ESPE Yearbook of Paediatric Endocrinology

The Journal of Clinical Endocrinology & Metabolism, 110(2), 406–416. 2025. doi.org/10.1210/clinem/dgae519

Brief Summary: This large original cohort study (n=832) evaluated the diagnostic utility of long-read sequencing (LRS) in patients with 21-hydroxylase deficiency (21-OHD). LRS was applied to 152 cases where conventional methods had failed, successfully detecting complex CYP21A2/TNXB chimeric variants and clarifying ambiguous multi-copy genotypes.

The CYP21A2 gene lies within the highly homologous RCCX module and frequently undergoes recombination with its pseudogene CYP21A1P, making genetic diagnosis of 21-OHD particularly challenging. In this study, LRS not only identified all previously known chimeric subtypes but also uncovered six additional CYP21A2 or TNXB chimeras missed by MLPA or Sanger sequencing. More than half of the 281 total chimeric alleles were detectable only by LRS, highlighting its unique diagnostic power. Thus, LRS emerges not merely as a complementary tool, but as an indispensable and potentially paradigm-shifting diagnostic technology.

The clinical relevance is also significant. CAH-X (TNXA/TNXB chimeras) was identified in 12.1% of patients, with biallelic carriers showing markedly more connective tissue features, such as joint hypermobility and skin hyperextensibility, compared to monoallelic carriers or CAH-X-negative individuals. This genotype-based stratification has direct implications for patient monitoring, genetic counseling, and multidisciplinary care planning.

Additionally, LRS enabled phasing of variants across duplicated CYP21A2 alleles in eight patients, allowing accurate parental origin assignment. Its ability to simultaneously detect pathogenic variants in CYP21A2 and in other key steroidogenesis genes (CYP11B1, CYP17A1, HSD3B2, STAR) supports its role as a comprehensive genotyping platform. Given its cost-effectiveness and added clinical value, the authors advocate LRS as a first-line diagnostic tool in CAH. Importantly, LRS also holds promise for other genetically complex adrenal disorders, such as P450 oxidoreductase deficiency and lipoid CAH, underscoring its expanding role in the molecular diagnosis of pediatric adrenal disorders. As long-read technologies become increasingly accessible, their routine integration into endocrine genetics workflows appears not only justified, but necessary.