Advocating Targeted Sequential Screening over Whole Exome Sequencing in 21-Hydroxylase Deficiency.

Objectives: Whole exome sequencing (WES) has emerged as the preferred method for diagnosing a range of Mendelian disorders. Nonetheless, the applicability of WES in genetic diagnosis of 21-hydroxylase deficiency (21-OHD) remains uncertain due to the intricacies involved in molecular analysis of the CYP21A2 gene.

Methods: In this case series, authors report the outcomes of couples or families who underwent WES followed by focused sequential strategy (FSS) targeting CYP21A2 gene hotspot mutations and targeted sequencing of genes associated with Congenital Adrenal Hyperplasia (CAH).

Results: This analysis revealed that WES, when compared to FSS, resulted in six false-negative findings out of seven subjects and one false-positive result. These results were corroborated through validation using Multiplex Ligation-Dependent Probe Amplification (MLPA) and Sanger sequencing.

Conclusions: One major limitation of exome sequencing lies in target enrichment, which often achieves less than 95% coverage of the regions of interest, potentially leading to false negatives. This challenge is particularly pronounced when deciphering the complex genetics of 21-OHD, characterized by intricate pseudogene-derived rearrangements and gene conversions. Additionally, next-generation sequencing (NGS) analysis of the CYP21A2 gene is not straightforward due to reads aligning to pseudogene regions, necessitating stringent computational pipelines with defined targets. However, simple genotyping assays have shown a high positive yield of pseudogene-derived mutations in over 80% of cases, while targeted NGS can be valuable in subjects with initially negative results. Therefore, WES is not recommended as the primary testing method for 21-OHD and may be better suited for rare forms of CAH once CYP21A2 mutations have been ruled out.