Significance: Sex development involves a precise spatiotemporal expression and interactions of numerous genetic factors, including the WT1 (Wilms tumor 1) gene. Complete and partial loss-of-function WT1 variants are associated with 46,XY disorders/differences of sex development (DSD). Some 46,XX individuals develop testis in absence of the testis-determining gene SRY . We describe a genotype/phenotype association where variants impacting the C-terminal zinc finger (ZF4) of WT1 cause testis development in 46,XX individuals. XX mice carrying a pathogenic variant of ZF4 display masculinization of the fetal gonads. Testis formation may be due to inappropriate interaction between the mutated WT1 and an essential ovarian determinant β-CATENIN. These data show that variants affecting a specific domain of a developmental transcription factor can switch organ fate.

The aim of this study was the molecular characterization of the AR gene as the cause of 46,XY disorder in our population. We studied 41, non related, 46,XY disorder of sexual differentiation index cases, having characteristics consistent with androgen insensivity syndrome (AIS). Genomic DNA was isolated from peripheral blood leukocytes of all patients and 25 family members from 17 non-related families. The AR gene analysis revealed an abnormal sequence in 58.5% of the index patients. All of the complete AIS (CAIS) cases were genetically confirmed, while in the partial form (PAIS) a mutation in AR was detected in only 13 (43.3%). Molecular studies revealed other affected or carrier relatives in 87% of the index cases. The AR mutations were found spread along the whole coding sequence, with a higher prevalence in the ligand binding domain. Nine out of 23 (39%) AR mutations were novel. In 17% of patients with detected AR mutations, somatic mosaicism was detected in leucocyte DNA. In our cohort, long-term follow up gender dysphoria, raised as male or female, was not found. Finally, in suspected PAIS, the identification of AR mutation occurred significantly less than in CAIS patients. Improved knowledge of the components of the AR complex and signaling network might contribute to long term outcome and genetic counseling in AIS patients.

Context: P450 oxidoreductase (POR) deficiency is a rare congenital adrenal hyperplasia with variable severity. The mechanisms of severe mutations and their full metabolic consequences, including drug metabolism, are not fully characterized. Objective: To define the clinical, biochemical, and molecular consequences of a novel homozygous POR missense mutation, p.Gly88Ser (G88S), identified in 4 unrelated Argentine families. Design: A translational study combining clinical case series analysis with comprehensive in vitro molecular and functional characterization of the novel protein variant. Setting: Tertiary pediatric endocrine centers in Argentina and Switzerland. Patients: We report 5 individuals (4 46,XY; 1 46,XX) from 4 unrelated families presenting with disorders of sex development and adrenal dysfunction. Main Outcome Measures: Clinical phenotypes, hormonal profiles, and POR gene sequencing. In vitro analysis of recombinant POR measured flavin content, reductase activity, and support of steroidogenic and drug-metabolizing P450s. Results: All patients were homozygous for the c.262G>A (p.G88S) mutation. This FMN binding domain variant caused protein instability with severe loss of FMN (<30%) and FAD (<15%) cofactors. Steroidogenic activities were virtually abolished (CYP21A2: 1.3%; CYP17A1 17,20-lyase: 5.5% of wild-type), explaining the clinical phenotype. Activities of major drug-metabolizing enzymes were also severely impaired (3%-9% of wild-type), establishing a “poor metabolizer” phenotype. Conclusion: The POR G88S mutation causes one of the most severe forms of PORD described, driven by dynamic protein instability and cofactor loss. It is a critical pharmacogenomic marker, and its recurrence in Argentina suggests a potential screening target.

Background: Persistent müllerian duct syndrome (PMDS) is characterized by the persistence of müllerian duct derivatives in otherwise normally virilized 46,XY males. Biallelic mutations of the anti-müllerian hormone (AMH) and AMH receptor type 2 (AMHR2) genes lead to PMDS type 1 and 2, respectively. Aim: The aims of the study were to report the clinical, hormonal, and genetic findings in a patient with PMDS and discuss surgical strategies to achieve successful orchidopexy. Results: A 4-year-old boy was evaluated after the incidental finding of müllerian derivates during laparoscopy for nonpalpable gonads. Karyotype was 46,XY and laboratory tests revealed normal serum gonadotropin and androgen levels but undetectable serum AMH levels. PMDS was suspected. Molecular analysis revealed a novel variant c.902_929del in exon 5 and a previously reported mutation (c.367C>T) in exon 1 of the AMH gene. Successful orchidopexy was performed in two sequential surgeries in which the müllerian duct structure was preserved and divided to protect the vascular supply to the gonads. Histological evaluation of the testicular biopsy showed mild signs of dysgenesis. Doppler ultrasound showed blood flow in both testes positioned in the scrotum 1.5 years after surgery. Conclusion: PMDS is a rare entity that requires a high index of suspicion (from surgeons) when evaluating a patient with bilateral cryptorchidism. Surgical treatment is challenging and long-term follow-up is essential. Histological evaluation of the testis deserves further investigation.

We hypothesized that DNA methylation is involved in human adrenal functional zonation. mRNAs expression and methylation pattern of RARB, NR4A1 and HSD3B2 genes in human adrenal tissues (HAT) and in pediatric virilizing adrenocortical tumors (VAT) were analyzed. For analysis of the results samples were divided into 3 age groups according to FeZ involution, pre and post-adrenarche ages. In all HAT, similar RARB mRNA was found including microdissected zona reticularis (ZR) and zona fasciculata, but HSD3B2 and NR4A1 mRNAs were lower in ZR (p < 0.05). NR4A1 and RARB promoters remained unmethylated in HAT and VAT. No adrenal zone-specific differences in NR4A1 methylation were observed. In summary, RARB was not associated with ZR-specific downregulation of HSD3B2 in postnatal human adrenocotical zonation. DNA methylation would not be involved in NR4A1 adrenocortical cell-type specific downregulation. Lack of CpG islands in HSD3B2 suggested that HSD3B2 ZR-specific downregulation would not be directly mediated by DNA methylation.