Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by a deficiency of enzymes involved in cortisol biosynthesis. More than 90% of the cases are due to 21-hydroxilase deficiency. This leads to the accumulation of 17-hydroxyprogesterone (17OHP) and allows diagnosis of this disorder in newborn screening (NS) programs. Premature newborns typically have higher levels than full-term newborns, making the use of age-appropriate cut-off values important for accurate interpretation. The aim of our study was to update the gestational age-adjusted cut-off values for 17OHP, the methodology currently used in Argentina by the jurisdictions participating in the National Program for the Strengthening of Early Detection of Congenital Diseases. Cut-off limits were obtained using percentiles of the distribution of 17OHP values for each gestational age on 53,230 dry blood spot (DBS) samples. Sensitivity, specificity, false positive rate, positive predictive value, and incidence were calculated. We successfully established gestational age-specific cut-off values for 17OHP. The analysis showed a sensitivity of 100%, specificity of 99.52%, a false positive rate of 0.49%, and a positive predictive value of 1.54%. The incidence of CAH was 1:13,308 live births. These findings underscore the importance of using population-appropriate cut-off limits. Such harmonization enables comparability among regional neonatal screening programs with the added benefit of decreasing the rate of recalls and associated costs.

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 four 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: Five individuals (four 46,XY; one 46,XX) from four 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. Conclusions: 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.