High-Resolution Variant Profiling of CAH-Associated Genes Using a Long-Read Sequencing Assay

BackgroundCongenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders primarily caused by mutations in CYP21A2 and CYP11B1. However, accurate genetic diagnosis remains challenging due to the high sequence homology between these genes and their corresponding homologs (CYP21A1P, CYP11B2). MethodsWe developed NanoCAH (Nanopore-based Comprehensive Analysis of CAH), a targeted long-read sequencing assay based on the Cyclone nanopore platform, CycloneSEQ G100-ER. This approach uses gene-specific long-range PCR to amplify CYP21A2 and CYP11B1, enabling detection of all classes of variants including single nucleotide variants (SNVs), copy number variants (CNVs), and gene conversions. ResultsA total of 59 samples were analyzed by NanoCAH, including 26 singletons and 11 trios. Among these samples, NanoCAH successfully detected all 85 variants previously identified by conventional MLPA and Sanger sequencing (100%, 85/85), including SNVs/Indels, deletions and duplications. Importantly, seven Exon 8 deletions and one Exon 8 duplication undetectable by MLPA were identified by NanoCAH assay. In addition, NanoCAH resolved a novel heterozygous 111-bp in-exon tandem duplication in CYP21A2 (c.65_175dup). NanoCAH further accurately resolved haplotypes in twenty cases without parental data, identifying 18 variants in trans and 2 in cis. These results demonstrate NanoCAHs robust capacity to detect complex variant types and provide precise haplotype resolution in a single assay. ConclusionsNanoCAH provides an accurate, cost-effective, and scalable solution for comprehensive CAH genotyping. Its ability to detect complex variant types and resolve haplotypes in a single assay highlights its potential as a first-line diagnostic tool in clinical genetics.