TRANSCRIPTOME AND WHOLE EXOME SEQUENCING TO ELUCIDATE DIFFERENT PHENOTYPES OF DISORDERS OF SEXUAL DEVELOPMENT IN 46,XY PATIENTS

Abstract

Conditions that present atypical genital and / or gonadal development leading to a disagreement among genetic, gonadal and phenotypic sex are classified as Sex Differentiation Disorders (DDS). Several genes are known to act in the pathways of sex determination and differentiation, such as: WT1, SRY, NR5A1, DMRT1, SOX9, GATA4, NR0B1, HAM, RA, SRD5A2, HSD17B3, among others. However, after screening for mutations in these genes, two important questions arise. The first of these concerns cases in which mutations are identified. In these cases, the main challenge is to investigate the biological effect they exert and what their real influence on the phenotypes would be. The second question to be addressed is that through gene-to-gene sequencing, about 60-70% of cases remain with undefined etiology. We propose this project, that will be divided into two parts, in attempt to address these two issues based on the results of previous studies. The first will be to analyze gene function in vitro by reporter gene expression and in vivo through the transcriptome of biopsy material from patients who have mutations at two nuclear receptors (RA and NR5A1), in order to investigate how these changes affect the regulation of genes, causing a possible imbalance on the pattern of gene expression, and correlate the observed effects with the different phenotypes presented by patients. In vitro studies on mammalian cell transfections will be performed, in addition RNA-seq and Real Time PCR, with RNA obtained from the gonads or genital tissues will be also studied. The second part aims to analyze the exome of 10 patients classified as idiopathic DDS 46,XY, in order to identify pathogenic variants and eventually reveal new genes involved in testicular differentiation. This work may contribute to a better understanding of the molecular mechanisms that may explain the phenotype of the patients, as well as, it will serve to implement the exome and transcriptome studies in our laboratory.