LAUSR

Abstract Genitourinary (GU) anomalies are among the most common categories of birth defects and consist of disorders of sexual differentiation and congenital anomalies of kidney and urinary tract (CAKUT). Disorders of sexual differentiation include hypospadias (1 in 125 male births), cryptorchidism (3-6% of full term male births) and ambiguous genitalia (1:2000-1:4000 births). Though the prevalence of GU anomalies is high, their causes are poorly understood. A genetic change contributing to disease phenotypes is a copy number variant (CNV; a micro-deletion or micro-duplication of a small chromosomal region). When CNVs occur in dosage-sensitive genes, a disease phenotype can arise. Using array comparative genomic hybridization (aCGH), adenylyl cyclase 2 (ADCY2) CNVs were identified in a patient with hypospadias and a patient with ambiguous genitalia. ADCY2 is a member of the adenylyl cyclase class III family of genes that are responsible for converting ATP into cAMP. cAMP, is involved in several essential processes required for GU development and function including testicular descent and androgen production. Through a retrospective literature and database (DECIPHER, ICSA, ClinVar), nine patients with GU abnormalities and ADCY2 CNVs were identified. These GU anomalies consisted of upper and lower tract defects. The incidence of ADCY2 CNVs was determined by qPCR in a cohort of 378 GU abnormal patients and 45 control patients. ADCY2 CNVs (duplications) were present in 1.59% of patients with GU anomalies and 0% of control patients. The incidence of ADCY2 CNVs in the general population (Database of genomic variants) is 0.65%. Expression of ADCY2 in the GU tract was localized to renal tubules, bladder muscle, urethra, and interstitial cells of the testis with the highest expression in the testes. This expression pattern suggests a role for ADCY2 in steroid biosynthesis. When Adcy2 was overexpressed in the murine Leydig cell line MLTC-,1 cAMP compared to control MLTC-1 cells (4342.20 pmol/µl vs 2231.59 pmol/µl, two-way ANOVA <0.05), steroidogenic acute regulatory factor (StAR) expression and testosterone (52.33 ng/dL vs. undetectable, two-way ANOVA <0.05) were increased. Notably, the increase in testosterone production occurred independent of human chorionic gonadotropin (hCG) stimulation suggesting a ligand-independent, constitutively active system. In contrast to control cells transfected with an empty vector, hCG administration to ADCY2 overexpressing cells induced a slight additional increase in testosterone production. The lack of a robust response to hCG led to examination of the luteinizing hormone receptor (LHR). LHR was downregulated and internalized in Adcy2 overexpressing cells upon hCG stimulus. These findings suggest that microduplications in humans encompassing ADCY2 may contributed to lower tract genitourinary birth defects.