GRAde

GRAde identifies chimeric CYP11B1/CYP11B2 genes and maps crossover breakpoints and gene conversion events underlying glucocorticoid‑remediable aldosteronism using long‑read sequence data.

Key Features:

• Target enrichment (PCR): Amplifies the genomic region containing potential CYP11B1/CYP11B2 chimeric sequences prior to sequencing.
• Sequencing technology: Uses long‑read sequencing data generated on the Oxford Nanopore MinION platform.
• Allele‑discriminating detection: Leverages nonidentical bases between CYP11B1 and CYP11B2 to discriminate parental and recombinant sequence segments.
• Crossover and gene conversion identification: Pinpoints crossover sites and detects potential gene conversion events within the CYP11B1/CYP11B2 locus.
• Chimera visualization: Produces visual representations of chimeric gene structures inferred from long reads.
• Cross‑validation support: Results can be compared with Sanger sequencing for validation of identified crossover regions.
• PCR artifact discrimination: Differentiates true genomic crossover patterns from PCR‑suggested chimeric artefacts.

Scientific Applications:

• Genetic diagnosis of GRA: Identification and characterization of chimeric CYP11B1/CYP11B2 genes responsible for glucocorticoid‑remediable aldosteronism.
• Breakpoint mapping: Precise mapping of crossover breakpoints in patient samples, demonstrated by identification of crossover patterns in 30 of 36 patients in a reported study.
• Gene conversion analysis: Detection of gene conversion events, including observation that 53.3% of cases exhibited gene conversion within intron 2 in the cited cohort.
• Detection of PCR false‑positives: Resolution of instances where PCR indicated chimeric genes but no crossover pattern was found (six such cases reported).

Methodology:

Long‑read sequences from Nanopore MinION of PCR‑amplified CYP11B1/CYP11B2 regions are analyzed by the GRAde pipeline using nonidentical base positions to pinpoint crossover sites, detect gene conversion events, and generate visualizations of chimeric forms.