ABSTRACT
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.
ABSTRACT
PURPOSE: Congenital Adrenal Hyperplasia (CAH) is one of the highly prevalent autosomal recessive endocrine disorders. The majority of CAH cases result from mutations in the CYP21A2 gene, leading to 21-hydroxylase deficiency. However, with the pseudogene-associated challenges in CYP21A2 gene analysis, routine genetic diagnostics and carrier screening in CAH are not a part of the first-tier investigations in a clinical setting. Furthermore, there is a lack of data on the carrier frequency for 21-OH deficiency. Therefore, this study is aimed at investigating the carrier frequency of common pseudogene derived CYP21A2 mutations in Southern India. METHODS: Recently, a cost-effective Allele-specific PCR based genotyping for CYP21A2 hotspot mutations has been demonstrated to be a highly specific and sensitive assay at the authors' center. Leveraging this approach, a total of 1034 healthy individuals from South India underwent screening to identify the carrier frequency of nine hotspot mutations in the CYP21A2 gene. RESULTS: In this study, it was observed that 9.76% of the subjects were carriers for one or more of the nine different CYP21A2 mutations. Among the carriers, the most common was the large 30 kb deletion, followed by II72N, E6 CLUS, and I2G mutations. CONCLUSION: We have identified a high prevalence of CYP21A2 mutation carriers in Southern India. These findings emphasize the importance of implementing and expanding cost-effective genetic diagnostics and carrier screening throughout India. Such initiatives would play a crucial role in managing the disease burden, enabling early intervention, and establishing guidelines for CAH newborn genetic screening in the country. This study represents the first carrier screening data on CYP21A2 hotspot mutations from India and is the largest study conducted till date in this context.