PD48-03: How far should we explore Hypospadias? Next-generation sequencing applied to a large cohort of hypospadiac patients.

Nicolas KALFA*, Anne BERGOUGNOUX, Pascal PHILIBERT, Nadège SERVANT-FAUCONNET, Montpellier, France, Alice FAURE, MARSEILLE, France, Jean BREAUD, Nice, France, Laura GASPARI, Charles SULTAN, Françoise PARIS, Vuthy EA, Montpellier, France

Introduction: Next-generation sequencing (NGS) is generally used for patients with severe disorders of sex development (DSD). However,
NGS has not been applied extensively for patients with hypospadias only and most affected children do not benefit from an etiological diagnosis. We aim to
evaluate the clinical usefulness of NGS for patients with hypospadias, regardless of severity.

Methods: Prospective multicenter research including 293 children with glandular to penoscrotal hypospadias (no undescended testis, no micropenis). After
excluding likely pathogenic AR variants by Sanger sequencing, an NGS panel tested 336 genes including unexplored candidates in 284 patients. Outcome
Measurement was the rate of pathogenic and likely pathogenic variants using REVEL, ClinVar and in-house tools.

Results: Likely pathogenic variants were identified in 16 (5.5%) patients with both Sanger sequencing and NGS taken into account. Some genes were related to
DSD (AR, NR5A1, HSD17B3, MAMLD1), but reverse-phenotyping revealed two syndromic disorders with midline defects (MID1) and alteration in the retinoic acid
signaling pathway (RARA). Coverage analysis revealed an 18q deletion. Identification of likely pathogenic variants increased with hypospadias severity. Other
variants of unknown significance (VUS) in genes implicated in hypogonadotropic hypogonadism, Noonan syndrome and genital tubercle development were also
identified. Genetic study mainly focused on exonic variants and most cases remain unexplained.

Conclusions: NGS reveals either minor forms of DSD, undiagnosed syndromes or candidate rare variants in new genes, indicating that even patients with mild
hypospadias benefit from advanced sequencing techniques. Early molecular diagnosis would help improve follow-up at puberty and medical counseling for initially
undiagnosed syndromes. Future studies will improve the diagnosis by investigating the contribution of VUS.

Source of Funding: Public funding from the National Reference Network for Rare Disease, Genital Development DSD DevGen.