Allele-Specific PCR and Next Generation Sequencing based genetic screening for Congenital Adrenal Hyperplasia in India

Abstract Purpose Genotyping CYP21A2 gene is known to be extremely challenging and is not utilized as a first tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating extended panel of genes implicated in CAH. This study aims to establish a comprehensive genetic screening strategy for five genes in CAH. Methods Allele-Specific Polymerase Chain Reaction (ASPCR) for hotspot mutations in CYP21A2 gene followed by targeted Next-Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes were carried out to screen 72 clinically diagnosed CAH subjects from India. Results Utilizing ASPCR, 88.7% (n = 55/62) of the subjects suspected with 21 hydroxylase deficiency were positive for CYP21A2 hotspot mutations. Utilizing NGS, the ASPCR assay was found to highly sensitive and specific for screening these hotspot mutations. Additionally, through targeted NGS, six study subjects were positive for other CYP21A2 variants: one with a novel c.1274G > T, three with c.1451G > C and one with c.143A > G variant. One subject was compound heterozygous for c.955C > T/c.1042G > A variants identified using ASPCR and NGS. One subject suspected for a simple virilizing 21-hydroxylase deficiency was positive for a CYP19A1:c.1142A > T variant. CYP11B1 variants (c.1201-1G > A, c.1200 + 1delG, c.412C > T, c.1024C > T, c.1012dup, c.623G > A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. Conclusion With an overall mutation-positivity rate of 97.2% (140/144 alleles), ASPCR followed by a multigene targeted NGS assay has shown to be highly sensitive and specific as a cost-effective and comprehensive diagnostic tool for CAH in a clinical setting.