CYP17A1 Pathogenic Variants in 26 Chinese Patients with 17α-Hydroxylase Deficiency by Targeted Long-read Sequencing.

Abstract

17α-hydroxylase/17,20-lyase deficiency (17-OHD) is a rare subtype of congenital adrenal hyperplasia (CAH) caused by homozygous or compound heterozygous pathogenic variants in the CYP17A1 gene. This study aimed to identify and characterize pathogenic variants in individuals with 17-OHD, and to classify and validate the pathogenicity of novel variants. Variants were identified via targeted long-read sequencing (TLRS) of the entire CYP17A1 gene in enrolled 17-OHD patients. The American College of Medical Genetics and Genomics guidelines were employed to assess the pathogenicity of novel variants. A minigene splicing assay was utilized to determine the impact of variants on RNA splicing. This study encompassed 26 patients with 17-OHD, detecting two trans pathogenic variants per patient using the TLRS method. A total of 20 pathogenic variants in the CYP17A1 were identified, with variant c.985_987delinsAA being the most frequent (28/52 alleles), followed by variant c.1459_1467del (4/52 alleles). Five novel variants including c.280T>C, c.470T>A, c.636_637del, c.866A>G, and c.1095del, were classified as pathogenic/likely pathogenic ones according to ACMG criteria. The minigene assay revealed c.866A>G in exon 5 causes a frameshift due to a 104 base pair deletion, while c.470T>A generates two transcripts, with vast majority spliced like the wild-type, and a small fraction lack 35 base pairs in the 5' flank of exon 3. The TLRS can determine the cis/trans orientation of two distant variants. Five novel pathogenic variants were reported, broadening the spectrum of CYP17A1 pathogenic variant. The variant c.866A>G, located deep in exon, affects gene function through mechanisms of aberrant splicing.