RESUMO
Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder due to enzyme defects in adrenal steroidogenesis. Several genes code for these enzymes, out of which mutations in the CYP21A2 gene resulting in 21 hydroxylase deficiency, contribute to the most common form of CAH. However, pseudogene imposed challenges complicate genotyping CYP21A2 gene, and there is also a lack of comprehensive molecular investigations in other genetic forms of CAH in India. Here, we describe a cost-effective, highly specific, and sensitive Allele Specific PCR (ASPCR) assay designed and optimized in-house to screen eight common pathogenic mutations in the CYP21A2 gene. We have also established and utilized a multiplex PCR assay for target enrichment and Next-generation sequencing (NGS) of CYP11B1, CYP17A1, POR, and CYP19A1 genes. Following preliminary amplification of the functional gene CYP21A2, ASPCR based genotyping of eight common mutations - P30L, I2G, 8BPdel, I172N, E6CLUS (I235N, V236E, M238K) V281L, Q318X, and R356W was carried out. These results were further validated using Sanger and Next-generation sequencing. Once optimized to be specific and sensitive, the advantage of ASPCR in CYP21A2 genotyping extends to provide genetic screening for both adult and paediatric subjects and carrier testing at a low cost and less time. Furthermore, multiplex PCR coupled NGS has shown to be cost-effective and robust for parallel multigene sequencing in CAH.