Genetic diagnosis of CYP21A2-related CAH: adaptive sampling long-read sequencing is an accurate and scalable solution.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder, commonly caused by variants in CYP21A2 (chr6p21.33), which encodes the 21-hydroxylase enzyme. Genetic diagnosis is challenging due to the high homology between CYP21A2 and its nearby pseudogene CYP21A1P. The current gold standard, PCR-based Sanger sequencing combined with multiplex ligation-dependent probe amplification (MLPA), is labor-intensive, costly, and amenable to PCR bias. Furthermore, it is not reliable in detecting complex structural variants, and it provides no information on whether variants are located on the same allele or not. The purpose of this study was to develop a method based on long-read sequencing (LRS) for accurate diagnostics of CYP21A2 variants and their phasing. Adaptive sampling (AS-)-LRS with chromosome 6 as region-of-interest was applied to DNA from 34 patients clinically diagnosed with CAH. To overcome mapping challenges in the highly homologous regions, we developed NanoCAH, a custom bioinformatic tool that accurately distinguishes between CYP21A2 and CYP21A1P reads. Using AS-LRS and NanoCAH, we genetically confirmed CYP21A2-associated CAH in 32 (94%) of the patients, including reliable phasing of the variants without the need for parental testing. AS-LRS clarified previously ambiguous findings, including the detection of chimeric genes, deletions, and missed variants. Compared to current gold standard methods, AS-LRS proved to be faster and more scalable, while providing greater accuracy in detecting variants within the CYP21A2 region. This makes AS-LRS a promising tool not only for CAH diagnosis but also for genetic testing in other regions with complex genomic architecture.