Novel perspectives of the genetic etiology of the 46, XY disorders of sex development

Author(s)

Nathália Lisboa Rosa Almeida Gomes

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2019-10-11

Examining board members

Berenice Bilharinho de Mendonça; Tânia Aparecida Sartori Sanchez Bachega; Larissa Garcia Gomes; Gil Guerra Júnior

• Advisor: Berenice Bilharinho de Mendonça

Abstract

Since the genetic etiology of the 46,XY disorders of sex development (DSD) is highly heterogeneous, obtaining a definitive molecular diagnosis by single gene test is challenging. A molecular diagnosis is identified in less than 50% of these patients who are studied by Sanger sequencing, especially those with gonadal dysgenesis (GD). In the last years, massively parallel sequencing (MPS) technologies have been proposed as an efficient sequencing approach by analyzing multiple genes at the same time, as well as allowing the identification of novel genes, as the DEAH-box helicase 37 (DHX37). This gene was previously identified in our cohort as a candidate gene for the embryonic testicular regression syndrome (ETRS), which is considered part of the spectrum of gonadal dysgenesis (GD). In this context, the objectives of this project were: 1) to investigate the molecular etiology of a cohort of patients with 46,XY DSD, identifying variants in genes previously known to be associated with DSD and also in DSD candidate genes; 2) identify novel candidate genes for the DSD etiology, using MPS by panel sequencing and by whole exome sequencing (WES), respectively. We designed an amplicon-based capture panel of 63 genes for targeted sequencing including genes already associated and candidate genes for the DSD etiology, including the DHX37. Sequencing was performed in the Illumina platform. The data was analyzed by an in-house pipeline in order to identify the missense and indels variants and for copy number variations. The variants were classified according to the American College of Medical Genetics. We studied 100 sporadic cases and 14 familial cases from 7 different families, including 45 index-cases with GD (12 of them had ETRS), 54 cases classified as DSD of unknown etiology and 8 cases with androgen synthesis defect. The sporadic cases were studied by target MPS and most of the familial cases were studied by WES. A genetic diagnosis was established in 20.5% of the patients. Pathogenic or likely pathogenic variants were identified in 6 of 45 index-cases (18%) with GD, 10 of 55 index-cases (18.5%) with DSD of unknown etiology and 6 of 8 patients (75%) suspected of having a testosterone synthesis defect, including variants in NR5A1, DHX37, WT1, AMH, MAP3K1, SRD5A2 E HSD17B3 genes. Variants of uncertain significance were identified in 12% of the cohort, highlighting variants in GATA4, MAP3K1, ESR2, CBX2.2. Two variants were identified in 5% of the patients, suggesting a digenic inheritance. The DHX37 variants (n=4) were the most frequent and were identified in four sporadic cases and in three familial cases from two unrelated families with variable phenotypes, including ETRS and partial GD, most of them without Mullerian derivatives. The variants were identified in two conserved domains of the protein (helicase C-terminal and ATP binding domain) and their frequency was enriched among patients with ETRS (44%) and GD (9%) from the cohort in comparison to the frequency of deleterious variants among controls (p < 0.001). The present findings reinforce the relevance of MPS in the 46,XY DSD investigation and indicate that DHX37 variants are a frequent cause of all GD spectrum of, especially for ETRS, being this gene important not only for the development but also for the maintenance of the testicular tissue (AU)

• FAPESP's process: 15/21948-8 - Molecular pathogenesis and characterization of monogenic developmental diseases: a route to Translational Medicine
• Grantee: Nathalia Lisboa Rosa Almeida Gomes
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)