SUN-166 Epigenetic Insights into Hypospadias: Methylation Analysis of Monozygotic Twins Discordant for SGA and Genital differentiation

Abstract Disclosure: B.L. Braga: None. R.L. Batista: None. A.D. Narcizo: None. A.D. Reis: None. M.F. Funari: None. M.Y. Nishi: None. S. Domenice: None. F.E. de Zegher: None. M. Maschietto: None. B.B. Mendonca: None. Background: The etiology of hypospadias is multifactorial, encompassing genetic, environmental, and epigenetic factors. This condition is 10 times more common among SGA individuals than in the general population. Increasing evidence suggests that IUGR may contribute to the development of hypospadias through epigenetic modifications affecting urogenital differentiation. Reports on monozygotic twins discordant for hypospadias consistently show that the affected twin has a lower birth weight, reinforcing the potential link between IUGR and altered epigenetic programming. This study aims to investigate the epigenetic landscape of monozygotic twins discordant for SGA status and hypospadias. Patients and methods: We reported on a pair of monozygotic twins (46,XY) who were born at term after a normal gestation. One boy (birth weight, 3.0 kg; length, 48 cm) had normal genitalia, whereas the other twin (birth weight, 1.7 kg; length, 43 cm) had proximal hypospadias with testes in labioscrotal folds; he had no other congenital abnormalities1. Whole-exome sequencing (WES) was performed on the sibling with hypospadias using SureSelectXT Human All Exon V6 kits. DNA methylation profiling from peripheral blood leukocytes was conducted on both twins using the Illumina MethylationEPIC BeadChip arrays. Probes with an absolute delta β-value ≥ 0.15 between twins were selected for further analysis. Genes were evaluated for enrichment pathways and biological processes using KEGG and Gene Ontology (GO) databases. Features with FDR <0.05 were considered significant. Results: WES and CNV analysis did not identify pathogenic variants associated with hypospadias. DNA methylation analysis revealed 4,436 differentially methylated probes (Δβ ≥ 0.15) between the twins. Enrichment pathways identified 33 pathways (FDR < 0.05, 221 genes), including cortisol synthesis and secretion, GnRH secretion, and estrogen signaling, suggesting hormonal influences on urogenital differentiation. Additionally, 36 enriched biological processes (FDR < 0.05, 266 genes) were identified, highlighting protein tyrosine kinase activity and receptor-mediated signaling, both critical for cell differentiation and development. Conclusion: Our findings reinforce the role of epigenetic modifications in urogenital development and highlight pathways relevant to hormone signaling. These results support the hypothesis that intrauterine growth restriction and epigenetic reprogramming contribute to the pathogenesis of hypospadias, opening avenues for further research into early-life environmental influences on genital differentiation. Reference: 1. Mendonca BB, Billerbeck AEC, de Zegher F. Nongenetic male pseudohermaphroditism and reduced prenatal growth. N Engl J Med. 2001;345(15):1135-1135. doi:10.1056/NEJM200110113451518 Presentation: Sunday, July 13, 2025