Abstract
Hypospadias is a common birth defect where the urethral opening forms on the ventral side of the penis. We performed integrative methylomic, genomic, and transcriptomic analyses to characterize sites of DNA methylation that influence genital development. In case–control and case-only epigenome-wide association studies (EWAS) of preputial tissue we identified 25 CpGs associated with hypospadias characteristics and used one-sample two stage least squares Mendelian randomization (2SLS MR) to show a causal relationship for 21 of the CpGs. The largest difference was 15.7% lower beta-value at cg14436889 among hypospadias cases than controls (EWAS P = 5.4e−7) and is likely causal (2SLS MR P = 9.8e−15). Integrative annotation using two-sample Mendelian randomization of these methylation regions highlight potentially causal roles of genes involved in germ layer differentiation (WDHD1, DNM1L, TULP3), beta-catenin signaling (PKP2, UBE2R2, TNKS), androgens (CYP4A11, CYP4A22, CYP4B1, CYP4X1, CYP4Z2P, EPHX1, CD33/SIGLEC3, SIGLEC5, SIGLEC7, KLK5, KLK7, KLK10, KLK13, KLK14), and reproductive traits (ACAA1, PLCD1, EFCAB4B, GMCL1, MKRN2, DNM1L, TEAD4, TSPAN9, KLK family). This study identified CpGs that remained differentially methylated after urogenital development and used the most relevant tissue sample available to study hypospadias. We identified multiple methylation sites and candidate genes that can be further evaluated for their roles in regulating urogenital development.
Highlights
Hypospadias is a common birth defect in boys characterized by the urethral opening forming on the ventral side of the penis rather than at the meatus
In the hypospadias epigenome-wide association study (EWAS) we adjusted for surrogate variables (SVs) to correct for differences between cases and controls, estimated cell types, Methylation principal components (mePCs), subject age, and tissue age
In methylome-wide analyses we identified differential methylation at 10 CpGs between hypospadias cases and controls (FDR < 0.20), 9 CpGs differentially methylated with chordee (FDR < 0.15), and 6 CpGs differentially methylated by severity (FDR < 0.15) (Table 2)
Summary
Hypospadias is a common birth defect in boys characterized by the urethral opening forming on the ventral side of the penis rather than at the meatus. Genome-wide association studies (GWAS) of hypospadias do not replicate variation in these candidate genes at the genome-wide level (P < 5e−8), rather they have identified other genomic regions that do not fully explain hypospadias r isk[10]. One previous epigenome-wide association study (EWAS) by Choudhry et al identified 14 CpGs associated with hypospadias (P < 1e−4) among a sample of 12 affected boys and 8 boys without h ypospadias[13]. No study to date has used an epigenome-wide approach to identify CpGs differentially methylated in relationship to chordee or hypospadias severity. We first conducted epigenome-wide association studies for hypospadias (case–control), chordee (case-only), and severity (case-only). We used genetic instrumental variable analysis for differentially methylated CpGs and genes within regions of differential methylation to characterize causal relationships in the etiology of multiple features of hypospadias development
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