression level of the at least one biomarker, or a combination thereof, compared to a reference value indicates muscle atrophy.

4. 4. The method of claim 3, comprising determining the expression level of one of the following combinations of biomarkers: ACTR6 and GIMAP2, ACTR6 and RCN2, GIMAP2 and RCN2, or ACTR6, GIMAP2 and RCN2.

5. 5. The method of claim 3 or 4, further comprising determining the expression level of at least one additional biomarker selected from RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2.

6. 1. An in vitro or ex vivo method for assessing the effectiveness of a treatment to reverse muscle atrophy in a subject, comprising: (i) determining the expression level of at least one biomarker selected from RCN2, ACTR6, GIMAP2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2 in a first blood sample from the subject obtained prior to said treatment or at an early stage of said treatment; and (ii) determining the expression level of the same at least one biomarker in a second blood sample from the subject obtained at a later stage or later during said treatment, wherein an expression level of the at least one biomarker measured in step (ii) that is greater than the expression level of the same at least one biomarker measured in step (i) indicates that the treatment is efficacious. A method comprising:

7. 1. An in vitro or ex vivo method for assisting in selecting, adapting or modifying treatment in a subject suffering from a disease, comprising: (i) assessing the presence of muscle atrophy in the subject by determining the expression level of at least one biomarker selected from RCN2 (SEQ ID NO: 3), ACTR6 (SEQ ID NO: 1), GIMAP2 (SEQ ID NO: 2), RPL22L1 (SEQ ID NO: 4), TRIAP1 (SEQ ID NO: 5), NIFK (SEQ ID NO: 6), APIP (SEQ ID NO: 7), GEMIN6 (SEQ ID NO: 8), RWDD1 (SEQ ID NO: 9), ZNF613 (SEQ ID NO: 10), BPNT1 (SEQ ID NO: 11), CCT2 (SEQ ID NO: 12) and LYRM2 (SEQ ID NO: 13) in a blood sample obtained from the subject; and (ii) taking into account the presence of muscle atrophy to select, adapt or modify treatment in a subject suffering from a disease A method comprising:

8. 8. The in vitro or ex vivo method of claim 7, further comprising the step (iii) of selecting a treatment for muscle atrophy, wherein the treatment comprises a drug (a pharmacological agent such as a glucocorticoid, an anti-inflammatory drug, etc.), a nutritional agent (a dietary supplement, ursolic acid, tomatidine, etc.), exercise, or a combination thereof.

9. 8. The in vitro or ex vivo method of claim 2, 6 or 7, wherein the expression level of said at least one biomarker is determined at the nucleic acid level.

10. 10. The in vitro or ex vivo method of claim 9, wherein the expression level of the at least one biomarker is determined using real-time reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), digital PCR, RNAseq, microarray, gene chip, nCounter gene expression assay, serial analysis of gene expression (SAGE), rapid analysis of gene expression (RAGE), nuclease protection assay, northern blotting, or any other equivalent gene expression detection technique.

11. 10. The in vitro or ex vivo method of claim 9, wherein the expression level of the at least one biomarker is determined using absolute quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) or digital PCR.

12. 8. The method of any one of claims 2, 6 or 7, wherein the subject is a human subject.

13. 8. The use according to claim 1 or the method according to any one of claims 2, 6 or 7, wherein the blood sample is selected from a peripheral blood, plasma and serum sample.

14. An oligonucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14-39.

15. A kit comprising at least two oligonucleotides described in claim 14 for amplifying at least one biomarker selected from RCN2, ACTR6, GIMAP2, RPL22L1, TRIAP1, NIFK, APIP, GEMIN6, RWDD1, ZNF613, BPNT1, CCT2 and LYRM2.

16. The kit of claim 15, further comprising reagents for carrying out an amplification reaction and / or instructions for use.

17. 17. Use of the kit of claim 16 on a blood sample for diagnosing muscle atrophy; for assessing the effectiveness of a treatment to reverse muscle atrophy; for assessing the onset or risk of onset of a disease in a subject and / or to assist in selecting, adapting or modifying a treatment in a subject suffering from a disease.

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