Abstract
Introduction: Despite the use of robust techniques for diagnosis, such as arrays and large-scale sequencing of patients with differences of sex development (DSD), the etiology of a great number of DSD patients remains unclear. Investigation of alternative signaling pathways and epigenetic factors is scarce in 46,XY DSD patients. The ZEB proteins have been related to the occurrence of hypospadias in humans, a feature often observed in the atypical genitalia of patients with 46,XY DSD. Additionally, miR-200c has been reported to regulate ZEB. Objective: To evaluate the expression of miR-200c in plasma samples of 46,XY DSD patients with unknown etiology. Methods: Plasma miR-200c of six adult 46,XY DSD patients with unknown etiology (age 18-33, mean 19±8) and 15 adult male controls (age 18-55, mean 29±10 yo) were analyzed. External Masculinization Score (EMS) was used to describe the undervirilization degree of patients’ external genitalia and to classify them in two groups with low EMS (LEMS: 0-4.9 points) and high EMS (HEMS: 5-10 points). All patients presented atypical genitalia with hypospadias. miR-200c was selected based on its targeting to ZEB1 and in silico analysis; miR-23a was used as internal normalization control. RNA was extracted from plasma samples with Magmax Mirvana Total RNA isolation kit. cDNA was synthesized using TaqMan Advanced miRNA cDNA Synthesis Kit and qPCR was performed using TaqMan Advanced miRNA. The data analysis of qPCR results of patients, of each individualized patient and also the EMS groups were compared with the control group by statistical test. Results: LEMS group presented lower expression values of miR-200c when compared to HEMS group (P=0.0001) and control group (p=0.0009), but no difference was observed when comparing HEMS group and controls, the two patients with lower miR-200c expression presented the lowest EMS (EMS- 3 and 3.5). Altogether, patients presented lower values of miR-200c, although not significantly (p=0.09). Discussion: These findings corroborate with previous literature data correlating miR200-c, ZEB1 and hypospadias. The regulatory loop of miR-200c/Zeb1 was previously demonstrated in rats with hypospadias, confirming that low expression of miR-200c induce a higher Zeb1 expression. The ZEB1 upregulation in penile tissue is positively correlated with the severity of hypospadias in animal models and humans. In the present study, 46,XY DSD patients with severe genital undervirilization had lower miR-200c expression in plasma. Conclusion: Plasma miRNA expression patterns may be a new strategy research in 46,XY DSD, contributing for understanding the processes involved in the external genitalia development.
SUN-709 MiR-200c Expression Profiles in Plasma of 46,XY DSD Patients of Unknown Etiology
