Methods of diagnosing the risk for spinal deformity progression in symptomatic subjects affected by idiopathic scoliosis and kits therefor

Abstract

The present disclosure provides a method of diagnosing the risk of spinal deformity progression in a subject symptomatic for an idiopathic scoliosis comprising measuring a level of expression of a plurality of miRNAs in a subject's sample, wherein a variation (higher, lower or similar to, depending on the miRNA) in the level of expression of each miRNA in the panel as compared to a corresponding reference level, is an indication that the subject's spinal deformity will progress.

Description

[0001] TITLE

[0002] METHODS OF DIAGNOSING THE RISK FOR SPINAL DEFORMITY PROGRESSION IN SYMPTOMATIC SUBJECTS AFFECTED BY IDIOPATHIC SCOLIOSIS AND KITS THEREFOR

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application is a PCT application Serial No PCT / CA2025 / * filed on October 30, 2025, and published in English under PCT Article 21 (2), which itself claims benefit of U.S. provisional application Serial No. 63 / 714,180, filed on October 31, 2025. All documents above are incorporated herein in their entirety by reference.

[0005] FIELD OF THE DISCLOSURE

[0006] The present disclosure relates to methods of diagnosing the risk for spinal deformity progression in subjects affected by idiopathic scoliosis and kits therefor. More specifically, the present disclosure is concerned with methods of diagnosing the risk for spinal deformity progression in subjects affected by idiopathic scoliosis (e.g., AIS) and kits therefor, comprising miRNAs.

[0007] REFERENCE TO SEQUENCE LISTING

[0008] Pursuant to 37 C.F.R. 1 .821 (c), a sequence listing is submitted herewith as an ASCII compliant text file named G12810- 00898 Sequence Listing, that was created on October 30, 2025, and has a size of 59 kilobytes. The content of the aforementioned file named G12810-00898 Sequence Listing is hereby incorporated.

[0009] BACKGROUND OF THE DISCLOSURE

[0010] Adolescent Idiopathic Scoliosis (AIS) is one of the most common childhood deformities worldwide. It is characterized by a tridimensional spinal deformity with unknown causes, representing both an immediate medical challenge and a chronic condition affecting individuals throughout their lives. It is the most common orthopedic condition requiring surgery in adolescents and affects 2-4% of this population. There is a variability in scoliosis progression in affected subjects and 85% of AIS cases occurring in individuals without familial antecedents of scoliosis (Cheng, J.C., et al. Adolescent idiopathic scoliosis. Nat Rev Dis Primers. 1 ,15030 (2015). There is currently no proven method or prognostic test to identify, at an early stage, symptomatic patients at risk of developing severe scoliosis who could benefit from growth-guided devices or minimally invasive non-fusion instrumentation surgeries. These innovative treatments must be performed at an early disease stage in younger patients to benefit from their growth potential.

[0011] Consequently, the application of current treatments, such as growth guided devices such as bracing, or surgical correction, is delayed until a significant deformity is detected or until a significant progression is clearly demonstrated, resulting in costly and less than optimal treatments.

[0012] Given the clinical heterogeneity of the disease, biological sex differences (higher prevalence in girls) and potential crosstalk with undefined environmental factors, a search for epigenetic factors has been undertaken.

[0013] There is a need for a method for identifying, at an early stage, symptomatic AIS subjects at risk of developing a severe scoliosis. The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0014] SUMMARY OF THE DISCLOSURE

[0015] The present disclosure provides the use of circulating microRNAs (miRNAs) as biomarkers to predict the risk of developing severe scoliosis (Cobb angle > 45°) in AIS. The present disclosure also discriminates symptomatic patients with a risk of moderate disease progression (Cobb angle between 25-44°) from those with a minimal progression potential (Cobb angle <25°) compared to matched healthy controls.

[0016] Blood samples and clinical data were collected from 116 AIS patients who were followed until skeletal maturity and stratified according to their clinical outcome. Genome-wide expression profiling of miRNAs was performed with plasma obtained at the time of diagnosis of AIS (mean age of 13.3 ± 1.7 years with a mean Cobb angle of 24.4°± 12.4°). This approach led to the identification of 16 circulating miRNAs that are upregulated in AIS patients who developed a severe scoliosis (Cobb angle >45°) at skeletal maturity compared to moderate and mild scoliosis groups (Cobb angle between 25°-44° and <25° respectively). After optimization and the application of Random Forest Models, a panel of six miRNAs (miR-1-3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b-3p) out of 16 enabled the development of an algorithm predicting the risk of developing a severe scoliosis with great accuracy (100%), sensitivity (100%) and specificity (100%). Sex-specific panels also enabled the development of an algorithm predicting the risk of developing a severe or moderate scoliosis with great accuracy (100%), sensitivity (100%) and specificity (100%).

[0017] These panels provide spinal deformity progression risk signatures for subjects symptomatic with scoliosis.

[0018] More specifically, in accordance with the present disclosure, there is provided the following items:

[0019] Item 1 . A method of diagnosing the risk of spinal deformity progression in a subject symptomatic for an idiopathic scoliosis comprising:

[0020] (a) measuring a level of expression of at least the following biomarkers:

[0021] (i) miR-1 -3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b-3p in a subject’s sample;

[0022] (ii) let-7f-5p, miR-18a-3p, miR-103a-3p, and miR-551b-3p in a female subject’s sample; or

[0023] (iii) miR-551 b-3p and miR-4665-5p in a male subject’s sample; wherein: in (i) a level of expression of each of miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b-3p higher than corresponding reference levels; in (ii) a level of expression of each of let-7f-5p and miR-103a-3p similar to a corresponding reference level, and a level of expression of each of miR-18a-3p and miR-551b-3p higher than a corresponding reference level; or in (iii) a level of expression of miR-551 b-3p higher than a corresponding reference level and a level of expression of miR-4665-5p lower than a corresponding reference level, is an indication that the subject’s spinal deformity will progress.

[0024] Item 2. The method of item 1 , wherein the subject is a human.

[0025] Item 3. The method of item 1 or 2, wherein the subject’s sample is blood, plasma or serum. In another specific embodiment the subject’s sample is osteoblasts. In specific embodiments, wherein the sample is osteoblasts, in (a) the biomarkers (i) are measured.

[0026] Item 4. The method of any one of items 1 to 3, wherein the subject is between 9 and 18 years old or between 9 and 17 years and has a Cobb angle of 10°.

[0027] Item 5. The method of any one of items 1 to 4, wherein in (a) the biomarkers (i) are measured.

[0028] Item 6. The method of item 5, wherein the subject is a female.

[0029] Item 7. The method of item 6, further comprising measuring a level of expression of miR-18a-3p, wherein a level of expression of miR-18a-3p higher than a corresponding reference level is a further indication that the subject’s spinal deformity will progress.

[0030] Item 8. The method of any one of items 1 to 3, wherein in (a) the biomarkers (ii) are measured.

[0031] Item 9. The method of any one of items 5 to 8, wherein the method provides an indication that the subject’s spinal deformity will reach a Cobb angle of 45°.

[0032] Item 10. The method of any one of items 5 to 10, wherein the subject in (a) is between 9 and 16 years old or between 9 and 15 years old or between 9 and 14 years old; and / or has a Cobb angle of 10°.

[0033] Item 11. The method of any one of items 1 to 4, wherein in (a) the biomarkers (iii) are measured.

[0034] Item 12. The method of item 11 , wherein the method provides an indication that the subject’s spinal deformity will increase of > 15 ° while not exceeding a final Cobb angle of 44 °.

[0035] Item 13. The method of any one of items 1 to 12, wherein the subject has adolescent idiopathic scoliosis.

[0036] Item 14. The method of any one of items 1 to 13, wherein the subject is Caucasian.

[0037] Item 15. The method of any one of items 1 to 14, further comprising (b) treating the subject with one or more of a growth guided device or surgery, preferably a surgery (e.g., non-fusion surgery) when the level of expression (i), (ii) or (iii) measured in (a) is indicative that the subject’s spinal deformity will progress.

[0038] Item 16. Kit comprising

[0039] (a) (i) reagents for specifically detecting each of miR-1 -3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-

[0040] 3p, and miR-148b-3p;

[0041] (ii) reagents for specifically detecting each of let-7f-5p, miR-18a-3p, miR-103a-3p, and miR-551 b-3p; or

[0042] (iii) reagents for specifically detecting each of miR-551 b-3p and miR-4665-5p; and (b) optionally (i) instructions for using the kit to diagnose the risk of spinal deformity progression in a subject symptomatic for an idiopathic scoliosis; (ii) at least one tool, reagent or medium for the isolation of the subject sample; or (iii) a combination of (i) and (ii).

[0043] Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In the appended drawings:

[0046] FIGs. 1A-0 present the top 15 circulating miRNAs upregulated in severe AIS cases. FIGs. 1A to 10 represent the expression analysis of let-7f-5p, miR-1 -3p, miR-18a-3p, miR-19a-3p, miR524 19b-3p, miR-103a-3p, miR-107, miR- 133b, miR-143-3p, miR-148a-3p, miR-148b-3p, 525 miR-152-3p, miR-214-3p, miR-551b-3p, and miR-576-5p respectively. A comparison of relative expression level for each miRNA was performed between the severe or progressor group (P) vs. the non-severe group, including moderate progressor + non progressor (MoP+NP) and the age- and sex-matched healthy control (HC) subjects. GraphPad™ Prism 9 was used to generate graphs and statistical analysis. Statistical significance between each group was determined using the T-test for two groups and ANOVA for more than two groups (* p-value<0.05, ** p-value<0.01, *** p value<0.001).

[0047] FIGs. 2A-0 present the top 15 miRNAs upregulated in AIS progressor group (P) compared to moderate (MOP) and non-progressor (NP) groups. FIGs. 2A to 20 represent the expression analysis of let-7f-5p, miR-1 -3p, miR-18a-3p, miR-19a-3p, miR-19b-3p, miR- 103a-3p, miR-107, miR-133b, miR-143-3p, miR-148a-3p, miR-148b-3p, miR-152-3p, miR-214-3p, miR-551 b-3p, and miR-576-5p respectively. These 15 miRNAs were upregulated in severe group (P) compared non-progressor (NP) groups and the age- and sex-matched healthy control (HC) subjects. Three (miR-19b- 3p, miR-103a-3p, miR-148b-3p) miRNAs were significantly upregulated between severe group (P) and moderate progressor group (MOP). GraphPad™ Prism 9 was used to generate graphs and statistical analysis. Statistical analysis. Statistical significance between each group was determined using the T-test for two groups and ANOVA for more than two groups (* p-value<0.05, ** p-value<0.01 , *** p-value<0.001).

[0048] FIGs. 3A-B. Elevation of circulating miR18a-3p is sex dependent in AIS progressors. Expression of MiR-18a-3p is upregulated explicitly in females classified in the severe (P) group. FIG. 3A shows the comparison of miR-18a-3p expression between the females exhibiting a severe scoliosis (FP group) with the female with mild-to-moderate scoliosis (FMOP+FNP group) and healthy subjects (FHC). FIG. 3B shows the comparison of miR-18a-3p expression in males exhibiting a severe scoliosis (MP group) compared to males classified in the non-severe group (FMOP+FNP). GraphPad™ Prism 9 was used to generate graphs and statistical analysis. Statistical significance between each group was determined using the T-test for two groups and ANOVA for more than two groups (* p-value<0.05).

[0049] FIGs. 4A-D. Circulating miRNAs downregulated in the AIS non-progressor males compared to healthy controls. FIGs. 4A-B illustrate miR-103a-3p expression in male and female patients respectively, with a severe downregulation only in males non-progressor (MNP). FIGs. 4C-D represent a similar comparison showing a significant down regulation of miR-107 in male non-progressor group (MNP). Of note, female patients and controls exhibited opposite behavior in terms of expression for both miRNAs. GraphPad™ Prism 9 was used to generate the graphs and perform the statistical analysis. To compare the statistical significance between each group, the T-test were used for two populations and the ANOVA test for more than two populations. (* p-value<0.05, ** p-value<0.01).

[0050] FIGs. 5A-D. Circulating miRNAs showing an upregulation in AIS moderate scoliosis progressor group. FIG. 5A shows an upregulation of miR-4665-5p only in male patients with AIS classified in the moderate progressor group (MMOP) compared to the other two groups, while FIG. 5B shows no difference among the female groups. FIGs. 5C and D represent a similar analysis with miR-551 b-5p in males and female patients respectively indicating a significant upregulation of this miRNA only in male progressor group (MP) and male moderate progressor group (MMOP). GraphPad™ Prism 9 was used to make graphs and statistical analysis. To compare the statistical significance between each group, we used the T-test for two populations and the ANOVA test for more than two populations. (* p-value<0.05, ** p-value<0.01, *** p-value<0.001).

[0051] FIGs. 6A-E. Receiver-operator curve (ROC) analysis for predicting scoliosis severity in symptomatic AIS patients. FIG. 6A represents the ROC curve generated by combining the relative expression (2<AACT)) values from six circulating miRNAs (miR-1-3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, miR-148b-3p) upregulated in AIS progressors (P) group in both sexes, which shows an accuracy, a specificity and sensitivity of 100%, with a ROC curve AUC = 1 .0. FIG. 6B represents the same approach with the inclusion of the 15 miRNAs upregulated in the AIS progressors (P) group and shows a lesser predictive performance with an accuracy of 90%, a specificity of 88%, a sensitivity of 100% and a ROC curve AUC = 0.94. FIG. 6C illustrates a ROC curve for predicting female progressor (FP) vs female non- progressors (FNP) with seven circulating miRNAs (miR-1-3p, miR-18a-3p, miR-19a-3p, miR-19b-3p, miR-133b, miR- 143-3p, miR-148b-3p) and shows an accuracy, specificity and sensitivity of 100%, with a ROC curve AUC = 1.0. FIG. 6D corresponds to a ROC curve for predicting female progressor (FP) vs female non-progressor (FNP) with four circulating miRNAs (let-7f-5p, miR-18a-3p, miR-103a-3p, miR-551 b-3p), and shows an accuracy, specificity and sensitivity of 100%, with a ROC curve AUC = 1 .0. FIG. 6E shows a ROC curve for predicting male moderate progressor (MMOP) vs male non-progressor (MNP), with an accuracy, specificity and sensitivity of 100%, with a ROC curve AUC = 1.0.

[0052] FIGs. 7A-B. Circulating miR-148b-3p is upregulated in osteoblasts of AIS surgical cases compared to non-scoliotic controls (trauma cases). FIG. 7A represents the expression of miR-148b-3p in plasma samples of AIS severe or progressor group (P) and AIS non-severe or non-progressor group (NP) compared to healthy controls (HC). FIG. 7B represents the expression of miR-148b-3p in osteoblast samples of surgical AIS cases compared to non-scoliotic trauma cases as non-scoliotic controls. GraphPad Prism™ 9 was used to generate graphs and statistical analysis. Statistical significance between each group was determined using the T-test for two groups and ANOVA for more than two groups (* p-value<0.05, ** p-value<0.01).

[0053] FIG. 8. Muscle, bone, cartilage disease related to our candidate miRNAs. Using the Ingenuity Pathway Analysis (IPA) software (QIAGEN Inc. software version 51,963,813), the inventors determined that 11 miRNAs (let-7f-5p, miR-1 -3p, miR-19a-3p, miR-19b- 3p, miR-103a-3p, miR-107, miR-143-3p, miR-148a-3p, miR-148b-3p, miR-152-3p, miR- 214- 3p) of the 15 candidate miRNAs have been reported to be related to muscle, bone, and cartilage related disease. These miRNAs have been reported in bone and mostly in muscle-related diseases.

[0054] FIG. 9. Predicted pathway and network of miRNAs of the present disclosure associated with AIS and scoliosis severity. The potential targets of these 15 miRNAs and previously discovered AIS severity associated miRNAs, including genes, molecules and their connection to idiopathic scoliosis or scoliosis phenotype, are shown in one integrated network. The 15 candidate miRNAs are represented in light blue; AIS severity associated miRNAs are in dark blue, their interaction with targets indicated with pink lines; the genes predicted to interact are in green; the diseases associated with miRNAs or genes are in light pink. The Ingenuity Pathway Analysis™ (IPA) software (QIAGEN Inc. software version 51 ,963,813) and manual curations were applied to construct the network.

[0055] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0056] Definitions

[0057] For clarity, definitions of the following terms in the context of the present disclosure are provided.

[0058] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0059] The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0060] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0061] The use of any and all examples, or exemplary language (“e.g.”, "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.

[0062] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.

[0063] Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).

[0064] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0065] Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.

[0066] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0067] Unless otherwise indicated, the techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0068] The present disclosure teaches that the levels (e.g., circulating levels) of one or more specific miRNAs in a sample of a subject that is symptomatic for idiopathic scoliosis (e.g., AIS) can be used for diagnosing the risk of spinal deformity progression (i.e. increase in the Cobb angle) in the subject. Typically, a subject is considered to be symptomatic for IS (e.g., AIS) if its Cobb angle is > 10° (hereinafter “symptomatic subject”). In specific embodiments, the levels (e.g., circulating levels) of one or more specific miRNAs in a sample of a subject that is symptomatic for idiopathic scoliosis (e.g., AIS) can be used for the diagnosis of the risk of severe spinal deformity progression (i.e. increase in the Cobb angle reaching 45°). Typically, spinal deformity in AIS can continue until subjects reach their skeletal maturity (Risser sign of 4 or 5) which generally occurs at or around 15-16 years old in women and around 17-18 years old in men.

[0069] More particularly, the age of skeletal maturity varies between girls and boys, particularly among Caucasians of European ancestry: For girls, skeletal maturity is initiated between 12 and 14 years, with a rapid growth spurt before the onset of menstruation, followed by quick stabilization. Girls are often considered skeletally mature around 15-16 years old. By this age, most girls have completed their growth spurts and achieved skeletal maturity. For boys, skeletal maturity occurs later, usually around 17-18 years old. Boys tend to have a longer growth period, so they continue to grow and develop for a few years longer than girls.

[0070] The Risser index is used to assess skeletal maturity by examining the ossification of the iliac crest (on a radiography), particularly in the context of scoliosis management. It is divided into five stages based on the progression of ossification:

[0071] Risser 0: No ossification. Risser 1 : Ossification in the lateral quarter of the iliac crest. Risser 2: Ossification extends to half of the iliac crest. Risser 3: Ossification covers three-quarters of the iliac crest. Risser 4: Complete ossification, but growth plates haven't fused. Risser 5: Complete fusion of the growth plates, indicating the end of growth.

[0072] In summary, girls reach skeletal maturity earlier than boys, and the Risser index is used to track the stages of iliac crest ossification to estimate remaining growth. MicroRNAs (miRNAs) are a class of small non-coding RNAs that suppress the translation and / or stability of specific mRNA targets by binding to 3’UTR sites of targeted mRNAs.

[0073] In specific embodiments, the levels of the one or more specific miRNAs are higher in the subject at risk than reference levels (e.g., levels in corresponding healthy subjects) and / or higher than in symptomatic subjects whose spinal deformity does not progress or only progresses slightly (i.e. reaches a final Cobb angle of <24°; called herein non progressor or NP) and / or higher than that in symptomatic subjects whose spinal deformity progresses moderately (i.e. reaches a final Cobb angle of 5° and s 44° inclusively; called herein moderate progressor or MoP).

[0074] The present disclosure provides a method of diagnosing the risk of spinal deformity progression in a subject symptomatic for an idiopathic scoliosis comprising: (a) measuring a level of expression of one 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, 13 or more, 14 or more, 15 or more, or all 16) of the biomarkers listed in Table VIII in a subject sample and comparing it to a reference level, wherein the result of the comparison for each of the one or more miRNA (i.e. higher than, similar to, or lower than the reference level) determines whether the subject is at risk of spinal deformity progression. In specific embodiments, the method uses any one of the combination of miRNAs of any one of (i) to (iii): (i) miR-1 -3p, miR-19a- 3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b-3p in a subject’s sample; (ii) let-7f-5p, miR-18a-3p, miR-103a-3p, and miR-551 b-3p in a female subject’s sample; or (iii)miR-551 b-3p and miR-4665-5p in a male subject’s sample, wherein in (i) a level of expression of each of miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b- 3p higher than those in a control subject sample; in (ii) a level of expression of each of let-7f-5p and miR-103a-3p similar to a corresponding reference level, and a level of expression of each of miR-18a-3p and miR-551 b-3p higher than a corresponding reference level; or in (iii) a level of expression of miR-551 b-3p higher than a corresponding reference level and a level of expression of miR-4665-5p lower than a corresponding reference level, is an indication that the subject’s spinal deformity will progress.

[0075] In accordance with the present disclosure, determining the expression level of miRNAs of the present disclosure can be achieved by any known methods for determining the expression level of a miRNA in a sample. Said methods comprise nucleic acid analyzing devices and methods which may utilize labelled molecules that specifically bind to the miRNAs in various assay formats. Said assays will develop a signal which is indicative for the presence or absence of a miRNA of the present disclosure. Moreover, the signal strength can, preferably, be correlated directly or indirectly (e.g. reverse- proportional) to the expression level of miRNAs present in a sample. Typically, hybridization- or PCR- based techniques for nucleic acid analysis are applied for quantitatively determining the expression level of a miRNA. Said techniques, preferably, use nucleic acid molecules that specifically hybridize to the target miRNA, as specific detection agents. Moreover, the PCR-based techniques also use nucleic acid molecules as oligonucleotide primers for specifically amplifying the target miRNA and, thus, further specific detection agents. Nucleic acid molecules that specifically bind to the target miRNA can be derived from the nucleic acid sequence of miRNAs by the skilled person using commonly known methods. Such sequences, typically, have a complementary nucleic acid sequence to the target sequence. Moreover, oligonucleotides suitable as primer nucleic acids can also be derived from the miRNA sequence to be amplified by the skilled artisan by the skilled person using commonly known methods. Further suitable methods comprise measuring a physical or chemical property specific for the miRNAs such as its precise molecular mass or NMR spectrum. Said methods comprise, preferably, biosensors, optical devices coupled to nucleic acid analyzers, nucleotide chips and, in particular, nucleic acid microarrays (surface to which probes that correspond in sequence to microRNAs of the present disclosure and can specifically hybridize thereto are bound), immunoassays, analytical devices such as mass- spectrometers, NMR- analyzers or chromatography devices. In specific embodiments, the expression level of miRNAs can be determined by a nucleic acid analysis technology and, more preferably, by a PCR-based technology. In particular, the expression level of miRNAs can be determined by real-time quantitative reverse transcription PCR (qPCR), most preferably, carried out as described in the accompanying Examples, herein.

[0076] Alternatively, the expression level of miRNAs may be determined by in situ hybridization technologies where a labeled probe is used in order to determine the miRNA molecules in a tissue sample. Quantification may be carried out by optically determining a label, such as a fluorescent or chemiluminescent label coupled to the detection probe. The expression level of miRNAs may also be determined by hybridization techniques using a target specific probe which subsequently binds to linker and enhancer probes such as the RNAscope technology. This technology may also be used in an in-situ approach on tissue samples. In order to evaluate the amount of bound labeled probes, flow-through cytometry may be applied.

[0077] In specific embodiments, the method of the present disclosure further comprises (a) obtaining miRNA expression data from the subject sample (e.g., blood sample such as serum or plasma): (i) extracting miRNAs from the sample; (ii) reverse transcribing the miRNAs extracted from the sample into cDNAs; (iii) amplifying the cDNAs; (iv) measuring expression levels of one or more of the miRNAs of the present disclosure (e.g., the panel / plurality of miRNA presented in any one of FIGs. 6A to 6E) in the amplified cDNAs; (v) detecting an expression level of the one or more miRNAs of the present disclosure (e.g., the panel / plurality of miRNA presented in any one of FIGs. 6A to 6E), as compared to a reference level for each of these one or more miRNAs.

[0078] The term “expression level” as used herein encompasses the absolute levels of miRNAs of the disclosure, their relative levels or concentration (e.g., normalized with the results of the spiked-in exogenous control, such as cel-mir-39a-3p or host endogenous hsa-miR-16-5p) as well as any value or parameter which correlates thereto or can be derived therefrom. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said miRNAs by direct measurements. Moreover, encompassed are all values or parameters which are obtained by indirect measurements specified elsewhere in this description, e.g., response levels determined from biochemical or biological read out systems in response to the miRNAs or intensity signals obtained from specifically bound ligands. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations.

[0079] The term “compared” or the comparison leading to the conclusion that an expression level of a miRNA is higher than, similar to or lower than as used herein encompasses comparing the amount of the one or more miRNAs of the present disclosure comprised by the subject sample with a reference level described elsewhere in this description. It is to be understood that comparing as used herein refers to a comparison of corresponding parameters or values, e.g., an absolute level is compared to an absolute reference level while a concentration or a relative level is compared to a reference concentration or relative level or an intensity signal obtained from a test sample is compared to the same type of intensity signal of a reference sample. The comparison expressed or implied in the method of the present disclosure may be carried out manually or computer assisted. For a computer assisted comparison, the value of the determined level may be compared to values corresponding to suitable reference levels which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output format. The reference is to be chosen so that either a difference or a similarity in the compared levels allows identifying those test subjects which belong into the group of subjects being susceptible to spinal deformity progression, and to certain treatments or follow up schedules are described herein.

[0080] In specific embodiments, the results obtained with the method of the present disclosure are further confirmed with other diagnostic methods for scoliosis (e.g., AIS) known in the art. For example, one or more of the methods described in WO 2008 / 119170; WO 2012 / 045176; WO 2014 / 201560; WO 2014 / 201557; WO 03 / 073102; WO 2015 / 032005; WO 2018 / 035606; and WO 2019 / 140518 can be used in addition to the method of the present disclosure.

[0081] As used herein the term “progression” in the context of spinal deformity progression refers to an increase in the Cobb angle.

[0082] The term “sample” as used herein is any type of biological sample (from a tested subject or from a control subject(s) (used to assess a reference level)) which may be used in the methods of the present disclosure to measure the level of one or more of the miRNAs of the present disclosure in a subject. MiRNAs are known to be expressed in blood or osteoblasts. As used herein, the term sample encompasses “cell sample” referring to a sample which originally comprised cells (e.g., osteoblasts.) from the subject. The term “blood sample” refers to whole blood sample, or to plasma or serum. The sample as used herein may be a crude sample or a purified sample, it may be processed to a nucleic acid sample.

[0083] Cells samples containing osteoblasts can be obtained intraoperatively with a standard procedure. Typically, they can be obtained intraoperatively from bone specimens originating from vertebrae in all AIS cases. Bony fragments are mechanically reduced in smaller pieces with a bone cutter in sterile conditions and incubated at 37°C in 5% CO2 in a 10-cm culture dish, in presence of alpha-MEM medium containing 10% fetal bovine serum (FBS; certified FBS, Invitrogen, Burlington, ON, Canada) and 1 % penicillin / streptomycin (Invitrogen). After a 30-day period, osteoblasts emerging from the bone pieces are separated at confluence from the remaining bone fragments by trypsinization.

[0084] As used herein the term “reference level” e.g., in the context of miRNA level (e.g., in blood) refers, unless otherwise specified, to a level range or a level considered normal in the art (e.g., spiked-in) or the level in one or more corresponding (i.e., gender and age matched) healthy subject, namely a subject that does not have a scoliosis.

[0085] In another embodiment, the term “reference level” refers to a level range or a level found in one or more age- and sex- (and preferably race-) matched scoliotic subjects whose spinal deformity did not progress between the first scoliosis assessment and skeletal maturity. In another embodiment, it refers to a level range or a level found in one or more age- and sex- (and preferably race-) matched scoliotic subjects whose spinal deformity moderately progressed between the first scoliosis assessment and skeletal maturity. In another embodiment, it refers to a level range or a level found in one or more age- and sex- (and preferably race-) matched scoliotic subject whose spinal deformity severely progressed between the first scoliosis assessment and skeletal maturity (i.e. achieved a Cobb angle 45°). In such cases, the reference level is designated “non progressor reference level”, “moderate progressor reference level” and “progressor reference level”, respectively.

[0086] The term “spiked-in” refers to a defined sequence nucleic acid species (such as an RNA species, sequence or transcript, e.g., Cel-mi R-39-3p) that is added to a sample during processing and used to assess the performance of a qPCR. “Spiked-in” refers to artificial sequences that can include standard or modified nucleotides such as locked nucleic acids (LNAs), peptide nucleic acids (PNA), or nucleic acid analogues (e.g., isoG, isoC, etc.). In some embodiments, the defined sequence nucleic acid comprises a sequence that is not likely to be found in the biological sample to be analyzed and is selected to have minimal self-hybridization and cross hybridization with other similar sequences in the set. Such spiked-in controls can be used to monitor qPCR quality, in terms of dynamic range, reproducibility, etc. Different spiked-in controls can be used to monitor different processes in a qPCR analysis. In some embodiments, the measured degree of hybridization between the spiked-in and the control probes is used to calibrate and normalize the hybridization measurements of the sample RNA or miRNA.

[0087] In specific embodiments, the method includes normalizing the amount of expression of the miRNA marker. The method includes measuring an amount of expression of commercially available spiked-in markers as references against the expression level of miRNA from the subject.

[0088] In specific embodiments, for interpretation of quantitative nucleic acid expression measurements, a normalizer may be needed to correct expression data for differences in sample input, RNA quality, and RT efficiency between samples. In some embodiments, to accurately assess whether increased miRNA is significant, the miRNA expression can be normalized to accurately compare levels of expression between samples, e.g., it is a baseline level against which expression is compared. In quantitative assays, such as for example, quantitative real-time Reverse Transcriptase- PCR (qRT-PCR or qPCR) normalization can be performed using spiked-in markers as references against the expression level of a miRNA under investigation. Normalization includes rendering the measurements of different arrays or PCR or in particular RT-PCR experiments comparable by reducing or removing the technical variability. Within these experiments there exists a multiplicity of sources capable of falsifying the measurements. Possible technical sources of interference are: different efficiency in reverse transcription, labeling or hybridization reactions, as well as problems with the arrays, batch effects in reagents, or lab-specific conditions. By normalization a more robust detection of miRNA expression can occur.

[0089] Typically, miRNA normalization involves use of spiked-in markers that have known fractional cycle number or crossing point. These are utilized as a reference, internal control or reference values in the quantification of miRNA expression. A spiked-in marker exhibits minimum change of expression and transcription across different miRNA samples and thus serves as a control, or reference, for the measurement of variable miRNA activities across different samples. In specific embodiments, fold changes or equivalents thereof for the miRNAs of the present disclosure are normalized to the spiked-in reference miRNAs.

[0090] Receiver Operating Characteristic (ROC) curves can be generated for individual miRNA levels and a linear combination of miRNA levels to determine the cutoff points that yielded the highest combined sensitivity and specificity for predicting spinal deformity progression.

[0091] As used herein the term “subject” is meant to refer to any mammal including human, mouse, rat, dog, chicken, cat, pig, monkey, horse, etc. In particular embodiments, it refers to a human (e.g., a child, adolescent (teenager) or adult which may benefit from any of the methods, compositions, kits or probes sets of the present disclosure). In embodiments, the subject is a female. In embodiments, the subject is a male. In embodiments, the subject has at least one family member which has been diagnosed with IS. In embodiments, the family member is a sibling.

[0092] In specific embodiments, the subject is a Caucasian. Ancestral backgrounds influence genetic makeup and can lead to the prevalence of specific genetic markers in certain ethnic or racial groups, such as miRNAs of the present disclosure. As used herein the term “Caucasian” is meant to refer to subjects of European ancestry. It includes populations in other parts of the world such as North America of European ancestry.

[0093] In specific embodiments, the subject is between 9 and 18 years old of 9 to 17 years old; and has a Cobb angle of 10°. In specific embodiments, the female subject is between 9 and 16 years old or between 9 and 15 years old or between 9 and 14 years old; and has a Cobb angle of 10°.

[0094] As used herein the term “higher'’ in the context of the level of a miRNA of the present disclosure in a subject sample (e.g., blood sample such as plasma or serum) compared to the (corresponding) reference level. In specific embodiments it refers to a level at least 5% higher than the reference level, 10% higher; at least 15% higher; at least 20% higher; at least 25% higher; at least 30% higher; at least 35% higher; at least 40% higher; at least 45% higher; at least 50% higher; at least 55% higher; at least 65% higher; at least 70% higher; at least 75% higher; at least 80% higher; at least 85% higher; at least 90% higher; at least 95% higher; at least 100% higher; at least 110% higher; at least 120% higher; at least 130% higher; at least 140% higher; at least 150% higher; at least 160% higher; at least 170% higher; at least 180% higher; at least 190% higher; at least 200% higher; at least 210% higher; at least 220% higher; at least 230% higher or more than the reference level.

[0095] As used herein the term “lower” in the context of the level of a miRNA of the present disclosure in a subject sample (e.g., blood sample such as plasma or serum) compared to the (corresponding) reference level. In specific embodiments it refers to a level at least 5% lower than the reference level, 10% lower; at least 15% lower; at least 20% lower; at least 25% lower; at least 30% lower; at least 35% lower; at least 40% lower; at least 45% lower; at least 50% lower; at least 55% lower; at least 65% lower; at least 70% lower; at least 75% lower; at least 80% lower; at least 85% lower; at least 90% lower; at least 95% lower; at least 100% lower; at least 110% lower; at least 120% lower; at least 130% lower; at least 140% lower; at least 150% lower; at least 160% lower; at least 170% lower; at least 180% lower; at least 190% lower; at least 200% lower; at least 210% lower; at least 220% lower; at least 230% lower or more than the reference level.

[0096] As used herein the term “similar to” in the context of the level of a miRNA of the present disclosure in a subject sample (e.g., blood sample such as plasma or serum) compared to the (corresponding) reference level, refers to a level identical to the reference level, less than 5% higher (i.e. 4% higher, or 3% higher, or 2% higher or 1% higher) than the reference level, or less than 5% lower (i.e. 4% lower, or 3% lower, or 2% lower or 1 % lower) than the reference level.

[0097] In another aspect, the present disclosure relates to a method of preventing or treating spinal deformity progression in a subject comprising: (i) identifying a subject at risk of spinal deformity progression using the method disclosed herein; and (ii) one or more of adapting an undergoing treatment, selecting a new treatment, determining the frequency of a specific treatment or follow-up schedule; and eventually administering the (adapted or new) treatment to the subject so as to prevent or treat IS.

[0098] Courses of action available from the moment a subject symptomatic for scoliosis (IS such as AIS) is identified / diagnosed as being at risk of spinal deformity progression (high or moderate) include one or more of the following treatments:

[0099] • Application of an external growth guided device which refers to a non-surgical intervention designed to control spinal curvature while allowing the spine to continue growing. These devices include bracing, which externally supports the spine to slow or stop the progression of scoliosis while accommodating natural growth.

[0100] • Non-fusion surgeries, which involve surgical procedures aimed at correcting spinal curvature without permanently fusing the vertebrae, thus preserving the mobility and growth potential of the spine. These surgeries are typically employed for more advanced cases or when external growth guided devices are insufficient. Examples of non-fusion surgery include vertebral body tethering (VBT), a minimally invasive procedure where flexible tethers are attached to the vertebrae, correcting the curvature as the spine grows, and the (dynamic) growing rod technique, where adjustable rods are surgically implanted and periodically lengthened to guide spine growth. Memory alloy staples (e.g., nitinol staples) can also be inserted across vertebrae to modulate growth and stabilize the spine. These surgeries aim to correct the deformity while allowing the spine to grow and maintain flexibility, unlike spinal fusion, which irreversibly fuses vertebrae and halts growth, leading to potential imbalances in the body as other parts, like arms and legs, continue to grow.

[0101] • Spinal fusion surgery, a more invasive option, involving permanently fusing vertebrae to correct and stabilize spinal curvature. However, this solution is less ideal for young adolescents, as it stops spinal growth, which can lead to body imbalances as other limbs continue to grow. This approach may create complications in symmetry and mobility, making it a less favorable choice unless necessary due to the severity of the condition.

[0102] • Prescribing diet to remove certain food products identified as contributors to scoliosis in certain subjects; administering therapeutic agent (e.g., neutralizing antibody specific to OPN, melatonin, selenium, PROTANDIM; HA supplements or HA-rich diet, antibody against CD44 etc.); prescribing postural exercises (e.g., massages, or low intensity pulsed ultrasound (LIPUS)); prescribing acupoint heat sensitive moxibustion, heat therapy with pad, thermal bath, or electroacupuncture; and / or prescribing a frequency for the foregoing one or more treatments. • Alternatively, or in addition to the one or more foregoing treatments, an available course of action is establishing or adapting follow-up schedule (e.g., increasing the number of follow-up visits to the doctor, during for example a 3-, 6- or 12-month period or more). It is recommended to more frequently examine subject who will be identified as at risk for spinal deformity progression versus those considered not at risk through the methods presented herein in order to better manage appointments and the waiting list, thus prioritizing patients who require closer medical follow-up.

[0103] As used herein, the treatment of a subject symptomatic for scoliosis (IS such as AIS) identified / diagnosed as being at risk of spinal deformity progression (high or moderate) by the method of the present disclosure may be one or more of the foregoing treatments or follow up. In specific embodiments, the treatment is non-fusion surgery, growth guided device or spinal fusion surgery, preferably non-fusion surgery.

[0104] Courses of action available from the moment a subject symptomatic for scoliosis (IS such as AIS) is identified / diagnosed as not being at risk of spinal deformity progression include one or more of the following:

[0105] • Application of an external growth guided device as defined above (e.g. bracing).

[0106] • Prescribing diet to remove certain food products identified as contributors to scoliosis in certain subjects; administering therapeutic agent (e.g., neutralizing antibody specific to OPN, melatonin, selenium, PROTANDIM; HA supplements or HA-rich diet, antibody against CD44 etc.); prescribing postural exercises (e.g., massages, or low intensity pulsed ultrasound (LIPUS)); prescribing acupoint heat sensitive moxibustion, heat therapy with pad, thermal bath, or electroacupuncture; and / or prescribing a frequency for the foregoing one or more treatments.

[0107] • Alternatively, or in addition to the one or more foregoing treatments, an available course of action is establishing or adapting follow-up schedule (e.g., decreasing the number of follow-up visits to the doctor, during for example a 3-, 6- or 12-month period ore more or decreasing the number of x-rays during for example a 3-, 6- or 12-month period or more).

[0108] As used herein the term “treating” or “treatment” in reference to scoliosis (e.g., IS or AIS) in a symptomatic subject is meant to refer to at least one of a reduction of Cobb angle in a preexisting spinal deformity; improvement of column mobility, preservation / maintenance of column mobility; improvement of equilibrium and balance in a specific plan; preservation / maintenance of equilibrium and balance in a specific plan; improvement of functionality in a specific plan; preservation / maintenance of functionality in a specific plan; cosmetic improvement; and combination of any of the above.

[0109] As used herein the term “preventing” or “prevention” in reference to scoliosis (e.g., IS or AIS) in a symptomatic subject is meant to refer to at least one of a reduction in the progression of a Cobb angle in a patient having a scoliosis; a reduction in the deterioration of column mobility; a reduction in the deterioration of equilibrium and balance in a specific plan; a reduction in the deterioration of functionality in a specific plan; a preservation / maintenance or a reduction in the deterioration of cosmetic improvement; and combination of any of the above. In another aspect, the present disclosure concerns a composition, kit or probes set for use in methods disclosed herein.

[0110] In another aspect, the present disclosure concerns a probes set (e.g., chip). Preferred probe sets are designed to detect expression of one or more (e.g., two, three, four, five, six, seven or eight or more) miRNAs of the present disclosure and provide information about the risk for spinal deformity progression. Particularly preferred probe sets comprise probes either labeled (e.g., fluorescent label, quencher, etc.) or unlabeled, that are capable of detecting the miRNAs of the present disclosure. Probe sets are particularly useful because they are smaller and cheaper than probe sets that are intended to detect as many miRNAs as possible in a particular genome. The probe sets are targeted at the detection of miRNAs that are informative about the risk for spinal deformity progression. Probe sets may also comprise a large or small number of probes that detect miRNAs that are not informative about the risk for spinal deformity progression. Such probes are useful as controls and for normalization (e.g., spiked-in markers). Probe sets may be a dry mixture or a mixture in solution. In some embodiments, probe sets can be affixed to a solid substrate to form an array of probes. It is anticipated that probe sets may also be useful for multiplex PCR. The probes may be nucleic acids (e.g., DNA, RNA, chemically modified forms of DNA and RNA), LNAs (Locked nucleic acids), or PNAs (Peptide nucleic acids), or any other polymeric compound capable of specifically interacting with the desired nucleic acid sequences.

[0111] In another aspect, the present disclosure concerns a kit for detecting a level of at least one (e.g., two, three, four, five, six, seven or eight or more) miRNA(s) in a subject sample and / or for determining whether the subject is at risk of developing IS comprising reagents for specifically detecting the at least one (e.g., two, three, four, five, six, seven or eight or more) miRNA(s) in the subject sample. The “reagents” may comprise, for example, one or more (e.g., two, three, four, five, six, seven or eight or more) oligonucleotide probes or primers or primers or a probes set as described herein. In other embodiments, the kit further comprises one or more of (a) at least one tool, reagent or media for the isolation of the subject sample (e.g., syringe) and (b) a sample (e.g., blood such as serum or plasma sample) from the subject.

[0112] In specific embodiments of the kit or probes set, each of the oligonucleotide probes or primers specific for detection of at least one (e.g., two, three, four, five, six, seven or eight or more) miRNA in the subject sample, is specific for a miRNA of Table IX. In more specific embodiments, the kit or probes set comprise oligonucleotide probes or primers specific for the detection of a panel / plurality of miRNAs as presented in any one of FIGs. 6A to 6E. In specific embodiments, the kit or probes set comprise oligonucleotide probes or primers specific for the detection of (i) miR-1 - 3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b-3p, and optionally miR-18a-3p; or (ii) let-7f-5p, miR-18a-3p, miR-103a-3p, and miR-551b-3p in a female subject’s sample; or (iii) miR-551b-3p and miR-4665-5p.

[0113] Oligonucleotides probes or primers specific for detection of the miRNAs of the present disclosure have a length of 18 to 23 nucleotide corresponding to the full-length miRNA of the present disclosure (e.g., 21 , 22 or 23 nucleotides) or to a portion thereof. These probes are complementary and specific to the target miRNAs of the present disclosure or to their cDNA and may specifically hybridize thereto under stringent conditions. Oligonucleotides probes or primers can be synthesized using methods well established in the field to ensure complementarity to the target RNA or cDNA sequence. Additionally, they can be modified to include fluorescent tags when used as probes, or transformed into chimeric oligonucleotides incorporating mixed DNA and RNA bases, 2'-O-methyl (2'-0Me) RNA, 2'-fluoro (2'-F) RNA, or other chemical modifications. These modifications can enhance probe stability, binding affinity, and specificity, particularly in qPCR assays. In specific embodiments, the oligonucleotides probes or primers specific for detection of the miRNAs of the present disclosure are synthesized based on sequences presented in Tables VIII and IX below.

[0114] In embodiments, there is provided a composition (e.g., a diagnostic composition) or assay mixture which is generated following one or more steps of the methods describe herein and which include a subject sample (e.g., cell sample, blood sample, plasma sample, serum sample, etc.) from the subject to be tested, and means or reagents for detecting the nucleic acids, and / or SNPs described herein. The preparation of such composition occurs while testing a subject’s sample for the risk of developing a more severe scoliosis; for aiding in the prevention and treatment of scoliosis including for determining the best treatment regimen; for adapting an undergoing treatment regimen; for selecting a new treatment regimen, for determining the frequency of a specific treatment regimen or follow-up schedule or for classifying the subject into a particular genetic group or endophenotype. Such compositions may be prepared using kits and / or probes set described herein.

[0115] In specific embodiments of the methods, kits, probes set, or composition of the present disclosure, one or more other miRNA can be used instead or in addition to those listed in Table IX and FIGs 6A-E.

[0116] In another aspect of the present disclosure includes a computer program suitable for implementing any of the methods of the present disclosure. In addition, a device comprising the above-mentioned computer program also forms part of the present disclosure as well as its use for the diagnostic / prognostic of the risk for spinal deformity progression in a subject symptomatic for scoliosis.

[0117] The present disclosure is illustrated in further detail by the following non-limiting examples.

[0118] EXAMPLE 1 : Material and methods

[0119] Study design and populations. This cross-sectional study is part of a larger longitudinal prospective study conducted in three pediatric spine centers. The participants were recruited from June 2002 to August 2013, and data collection was completed in June 2016. Healthy controls (HC) without a family antecedent of scoliosis were recruited during the same period from primary and high schools from the Greater Montreal’s area. The inclusion criteria concerned children aged 13.3 ± 1 .7 years enrolled at their initial appointment at the scoliosis clinic with a history and physical examination consistent with AIS diagnosis. The diagnosis was confirmed by radiography with a minimum curvature in the coronal plane of 10° (mean Cobb angle value of 24.4 ± 12.4), showed by a standing postero-anterior spinal radiograph, by the Cobb method with vertebral rotation and without any known congenital or genetic disorder. All participants were skeletally immature with a Risser sign between 0 and 2, no prior treatment, and female participants were either at pre- menarche or less than one-year post-men arche. All AIS patients were followed until they reached their skeletal maturity (Risser sign of 4 or 5) or underwent surgery for a spinal deformity correction. 116 AIS patients met the eligibility criteria, which allowed their stratification according to their clinical outcome. The AIS patients were categorized into three groups. The first group developed over time a severe scoliosis (final Cobb angle 45°) requiring corrective spinal surgery and represents severe disease progressors (P). The second group was considered moderate progressors exhibiting moderate curve progression (MOP) exhibiting a scoliosis with a final Cobb angle varying between 25° and 44° inclusively at skeletal maturity and avoided surgery. The third group, termed non progressors (NP), represents individuals who did not exhibit a significant spinal deformity progression with a final Cobb angle < 15° at skeletal maturity. Age- and sex-matched healthy control (HC) subjects were recruited from primary and high schools and were also physically examined by a seasoned spine surgeon to rule out any form of scoliosis before enrolment. All the HC subjects were interviewed about their familial antecedents, and those with a familial history of AIS, secondary or syndromic scoliosis were excluded from the study. This study was approved by the institutional review boards of Sainte- Justine University Hospital (protocols #2018-1935), The Montreal Children’s Hospital, The Shriners Hospital for Children and McGill University as well as by the Affluent and Montreal English School Boards. Written informed consents were given by parents or legal guardians and assents were obtained from all minors. All methods were carried out by relevant guidelines and human ethic regulations.

[0120] Blood specimen collection. Peripheral blood samples of participants were collected in EDTA-treated tubes and centrifuged at 11 ,000 x g for 10 minutes. Derived plasma samples were aliquoted and stored at -80°C until analysis.

[0121] RNA extraction formicroRNA array analysis. MiRNAs were extracted from plasma samples obtained from AIS patients and matched healthy controls. Plasma samples were thawed and centrifugated at 17,000 x g for 15 minutes at 4°C. The RNA was extracted using the NucleoSpin™ miRNA Plasma kit (Macherey Nagel) according to the manufacturer's instructions. For miRNA extraction from human osteoblasts, the mirVana™ PARIS extraction kit was used and host miR-16-5p was measured as an internal control for expression analysis in AIS patients (surgical cases) and non- scoliotic controls (trauma cases).

[0122] Microarray analysis. A genome-wide expression profiling was performed for each participant in the discovery cohort at Oaklabs (Hennigsdorf, Germany), using the Agilent™ expression array-Human miRNA 8x60K (Agilent Technologies, Santa Clara, CA, USA) harboring 2549 mature human miRNAs. Only miRNAs meeting the criteria both p-value threshold is 0.05 and the Iog2 (Fold Change) > 0.6 were selected.

[0123] Validation of candidate miRNAs in replication cohort by qPCR. The plasma samples from the biobank cohort were thawed on ice for 15 minutes, followed by centrifugation for 15 minutes at 17,000x g at 4°C to remove any remaining cellular debris. RNA extraction with enrichment of small RNAs was performed using the mirVana™ PARIS extraction kit (mirVana PARIS RNA and Native Protein Purification Kit, Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer's instructions. 75pil of eluent solution was used to elute the RNAs from the filter cartridge, and RNA samples were stored at -80°C. As a spike-in control, 50 nmol of Cel-miR-39-3p synthetic oligonucleotide RNA with the sequence: 5’-UCACCGGGUGUAAAUCAGCUUG-3’ (SEQ ID NO: 1) (Thermo Fisher Scientific) was added to the plasma after addition of a denaturing solution. For osteoblast samples, endogenous host miRNA miR-16-5p 5’ with the sequence: UAGCAGCACGUAAAUAUUGGCG-3’ (SEQ ID NO: 2) was used as a control.

[0124] Complementary DNA (cDNA) synthesis, qPCR miRNA detection and quantification. cDNA was synthesized from the extracted miRNA samples using a PCR thermocycler (T3000 Thermocycler™, Biometra, Montreal Biotech Inc., Montreal, QC, Canada) and the TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific) by following manufacturer's instructions. The TaqMan™ Advanced miRNA cDNA Synthesis Kit uses a 3' poly-A tail and a 5' ligation of an adapter sequence to extend the mature miRNAs present in the sample at both ends before reverse transcription. The universal RT primers recognize the universal sequences present at the extended 5' and 3' ends of the mature miRNAs and all mature miRNAs in the sample are transcribed into cDNA. The resulting cDNA samples were stored at -20°C. The synthesized cDNA was the template for qPCR using the TaqMan™ Advanced miRNA Assays (Thermo Fisher Scientific) and probes for each miRNA. The qPCR reaction was performed using the QuantStudio™ 3 instrument (Thermo Fisher Scientific). The qPCR was performed in duplicate for each sample, and the mean of the obtained cycle thresholds (CT) was used for calculations. qPCR data analysis. The results of each miRNA for each sample were normalized with the results of the exogenous control, namely cel-mir-39a-3p for plasma samples, and miR-16-5p for osteoblast samples. ACysample is the individual expression difference (miRNA of interest in subject / patient vs control miRNA) = CT miR - CT control. Mean ACT control is the mean of ACT for all the control samples. AACT is a comparison of patients / subject ACT sample with the control ACT. The AACT was calculated for each sample as follows, AACT = ACT sample - mean ACT control. The Fold change represents the relative expression level of each miRNA in the subject between each subject and the mean of controls and was calculated as 2<-MCT).

[0125] Construction of gene pathways and networks targeted by dysreguiated miRNAs in AiS. The potential targets of miRNAs of interest, including genes, and diseases were primarily identified through the previous studies. The connections (interactions) of the miRNAs and their targets were constructed based on the Ingenuity Knowledge Base using the Ingenuity Pathway Analysis™ (IPA) software (QIAGEN Inc. software version 51963813).

[0126] Machine teaming and statistical analyses. Random Forest Model (RFM) was applied to build a model that could predict and differentiate between non-severe and severe groups. The data was randomly separated into training data set (80%) and testing data set (20%): the training data set was utilized to train the RFM and the testing data was used to assess the model. To evaluate the RFM, different measures were used to achieve the performance of the predictive algorithm such as accuracy, specificity, sensitivity, and receiver operating characteristic (ROC) curves. The ROC curves showed the trade-off between sensitivity and specificity, and the area under the curve (AUC) was used as an index for evaluating the predictive performance of the constructed miRNA panel.

[0127] Osteoblasts. Osteoblasts were obtained intraoperatively from bone specimens originating from vertebrae in all AIS cases (varying from T3 to L4 according to the surgical procedure performed) with the exception of trauma cases used as non-scoliotic controls, where bone fragments were obtained from humerus, tibia or femur. Bony fragments were mechanically reduced in smaller pieces with a bone cutter in sterile conditions and incubated at 37°C in 5% CO2 in a 10-cm culture dish, in presence of modified minimum essential medium (alpha-MEM medium Wisent, Saint-Bruno, QC, Canada) containing 10% fetal bovine serum (FBS; certified FBS, Thermo Fisher Scientific, Waltham, MA, USA) and 1 % penicillin / streptomycin (Thermo Fisher Scientific). Culture media was renewed every three days and cells were allowed to grow until confluence. After a 30-day period, osteoblasts emerging from the bone pieces were separated at confluence from the remaining bone fragments by trypsinization. Statistics Multiple comparisons of means were performed with one-way ANOVA and only. Only P values < 0.05 were considered significant.

[0128] EXAMPLE 2: Study populations

[0129] The clinical and demographic characteristics of the discovery cohort are reported in Table I, below. Stratification by scoliosis severity was determined only in the participants who have completed their longitudinal follow-up and reached their skeletal maturity. This discovery cohort consisted of 34 AIS patients (21 F / 13M) classified as spinal deformity progressors (P), 35 AIS patients (20F / 15M) classified as non-progressors (NP), and 14 matched healthy controls (9F / 5M). Table II below shows the characteristics of the initial validation cohort used to assess the capacity of the inventors’ qPCR probes to amplify the candidate miRNAs of the disclosure. This second cohort was composed of six female AIS patients classified as spinal deformity progressors (P), six female AIS patients classified as non-progressors (NP), and six matched healthy controls (5F / 1 M). Table III below shows the characteristics of the replication cohort, which was composed of 15 AIS patients (11 F / 4M) classified as spinal deformity progressors (P), 20 AIS patients (11 F / 9M) classified as non-progressors, and 10 matched healthy controls (4F / 6M).

[0130] Table I. Clinical and demographic characteristics of participants - discovery cohort

[0131] Data are expressed as a mean ± standard deviation. P=progressors; NP=non- progressors; MOP=moderate progressors; HC=healthy controls; F=females and M=males.

[0132] Table II. Summarized clinical and demographic characteristics of participants - validation cohort 1

[0133] Data are expressed as a mean ± standard deviation. P=progressors; NP=non- progressors; HC=healthy controls; F=females and M=males.

[0134] Table III. Summarized clinical and demographic characteristics of participants - validation cohort 2

[0135] Data are expressed as a mean ± standard deviation. P=progressors; NP=non- progressors; MOP=moderate progressors; HC=healthy controls; F=females and M=males. able IV. Clinical and demographic characteristics of participants - validation cohort 1

[0136] able V. Clinical and demographic characteristics of participants - validation cohort 2

[0137]

[0138] EXAMPLE 3: Subclassification of AIS patients with scoliosis progression less than 45°

[0139] Initially, all patients diagnosed with AIS and exhibiting a spinal deformity less than 45° (considering only the main curve) at skeletal maturity were grouped in the non-progressor (NP) group. However, it was observed that these patients could be categorized into two distinct groups. Indeed, some patients displayed a Cobb angle progression of more than 15° over time, leading the inventors to classify them as moderate progressors (MOP). Conversely, patients exhibiting a progression of less than 15° were classified as non-progressors (NP). This refined stratification (See Table VI below) was applied during the replication cohort, resulting in the classification of 9 patients in the MOP group (6F / 3M) and 11 (5F / 6M) in the NP group.

[0140] Table VI - Refined stratification of subjects

[0141] EXAMPLE 4: Identification of circulating miRNAs associated with AIS

[0142] A comprehensive genome-wide expression profiling of circulating miRNAs was conducted using plasma samples from the discovery cohort. The Agilent™ expression array-Human miRNA 8x60K chips was employed to identify candidate circulating miRNAs differentially expressed in AIS patients compared to HC group. The initial screening revealed 90 candidate miRNAs exhibiting the most significant fold change with a P value <0.05, listed in Table VII below.

[0143] Table VII. List of circulating miRNAs differentially expressed in AIS and identified by miRNA array (discovery cohort).

[0144] EXAMPLE 5: Validation and replication assays of identified circulating miRNAs

[0145] Selected candidate miRNAs were validated initially by RT-qPCR using a distinct small cohort of AIS patients (n=12) and HC (n=6) (Table II, above). The fold difference in miRNA expression between each patient and the mean of HC group was calculated as 2-AACT.

[0146] Of note, only 70 miRNAs out of 90 initially discovered using the Agilent™ microarray (Table VIII below) could be amplified. Subsequently, these 70 miRNAs were validated in a larger cohort comprising 35 AIS patients and 10 matched HC (Table III, above).

[0147] Table VIII. List of circulating miRNAs associated with AIS and validated by qPCR (validation cohort 1)

[0148] Among the 70 candidate miRNAs, 16 miRNAs (let-7f-5p, miR-1 -3p, miR-18a-3p, miR-19a-3p, miR-19b-3p, miR-103a- 3p, miR-107, miR-133b, miR-143-3p, miR-148a-3p, miR-148b-3p, miR-152-3p, miR-214-3p, miR-551b-3p, miR-576- 5p and miR-4665-5p, listed in Table IX below) were significantly upregulated in AIS P group developing a severe scoliosis (Cobb angle ^45°) compared to MOP+NP group developing mild-to-moderate scoliosis (Cobb angle 10°- 44°) (FIGs. 1A-0 and FIGs. 2A-O).

[0149] Table IX: miRNAs significantly upregulated in P group developing a severe scoliosis (Cobb angle ^45°) including known polymorphisms thereof

[0150]

[0151] Table X: Reverse, complements and reverse-complements oligonucleotides for miRNAs of Table IX

[0152]

[0153]

[0154] EXAMPLE 6: Sex-specific differences in circulating miRNA expression

[0155] Given the higher prevalence of AIS in females compared to males, a more in-depth investigation was conducted into how biological sex impacts the expression profile of these miRNAs. Intriguingly, distinct expression patterns were observed depending on the severity of the disease in each sex. In females, one of the 65 miRNAs, miR-18a-3p, was significantly upregulated in the female severe group (FP) compared to the females classified in the non-severe group (FMOP+FNP), as shown in FIGs. 3A-B. On the other hand, in males, two miRNAs including miR-103a-3p, and miR- 107 were significantly downregulated in the male non-progressor group (MNP) compared to healthy controls (FIGs. 4A-D).

[0156] Additionally, miR-4665-5p and miR-551b-3p displayed significant upregulation in the male moderate progressor group (MMOP) compared to the male non-progressor group (MNP) (FIGs. 5A-D). It is noteworthy to mention that miR-4665- 5p, although not initially included in the top 15 miRNAs, emerged as a key miRNA associated with moderate spinal deformity progression in male patients with AIS.

[0157] EXAMPLE 7: AIS-associated miRNAs in osteoblast

[0158] Osteoblast samples were derived and obtained by intraoperative bone biopsies of 11 female AIS surgical cases with a mean Cobb angle of 59.7° ± 10.5° and three female non-scoliotic trauma cases as controls, as reported in Table XI below.

[0159] Table XI: Clinical and demographic characteristics of surgical AIS cases and control cases used for validation of osteoblast miRNAs

[0160] The data are represented as mean ± standard deviation.

[0161] MiRNA expression analysis in patients and healthy controls was conducted as described in Example 1 .

[0162] Five microRNAs (miR-103a-3p, miR-107, miR-148a-3p, miR-148b-3p, and miR-152-3p) were tested in human osteoblasts (AIS vs non-scoliotic trauma controls). The elevation of miR-148b-3p was confirmed in osteoblasts of subjects with severe scoliosis (FIGs. 7A and B).

[0163] EXAMPLE 8: AIS-associated circulating miRNAs and prediction of scoliosis severity

[0164] The clinical utility of the panel of 15 circulating miRNAs was evaluated to predict the development of a severe scoliosis using the Random Forest Model (RFM). RFM was first applied to a randomly selected training dataset representing 80% of all the AIS cases presented in Example 2. The performance of the models was then challenged using the remaining 20% of these AIS cases as testing dataset. Receiver operating characteristic curves (ROC curves) were generated, and the following results were obtained. When classifying patients with severe scoliosis (P) versus patients with mild scoliosis (NP), a panel of six miRNAs (miR-1-3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, miR- 148b-3p) showed an accuracy, specificity, and sensitivity of 100%, with a ROC curve AUC = 1.0 in predicting the risk of developing a severe scoliosis in symptomatic patients from both sexes (FIG. 6A).

[0165] The inclusion of all 15 miRNAs in the algorithm slightly reduced the accuracy (90%) and specificity (88%) of the test, without affecting its sensitivity (100%) (FIG. 6B).

[0166] In the context of sex-specific miRNAs, two additional panels of miRNAs can predict female curve progression with the accuracy, specificity and the sensitivity of 100% and a ROC AUC = 1 .0. The first panel includes seven miRNAs (miR- 1-3p, miR-18a-3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, miR-148b-3p) (FIG. 6C). The second panel includes four miRNAs (let-7f-5p, miR-18a-3p, miR-103a-3p, miR-551 b-3p) (FIG. 6D). Also, two miRNAs (miR-551b-3p and miR-4665-5p) were identified predicting moderate spinal deformity progression in males with a similar accuracy, specificity and the sensitivity of 100% and a ROC AUC = 1.0 (FIG. 6E).

[0167] EXAMPLE 9: Gene pathways and network analysis

[0168] A systematic gene pathway and network analysis was first applied to better understand the mechanistic contribution of the miRNAs of the disclosure in AIS pathogenesis. A gene pathway analyses using the Ingenuity Pathway Analysis (IPA) software revealed that a majority of the miRNAs of the present disclosure were primarily involved in muscle dystrophy, skeletal muscular disorders, muscle overgrowth, cardiac muscle disorders, progressive dystrophy, and other muscles related diseases (FIG. 8). Hence, two of the miRNAs of the disclosure (miR-1 -3p and miR-133b) have been previously identified as myomiRs (Horak, 2016). A hybrid approach of combining IPA and manual curations was then applied. The literature was first searched, and genes and molecules reported to be associated with AIS or scoliosis were manually identified. Connections with each of the miRNAs disclosed herein were then built, and these genes and functions based on the IPA experimentally observed Ingenuity Knowledge Base. This comprehensive analysis allowed the inventors to construct more complete networks that connected each miRNA to its targets (e.g., AIS-related genes) that could play critical roles in the pathogenesis of AIS. Using the IPA and manual curation hybrid approach, a larger and more complete network connecting these 15 miRNAs and their key genes, and disease targets were further constructed. Similar approach by integrating previous scoliosis severity associated miRNAs from the literature showed that these miRNAs (miR-96-5p, miR-145-5p, miR-151 a-3p) share common targets with the targets of the candidate miRNAs of the present disclosure (FIG. 9).

[0169] The scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

1. CLAIMS:1 . A method of diagnosing the risk of spinal deformity progression in a subject symptomatic for an idiopathic scoliosis comprising:(a) measuring a level of expression of at least the following biomarkers:(i) miR-1 -3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b-3p in a subject’s sample;(ii) let-7f-5p, miR-18a-3p, miR-103a-3p, and miR-551 b-3p in a female subject’s sample; or(iii) miR-551 b-3p and miR-4665-5p in a male subject’s sample; wherein: in (i) a level of expression of each of miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b-3p higher than corresponding reference levels; in (ii) a level of expression of each of let-7f-5p and miR-103a-3p similar to a corresponding reference level, and a level of expression of each of miR-18a-3p and miR-551b-3p higher than a corresponding reference level; or in (iii) a level of expression of miR-551 b-3p higher than a corresponding reference level and a level of expression of miR-4665-5p lower than a corresponding reference level, is an indication that the subject’s spinal deformity will progress.

2. The method of claim 1 , wherein the subject is a human.

3. The method of claim 1 or 2, wherein the subject’s sample is blood, plasma or serum.

4. The method of any one of claims 1 to 3, wherein the subject is between 9 and 18 years old or between 9 and17 years and has a Cobb angle of 10°.

5. The method of any one of claims 1 to 4, wherein in (a) the biomarkers (i) are measured.

6. The method of claim 5, wherein the subject is a female.

7. The method of claim 6, further comprising measuring a level of expression of miR-18a-3p, wherein a level of expression of miR-18a-3p higher than a corresponding reference level is a further indication that the subject’s spinal deformity will progress.

8. The method of any one of claims 1 to 3, wherein in (a) the biomarkers (ii) are measured.

9. The method of any one of claims 5 to 8, wherein the method provides an indication that the subject’s spinal deformity will reach a Cobb angle of 45°.

10. The method of any one of claims 5 to 10, wherein the subject in (a) is between 9 and 16 years old or between 9 and 15 years old or between 9 and 14 years old; and / or has a Cobb angle of 10°.11 . The method of any one of claims 1 to 4, wherein in (a) the biomarkers (iii) are measured.

12. The method of claim 11 , wherein the method provides an indication that the subject’s spinal deformity will increase of > 15 ° while not exceeding a final Cobb angle of 44 °.

13. The method of any one of claims 1 to 12, wherein the subject has adolescent idiopathic scoliosis.

14. The method of any one of claims 1 to 13, wherein the subject is Caucasian.

15. The method of any one of claims 1 to 14, further comprising (b) treating the subject with one or more of a growth guided device or surgery, preferably a surgery (e.g., non-fusion surgery) when the level of expression (i), (ii) or(iii) measured in (a) is indicative that the subject’s spinal deformity will progress.

16. Kit comprising:(a) (i) reagents for specifically detecting each of miR-1 -3p, miR-19a-3p, miR-19b-3p, miR-133b, miR-143-3p, and miR-148b-3p;(ii) reagents for specifically detecting each of let-7f-5p, miR-18a-3p, miR-103a-3p, and miR-551 b-3p; or(iii) reagents for specifically detecting each of miR-551 b-3p and miR-4665-5p; and(b) optionally (i) instructions for using the kit to diagnose the risk of spinal deformity progression in a subject symptomatic for an idiopathic scoliosis; (ii) at least one tool, reagent or media for the isolation of the subject sample; or (iii) a combination of (i) and (ii).

Images