Long-Read Sequencing Solves Complex Structure of CYP21A2 in a Large 21-Hydroxylase Deficiency Cohort

Abstract Context Genetic testing for 21-hydroxylase deficiency (21-OHD) is always challenging. The current approaches of short-read sequencing and multiplex ligation-dependent probe amplification (MLPA) are insufficient for the detection of chimeric genes or complicated variants from multiple copies. Recently developed long-read sequencing (LRS) can solve this problem. Objective To investigate the clinical utility of LRS in precision diagnosis of 21-OHD. Methods In the cohort of 832 patients with 21-OHD, the current approaches provided the precise molecular diagnosis for 81.7% (680/832) of cases. LRS was performed to solve the remaining 144 cases with complex chimeric variants and 8 cases with variants from multiple copies. Clinical manifestations in patients with continuous deletions of CYP21A2 extending to TNXB (namely CAH-X) were further evaluated. Results Using LRS in combination with previous genetic test results, a total of 16.9% (281/1664) CYP21A1P/CYP21A2 or TNXA/TNXB chimeric alleles were identified in 832 patients, with CYP21A1P/CYP21A2 accounting for 10.4% and TNXA/TNXB for 6.5%. The top 3 common chimeras were CYP21 CH-1, TNX CH-1, and TNX CH-2, accounting for 77.2% (217/281) of all chimeric alleles. The 8 patients with variants on multiple copies of CYP21A2 were accurately identified with LRS. The prevalence of CAH-X in our cohort was 12.1%, and a high frequency of connective tissue-related symptoms was observed in CAH-X patients. Conclusion LRS can detect all types of CYP21A2 variants, including complex chimeras and pathogenic variants on multiple copies in patients with 21-OHD, which could be utilized as a first-tier routine test for the precision diagnosis and categorization of congenital adrenal hyperplasia.