Evolution of molecular diagnostic strategies for 21-hydroxylase deficiency: from classical methods to advanced genomic techniques

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most technically challenging monogenic conditions for molecular diagnosis, owing to the complex genomic architecture of the CYP21A2 locus and the extensive homology between the functional gene and its pseudogene. Over the past decades, diagnostic approaches have evolved from locus-specific assays, such as allele-specific polymerase chain reaction, to the combined use of Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA), which together constituted the historical gold-standard approach strategy capable of identifying pathogenic variants, deletions and chimeric alleles. Although short-read next-generation sequencing expanded variant detection and enabled simultaneous analysis of multiple adrenal genes, its performance remains limited within the RCCX region due to misalignment artefacts and inability to resolve complex structural rearrangements. Recently, long-read sequencing (LRS) has emerged as a single-platform technology capable of resolving the CYP21A2–CYP21A1P module, accurately detecting all variant classes and directly determining cis/trans phase. Comparative studies demonstrated complete concordance with standard methods while revealing additional rearrangements previously undetectable, positioning LRS as a future reference method for high-resolution genotyping. These advances extend beyond diagnostic refinement to impact population-level strategies, where integration of molecular testing into newborn screening algorithms reduces false positives, accelerates referral pathways, and enhances salt-wasting risk prediction. In this review, we summarized the historical development, technical limitations, and clinical implications of current and emerging molecular approaches for CAH diagnosis. We highlight how LRS and integrative analytical tools are reshaping clinical practice, refining genetic counselling, and guiding personalized therapeutic strategies. Collectively, these innovations represent a decisive step toward precision Endocrinology.