smallRNA sequencing of circulating miRNAs in blood from LGMD patients

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of progressive and genetic neuromuscular disorders that involve weakness and wasting of predominantly pelvic and shoulder girdle muscles. More than 30 subtypes have been identified, with variable phenotype. In the early stages of the disease, these LGMD share common clinical and histopathological characteristics among different subtypes and with other neuromuscular disorders, complicating the disease-subtype identification and thus, lengthening the final diagnosis of the disease. In the present study, we try to identify by a non-invasive method a molecular signature including biochemical and epigenetic parameters with a potential value for LGMD patient prognosis and stratification. Circulating miRNome was obtained by smallRNA-seq in plasma samples from LGMD patients (n=6) and matched controls (n=6). Over-representation analysis (ORA) using GO terms and KEGG database was used to study the functional enrichment of target genes. Biochemical parameters related to calcium metabolism, hormonal status, and muscle metabolism were also analyzed in these patients. Thirteen differentially expressed miRs (FDR < 0.05) were able to separate each clinical group by hierarchical clustering. Indeed, most of differentially expressed miRs were up-regulated. Functional enrichment analysis of the target genes for the dysregulated miRs revealed that targets of up-regulated miRs modulated cell cycle and cancer related pathways, while targets of down-regulated miRs were involved in muscle tissue development, regeneration and senescence. Four of the circulating miRs (miR-19b-3p, miR-192-5p, miR-122-5p and miR 323-3p) were further validated by qPCR in LGMD and in the more frequent muscular dystrophies: Duchenne (DMD)(n=5) and facioscapulohumeral muscular dystrophy (FSHD)(n=4). Receiver operating characteristic curve analysis revealed high AUC values for selected miRs (1.0 for miR-19b-3p, miR-192-5p and miR-122-5p; 0.75 for miR-323-3p) suggesting that these miRs could be good biomarker candidates for LGMD. The covariates gender, age at disease onset, and ambulation did not correlate with the expression levels of these miRs. However, differential expression of miR-122-5p (=10-60 fold, p< 0.0001), miR-192-5p (=10-60 fold, p< 0.0001) and miR-323-3p (= 2-10 fold, p<0.05) levels compared to matched controls could discriminate LGMD from DMD or FSHD dystrophies. In addition, a strong correlation between some of these circulating miRs and biochemical variables such as CK, vitamin D3, ALP and PTH was found only in LGMD patients. Overall design: A total of six patients diagnosed of LGMD at Hospital Universitari I Politècnic La Fe de Valencia (Spain) were used for the miRNome analysis. A cohort of patients of DMD (n=5) and patients affected by FSHD (n=4) were also included for further validation of results obtained by smallRNA-seq and to compare the miRs differentially expressed in LGMD with other neuromuscular diseases. Informed written consent was obtained from all participants. Blood samples were taken by venipuncture in the Hospital Universitari I Politècnic La Fe and immediately transported, processed, and stored according to the standard operating procedures in the CIBERER Biobank for further research (www.ciberer-biobank.es).Age and gender-matched controls (n=15) for all groups were provided by IBSP-CV Biobank and processed in the same way. The selection process and all experimental methods were carried out in accordance with the relevant clinical guidelines, following standard operation procedures, and with the approval of the ethics and scientific committees (Ethics Committee from the Hospital Universitari I Politècnic La Fe, Registry number 2019/0094).