High-fidelity long-read sequencing reveals a complex RCCX locus at the single-nucleotide level in Korean patients with congenital adrenal hyperplasia

Background The RCCX locus, resulting from segmental duplication, exhibits extensive sequence identity and modular variations because of unequal crossover events, leading to copy number variations and the formation of chimeric genes between active and pseudogenes. Precise characterization of this locus is essential for molecular diagnosis, because aberrations within this region can cause congenital adrenal hyperplasia (CAH), an autosomal recessive disorder. However, the intricate modular structures, comprising mono-, bi-, and tri-modular haplotypes, have posed challenges to accurate analysis using conventional sequencing technologies. Methods In this study, we analyzed the segmentally duplicated RCCX locus using a pangenome-based approach. High-fidelity long-read sequencing data were produced to generate high-quality personalized genome assemblies from patients with CAH. Their RCCX haplotypes were fully resolved at the single-nucleotide level, and compared with those from non-patient populations. Results We generated high-fidelity long-read sequencing data and constructed 26 phased genome assemblies from 13 Korean patients with CAH, achieving single-nucleotide resolution analysis of the RCCX locus. Furthermore, by integrating human draft pangenome reference data, we analyzed non-patient cohorts and identified characteristic variations unique to the Korean patient population and novel structural variants formed through DNA damage and homologous recombination. Conclusions Our findings provide a key methodological framework for resolving segmentally duplicated genomic regions associated with genetic disorders. This study is expected to serve as a foundation for the precise molecular diagnosis of patients with CAH.