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

Area of Science:

• Genetics
• Molecular Biology
• Endocrinology

Background:

• Whole exome sequencing (WES) is a common diagnostic tool for Mendelian disorders.
• The genetic complexity of 21-hydroxylase deficiency (21-OHD), involving the CYP21A2 gene, poses challenges for WES applicability.
• Accurate genetic diagnosis is crucial for managing 21-OHD and Congenital Adrenal Hyperplasia (CAH).

Purpose of the Study:

• To evaluate the diagnostic utility of WES compared to a focused sequential strategy (FSS) for 21-OHD.
• To identify the limitations of WES in detecting CYP21A2 gene mutations.
• To determine the optimal genetic testing approach for 21-OHD.

Main Methods:

• Case series analysis of patients undergoing WES.
• Focused sequential strategy (FSS) targeting CYP21A2 gene hotspot mutations.
• Targeted sequencing of Congenital Adrenal Hyperplasia (CAH)-associated genes.
• Validation using Multiplex Ligation-Dependent Probe Amplification (MLPA) and Sanger sequencing.

Main Results:

• WES demonstrated a high rate of false negatives (6/7) and one false positive compared to FSS for 21-OHD.
• Limitations in WES target enrichment (<95% coverage) contribute to false negatives.
• CYP21A2 gene analysis is complicated by pseudogene rearrangements and gene conversions, affecting WES accuracy.

Conclusions:

• WES is not recommended as a primary diagnostic method for 21-OHD due to significant limitations in accuracy.
• Targeted sequencing and genotyping assays are more effective for detecting CYP21A2 mutations, especially pseudogene-derived ones.
• WES may be considered for rare forms of CAH after CYP21A2 mutations are ruled out by targeted methods.