Improved genetic characterization of congenital adrenal hyperplasia by long-read sequencing compared with multiplex ligation-dependent probe amplification plus Sanger sequencing

Genetic analysis of congenital adrenal hyperplasia (CAH) has been challenging due to high homology between CYP21A2 and its pseudogene CYP21A1P. This study aimed to evaluate the clinical utility of long-read sequencing (LRS) in diagnosis of CAH due to 21-hydroxylase deficiency by comparing with multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing. In this retrospective study, 69 samples including 49 probands from 47 families with high-risk of CAH were enrolled and blindly subjected to detection of CAH by LRS. The genotype results were compared with control methods, and discordant samples were validated by additional Sanger sequencing. LRS successfully identified biallelic variants of CYP21A2 in the 39 probands diagnosed as CAH. The remaining 10 probands were not CAH patients. Additionally, LRS directly identified two pathogenic SNVs (c.293-13C/A>G and c.955C>T) in the presence of interference caused by nearby indels. The cis-trans configuration of two or more single-nucleotide variants and indels (SNVs/indels) identified in 18 samples was directly determined by LRS without family analysis. Eight CYP21A1P/A2 or TNXA/B deletion chimeras comprised of five subtypes were identified and the junction sites were precisely determined. Moreover, LRS determined the exact genotype in two probands that had three heterozygous SNVs/indels and duplication, which could not be clarified by control methods. These findings highlight that LRS could assist in more accurate genotype imputation and more precise CAH diagnosis.