Genome-Wide Profiling of Circulating MicroRNAs in Adolescent Idiopathic Scoliosis

Adolescent Idiopathic Scoliosis (AIS) is the most common orthopedic condition requiring surgery, affecting 4% of adolescents. There is currently no proven method or prognostic test to identify 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. In this prospective cross-sectional study, we investigated the clinical utility of circulating microRNAs (miRNAs), an important class of small non-coding RNA, as biomarkers to predict the risk of developing severe scoliosis in AIS. 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 15 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 15 led us to develop an algorithm predicting the risk of developing a severe scoliosis with great accuracy (100%), sensitivity (100%) and specificity (100%). Having a scoliosis predictive bioassay and decision-making tools to predict curve progression in order to find the best treatment plan will undoubtedly transform the orthopedic care system in the field of pediatric scoliosis by integrating innovative precision medicine approaches. In addition, investigation of genes targeted by these miRNAs could fill our gaps in our understanding of AIS pathogenesis and reveal new actionable targets. Study population: This cross-sectional study is part of a larger longitudinal prospective study conducted in three pediatric spine centers. 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. 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 (21F/13M) classified as spinal deformity progressors (P), 35 AIS patients (20F/15M) classified as non-progressors (NP), and 14 matched healthy controls (9F/5M). A second subgroup was established to assess the capacity of our qPCR probes to amplify our candidate miRNAs, 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/ 1M). Finally a replication cohort, which was composed of 15 AIS patients (11F/4M) classified as spinal deformity progressors (P), 20 AIS patients (11F/9M) classified as non-progressors, and 10 matched healthy controls (4F/6M) was used. Identification of circulating miRNAs associated with AIS: We conducted a comprehensive genome-wide expression profiling of circulating miRNAs using plasma samples from our discovery cohort. We employed the Agilent expression array-Human miRNA 8x60K chips to identify candidate circulating miRNAs differentially expressed in AIS patients compared to HC group. Validation and replication assays of identified circulating miRNAs: Selected candidate miRNAs were validated initially by RT-qPCR using a distinct small cohort of AIS patients (n=12) and HC (n=6). Of note, we were able to amplify only 70 miRNAs out of 90 initially discovered using the Agilent microarray. Subsequently, these 70 miRNAs were validated in a larger cohort comprising 35 AIS patients and 10 matched HC. Study population: This cross-sectional study is part of a larger longitudinal prospective study conducted in three pediatric spine centers. 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. 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 (21F/13M) classified as spinal deformity progressors (P), 35 AIS patients (20F/15M) classified as non-progressors (NP), and 14 matched healthy controls (9F/5M). A second subgroup was established to assess the capacity of our qPCR probes to amplify our candidate miRNAs, 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/ 1M). Finally a replication cohort, which was composed of 15 AIS patients (11F/4M) classified as spinal deformity progressors (P), 20 AIS patients (11F/9M) classified as non-progressors, and 10 matched healthy controls (4F/6M) was used. Identification of circulating miRNAs associated with AIS: We conducted a comprehensive genome-wide expression profiling of circulating miRNAs using plasma samples from our discovery cohort. We employed the Agilent expression array-Human miRNA 8x60K chips to identify candidate circulating miRNAs differentially expressed in AIS patients compared to HC group. Validation and replication assays of identified circulating miRNAs: Selected candidate miRNAs were validated initially by RT-qPCR using a distinct small cohort of AIS patients (n=12) and HC (n=6). Of note, we were able to amplify only 70 miRNAs out of 90 initially discovered using the Agilent microarray. Subsequently, these 70 miRNAs were validated in a larger cohort comprising 35 AIS patients and 10 matched HC.