21-hydroxylase deficiency (21-OHD) represents the most common form of congenital adrenal hyperplasia (CAH) due to CYP21A2 gene pathogenic variants. Τhe aim of this study was the identification of CYP21A2 variants in 500 subjects of Greek origin with a suspicion of 21-OHD and, by using the existing hormonal assessment and genotypes of the 500 subjects tested, to identify a biomarker that could differentiate between the heterozygotes and the cases with no pathogenic variants identified. Five hundred subjects with clinical suspicion of 21-OHD underwent CYP21A2 gene sequencing and Multiplex Ligation Dependent Probe Amplification (MLPA). Genetic diagnosis was achieved in 27.4% of the subjects tested, most of which presented with the non-classic form (NC) of 21-OHD. Heterozygotes accounted for 42.6% of cases, whereas no pathogenic variants were identified in 27% of cases. De novo aberrations, duplications, and five novel variants were also identified. Statistical analysis revealed that the difference between the basal and 60′ post-ACTH stimulation 17-hydroxyprogesterone concentrations (Δ17-OHP^60-0) could be a potential biomarker (p < 0.05) distinguishing the heterozygotes from the cases with no pathogenic variants identified, although no clear cut-off value could be set. Further analysis revealed overlapping clinical manifestations among all the subjects tested. The presented phenotypic traits of the subjects tested and the inability to identify a discriminative biochemical marker highlight the importance of comprehensive CYP21A2 genotyping to ascertain the correct genetic diagnosis and proper genetic counselling.

Background: Although Short Read Next-Generation Sequencing (SR-NGS) is widely employed in diagnosis, its application in CYP21A2 genotyping remains limited due to its high sequence homology with its pseudogene, CYP21A1P. Herein, we present (a) a complete pipeline for the diagnostic use of SR-NGS in CYP21A2 genotyping following its assessment; (b) two distinct in-house bioinformatics pipelines for variant calling; and (c) the results by implementing this pipeline in diagnosis. Method s: A total of 221 subjects were studied, comprising a pilot group (n = 21), recruited for assessment of the assay, and a study group (n = 200) categorized in three subgroups, referred for CYP21A2 genotyping. Both groups underwent SR-NGS. Two different bioinformatics algorithms for variant calling were applied and variant filtration was performed using VarAFT (v2.17). In the study group, MLPA was additionally employed. Results: The SR-NGS assay, employing GATK HaplotypeCaller, demonstrated 100% sensitivity and specificity when compared to Sanger Sequencing; however, complex CYP21A2 rearrangements cannot be detected. In the study group, pathogenic variants were identified in 52.7%, 100% and 25% of cases in subgroups (a), (b) and (c) respectively, whereas gene duplications accounted for 12.3% (7/57) of subjects tested. Conclusions: This study provides a comprehensive protocol for the use of SR-NGS in a CYP21A2 diagnostic genotyping, integrating complementary bioinformatics pipelines and MLPA for copy number analysis.