A Comprehensive Pipeline for the Use of Short Read Next-Generation Sequencing (SR-NGS) in CYP21A2 Diagnostic...

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. Methods: 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.