Prospective CYP21A2 Genotype Analysis and Steroidomic Profiling in Indian Women Evaluated for PCOS/PMOS

Abstract Purpose Data on the prevalence of genetically confirmed non-classic congenital adrenal hyperplasia (NCCAH) and CYP21A2 heterozygous carriers (HTZ-C) among Indian women with Polycystic ovarian syndrome (PCOS)/Polyendocrine metabolic ovarian syndrome (PMOS) are scarce. We aimed to determine their prevalence and evaluate ACTH-stimulated steroid profiling as a tool for triaging women for genetic testing. Methods We prospectively enrolled 100 women with PCOS/PMOS diagnosed according to the Rotterdam criteria. All participants underwent ACTH-stimulated steroid profiling using liquid chromatography–tandem mass spectrometry (LC–MS/MS), followed by comprehensive CYP21A2 sequencing and multiplex ligation-dependent probe amplification (MLPA) to identify sequence variants and copy-number changes. Results Combined CYP21A2 sequencing and MLPA identified NCCAH in 1% and HTZ-C in 5% of participants. The one participant with NCCAH had p.Pro31Leu/30-kb deletion. The HTZ-C variants included 5′UTR, 3′UTR, p.Ile173Asn, p.Arg357Trp, and a 30-kb deletion, with non-classic and classic variants accounting for 2% and 3%, respectively. The p.Val282Leu variant was notably absent. Sequencing demonstrated 100% sensitivity and 88.4% specificity, whereas MLPA demonstrated 50% sensitivity and 100% specificity. An ACTH-stimulated [100 × (21-deoxycortisol + 17-hydroxyprogesterone)/corticosterone] ratio >16.56 demonstrated 100% sensitivity and 96.8% specificity for identifying participants with NCCAH or CYP21A2 heterozygosity. Conclusion The yield of classic CYP21A2 variants was similar to global reports, while the low yield of non-classic variants and absence of p.Val282Leu highlight important ethnic differences. ACTH-stimulated LC–MS/MS steroid profiling may aid in triaging for CYP21A2 sequencing with or without MLPA.