Detection and characterization of the types of CYP21A1P/CYP21A2 and TNXA/TNXB fused genes by long-read sequencing among children with Steroid 21-hydroxylase deficiency

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To assess the diagnostic efficiency of long-read sequencing (LRS) for the determination of CYP21A1P/CYP21A2 and TNXA/TNXB fusion genotypes among children with 21-hydroxylase deficiency (21-OHD) and explore their clinical characteristics. LRS sequencing was carried out on 30 children diagnosed with 21-OHD at the Department of Endocrinology, Fujian Children's Hospital between November 2022 and September 2023 by clinical symptoms or conventional Sanger sequencing combined with multiple ligation-dependent probe amplification (MLPA). The results of the two methods were compared. Clinical data of the children were collected and analyzed. This study has been approved by the Medical Ethics Committee of the Fujian Children's Hospital (Ethic No. 2022ETKLR10024). Of the 30 children with 21-OHD, 11 (36.7%) were found to carry CYP21A1P/CYP21A2 and TNXA/TNXB fusion genes by LRS. The most common type of fused CYP21A1P/CYP21A2 gene was CH-1 (72.7%), and 1 (3.3%) was found to harbor TNXA/TNXB CH-1. Eleven cases (36.7%) were found to carry large deletions by Sanger sequencing combined with MLPA, with the most common one being CYP21A2 exons 1-3 del (72.7%), which was followed by CYP21A2 exons 1-7 del (18.2%). Follow up of 11 patients carrying a fusion gene revealed that 6 were sale wasting (SW) types, 5 were simple virilizing (SV) types, whilst no non-classical (NC) type was found. Four girls had presented with central precocious puberty (CPP). One child carrying TNXA/TNXB CH-1 had presented with CAH-X syndrome. Compared with Sanger sequencing combined with MLPA detection method, LRS sequencing was able to differentiate the subtypes of CYP21A1P/CYP21A2 and TNXA/TNXB fusion genes, pinpoint the breakpoints of the deletions, and directly determine the cis-trans position without the need to analyze the genotype of the pedigree members, which has provided a reliable method for the typing of 21-OHD. As some fusion genes may retain 21-hydroxylase activity, female carriers may have a higher incidence of CPP.