Sequential sequencing reveals the architecture and complexity of genomic variants in patients with Alport syndrome.

Alport syndrome (AS) is a prevalent inherited kidney disorder mainly caused by mutations in COL4A3, COL4A4, and COL4A5 genes. To elucidate the genetic variants of AS, we implemented a sequential sequencing strategy within a Chinese cohort of 555 patients, comprising whole-exome sequencing (WES) for all participants, followed by whole-genome sequencing (WGS), RNA sequencing (RNA-seq), and nanopore long-read sequencing (NLR-seq) for selected individuals. We identify 431 distinct variants in 509 (91.7%) patients, with 42.2% being novel. Beyond WES, additional sequencing approaches resolve 23 patients with noncoding, copy number, or structural variants. Notably, noncoding variants account for 16.2% of detected variants and exhibit ethnic-specific mutagenesis patterns. More interestingly, NLR-seq uncovers two novel types of structural variants, namely large insertions in intronic regions and complex duplication-inversion variants. This study provides deeper insights into the genetic architecture of AS and proposes a research paradigm for improving the genetic diagnosis of inherited diseases.