Plasmatic expression profiles of microRNAs miR-200c and miR-210 in patients with differences of sexual development (DSD) 46,XY of unknown etiology

Introduction: Differences of Sexual Development (DSD) affect 1: 1000-4500 live births and are defined as changes in which chromosomal, gonadal or genital development are atypical. Patients with 46,XY DDS have a broad phenotypic spectrum and many have atypical genitalia at birth. The diagnostic evaluation of a patient with DSD includes hormonal measurements, radiological, cytogenetic and molecular studies. However, a significant percentage of patients remain without etiology and are classified as DSD of unknown etiology. This fact suggests that epigenetic mechanisms, i.e. not restricted to DNA and, therefore, not contemplated by usual molecular analyzes, such as Sanger and Exoma sequencing, may play a role in its development. Objectives: In order to better evaluate these patients with unknown etiology, this project investigated the plasma expression profile of microRNAs (miRNAs), small non-coding protein RNAs, which bind complementarily to the 3 ?UTR region of target mRNA, blocking the protein synthesis. Methodology: In silico and literature analysis were used to select miRNAs miR-34c, miR-200c, miR-210, and references miR-23a and miR-451. RNA was extracted from the plasma of patients and controls with the MagMAX mirVana Total RNA kit (Thermofisher) after analysis of hemolysis by absorbance at 414 nm. RT-qPCR reaction was performed with the TaqMan Advanced miRNA cDNA and Advanced miRNA Assay kits (Thermofisher). The data were analyzed by ?Ct and 2-??Ct. Patients and controls were divided into two groups, prepubertal (according to testicular volume <3ml and Testosterone level <30ng/dL), and post- pubertal group. Additionally, patients were subdivided into 2 subgroups according to the External Masculinization Score (EMS), into Poorly Virilizated (PVG, score<5) and Moderatly Virilizated (MVG, score>5). For age expression analysis controls were grouped as follows: 1- 12 years, 13-20 years, 21-30 years, 31-40 years and 41-55 years. Results: Plasma expression of miR-200c was higher in the groups aged 13-20 to 41-55 years than in the group aged 1-12 (ANOVA p=0.0067). In addition, all groups over the age of 13 had individually higher averages of miR-200c than the group aged 1-12 (p<0.05). ANOVA analysis of post-pubertal PVG, MVG and controls showed a statistical significance p=0.0013, revealing a differential expression of miR-200c between these groups. The highest mean of miR-200c expression was observed in post pubertal MVG group of patients, the Tukey\'s post-test between MVG and PVG showed p=0.01. Post pubertal patients had higher miR-200c expression than prepubertal patients (p=0.002). Post pubertal PVG patients showed miR-200c expression values similar to those of prepubertal 46, XY DSD and prepubertal controls. In relation to miR-210, ANOVA analysis of post pubertal PVG, MVG and controls showed statistical significance of p=0.005, with MVG and PVG presenting means of expression superior to the control group (p=0.002). The same pattern was observed in relation to the prepubertal group (p = 0.022). Conclusion: In several tissues, a negative feedback loop in which miR-200c inhibits ZEB1 expression has been demonstrated, and has been associated with modulation of the epithelial-mesenchymal transition (EMT). Elevated levels of ZEB1 associated with low levels of miR-200c have been observed in the genital skin of adult male individuals and rats with hypospadias, reinforcing a potential role of miR-200c in the development of hypospadias. Our findings provide new evidence on circulating miR-200c, which may represent a new strategy for research on 46,XY DSD, and contribute to better understand the development processes of the male external genitalia. (AU)